ABCG9 ABCG11 and ABCG14 ABC transporters are requiredfor vascular development in Arabidopsis
Rozenn Le Hir123 Clement Sorin2 Dipankar Chakraborti1dagger Thomas Moritz1 Hubert Schaller4 Frederique Tellier34
Stephanie Robert1 Halima Morin5 Laszlo Bako2 and Catherine Bellini123
1Umea Plant Science Centre Department of Forest Genetics and Plant Physiology Swedish University of Agricultural
Sciences Sndash90187 Umea Sweden2Umea Plant Science Centre Department of Plant Physiology Umea University Sndash90183 Umea Sweden3UMR 1318 AgroParisTech Institut Jean-Pierre Bourgin Institut National de la Recherche Agronomique Centre de Versailles
RD10 78026 Versailles Cedex France4Departements Reseaux Metaboliques Vegetaux Institut de Biologie Moleculaire des Plantes Centre National de la Recher-
che ScientifiqueUniversite de Strasbourg 28 rue Goethe 67083 Strasbourg Cedex France and5Plateforme de Cytologie et Imagerie Vegetale UMR 1318 Institut National de la Recherche Agronomique Centre de
Versailles 78026 Versailles Cedex France
Received 27 January 2012 revised 11 September 2013 accepted 17 September 2013 published online 5 November 2013
For correspondence (e-mail rozennle-hirversaillesinrafr)daggerPresent address Department of Biotechnology St Xavierrsquos College 700016 Calcutta India
SUMMARY
In order to obtain insights into the regulatory pathways controlling phloem development we characterized
three genes encoding membrane proteins from the G sub-family of ABC transporters (ABCG9 ABCG11 and
ABCG14) whose expression in the phloem has been confirmed Mutations in the genes encoding these
dimerizing lsquohalf transportersrsquo are semi-dominant and result in vascular patterning defects in cotyledons and
the floral stem Co-immunoprecipitation and bimolecular fluorescence complementation experiments dem-
onstrated that these proteins dimerize either by flexible pairing (ABCG11 and ABCG9) or by forming strict
heterodimers (ABCG14) In addition metabolome analyses and measurement of sterol ester contents in the
mutants suggested that ABCG9 ABCG11 and ABCG14 are involved in lipidsterol homeostasis regulation
Our results show that these three ABCG genes are required for proper vascular development in Arabidopsis
thaliana
Keywords vascular development ABC transporters ABCG dimer lipidsterol homeostasis Arabidopsis
thaliana
INTRODUCTION
In higher plants vascular bundles constitute a network
connecting various parts of the plant They provide both
mechanical support and conduits for the distribution of
compounds required for proper growth and defense Each
bundle comprises two highly specialized conductive
tissues xylem and phloem The xylem is responsible for
the transport of water and mineral nutrients via water
potential gradients Carbon sourcesink relationships and
energetic loading processes mobilize the movement of
nutrients defense compounds and informational signals
through sieve elements companion cells and parenchyma
cells of the phloem (Turgeon and Wolf 2009) Phloem
physiology has been studied for over two centuries and
transcript profiling in several plant species has recently
provided insights into phloem-specialized functions (Le Hir
et al 2008 Turgeon and Wolf 2009) However a compre-
hensive understanding of phloem development transport
and signaling is still lacking
There are more than 3000 known members of the ABC
protein superfamily in all extant phyla and these proteins
function in the transport of a wide variety of compounds
including hormones mineral ions lipids peptides second-
ary metabolites and xenobiotics (Jungwirth and Kuchler
2006) In plants the ABC superfamily is divided into eight
sub-families (ABCAndashH) and ABC proteins are highly abun-
dant with more than 120 isoforms in Arabidopsis thaliana
and Oryza sativa (rice) alone (Verrier et al 2008) ABC
transporters share a common architecture consisting of
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd
811
The Plant Journal (2013) 76 811ndash824 doi 101111tpj12334
two transmembrane domains (TMD) housing membrane
transit sites and two nucleotide bindinghydrolysis
domains (NBD) that provide the energetic basis for sub-
strate movement (Kneurooller and Murphy 2011) The ABCG
sub-class exhibits a TMDndashNBDndashTMDndashNBD architecture
and is divided into plantfungal-specific pleiotropic drug
resistance (PDR) full-length transporters and the eukaryotic
white brown complex (WBC) half-size transporters
that function as homo- or heterodimers to create the
TMDndashNBDndashTMDndashNBD structure (Verrier et al 2008) In
Arabidopsis the 28 half-size ABCG proteins comprise the
largest ABC sub-class with mechanistic diversity and sub-
strate specificity increased by the necessity for dimeriza-
tion for functionality (Verrier et al 2008 McFarlane et al
2010 Zhang et al 2010)
Arabidopsis ABCG11 (COF1DSOWBC11) and ABCG12
(CER5WBC12) have been shown to be required for the
export of various cuticular lipids (Pighin et al 2004 Bird
et al 2007 Luo et al 2007 Panikashvili et al 2007 2010
Ukitsu et al 2007) More recently it was shown that
ABCG26 (WBC27) plays an important role in the transport
of sporopollenin precursors (Choi et al 2010 Quilichini
et al 2010 Dou et al 2011 Kuromori et al 2011a) and
ABCG13 (WBC13) is required for flower cuticle secretion
and petal epidermis patterning (Panikashvili et al 2011) In
addition two ABCG half-size transporters (ABCG25WBC25
and ABCG22WBC22) are directly or indirectly involved in
ABA (abscisic acid) transport and responses (Kuromori
et al 2010 2011b) Here we show that three of the ABCG
family members ABCG9 ABCG11 and ABCG14 interact
physically with each other and are required for vascular
patterning in Arabidopsis
RESULTS
ABCG9 ABCG11 and ABCG14 are expressed in the
vascular system of Arabidopsis
In an effort to identify structural components of the
phloem infrastructure we searched several transcriptomic
datasets (Hertzberg et al 2001 Vilaine et al 2003 Schrader
et al 2004 Zhao et al 2005) for genes that are highly
expressed in the phloem and that encode transporters
Prominent in this selected group were genes encoding the
ABCG11 and ABCG14 lsquohalf transportersrsquo of the G sub-class
of ABC transporters A third ABCG transporter ABCG9 was
added to this group as it is closely related to ABCG14 (Ver-
rier et al 2008) We first measured their expression levels
in various organs by quantitative PCR (Figure S1andashc) Tran-
scripts for ABCG9 ABCG11 and ABCG14 were found in all
organs analyzed with an overall higher relative transcript
amount in the aerial parts compared with the root (Figure
S1andashc) For further analysis of the spatial expression pat-
tern each ABCG promoter was used to drive expression of
the GUS reporter gene ABCG9proGUS ABCG11proGUS
and ABCG14proGUS were visualized in the vascular sys-
tem of the cotyledons (Figure 1aei) The ABCG9proGUS
signal was restricted to the petiole main vein (Figure 1a)
GUS signals were also observed in phloem cells of the
flower stem for ABCG9proGUS ABCG11proGUS and
ABCG14proGUS (Figure 1bfj) and ABCG11proGUS sig-
nals were also observed in the cortical cells and interfasci-
cular fibers (Figure 1f) In 4-week-old rosette leaves
ABCG9proGUS (Figure 1c) ABCG11proGUS (Figure 1g)
and ABCG14proGUS (Figure 1k) were expressed in the
vascular system In addition expression of ABCG11pro
GUS was identified in the rosette leaf epidermis (Fig-
ure 1g) In roots ABCG11proGUS expression was detected
in lateral root primordia (Figure 1h) whereas expression
of ABCG9proGUS and ABCG14proGUS was observed in
the central cylinder (Figure 1di) Overall ABCG9 and
ABCG14 expression patterns were found to be restricted
to vascular bundles whereas in accordance with previous
results ABCG11 has a broader localization (Bird et al
2007 Luo et al 2007 Panikashvili et al 2007 Ukitsu
et al 2007)
Mutations in ABCG9 ABCG11 and ABCG14 are
responsible for defects in plant growth
Two TndashDNA insertion lines were identified and confirmed
in the first exon (SALK_047133) and the second exon
(GABI_353B01) of the ABCG9 gene (Figure 2a) The respec-
tive mutants were confirmed to be a weak (abcg9ndash1) and
null (abcg9ndash2) allele mutant (Figure 2de and Figure S1d)
Three independent mutations were confirmed in the
ABCG11 gene (abcg11ndash6 abcg11ndash7 and abcg11ndash8 Fig-
ure 2b) and were shown to be a weak allele mutant
(abcg11ndash6 SALK_096377 Figure 2f and Figure S1d) and
two knockout mutants (abcg11ndash7 and abcg11ndash8
GABI_590C03 and GABI_728E03 respectively Figure 2gh
and Figure S1d) Only one TndashDNA insertion was identified
in the promoter region of the ABCG14 gene (SALK_036952
Figure 2c) A reduced amount of full-length transcript was
amplified from the corresponding abcg14ndash1 mutant (Fig-
ure 2i and Figure S1d) Hence it was deemed to be a weak
allele mutant
The general phenotype of the various mutant lines was
assessed in both 20-day-old and 4-week-old plants grown
in soil (Figure 3andashn) Under these conditions no clear
phenotypic divergence from wild-type was observed for
the weak allele abcg14ndash1 (Figure 3hn) but a mild pheno-
type was observed for the single mutant abcg9ndash2 (10
smaller than the wild-type) (Figure 3cn) Interestingly the
double mutants abcg9ndash1 abcg14ndash1 (Figure 3jn and Figure
S1d) and abcg9ndash2 abcg14ndash1 (Figure 3kn and Figure S1d)
displayed a stronger phenotype (27 and 59 smaller than
the wild-type respectively) than the single mutants sug-
gesting an additive effect of these mutations Complemen-
tation of abcg9ndash2 and abcg14ndash1 mutants by hemagglutinin
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
812 Rozenn Le Hir et al
(HA)-tagged versions of ABCG9 and ABCG14 respectively
led us to conclude that these mutations were indeed
responsible for the observed phenotypes (Figure 3din
and Figure S1d) On the other hand the abcg11ndash7 homozy-
gous plants showed severe morphological abnormalities
similar to previously described abcg11 alleles (Bird et al
2007 Luo et al 2007 Panikashvili et al 2007 Ukitsu et al
2007) They were dwarf with unexpanded fused leaves
(Figure 3g and Figure S1d) Strikingly abcg11-7 plants car-
rying the mutation in ABCG11 gene in a heterozygous state
were 32 smaller than the wild-type plants on average
(Figure 3fn and Figure S1d) Finally abcg9-2abcg11-
7abcg14-1 mutants displayed a stronger phenotype than
abcg11ndash7 heterozygous plants (Figure 3ln) suggesting
that these mutations are additive Moreover the triple
homozygous mutants were as small as the abcg11ndash7
homozygous plants and displayed the same developmen-
tal abnormalities (Figure 3gmn)
Mutations in ABCG9 ABCG11 and ABCG14 affect vascular
patterning
Because these three genes are expressed in the vascular
system alleles of abcg9 abcg11 and abcg14 as well as
multiple mutants were analyzed for evidence of altered
vascular development by characterization of cotyledon
venation patterns in young seedlings (Cnops et al 2006)
The complexity of the vascular pattern was assessed in
terms of the number of secondary vein loops originating
from the mid-vein (Figure 4) In wild-type (Colndash0) seed-
lings 94 of the cotyledon venation pattern was between
classes III and V [from two loops plus the start of two
others (class III) up to four loops (class V) Figure 4] with
41 representing the most complex pattern (class V) The
double mutants abcg9ndash1abcg14ndash1 and abcg9ndash2abcg14ndash1
displayed a more complex venation pattern than the wild-
type (with respectively 100 and 96 of the cotyledon
(a) (b) (c) (d)
(e) (f) (g) (h)
(i) (j) (k) (l)
Figure 1 Expression patterns of ABCG genes
Expression patterns of ABCG9 ABCG11 and ABCG14 in cotyledons of 10-day-old seedlings (a e i respectively) the floral stem of 4-week-old plants (b f j
respectively) rosette leaves (c g k respectively) and roots (d h l respectively) co cortex e epidermis if interfascicular fibers ph phloem xy xylem Scale
bar = 100 lm
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 813
vascular pattern between classes III and V) The other
mutant lines showed a significantly less complex vena-
tion pattern than the wild-type (Figure 4) The single
mutants also differed significantly with respect to the loss
of complexity The abcg11ndash7 line displayed the least com-
plex vascular system (only 38 of the venation patterns
were between classes III and V) followed by abcg14ndash1
and then abcg9ndash2 (73 and 86 respectively) (Figure 4)
Introduction of HA-tagged versions of the ABCG9 and
ABCG14 proteins in the corresponding mutant back-
ground (abcg9ndash2 and abcg14ndash1) complemented the loss
of vascular complexity (Figure 4) confirming that the
phenotype was indeed due to defective expression of
ABCG9 and ABCG14 genes and the tagged proteins were
functional Interestingly the cotyledons of the triple
mutant displayed a significantly less complex vascular
system than that of the single mutants (Figure 4) In addi-
tion to the reduced vascular complexity in abcg11 homo-
zygous and heterozygous mutants the presence of open
vascular loops indicated a defect in vascular continuity
(Figure 4) Interestingly the continuity was restored in the
triple homozygous mutant (Figure 4)
Subsequently we characterized vascular bundles in the
floral stem of 4-week-old plants grown in soil (Table 1)
Under our conditions the wild-type flower stems dis-
played a vascular bundle density of 592 vascular bundles
per mm (Table 1) No significant variation in the number
of vascular bundles was observed in the single mutants
and complemented lines (Table 1) In contrast to the
increased complexity of the cotyledon venation pattern
(Figure 4) significantly fewer vascular bundles were pres-
ent in abcg9ndash2 abcg14ndash1 and abcg9ndash2 abcg11ndash7 abcg14ndash1
mutants (828 and 76 vascular bundles respectively)
(Table 1) Consistent with the smaller plant stature of
these double and triple mutants (Figure 1n) the diameter
of the floral stem was reduced (Table 1) However the
number of vascular bundles could not be explained by a
reduction of the floral stem diameter as the abcg11ndash7
mutant displayed the same number of vascular bundles
than the wild-type but its floral stem diameter was
strongly reduced (Table 1) Altogether these results
demonstrate that ABCG9 ABCG11 and ABCG14 play an
important role in vascular patterning during Arabidopsis
plant development
ABCG9 and ABCG14 are plasma membrane-localized
proteins that interact physically to form homo- andor
heterodimers with ABCG11
Because ABCG half transporters are thought to require
dimerization for functionality we investigated whether
ABCG9 ABCG11 and ABCG14 interact physically with
themselves andor other ABCG isoforms We first investi-
gated the subcellular localization of ABCG9 and ABCG14
after transfection of Arabidopsis mesophyll protoplasts
prepared from transgenic seedlings expressing the plasma
membrane marker low temperature induced protein 6b
(GFPndashLTi6b) (Cutler et al 2000) with constructs encoding
transcriptional fusions with red fluorescent protein (RFP)
In both cases the GFP fluorescent signal (Figure 5ae) co-
localized with the RFP fluorescent signal (Figure 5bf) at
the plasma membrane of transformed cells (Figure 5bh)
indicating that both ABCG9 and ABCG14 localize to the
plasma membrane like ABCG11 (Bird et al 2007 Luo
et al 2007 Panikashvili et al 2007 Ukitsu et al 2007) In
addition immunolocalization of a HA-tagged version of
ABCG9 and ABCG14 in their respective complemented
lines confirmed that these two proteins are localized to
the plasma membrane in planta (Figure 5ij) Next
(a)
(b)
(c)
(d) (e)
(f) (g)
(h) (i)
Figure 2 TndashDNA insertion lines for ABCG genes
(andashc) TndashDNA positions in ABCG9 ABCG11 and ABCG14 respectively Black
boxes promoters dark gray boxes 5prime and 3prime untranslated regions light gray
boxes exons lines introns
(dndashi) The presence of full-length mRNA was checked in abcg mutants as
described in Experimental procedures The elongation factor EF1a was used
as an internal control
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
814 Rozenn Le Hir et al
Arabidopsis protoplasts were transfected with open read-
ing frames encoding individual cndashMyc- or HA-tagged
versions of the three ABCGs and the identity of the epi-
tope-tagged proteins was verified by SDSndashPAGE and
Western blotting using anti-HA and anti-c-Myc antibodies
In all cases only one band of approximately 75 kDa cor-
responding to the predicted molecular mass of the pro-
teins in the Plant Membrane Protein Database (Schwacke
et al 2003) was obtained (Figure 5kndashp lane 1 and Figure
S2andashf lane 1) Subsequently protoplasts were co-trans-
fected with cndashMyc- and HA-tagged isoforms immunopre-
cipitated with antisera against one of the epitope tags
and probed with antisera directed against the alternative
epitope tag in Western blots of SDSndashPAGE gels in order
to determine whether homodimers or heterodimers were
formed (Figure 5kndashp) No ABCG14 homodimers or
ABCG9ndashABCG14 heterodimers were detected (Figure 5m
lane 4 and Figure 5o lane 4) In contrast ABCG11 formed
an apparent homodimer as described previously (McFar-
lane et al 2010) ABCG9 also formed an apparent homod-
imer ABCG14 formed an apparent heterodimer with
ABCG11 and ABCG9 formed an apparent heterodimer
with ABCG11 (Figure 5klnp lane 4) These interactions
were confirmed in a plant-based in vivo system using the
bimolecular fluorescence complementation assay (Figure
S3andashl) We therefore concluded that ABCG14 forms a strict
heterodimer with ABCG11 whereas ABCG11 and ABCG9
can both hetero- and homodimerize
Cuticular and epicuticular lipid precursor content as well as
cuticle integrity are not altered in abcg9ndash2 and abcg14ndash1
mutants
ABCG11 is known to be required for cuticle integrity (Bird
et al 2007 Luo et al 2007 Panikashvili et al 2007 Ukitsu
et al 2007) and because ABCG11 interacts physically with
ABCG9 and ABCG14 we looked for potential defects in
very long chain fatty acids (VLCFAs) which are among the
precursors of cuticular and epicuticular lipids (Roudier
et al 2010) as well as for possible cuticle defects in
abcg9ndash2 and abcg14ndash1 mutants
Quantification of fatty acids (from 160 to 180) as well as
VLCFAs (from 200 to 240) in apical parts of abcg9ndash2 and
abcg14ndash1 seedlings revealed no differences compared to
wild-type levels (Figure 6ab) It has previously been
(a) (b) (c) (d)
(e) (f) (g) (h)
(i)
(j)
(i)
(k) (l) (m)
(n)
Figure 3 Phenotype of abcg mutants grown in
soil
(andashm) Twenty-day-old plants grown in soil
Scale bar = 1 cm
(n) Green areas of 4-week-old plants Values
represent means SE from 20 individual
plants The experiment was repeated twice with
similar results Asterisks indicate statistically
significant differences between the wild-type
and mutants (P lt 005 Studentrsquos t test n = 20)
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 815
shown that when the cuticle is altered toluidine blue (TB)
permeates the epidermal surface (Tanaka et al 2004)
Therefore we used TB to check for cuticle defects in the
mutants After treatment with TB no staining was
observed in the wild-type abcg11+ or the abcg9ndash2 and
abcg14ndash1 mutants (Figure 6cef) whereas abcg11ndash7
homozygous seedlings showed patches of blue staining
confirming the cuticle alteration previously described
(Figure 6d) (Ukitsu et al 2007) These observations sug-
gest that unlike mutations in ABCG11 mutations in
ABGC9 or ABCG14 do not affect cuticle formation
abcg9ndash2 and abcg14ndash1 mutants are defective in sterol
composition
To obtain an insight into the potential substrates trans-
ported by these ABCG transporters we analyzed the shoot
and root metabolome of single mutants Orthogonal partial
least-squares discriminant analysis was applied to the
GCMS data in order to classify the profiles of the geno-
types In both shoots (Figure 7andashc) and roots (Figure S4andash
c) homozygous abcg11ndash7 abcg9ndash1 abcg9ndash2 and abcg14ndash1
mutants exhibited very different metabolomic profiles from
the wild-type Interestingly the heterozygous abcg11ndash7+
and abcg9ndash2+ plants had metabolomic profiles intermedi-
ate between the wild-type and their respective homozy-
gous mutant plants (Figure 7ab and Figure S4ab) These
results suggest that a mutation in one copy of ABCG11 or
ABCG9 is sufficient to significantly affect the physiology of
the plant even if it does not dramatically affect visible
aspects of its phenotype
The metabolites showing the most significant differ-
ences were identified by comparing their retention indices
and mass spectra with entries in publicly available reten-
tion libraries (Schauer et al 2005) For all the genotypes
analyzed the metabolites identified as being significantly
different from the wild-type belonged to three main clas-
ses amino acids carbohydrates and lipidssterols The
levels of these compounds were almost all reduced in
abcg9ndash2 and abcg14ndash1 but in excess in abcg11ndash7 (Table 2
and Appendix S1)
Because our study focused on vascular development in
aerial tissues only data obtained for the apical parts are
described in detail here (Table 2) Root data are presented
in Appendix S1 Whereas many neutral amino acids were
significantly depleted in abcg9ndash2 and abcg14ndash1 aerial
Figure 4 Cotyledon venation pattern in abcg mutant lines
Venation complexity and continuity of each examined class of mutant and
wild-type seedlings Values in brackets indicate the percentage contribution
of each class Asterisks indicate statistically significant differences between
the wild-type and mutants (P lt 005 Pearsonrsquos v2 test with Monte Carlo
permutations N = 10 000)
Table 1 Distribution of vascular bundles in the floral stem
Genotype Vascular bundle number Floral stem diameter (mm) Vascular bundle density Total
Col-0 930 105 157 020 592 049 10abcg11-7+ 914 134 140 080 652 042 12abcg11-7 902 115 051 004 176 182 8abcg14-1 828 049 145 018 632 0049 1135SHA-ABCG14 abcg14-1 940 089 170 016 554 058 12abcg9-1 957 053 160 006 586 031 7abcg9-2 875 103 162 010 553 046 835SHA-ABCG9 abcg9-2 933 051 174 010 606 026 8abcg9-1 abcg14-1 871 048 154 003 565 030 8abcg9-2abcg14-1 828 049 124 015 674 084 8abcg9-2abcg14-1abcg11-7
760 054 063 005 1203 169 9
Statistically significant differences between the wild-type and mutants (P lt 005 Studentrsquos t test)
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
816 Rozenn Le Hir et al
tissues (nine and eight out of 20 neutral amino acids
respectively) only two amino acids (glutamine and gluta-
mate) associated with Arabidopsis phloem cells (Schad
et al 2005) were different in abcg11ndash7 (Table 2) Xylose
and the non-reducing sugars trehalose and raffinose were
also decreased in abcg14ndash1 (Table 2) whereas glucose
fructose galactose maltose xylose were significantly dif-
ferent in abg9ndash2 and abcg11ndash7 (reduced and increased
respectively compared to wild-type) (Table 2)
Levels of sterols (campesterol 24ndashmethylene cholesterol
and sitosterol) saturated and unsaturated fatty acids (lino-
leic acid lauric acid palmitic acid and triacontanoic acid)
glycerolipids (monoacylglycerol) and lipid metabolism
intermediates (glycerol glycerol-3ndashphosphate and inositol-
1ndashphosphate) were also significantly different from wild-
type in abcg mutants (Table 2) The abcg9ndash2 mutant
showed 30 lower levels of 24ndashmethylene cholesterol
compared to the wild-type (Appendix S1) In the abcg14ndash1
mutant 50 and 35 decreases of 24ndashmethylene cholesterol
and sitosterol levels were measured respectively (Appen-
dix S1) Finally campesterol showed a 50 increase in the
abcg11ndash7 mutant (Appendix S1) To extend this analysis
measurements of conjugated sterols [sterol ester (SE)
sterol glucoside and acylated sterol glucoside] were also
performed When seedlings were grown in vitro no differ-
ence in the sterol glucoside and acylated sterol glucoside
contents was detected between the wild-type and the vari-
ous mutant lines (Figure S4d) However there was a ten-
dency towards a reduced SE content in the double mutant
abcg9ndash2 abcg14ndash1 compared to the wild-type (Figure S4d)
This observation prompted us to examine variations in the
SE content in older plants (4-week-old plants grown in soil
in the greenhouse) At this developmental stage no differ-
ence in the free sterol (FS) content was observed between
the wild-type and the various mutant lines (Figure 7d) but
the fraction comprising FS and SE was significantly
(a) (b) (c) (d)
(e) (f) (g) (h)
(i) (j)
(k) (l) (m)
(n) (o) (p)
Figure 5 Subcellular localization and physical interactions of ABCG9 ABCG11 and ABCG14
(andashh) ABCG9 and ABCG14 co-localize to the plasma membrane when transiently expressed in Arabidopsis mesophyll protoplasts of the GFPndashLTi6b marker line
(Cutler et al 2000)
(a e) Cyan fluorescence of GFPndashLTi6b(b f) Purple fluorescence from mRFPndashABCG9 and mRFPndashABCG14 respectively
(c g) Yellow auto-fluorescence of the chloroplasts
(d h) Merged images of the three channels Scale bar = 10 lm
(i j) Confocal images of immunofluorescence staining with anti-HA antibodies in root cells of abcg9ndash2 and abcg14ndash1 mutants complemented with HA-tagged
ABCG9 (i) and HA-tagged ABCG14 (j) Scale bars = 10 lm
(kndashp) Proteins were extracted from protoplasts co-transfected with HA- or cndashMyc-tagged versions of ABCGs (lanes 1 and 4) or transfected with a single plasmid
containing the HA-tagged version (lane 2) or the c-Myc-tagged version (lane 3) In lanes 2ndash4 proteins were immunoprecipitated with the anti-c-Myc antibody
and subjected to anti-HA protein gel-blot analysis to reveal the other partner of the dimer Transient co-expression of (k) HAndashABCG9cndashMycndashABCG9 (l) HAndashABCG11cndashMycndashABCG11 (m) HAndashABCG14cndashMycndashABCG14 (n) HAndashABCG14cndashMycndashABCG11 (o) HAndashABCG14cndashMycndashABCG9 and (p) HAndashABCG11cndashMycndashABCG9
in wild-type protoplasts
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 817
reduced probably accounting for the difference in the SE
content between the wild-type and the abcg9ndash2 abcg14ndash1
double mutant (Figure 7e)
(a)
(b)
(c) (d) (e)
(f) (g)
Figure 6 Long chain and very long chain fatty acid content and cuticle
integrity in abcg9ndash2 and abcg14ndash1 mutants
(a b) Fatty acid (a) and VLCFA content (b) in abcg9ndash2 and abcg14ndash1 mutants
in apical parts of 10-day-old seedlings (three biological replicates each of
approximately 5 mg dry weight were used)
(cndashf) Toluidine blue staining patterns of abcg mutants in 7-day-old seed-
lings Scale bars = 3 cm
(a)
(b)
(c)
(d) (e)
Figure 7 Metabolome analysis and measurements of sterol content in abcg
mutants
(andashc) Results of orthogonal partial least-squares discriminant analysis of
global metabolite contents of shoots of 7-day-old seedlings from wild-type
(black squares) abcg11ndash7+ (gray triangles) abcg11ndash7 (white stars) abcg9ndash1(white hexagons) abcg9ndash2+ (gray hexagons) abcg9ndash2 (white hexagons)
and abcg14ndash1 (black circles) seedlings (five biological replicates of 10 mg
each were used for the analysis)
(d e) Free sterol (FS) and sterol ester (SE) content of wild-type (white bars)
abcg9ndash2 (light gray bars) abcg14ndash1 (dark gray bars) and abcg9ndash2 abcg14ndash1(black bars) Error bars indicate standard deviations obtained from three
independent biological replicates The asterisk indicates a statistically signif-
icant difference between the wild-type and the mutant (P lt 005 Studentrsquos
t test)
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
818 Rozenn Le Hir et al
DISCUSSION
ABCG9 and ABCG14 are two plasma membrane proteins
that interact physically in multiple homo-heterodimer
combinations
In this study we showed that like the previously described
ABCG11 (Pighin et al 2004 Bird et al 2007 Luo et al
2007 Panikashvili et al 2007 2010 Ukitsu et al 2007)
ABCG9 and ABCG14 localize to the plasma membrane
Based on co-immunoprecipitation of tagged proteins and
the bimolecular fluorescence complementation assay we
demonstrated that ABCG9 interacts with ABCG11 to form a
heterodimer and like ABCG11 (McFarlane et al 2010) can
homodimerize In contrast ABCG14 interacts only with
ABCG11 forming an obligate heterodimer This extends
the number of potential partners of ABCG11 to three as it
was previously shown to dimerize with the ABCG12CER5
protein (McFarlane et al 2010) Similarly ABCG9 under-
goes flexible dimerization to form either homo- or hetero-
dimers suggesting that ABCGs may be promiscuous
proteins with different functions depending on their inter-
acting partner andor expression profile
Mutations in ABCG genes are semi-dominant
In the last few years several Arabidopsis mutants with
altered expression of various ABCG genes such as ABCG11
(Bird et al 2007 Luo et al 2007 Panikashvili et al 2007
Ukitsu et al 2007) ABCG12CER5 (Pighin et al 2004)
ABCG13 (Panikashvili et al 2011) ABCG22 (Kuromori
et al 2011a) ABCG25 (Kuromori et al 2010) and ABCG26
(Choi et al 2010 Quilichini et al 2010 Dou et al 2011
Kuromori et al 2011b) have been characterized but even
though these half-size transporters require dimerization to
become functional all the mutations were described as
recessive Here we demonstrated that mutations in these
genes have semi-dominant effects Indeed the metabolome
analyses showed that abcg9ndash2+ and abcg11ndash7+ heterozy-
gous seedlings have a profile intermediate between that of
wild-type and homozygous seedlings The possibility of
dominant-negative effects was excluded because no
Table 2 Shoot metabolites of 10-day-old abcg9ndash2 abcg11ndash7 and abcg14ndash1 seedlings whose levels were significantly different from wild-type
Group
Metabolites that differed between mutant and wild-type
abcg9ndash2Colndash0 abcg11ndash7Colndash0 abcg14ndash1Colndash0
Amino acids Glycine Glutamate AlanineAlanine Glutamine ValineValine ThreonineThreonine GABASerine Prolinebndashalanine IsoleucineIsoleucine SerineGABA bndashalanineProline
Carbohydrates Glucose Glucose ErythritolFructose Fructose TrehaloseXylose Galactose XyloseMaltose RaffinoseSucrose
Lipids and intermediates 24ndashmethylene-cholesterol Campesterol Linoleic acidMonoacylglycerol Glycerol-3ndashphosphate Triacontanoic acidPalmitic acid Inositol-1ndashphosphate GlycerolGlycerol-3ndashphosphate Lauric acidInositol-1ndashphosphate 24ndashmethylene-cholesterol
SitosterolGlycerol-3ndashphosphate
TCA cycle and others Phosphoric acid Phosphate compound Glyceric acid24ndashdihydroxybutanoic acid Dehydroascorbic acid Fumaric acidGlyceric acid Malic acid Threonic acid-14ndashlactoneFumaric acid 2ndashoxoglutaric acid Threonic acidThreonic acid bndashhydroxy-bndashmethylglutaric acid Shikimic acidThreonic acid-14ndashlactone Gluconic acid lactone-like Succinic acidShikimic acid Phytol2ndashoxoglutaric acid Ethanolamine
Nicotinic acid
For abcg9ndash2 and abcg14ndash1 the levels of the compounds were reduced whereas they were increased in abcg11ndash7
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 819
truncated transcript was found for either abcg9ndash2 or
abcg11ndash7 mutants Hence abcg9ndash2 and abcg11ndash7 are true
null alleles These observations are consistent with the
requirement for homo- or heterodimerization of the ABCG
proteins to form functional transporters In the heterozy-
gous plants ABCG11 transcript levels were 50 lower than
in wild-type counterparts suggesting that ABCG11 protein
and thus corresponding homo- and heterodimers were
likely to be less abundant which may also explain the meta-
bolic perturbations and consequent phenotypic modifica-
tions These findings also suggest that wild-type plants are
not saturated by ABCG11 proteins as a 50 reduction in
protein content appears to be sufficient to alter the physiol-
ogy of the heterozygous mutant plants This hypothesis is
strengthened by the observation that mutants carrying the
weak abcg11ndash6 allele in which there was only a 25 reduc-
tion in ABCG11 transcript levels showed a similar pheno-
type to the heterozygous abcg11ndash7+ and abcg11ndash8+
plants Similarly the abcg9ndash1 weak allele mutant had a very
similar phenotype to the abcg9ndash2+ heterozygous seedlings
suggesting that considering the mode of action of these
proteins the semi-dominant effects that we observed are
likely to be a general feature among the ABCG genes
ABCG9 ABCG11 and ABCG14 transporters are involved in
vascular system development in Arabidopsis
We showed that the ABCG11 ABCG9 and ABCG14 promot-
ers drove expression in the vascular system of Arabidopsis
cotyledons and rosette leaves and more precisely in the
phloem of flower stems In addition mutations in ABCG9
ABCG11 and ABCG14 genes altered vascular development
in cotyledons and the floral stem Similar vascular localiza-
tion of ABCG11 has already been described by Panikashvili
et al (20072010) who showed that in addition to the epi-
dermis ABCG11 is expressed in the vasculature as early as
during embryo development and then throughout plant
development They showed that the vascular pattern in
leaves of the homozygote mutant seedlings was less com-
plex than in the wild-type and discontinuous (Panikashvili
et al 2007) The present article describes a mutant that
has both cuticle and vascular system patterning defects
although it has been previously shown that mutants
altered in cuticle formation have dramatically reduced
growth and altered leaf morphology (Jenks et al 1996)
which may account for vascular defects Analysis of the
abcg11+ heterozygous seedlings that have vascular
defects but apparently no cuticle alteration suggested that
these phenotypes are probably uncoupled Because
ABCG11 dimerizes with several members of its sub-family
it seems reasonable to postulate that it is involved in the
transport of different compounds depending on its partner
Here we provide evidence that ABCG11 in addition to
forming dimers with ABCG12 (McFarlane et al 2010) in
epidermal cells is likely to dimerize with ABCG9 and
ABCG14 in phloem cells suggesting functional compart-
mentalization of these proteins and a possible role in vas-
cular system patterning in Arabidopsis
Metabolite profiling suggests that ABCG9 ABCG11 and
ABCG14 are involved in sterollipid homeostasis
We investigated in more depth the metabolic changes
induced by mutations in ABCG9 ABCG11 and ABCG14
We found that levels of metabolites of several classes
including lipids sugars and amino acids were altered in
both the heterozygotes and homozygotes and that the
profiles in the root and apical parts differed suggesting
that ABCGs may play different roles in the roots and shoot
Interestingly the modifications of amino acid and sugar
contents observed in the metabolic profiles of the abcg
mutants suggest an impaired carbonnitrogen balance that
may be directly or indirectly linked to the vascular defects
leading to possible problems with phloem loading or
unloading Saturated and unsaturated fatty acids and sterol
contents were also altered in the mutants Patterning
defects in the vascular system are often observed in
mutants altered in hormone homeostasis such as for
auxin or brassinosteroids (Kaneda et al 2011) and direct
or indirect modifications of sterol homeostasis are thought
to be one of the primary causes of these downstream
effects (Pullen et al 2010) The detected defects in sterol
homeostasis may explain the abnormal vascular develop-
ment observed in abcg9 abcg11 and abcg14 mutants
Accordingly several sterol-deficient mutants such as fac-
kelhydra (Jang et al 2000) and cpv1 (Carland et al 2002)
have also been shown to exhibit severe defects in vascular
development It has been suggested that the bushy habit
and low fertility of cpv1smt2 mutants may be due to an
increased campesterolsitosterol ratio rather than sterol
deficiency per se (Clouse 2002) This ratio is controlled by
the activity of SMT2 (STEROL METHYLTRANSFERASE2)
Interestingly strong down-regulation of SMT2 expression
was observed in all the mutant backgrounds (Figure S5)
and the metabolic data indicated a deficit in 24ndashmethylene
cholesterol (precursor of both campesterol and sitosterol)
andor sitosterol in abcg9 and abcg14 mutants respec-
tively In addition the significant decrease in sterol ester
content in the double mutant abcg9ndash2 abcg14ndash1 correlated
with down-regulation of expression of the PHOSPHOLIPID
STEROL ACYLTRANSFERASE1 (PSAT1) gene encoding the
enzyme that is mainly responsible for SE synthesis (Bou-
vier-Nave et al 2010) and is proposed to be a control step
in sterol homeostasis (Kopischke et al 2013) Altogether
these results suggest that the vascular phenotype
observed in abcg mutants may be linked to modification of
sterol homeostasis
Therefore we postulate that the various dimer combina-
tions between ABCG9 ABCG11 and ABCG14 may
participate in the transport of squalene-derived metabolites
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
820 Rozenn Le Hir et al
such as sterol andor sterol conjugates via the phloem sap
The presence of sterols in phloem sap has been observed in
various species such as barley (Hordeum vulgare Lehrer
et al 2000) and fava bean (Vicia faba Bouvaine et al
2012) and indirect proof of the presence of sterols in
phloem sap has been obtained by analysis of the phytos-
terol content in exclusively phloem-feeding aphids that are
unable to synthetize them directly (Behmer et al 2011) In
addition the evidence for long-distance transport of sterol
precursors has been supported by observations that sterol
esters accumulate in organs distal to the site of application
of exogenous precursors (Bouvier-Nave et al 2010) As
sterols have been proposed to act as signal molecules in
the control of developmental processes such as vascular
patterning (Carland et al 2002 Pullen et al 2010) modifi-
cations of sterol homeostasis may affect development of
the vascular system in the abcgmutants described here
In conclusion we propose that ABCG9 ABCG11 and
ABCG14 are important for lipidsterol homeostasis Thus
these three proteins are likely to be involved in the control
of various aspects of plant development andor physiol-
ogy including patterning of the vascular system
EXPERIMENTAL PROCEDURES
Plant material and growth conditions
Insertion lines in ABCG9 (SALK_047133 and GABI_353B01)ABCG11 (SALK_096377 and GABI_590C03) and ABCG14 (SALK_036952) genes were obtained from the European ArabidopsisStock Centre (httparabidopsisinfo) Heterozygoushomozygousplants were screened using gene-specific primers in combinationwith the specific primer for the left border of the TndashDNA insertion(Table S1)
Plants were grown in soil or in vitro as described previously(Sorin et al 2005) Arabidopsis Colndash0 cell cultures were main-tained by weekly sub-culturing in MS medium (Murashige andSkoog 1962) supplemented with 240 lg l1 24ndashdichlorophenoxy-acetic acid and 14 lg l1 kinetin and were grown under 12 h light12 h dark cycles at 25 lE m2 sec1 and with 23degC day and nighttemperatures
Morphological characterization and GUS staining
To study the venation pattern cotyledons of 7-day-old seedlingsand rosette leaves of 4-week-old plants were cleared in a buffercontaining chloral hydrate glycerol and water (831 wvv) afterwhich 200 cotyledons per genotype were analyzed and the per-centage showing particular levels of complexity or discontinuity invascularity was estimated as described by Cnops et al (2006)
Flower stem segments (1ndash2 cm long) were removed from ten3ndash4-week-old plants grown in soil embedded in 8 agarose andsectioned using a VT100S vibratome (Leica httpwwwleicacom)The cross-sections were then stained with safraninalcian blue for1ndash2 min (Sigma httpwwwsigmaaldrichcom) rinsed in waterand mounted in 50 glycerol
Histochemical GUS assays were performed as described previ-ously (Sorin et al 2005) All observations were performed usingan Axioplan light microscope equipped with an Axiocam cameraand Axiovision software (Zeiss httpwwwzeisscom)
To observe potential cuticle defects in the various mutant linesa TB test was performed by immersing in vitro-grown plantlets ina 005 wv TB solution for 2 min as described by Tanaka et al(2004)
Statistical analyses
Statistical analyses of differences between-genotype means andvariance for the studied morphological traits were performedusing Studentrsquos t test (GraphPad Prism version 50 httpwwwgraphpadcom) In the venation pattern experiment thesignificance of differences between wild-type and mutants wasassessed using R (R Development Core Team 2010) and Pearsonrsquosv2 test with Monte Carlo permutations (N = 10 000)
Plasmid constructs
The native ABCG promoters and open reading frame of ABCG11were amplified from genomic DNA or cDNA extracted from 7-day-old Arabidopsis seedlings using Phusion high-fidelity DNApolymerase (Finnzyme wwwthermoscientificbiocomfinnzymes)according to the manufacturerrsquos instructions with gene-specificprimers (Table S1) The PCR products were cloned into pENTRDndashTOPO (Invitrogen httpwwwinvitrogencom) and recombinedinto pKGWFS7 (Karimi et al 2002) to create ABCGproGUS Allrecombinations were performed using the Gateway LR Clonase
enzyme mix (Invitrogen) At each cloning step the sequenceswere confirmed by sequencing and all the constructs weretransferred into Agrobacterium strain GV3101pMP90 (C58C1)Wild-type or abcg11ndash7+ heterozygous plants were transformed byfloral dipping (Clough and Bent 1998) For ABCGGUS constructsthe expression pattern was checked in the T2 progeny of 10ndash15independent transgenic lines and one representative homozygousline was used for further characterization
Tagged protein constructs and protoplast transformation
Epitope-tagged versions of ABCG9 ABCG11 and ABCG14 proteinswere produced in pRT104ndash3xHA and pRT104ndash3xMyc plasmids(Feurouleuroop et al 2005) and pSAT6-mRFP (Citovsky et al 2006) Allthese plasmids have a 35S promoter sequence upstream of themulti-cloning site The open reading frames of ABCG11 andABCG14 and the genomic clone of ABCG9 were amplified fromcDNA or genomic DNA extracted from 7-day-old Arabidopsisseedlings using Phusion high-fidelity DNA polymerase (Finnzyme)according to the manufacturerrsquos instructions with gene-specificprimers carrying EcoRI or SalI restriction sites to facilitate subse-quent cloning (Table S1) The products obtained after PCR weredigested by EcoRI and SalI prior to ligation into appropriate plas-mids that had previously been cut open with the same restrictionenzymes Constructs were verified by sequencing Protoplastsfrom Arabidopsis cell cultures or 14-day-old Arabidopsis seedlingswere prepared and transformed as previously described (Meski-ene et al 2003 Zhai et al 2009) For co-immunoprecipitation andsubcellular localization assays 100 000 and 50 000 protoplastcells respectively were transfected with 15 and 10 ll of plasmidsrespectively (containing between 5 and 75 lg of each construct)and the resulting transformed protoplasts were processed afterincubation in the dark at room temperature for 16ndash18 and 24 hrespectively
Whole-mount immunolocalization
The HA-tagged versions of ABCG9 and ABCG14 were detected in7-day-old in vitro-grown Arabidopsis seedlings as described inMethods S1 Anti-HA antibody 12CA5 (Roche httpwwwroche
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 821
com) (1100 dilution) combined with Alexa Fluor 488-conjugatedsecondary antibody (1100 dilution) were used
Confocal laser-scanning microscopy
Images were collected using an SP2 confocal microscope (Leica)RFP and Alexa Fluor 488 were detected using laser lines at 561and 488 nm respectively according to the manufacturerrsquos set-tings RFP fluorescence emission was detected at 550ndash600 nmand Alexa Fluor 488 and GFP fluorescence emission were detectedat 495ndash545 nm For subcellular localization in protoplasts imageswere coded in cyan (GFP) magenta (RFP) and yellow (auto-fluo-rescence) resulting in purple coloration for co-localization inmerged images For the immunolocalization experiment imageswere coded in green (GFP) A water-corrected 209 objective (HCPlan-Apochromat 20907 ImmKor Leica) was used Images werecropped using Adobe Photoshop CS2 and assembled using AdobeIllustrator CS2 software (Abode httpwwwabodecom) Eachimage shown represents a single focal plan
Co-immunoprecipitation assays
Transformed protoplast proteins were extracted using 50 ll lysisbuffer containing 25 mM TrisHCl pH 78 10 mM MgCl2 75 mM
NaCl 5 mM EGTA 1 mM benzamidine 1 mM dithiothreitol 10glycerol 1 Triton Xndash100 and 19 protein inhibitor cocktail(Sigma httpsigmaaldrichcom) The transformed protoplast sus-pension was frozen in liquid nitrogen then thawed on ice andsubjected to centrifugation for 5 min at 150 g Two microliters ofanti-Myc (9E10 Covance httpwwwcovancecom) or anti-HA(16B12 Covance) antibodies were added to the extracts and themixture was incubated for 2 h at 4degC on a rotating wheel Immu-nocomplexes were captured by incubation with 10 ll Protein GndashSepharose beads for a further 2 h at 4degC on a rotating wheel afterwhich they were washed three times for 5 min each in 25 mM
NaPi 150 mM NaCl 5 glycerol and 02 Igepal CAndash630 buffer(Sigma) and then eluted using 40 ll SDS sample buffer at 37degCfor 30 min Co-immunoprecipitation of Myc- and HA-tagged pro-teins was revealed by SDSndashPAGE followed by Western blottingusing both anti-Myc (Invitrogen) and anti-HA (3F10 Roche) anti-bodies at 12000 dilution
RNA extraction and cDNA preparation
To quantify the relative amounts of the ABCG transcriptsexpressed in various organs three rosette leaves all the cau-line leaves the bottom 5 cm of the floral stem all closed flow-ers and all green siliques were harvested from each of threeindependent 4-week-old Arabidopsis plants of each genotypegrown in soil For the other experiments 10-day-old seedlingsgrown in vitro were harvested All the material was directly fro-zen in liquid nitrogen and stored at 80degC prior to RNA extrac-tion Samples were then disrupted using a MM 301 vibrationmill (Retsch GmbH httpwwwretschcom) at a frequency of25 Hz for 2 min with 3 mm tungsten carbide beads (Qiagenhttpwwwqiagencom) Total RNA was isolated from thismaterial using TRIzol (Life Technologies httplifetechnologiescom) RNA quantity and quality were measured using a Nano-Drop ND 1000 (Thermo Scientific httpwwwthermoscientificcom) All samples with 260280 nm and 260230 nm absorbanceratios lower than 18 were discarded and RNA was re-extractedDNase treatment was applied to 5 lg samples of total RNAusing a DNase I kit (Fermentas httpwwwfermentascom)Double-stranded cDNA was synthesized from 5 lg total RNAusing a SuperScript reverse transcriptase kit (Invitrogen) witholigo(dT)12 primer Finally RNase treatment was applied to the
samples using Escherichia coli RNAse H (Fermentas) The cDNAsamples obtained were diluted 30-fold (except for abcg9 linesamples which were diluted 10-fold) prior to use either toverify the absence of full-length transcripts in putative knockoutmutants or to quantify gene expression by quantitative PCR Allprimers used were designed using the Primer3 program (Rozenand Skaletsky 2000) and their sequences are listed inTable S1
QUANTITATIVE REAL-TIME PCR
Quantitative PCR amplifications were performed in 20 llmixtures containing 10 ll 59 SYBR Green I master mix
(Roche) 02 ll forward and reverse primer (30 lM each)
48 ll sterile water and 5 ll cDNA The BiondashRad CFX96
real-time PCR detection system (httpwwwbio-radcom)
was used for quantification of expression of the sterol
biosynthesis transcripts and the ABCG transcripts in the
various mutant backgrounds Finally the Roche LightCy-
cler 480 system (Roche) was used to quantify the rela-
tive abundance of ABCG transcripts in various organs
Four genes were considered as potential reference genes
[APT1 (At1g27450) TIP41 (At4g34270) EF1a (At5g60390)
and UBQ10 (At4g05320)] and their suitability was tested
using the geNorm algorithm (Vandesompele et al 2002)
to determine the most appropriate one for each experi-
ment A more detailed procedure following the Minimum
Information for Publication of Quantitative Real-Time PCR
Experiments guidelines (Bustin et al 2009) is described
in Methods S2 and the annealing temperature and
efficiencies of the primer pairs used are shown in
Table S2
VLCFA quantification
Approximately 5 mg dry weight of 10-day-old in vitro
Arabidopsis seedlings were analyzed for VLCFA quantifica-
tion as described by Li et al (2006)
Sterol extraction and GC-FID characterization
A pool of 10-day-old seedlings grown in vitro or rosettes
from 4-week-old plants grown in soil were lyophilized prior
to extraction Sterol extraction and isolation was per-
formed as described previously (Bouvier-Nave et al 2010)
Gas Chromatography-Flame Ionization Detector (GC-FID)
characterization was performed as described by Silvestro
et al (2013) To quantify the total amounts of free sterols
and sterol esters non-saponifiable lipids were extracted
using nndashhexane and sterols were derivatized as sterols ace-
tates Quantification was achieved using lupenyl-3 28ndashdi-
acetate as an internal standard
Metabolome analysis
To obtain metabolome profiles associated with wild-type
abcg11ndash7+ abcg11ndash7 abcg9ndash1 abcg9ndash2 and abcg14ndash1
genotypes 10-day-old seedlings representing each geno-
type grown in vitro as described above were harvested
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
822 Rozenn Le Hir et al
and divided into shoot and root samples In the case of
abcg9ndash2 the phenotype of the heterozygote abcg9ndash2+
was indistinguishable from the homozygote phenotype
and therefore heterozygous and homozygous seedlings
from a segregating population were pooled Hence in this
particular experiment abcg9ndash2+ refers to a mixed popula-
tion comprising two-thirds heterozygotes and one-third
homozygotes
Metabolites in five samples of shoot and root parts of
seedlings of each genotype (10 mg fresh weight per sam-
ple) were then extracted analyzed by GCMS and the
acquired data were processed as previously described
(Gullberg et al 2004 Jonsson et al 2005) All multivariate
statistical investigations (partial component analysis and
orthogonal partial least-squares discriminant analysis) for
between-genotype similarities and differences in general
metabolite profiles were performed using Simca software
(Umetrics httpwwwumetricscom)
ACKNOWLEDGEMENTS
We thank A Murphy (Plant Science and Landscape Archi-
tecture University of Maryland MD USA) for stimulating
discussions and critical reading of the manuscript I Carl-
son (Umea Plant Science Center Department of Forest
Genetics and Plant Physiology Swedish University of Agri-
cultural Sciences Umea Sweden) for technical assistance
and S Vernhettes for kindly donating seeds of the
transgenic line carrying the plasma membrane marker
GFPndashLTi6b We also thank the European Arabidopsis Stock
Centre and the GABI-Kat stock centre for providing seeds
This work was supported by allocations granted to CB by
the Swedish Research Council for Agriculture the Swedish
Foundation for Strategic Research the Swedish Research
Council for Research and Innovation for Sustainable
Growth the K amp A Wallenberg Foundation and the Carl
Trygger Foundation
SUPPORTING INFORMATION
Additional Supporting Information may be found in the onlineversion of this articleAppendix S1 Raw data quantification for compounds identified inthe metabolite profiling by GCMS
Figure S1 Relative expression of ABCG genes in various organsand mutant backgroundsFigure S2 Co-immunoprecipitation controlsFigure S3 ABCG9 and ABCG14 homo- andor heterodimerize withABCG11Figure S4 Complementary data on metabolome analysis and con-jugated sterol contentFigure S5 Relative expression of genes related to sterol homeo-stasisMethods S1 Method for whole-mount immunolocalizationMethods S2 Method for quantitative PCR
Table S1 List of primers used in this studyTable S2 Annealing temperature and efficiencies for the primerspairs used in this study
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Behmer ST Grebenok RJ and Douglas AE (2011) Plant sterols and host
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Wu XW Yephremov A and Samuels L (2007) Characterization of Ara-
bidopsis ABCG11WBC11 an ATP binding cassette (ABC) transporter that
is required for cuticular lipid secretion Plant J 52 485ndash498Bouvaine S Behmer ST Lin GG Faure MndashL Grebenok RJ and
Douglas AE (2012) The physiology of sterol nutrition in the pea aphid
Acyrthosiphon pisum J Insect Physiol 58 1383ndash1389Bouvier-Nave P Berna A Noiriel A Compagnon V Carlsson AS
Banas A Stymne S and Schaller H (2010) Involvement of the phos-
pholipid sterol acyltransferase1 in plant sterol homeostasis and leaf
senescence Plant Physiol 152 107ndash119Bustin SA Benes V Garson JA Hellemans J Huggett J Kubista
M Mueller R Nolan T Pfaffl MW and Shipley GL (2009) The MIQE
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Carland FM Fujioka S Takatsuto S Yoshida S and Nelson T (2002)
The identification of CVP1 reveals a role for sterols in vascular pattern-
ing Plant Cell 14 2045ndash2058Choi H Jin JY Choi S Hwang JU Kim YY Suh MC and Lee Y
(2010) An ABCGWBC-type ABC transporter is essential for transport of
sporopollenin precursors for exine formation in developing pollen Plant
J 65 181ndash193Citovsky V Lee LY Vyas S Glick E Chen MH Vainstein A Gafni
Y Gelvin SB and Tzfira T (2006) Subcellular localization of interacting
proteins by bimolecular fluorescence complementation in planta J Mol
Biol 362 1120ndash1131Clough SJ and Bent AF (1998) Floral dip a simplified method for Agro-
bacterium-mediated transformation of Arabidopsis thaliana Plant J 16
735ndash743Clouse SD (2002) Arabidopsis mutants reveal multiple roles for sterols in
plant development Plant Cell 14 1995ndash2000Cnops G Neyt P Raes J et al (2006) The TORNADO1 and TORNADO2
genes function in several patterning processes during early leaf develop-
ment in Arabidopsis thaliana Plant Cell 18 852ndash866Cutler SR Ehrhardt DW Griffitts JS and Somerville CR (2000) Random
GFPcDNA fusions enable visualization of subcellular structures in cells of
Arabidopsis at a high frequency Proc Natl Acad Sci USA 97 3718ndash3723Dou XY Yang KZ Zhang Y Wang W Liu XL Chen LQ Zhang
XQ and Ye D (2011) WBC27 an adenosine tri-phosphate-binding cas-
sette protein controls pollen wall formation and patterning in Arabidop-
sis J Integr Plant Biol 53 74ndash88Feurouleuroop K Pettko-Szandtner A Magyar Z Miskolczi P Kondorosi E
Dudits D and Bako L (2005) The Medicago CDKC1ndashCYCLINT1 kinase
complex phosphorylates the carboxy-terminal domain of RNA polymer-
ase II and promotes transcription Plant J 42 810ndash820Gullberg J Jonsson P Nordstrom A Sjostrom M and Moritz T (2004)
Design of experiments an efficient strategy to identify factors influenc-
ing extraction and derivatization of Arabidopsis thaliana samples in met-
abolomic studies with gas chromatographymass spectrometry Anal
Biochem 331 283ndash295Hertzberg M Aspeborg H Schrader J et al (2001) A transcriptional
roadmap to wood formation Proc Natl Acad Sci USA 98 14732ndash14737Jang JC Fujioka S Tasaka M Seto H Takatsuto S Ishii A Aida M
Yoshida S and Sheen J (2000) A critical role of sterols in embryonic
patterning and meristem programming revealed by the fackel mutants of
Arabidopsis thaliana Genes Dev 14 1485ndash1497Jenks MA Rashotte AM Tuttle HA and Feldmann KA (1996) Mutants
in Arabidopsis thaliana altered in epicuticular wax and leaf morphology
Plant Physiol 110 377ndash385Jonsson P Johansson AI Gullberg J Trygg J A J Grung B Markl-
und S Sjostrom M Antti H and Moritz T (2005) High-throughput
data analysis for detecting and identifying differences between samples
in GCMS-based metabolomic analyses Anal Chem 77 5635ndash5642Jungwirth H and Kuchler K (2006) Yeast ABC transporters ndash a tale of sex
stress drugs and aging FEBS Lett 580 1131ndash1138Kaneda M Schuetz M Lin B Chanis C Hamberger B Western T
Ehlting J and Samuels A (2011) ABC transporters coordinately
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ABCG proteins and vascular development 823
expressed during lignification of Arabidopsis stems include a set of AB-
CBs associated with auxin transport J Exp Bot 62 2063
Karimi M Inze D and Depicker A (2002) GATEWAYTMvectors for Agrobac-
terium-mediated plant transformation Trends Plant Sci 7 193ndash195Kneurooller A and Murphy A (2011) ABC transporters and their function at the
plasma membrane In The Plant Plasma Membrane Plant Cell Mono-
graphs vol 19 (Murphy AS Peer W and Schulz B eds) New York
NY Springer pp 353ndash377Kopischke M Westphal L Schneeberger K Clark R Ossowski S
Wewer V Fuchs R Landtag J Hause G and Deuroormann P (2013)
Impaired sterol ester synthesis alters the response of Arabidopsis thali-
ana to Phytophthora infestans Plant J 73 456ndash468Kuromori T Miyaji T Yabuuchi H Shimizu H Sugimoto E Kamiya
A Moriyama Y and Shinozaki K (2010) ABC transporter AtABCG25 is
involved in abscisic acid transport and responses Proc Natl Acad Sci
USA 107 2361ndash2366Kuromori T Itoh T Sugimoto E and Shinozaki K (2011a) Arabidopsis
mutant of AtABCG26 an ABC transporter gene is defective in pollen
maturation J Plant Physiol 168 2001ndash2005Kuromori T Sugimoto E and Shinozaki K (2011b) Arabidopsis mutants
of AtABCG22 an ABC transporter gene increase transpiration and
drought susceptibility Plant J 67 885ndash894Le Hir R Beneteau J Bellini C Vilaine F and Dinant S (2008) Gene
expression profiling keys for investigating phloem functions Trends
Plant Sci 13 273ndash280Lehrer AT Dugassa-Gobena D Vidal S and Seifert K (2000) Transport
of resistance-inducing sterols in phloem sap of barley Z Naturforsch C
55 948ndash952Li Y Beisson F Pollard M and Ohlrogge J (2006) Oil content of Arabid-
opsis seeds the influence of seed anatomy light and plant-to-plant vari-
ation Phytochemistry 67 904ndash915Luo B Xue XndashY Hu WndashL Wang LndashJ and Chen XndashY (2007) An ABC
transporter gene of Arabidopsis thaliana AtWBC11 is involved in cuticle
development and prevention of organ fusion Plant Cell Physiol 48
1790ndash1802McFarlane HE Shin JJH Bird DA and Samuels AL (2010)
Arabidopsis ABCG transporters which are required for export of diverse
cuticular lipids dimerize in different combinations Plant Cell 22 3066ndash3075
Meskiene I Baudouin E Schweighofer A Liwosz A Jonak C Rodri-
guez PL Jelinek H and Hirt H (2003) Stress-induced protein phospha-
tase 2C is a negative regulator of a mitogen-activated protein kinase
J Biol Chem 278 18945ndash18952Murashige T and Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures Physiol Plant 15 473ndash497Panikashvili D Savaldi-Goldstein S Mandel T Yifhar T Franke RB
Hofer R Schreiber L Chory J and Aharoni A (2007) The Arabidopsis
DESPERADOAtWBC11 transporter is required for cutin and wax secre-
tion Plant Physiol 145 1345ndash1360Panikashvili D Shi JX Bocobza S Franke RB Schreiber L and Aha-
roni A (2010) The Arabidopsis DSOABCG11 transporter affects cutin
metabolism in reproductive organs and suberin in roots Mol Plant 3
563ndash575Panikashvili D Shi JX Schreiber L and Aharoni A (2011) The Arabid-
opsis ABCG13 transporter is required for flower cuticle secretion and pat-
terning of the petal epidermis New Phytol 190 113ndash124Pighin JA Zheng H Balakshin LJ Goodman IP Western TL Jetter
R Kunst L and Samuels AL (2004) Plant cuticular lipid export requires
an ABC transporter Science 306 702ndash704Pullen M Clark N Zarinkamar F Topping J and Lindsey K (2010)
Analysis of vascular development in the hydra sterol biosynthetic
mutants of Arabidopsis PLoS ONE 5 e12227
Quilichini TD Friedmann MC Samuels AL and Douglas CJ (2010)
ATP-binding cassette transporter G26 is required for male fertility and
pollen exine formation in Arabidopsis Plant Physiol 154 678ndash690R Development Core Team (2010) R A Language and Environment for
Statistical Computing Vienna Austria R Foundation for Statistical
Computing
Roudier F Gissot L Beaudoin F Haslam R Michaelson L Marion J
Molino D Lima A Bach L and Morin H (2010) Very-long-chain fatty
acids are involved in polar auxin transport and developmental patterning
in Arabidopsis Plant Cell 22 364ndash375Rozen S and Skaletsky HJ (2000) Primer3 on the WWW for general users
and for biologist programmers Methods Mol Biol 132 365ndash386Schad M Lipton MS Giavalisco P Smith RD and Kehr J (2005) Eval-
uation of two-dimensional electrophoresis and liquid chromatography
tandem mass spectrometry for tissue-specific protein profiling of
laser-microdissected plant samples Electrophoresis 26 2729ndash2738Schauer N Steinhauser D Strelkov S et al (2005) GCndashMS libraries for
the rapid identification of metabolites in complex biological samples
FEBS Lett 579 1332ndash1337Schrader J Nilsson J Mellerowicz EJ Berglund A Nilsson P Hertz-
berg M and Sandberg G (2004) A high-resolution transcript profile
across the wood forming meristem of poplar identifies potential regula-
tors of cambial stem cell identity Plant Cell 16 2278ndash2292Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desi-
mone M Frommer WB Fleurougge UndashI and Kunze R (2003) ARAMEM-
NON a novel database for Arabidopsis integral membrane proteins
Plant Physiol 131 16ndash26Silvestro D Andersen TG Schaller H and Jensen PE (2013) Plant ste-
rol metabolism D7ndashsterol-C5ndashdesaturase (STE1DWARF7) D57ndashste-rol-D7ndashreductase (DWARF5) and D24ndashsterol-D24ndashreductase (DIMINUTO
DWARF1) show multiple subcellular localizations in Arabidopsis thaliana
(Heynh) L PLoS ONE 8 e56429
Sorin C Bussell JD Camus I Ljung K Kowalczyk M Geiss G McKh-
ann H Garcion C Vaucheret H and Sandberg G (2005) Auxin and
light control of adventitious rooting in Arabidopsis require ARGONA-
UTE1 Plant Cell 17 1343ndash1359Tanaka T Tanaka H Machida C Watanabe M and Machida Y (2004) A
new method for rapid visualization of defects in leaf cuticle reveals five
intrinsic patterns of surface defects in Arabidopsis Plant J 37 139ndash146Turgeon R and Wolf S (2009) Phloem transport cellular pathways and
molecular trafficking Annu Rev Plant Biol 60 207ndash221Ukitsu H Kuromori T Toyooka K et al (2007) Cytological and biochemi-
cal analysis of COF1 an Arabidopsis mutant of an ABC transporter gene
Plant Cell Physiol 48 1524ndash1533Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De
Paepe A and Speleman F (2002) Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biol 3 research00340031-00340011Verrier PJ Bird D Burla B et al (2008) Plant ABC proteins ndash a unified
nomenclature andupdated inventory Trends Plant Sci 13 151ndash159Vilaine F Palauqui JC Amselem J Kusiak C Lemoine R and Dinant
S (2003) Towards deciphering phloem a transcriptome analysis of the
phloem of Apium graveolens Plant J 36 67ndash81Zhai Z Sooksa-nguan T and Vatamaniuk OK (2009) Establishing RNA
interference as a reverse-genetic approach for gene functional analysis
in protoplasts Plant Physiol 149 642ndash652Zhang Q Blaylock LA and Harrison MJ (2010) Two Medicago truncatula
half-ABC transporters are essential for arbuscule development in
arbuscular mycorrhizal symbiosis Plant Cell 22 1483
Zhao C Craig JC Petzold HE Dickerman AW and Beers EP (2005)
The xylem and phloem transcriptomes from secondary tissues of the
Arabidopsis root-hypocotyl Plant Physiol 138 803ndash818
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
824 Rozenn Le Hir et al
two transmembrane domains (TMD) housing membrane
transit sites and two nucleotide bindinghydrolysis
domains (NBD) that provide the energetic basis for sub-
strate movement (Kneurooller and Murphy 2011) The ABCG
sub-class exhibits a TMDndashNBDndashTMDndashNBD architecture
and is divided into plantfungal-specific pleiotropic drug
resistance (PDR) full-length transporters and the eukaryotic
white brown complex (WBC) half-size transporters
that function as homo- or heterodimers to create the
TMDndashNBDndashTMDndashNBD structure (Verrier et al 2008) In
Arabidopsis the 28 half-size ABCG proteins comprise the
largest ABC sub-class with mechanistic diversity and sub-
strate specificity increased by the necessity for dimeriza-
tion for functionality (Verrier et al 2008 McFarlane et al
2010 Zhang et al 2010)
Arabidopsis ABCG11 (COF1DSOWBC11) and ABCG12
(CER5WBC12) have been shown to be required for the
export of various cuticular lipids (Pighin et al 2004 Bird
et al 2007 Luo et al 2007 Panikashvili et al 2007 2010
Ukitsu et al 2007) More recently it was shown that
ABCG26 (WBC27) plays an important role in the transport
of sporopollenin precursors (Choi et al 2010 Quilichini
et al 2010 Dou et al 2011 Kuromori et al 2011a) and
ABCG13 (WBC13) is required for flower cuticle secretion
and petal epidermis patterning (Panikashvili et al 2011) In
addition two ABCG half-size transporters (ABCG25WBC25
and ABCG22WBC22) are directly or indirectly involved in
ABA (abscisic acid) transport and responses (Kuromori
et al 2010 2011b) Here we show that three of the ABCG
family members ABCG9 ABCG11 and ABCG14 interact
physically with each other and are required for vascular
patterning in Arabidopsis
RESULTS
ABCG9 ABCG11 and ABCG14 are expressed in the
vascular system of Arabidopsis
In an effort to identify structural components of the
phloem infrastructure we searched several transcriptomic
datasets (Hertzberg et al 2001 Vilaine et al 2003 Schrader
et al 2004 Zhao et al 2005) for genes that are highly
expressed in the phloem and that encode transporters
Prominent in this selected group were genes encoding the
ABCG11 and ABCG14 lsquohalf transportersrsquo of the G sub-class
of ABC transporters A third ABCG transporter ABCG9 was
added to this group as it is closely related to ABCG14 (Ver-
rier et al 2008) We first measured their expression levels
in various organs by quantitative PCR (Figure S1andashc) Tran-
scripts for ABCG9 ABCG11 and ABCG14 were found in all
organs analyzed with an overall higher relative transcript
amount in the aerial parts compared with the root (Figure
S1andashc) For further analysis of the spatial expression pat-
tern each ABCG promoter was used to drive expression of
the GUS reporter gene ABCG9proGUS ABCG11proGUS
and ABCG14proGUS were visualized in the vascular sys-
tem of the cotyledons (Figure 1aei) The ABCG9proGUS
signal was restricted to the petiole main vein (Figure 1a)
GUS signals were also observed in phloem cells of the
flower stem for ABCG9proGUS ABCG11proGUS and
ABCG14proGUS (Figure 1bfj) and ABCG11proGUS sig-
nals were also observed in the cortical cells and interfasci-
cular fibers (Figure 1f) In 4-week-old rosette leaves
ABCG9proGUS (Figure 1c) ABCG11proGUS (Figure 1g)
and ABCG14proGUS (Figure 1k) were expressed in the
vascular system In addition expression of ABCG11pro
GUS was identified in the rosette leaf epidermis (Fig-
ure 1g) In roots ABCG11proGUS expression was detected
in lateral root primordia (Figure 1h) whereas expression
of ABCG9proGUS and ABCG14proGUS was observed in
the central cylinder (Figure 1di) Overall ABCG9 and
ABCG14 expression patterns were found to be restricted
to vascular bundles whereas in accordance with previous
results ABCG11 has a broader localization (Bird et al
2007 Luo et al 2007 Panikashvili et al 2007 Ukitsu
et al 2007)
Mutations in ABCG9 ABCG11 and ABCG14 are
responsible for defects in plant growth
Two TndashDNA insertion lines were identified and confirmed
in the first exon (SALK_047133) and the second exon
(GABI_353B01) of the ABCG9 gene (Figure 2a) The respec-
tive mutants were confirmed to be a weak (abcg9ndash1) and
null (abcg9ndash2) allele mutant (Figure 2de and Figure S1d)
Three independent mutations were confirmed in the
ABCG11 gene (abcg11ndash6 abcg11ndash7 and abcg11ndash8 Fig-
ure 2b) and were shown to be a weak allele mutant
(abcg11ndash6 SALK_096377 Figure 2f and Figure S1d) and
two knockout mutants (abcg11ndash7 and abcg11ndash8
GABI_590C03 and GABI_728E03 respectively Figure 2gh
and Figure S1d) Only one TndashDNA insertion was identified
in the promoter region of the ABCG14 gene (SALK_036952
Figure 2c) A reduced amount of full-length transcript was
amplified from the corresponding abcg14ndash1 mutant (Fig-
ure 2i and Figure S1d) Hence it was deemed to be a weak
allele mutant
The general phenotype of the various mutant lines was
assessed in both 20-day-old and 4-week-old plants grown
in soil (Figure 3andashn) Under these conditions no clear
phenotypic divergence from wild-type was observed for
the weak allele abcg14ndash1 (Figure 3hn) but a mild pheno-
type was observed for the single mutant abcg9ndash2 (10
smaller than the wild-type) (Figure 3cn) Interestingly the
double mutants abcg9ndash1 abcg14ndash1 (Figure 3jn and Figure
S1d) and abcg9ndash2 abcg14ndash1 (Figure 3kn and Figure S1d)
displayed a stronger phenotype (27 and 59 smaller than
the wild-type respectively) than the single mutants sug-
gesting an additive effect of these mutations Complemen-
tation of abcg9ndash2 and abcg14ndash1 mutants by hemagglutinin
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
812 Rozenn Le Hir et al
(HA)-tagged versions of ABCG9 and ABCG14 respectively
led us to conclude that these mutations were indeed
responsible for the observed phenotypes (Figure 3din
and Figure S1d) On the other hand the abcg11ndash7 homozy-
gous plants showed severe morphological abnormalities
similar to previously described abcg11 alleles (Bird et al
2007 Luo et al 2007 Panikashvili et al 2007 Ukitsu et al
2007) They were dwarf with unexpanded fused leaves
(Figure 3g and Figure S1d) Strikingly abcg11-7 plants car-
rying the mutation in ABCG11 gene in a heterozygous state
were 32 smaller than the wild-type plants on average
(Figure 3fn and Figure S1d) Finally abcg9-2abcg11-
7abcg14-1 mutants displayed a stronger phenotype than
abcg11ndash7 heterozygous plants (Figure 3ln) suggesting
that these mutations are additive Moreover the triple
homozygous mutants were as small as the abcg11ndash7
homozygous plants and displayed the same developmen-
tal abnormalities (Figure 3gmn)
Mutations in ABCG9 ABCG11 and ABCG14 affect vascular
patterning
Because these three genes are expressed in the vascular
system alleles of abcg9 abcg11 and abcg14 as well as
multiple mutants were analyzed for evidence of altered
vascular development by characterization of cotyledon
venation patterns in young seedlings (Cnops et al 2006)
The complexity of the vascular pattern was assessed in
terms of the number of secondary vein loops originating
from the mid-vein (Figure 4) In wild-type (Colndash0) seed-
lings 94 of the cotyledon venation pattern was between
classes III and V [from two loops plus the start of two
others (class III) up to four loops (class V) Figure 4] with
41 representing the most complex pattern (class V) The
double mutants abcg9ndash1abcg14ndash1 and abcg9ndash2abcg14ndash1
displayed a more complex venation pattern than the wild-
type (with respectively 100 and 96 of the cotyledon
(a) (b) (c) (d)
(e) (f) (g) (h)
(i) (j) (k) (l)
Figure 1 Expression patterns of ABCG genes
Expression patterns of ABCG9 ABCG11 and ABCG14 in cotyledons of 10-day-old seedlings (a e i respectively) the floral stem of 4-week-old plants (b f j
respectively) rosette leaves (c g k respectively) and roots (d h l respectively) co cortex e epidermis if interfascicular fibers ph phloem xy xylem Scale
bar = 100 lm
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 813
vascular pattern between classes III and V) The other
mutant lines showed a significantly less complex vena-
tion pattern than the wild-type (Figure 4) The single
mutants also differed significantly with respect to the loss
of complexity The abcg11ndash7 line displayed the least com-
plex vascular system (only 38 of the venation patterns
were between classes III and V) followed by abcg14ndash1
and then abcg9ndash2 (73 and 86 respectively) (Figure 4)
Introduction of HA-tagged versions of the ABCG9 and
ABCG14 proteins in the corresponding mutant back-
ground (abcg9ndash2 and abcg14ndash1) complemented the loss
of vascular complexity (Figure 4) confirming that the
phenotype was indeed due to defective expression of
ABCG9 and ABCG14 genes and the tagged proteins were
functional Interestingly the cotyledons of the triple
mutant displayed a significantly less complex vascular
system than that of the single mutants (Figure 4) In addi-
tion to the reduced vascular complexity in abcg11 homo-
zygous and heterozygous mutants the presence of open
vascular loops indicated a defect in vascular continuity
(Figure 4) Interestingly the continuity was restored in the
triple homozygous mutant (Figure 4)
Subsequently we characterized vascular bundles in the
floral stem of 4-week-old plants grown in soil (Table 1)
Under our conditions the wild-type flower stems dis-
played a vascular bundle density of 592 vascular bundles
per mm (Table 1) No significant variation in the number
of vascular bundles was observed in the single mutants
and complemented lines (Table 1) In contrast to the
increased complexity of the cotyledon venation pattern
(Figure 4) significantly fewer vascular bundles were pres-
ent in abcg9ndash2 abcg14ndash1 and abcg9ndash2 abcg11ndash7 abcg14ndash1
mutants (828 and 76 vascular bundles respectively)
(Table 1) Consistent with the smaller plant stature of
these double and triple mutants (Figure 1n) the diameter
of the floral stem was reduced (Table 1) However the
number of vascular bundles could not be explained by a
reduction of the floral stem diameter as the abcg11ndash7
mutant displayed the same number of vascular bundles
than the wild-type but its floral stem diameter was
strongly reduced (Table 1) Altogether these results
demonstrate that ABCG9 ABCG11 and ABCG14 play an
important role in vascular patterning during Arabidopsis
plant development
ABCG9 and ABCG14 are plasma membrane-localized
proteins that interact physically to form homo- andor
heterodimers with ABCG11
Because ABCG half transporters are thought to require
dimerization for functionality we investigated whether
ABCG9 ABCG11 and ABCG14 interact physically with
themselves andor other ABCG isoforms We first investi-
gated the subcellular localization of ABCG9 and ABCG14
after transfection of Arabidopsis mesophyll protoplasts
prepared from transgenic seedlings expressing the plasma
membrane marker low temperature induced protein 6b
(GFPndashLTi6b) (Cutler et al 2000) with constructs encoding
transcriptional fusions with red fluorescent protein (RFP)
In both cases the GFP fluorescent signal (Figure 5ae) co-
localized with the RFP fluorescent signal (Figure 5bf) at
the plasma membrane of transformed cells (Figure 5bh)
indicating that both ABCG9 and ABCG14 localize to the
plasma membrane like ABCG11 (Bird et al 2007 Luo
et al 2007 Panikashvili et al 2007 Ukitsu et al 2007) In
addition immunolocalization of a HA-tagged version of
ABCG9 and ABCG14 in their respective complemented
lines confirmed that these two proteins are localized to
the plasma membrane in planta (Figure 5ij) Next
(a)
(b)
(c)
(d) (e)
(f) (g)
(h) (i)
Figure 2 TndashDNA insertion lines for ABCG genes
(andashc) TndashDNA positions in ABCG9 ABCG11 and ABCG14 respectively Black
boxes promoters dark gray boxes 5prime and 3prime untranslated regions light gray
boxes exons lines introns
(dndashi) The presence of full-length mRNA was checked in abcg mutants as
described in Experimental procedures The elongation factor EF1a was used
as an internal control
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
814 Rozenn Le Hir et al
Arabidopsis protoplasts were transfected with open read-
ing frames encoding individual cndashMyc- or HA-tagged
versions of the three ABCGs and the identity of the epi-
tope-tagged proteins was verified by SDSndashPAGE and
Western blotting using anti-HA and anti-c-Myc antibodies
In all cases only one band of approximately 75 kDa cor-
responding to the predicted molecular mass of the pro-
teins in the Plant Membrane Protein Database (Schwacke
et al 2003) was obtained (Figure 5kndashp lane 1 and Figure
S2andashf lane 1) Subsequently protoplasts were co-trans-
fected with cndashMyc- and HA-tagged isoforms immunopre-
cipitated with antisera against one of the epitope tags
and probed with antisera directed against the alternative
epitope tag in Western blots of SDSndashPAGE gels in order
to determine whether homodimers or heterodimers were
formed (Figure 5kndashp) No ABCG14 homodimers or
ABCG9ndashABCG14 heterodimers were detected (Figure 5m
lane 4 and Figure 5o lane 4) In contrast ABCG11 formed
an apparent homodimer as described previously (McFar-
lane et al 2010) ABCG9 also formed an apparent homod-
imer ABCG14 formed an apparent heterodimer with
ABCG11 and ABCG9 formed an apparent heterodimer
with ABCG11 (Figure 5klnp lane 4) These interactions
were confirmed in a plant-based in vivo system using the
bimolecular fluorescence complementation assay (Figure
S3andashl) We therefore concluded that ABCG14 forms a strict
heterodimer with ABCG11 whereas ABCG11 and ABCG9
can both hetero- and homodimerize
Cuticular and epicuticular lipid precursor content as well as
cuticle integrity are not altered in abcg9ndash2 and abcg14ndash1
mutants
ABCG11 is known to be required for cuticle integrity (Bird
et al 2007 Luo et al 2007 Panikashvili et al 2007 Ukitsu
et al 2007) and because ABCG11 interacts physically with
ABCG9 and ABCG14 we looked for potential defects in
very long chain fatty acids (VLCFAs) which are among the
precursors of cuticular and epicuticular lipids (Roudier
et al 2010) as well as for possible cuticle defects in
abcg9ndash2 and abcg14ndash1 mutants
Quantification of fatty acids (from 160 to 180) as well as
VLCFAs (from 200 to 240) in apical parts of abcg9ndash2 and
abcg14ndash1 seedlings revealed no differences compared to
wild-type levels (Figure 6ab) It has previously been
(a) (b) (c) (d)
(e) (f) (g) (h)
(i)
(j)
(i)
(k) (l) (m)
(n)
Figure 3 Phenotype of abcg mutants grown in
soil
(andashm) Twenty-day-old plants grown in soil
Scale bar = 1 cm
(n) Green areas of 4-week-old plants Values
represent means SE from 20 individual
plants The experiment was repeated twice with
similar results Asterisks indicate statistically
significant differences between the wild-type
and mutants (P lt 005 Studentrsquos t test n = 20)
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 815
shown that when the cuticle is altered toluidine blue (TB)
permeates the epidermal surface (Tanaka et al 2004)
Therefore we used TB to check for cuticle defects in the
mutants After treatment with TB no staining was
observed in the wild-type abcg11+ or the abcg9ndash2 and
abcg14ndash1 mutants (Figure 6cef) whereas abcg11ndash7
homozygous seedlings showed patches of blue staining
confirming the cuticle alteration previously described
(Figure 6d) (Ukitsu et al 2007) These observations sug-
gest that unlike mutations in ABCG11 mutations in
ABGC9 or ABCG14 do not affect cuticle formation
abcg9ndash2 and abcg14ndash1 mutants are defective in sterol
composition
To obtain an insight into the potential substrates trans-
ported by these ABCG transporters we analyzed the shoot
and root metabolome of single mutants Orthogonal partial
least-squares discriminant analysis was applied to the
GCMS data in order to classify the profiles of the geno-
types In both shoots (Figure 7andashc) and roots (Figure S4andash
c) homozygous abcg11ndash7 abcg9ndash1 abcg9ndash2 and abcg14ndash1
mutants exhibited very different metabolomic profiles from
the wild-type Interestingly the heterozygous abcg11ndash7+
and abcg9ndash2+ plants had metabolomic profiles intermedi-
ate between the wild-type and their respective homozy-
gous mutant plants (Figure 7ab and Figure S4ab) These
results suggest that a mutation in one copy of ABCG11 or
ABCG9 is sufficient to significantly affect the physiology of
the plant even if it does not dramatically affect visible
aspects of its phenotype
The metabolites showing the most significant differ-
ences were identified by comparing their retention indices
and mass spectra with entries in publicly available reten-
tion libraries (Schauer et al 2005) For all the genotypes
analyzed the metabolites identified as being significantly
different from the wild-type belonged to three main clas-
ses amino acids carbohydrates and lipidssterols The
levels of these compounds were almost all reduced in
abcg9ndash2 and abcg14ndash1 but in excess in abcg11ndash7 (Table 2
and Appendix S1)
Because our study focused on vascular development in
aerial tissues only data obtained for the apical parts are
described in detail here (Table 2) Root data are presented
in Appendix S1 Whereas many neutral amino acids were
significantly depleted in abcg9ndash2 and abcg14ndash1 aerial
Figure 4 Cotyledon venation pattern in abcg mutant lines
Venation complexity and continuity of each examined class of mutant and
wild-type seedlings Values in brackets indicate the percentage contribution
of each class Asterisks indicate statistically significant differences between
the wild-type and mutants (P lt 005 Pearsonrsquos v2 test with Monte Carlo
permutations N = 10 000)
Table 1 Distribution of vascular bundles in the floral stem
Genotype Vascular bundle number Floral stem diameter (mm) Vascular bundle density Total
Col-0 930 105 157 020 592 049 10abcg11-7+ 914 134 140 080 652 042 12abcg11-7 902 115 051 004 176 182 8abcg14-1 828 049 145 018 632 0049 1135SHA-ABCG14 abcg14-1 940 089 170 016 554 058 12abcg9-1 957 053 160 006 586 031 7abcg9-2 875 103 162 010 553 046 835SHA-ABCG9 abcg9-2 933 051 174 010 606 026 8abcg9-1 abcg14-1 871 048 154 003 565 030 8abcg9-2abcg14-1 828 049 124 015 674 084 8abcg9-2abcg14-1abcg11-7
760 054 063 005 1203 169 9
Statistically significant differences between the wild-type and mutants (P lt 005 Studentrsquos t test)
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
816 Rozenn Le Hir et al
tissues (nine and eight out of 20 neutral amino acids
respectively) only two amino acids (glutamine and gluta-
mate) associated with Arabidopsis phloem cells (Schad
et al 2005) were different in abcg11ndash7 (Table 2) Xylose
and the non-reducing sugars trehalose and raffinose were
also decreased in abcg14ndash1 (Table 2) whereas glucose
fructose galactose maltose xylose were significantly dif-
ferent in abg9ndash2 and abcg11ndash7 (reduced and increased
respectively compared to wild-type) (Table 2)
Levels of sterols (campesterol 24ndashmethylene cholesterol
and sitosterol) saturated and unsaturated fatty acids (lino-
leic acid lauric acid palmitic acid and triacontanoic acid)
glycerolipids (monoacylglycerol) and lipid metabolism
intermediates (glycerol glycerol-3ndashphosphate and inositol-
1ndashphosphate) were also significantly different from wild-
type in abcg mutants (Table 2) The abcg9ndash2 mutant
showed 30 lower levels of 24ndashmethylene cholesterol
compared to the wild-type (Appendix S1) In the abcg14ndash1
mutant 50 and 35 decreases of 24ndashmethylene cholesterol
and sitosterol levels were measured respectively (Appen-
dix S1) Finally campesterol showed a 50 increase in the
abcg11ndash7 mutant (Appendix S1) To extend this analysis
measurements of conjugated sterols [sterol ester (SE)
sterol glucoside and acylated sterol glucoside] were also
performed When seedlings were grown in vitro no differ-
ence in the sterol glucoside and acylated sterol glucoside
contents was detected between the wild-type and the vari-
ous mutant lines (Figure S4d) However there was a ten-
dency towards a reduced SE content in the double mutant
abcg9ndash2 abcg14ndash1 compared to the wild-type (Figure S4d)
This observation prompted us to examine variations in the
SE content in older plants (4-week-old plants grown in soil
in the greenhouse) At this developmental stage no differ-
ence in the free sterol (FS) content was observed between
the wild-type and the various mutant lines (Figure 7d) but
the fraction comprising FS and SE was significantly
(a) (b) (c) (d)
(e) (f) (g) (h)
(i) (j)
(k) (l) (m)
(n) (o) (p)
Figure 5 Subcellular localization and physical interactions of ABCG9 ABCG11 and ABCG14
(andashh) ABCG9 and ABCG14 co-localize to the plasma membrane when transiently expressed in Arabidopsis mesophyll protoplasts of the GFPndashLTi6b marker line
(Cutler et al 2000)
(a e) Cyan fluorescence of GFPndashLTi6b(b f) Purple fluorescence from mRFPndashABCG9 and mRFPndashABCG14 respectively
(c g) Yellow auto-fluorescence of the chloroplasts
(d h) Merged images of the three channels Scale bar = 10 lm
(i j) Confocal images of immunofluorescence staining with anti-HA antibodies in root cells of abcg9ndash2 and abcg14ndash1 mutants complemented with HA-tagged
ABCG9 (i) and HA-tagged ABCG14 (j) Scale bars = 10 lm
(kndashp) Proteins were extracted from protoplasts co-transfected with HA- or cndashMyc-tagged versions of ABCGs (lanes 1 and 4) or transfected with a single plasmid
containing the HA-tagged version (lane 2) or the c-Myc-tagged version (lane 3) In lanes 2ndash4 proteins were immunoprecipitated with the anti-c-Myc antibody
and subjected to anti-HA protein gel-blot analysis to reveal the other partner of the dimer Transient co-expression of (k) HAndashABCG9cndashMycndashABCG9 (l) HAndashABCG11cndashMycndashABCG11 (m) HAndashABCG14cndashMycndashABCG14 (n) HAndashABCG14cndashMycndashABCG11 (o) HAndashABCG14cndashMycndashABCG9 and (p) HAndashABCG11cndashMycndashABCG9
in wild-type protoplasts
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 817
reduced probably accounting for the difference in the SE
content between the wild-type and the abcg9ndash2 abcg14ndash1
double mutant (Figure 7e)
(a)
(b)
(c) (d) (e)
(f) (g)
Figure 6 Long chain and very long chain fatty acid content and cuticle
integrity in abcg9ndash2 and abcg14ndash1 mutants
(a b) Fatty acid (a) and VLCFA content (b) in abcg9ndash2 and abcg14ndash1 mutants
in apical parts of 10-day-old seedlings (three biological replicates each of
approximately 5 mg dry weight were used)
(cndashf) Toluidine blue staining patterns of abcg mutants in 7-day-old seed-
lings Scale bars = 3 cm
(a)
(b)
(c)
(d) (e)
Figure 7 Metabolome analysis and measurements of sterol content in abcg
mutants
(andashc) Results of orthogonal partial least-squares discriminant analysis of
global metabolite contents of shoots of 7-day-old seedlings from wild-type
(black squares) abcg11ndash7+ (gray triangles) abcg11ndash7 (white stars) abcg9ndash1(white hexagons) abcg9ndash2+ (gray hexagons) abcg9ndash2 (white hexagons)
and abcg14ndash1 (black circles) seedlings (five biological replicates of 10 mg
each were used for the analysis)
(d e) Free sterol (FS) and sterol ester (SE) content of wild-type (white bars)
abcg9ndash2 (light gray bars) abcg14ndash1 (dark gray bars) and abcg9ndash2 abcg14ndash1(black bars) Error bars indicate standard deviations obtained from three
independent biological replicates The asterisk indicates a statistically signif-
icant difference between the wild-type and the mutant (P lt 005 Studentrsquos
t test)
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
818 Rozenn Le Hir et al
DISCUSSION
ABCG9 and ABCG14 are two plasma membrane proteins
that interact physically in multiple homo-heterodimer
combinations
In this study we showed that like the previously described
ABCG11 (Pighin et al 2004 Bird et al 2007 Luo et al
2007 Panikashvili et al 2007 2010 Ukitsu et al 2007)
ABCG9 and ABCG14 localize to the plasma membrane
Based on co-immunoprecipitation of tagged proteins and
the bimolecular fluorescence complementation assay we
demonstrated that ABCG9 interacts with ABCG11 to form a
heterodimer and like ABCG11 (McFarlane et al 2010) can
homodimerize In contrast ABCG14 interacts only with
ABCG11 forming an obligate heterodimer This extends
the number of potential partners of ABCG11 to three as it
was previously shown to dimerize with the ABCG12CER5
protein (McFarlane et al 2010) Similarly ABCG9 under-
goes flexible dimerization to form either homo- or hetero-
dimers suggesting that ABCGs may be promiscuous
proteins with different functions depending on their inter-
acting partner andor expression profile
Mutations in ABCG genes are semi-dominant
In the last few years several Arabidopsis mutants with
altered expression of various ABCG genes such as ABCG11
(Bird et al 2007 Luo et al 2007 Panikashvili et al 2007
Ukitsu et al 2007) ABCG12CER5 (Pighin et al 2004)
ABCG13 (Panikashvili et al 2011) ABCG22 (Kuromori
et al 2011a) ABCG25 (Kuromori et al 2010) and ABCG26
(Choi et al 2010 Quilichini et al 2010 Dou et al 2011
Kuromori et al 2011b) have been characterized but even
though these half-size transporters require dimerization to
become functional all the mutations were described as
recessive Here we demonstrated that mutations in these
genes have semi-dominant effects Indeed the metabolome
analyses showed that abcg9ndash2+ and abcg11ndash7+ heterozy-
gous seedlings have a profile intermediate between that of
wild-type and homozygous seedlings The possibility of
dominant-negative effects was excluded because no
Table 2 Shoot metabolites of 10-day-old abcg9ndash2 abcg11ndash7 and abcg14ndash1 seedlings whose levels were significantly different from wild-type
Group
Metabolites that differed between mutant and wild-type
abcg9ndash2Colndash0 abcg11ndash7Colndash0 abcg14ndash1Colndash0
Amino acids Glycine Glutamate AlanineAlanine Glutamine ValineValine ThreonineThreonine GABASerine Prolinebndashalanine IsoleucineIsoleucine SerineGABA bndashalanineProline
Carbohydrates Glucose Glucose ErythritolFructose Fructose TrehaloseXylose Galactose XyloseMaltose RaffinoseSucrose
Lipids and intermediates 24ndashmethylene-cholesterol Campesterol Linoleic acidMonoacylglycerol Glycerol-3ndashphosphate Triacontanoic acidPalmitic acid Inositol-1ndashphosphate GlycerolGlycerol-3ndashphosphate Lauric acidInositol-1ndashphosphate 24ndashmethylene-cholesterol
SitosterolGlycerol-3ndashphosphate
TCA cycle and others Phosphoric acid Phosphate compound Glyceric acid24ndashdihydroxybutanoic acid Dehydroascorbic acid Fumaric acidGlyceric acid Malic acid Threonic acid-14ndashlactoneFumaric acid 2ndashoxoglutaric acid Threonic acidThreonic acid bndashhydroxy-bndashmethylglutaric acid Shikimic acidThreonic acid-14ndashlactone Gluconic acid lactone-like Succinic acidShikimic acid Phytol2ndashoxoglutaric acid Ethanolamine
Nicotinic acid
For abcg9ndash2 and abcg14ndash1 the levels of the compounds were reduced whereas they were increased in abcg11ndash7
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 819
truncated transcript was found for either abcg9ndash2 or
abcg11ndash7 mutants Hence abcg9ndash2 and abcg11ndash7 are true
null alleles These observations are consistent with the
requirement for homo- or heterodimerization of the ABCG
proteins to form functional transporters In the heterozy-
gous plants ABCG11 transcript levels were 50 lower than
in wild-type counterparts suggesting that ABCG11 protein
and thus corresponding homo- and heterodimers were
likely to be less abundant which may also explain the meta-
bolic perturbations and consequent phenotypic modifica-
tions These findings also suggest that wild-type plants are
not saturated by ABCG11 proteins as a 50 reduction in
protein content appears to be sufficient to alter the physiol-
ogy of the heterozygous mutant plants This hypothesis is
strengthened by the observation that mutants carrying the
weak abcg11ndash6 allele in which there was only a 25 reduc-
tion in ABCG11 transcript levels showed a similar pheno-
type to the heterozygous abcg11ndash7+ and abcg11ndash8+
plants Similarly the abcg9ndash1 weak allele mutant had a very
similar phenotype to the abcg9ndash2+ heterozygous seedlings
suggesting that considering the mode of action of these
proteins the semi-dominant effects that we observed are
likely to be a general feature among the ABCG genes
ABCG9 ABCG11 and ABCG14 transporters are involved in
vascular system development in Arabidopsis
We showed that the ABCG11 ABCG9 and ABCG14 promot-
ers drove expression in the vascular system of Arabidopsis
cotyledons and rosette leaves and more precisely in the
phloem of flower stems In addition mutations in ABCG9
ABCG11 and ABCG14 genes altered vascular development
in cotyledons and the floral stem Similar vascular localiza-
tion of ABCG11 has already been described by Panikashvili
et al (20072010) who showed that in addition to the epi-
dermis ABCG11 is expressed in the vasculature as early as
during embryo development and then throughout plant
development They showed that the vascular pattern in
leaves of the homozygote mutant seedlings was less com-
plex than in the wild-type and discontinuous (Panikashvili
et al 2007) The present article describes a mutant that
has both cuticle and vascular system patterning defects
although it has been previously shown that mutants
altered in cuticle formation have dramatically reduced
growth and altered leaf morphology (Jenks et al 1996)
which may account for vascular defects Analysis of the
abcg11+ heterozygous seedlings that have vascular
defects but apparently no cuticle alteration suggested that
these phenotypes are probably uncoupled Because
ABCG11 dimerizes with several members of its sub-family
it seems reasonable to postulate that it is involved in the
transport of different compounds depending on its partner
Here we provide evidence that ABCG11 in addition to
forming dimers with ABCG12 (McFarlane et al 2010) in
epidermal cells is likely to dimerize with ABCG9 and
ABCG14 in phloem cells suggesting functional compart-
mentalization of these proteins and a possible role in vas-
cular system patterning in Arabidopsis
Metabolite profiling suggests that ABCG9 ABCG11 and
ABCG14 are involved in sterollipid homeostasis
We investigated in more depth the metabolic changes
induced by mutations in ABCG9 ABCG11 and ABCG14
We found that levels of metabolites of several classes
including lipids sugars and amino acids were altered in
both the heterozygotes and homozygotes and that the
profiles in the root and apical parts differed suggesting
that ABCGs may play different roles in the roots and shoot
Interestingly the modifications of amino acid and sugar
contents observed in the metabolic profiles of the abcg
mutants suggest an impaired carbonnitrogen balance that
may be directly or indirectly linked to the vascular defects
leading to possible problems with phloem loading or
unloading Saturated and unsaturated fatty acids and sterol
contents were also altered in the mutants Patterning
defects in the vascular system are often observed in
mutants altered in hormone homeostasis such as for
auxin or brassinosteroids (Kaneda et al 2011) and direct
or indirect modifications of sterol homeostasis are thought
to be one of the primary causes of these downstream
effects (Pullen et al 2010) The detected defects in sterol
homeostasis may explain the abnormal vascular develop-
ment observed in abcg9 abcg11 and abcg14 mutants
Accordingly several sterol-deficient mutants such as fac-
kelhydra (Jang et al 2000) and cpv1 (Carland et al 2002)
have also been shown to exhibit severe defects in vascular
development It has been suggested that the bushy habit
and low fertility of cpv1smt2 mutants may be due to an
increased campesterolsitosterol ratio rather than sterol
deficiency per se (Clouse 2002) This ratio is controlled by
the activity of SMT2 (STEROL METHYLTRANSFERASE2)
Interestingly strong down-regulation of SMT2 expression
was observed in all the mutant backgrounds (Figure S5)
and the metabolic data indicated a deficit in 24ndashmethylene
cholesterol (precursor of both campesterol and sitosterol)
andor sitosterol in abcg9 and abcg14 mutants respec-
tively In addition the significant decrease in sterol ester
content in the double mutant abcg9ndash2 abcg14ndash1 correlated
with down-regulation of expression of the PHOSPHOLIPID
STEROL ACYLTRANSFERASE1 (PSAT1) gene encoding the
enzyme that is mainly responsible for SE synthesis (Bou-
vier-Nave et al 2010) and is proposed to be a control step
in sterol homeostasis (Kopischke et al 2013) Altogether
these results suggest that the vascular phenotype
observed in abcg mutants may be linked to modification of
sterol homeostasis
Therefore we postulate that the various dimer combina-
tions between ABCG9 ABCG11 and ABCG14 may
participate in the transport of squalene-derived metabolites
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
820 Rozenn Le Hir et al
such as sterol andor sterol conjugates via the phloem sap
The presence of sterols in phloem sap has been observed in
various species such as barley (Hordeum vulgare Lehrer
et al 2000) and fava bean (Vicia faba Bouvaine et al
2012) and indirect proof of the presence of sterols in
phloem sap has been obtained by analysis of the phytos-
terol content in exclusively phloem-feeding aphids that are
unable to synthetize them directly (Behmer et al 2011) In
addition the evidence for long-distance transport of sterol
precursors has been supported by observations that sterol
esters accumulate in organs distal to the site of application
of exogenous precursors (Bouvier-Nave et al 2010) As
sterols have been proposed to act as signal molecules in
the control of developmental processes such as vascular
patterning (Carland et al 2002 Pullen et al 2010) modifi-
cations of sterol homeostasis may affect development of
the vascular system in the abcgmutants described here
In conclusion we propose that ABCG9 ABCG11 and
ABCG14 are important for lipidsterol homeostasis Thus
these three proteins are likely to be involved in the control
of various aspects of plant development andor physiol-
ogy including patterning of the vascular system
EXPERIMENTAL PROCEDURES
Plant material and growth conditions
Insertion lines in ABCG9 (SALK_047133 and GABI_353B01)ABCG11 (SALK_096377 and GABI_590C03) and ABCG14 (SALK_036952) genes were obtained from the European ArabidopsisStock Centre (httparabidopsisinfo) Heterozygoushomozygousplants were screened using gene-specific primers in combinationwith the specific primer for the left border of the TndashDNA insertion(Table S1)
Plants were grown in soil or in vitro as described previously(Sorin et al 2005) Arabidopsis Colndash0 cell cultures were main-tained by weekly sub-culturing in MS medium (Murashige andSkoog 1962) supplemented with 240 lg l1 24ndashdichlorophenoxy-acetic acid and 14 lg l1 kinetin and were grown under 12 h light12 h dark cycles at 25 lE m2 sec1 and with 23degC day and nighttemperatures
Morphological characterization and GUS staining
To study the venation pattern cotyledons of 7-day-old seedlingsand rosette leaves of 4-week-old plants were cleared in a buffercontaining chloral hydrate glycerol and water (831 wvv) afterwhich 200 cotyledons per genotype were analyzed and the per-centage showing particular levels of complexity or discontinuity invascularity was estimated as described by Cnops et al (2006)
Flower stem segments (1ndash2 cm long) were removed from ten3ndash4-week-old plants grown in soil embedded in 8 agarose andsectioned using a VT100S vibratome (Leica httpwwwleicacom)The cross-sections were then stained with safraninalcian blue for1ndash2 min (Sigma httpwwwsigmaaldrichcom) rinsed in waterand mounted in 50 glycerol
Histochemical GUS assays were performed as described previ-ously (Sorin et al 2005) All observations were performed usingan Axioplan light microscope equipped with an Axiocam cameraand Axiovision software (Zeiss httpwwwzeisscom)
To observe potential cuticle defects in the various mutant linesa TB test was performed by immersing in vitro-grown plantlets ina 005 wv TB solution for 2 min as described by Tanaka et al(2004)
Statistical analyses
Statistical analyses of differences between-genotype means andvariance for the studied morphological traits were performedusing Studentrsquos t test (GraphPad Prism version 50 httpwwwgraphpadcom) In the venation pattern experiment thesignificance of differences between wild-type and mutants wasassessed using R (R Development Core Team 2010) and Pearsonrsquosv2 test with Monte Carlo permutations (N = 10 000)
Plasmid constructs
The native ABCG promoters and open reading frame of ABCG11were amplified from genomic DNA or cDNA extracted from 7-day-old Arabidopsis seedlings using Phusion high-fidelity DNApolymerase (Finnzyme wwwthermoscientificbiocomfinnzymes)according to the manufacturerrsquos instructions with gene-specificprimers (Table S1) The PCR products were cloned into pENTRDndashTOPO (Invitrogen httpwwwinvitrogencom) and recombinedinto pKGWFS7 (Karimi et al 2002) to create ABCGproGUS Allrecombinations were performed using the Gateway LR Clonase
enzyme mix (Invitrogen) At each cloning step the sequenceswere confirmed by sequencing and all the constructs weretransferred into Agrobacterium strain GV3101pMP90 (C58C1)Wild-type or abcg11ndash7+ heterozygous plants were transformed byfloral dipping (Clough and Bent 1998) For ABCGGUS constructsthe expression pattern was checked in the T2 progeny of 10ndash15independent transgenic lines and one representative homozygousline was used for further characterization
Tagged protein constructs and protoplast transformation
Epitope-tagged versions of ABCG9 ABCG11 and ABCG14 proteinswere produced in pRT104ndash3xHA and pRT104ndash3xMyc plasmids(Feurouleuroop et al 2005) and pSAT6-mRFP (Citovsky et al 2006) Allthese plasmids have a 35S promoter sequence upstream of themulti-cloning site The open reading frames of ABCG11 andABCG14 and the genomic clone of ABCG9 were amplified fromcDNA or genomic DNA extracted from 7-day-old Arabidopsisseedlings using Phusion high-fidelity DNA polymerase (Finnzyme)according to the manufacturerrsquos instructions with gene-specificprimers carrying EcoRI or SalI restriction sites to facilitate subse-quent cloning (Table S1) The products obtained after PCR weredigested by EcoRI and SalI prior to ligation into appropriate plas-mids that had previously been cut open with the same restrictionenzymes Constructs were verified by sequencing Protoplastsfrom Arabidopsis cell cultures or 14-day-old Arabidopsis seedlingswere prepared and transformed as previously described (Meski-ene et al 2003 Zhai et al 2009) For co-immunoprecipitation andsubcellular localization assays 100 000 and 50 000 protoplastcells respectively were transfected with 15 and 10 ll of plasmidsrespectively (containing between 5 and 75 lg of each construct)and the resulting transformed protoplasts were processed afterincubation in the dark at room temperature for 16ndash18 and 24 hrespectively
Whole-mount immunolocalization
The HA-tagged versions of ABCG9 and ABCG14 were detected in7-day-old in vitro-grown Arabidopsis seedlings as described inMethods S1 Anti-HA antibody 12CA5 (Roche httpwwwroche
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 821
com) (1100 dilution) combined with Alexa Fluor 488-conjugatedsecondary antibody (1100 dilution) were used
Confocal laser-scanning microscopy
Images were collected using an SP2 confocal microscope (Leica)RFP and Alexa Fluor 488 were detected using laser lines at 561and 488 nm respectively according to the manufacturerrsquos set-tings RFP fluorescence emission was detected at 550ndash600 nmand Alexa Fluor 488 and GFP fluorescence emission were detectedat 495ndash545 nm For subcellular localization in protoplasts imageswere coded in cyan (GFP) magenta (RFP) and yellow (auto-fluo-rescence) resulting in purple coloration for co-localization inmerged images For the immunolocalization experiment imageswere coded in green (GFP) A water-corrected 209 objective (HCPlan-Apochromat 20907 ImmKor Leica) was used Images werecropped using Adobe Photoshop CS2 and assembled using AdobeIllustrator CS2 software (Abode httpwwwabodecom) Eachimage shown represents a single focal plan
Co-immunoprecipitation assays
Transformed protoplast proteins were extracted using 50 ll lysisbuffer containing 25 mM TrisHCl pH 78 10 mM MgCl2 75 mM
NaCl 5 mM EGTA 1 mM benzamidine 1 mM dithiothreitol 10glycerol 1 Triton Xndash100 and 19 protein inhibitor cocktail(Sigma httpsigmaaldrichcom) The transformed protoplast sus-pension was frozen in liquid nitrogen then thawed on ice andsubjected to centrifugation for 5 min at 150 g Two microliters ofanti-Myc (9E10 Covance httpwwwcovancecom) or anti-HA(16B12 Covance) antibodies were added to the extracts and themixture was incubated for 2 h at 4degC on a rotating wheel Immu-nocomplexes were captured by incubation with 10 ll Protein GndashSepharose beads for a further 2 h at 4degC on a rotating wheel afterwhich they were washed three times for 5 min each in 25 mM
NaPi 150 mM NaCl 5 glycerol and 02 Igepal CAndash630 buffer(Sigma) and then eluted using 40 ll SDS sample buffer at 37degCfor 30 min Co-immunoprecipitation of Myc- and HA-tagged pro-teins was revealed by SDSndashPAGE followed by Western blottingusing both anti-Myc (Invitrogen) and anti-HA (3F10 Roche) anti-bodies at 12000 dilution
RNA extraction and cDNA preparation
To quantify the relative amounts of the ABCG transcriptsexpressed in various organs three rosette leaves all the cau-line leaves the bottom 5 cm of the floral stem all closed flow-ers and all green siliques were harvested from each of threeindependent 4-week-old Arabidopsis plants of each genotypegrown in soil For the other experiments 10-day-old seedlingsgrown in vitro were harvested All the material was directly fro-zen in liquid nitrogen and stored at 80degC prior to RNA extrac-tion Samples were then disrupted using a MM 301 vibrationmill (Retsch GmbH httpwwwretschcom) at a frequency of25 Hz for 2 min with 3 mm tungsten carbide beads (Qiagenhttpwwwqiagencom) Total RNA was isolated from thismaterial using TRIzol (Life Technologies httplifetechnologiescom) RNA quantity and quality were measured using a Nano-Drop ND 1000 (Thermo Scientific httpwwwthermoscientificcom) All samples with 260280 nm and 260230 nm absorbanceratios lower than 18 were discarded and RNA was re-extractedDNase treatment was applied to 5 lg samples of total RNAusing a DNase I kit (Fermentas httpwwwfermentascom)Double-stranded cDNA was synthesized from 5 lg total RNAusing a SuperScript reverse transcriptase kit (Invitrogen) witholigo(dT)12 primer Finally RNase treatment was applied to the
samples using Escherichia coli RNAse H (Fermentas) The cDNAsamples obtained were diluted 30-fold (except for abcg9 linesamples which were diluted 10-fold) prior to use either toverify the absence of full-length transcripts in putative knockoutmutants or to quantify gene expression by quantitative PCR Allprimers used were designed using the Primer3 program (Rozenand Skaletsky 2000) and their sequences are listed inTable S1
QUANTITATIVE REAL-TIME PCR
Quantitative PCR amplifications were performed in 20 llmixtures containing 10 ll 59 SYBR Green I master mix
(Roche) 02 ll forward and reverse primer (30 lM each)
48 ll sterile water and 5 ll cDNA The BiondashRad CFX96
real-time PCR detection system (httpwwwbio-radcom)
was used for quantification of expression of the sterol
biosynthesis transcripts and the ABCG transcripts in the
various mutant backgrounds Finally the Roche LightCy-
cler 480 system (Roche) was used to quantify the rela-
tive abundance of ABCG transcripts in various organs
Four genes were considered as potential reference genes
[APT1 (At1g27450) TIP41 (At4g34270) EF1a (At5g60390)
and UBQ10 (At4g05320)] and their suitability was tested
using the geNorm algorithm (Vandesompele et al 2002)
to determine the most appropriate one for each experi-
ment A more detailed procedure following the Minimum
Information for Publication of Quantitative Real-Time PCR
Experiments guidelines (Bustin et al 2009) is described
in Methods S2 and the annealing temperature and
efficiencies of the primer pairs used are shown in
Table S2
VLCFA quantification
Approximately 5 mg dry weight of 10-day-old in vitro
Arabidopsis seedlings were analyzed for VLCFA quantifica-
tion as described by Li et al (2006)
Sterol extraction and GC-FID characterization
A pool of 10-day-old seedlings grown in vitro or rosettes
from 4-week-old plants grown in soil were lyophilized prior
to extraction Sterol extraction and isolation was per-
formed as described previously (Bouvier-Nave et al 2010)
Gas Chromatography-Flame Ionization Detector (GC-FID)
characterization was performed as described by Silvestro
et al (2013) To quantify the total amounts of free sterols
and sterol esters non-saponifiable lipids were extracted
using nndashhexane and sterols were derivatized as sterols ace-
tates Quantification was achieved using lupenyl-3 28ndashdi-
acetate as an internal standard
Metabolome analysis
To obtain metabolome profiles associated with wild-type
abcg11ndash7+ abcg11ndash7 abcg9ndash1 abcg9ndash2 and abcg14ndash1
genotypes 10-day-old seedlings representing each geno-
type grown in vitro as described above were harvested
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
822 Rozenn Le Hir et al
and divided into shoot and root samples In the case of
abcg9ndash2 the phenotype of the heterozygote abcg9ndash2+
was indistinguishable from the homozygote phenotype
and therefore heterozygous and homozygous seedlings
from a segregating population were pooled Hence in this
particular experiment abcg9ndash2+ refers to a mixed popula-
tion comprising two-thirds heterozygotes and one-third
homozygotes
Metabolites in five samples of shoot and root parts of
seedlings of each genotype (10 mg fresh weight per sam-
ple) were then extracted analyzed by GCMS and the
acquired data were processed as previously described
(Gullberg et al 2004 Jonsson et al 2005) All multivariate
statistical investigations (partial component analysis and
orthogonal partial least-squares discriminant analysis) for
between-genotype similarities and differences in general
metabolite profiles were performed using Simca software
(Umetrics httpwwwumetricscom)
ACKNOWLEDGEMENTS
We thank A Murphy (Plant Science and Landscape Archi-
tecture University of Maryland MD USA) for stimulating
discussions and critical reading of the manuscript I Carl-
son (Umea Plant Science Center Department of Forest
Genetics and Plant Physiology Swedish University of Agri-
cultural Sciences Umea Sweden) for technical assistance
and S Vernhettes for kindly donating seeds of the
transgenic line carrying the plasma membrane marker
GFPndashLTi6b We also thank the European Arabidopsis Stock
Centre and the GABI-Kat stock centre for providing seeds
This work was supported by allocations granted to CB by
the Swedish Research Council for Agriculture the Swedish
Foundation for Strategic Research the Swedish Research
Council for Research and Innovation for Sustainable
Growth the K amp A Wallenberg Foundation and the Carl
Trygger Foundation
SUPPORTING INFORMATION
Additional Supporting Information may be found in the onlineversion of this articleAppendix S1 Raw data quantification for compounds identified inthe metabolite profiling by GCMS
Figure S1 Relative expression of ABCG genes in various organsand mutant backgroundsFigure S2 Co-immunoprecipitation controlsFigure S3 ABCG9 and ABCG14 homo- andor heterodimerize withABCG11Figure S4 Complementary data on metabolome analysis and con-jugated sterol contentFigure S5 Relative expression of genes related to sterol homeo-stasisMethods S1 Method for whole-mount immunolocalizationMethods S2 Method for quantitative PCR
Table S1 List of primers used in this studyTable S2 Annealing temperature and efficiencies for the primerspairs used in this study
REFERENCES
Behmer ST Grebenok RJ and Douglas AE (2011) Plant sterols and host
plant suitability for a phloem-feeding insect Funct Ecol 25 484ndash491Bird D Beisson F Brigham A Shin J Greer S Jetter R Kunst L
Wu XW Yephremov A and Samuels L (2007) Characterization of Ara-
bidopsis ABCG11WBC11 an ATP binding cassette (ABC) transporter that
is required for cuticular lipid secretion Plant J 52 485ndash498Bouvaine S Behmer ST Lin GG Faure MndashL Grebenok RJ and
Douglas AE (2012) The physiology of sterol nutrition in the pea aphid
Acyrthosiphon pisum J Insect Physiol 58 1383ndash1389Bouvier-Nave P Berna A Noiriel A Compagnon V Carlsson AS
Banas A Stymne S and Schaller H (2010) Involvement of the phos-
pholipid sterol acyltransferase1 in plant sterol homeostasis and leaf
senescence Plant Physiol 152 107ndash119Bustin SA Benes V Garson JA Hellemans J Huggett J Kubista
M Mueller R Nolan T Pfaffl MW and Shipley GL (2009) The MIQE
guidelines Minimum information for publication of quantitative real--
time PCR experiments Clin Chem 55 611
Carland FM Fujioka S Takatsuto S Yoshida S and Nelson T (2002)
The identification of CVP1 reveals a role for sterols in vascular pattern-
ing Plant Cell 14 2045ndash2058Choi H Jin JY Choi S Hwang JU Kim YY Suh MC and Lee Y
(2010) An ABCGWBC-type ABC transporter is essential for transport of
sporopollenin precursors for exine formation in developing pollen Plant
J 65 181ndash193Citovsky V Lee LY Vyas S Glick E Chen MH Vainstein A Gafni
Y Gelvin SB and Tzfira T (2006) Subcellular localization of interacting
proteins by bimolecular fluorescence complementation in planta J Mol
Biol 362 1120ndash1131Clough SJ and Bent AF (1998) Floral dip a simplified method for Agro-
bacterium-mediated transformation of Arabidopsis thaliana Plant J 16
735ndash743Clouse SD (2002) Arabidopsis mutants reveal multiple roles for sterols in
plant development Plant Cell 14 1995ndash2000Cnops G Neyt P Raes J et al (2006) The TORNADO1 and TORNADO2
genes function in several patterning processes during early leaf develop-
ment in Arabidopsis thaliana Plant Cell 18 852ndash866Cutler SR Ehrhardt DW Griffitts JS and Somerville CR (2000) Random
GFPcDNA fusions enable visualization of subcellular structures in cells of
Arabidopsis at a high frequency Proc Natl Acad Sci USA 97 3718ndash3723Dou XY Yang KZ Zhang Y Wang W Liu XL Chen LQ Zhang
XQ and Ye D (2011) WBC27 an adenosine tri-phosphate-binding cas-
sette protein controls pollen wall formation and patterning in Arabidop-
sis J Integr Plant Biol 53 74ndash88Feurouleuroop K Pettko-Szandtner A Magyar Z Miskolczi P Kondorosi E
Dudits D and Bako L (2005) The Medicago CDKC1ndashCYCLINT1 kinase
complex phosphorylates the carboxy-terminal domain of RNA polymer-
ase II and promotes transcription Plant J 42 810ndash820Gullberg J Jonsson P Nordstrom A Sjostrom M and Moritz T (2004)
Design of experiments an efficient strategy to identify factors influenc-
ing extraction and derivatization of Arabidopsis thaliana samples in met-
abolomic studies with gas chromatographymass spectrometry Anal
Biochem 331 283ndash295Hertzberg M Aspeborg H Schrader J et al (2001) A transcriptional
roadmap to wood formation Proc Natl Acad Sci USA 98 14732ndash14737Jang JC Fujioka S Tasaka M Seto H Takatsuto S Ishii A Aida M
Yoshida S and Sheen J (2000) A critical role of sterols in embryonic
patterning and meristem programming revealed by the fackel mutants of
Arabidopsis thaliana Genes Dev 14 1485ndash1497Jenks MA Rashotte AM Tuttle HA and Feldmann KA (1996) Mutants
in Arabidopsis thaliana altered in epicuticular wax and leaf morphology
Plant Physiol 110 377ndash385Jonsson P Johansson AI Gullberg J Trygg J A J Grung B Markl-
und S Sjostrom M Antti H and Moritz T (2005) High-throughput
data analysis for detecting and identifying differences between samples
in GCMS-based metabolomic analyses Anal Chem 77 5635ndash5642Jungwirth H and Kuchler K (2006) Yeast ABC transporters ndash a tale of sex
stress drugs and aging FEBS Lett 580 1131ndash1138Kaneda M Schuetz M Lin B Chanis C Hamberger B Western T
Ehlting J and Samuels A (2011) ABC transporters coordinately
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 823
expressed during lignification of Arabidopsis stems include a set of AB-
CBs associated with auxin transport J Exp Bot 62 2063
Karimi M Inze D and Depicker A (2002) GATEWAYTMvectors for Agrobac-
terium-mediated plant transformation Trends Plant Sci 7 193ndash195Kneurooller A and Murphy A (2011) ABC transporters and their function at the
plasma membrane In The Plant Plasma Membrane Plant Cell Mono-
graphs vol 19 (Murphy AS Peer W and Schulz B eds) New York
NY Springer pp 353ndash377Kopischke M Westphal L Schneeberger K Clark R Ossowski S
Wewer V Fuchs R Landtag J Hause G and Deuroormann P (2013)
Impaired sterol ester synthesis alters the response of Arabidopsis thali-
ana to Phytophthora infestans Plant J 73 456ndash468Kuromori T Miyaji T Yabuuchi H Shimizu H Sugimoto E Kamiya
A Moriyama Y and Shinozaki K (2010) ABC transporter AtABCG25 is
involved in abscisic acid transport and responses Proc Natl Acad Sci
USA 107 2361ndash2366Kuromori T Itoh T Sugimoto E and Shinozaki K (2011a) Arabidopsis
mutant of AtABCG26 an ABC transporter gene is defective in pollen
maturation J Plant Physiol 168 2001ndash2005Kuromori T Sugimoto E and Shinozaki K (2011b) Arabidopsis mutants
of AtABCG22 an ABC transporter gene increase transpiration and
drought susceptibility Plant J 67 885ndash894Le Hir R Beneteau J Bellini C Vilaine F and Dinant S (2008) Gene
expression profiling keys for investigating phloem functions Trends
Plant Sci 13 273ndash280Lehrer AT Dugassa-Gobena D Vidal S and Seifert K (2000) Transport
of resistance-inducing sterols in phloem sap of barley Z Naturforsch C
55 948ndash952Li Y Beisson F Pollard M and Ohlrogge J (2006) Oil content of Arabid-
opsis seeds the influence of seed anatomy light and plant-to-plant vari-
ation Phytochemistry 67 904ndash915Luo B Xue XndashY Hu WndashL Wang LndashJ and Chen XndashY (2007) An ABC
transporter gene of Arabidopsis thaliana AtWBC11 is involved in cuticle
development and prevention of organ fusion Plant Cell Physiol 48
1790ndash1802McFarlane HE Shin JJH Bird DA and Samuels AL (2010)
Arabidopsis ABCG transporters which are required for export of diverse
cuticular lipids dimerize in different combinations Plant Cell 22 3066ndash3075
Meskiene I Baudouin E Schweighofer A Liwosz A Jonak C Rodri-
guez PL Jelinek H and Hirt H (2003) Stress-induced protein phospha-
tase 2C is a negative regulator of a mitogen-activated protein kinase
J Biol Chem 278 18945ndash18952Murashige T and Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures Physiol Plant 15 473ndash497Panikashvili D Savaldi-Goldstein S Mandel T Yifhar T Franke RB
Hofer R Schreiber L Chory J and Aharoni A (2007) The Arabidopsis
DESPERADOAtWBC11 transporter is required for cutin and wax secre-
tion Plant Physiol 145 1345ndash1360Panikashvili D Shi JX Bocobza S Franke RB Schreiber L and Aha-
roni A (2010) The Arabidopsis DSOABCG11 transporter affects cutin
metabolism in reproductive organs and suberin in roots Mol Plant 3
563ndash575Panikashvili D Shi JX Schreiber L and Aharoni A (2011) The Arabid-
opsis ABCG13 transporter is required for flower cuticle secretion and pat-
terning of the petal epidermis New Phytol 190 113ndash124Pighin JA Zheng H Balakshin LJ Goodman IP Western TL Jetter
R Kunst L and Samuels AL (2004) Plant cuticular lipid export requires
an ABC transporter Science 306 702ndash704Pullen M Clark N Zarinkamar F Topping J and Lindsey K (2010)
Analysis of vascular development in the hydra sterol biosynthetic
mutants of Arabidopsis PLoS ONE 5 e12227
Quilichini TD Friedmann MC Samuels AL and Douglas CJ (2010)
ATP-binding cassette transporter G26 is required for male fertility and
pollen exine formation in Arabidopsis Plant Physiol 154 678ndash690R Development Core Team (2010) R A Language and Environment for
Statistical Computing Vienna Austria R Foundation for Statistical
Computing
Roudier F Gissot L Beaudoin F Haslam R Michaelson L Marion J
Molino D Lima A Bach L and Morin H (2010) Very-long-chain fatty
acids are involved in polar auxin transport and developmental patterning
in Arabidopsis Plant Cell 22 364ndash375Rozen S and Skaletsky HJ (2000) Primer3 on the WWW for general users
and for biologist programmers Methods Mol Biol 132 365ndash386Schad M Lipton MS Giavalisco P Smith RD and Kehr J (2005) Eval-
uation of two-dimensional electrophoresis and liquid chromatography
tandem mass spectrometry for tissue-specific protein profiling of
laser-microdissected plant samples Electrophoresis 26 2729ndash2738Schauer N Steinhauser D Strelkov S et al (2005) GCndashMS libraries for
the rapid identification of metabolites in complex biological samples
FEBS Lett 579 1332ndash1337Schrader J Nilsson J Mellerowicz EJ Berglund A Nilsson P Hertz-
berg M and Sandberg G (2004) A high-resolution transcript profile
across the wood forming meristem of poplar identifies potential regula-
tors of cambial stem cell identity Plant Cell 16 2278ndash2292Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desi-
mone M Frommer WB Fleurougge UndashI and Kunze R (2003) ARAMEM-
NON a novel database for Arabidopsis integral membrane proteins
Plant Physiol 131 16ndash26Silvestro D Andersen TG Schaller H and Jensen PE (2013) Plant ste-
rol metabolism D7ndashsterol-C5ndashdesaturase (STE1DWARF7) D57ndashste-rol-D7ndashreductase (DWARF5) and D24ndashsterol-D24ndashreductase (DIMINUTO
DWARF1) show multiple subcellular localizations in Arabidopsis thaliana
(Heynh) L PLoS ONE 8 e56429
Sorin C Bussell JD Camus I Ljung K Kowalczyk M Geiss G McKh-
ann H Garcion C Vaucheret H and Sandberg G (2005) Auxin and
light control of adventitious rooting in Arabidopsis require ARGONA-
UTE1 Plant Cell 17 1343ndash1359Tanaka T Tanaka H Machida C Watanabe M and Machida Y (2004) A
new method for rapid visualization of defects in leaf cuticle reveals five
intrinsic patterns of surface defects in Arabidopsis Plant J 37 139ndash146Turgeon R and Wolf S (2009) Phloem transport cellular pathways and
molecular trafficking Annu Rev Plant Biol 60 207ndash221Ukitsu H Kuromori T Toyooka K et al (2007) Cytological and biochemi-
cal analysis of COF1 an Arabidopsis mutant of an ABC transporter gene
Plant Cell Physiol 48 1524ndash1533Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De
Paepe A and Speleman F (2002) Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biol 3 research00340031-00340011Verrier PJ Bird D Burla B et al (2008) Plant ABC proteins ndash a unified
nomenclature andupdated inventory Trends Plant Sci 13 151ndash159Vilaine F Palauqui JC Amselem J Kusiak C Lemoine R and Dinant
S (2003) Towards deciphering phloem a transcriptome analysis of the
phloem of Apium graveolens Plant J 36 67ndash81Zhai Z Sooksa-nguan T and Vatamaniuk OK (2009) Establishing RNA
interference as a reverse-genetic approach for gene functional analysis
in protoplasts Plant Physiol 149 642ndash652Zhang Q Blaylock LA and Harrison MJ (2010) Two Medicago truncatula
half-ABC transporters are essential for arbuscule development in
arbuscular mycorrhizal symbiosis Plant Cell 22 1483
Zhao C Craig JC Petzold HE Dickerman AW and Beers EP (2005)
The xylem and phloem transcriptomes from secondary tissues of the
Arabidopsis root-hypocotyl Plant Physiol 138 803ndash818
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
824 Rozenn Le Hir et al
(HA)-tagged versions of ABCG9 and ABCG14 respectively
led us to conclude that these mutations were indeed
responsible for the observed phenotypes (Figure 3din
and Figure S1d) On the other hand the abcg11ndash7 homozy-
gous plants showed severe morphological abnormalities
similar to previously described abcg11 alleles (Bird et al
2007 Luo et al 2007 Panikashvili et al 2007 Ukitsu et al
2007) They were dwarf with unexpanded fused leaves
(Figure 3g and Figure S1d) Strikingly abcg11-7 plants car-
rying the mutation in ABCG11 gene in a heterozygous state
were 32 smaller than the wild-type plants on average
(Figure 3fn and Figure S1d) Finally abcg9-2abcg11-
7abcg14-1 mutants displayed a stronger phenotype than
abcg11ndash7 heterozygous plants (Figure 3ln) suggesting
that these mutations are additive Moreover the triple
homozygous mutants were as small as the abcg11ndash7
homozygous plants and displayed the same developmen-
tal abnormalities (Figure 3gmn)
Mutations in ABCG9 ABCG11 and ABCG14 affect vascular
patterning
Because these three genes are expressed in the vascular
system alleles of abcg9 abcg11 and abcg14 as well as
multiple mutants were analyzed for evidence of altered
vascular development by characterization of cotyledon
venation patterns in young seedlings (Cnops et al 2006)
The complexity of the vascular pattern was assessed in
terms of the number of secondary vein loops originating
from the mid-vein (Figure 4) In wild-type (Colndash0) seed-
lings 94 of the cotyledon venation pattern was between
classes III and V [from two loops plus the start of two
others (class III) up to four loops (class V) Figure 4] with
41 representing the most complex pattern (class V) The
double mutants abcg9ndash1abcg14ndash1 and abcg9ndash2abcg14ndash1
displayed a more complex venation pattern than the wild-
type (with respectively 100 and 96 of the cotyledon
(a) (b) (c) (d)
(e) (f) (g) (h)
(i) (j) (k) (l)
Figure 1 Expression patterns of ABCG genes
Expression patterns of ABCG9 ABCG11 and ABCG14 in cotyledons of 10-day-old seedlings (a e i respectively) the floral stem of 4-week-old plants (b f j
respectively) rosette leaves (c g k respectively) and roots (d h l respectively) co cortex e epidermis if interfascicular fibers ph phloem xy xylem Scale
bar = 100 lm
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 813
vascular pattern between classes III and V) The other
mutant lines showed a significantly less complex vena-
tion pattern than the wild-type (Figure 4) The single
mutants also differed significantly with respect to the loss
of complexity The abcg11ndash7 line displayed the least com-
plex vascular system (only 38 of the venation patterns
were between classes III and V) followed by abcg14ndash1
and then abcg9ndash2 (73 and 86 respectively) (Figure 4)
Introduction of HA-tagged versions of the ABCG9 and
ABCG14 proteins in the corresponding mutant back-
ground (abcg9ndash2 and abcg14ndash1) complemented the loss
of vascular complexity (Figure 4) confirming that the
phenotype was indeed due to defective expression of
ABCG9 and ABCG14 genes and the tagged proteins were
functional Interestingly the cotyledons of the triple
mutant displayed a significantly less complex vascular
system than that of the single mutants (Figure 4) In addi-
tion to the reduced vascular complexity in abcg11 homo-
zygous and heterozygous mutants the presence of open
vascular loops indicated a defect in vascular continuity
(Figure 4) Interestingly the continuity was restored in the
triple homozygous mutant (Figure 4)
Subsequently we characterized vascular bundles in the
floral stem of 4-week-old plants grown in soil (Table 1)
Under our conditions the wild-type flower stems dis-
played a vascular bundle density of 592 vascular bundles
per mm (Table 1) No significant variation in the number
of vascular bundles was observed in the single mutants
and complemented lines (Table 1) In contrast to the
increased complexity of the cotyledon venation pattern
(Figure 4) significantly fewer vascular bundles were pres-
ent in abcg9ndash2 abcg14ndash1 and abcg9ndash2 abcg11ndash7 abcg14ndash1
mutants (828 and 76 vascular bundles respectively)
(Table 1) Consistent with the smaller plant stature of
these double and triple mutants (Figure 1n) the diameter
of the floral stem was reduced (Table 1) However the
number of vascular bundles could not be explained by a
reduction of the floral stem diameter as the abcg11ndash7
mutant displayed the same number of vascular bundles
than the wild-type but its floral stem diameter was
strongly reduced (Table 1) Altogether these results
demonstrate that ABCG9 ABCG11 and ABCG14 play an
important role in vascular patterning during Arabidopsis
plant development
ABCG9 and ABCG14 are plasma membrane-localized
proteins that interact physically to form homo- andor
heterodimers with ABCG11
Because ABCG half transporters are thought to require
dimerization for functionality we investigated whether
ABCG9 ABCG11 and ABCG14 interact physically with
themselves andor other ABCG isoforms We first investi-
gated the subcellular localization of ABCG9 and ABCG14
after transfection of Arabidopsis mesophyll protoplasts
prepared from transgenic seedlings expressing the plasma
membrane marker low temperature induced protein 6b
(GFPndashLTi6b) (Cutler et al 2000) with constructs encoding
transcriptional fusions with red fluorescent protein (RFP)
In both cases the GFP fluorescent signal (Figure 5ae) co-
localized with the RFP fluorescent signal (Figure 5bf) at
the plasma membrane of transformed cells (Figure 5bh)
indicating that both ABCG9 and ABCG14 localize to the
plasma membrane like ABCG11 (Bird et al 2007 Luo
et al 2007 Panikashvili et al 2007 Ukitsu et al 2007) In
addition immunolocalization of a HA-tagged version of
ABCG9 and ABCG14 in their respective complemented
lines confirmed that these two proteins are localized to
the plasma membrane in planta (Figure 5ij) Next
(a)
(b)
(c)
(d) (e)
(f) (g)
(h) (i)
Figure 2 TndashDNA insertion lines for ABCG genes
(andashc) TndashDNA positions in ABCG9 ABCG11 and ABCG14 respectively Black
boxes promoters dark gray boxes 5prime and 3prime untranslated regions light gray
boxes exons lines introns
(dndashi) The presence of full-length mRNA was checked in abcg mutants as
described in Experimental procedures The elongation factor EF1a was used
as an internal control
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
814 Rozenn Le Hir et al
Arabidopsis protoplasts were transfected with open read-
ing frames encoding individual cndashMyc- or HA-tagged
versions of the three ABCGs and the identity of the epi-
tope-tagged proteins was verified by SDSndashPAGE and
Western blotting using anti-HA and anti-c-Myc antibodies
In all cases only one band of approximately 75 kDa cor-
responding to the predicted molecular mass of the pro-
teins in the Plant Membrane Protein Database (Schwacke
et al 2003) was obtained (Figure 5kndashp lane 1 and Figure
S2andashf lane 1) Subsequently protoplasts were co-trans-
fected with cndashMyc- and HA-tagged isoforms immunopre-
cipitated with antisera against one of the epitope tags
and probed with antisera directed against the alternative
epitope tag in Western blots of SDSndashPAGE gels in order
to determine whether homodimers or heterodimers were
formed (Figure 5kndashp) No ABCG14 homodimers or
ABCG9ndashABCG14 heterodimers were detected (Figure 5m
lane 4 and Figure 5o lane 4) In contrast ABCG11 formed
an apparent homodimer as described previously (McFar-
lane et al 2010) ABCG9 also formed an apparent homod-
imer ABCG14 formed an apparent heterodimer with
ABCG11 and ABCG9 formed an apparent heterodimer
with ABCG11 (Figure 5klnp lane 4) These interactions
were confirmed in a plant-based in vivo system using the
bimolecular fluorescence complementation assay (Figure
S3andashl) We therefore concluded that ABCG14 forms a strict
heterodimer with ABCG11 whereas ABCG11 and ABCG9
can both hetero- and homodimerize
Cuticular and epicuticular lipid precursor content as well as
cuticle integrity are not altered in abcg9ndash2 and abcg14ndash1
mutants
ABCG11 is known to be required for cuticle integrity (Bird
et al 2007 Luo et al 2007 Panikashvili et al 2007 Ukitsu
et al 2007) and because ABCG11 interacts physically with
ABCG9 and ABCG14 we looked for potential defects in
very long chain fatty acids (VLCFAs) which are among the
precursors of cuticular and epicuticular lipids (Roudier
et al 2010) as well as for possible cuticle defects in
abcg9ndash2 and abcg14ndash1 mutants
Quantification of fatty acids (from 160 to 180) as well as
VLCFAs (from 200 to 240) in apical parts of abcg9ndash2 and
abcg14ndash1 seedlings revealed no differences compared to
wild-type levels (Figure 6ab) It has previously been
(a) (b) (c) (d)
(e) (f) (g) (h)
(i)
(j)
(i)
(k) (l) (m)
(n)
Figure 3 Phenotype of abcg mutants grown in
soil
(andashm) Twenty-day-old plants grown in soil
Scale bar = 1 cm
(n) Green areas of 4-week-old plants Values
represent means SE from 20 individual
plants The experiment was repeated twice with
similar results Asterisks indicate statistically
significant differences between the wild-type
and mutants (P lt 005 Studentrsquos t test n = 20)
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 815
shown that when the cuticle is altered toluidine blue (TB)
permeates the epidermal surface (Tanaka et al 2004)
Therefore we used TB to check for cuticle defects in the
mutants After treatment with TB no staining was
observed in the wild-type abcg11+ or the abcg9ndash2 and
abcg14ndash1 mutants (Figure 6cef) whereas abcg11ndash7
homozygous seedlings showed patches of blue staining
confirming the cuticle alteration previously described
(Figure 6d) (Ukitsu et al 2007) These observations sug-
gest that unlike mutations in ABCG11 mutations in
ABGC9 or ABCG14 do not affect cuticle formation
abcg9ndash2 and abcg14ndash1 mutants are defective in sterol
composition
To obtain an insight into the potential substrates trans-
ported by these ABCG transporters we analyzed the shoot
and root metabolome of single mutants Orthogonal partial
least-squares discriminant analysis was applied to the
GCMS data in order to classify the profiles of the geno-
types In both shoots (Figure 7andashc) and roots (Figure S4andash
c) homozygous abcg11ndash7 abcg9ndash1 abcg9ndash2 and abcg14ndash1
mutants exhibited very different metabolomic profiles from
the wild-type Interestingly the heterozygous abcg11ndash7+
and abcg9ndash2+ plants had metabolomic profiles intermedi-
ate between the wild-type and their respective homozy-
gous mutant plants (Figure 7ab and Figure S4ab) These
results suggest that a mutation in one copy of ABCG11 or
ABCG9 is sufficient to significantly affect the physiology of
the plant even if it does not dramatically affect visible
aspects of its phenotype
The metabolites showing the most significant differ-
ences were identified by comparing their retention indices
and mass spectra with entries in publicly available reten-
tion libraries (Schauer et al 2005) For all the genotypes
analyzed the metabolites identified as being significantly
different from the wild-type belonged to three main clas-
ses amino acids carbohydrates and lipidssterols The
levels of these compounds were almost all reduced in
abcg9ndash2 and abcg14ndash1 but in excess in abcg11ndash7 (Table 2
and Appendix S1)
Because our study focused on vascular development in
aerial tissues only data obtained for the apical parts are
described in detail here (Table 2) Root data are presented
in Appendix S1 Whereas many neutral amino acids were
significantly depleted in abcg9ndash2 and abcg14ndash1 aerial
Figure 4 Cotyledon venation pattern in abcg mutant lines
Venation complexity and continuity of each examined class of mutant and
wild-type seedlings Values in brackets indicate the percentage contribution
of each class Asterisks indicate statistically significant differences between
the wild-type and mutants (P lt 005 Pearsonrsquos v2 test with Monte Carlo
permutations N = 10 000)
Table 1 Distribution of vascular bundles in the floral stem
Genotype Vascular bundle number Floral stem diameter (mm) Vascular bundle density Total
Col-0 930 105 157 020 592 049 10abcg11-7+ 914 134 140 080 652 042 12abcg11-7 902 115 051 004 176 182 8abcg14-1 828 049 145 018 632 0049 1135SHA-ABCG14 abcg14-1 940 089 170 016 554 058 12abcg9-1 957 053 160 006 586 031 7abcg9-2 875 103 162 010 553 046 835SHA-ABCG9 abcg9-2 933 051 174 010 606 026 8abcg9-1 abcg14-1 871 048 154 003 565 030 8abcg9-2abcg14-1 828 049 124 015 674 084 8abcg9-2abcg14-1abcg11-7
760 054 063 005 1203 169 9
Statistically significant differences between the wild-type and mutants (P lt 005 Studentrsquos t test)
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
816 Rozenn Le Hir et al
tissues (nine and eight out of 20 neutral amino acids
respectively) only two amino acids (glutamine and gluta-
mate) associated with Arabidopsis phloem cells (Schad
et al 2005) were different in abcg11ndash7 (Table 2) Xylose
and the non-reducing sugars trehalose and raffinose were
also decreased in abcg14ndash1 (Table 2) whereas glucose
fructose galactose maltose xylose were significantly dif-
ferent in abg9ndash2 and abcg11ndash7 (reduced and increased
respectively compared to wild-type) (Table 2)
Levels of sterols (campesterol 24ndashmethylene cholesterol
and sitosterol) saturated and unsaturated fatty acids (lino-
leic acid lauric acid palmitic acid and triacontanoic acid)
glycerolipids (monoacylglycerol) and lipid metabolism
intermediates (glycerol glycerol-3ndashphosphate and inositol-
1ndashphosphate) were also significantly different from wild-
type in abcg mutants (Table 2) The abcg9ndash2 mutant
showed 30 lower levels of 24ndashmethylene cholesterol
compared to the wild-type (Appendix S1) In the abcg14ndash1
mutant 50 and 35 decreases of 24ndashmethylene cholesterol
and sitosterol levels were measured respectively (Appen-
dix S1) Finally campesterol showed a 50 increase in the
abcg11ndash7 mutant (Appendix S1) To extend this analysis
measurements of conjugated sterols [sterol ester (SE)
sterol glucoside and acylated sterol glucoside] were also
performed When seedlings were grown in vitro no differ-
ence in the sterol glucoside and acylated sterol glucoside
contents was detected between the wild-type and the vari-
ous mutant lines (Figure S4d) However there was a ten-
dency towards a reduced SE content in the double mutant
abcg9ndash2 abcg14ndash1 compared to the wild-type (Figure S4d)
This observation prompted us to examine variations in the
SE content in older plants (4-week-old plants grown in soil
in the greenhouse) At this developmental stage no differ-
ence in the free sterol (FS) content was observed between
the wild-type and the various mutant lines (Figure 7d) but
the fraction comprising FS and SE was significantly
(a) (b) (c) (d)
(e) (f) (g) (h)
(i) (j)
(k) (l) (m)
(n) (o) (p)
Figure 5 Subcellular localization and physical interactions of ABCG9 ABCG11 and ABCG14
(andashh) ABCG9 and ABCG14 co-localize to the plasma membrane when transiently expressed in Arabidopsis mesophyll protoplasts of the GFPndashLTi6b marker line
(Cutler et al 2000)
(a e) Cyan fluorescence of GFPndashLTi6b(b f) Purple fluorescence from mRFPndashABCG9 and mRFPndashABCG14 respectively
(c g) Yellow auto-fluorescence of the chloroplasts
(d h) Merged images of the three channels Scale bar = 10 lm
(i j) Confocal images of immunofluorescence staining with anti-HA antibodies in root cells of abcg9ndash2 and abcg14ndash1 mutants complemented with HA-tagged
ABCG9 (i) and HA-tagged ABCG14 (j) Scale bars = 10 lm
(kndashp) Proteins were extracted from protoplasts co-transfected with HA- or cndashMyc-tagged versions of ABCGs (lanes 1 and 4) or transfected with a single plasmid
containing the HA-tagged version (lane 2) or the c-Myc-tagged version (lane 3) In lanes 2ndash4 proteins were immunoprecipitated with the anti-c-Myc antibody
and subjected to anti-HA protein gel-blot analysis to reveal the other partner of the dimer Transient co-expression of (k) HAndashABCG9cndashMycndashABCG9 (l) HAndashABCG11cndashMycndashABCG11 (m) HAndashABCG14cndashMycndashABCG14 (n) HAndashABCG14cndashMycndashABCG11 (o) HAndashABCG14cndashMycndashABCG9 and (p) HAndashABCG11cndashMycndashABCG9
in wild-type protoplasts
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 817
reduced probably accounting for the difference in the SE
content between the wild-type and the abcg9ndash2 abcg14ndash1
double mutant (Figure 7e)
(a)
(b)
(c) (d) (e)
(f) (g)
Figure 6 Long chain and very long chain fatty acid content and cuticle
integrity in abcg9ndash2 and abcg14ndash1 mutants
(a b) Fatty acid (a) and VLCFA content (b) in abcg9ndash2 and abcg14ndash1 mutants
in apical parts of 10-day-old seedlings (three biological replicates each of
approximately 5 mg dry weight were used)
(cndashf) Toluidine blue staining patterns of abcg mutants in 7-day-old seed-
lings Scale bars = 3 cm
(a)
(b)
(c)
(d) (e)
Figure 7 Metabolome analysis and measurements of sterol content in abcg
mutants
(andashc) Results of orthogonal partial least-squares discriminant analysis of
global metabolite contents of shoots of 7-day-old seedlings from wild-type
(black squares) abcg11ndash7+ (gray triangles) abcg11ndash7 (white stars) abcg9ndash1(white hexagons) abcg9ndash2+ (gray hexagons) abcg9ndash2 (white hexagons)
and abcg14ndash1 (black circles) seedlings (five biological replicates of 10 mg
each were used for the analysis)
(d e) Free sterol (FS) and sterol ester (SE) content of wild-type (white bars)
abcg9ndash2 (light gray bars) abcg14ndash1 (dark gray bars) and abcg9ndash2 abcg14ndash1(black bars) Error bars indicate standard deviations obtained from three
independent biological replicates The asterisk indicates a statistically signif-
icant difference between the wild-type and the mutant (P lt 005 Studentrsquos
t test)
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
818 Rozenn Le Hir et al
DISCUSSION
ABCG9 and ABCG14 are two plasma membrane proteins
that interact physically in multiple homo-heterodimer
combinations
In this study we showed that like the previously described
ABCG11 (Pighin et al 2004 Bird et al 2007 Luo et al
2007 Panikashvili et al 2007 2010 Ukitsu et al 2007)
ABCG9 and ABCG14 localize to the plasma membrane
Based on co-immunoprecipitation of tagged proteins and
the bimolecular fluorescence complementation assay we
demonstrated that ABCG9 interacts with ABCG11 to form a
heterodimer and like ABCG11 (McFarlane et al 2010) can
homodimerize In contrast ABCG14 interacts only with
ABCG11 forming an obligate heterodimer This extends
the number of potential partners of ABCG11 to three as it
was previously shown to dimerize with the ABCG12CER5
protein (McFarlane et al 2010) Similarly ABCG9 under-
goes flexible dimerization to form either homo- or hetero-
dimers suggesting that ABCGs may be promiscuous
proteins with different functions depending on their inter-
acting partner andor expression profile
Mutations in ABCG genes are semi-dominant
In the last few years several Arabidopsis mutants with
altered expression of various ABCG genes such as ABCG11
(Bird et al 2007 Luo et al 2007 Panikashvili et al 2007
Ukitsu et al 2007) ABCG12CER5 (Pighin et al 2004)
ABCG13 (Panikashvili et al 2011) ABCG22 (Kuromori
et al 2011a) ABCG25 (Kuromori et al 2010) and ABCG26
(Choi et al 2010 Quilichini et al 2010 Dou et al 2011
Kuromori et al 2011b) have been characterized but even
though these half-size transporters require dimerization to
become functional all the mutations were described as
recessive Here we demonstrated that mutations in these
genes have semi-dominant effects Indeed the metabolome
analyses showed that abcg9ndash2+ and abcg11ndash7+ heterozy-
gous seedlings have a profile intermediate between that of
wild-type and homozygous seedlings The possibility of
dominant-negative effects was excluded because no
Table 2 Shoot metabolites of 10-day-old abcg9ndash2 abcg11ndash7 and abcg14ndash1 seedlings whose levels were significantly different from wild-type
Group
Metabolites that differed between mutant and wild-type
abcg9ndash2Colndash0 abcg11ndash7Colndash0 abcg14ndash1Colndash0
Amino acids Glycine Glutamate AlanineAlanine Glutamine ValineValine ThreonineThreonine GABASerine Prolinebndashalanine IsoleucineIsoleucine SerineGABA bndashalanineProline
Carbohydrates Glucose Glucose ErythritolFructose Fructose TrehaloseXylose Galactose XyloseMaltose RaffinoseSucrose
Lipids and intermediates 24ndashmethylene-cholesterol Campesterol Linoleic acidMonoacylglycerol Glycerol-3ndashphosphate Triacontanoic acidPalmitic acid Inositol-1ndashphosphate GlycerolGlycerol-3ndashphosphate Lauric acidInositol-1ndashphosphate 24ndashmethylene-cholesterol
SitosterolGlycerol-3ndashphosphate
TCA cycle and others Phosphoric acid Phosphate compound Glyceric acid24ndashdihydroxybutanoic acid Dehydroascorbic acid Fumaric acidGlyceric acid Malic acid Threonic acid-14ndashlactoneFumaric acid 2ndashoxoglutaric acid Threonic acidThreonic acid bndashhydroxy-bndashmethylglutaric acid Shikimic acidThreonic acid-14ndashlactone Gluconic acid lactone-like Succinic acidShikimic acid Phytol2ndashoxoglutaric acid Ethanolamine
Nicotinic acid
For abcg9ndash2 and abcg14ndash1 the levels of the compounds were reduced whereas they were increased in abcg11ndash7
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 819
truncated transcript was found for either abcg9ndash2 or
abcg11ndash7 mutants Hence abcg9ndash2 and abcg11ndash7 are true
null alleles These observations are consistent with the
requirement for homo- or heterodimerization of the ABCG
proteins to form functional transporters In the heterozy-
gous plants ABCG11 transcript levels were 50 lower than
in wild-type counterparts suggesting that ABCG11 protein
and thus corresponding homo- and heterodimers were
likely to be less abundant which may also explain the meta-
bolic perturbations and consequent phenotypic modifica-
tions These findings also suggest that wild-type plants are
not saturated by ABCG11 proteins as a 50 reduction in
protein content appears to be sufficient to alter the physiol-
ogy of the heterozygous mutant plants This hypothesis is
strengthened by the observation that mutants carrying the
weak abcg11ndash6 allele in which there was only a 25 reduc-
tion in ABCG11 transcript levels showed a similar pheno-
type to the heterozygous abcg11ndash7+ and abcg11ndash8+
plants Similarly the abcg9ndash1 weak allele mutant had a very
similar phenotype to the abcg9ndash2+ heterozygous seedlings
suggesting that considering the mode of action of these
proteins the semi-dominant effects that we observed are
likely to be a general feature among the ABCG genes
ABCG9 ABCG11 and ABCG14 transporters are involved in
vascular system development in Arabidopsis
We showed that the ABCG11 ABCG9 and ABCG14 promot-
ers drove expression in the vascular system of Arabidopsis
cotyledons and rosette leaves and more precisely in the
phloem of flower stems In addition mutations in ABCG9
ABCG11 and ABCG14 genes altered vascular development
in cotyledons and the floral stem Similar vascular localiza-
tion of ABCG11 has already been described by Panikashvili
et al (20072010) who showed that in addition to the epi-
dermis ABCG11 is expressed in the vasculature as early as
during embryo development and then throughout plant
development They showed that the vascular pattern in
leaves of the homozygote mutant seedlings was less com-
plex than in the wild-type and discontinuous (Panikashvili
et al 2007) The present article describes a mutant that
has both cuticle and vascular system patterning defects
although it has been previously shown that mutants
altered in cuticle formation have dramatically reduced
growth and altered leaf morphology (Jenks et al 1996)
which may account for vascular defects Analysis of the
abcg11+ heterozygous seedlings that have vascular
defects but apparently no cuticle alteration suggested that
these phenotypes are probably uncoupled Because
ABCG11 dimerizes with several members of its sub-family
it seems reasonable to postulate that it is involved in the
transport of different compounds depending on its partner
Here we provide evidence that ABCG11 in addition to
forming dimers with ABCG12 (McFarlane et al 2010) in
epidermal cells is likely to dimerize with ABCG9 and
ABCG14 in phloem cells suggesting functional compart-
mentalization of these proteins and a possible role in vas-
cular system patterning in Arabidopsis
Metabolite profiling suggests that ABCG9 ABCG11 and
ABCG14 are involved in sterollipid homeostasis
We investigated in more depth the metabolic changes
induced by mutations in ABCG9 ABCG11 and ABCG14
We found that levels of metabolites of several classes
including lipids sugars and amino acids were altered in
both the heterozygotes and homozygotes and that the
profiles in the root and apical parts differed suggesting
that ABCGs may play different roles in the roots and shoot
Interestingly the modifications of amino acid and sugar
contents observed in the metabolic profiles of the abcg
mutants suggest an impaired carbonnitrogen balance that
may be directly or indirectly linked to the vascular defects
leading to possible problems with phloem loading or
unloading Saturated and unsaturated fatty acids and sterol
contents were also altered in the mutants Patterning
defects in the vascular system are often observed in
mutants altered in hormone homeostasis such as for
auxin or brassinosteroids (Kaneda et al 2011) and direct
or indirect modifications of sterol homeostasis are thought
to be one of the primary causes of these downstream
effects (Pullen et al 2010) The detected defects in sterol
homeostasis may explain the abnormal vascular develop-
ment observed in abcg9 abcg11 and abcg14 mutants
Accordingly several sterol-deficient mutants such as fac-
kelhydra (Jang et al 2000) and cpv1 (Carland et al 2002)
have also been shown to exhibit severe defects in vascular
development It has been suggested that the bushy habit
and low fertility of cpv1smt2 mutants may be due to an
increased campesterolsitosterol ratio rather than sterol
deficiency per se (Clouse 2002) This ratio is controlled by
the activity of SMT2 (STEROL METHYLTRANSFERASE2)
Interestingly strong down-regulation of SMT2 expression
was observed in all the mutant backgrounds (Figure S5)
and the metabolic data indicated a deficit in 24ndashmethylene
cholesterol (precursor of both campesterol and sitosterol)
andor sitosterol in abcg9 and abcg14 mutants respec-
tively In addition the significant decrease in sterol ester
content in the double mutant abcg9ndash2 abcg14ndash1 correlated
with down-regulation of expression of the PHOSPHOLIPID
STEROL ACYLTRANSFERASE1 (PSAT1) gene encoding the
enzyme that is mainly responsible for SE synthesis (Bou-
vier-Nave et al 2010) and is proposed to be a control step
in sterol homeostasis (Kopischke et al 2013) Altogether
these results suggest that the vascular phenotype
observed in abcg mutants may be linked to modification of
sterol homeostasis
Therefore we postulate that the various dimer combina-
tions between ABCG9 ABCG11 and ABCG14 may
participate in the transport of squalene-derived metabolites
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
820 Rozenn Le Hir et al
such as sterol andor sterol conjugates via the phloem sap
The presence of sterols in phloem sap has been observed in
various species such as barley (Hordeum vulgare Lehrer
et al 2000) and fava bean (Vicia faba Bouvaine et al
2012) and indirect proof of the presence of sterols in
phloem sap has been obtained by analysis of the phytos-
terol content in exclusively phloem-feeding aphids that are
unable to synthetize them directly (Behmer et al 2011) In
addition the evidence for long-distance transport of sterol
precursors has been supported by observations that sterol
esters accumulate in organs distal to the site of application
of exogenous precursors (Bouvier-Nave et al 2010) As
sterols have been proposed to act as signal molecules in
the control of developmental processes such as vascular
patterning (Carland et al 2002 Pullen et al 2010) modifi-
cations of sterol homeostasis may affect development of
the vascular system in the abcgmutants described here
In conclusion we propose that ABCG9 ABCG11 and
ABCG14 are important for lipidsterol homeostasis Thus
these three proteins are likely to be involved in the control
of various aspects of plant development andor physiol-
ogy including patterning of the vascular system
EXPERIMENTAL PROCEDURES
Plant material and growth conditions
Insertion lines in ABCG9 (SALK_047133 and GABI_353B01)ABCG11 (SALK_096377 and GABI_590C03) and ABCG14 (SALK_036952) genes were obtained from the European ArabidopsisStock Centre (httparabidopsisinfo) Heterozygoushomozygousplants were screened using gene-specific primers in combinationwith the specific primer for the left border of the TndashDNA insertion(Table S1)
Plants were grown in soil or in vitro as described previously(Sorin et al 2005) Arabidopsis Colndash0 cell cultures were main-tained by weekly sub-culturing in MS medium (Murashige andSkoog 1962) supplemented with 240 lg l1 24ndashdichlorophenoxy-acetic acid and 14 lg l1 kinetin and were grown under 12 h light12 h dark cycles at 25 lE m2 sec1 and with 23degC day and nighttemperatures
Morphological characterization and GUS staining
To study the venation pattern cotyledons of 7-day-old seedlingsand rosette leaves of 4-week-old plants were cleared in a buffercontaining chloral hydrate glycerol and water (831 wvv) afterwhich 200 cotyledons per genotype were analyzed and the per-centage showing particular levels of complexity or discontinuity invascularity was estimated as described by Cnops et al (2006)
Flower stem segments (1ndash2 cm long) were removed from ten3ndash4-week-old plants grown in soil embedded in 8 agarose andsectioned using a VT100S vibratome (Leica httpwwwleicacom)The cross-sections were then stained with safraninalcian blue for1ndash2 min (Sigma httpwwwsigmaaldrichcom) rinsed in waterand mounted in 50 glycerol
Histochemical GUS assays were performed as described previ-ously (Sorin et al 2005) All observations were performed usingan Axioplan light microscope equipped with an Axiocam cameraand Axiovision software (Zeiss httpwwwzeisscom)
To observe potential cuticle defects in the various mutant linesa TB test was performed by immersing in vitro-grown plantlets ina 005 wv TB solution for 2 min as described by Tanaka et al(2004)
Statistical analyses
Statistical analyses of differences between-genotype means andvariance for the studied morphological traits were performedusing Studentrsquos t test (GraphPad Prism version 50 httpwwwgraphpadcom) In the venation pattern experiment thesignificance of differences between wild-type and mutants wasassessed using R (R Development Core Team 2010) and Pearsonrsquosv2 test with Monte Carlo permutations (N = 10 000)
Plasmid constructs
The native ABCG promoters and open reading frame of ABCG11were amplified from genomic DNA or cDNA extracted from 7-day-old Arabidopsis seedlings using Phusion high-fidelity DNApolymerase (Finnzyme wwwthermoscientificbiocomfinnzymes)according to the manufacturerrsquos instructions with gene-specificprimers (Table S1) The PCR products were cloned into pENTRDndashTOPO (Invitrogen httpwwwinvitrogencom) and recombinedinto pKGWFS7 (Karimi et al 2002) to create ABCGproGUS Allrecombinations were performed using the Gateway LR Clonase
enzyme mix (Invitrogen) At each cloning step the sequenceswere confirmed by sequencing and all the constructs weretransferred into Agrobacterium strain GV3101pMP90 (C58C1)Wild-type or abcg11ndash7+ heterozygous plants were transformed byfloral dipping (Clough and Bent 1998) For ABCGGUS constructsthe expression pattern was checked in the T2 progeny of 10ndash15independent transgenic lines and one representative homozygousline was used for further characterization
Tagged protein constructs and protoplast transformation
Epitope-tagged versions of ABCG9 ABCG11 and ABCG14 proteinswere produced in pRT104ndash3xHA and pRT104ndash3xMyc plasmids(Feurouleuroop et al 2005) and pSAT6-mRFP (Citovsky et al 2006) Allthese plasmids have a 35S promoter sequence upstream of themulti-cloning site The open reading frames of ABCG11 andABCG14 and the genomic clone of ABCG9 were amplified fromcDNA or genomic DNA extracted from 7-day-old Arabidopsisseedlings using Phusion high-fidelity DNA polymerase (Finnzyme)according to the manufacturerrsquos instructions with gene-specificprimers carrying EcoRI or SalI restriction sites to facilitate subse-quent cloning (Table S1) The products obtained after PCR weredigested by EcoRI and SalI prior to ligation into appropriate plas-mids that had previously been cut open with the same restrictionenzymes Constructs were verified by sequencing Protoplastsfrom Arabidopsis cell cultures or 14-day-old Arabidopsis seedlingswere prepared and transformed as previously described (Meski-ene et al 2003 Zhai et al 2009) For co-immunoprecipitation andsubcellular localization assays 100 000 and 50 000 protoplastcells respectively were transfected with 15 and 10 ll of plasmidsrespectively (containing between 5 and 75 lg of each construct)and the resulting transformed protoplasts were processed afterincubation in the dark at room temperature for 16ndash18 and 24 hrespectively
Whole-mount immunolocalization
The HA-tagged versions of ABCG9 and ABCG14 were detected in7-day-old in vitro-grown Arabidopsis seedlings as described inMethods S1 Anti-HA antibody 12CA5 (Roche httpwwwroche
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 821
com) (1100 dilution) combined with Alexa Fluor 488-conjugatedsecondary antibody (1100 dilution) were used
Confocal laser-scanning microscopy
Images were collected using an SP2 confocal microscope (Leica)RFP and Alexa Fluor 488 were detected using laser lines at 561and 488 nm respectively according to the manufacturerrsquos set-tings RFP fluorescence emission was detected at 550ndash600 nmand Alexa Fluor 488 and GFP fluorescence emission were detectedat 495ndash545 nm For subcellular localization in protoplasts imageswere coded in cyan (GFP) magenta (RFP) and yellow (auto-fluo-rescence) resulting in purple coloration for co-localization inmerged images For the immunolocalization experiment imageswere coded in green (GFP) A water-corrected 209 objective (HCPlan-Apochromat 20907 ImmKor Leica) was used Images werecropped using Adobe Photoshop CS2 and assembled using AdobeIllustrator CS2 software (Abode httpwwwabodecom) Eachimage shown represents a single focal plan
Co-immunoprecipitation assays
Transformed protoplast proteins were extracted using 50 ll lysisbuffer containing 25 mM TrisHCl pH 78 10 mM MgCl2 75 mM
NaCl 5 mM EGTA 1 mM benzamidine 1 mM dithiothreitol 10glycerol 1 Triton Xndash100 and 19 protein inhibitor cocktail(Sigma httpsigmaaldrichcom) The transformed protoplast sus-pension was frozen in liquid nitrogen then thawed on ice andsubjected to centrifugation for 5 min at 150 g Two microliters ofanti-Myc (9E10 Covance httpwwwcovancecom) or anti-HA(16B12 Covance) antibodies were added to the extracts and themixture was incubated for 2 h at 4degC on a rotating wheel Immu-nocomplexes were captured by incubation with 10 ll Protein GndashSepharose beads for a further 2 h at 4degC on a rotating wheel afterwhich they were washed three times for 5 min each in 25 mM
NaPi 150 mM NaCl 5 glycerol and 02 Igepal CAndash630 buffer(Sigma) and then eluted using 40 ll SDS sample buffer at 37degCfor 30 min Co-immunoprecipitation of Myc- and HA-tagged pro-teins was revealed by SDSndashPAGE followed by Western blottingusing both anti-Myc (Invitrogen) and anti-HA (3F10 Roche) anti-bodies at 12000 dilution
RNA extraction and cDNA preparation
To quantify the relative amounts of the ABCG transcriptsexpressed in various organs three rosette leaves all the cau-line leaves the bottom 5 cm of the floral stem all closed flow-ers and all green siliques were harvested from each of threeindependent 4-week-old Arabidopsis plants of each genotypegrown in soil For the other experiments 10-day-old seedlingsgrown in vitro were harvested All the material was directly fro-zen in liquid nitrogen and stored at 80degC prior to RNA extrac-tion Samples were then disrupted using a MM 301 vibrationmill (Retsch GmbH httpwwwretschcom) at a frequency of25 Hz for 2 min with 3 mm tungsten carbide beads (Qiagenhttpwwwqiagencom) Total RNA was isolated from thismaterial using TRIzol (Life Technologies httplifetechnologiescom) RNA quantity and quality were measured using a Nano-Drop ND 1000 (Thermo Scientific httpwwwthermoscientificcom) All samples with 260280 nm and 260230 nm absorbanceratios lower than 18 were discarded and RNA was re-extractedDNase treatment was applied to 5 lg samples of total RNAusing a DNase I kit (Fermentas httpwwwfermentascom)Double-stranded cDNA was synthesized from 5 lg total RNAusing a SuperScript reverse transcriptase kit (Invitrogen) witholigo(dT)12 primer Finally RNase treatment was applied to the
samples using Escherichia coli RNAse H (Fermentas) The cDNAsamples obtained were diluted 30-fold (except for abcg9 linesamples which were diluted 10-fold) prior to use either toverify the absence of full-length transcripts in putative knockoutmutants or to quantify gene expression by quantitative PCR Allprimers used were designed using the Primer3 program (Rozenand Skaletsky 2000) and their sequences are listed inTable S1
QUANTITATIVE REAL-TIME PCR
Quantitative PCR amplifications were performed in 20 llmixtures containing 10 ll 59 SYBR Green I master mix
(Roche) 02 ll forward and reverse primer (30 lM each)
48 ll sterile water and 5 ll cDNA The BiondashRad CFX96
real-time PCR detection system (httpwwwbio-radcom)
was used for quantification of expression of the sterol
biosynthesis transcripts and the ABCG transcripts in the
various mutant backgrounds Finally the Roche LightCy-
cler 480 system (Roche) was used to quantify the rela-
tive abundance of ABCG transcripts in various organs
Four genes were considered as potential reference genes
[APT1 (At1g27450) TIP41 (At4g34270) EF1a (At5g60390)
and UBQ10 (At4g05320)] and their suitability was tested
using the geNorm algorithm (Vandesompele et al 2002)
to determine the most appropriate one for each experi-
ment A more detailed procedure following the Minimum
Information for Publication of Quantitative Real-Time PCR
Experiments guidelines (Bustin et al 2009) is described
in Methods S2 and the annealing temperature and
efficiencies of the primer pairs used are shown in
Table S2
VLCFA quantification
Approximately 5 mg dry weight of 10-day-old in vitro
Arabidopsis seedlings were analyzed for VLCFA quantifica-
tion as described by Li et al (2006)
Sterol extraction and GC-FID characterization
A pool of 10-day-old seedlings grown in vitro or rosettes
from 4-week-old plants grown in soil were lyophilized prior
to extraction Sterol extraction and isolation was per-
formed as described previously (Bouvier-Nave et al 2010)
Gas Chromatography-Flame Ionization Detector (GC-FID)
characterization was performed as described by Silvestro
et al (2013) To quantify the total amounts of free sterols
and sterol esters non-saponifiable lipids were extracted
using nndashhexane and sterols were derivatized as sterols ace-
tates Quantification was achieved using lupenyl-3 28ndashdi-
acetate as an internal standard
Metabolome analysis
To obtain metabolome profiles associated with wild-type
abcg11ndash7+ abcg11ndash7 abcg9ndash1 abcg9ndash2 and abcg14ndash1
genotypes 10-day-old seedlings representing each geno-
type grown in vitro as described above were harvested
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
822 Rozenn Le Hir et al
and divided into shoot and root samples In the case of
abcg9ndash2 the phenotype of the heterozygote abcg9ndash2+
was indistinguishable from the homozygote phenotype
and therefore heterozygous and homozygous seedlings
from a segregating population were pooled Hence in this
particular experiment abcg9ndash2+ refers to a mixed popula-
tion comprising two-thirds heterozygotes and one-third
homozygotes
Metabolites in five samples of shoot and root parts of
seedlings of each genotype (10 mg fresh weight per sam-
ple) were then extracted analyzed by GCMS and the
acquired data were processed as previously described
(Gullberg et al 2004 Jonsson et al 2005) All multivariate
statistical investigations (partial component analysis and
orthogonal partial least-squares discriminant analysis) for
between-genotype similarities and differences in general
metabolite profiles were performed using Simca software
(Umetrics httpwwwumetricscom)
ACKNOWLEDGEMENTS
We thank A Murphy (Plant Science and Landscape Archi-
tecture University of Maryland MD USA) for stimulating
discussions and critical reading of the manuscript I Carl-
son (Umea Plant Science Center Department of Forest
Genetics and Plant Physiology Swedish University of Agri-
cultural Sciences Umea Sweden) for technical assistance
and S Vernhettes for kindly donating seeds of the
transgenic line carrying the plasma membrane marker
GFPndashLTi6b We also thank the European Arabidopsis Stock
Centre and the GABI-Kat stock centre for providing seeds
This work was supported by allocations granted to CB by
the Swedish Research Council for Agriculture the Swedish
Foundation for Strategic Research the Swedish Research
Council for Research and Innovation for Sustainable
Growth the K amp A Wallenberg Foundation and the Carl
Trygger Foundation
SUPPORTING INFORMATION
Additional Supporting Information may be found in the onlineversion of this articleAppendix S1 Raw data quantification for compounds identified inthe metabolite profiling by GCMS
Figure S1 Relative expression of ABCG genes in various organsand mutant backgroundsFigure S2 Co-immunoprecipitation controlsFigure S3 ABCG9 and ABCG14 homo- andor heterodimerize withABCG11Figure S4 Complementary data on metabolome analysis and con-jugated sterol contentFigure S5 Relative expression of genes related to sterol homeo-stasisMethods S1 Method for whole-mount immunolocalizationMethods S2 Method for quantitative PCR
Table S1 List of primers used in this studyTable S2 Annealing temperature and efficiencies for the primerspairs used in this study
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Wu XW Yephremov A and Samuels L (2007) Characterization of Ara-
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Douglas AE (2012) The physiology of sterol nutrition in the pea aphid
Acyrthosiphon pisum J Insect Physiol 58 1383ndash1389Bouvier-Nave P Berna A Noiriel A Compagnon V Carlsson AS
Banas A Stymne S and Schaller H (2010) Involvement of the phos-
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M Mueller R Nolan T Pfaffl MW and Shipley GL (2009) The MIQE
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Carland FM Fujioka S Takatsuto S Yoshida S and Nelson T (2002)
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sporopollenin precursors for exine formation in developing pollen Plant
J 65 181ndash193Citovsky V Lee LY Vyas S Glick E Chen MH Vainstein A Gafni
Y Gelvin SB and Tzfira T (2006) Subcellular localization of interacting
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Biol 362 1120ndash1131Clough SJ and Bent AF (1998) Floral dip a simplified method for Agro-
bacterium-mediated transformation of Arabidopsis thaliana Plant J 16
735ndash743Clouse SD (2002) Arabidopsis mutants reveal multiple roles for sterols in
plant development Plant Cell 14 1995ndash2000Cnops G Neyt P Raes J et al (2006) The TORNADO1 and TORNADO2
genes function in several patterning processes during early leaf develop-
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GFPcDNA fusions enable visualization of subcellular structures in cells of
Arabidopsis at a high frequency Proc Natl Acad Sci USA 97 3718ndash3723Dou XY Yang KZ Zhang Y Wang W Liu XL Chen LQ Zhang
XQ and Ye D (2011) WBC27 an adenosine tri-phosphate-binding cas-
sette protein controls pollen wall formation and patterning in Arabidop-
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Dudits D and Bako L (2005) The Medicago CDKC1ndashCYCLINT1 kinase
complex phosphorylates the carboxy-terminal domain of RNA polymer-
ase II and promotes transcription Plant J 42 810ndash820Gullberg J Jonsson P Nordstrom A Sjostrom M and Moritz T (2004)
Design of experiments an efficient strategy to identify factors influenc-
ing extraction and derivatization of Arabidopsis thaliana samples in met-
abolomic studies with gas chromatographymass spectrometry Anal
Biochem 331 283ndash295Hertzberg M Aspeborg H Schrader J et al (2001) A transcriptional
roadmap to wood formation Proc Natl Acad Sci USA 98 14732ndash14737Jang JC Fujioka S Tasaka M Seto H Takatsuto S Ishii A Aida M
Yoshida S and Sheen J (2000) A critical role of sterols in embryonic
patterning and meristem programming revealed by the fackel mutants of
Arabidopsis thaliana Genes Dev 14 1485ndash1497Jenks MA Rashotte AM Tuttle HA and Feldmann KA (1996) Mutants
in Arabidopsis thaliana altered in epicuticular wax and leaf morphology
Plant Physiol 110 377ndash385Jonsson P Johansson AI Gullberg J Trygg J A J Grung B Markl-
und S Sjostrom M Antti H and Moritz T (2005) High-throughput
data analysis for detecting and identifying differences between samples
in GCMS-based metabolomic analyses Anal Chem 77 5635ndash5642Jungwirth H and Kuchler K (2006) Yeast ABC transporters ndash a tale of sex
stress drugs and aging FEBS Lett 580 1131ndash1138Kaneda M Schuetz M Lin B Chanis C Hamberger B Western T
Ehlting J and Samuels A (2011) ABC transporters coordinately
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ABCG proteins and vascular development 823
expressed during lignification of Arabidopsis stems include a set of AB-
CBs associated with auxin transport J Exp Bot 62 2063
Karimi M Inze D and Depicker A (2002) GATEWAYTMvectors for Agrobac-
terium-mediated plant transformation Trends Plant Sci 7 193ndash195Kneurooller A and Murphy A (2011) ABC transporters and their function at the
plasma membrane In The Plant Plasma Membrane Plant Cell Mono-
graphs vol 19 (Murphy AS Peer W and Schulz B eds) New York
NY Springer pp 353ndash377Kopischke M Westphal L Schneeberger K Clark R Ossowski S
Wewer V Fuchs R Landtag J Hause G and Deuroormann P (2013)
Impaired sterol ester synthesis alters the response of Arabidopsis thali-
ana to Phytophthora infestans Plant J 73 456ndash468Kuromori T Miyaji T Yabuuchi H Shimizu H Sugimoto E Kamiya
A Moriyama Y and Shinozaki K (2010) ABC transporter AtABCG25 is
involved in abscisic acid transport and responses Proc Natl Acad Sci
USA 107 2361ndash2366Kuromori T Itoh T Sugimoto E and Shinozaki K (2011a) Arabidopsis
mutant of AtABCG26 an ABC transporter gene is defective in pollen
maturation J Plant Physiol 168 2001ndash2005Kuromori T Sugimoto E and Shinozaki K (2011b) Arabidopsis mutants
of AtABCG22 an ABC transporter gene increase transpiration and
drought susceptibility Plant J 67 885ndash894Le Hir R Beneteau J Bellini C Vilaine F and Dinant S (2008) Gene
expression profiling keys for investigating phloem functions Trends
Plant Sci 13 273ndash280Lehrer AT Dugassa-Gobena D Vidal S and Seifert K (2000) Transport
of resistance-inducing sterols in phloem sap of barley Z Naturforsch C
55 948ndash952Li Y Beisson F Pollard M and Ohlrogge J (2006) Oil content of Arabid-
opsis seeds the influence of seed anatomy light and plant-to-plant vari-
ation Phytochemistry 67 904ndash915Luo B Xue XndashY Hu WndashL Wang LndashJ and Chen XndashY (2007) An ABC
transporter gene of Arabidopsis thaliana AtWBC11 is involved in cuticle
development and prevention of organ fusion Plant Cell Physiol 48
1790ndash1802McFarlane HE Shin JJH Bird DA and Samuels AL (2010)
Arabidopsis ABCG transporters which are required for export of diverse
cuticular lipids dimerize in different combinations Plant Cell 22 3066ndash3075
Meskiene I Baudouin E Schweighofer A Liwosz A Jonak C Rodri-
guez PL Jelinek H and Hirt H (2003) Stress-induced protein phospha-
tase 2C is a negative regulator of a mitogen-activated protein kinase
J Biol Chem 278 18945ndash18952Murashige T and Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures Physiol Plant 15 473ndash497Panikashvili D Savaldi-Goldstein S Mandel T Yifhar T Franke RB
Hofer R Schreiber L Chory J and Aharoni A (2007) The Arabidopsis
DESPERADOAtWBC11 transporter is required for cutin and wax secre-
tion Plant Physiol 145 1345ndash1360Panikashvili D Shi JX Bocobza S Franke RB Schreiber L and Aha-
roni A (2010) The Arabidopsis DSOABCG11 transporter affects cutin
metabolism in reproductive organs and suberin in roots Mol Plant 3
563ndash575Panikashvili D Shi JX Schreiber L and Aharoni A (2011) The Arabid-
opsis ABCG13 transporter is required for flower cuticle secretion and pat-
terning of the petal epidermis New Phytol 190 113ndash124Pighin JA Zheng H Balakshin LJ Goodman IP Western TL Jetter
R Kunst L and Samuels AL (2004) Plant cuticular lipid export requires
an ABC transporter Science 306 702ndash704Pullen M Clark N Zarinkamar F Topping J and Lindsey K (2010)
Analysis of vascular development in the hydra sterol biosynthetic
mutants of Arabidopsis PLoS ONE 5 e12227
Quilichini TD Friedmann MC Samuels AL and Douglas CJ (2010)
ATP-binding cassette transporter G26 is required for male fertility and
pollen exine formation in Arabidopsis Plant Physiol 154 678ndash690R Development Core Team (2010) R A Language and Environment for
Statistical Computing Vienna Austria R Foundation for Statistical
Computing
Roudier F Gissot L Beaudoin F Haslam R Michaelson L Marion J
Molino D Lima A Bach L and Morin H (2010) Very-long-chain fatty
acids are involved in polar auxin transport and developmental patterning
in Arabidopsis Plant Cell 22 364ndash375Rozen S and Skaletsky HJ (2000) Primer3 on the WWW for general users
and for biologist programmers Methods Mol Biol 132 365ndash386Schad M Lipton MS Giavalisco P Smith RD and Kehr J (2005) Eval-
uation of two-dimensional electrophoresis and liquid chromatography
tandem mass spectrometry for tissue-specific protein profiling of
laser-microdissected plant samples Electrophoresis 26 2729ndash2738Schauer N Steinhauser D Strelkov S et al (2005) GCndashMS libraries for
the rapid identification of metabolites in complex biological samples
FEBS Lett 579 1332ndash1337Schrader J Nilsson J Mellerowicz EJ Berglund A Nilsson P Hertz-
berg M and Sandberg G (2004) A high-resolution transcript profile
across the wood forming meristem of poplar identifies potential regula-
tors of cambial stem cell identity Plant Cell 16 2278ndash2292Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desi-
mone M Frommer WB Fleurougge UndashI and Kunze R (2003) ARAMEM-
NON a novel database for Arabidopsis integral membrane proteins
Plant Physiol 131 16ndash26Silvestro D Andersen TG Schaller H and Jensen PE (2013) Plant ste-
rol metabolism D7ndashsterol-C5ndashdesaturase (STE1DWARF7) D57ndashste-rol-D7ndashreductase (DWARF5) and D24ndashsterol-D24ndashreductase (DIMINUTO
DWARF1) show multiple subcellular localizations in Arabidopsis thaliana
(Heynh) L PLoS ONE 8 e56429
Sorin C Bussell JD Camus I Ljung K Kowalczyk M Geiss G McKh-
ann H Garcion C Vaucheret H and Sandberg G (2005) Auxin and
light control of adventitious rooting in Arabidopsis require ARGONA-
UTE1 Plant Cell 17 1343ndash1359Tanaka T Tanaka H Machida C Watanabe M and Machida Y (2004) A
new method for rapid visualization of defects in leaf cuticle reveals five
intrinsic patterns of surface defects in Arabidopsis Plant J 37 139ndash146Turgeon R and Wolf S (2009) Phloem transport cellular pathways and
molecular trafficking Annu Rev Plant Biol 60 207ndash221Ukitsu H Kuromori T Toyooka K et al (2007) Cytological and biochemi-
cal analysis of COF1 an Arabidopsis mutant of an ABC transporter gene
Plant Cell Physiol 48 1524ndash1533Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De
Paepe A and Speleman F (2002) Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biol 3 research00340031-00340011Verrier PJ Bird D Burla B et al (2008) Plant ABC proteins ndash a unified
nomenclature andupdated inventory Trends Plant Sci 13 151ndash159Vilaine F Palauqui JC Amselem J Kusiak C Lemoine R and Dinant
S (2003) Towards deciphering phloem a transcriptome analysis of the
phloem of Apium graveolens Plant J 36 67ndash81Zhai Z Sooksa-nguan T and Vatamaniuk OK (2009) Establishing RNA
interference as a reverse-genetic approach for gene functional analysis
in protoplasts Plant Physiol 149 642ndash652Zhang Q Blaylock LA and Harrison MJ (2010) Two Medicago truncatula
half-ABC transporters are essential for arbuscule development in
arbuscular mycorrhizal symbiosis Plant Cell 22 1483
Zhao C Craig JC Petzold HE Dickerman AW and Beers EP (2005)
The xylem and phloem transcriptomes from secondary tissues of the
Arabidopsis root-hypocotyl Plant Physiol 138 803ndash818
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
824 Rozenn Le Hir et al
vascular pattern between classes III and V) The other
mutant lines showed a significantly less complex vena-
tion pattern than the wild-type (Figure 4) The single
mutants also differed significantly with respect to the loss
of complexity The abcg11ndash7 line displayed the least com-
plex vascular system (only 38 of the venation patterns
were between classes III and V) followed by abcg14ndash1
and then abcg9ndash2 (73 and 86 respectively) (Figure 4)
Introduction of HA-tagged versions of the ABCG9 and
ABCG14 proteins in the corresponding mutant back-
ground (abcg9ndash2 and abcg14ndash1) complemented the loss
of vascular complexity (Figure 4) confirming that the
phenotype was indeed due to defective expression of
ABCG9 and ABCG14 genes and the tagged proteins were
functional Interestingly the cotyledons of the triple
mutant displayed a significantly less complex vascular
system than that of the single mutants (Figure 4) In addi-
tion to the reduced vascular complexity in abcg11 homo-
zygous and heterozygous mutants the presence of open
vascular loops indicated a defect in vascular continuity
(Figure 4) Interestingly the continuity was restored in the
triple homozygous mutant (Figure 4)
Subsequently we characterized vascular bundles in the
floral stem of 4-week-old plants grown in soil (Table 1)
Under our conditions the wild-type flower stems dis-
played a vascular bundle density of 592 vascular bundles
per mm (Table 1) No significant variation in the number
of vascular bundles was observed in the single mutants
and complemented lines (Table 1) In contrast to the
increased complexity of the cotyledon venation pattern
(Figure 4) significantly fewer vascular bundles were pres-
ent in abcg9ndash2 abcg14ndash1 and abcg9ndash2 abcg11ndash7 abcg14ndash1
mutants (828 and 76 vascular bundles respectively)
(Table 1) Consistent with the smaller plant stature of
these double and triple mutants (Figure 1n) the diameter
of the floral stem was reduced (Table 1) However the
number of vascular bundles could not be explained by a
reduction of the floral stem diameter as the abcg11ndash7
mutant displayed the same number of vascular bundles
than the wild-type but its floral stem diameter was
strongly reduced (Table 1) Altogether these results
demonstrate that ABCG9 ABCG11 and ABCG14 play an
important role in vascular patterning during Arabidopsis
plant development
ABCG9 and ABCG14 are plasma membrane-localized
proteins that interact physically to form homo- andor
heterodimers with ABCG11
Because ABCG half transporters are thought to require
dimerization for functionality we investigated whether
ABCG9 ABCG11 and ABCG14 interact physically with
themselves andor other ABCG isoforms We first investi-
gated the subcellular localization of ABCG9 and ABCG14
after transfection of Arabidopsis mesophyll protoplasts
prepared from transgenic seedlings expressing the plasma
membrane marker low temperature induced protein 6b
(GFPndashLTi6b) (Cutler et al 2000) with constructs encoding
transcriptional fusions with red fluorescent protein (RFP)
In both cases the GFP fluorescent signal (Figure 5ae) co-
localized with the RFP fluorescent signal (Figure 5bf) at
the plasma membrane of transformed cells (Figure 5bh)
indicating that both ABCG9 and ABCG14 localize to the
plasma membrane like ABCG11 (Bird et al 2007 Luo
et al 2007 Panikashvili et al 2007 Ukitsu et al 2007) In
addition immunolocalization of a HA-tagged version of
ABCG9 and ABCG14 in their respective complemented
lines confirmed that these two proteins are localized to
the plasma membrane in planta (Figure 5ij) Next
(a)
(b)
(c)
(d) (e)
(f) (g)
(h) (i)
Figure 2 TndashDNA insertion lines for ABCG genes
(andashc) TndashDNA positions in ABCG9 ABCG11 and ABCG14 respectively Black
boxes promoters dark gray boxes 5prime and 3prime untranslated regions light gray
boxes exons lines introns
(dndashi) The presence of full-length mRNA was checked in abcg mutants as
described in Experimental procedures The elongation factor EF1a was used
as an internal control
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
814 Rozenn Le Hir et al
Arabidopsis protoplasts were transfected with open read-
ing frames encoding individual cndashMyc- or HA-tagged
versions of the three ABCGs and the identity of the epi-
tope-tagged proteins was verified by SDSndashPAGE and
Western blotting using anti-HA and anti-c-Myc antibodies
In all cases only one band of approximately 75 kDa cor-
responding to the predicted molecular mass of the pro-
teins in the Plant Membrane Protein Database (Schwacke
et al 2003) was obtained (Figure 5kndashp lane 1 and Figure
S2andashf lane 1) Subsequently protoplasts were co-trans-
fected with cndashMyc- and HA-tagged isoforms immunopre-
cipitated with antisera against one of the epitope tags
and probed with antisera directed against the alternative
epitope tag in Western blots of SDSndashPAGE gels in order
to determine whether homodimers or heterodimers were
formed (Figure 5kndashp) No ABCG14 homodimers or
ABCG9ndashABCG14 heterodimers were detected (Figure 5m
lane 4 and Figure 5o lane 4) In contrast ABCG11 formed
an apparent homodimer as described previously (McFar-
lane et al 2010) ABCG9 also formed an apparent homod-
imer ABCG14 formed an apparent heterodimer with
ABCG11 and ABCG9 formed an apparent heterodimer
with ABCG11 (Figure 5klnp lane 4) These interactions
were confirmed in a plant-based in vivo system using the
bimolecular fluorescence complementation assay (Figure
S3andashl) We therefore concluded that ABCG14 forms a strict
heterodimer with ABCG11 whereas ABCG11 and ABCG9
can both hetero- and homodimerize
Cuticular and epicuticular lipid precursor content as well as
cuticle integrity are not altered in abcg9ndash2 and abcg14ndash1
mutants
ABCG11 is known to be required for cuticle integrity (Bird
et al 2007 Luo et al 2007 Panikashvili et al 2007 Ukitsu
et al 2007) and because ABCG11 interacts physically with
ABCG9 and ABCG14 we looked for potential defects in
very long chain fatty acids (VLCFAs) which are among the
precursors of cuticular and epicuticular lipids (Roudier
et al 2010) as well as for possible cuticle defects in
abcg9ndash2 and abcg14ndash1 mutants
Quantification of fatty acids (from 160 to 180) as well as
VLCFAs (from 200 to 240) in apical parts of abcg9ndash2 and
abcg14ndash1 seedlings revealed no differences compared to
wild-type levels (Figure 6ab) It has previously been
(a) (b) (c) (d)
(e) (f) (g) (h)
(i)
(j)
(i)
(k) (l) (m)
(n)
Figure 3 Phenotype of abcg mutants grown in
soil
(andashm) Twenty-day-old plants grown in soil
Scale bar = 1 cm
(n) Green areas of 4-week-old plants Values
represent means SE from 20 individual
plants The experiment was repeated twice with
similar results Asterisks indicate statistically
significant differences between the wild-type
and mutants (P lt 005 Studentrsquos t test n = 20)
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 815
shown that when the cuticle is altered toluidine blue (TB)
permeates the epidermal surface (Tanaka et al 2004)
Therefore we used TB to check for cuticle defects in the
mutants After treatment with TB no staining was
observed in the wild-type abcg11+ or the abcg9ndash2 and
abcg14ndash1 mutants (Figure 6cef) whereas abcg11ndash7
homozygous seedlings showed patches of blue staining
confirming the cuticle alteration previously described
(Figure 6d) (Ukitsu et al 2007) These observations sug-
gest that unlike mutations in ABCG11 mutations in
ABGC9 or ABCG14 do not affect cuticle formation
abcg9ndash2 and abcg14ndash1 mutants are defective in sterol
composition
To obtain an insight into the potential substrates trans-
ported by these ABCG transporters we analyzed the shoot
and root metabolome of single mutants Orthogonal partial
least-squares discriminant analysis was applied to the
GCMS data in order to classify the profiles of the geno-
types In both shoots (Figure 7andashc) and roots (Figure S4andash
c) homozygous abcg11ndash7 abcg9ndash1 abcg9ndash2 and abcg14ndash1
mutants exhibited very different metabolomic profiles from
the wild-type Interestingly the heterozygous abcg11ndash7+
and abcg9ndash2+ plants had metabolomic profiles intermedi-
ate between the wild-type and their respective homozy-
gous mutant plants (Figure 7ab and Figure S4ab) These
results suggest that a mutation in one copy of ABCG11 or
ABCG9 is sufficient to significantly affect the physiology of
the plant even if it does not dramatically affect visible
aspects of its phenotype
The metabolites showing the most significant differ-
ences were identified by comparing their retention indices
and mass spectra with entries in publicly available reten-
tion libraries (Schauer et al 2005) For all the genotypes
analyzed the metabolites identified as being significantly
different from the wild-type belonged to three main clas-
ses amino acids carbohydrates and lipidssterols The
levels of these compounds were almost all reduced in
abcg9ndash2 and abcg14ndash1 but in excess in abcg11ndash7 (Table 2
and Appendix S1)
Because our study focused on vascular development in
aerial tissues only data obtained for the apical parts are
described in detail here (Table 2) Root data are presented
in Appendix S1 Whereas many neutral amino acids were
significantly depleted in abcg9ndash2 and abcg14ndash1 aerial
Figure 4 Cotyledon venation pattern in abcg mutant lines
Venation complexity and continuity of each examined class of mutant and
wild-type seedlings Values in brackets indicate the percentage contribution
of each class Asterisks indicate statistically significant differences between
the wild-type and mutants (P lt 005 Pearsonrsquos v2 test with Monte Carlo
permutations N = 10 000)
Table 1 Distribution of vascular bundles in the floral stem
Genotype Vascular bundle number Floral stem diameter (mm) Vascular bundle density Total
Col-0 930 105 157 020 592 049 10abcg11-7+ 914 134 140 080 652 042 12abcg11-7 902 115 051 004 176 182 8abcg14-1 828 049 145 018 632 0049 1135SHA-ABCG14 abcg14-1 940 089 170 016 554 058 12abcg9-1 957 053 160 006 586 031 7abcg9-2 875 103 162 010 553 046 835SHA-ABCG9 abcg9-2 933 051 174 010 606 026 8abcg9-1 abcg14-1 871 048 154 003 565 030 8abcg9-2abcg14-1 828 049 124 015 674 084 8abcg9-2abcg14-1abcg11-7
760 054 063 005 1203 169 9
Statistically significant differences between the wild-type and mutants (P lt 005 Studentrsquos t test)
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
816 Rozenn Le Hir et al
tissues (nine and eight out of 20 neutral amino acids
respectively) only two amino acids (glutamine and gluta-
mate) associated with Arabidopsis phloem cells (Schad
et al 2005) were different in abcg11ndash7 (Table 2) Xylose
and the non-reducing sugars trehalose and raffinose were
also decreased in abcg14ndash1 (Table 2) whereas glucose
fructose galactose maltose xylose were significantly dif-
ferent in abg9ndash2 and abcg11ndash7 (reduced and increased
respectively compared to wild-type) (Table 2)
Levels of sterols (campesterol 24ndashmethylene cholesterol
and sitosterol) saturated and unsaturated fatty acids (lino-
leic acid lauric acid palmitic acid and triacontanoic acid)
glycerolipids (monoacylglycerol) and lipid metabolism
intermediates (glycerol glycerol-3ndashphosphate and inositol-
1ndashphosphate) were also significantly different from wild-
type in abcg mutants (Table 2) The abcg9ndash2 mutant
showed 30 lower levels of 24ndashmethylene cholesterol
compared to the wild-type (Appendix S1) In the abcg14ndash1
mutant 50 and 35 decreases of 24ndashmethylene cholesterol
and sitosterol levels were measured respectively (Appen-
dix S1) Finally campesterol showed a 50 increase in the
abcg11ndash7 mutant (Appendix S1) To extend this analysis
measurements of conjugated sterols [sterol ester (SE)
sterol glucoside and acylated sterol glucoside] were also
performed When seedlings were grown in vitro no differ-
ence in the sterol glucoside and acylated sterol glucoside
contents was detected between the wild-type and the vari-
ous mutant lines (Figure S4d) However there was a ten-
dency towards a reduced SE content in the double mutant
abcg9ndash2 abcg14ndash1 compared to the wild-type (Figure S4d)
This observation prompted us to examine variations in the
SE content in older plants (4-week-old plants grown in soil
in the greenhouse) At this developmental stage no differ-
ence in the free sterol (FS) content was observed between
the wild-type and the various mutant lines (Figure 7d) but
the fraction comprising FS and SE was significantly
(a) (b) (c) (d)
(e) (f) (g) (h)
(i) (j)
(k) (l) (m)
(n) (o) (p)
Figure 5 Subcellular localization and physical interactions of ABCG9 ABCG11 and ABCG14
(andashh) ABCG9 and ABCG14 co-localize to the plasma membrane when transiently expressed in Arabidopsis mesophyll protoplasts of the GFPndashLTi6b marker line
(Cutler et al 2000)
(a e) Cyan fluorescence of GFPndashLTi6b(b f) Purple fluorescence from mRFPndashABCG9 and mRFPndashABCG14 respectively
(c g) Yellow auto-fluorescence of the chloroplasts
(d h) Merged images of the three channels Scale bar = 10 lm
(i j) Confocal images of immunofluorescence staining with anti-HA antibodies in root cells of abcg9ndash2 and abcg14ndash1 mutants complemented with HA-tagged
ABCG9 (i) and HA-tagged ABCG14 (j) Scale bars = 10 lm
(kndashp) Proteins were extracted from protoplasts co-transfected with HA- or cndashMyc-tagged versions of ABCGs (lanes 1 and 4) or transfected with a single plasmid
containing the HA-tagged version (lane 2) or the c-Myc-tagged version (lane 3) In lanes 2ndash4 proteins were immunoprecipitated with the anti-c-Myc antibody
and subjected to anti-HA protein gel-blot analysis to reveal the other partner of the dimer Transient co-expression of (k) HAndashABCG9cndashMycndashABCG9 (l) HAndashABCG11cndashMycndashABCG11 (m) HAndashABCG14cndashMycndashABCG14 (n) HAndashABCG14cndashMycndashABCG11 (o) HAndashABCG14cndashMycndashABCG9 and (p) HAndashABCG11cndashMycndashABCG9
in wild-type protoplasts
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 817
reduced probably accounting for the difference in the SE
content between the wild-type and the abcg9ndash2 abcg14ndash1
double mutant (Figure 7e)
(a)
(b)
(c) (d) (e)
(f) (g)
Figure 6 Long chain and very long chain fatty acid content and cuticle
integrity in abcg9ndash2 and abcg14ndash1 mutants
(a b) Fatty acid (a) and VLCFA content (b) in abcg9ndash2 and abcg14ndash1 mutants
in apical parts of 10-day-old seedlings (three biological replicates each of
approximately 5 mg dry weight were used)
(cndashf) Toluidine blue staining patterns of abcg mutants in 7-day-old seed-
lings Scale bars = 3 cm
(a)
(b)
(c)
(d) (e)
Figure 7 Metabolome analysis and measurements of sterol content in abcg
mutants
(andashc) Results of orthogonal partial least-squares discriminant analysis of
global metabolite contents of shoots of 7-day-old seedlings from wild-type
(black squares) abcg11ndash7+ (gray triangles) abcg11ndash7 (white stars) abcg9ndash1(white hexagons) abcg9ndash2+ (gray hexagons) abcg9ndash2 (white hexagons)
and abcg14ndash1 (black circles) seedlings (five biological replicates of 10 mg
each were used for the analysis)
(d e) Free sterol (FS) and sterol ester (SE) content of wild-type (white bars)
abcg9ndash2 (light gray bars) abcg14ndash1 (dark gray bars) and abcg9ndash2 abcg14ndash1(black bars) Error bars indicate standard deviations obtained from three
independent biological replicates The asterisk indicates a statistically signif-
icant difference between the wild-type and the mutant (P lt 005 Studentrsquos
t test)
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
818 Rozenn Le Hir et al
DISCUSSION
ABCG9 and ABCG14 are two plasma membrane proteins
that interact physically in multiple homo-heterodimer
combinations
In this study we showed that like the previously described
ABCG11 (Pighin et al 2004 Bird et al 2007 Luo et al
2007 Panikashvili et al 2007 2010 Ukitsu et al 2007)
ABCG9 and ABCG14 localize to the plasma membrane
Based on co-immunoprecipitation of tagged proteins and
the bimolecular fluorescence complementation assay we
demonstrated that ABCG9 interacts with ABCG11 to form a
heterodimer and like ABCG11 (McFarlane et al 2010) can
homodimerize In contrast ABCG14 interacts only with
ABCG11 forming an obligate heterodimer This extends
the number of potential partners of ABCG11 to three as it
was previously shown to dimerize with the ABCG12CER5
protein (McFarlane et al 2010) Similarly ABCG9 under-
goes flexible dimerization to form either homo- or hetero-
dimers suggesting that ABCGs may be promiscuous
proteins with different functions depending on their inter-
acting partner andor expression profile
Mutations in ABCG genes are semi-dominant
In the last few years several Arabidopsis mutants with
altered expression of various ABCG genes such as ABCG11
(Bird et al 2007 Luo et al 2007 Panikashvili et al 2007
Ukitsu et al 2007) ABCG12CER5 (Pighin et al 2004)
ABCG13 (Panikashvili et al 2011) ABCG22 (Kuromori
et al 2011a) ABCG25 (Kuromori et al 2010) and ABCG26
(Choi et al 2010 Quilichini et al 2010 Dou et al 2011
Kuromori et al 2011b) have been characterized but even
though these half-size transporters require dimerization to
become functional all the mutations were described as
recessive Here we demonstrated that mutations in these
genes have semi-dominant effects Indeed the metabolome
analyses showed that abcg9ndash2+ and abcg11ndash7+ heterozy-
gous seedlings have a profile intermediate between that of
wild-type and homozygous seedlings The possibility of
dominant-negative effects was excluded because no
Table 2 Shoot metabolites of 10-day-old abcg9ndash2 abcg11ndash7 and abcg14ndash1 seedlings whose levels were significantly different from wild-type
Group
Metabolites that differed between mutant and wild-type
abcg9ndash2Colndash0 abcg11ndash7Colndash0 abcg14ndash1Colndash0
Amino acids Glycine Glutamate AlanineAlanine Glutamine ValineValine ThreonineThreonine GABASerine Prolinebndashalanine IsoleucineIsoleucine SerineGABA bndashalanineProline
Carbohydrates Glucose Glucose ErythritolFructose Fructose TrehaloseXylose Galactose XyloseMaltose RaffinoseSucrose
Lipids and intermediates 24ndashmethylene-cholesterol Campesterol Linoleic acidMonoacylglycerol Glycerol-3ndashphosphate Triacontanoic acidPalmitic acid Inositol-1ndashphosphate GlycerolGlycerol-3ndashphosphate Lauric acidInositol-1ndashphosphate 24ndashmethylene-cholesterol
SitosterolGlycerol-3ndashphosphate
TCA cycle and others Phosphoric acid Phosphate compound Glyceric acid24ndashdihydroxybutanoic acid Dehydroascorbic acid Fumaric acidGlyceric acid Malic acid Threonic acid-14ndashlactoneFumaric acid 2ndashoxoglutaric acid Threonic acidThreonic acid bndashhydroxy-bndashmethylglutaric acid Shikimic acidThreonic acid-14ndashlactone Gluconic acid lactone-like Succinic acidShikimic acid Phytol2ndashoxoglutaric acid Ethanolamine
Nicotinic acid
For abcg9ndash2 and abcg14ndash1 the levels of the compounds were reduced whereas they were increased in abcg11ndash7
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 819
truncated transcript was found for either abcg9ndash2 or
abcg11ndash7 mutants Hence abcg9ndash2 and abcg11ndash7 are true
null alleles These observations are consistent with the
requirement for homo- or heterodimerization of the ABCG
proteins to form functional transporters In the heterozy-
gous plants ABCG11 transcript levels were 50 lower than
in wild-type counterparts suggesting that ABCG11 protein
and thus corresponding homo- and heterodimers were
likely to be less abundant which may also explain the meta-
bolic perturbations and consequent phenotypic modifica-
tions These findings also suggest that wild-type plants are
not saturated by ABCG11 proteins as a 50 reduction in
protein content appears to be sufficient to alter the physiol-
ogy of the heterozygous mutant plants This hypothesis is
strengthened by the observation that mutants carrying the
weak abcg11ndash6 allele in which there was only a 25 reduc-
tion in ABCG11 transcript levels showed a similar pheno-
type to the heterozygous abcg11ndash7+ and abcg11ndash8+
plants Similarly the abcg9ndash1 weak allele mutant had a very
similar phenotype to the abcg9ndash2+ heterozygous seedlings
suggesting that considering the mode of action of these
proteins the semi-dominant effects that we observed are
likely to be a general feature among the ABCG genes
ABCG9 ABCG11 and ABCG14 transporters are involved in
vascular system development in Arabidopsis
We showed that the ABCG11 ABCG9 and ABCG14 promot-
ers drove expression in the vascular system of Arabidopsis
cotyledons and rosette leaves and more precisely in the
phloem of flower stems In addition mutations in ABCG9
ABCG11 and ABCG14 genes altered vascular development
in cotyledons and the floral stem Similar vascular localiza-
tion of ABCG11 has already been described by Panikashvili
et al (20072010) who showed that in addition to the epi-
dermis ABCG11 is expressed in the vasculature as early as
during embryo development and then throughout plant
development They showed that the vascular pattern in
leaves of the homozygote mutant seedlings was less com-
plex than in the wild-type and discontinuous (Panikashvili
et al 2007) The present article describes a mutant that
has both cuticle and vascular system patterning defects
although it has been previously shown that mutants
altered in cuticle formation have dramatically reduced
growth and altered leaf morphology (Jenks et al 1996)
which may account for vascular defects Analysis of the
abcg11+ heterozygous seedlings that have vascular
defects but apparently no cuticle alteration suggested that
these phenotypes are probably uncoupled Because
ABCG11 dimerizes with several members of its sub-family
it seems reasonable to postulate that it is involved in the
transport of different compounds depending on its partner
Here we provide evidence that ABCG11 in addition to
forming dimers with ABCG12 (McFarlane et al 2010) in
epidermal cells is likely to dimerize with ABCG9 and
ABCG14 in phloem cells suggesting functional compart-
mentalization of these proteins and a possible role in vas-
cular system patterning in Arabidopsis
Metabolite profiling suggests that ABCG9 ABCG11 and
ABCG14 are involved in sterollipid homeostasis
We investigated in more depth the metabolic changes
induced by mutations in ABCG9 ABCG11 and ABCG14
We found that levels of metabolites of several classes
including lipids sugars and amino acids were altered in
both the heterozygotes and homozygotes and that the
profiles in the root and apical parts differed suggesting
that ABCGs may play different roles in the roots and shoot
Interestingly the modifications of amino acid and sugar
contents observed in the metabolic profiles of the abcg
mutants suggest an impaired carbonnitrogen balance that
may be directly or indirectly linked to the vascular defects
leading to possible problems with phloem loading or
unloading Saturated and unsaturated fatty acids and sterol
contents were also altered in the mutants Patterning
defects in the vascular system are often observed in
mutants altered in hormone homeostasis such as for
auxin or brassinosteroids (Kaneda et al 2011) and direct
or indirect modifications of sterol homeostasis are thought
to be one of the primary causes of these downstream
effects (Pullen et al 2010) The detected defects in sterol
homeostasis may explain the abnormal vascular develop-
ment observed in abcg9 abcg11 and abcg14 mutants
Accordingly several sterol-deficient mutants such as fac-
kelhydra (Jang et al 2000) and cpv1 (Carland et al 2002)
have also been shown to exhibit severe defects in vascular
development It has been suggested that the bushy habit
and low fertility of cpv1smt2 mutants may be due to an
increased campesterolsitosterol ratio rather than sterol
deficiency per se (Clouse 2002) This ratio is controlled by
the activity of SMT2 (STEROL METHYLTRANSFERASE2)
Interestingly strong down-regulation of SMT2 expression
was observed in all the mutant backgrounds (Figure S5)
and the metabolic data indicated a deficit in 24ndashmethylene
cholesterol (precursor of both campesterol and sitosterol)
andor sitosterol in abcg9 and abcg14 mutants respec-
tively In addition the significant decrease in sterol ester
content in the double mutant abcg9ndash2 abcg14ndash1 correlated
with down-regulation of expression of the PHOSPHOLIPID
STEROL ACYLTRANSFERASE1 (PSAT1) gene encoding the
enzyme that is mainly responsible for SE synthesis (Bou-
vier-Nave et al 2010) and is proposed to be a control step
in sterol homeostasis (Kopischke et al 2013) Altogether
these results suggest that the vascular phenotype
observed in abcg mutants may be linked to modification of
sterol homeostasis
Therefore we postulate that the various dimer combina-
tions between ABCG9 ABCG11 and ABCG14 may
participate in the transport of squalene-derived metabolites
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
820 Rozenn Le Hir et al
such as sterol andor sterol conjugates via the phloem sap
The presence of sterols in phloem sap has been observed in
various species such as barley (Hordeum vulgare Lehrer
et al 2000) and fava bean (Vicia faba Bouvaine et al
2012) and indirect proof of the presence of sterols in
phloem sap has been obtained by analysis of the phytos-
terol content in exclusively phloem-feeding aphids that are
unable to synthetize them directly (Behmer et al 2011) In
addition the evidence for long-distance transport of sterol
precursors has been supported by observations that sterol
esters accumulate in organs distal to the site of application
of exogenous precursors (Bouvier-Nave et al 2010) As
sterols have been proposed to act as signal molecules in
the control of developmental processes such as vascular
patterning (Carland et al 2002 Pullen et al 2010) modifi-
cations of sterol homeostasis may affect development of
the vascular system in the abcgmutants described here
In conclusion we propose that ABCG9 ABCG11 and
ABCG14 are important for lipidsterol homeostasis Thus
these three proteins are likely to be involved in the control
of various aspects of plant development andor physiol-
ogy including patterning of the vascular system
EXPERIMENTAL PROCEDURES
Plant material and growth conditions
Insertion lines in ABCG9 (SALK_047133 and GABI_353B01)ABCG11 (SALK_096377 and GABI_590C03) and ABCG14 (SALK_036952) genes were obtained from the European ArabidopsisStock Centre (httparabidopsisinfo) Heterozygoushomozygousplants were screened using gene-specific primers in combinationwith the specific primer for the left border of the TndashDNA insertion(Table S1)
Plants were grown in soil or in vitro as described previously(Sorin et al 2005) Arabidopsis Colndash0 cell cultures were main-tained by weekly sub-culturing in MS medium (Murashige andSkoog 1962) supplemented with 240 lg l1 24ndashdichlorophenoxy-acetic acid and 14 lg l1 kinetin and were grown under 12 h light12 h dark cycles at 25 lE m2 sec1 and with 23degC day and nighttemperatures
Morphological characterization and GUS staining
To study the venation pattern cotyledons of 7-day-old seedlingsand rosette leaves of 4-week-old plants were cleared in a buffercontaining chloral hydrate glycerol and water (831 wvv) afterwhich 200 cotyledons per genotype were analyzed and the per-centage showing particular levels of complexity or discontinuity invascularity was estimated as described by Cnops et al (2006)
Flower stem segments (1ndash2 cm long) were removed from ten3ndash4-week-old plants grown in soil embedded in 8 agarose andsectioned using a VT100S vibratome (Leica httpwwwleicacom)The cross-sections were then stained with safraninalcian blue for1ndash2 min (Sigma httpwwwsigmaaldrichcom) rinsed in waterand mounted in 50 glycerol
Histochemical GUS assays were performed as described previ-ously (Sorin et al 2005) All observations were performed usingan Axioplan light microscope equipped with an Axiocam cameraand Axiovision software (Zeiss httpwwwzeisscom)
To observe potential cuticle defects in the various mutant linesa TB test was performed by immersing in vitro-grown plantlets ina 005 wv TB solution for 2 min as described by Tanaka et al(2004)
Statistical analyses
Statistical analyses of differences between-genotype means andvariance for the studied morphological traits were performedusing Studentrsquos t test (GraphPad Prism version 50 httpwwwgraphpadcom) In the venation pattern experiment thesignificance of differences between wild-type and mutants wasassessed using R (R Development Core Team 2010) and Pearsonrsquosv2 test with Monte Carlo permutations (N = 10 000)
Plasmid constructs
The native ABCG promoters and open reading frame of ABCG11were amplified from genomic DNA or cDNA extracted from 7-day-old Arabidopsis seedlings using Phusion high-fidelity DNApolymerase (Finnzyme wwwthermoscientificbiocomfinnzymes)according to the manufacturerrsquos instructions with gene-specificprimers (Table S1) The PCR products were cloned into pENTRDndashTOPO (Invitrogen httpwwwinvitrogencom) and recombinedinto pKGWFS7 (Karimi et al 2002) to create ABCGproGUS Allrecombinations were performed using the Gateway LR Clonase
enzyme mix (Invitrogen) At each cloning step the sequenceswere confirmed by sequencing and all the constructs weretransferred into Agrobacterium strain GV3101pMP90 (C58C1)Wild-type or abcg11ndash7+ heterozygous plants were transformed byfloral dipping (Clough and Bent 1998) For ABCGGUS constructsthe expression pattern was checked in the T2 progeny of 10ndash15independent transgenic lines and one representative homozygousline was used for further characterization
Tagged protein constructs and protoplast transformation
Epitope-tagged versions of ABCG9 ABCG11 and ABCG14 proteinswere produced in pRT104ndash3xHA and pRT104ndash3xMyc plasmids(Feurouleuroop et al 2005) and pSAT6-mRFP (Citovsky et al 2006) Allthese plasmids have a 35S promoter sequence upstream of themulti-cloning site The open reading frames of ABCG11 andABCG14 and the genomic clone of ABCG9 were amplified fromcDNA or genomic DNA extracted from 7-day-old Arabidopsisseedlings using Phusion high-fidelity DNA polymerase (Finnzyme)according to the manufacturerrsquos instructions with gene-specificprimers carrying EcoRI or SalI restriction sites to facilitate subse-quent cloning (Table S1) The products obtained after PCR weredigested by EcoRI and SalI prior to ligation into appropriate plas-mids that had previously been cut open with the same restrictionenzymes Constructs were verified by sequencing Protoplastsfrom Arabidopsis cell cultures or 14-day-old Arabidopsis seedlingswere prepared and transformed as previously described (Meski-ene et al 2003 Zhai et al 2009) For co-immunoprecipitation andsubcellular localization assays 100 000 and 50 000 protoplastcells respectively were transfected with 15 and 10 ll of plasmidsrespectively (containing between 5 and 75 lg of each construct)and the resulting transformed protoplasts were processed afterincubation in the dark at room temperature for 16ndash18 and 24 hrespectively
Whole-mount immunolocalization
The HA-tagged versions of ABCG9 and ABCG14 were detected in7-day-old in vitro-grown Arabidopsis seedlings as described inMethods S1 Anti-HA antibody 12CA5 (Roche httpwwwroche
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 821
com) (1100 dilution) combined with Alexa Fluor 488-conjugatedsecondary antibody (1100 dilution) were used
Confocal laser-scanning microscopy
Images were collected using an SP2 confocal microscope (Leica)RFP and Alexa Fluor 488 were detected using laser lines at 561and 488 nm respectively according to the manufacturerrsquos set-tings RFP fluorescence emission was detected at 550ndash600 nmand Alexa Fluor 488 and GFP fluorescence emission were detectedat 495ndash545 nm For subcellular localization in protoplasts imageswere coded in cyan (GFP) magenta (RFP) and yellow (auto-fluo-rescence) resulting in purple coloration for co-localization inmerged images For the immunolocalization experiment imageswere coded in green (GFP) A water-corrected 209 objective (HCPlan-Apochromat 20907 ImmKor Leica) was used Images werecropped using Adobe Photoshop CS2 and assembled using AdobeIllustrator CS2 software (Abode httpwwwabodecom) Eachimage shown represents a single focal plan
Co-immunoprecipitation assays
Transformed protoplast proteins were extracted using 50 ll lysisbuffer containing 25 mM TrisHCl pH 78 10 mM MgCl2 75 mM
NaCl 5 mM EGTA 1 mM benzamidine 1 mM dithiothreitol 10glycerol 1 Triton Xndash100 and 19 protein inhibitor cocktail(Sigma httpsigmaaldrichcom) The transformed protoplast sus-pension was frozen in liquid nitrogen then thawed on ice andsubjected to centrifugation for 5 min at 150 g Two microliters ofanti-Myc (9E10 Covance httpwwwcovancecom) or anti-HA(16B12 Covance) antibodies were added to the extracts and themixture was incubated for 2 h at 4degC on a rotating wheel Immu-nocomplexes were captured by incubation with 10 ll Protein GndashSepharose beads for a further 2 h at 4degC on a rotating wheel afterwhich they were washed three times for 5 min each in 25 mM
NaPi 150 mM NaCl 5 glycerol and 02 Igepal CAndash630 buffer(Sigma) and then eluted using 40 ll SDS sample buffer at 37degCfor 30 min Co-immunoprecipitation of Myc- and HA-tagged pro-teins was revealed by SDSndashPAGE followed by Western blottingusing both anti-Myc (Invitrogen) and anti-HA (3F10 Roche) anti-bodies at 12000 dilution
RNA extraction and cDNA preparation
To quantify the relative amounts of the ABCG transcriptsexpressed in various organs three rosette leaves all the cau-line leaves the bottom 5 cm of the floral stem all closed flow-ers and all green siliques were harvested from each of threeindependent 4-week-old Arabidopsis plants of each genotypegrown in soil For the other experiments 10-day-old seedlingsgrown in vitro were harvested All the material was directly fro-zen in liquid nitrogen and stored at 80degC prior to RNA extrac-tion Samples were then disrupted using a MM 301 vibrationmill (Retsch GmbH httpwwwretschcom) at a frequency of25 Hz for 2 min with 3 mm tungsten carbide beads (Qiagenhttpwwwqiagencom) Total RNA was isolated from thismaterial using TRIzol (Life Technologies httplifetechnologiescom) RNA quantity and quality were measured using a Nano-Drop ND 1000 (Thermo Scientific httpwwwthermoscientificcom) All samples with 260280 nm and 260230 nm absorbanceratios lower than 18 were discarded and RNA was re-extractedDNase treatment was applied to 5 lg samples of total RNAusing a DNase I kit (Fermentas httpwwwfermentascom)Double-stranded cDNA was synthesized from 5 lg total RNAusing a SuperScript reverse transcriptase kit (Invitrogen) witholigo(dT)12 primer Finally RNase treatment was applied to the
samples using Escherichia coli RNAse H (Fermentas) The cDNAsamples obtained were diluted 30-fold (except for abcg9 linesamples which were diluted 10-fold) prior to use either toverify the absence of full-length transcripts in putative knockoutmutants or to quantify gene expression by quantitative PCR Allprimers used were designed using the Primer3 program (Rozenand Skaletsky 2000) and their sequences are listed inTable S1
QUANTITATIVE REAL-TIME PCR
Quantitative PCR amplifications were performed in 20 llmixtures containing 10 ll 59 SYBR Green I master mix
(Roche) 02 ll forward and reverse primer (30 lM each)
48 ll sterile water and 5 ll cDNA The BiondashRad CFX96
real-time PCR detection system (httpwwwbio-radcom)
was used for quantification of expression of the sterol
biosynthesis transcripts and the ABCG transcripts in the
various mutant backgrounds Finally the Roche LightCy-
cler 480 system (Roche) was used to quantify the rela-
tive abundance of ABCG transcripts in various organs
Four genes were considered as potential reference genes
[APT1 (At1g27450) TIP41 (At4g34270) EF1a (At5g60390)
and UBQ10 (At4g05320)] and their suitability was tested
using the geNorm algorithm (Vandesompele et al 2002)
to determine the most appropriate one for each experi-
ment A more detailed procedure following the Minimum
Information for Publication of Quantitative Real-Time PCR
Experiments guidelines (Bustin et al 2009) is described
in Methods S2 and the annealing temperature and
efficiencies of the primer pairs used are shown in
Table S2
VLCFA quantification
Approximately 5 mg dry weight of 10-day-old in vitro
Arabidopsis seedlings were analyzed for VLCFA quantifica-
tion as described by Li et al (2006)
Sterol extraction and GC-FID characterization
A pool of 10-day-old seedlings grown in vitro or rosettes
from 4-week-old plants grown in soil were lyophilized prior
to extraction Sterol extraction and isolation was per-
formed as described previously (Bouvier-Nave et al 2010)
Gas Chromatography-Flame Ionization Detector (GC-FID)
characterization was performed as described by Silvestro
et al (2013) To quantify the total amounts of free sterols
and sterol esters non-saponifiable lipids were extracted
using nndashhexane and sterols were derivatized as sterols ace-
tates Quantification was achieved using lupenyl-3 28ndashdi-
acetate as an internal standard
Metabolome analysis
To obtain metabolome profiles associated with wild-type
abcg11ndash7+ abcg11ndash7 abcg9ndash1 abcg9ndash2 and abcg14ndash1
genotypes 10-day-old seedlings representing each geno-
type grown in vitro as described above were harvested
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
822 Rozenn Le Hir et al
and divided into shoot and root samples In the case of
abcg9ndash2 the phenotype of the heterozygote abcg9ndash2+
was indistinguishable from the homozygote phenotype
and therefore heterozygous and homozygous seedlings
from a segregating population were pooled Hence in this
particular experiment abcg9ndash2+ refers to a mixed popula-
tion comprising two-thirds heterozygotes and one-third
homozygotes
Metabolites in five samples of shoot and root parts of
seedlings of each genotype (10 mg fresh weight per sam-
ple) were then extracted analyzed by GCMS and the
acquired data were processed as previously described
(Gullberg et al 2004 Jonsson et al 2005) All multivariate
statistical investigations (partial component analysis and
orthogonal partial least-squares discriminant analysis) for
between-genotype similarities and differences in general
metabolite profiles were performed using Simca software
(Umetrics httpwwwumetricscom)
ACKNOWLEDGEMENTS
We thank A Murphy (Plant Science and Landscape Archi-
tecture University of Maryland MD USA) for stimulating
discussions and critical reading of the manuscript I Carl-
son (Umea Plant Science Center Department of Forest
Genetics and Plant Physiology Swedish University of Agri-
cultural Sciences Umea Sweden) for technical assistance
and S Vernhettes for kindly donating seeds of the
transgenic line carrying the plasma membrane marker
GFPndashLTi6b We also thank the European Arabidopsis Stock
Centre and the GABI-Kat stock centre for providing seeds
This work was supported by allocations granted to CB by
the Swedish Research Council for Agriculture the Swedish
Foundation for Strategic Research the Swedish Research
Council for Research and Innovation for Sustainable
Growth the K amp A Wallenberg Foundation and the Carl
Trygger Foundation
SUPPORTING INFORMATION
Additional Supporting Information may be found in the onlineversion of this articleAppendix S1 Raw data quantification for compounds identified inthe metabolite profiling by GCMS
Figure S1 Relative expression of ABCG genes in various organsand mutant backgroundsFigure S2 Co-immunoprecipitation controlsFigure S3 ABCG9 and ABCG14 homo- andor heterodimerize withABCG11Figure S4 Complementary data on metabolome analysis and con-jugated sterol contentFigure S5 Relative expression of genes related to sterol homeo-stasisMethods S1 Method for whole-mount immunolocalizationMethods S2 Method for quantitative PCR
Table S1 List of primers used in this studyTable S2 Annealing temperature and efficiencies for the primerspairs used in this study
REFERENCES
Behmer ST Grebenok RJ and Douglas AE (2011) Plant sterols and host
plant suitability for a phloem-feeding insect Funct Ecol 25 484ndash491Bird D Beisson F Brigham A Shin J Greer S Jetter R Kunst L
Wu XW Yephremov A and Samuels L (2007) Characterization of Ara-
bidopsis ABCG11WBC11 an ATP binding cassette (ABC) transporter that
is required for cuticular lipid secretion Plant J 52 485ndash498Bouvaine S Behmer ST Lin GG Faure MndashL Grebenok RJ and
Douglas AE (2012) The physiology of sterol nutrition in the pea aphid
Acyrthosiphon pisum J Insect Physiol 58 1383ndash1389Bouvier-Nave P Berna A Noiriel A Compagnon V Carlsson AS
Banas A Stymne S and Schaller H (2010) Involvement of the phos-
pholipid sterol acyltransferase1 in plant sterol homeostasis and leaf
senescence Plant Physiol 152 107ndash119Bustin SA Benes V Garson JA Hellemans J Huggett J Kubista
M Mueller R Nolan T Pfaffl MW and Shipley GL (2009) The MIQE
guidelines Minimum information for publication of quantitative real--
time PCR experiments Clin Chem 55 611
Carland FM Fujioka S Takatsuto S Yoshida S and Nelson T (2002)
The identification of CVP1 reveals a role for sterols in vascular pattern-
ing Plant Cell 14 2045ndash2058Choi H Jin JY Choi S Hwang JU Kim YY Suh MC and Lee Y
(2010) An ABCGWBC-type ABC transporter is essential for transport of
sporopollenin precursors for exine formation in developing pollen Plant
J 65 181ndash193Citovsky V Lee LY Vyas S Glick E Chen MH Vainstein A Gafni
Y Gelvin SB and Tzfira T (2006) Subcellular localization of interacting
proteins by bimolecular fluorescence complementation in planta J Mol
Biol 362 1120ndash1131Clough SJ and Bent AF (1998) Floral dip a simplified method for Agro-
bacterium-mediated transformation of Arabidopsis thaliana Plant J 16
735ndash743Clouse SD (2002) Arabidopsis mutants reveal multiple roles for sterols in
plant development Plant Cell 14 1995ndash2000Cnops G Neyt P Raes J et al (2006) The TORNADO1 and TORNADO2
genes function in several patterning processes during early leaf develop-
ment in Arabidopsis thaliana Plant Cell 18 852ndash866Cutler SR Ehrhardt DW Griffitts JS and Somerville CR (2000) Random
GFPcDNA fusions enable visualization of subcellular structures in cells of
Arabidopsis at a high frequency Proc Natl Acad Sci USA 97 3718ndash3723Dou XY Yang KZ Zhang Y Wang W Liu XL Chen LQ Zhang
XQ and Ye D (2011) WBC27 an adenosine tri-phosphate-binding cas-
sette protein controls pollen wall formation and patterning in Arabidop-
sis J Integr Plant Biol 53 74ndash88Feurouleuroop K Pettko-Szandtner A Magyar Z Miskolczi P Kondorosi E
Dudits D and Bako L (2005) The Medicago CDKC1ndashCYCLINT1 kinase
complex phosphorylates the carboxy-terminal domain of RNA polymer-
ase II and promotes transcription Plant J 42 810ndash820Gullberg J Jonsson P Nordstrom A Sjostrom M and Moritz T (2004)
Design of experiments an efficient strategy to identify factors influenc-
ing extraction and derivatization of Arabidopsis thaliana samples in met-
abolomic studies with gas chromatographymass spectrometry Anal
Biochem 331 283ndash295Hertzberg M Aspeborg H Schrader J et al (2001) A transcriptional
roadmap to wood formation Proc Natl Acad Sci USA 98 14732ndash14737Jang JC Fujioka S Tasaka M Seto H Takatsuto S Ishii A Aida M
Yoshida S and Sheen J (2000) A critical role of sterols in embryonic
patterning and meristem programming revealed by the fackel mutants of
Arabidopsis thaliana Genes Dev 14 1485ndash1497Jenks MA Rashotte AM Tuttle HA and Feldmann KA (1996) Mutants
in Arabidopsis thaliana altered in epicuticular wax and leaf morphology
Plant Physiol 110 377ndash385Jonsson P Johansson AI Gullberg J Trygg J A J Grung B Markl-
und S Sjostrom M Antti H and Moritz T (2005) High-throughput
data analysis for detecting and identifying differences between samples
in GCMS-based metabolomic analyses Anal Chem 77 5635ndash5642Jungwirth H and Kuchler K (2006) Yeast ABC transporters ndash a tale of sex
stress drugs and aging FEBS Lett 580 1131ndash1138Kaneda M Schuetz M Lin B Chanis C Hamberger B Western T
Ehlting J and Samuels A (2011) ABC transporters coordinately
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ABCG proteins and vascular development 823
expressed during lignification of Arabidopsis stems include a set of AB-
CBs associated with auxin transport J Exp Bot 62 2063
Karimi M Inze D and Depicker A (2002) GATEWAYTMvectors for Agrobac-
terium-mediated plant transformation Trends Plant Sci 7 193ndash195Kneurooller A and Murphy A (2011) ABC transporters and their function at the
plasma membrane In The Plant Plasma Membrane Plant Cell Mono-
graphs vol 19 (Murphy AS Peer W and Schulz B eds) New York
NY Springer pp 353ndash377Kopischke M Westphal L Schneeberger K Clark R Ossowski S
Wewer V Fuchs R Landtag J Hause G and Deuroormann P (2013)
Impaired sterol ester synthesis alters the response of Arabidopsis thali-
ana to Phytophthora infestans Plant J 73 456ndash468Kuromori T Miyaji T Yabuuchi H Shimizu H Sugimoto E Kamiya
A Moriyama Y and Shinozaki K (2010) ABC transporter AtABCG25 is
involved in abscisic acid transport and responses Proc Natl Acad Sci
USA 107 2361ndash2366Kuromori T Itoh T Sugimoto E and Shinozaki K (2011a) Arabidopsis
mutant of AtABCG26 an ABC transporter gene is defective in pollen
maturation J Plant Physiol 168 2001ndash2005Kuromori T Sugimoto E and Shinozaki K (2011b) Arabidopsis mutants
of AtABCG22 an ABC transporter gene increase transpiration and
drought susceptibility Plant J 67 885ndash894Le Hir R Beneteau J Bellini C Vilaine F and Dinant S (2008) Gene
expression profiling keys for investigating phloem functions Trends
Plant Sci 13 273ndash280Lehrer AT Dugassa-Gobena D Vidal S and Seifert K (2000) Transport
of resistance-inducing sterols in phloem sap of barley Z Naturforsch C
55 948ndash952Li Y Beisson F Pollard M and Ohlrogge J (2006) Oil content of Arabid-
opsis seeds the influence of seed anatomy light and plant-to-plant vari-
ation Phytochemistry 67 904ndash915Luo B Xue XndashY Hu WndashL Wang LndashJ and Chen XndashY (2007) An ABC
transporter gene of Arabidopsis thaliana AtWBC11 is involved in cuticle
development and prevention of organ fusion Plant Cell Physiol 48
1790ndash1802McFarlane HE Shin JJH Bird DA and Samuels AL (2010)
Arabidopsis ABCG transporters which are required for export of diverse
cuticular lipids dimerize in different combinations Plant Cell 22 3066ndash3075
Meskiene I Baudouin E Schweighofer A Liwosz A Jonak C Rodri-
guez PL Jelinek H and Hirt H (2003) Stress-induced protein phospha-
tase 2C is a negative regulator of a mitogen-activated protein kinase
J Biol Chem 278 18945ndash18952Murashige T and Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures Physiol Plant 15 473ndash497Panikashvili D Savaldi-Goldstein S Mandel T Yifhar T Franke RB
Hofer R Schreiber L Chory J and Aharoni A (2007) The Arabidopsis
DESPERADOAtWBC11 transporter is required for cutin and wax secre-
tion Plant Physiol 145 1345ndash1360Panikashvili D Shi JX Bocobza S Franke RB Schreiber L and Aha-
roni A (2010) The Arabidopsis DSOABCG11 transporter affects cutin
metabolism in reproductive organs and suberin in roots Mol Plant 3
563ndash575Panikashvili D Shi JX Schreiber L and Aharoni A (2011) The Arabid-
opsis ABCG13 transporter is required for flower cuticle secretion and pat-
terning of the petal epidermis New Phytol 190 113ndash124Pighin JA Zheng H Balakshin LJ Goodman IP Western TL Jetter
R Kunst L and Samuels AL (2004) Plant cuticular lipid export requires
an ABC transporter Science 306 702ndash704Pullen M Clark N Zarinkamar F Topping J and Lindsey K (2010)
Analysis of vascular development in the hydra sterol biosynthetic
mutants of Arabidopsis PLoS ONE 5 e12227
Quilichini TD Friedmann MC Samuels AL and Douglas CJ (2010)
ATP-binding cassette transporter G26 is required for male fertility and
pollen exine formation in Arabidopsis Plant Physiol 154 678ndash690R Development Core Team (2010) R A Language and Environment for
Statistical Computing Vienna Austria R Foundation for Statistical
Computing
Roudier F Gissot L Beaudoin F Haslam R Michaelson L Marion J
Molino D Lima A Bach L and Morin H (2010) Very-long-chain fatty
acids are involved in polar auxin transport and developmental patterning
in Arabidopsis Plant Cell 22 364ndash375Rozen S and Skaletsky HJ (2000) Primer3 on the WWW for general users
and for biologist programmers Methods Mol Biol 132 365ndash386Schad M Lipton MS Giavalisco P Smith RD and Kehr J (2005) Eval-
uation of two-dimensional electrophoresis and liquid chromatography
tandem mass spectrometry for tissue-specific protein profiling of
laser-microdissected plant samples Electrophoresis 26 2729ndash2738Schauer N Steinhauser D Strelkov S et al (2005) GCndashMS libraries for
the rapid identification of metabolites in complex biological samples
FEBS Lett 579 1332ndash1337Schrader J Nilsson J Mellerowicz EJ Berglund A Nilsson P Hertz-
berg M and Sandberg G (2004) A high-resolution transcript profile
across the wood forming meristem of poplar identifies potential regula-
tors of cambial stem cell identity Plant Cell 16 2278ndash2292Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desi-
mone M Frommer WB Fleurougge UndashI and Kunze R (2003) ARAMEM-
NON a novel database for Arabidopsis integral membrane proteins
Plant Physiol 131 16ndash26Silvestro D Andersen TG Schaller H and Jensen PE (2013) Plant ste-
rol metabolism D7ndashsterol-C5ndashdesaturase (STE1DWARF7) D57ndashste-rol-D7ndashreductase (DWARF5) and D24ndashsterol-D24ndashreductase (DIMINUTO
DWARF1) show multiple subcellular localizations in Arabidopsis thaliana
(Heynh) L PLoS ONE 8 e56429
Sorin C Bussell JD Camus I Ljung K Kowalczyk M Geiss G McKh-
ann H Garcion C Vaucheret H and Sandberg G (2005) Auxin and
light control of adventitious rooting in Arabidopsis require ARGONA-
UTE1 Plant Cell 17 1343ndash1359Tanaka T Tanaka H Machida C Watanabe M and Machida Y (2004) A
new method for rapid visualization of defects in leaf cuticle reveals five
intrinsic patterns of surface defects in Arabidopsis Plant J 37 139ndash146Turgeon R and Wolf S (2009) Phloem transport cellular pathways and
molecular trafficking Annu Rev Plant Biol 60 207ndash221Ukitsu H Kuromori T Toyooka K et al (2007) Cytological and biochemi-
cal analysis of COF1 an Arabidopsis mutant of an ABC transporter gene
Plant Cell Physiol 48 1524ndash1533Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De
Paepe A and Speleman F (2002) Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biol 3 research00340031-00340011Verrier PJ Bird D Burla B et al (2008) Plant ABC proteins ndash a unified
nomenclature andupdated inventory Trends Plant Sci 13 151ndash159Vilaine F Palauqui JC Amselem J Kusiak C Lemoine R and Dinant
S (2003) Towards deciphering phloem a transcriptome analysis of the
phloem of Apium graveolens Plant J 36 67ndash81Zhai Z Sooksa-nguan T and Vatamaniuk OK (2009) Establishing RNA
interference as a reverse-genetic approach for gene functional analysis
in protoplasts Plant Physiol 149 642ndash652Zhang Q Blaylock LA and Harrison MJ (2010) Two Medicago truncatula
half-ABC transporters are essential for arbuscule development in
arbuscular mycorrhizal symbiosis Plant Cell 22 1483
Zhao C Craig JC Petzold HE Dickerman AW and Beers EP (2005)
The xylem and phloem transcriptomes from secondary tissues of the
Arabidopsis root-hypocotyl Plant Physiol 138 803ndash818
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
824 Rozenn Le Hir et al
Arabidopsis protoplasts were transfected with open read-
ing frames encoding individual cndashMyc- or HA-tagged
versions of the three ABCGs and the identity of the epi-
tope-tagged proteins was verified by SDSndashPAGE and
Western blotting using anti-HA and anti-c-Myc antibodies
In all cases only one band of approximately 75 kDa cor-
responding to the predicted molecular mass of the pro-
teins in the Plant Membrane Protein Database (Schwacke
et al 2003) was obtained (Figure 5kndashp lane 1 and Figure
S2andashf lane 1) Subsequently protoplasts were co-trans-
fected with cndashMyc- and HA-tagged isoforms immunopre-
cipitated with antisera against one of the epitope tags
and probed with antisera directed against the alternative
epitope tag in Western blots of SDSndashPAGE gels in order
to determine whether homodimers or heterodimers were
formed (Figure 5kndashp) No ABCG14 homodimers or
ABCG9ndashABCG14 heterodimers were detected (Figure 5m
lane 4 and Figure 5o lane 4) In contrast ABCG11 formed
an apparent homodimer as described previously (McFar-
lane et al 2010) ABCG9 also formed an apparent homod-
imer ABCG14 formed an apparent heterodimer with
ABCG11 and ABCG9 formed an apparent heterodimer
with ABCG11 (Figure 5klnp lane 4) These interactions
were confirmed in a plant-based in vivo system using the
bimolecular fluorescence complementation assay (Figure
S3andashl) We therefore concluded that ABCG14 forms a strict
heterodimer with ABCG11 whereas ABCG11 and ABCG9
can both hetero- and homodimerize
Cuticular and epicuticular lipid precursor content as well as
cuticle integrity are not altered in abcg9ndash2 and abcg14ndash1
mutants
ABCG11 is known to be required for cuticle integrity (Bird
et al 2007 Luo et al 2007 Panikashvili et al 2007 Ukitsu
et al 2007) and because ABCG11 interacts physically with
ABCG9 and ABCG14 we looked for potential defects in
very long chain fatty acids (VLCFAs) which are among the
precursors of cuticular and epicuticular lipids (Roudier
et al 2010) as well as for possible cuticle defects in
abcg9ndash2 and abcg14ndash1 mutants
Quantification of fatty acids (from 160 to 180) as well as
VLCFAs (from 200 to 240) in apical parts of abcg9ndash2 and
abcg14ndash1 seedlings revealed no differences compared to
wild-type levels (Figure 6ab) It has previously been
(a) (b) (c) (d)
(e) (f) (g) (h)
(i)
(j)
(i)
(k) (l) (m)
(n)
Figure 3 Phenotype of abcg mutants grown in
soil
(andashm) Twenty-day-old plants grown in soil
Scale bar = 1 cm
(n) Green areas of 4-week-old plants Values
represent means SE from 20 individual
plants The experiment was repeated twice with
similar results Asterisks indicate statistically
significant differences between the wild-type
and mutants (P lt 005 Studentrsquos t test n = 20)
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 815
shown that when the cuticle is altered toluidine blue (TB)
permeates the epidermal surface (Tanaka et al 2004)
Therefore we used TB to check for cuticle defects in the
mutants After treatment with TB no staining was
observed in the wild-type abcg11+ or the abcg9ndash2 and
abcg14ndash1 mutants (Figure 6cef) whereas abcg11ndash7
homozygous seedlings showed patches of blue staining
confirming the cuticle alteration previously described
(Figure 6d) (Ukitsu et al 2007) These observations sug-
gest that unlike mutations in ABCG11 mutations in
ABGC9 or ABCG14 do not affect cuticle formation
abcg9ndash2 and abcg14ndash1 mutants are defective in sterol
composition
To obtain an insight into the potential substrates trans-
ported by these ABCG transporters we analyzed the shoot
and root metabolome of single mutants Orthogonal partial
least-squares discriminant analysis was applied to the
GCMS data in order to classify the profiles of the geno-
types In both shoots (Figure 7andashc) and roots (Figure S4andash
c) homozygous abcg11ndash7 abcg9ndash1 abcg9ndash2 and abcg14ndash1
mutants exhibited very different metabolomic profiles from
the wild-type Interestingly the heterozygous abcg11ndash7+
and abcg9ndash2+ plants had metabolomic profiles intermedi-
ate between the wild-type and their respective homozy-
gous mutant plants (Figure 7ab and Figure S4ab) These
results suggest that a mutation in one copy of ABCG11 or
ABCG9 is sufficient to significantly affect the physiology of
the plant even if it does not dramatically affect visible
aspects of its phenotype
The metabolites showing the most significant differ-
ences were identified by comparing their retention indices
and mass spectra with entries in publicly available reten-
tion libraries (Schauer et al 2005) For all the genotypes
analyzed the metabolites identified as being significantly
different from the wild-type belonged to three main clas-
ses amino acids carbohydrates and lipidssterols The
levels of these compounds were almost all reduced in
abcg9ndash2 and abcg14ndash1 but in excess in abcg11ndash7 (Table 2
and Appendix S1)
Because our study focused on vascular development in
aerial tissues only data obtained for the apical parts are
described in detail here (Table 2) Root data are presented
in Appendix S1 Whereas many neutral amino acids were
significantly depleted in abcg9ndash2 and abcg14ndash1 aerial
Figure 4 Cotyledon venation pattern in abcg mutant lines
Venation complexity and continuity of each examined class of mutant and
wild-type seedlings Values in brackets indicate the percentage contribution
of each class Asterisks indicate statistically significant differences between
the wild-type and mutants (P lt 005 Pearsonrsquos v2 test with Monte Carlo
permutations N = 10 000)
Table 1 Distribution of vascular bundles in the floral stem
Genotype Vascular bundle number Floral stem diameter (mm) Vascular bundle density Total
Col-0 930 105 157 020 592 049 10abcg11-7+ 914 134 140 080 652 042 12abcg11-7 902 115 051 004 176 182 8abcg14-1 828 049 145 018 632 0049 1135SHA-ABCG14 abcg14-1 940 089 170 016 554 058 12abcg9-1 957 053 160 006 586 031 7abcg9-2 875 103 162 010 553 046 835SHA-ABCG9 abcg9-2 933 051 174 010 606 026 8abcg9-1 abcg14-1 871 048 154 003 565 030 8abcg9-2abcg14-1 828 049 124 015 674 084 8abcg9-2abcg14-1abcg11-7
760 054 063 005 1203 169 9
Statistically significant differences between the wild-type and mutants (P lt 005 Studentrsquos t test)
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
816 Rozenn Le Hir et al
tissues (nine and eight out of 20 neutral amino acids
respectively) only two amino acids (glutamine and gluta-
mate) associated with Arabidopsis phloem cells (Schad
et al 2005) were different in abcg11ndash7 (Table 2) Xylose
and the non-reducing sugars trehalose and raffinose were
also decreased in abcg14ndash1 (Table 2) whereas glucose
fructose galactose maltose xylose were significantly dif-
ferent in abg9ndash2 and abcg11ndash7 (reduced and increased
respectively compared to wild-type) (Table 2)
Levels of sterols (campesterol 24ndashmethylene cholesterol
and sitosterol) saturated and unsaturated fatty acids (lino-
leic acid lauric acid palmitic acid and triacontanoic acid)
glycerolipids (monoacylglycerol) and lipid metabolism
intermediates (glycerol glycerol-3ndashphosphate and inositol-
1ndashphosphate) were also significantly different from wild-
type in abcg mutants (Table 2) The abcg9ndash2 mutant
showed 30 lower levels of 24ndashmethylene cholesterol
compared to the wild-type (Appendix S1) In the abcg14ndash1
mutant 50 and 35 decreases of 24ndashmethylene cholesterol
and sitosterol levels were measured respectively (Appen-
dix S1) Finally campesterol showed a 50 increase in the
abcg11ndash7 mutant (Appendix S1) To extend this analysis
measurements of conjugated sterols [sterol ester (SE)
sterol glucoside and acylated sterol glucoside] were also
performed When seedlings were grown in vitro no differ-
ence in the sterol glucoside and acylated sterol glucoside
contents was detected between the wild-type and the vari-
ous mutant lines (Figure S4d) However there was a ten-
dency towards a reduced SE content in the double mutant
abcg9ndash2 abcg14ndash1 compared to the wild-type (Figure S4d)
This observation prompted us to examine variations in the
SE content in older plants (4-week-old plants grown in soil
in the greenhouse) At this developmental stage no differ-
ence in the free sterol (FS) content was observed between
the wild-type and the various mutant lines (Figure 7d) but
the fraction comprising FS and SE was significantly
(a) (b) (c) (d)
(e) (f) (g) (h)
(i) (j)
(k) (l) (m)
(n) (o) (p)
Figure 5 Subcellular localization and physical interactions of ABCG9 ABCG11 and ABCG14
(andashh) ABCG9 and ABCG14 co-localize to the plasma membrane when transiently expressed in Arabidopsis mesophyll protoplasts of the GFPndashLTi6b marker line
(Cutler et al 2000)
(a e) Cyan fluorescence of GFPndashLTi6b(b f) Purple fluorescence from mRFPndashABCG9 and mRFPndashABCG14 respectively
(c g) Yellow auto-fluorescence of the chloroplasts
(d h) Merged images of the three channels Scale bar = 10 lm
(i j) Confocal images of immunofluorescence staining with anti-HA antibodies in root cells of abcg9ndash2 and abcg14ndash1 mutants complemented with HA-tagged
ABCG9 (i) and HA-tagged ABCG14 (j) Scale bars = 10 lm
(kndashp) Proteins were extracted from protoplasts co-transfected with HA- or cndashMyc-tagged versions of ABCGs (lanes 1 and 4) or transfected with a single plasmid
containing the HA-tagged version (lane 2) or the c-Myc-tagged version (lane 3) In lanes 2ndash4 proteins were immunoprecipitated with the anti-c-Myc antibody
and subjected to anti-HA protein gel-blot analysis to reveal the other partner of the dimer Transient co-expression of (k) HAndashABCG9cndashMycndashABCG9 (l) HAndashABCG11cndashMycndashABCG11 (m) HAndashABCG14cndashMycndashABCG14 (n) HAndashABCG14cndashMycndashABCG11 (o) HAndashABCG14cndashMycndashABCG9 and (p) HAndashABCG11cndashMycndashABCG9
in wild-type protoplasts
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 817
reduced probably accounting for the difference in the SE
content between the wild-type and the abcg9ndash2 abcg14ndash1
double mutant (Figure 7e)
(a)
(b)
(c) (d) (e)
(f) (g)
Figure 6 Long chain and very long chain fatty acid content and cuticle
integrity in abcg9ndash2 and abcg14ndash1 mutants
(a b) Fatty acid (a) and VLCFA content (b) in abcg9ndash2 and abcg14ndash1 mutants
in apical parts of 10-day-old seedlings (three biological replicates each of
approximately 5 mg dry weight were used)
(cndashf) Toluidine blue staining patterns of abcg mutants in 7-day-old seed-
lings Scale bars = 3 cm
(a)
(b)
(c)
(d) (e)
Figure 7 Metabolome analysis and measurements of sterol content in abcg
mutants
(andashc) Results of orthogonal partial least-squares discriminant analysis of
global metabolite contents of shoots of 7-day-old seedlings from wild-type
(black squares) abcg11ndash7+ (gray triangles) abcg11ndash7 (white stars) abcg9ndash1(white hexagons) abcg9ndash2+ (gray hexagons) abcg9ndash2 (white hexagons)
and abcg14ndash1 (black circles) seedlings (five biological replicates of 10 mg
each were used for the analysis)
(d e) Free sterol (FS) and sterol ester (SE) content of wild-type (white bars)
abcg9ndash2 (light gray bars) abcg14ndash1 (dark gray bars) and abcg9ndash2 abcg14ndash1(black bars) Error bars indicate standard deviations obtained from three
independent biological replicates The asterisk indicates a statistically signif-
icant difference between the wild-type and the mutant (P lt 005 Studentrsquos
t test)
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
818 Rozenn Le Hir et al
DISCUSSION
ABCG9 and ABCG14 are two plasma membrane proteins
that interact physically in multiple homo-heterodimer
combinations
In this study we showed that like the previously described
ABCG11 (Pighin et al 2004 Bird et al 2007 Luo et al
2007 Panikashvili et al 2007 2010 Ukitsu et al 2007)
ABCG9 and ABCG14 localize to the plasma membrane
Based on co-immunoprecipitation of tagged proteins and
the bimolecular fluorescence complementation assay we
demonstrated that ABCG9 interacts with ABCG11 to form a
heterodimer and like ABCG11 (McFarlane et al 2010) can
homodimerize In contrast ABCG14 interacts only with
ABCG11 forming an obligate heterodimer This extends
the number of potential partners of ABCG11 to three as it
was previously shown to dimerize with the ABCG12CER5
protein (McFarlane et al 2010) Similarly ABCG9 under-
goes flexible dimerization to form either homo- or hetero-
dimers suggesting that ABCGs may be promiscuous
proteins with different functions depending on their inter-
acting partner andor expression profile
Mutations in ABCG genes are semi-dominant
In the last few years several Arabidopsis mutants with
altered expression of various ABCG genes such as ABCG11
(Bird et al 2007 Luo et al 2007 Panikashvili et al 2007
Ukitsu et al 2007) ABCG12CER5 (Pighin et al 2004)
ABCG13 (Panikashvili et al 2011) ABCG22 (Kuromori
et al 2011a) ABCG25 (Kuromori et al 2010) and ABCG26
(Choi et al 2010 Quilichini et al 2010 Dou et al 2011
Kuromori et al 2011b) have been characterized but even
though these half-size transporters require dimerization to
become functional all the mutations were described as
recessive Here we demonstrated that mutations in these
genes have semi-dominant effects Indeed the metabolome
analyses showed that abcg9ndash2+ and abcg11ndash7+ heterozy-
gous seedlings have a profile intermediate between that of
wild-type and homozygous seedlings The possibility of
dominant-negative effects was excluded because no
Table 2 Shoot metabolites of 10-day-old abcg9ndash2 abcg11ndash7 and abcg14ndash1 seedlings whose levels were significantly different from wild-type
Group
Metabolites that differed between mutant and wild-type
abcg9ndash2Colndash0 abcg11ndash7Colndash0 abcg14ndash1Colndash0
Amino acids Glycine Glutamate AlanineAlanine Glutamine ValineValine ThreonineThreonine GABASerine Prolinebndashalanine IsoleucineIsoleucine SerineGABA bndashalanineProline
Carbohydrates Glucose Glucose ErythritolFructose Fructose TrehaloseXylose Galactose XyloseMaltose RaffinoseSucrose
Lipids and intermediates 24ndashmethylene-cholesterol Campesterol Linoleic acidMonoacylglycerol Glycerol-3ndashphosphate Triacontanoic acidPalmitic acid Inositol-1ndashphosphate GlycerolGlycerol-3ndashphosphate Lauric acidInositol-1ndashphosphate 24ndashmethylene-cholesterol
SitosterolGlycerol-3ndashphosphate
TCA cycle and others Phosphoric acid Phosphate compound Glyceric acid24ndashdihydroxybutanoic acid Dehydroascorbic acid Fumaric acidGlyceric acid Malic acid Threonic acid-14ndashlactoneFumaric acid 2ndashoxoglutaric acid Threonic acidThreonic acid bndashhydroxy-bndashmethylglutaric acid Shikimic acidThreonic acid-14ndashlactone Gluconic acid lactone-like Succinic acidShikimic acid Phytol2ndashoxoglutaric acid Ethanolamine
Nicotinic acid
For abcg9ndash2 and abcg14ndash1 the levels of the compounds were reduced whereas they were increased in abcg11ndash7
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 819
truncated transcript was found for either abcg9ndash2 or
abcg11ndash7 mutants Hence abcg9ndash2 and abcg11ndash7 are true
null alleles These observations are consistent with the
requirement for homo- or heterodimerization of the ABCG
proteins to form functional transporters In the heterozy-
gous plants ABCG11 transcript levels were 50 lower than
in wild-type counterparts suggesting that ABCG11 protein
and thus corresponding homo- and heterodimers were
likely to be less abundant which may also explain the meta-
bolic perturbations and consequent phenotypic modifica-
tions These findings also suggest that wild-type plants are
not saturated by ABCG11 proteins as a 50 reduction in
protein content appears to be sufficient to alter the physiol-
ogy of the heterozygous mutant plants This hypothesis is
strengthened by the observation that mutants carrying the
weak abcg11ndash6 allele in which there was only a 25 reduc-
tion in ABCG11 transcript levels showed a similar pheno-
type to the heterozygous abcg11ndash7+ and abcg11ndash8+
plants Similarly the abcg9ndash1 weak allele mutant had a very
similar phenotype to the abcg9ndash2+ heterozygous seedlings
suggesting that considering the mode of action of these
proteins the semi-dominant effects that we observed are
likely to be a general feature among the ABCG genes
ABCG9 ABCG11 and ABCG14 transporters are involved in
vascular system development in Arabidopsis
We showed that the ABCG11 ABCG9 and ABCG14 promot-
ers drove expression in the vascular system of Arabidopsis
cotyledons and rosette leaves and more precisely in the
phloem of flower stems In addition mutations in ABCG9
ABCG11 and ABCG14 genes altered vascular development
in cotyledons and the floral stem Similar vascular localiza-
tion of ABCG11 has already been described by Panikashvili
et al (20072010) who showed that in addition to the epi-
dermis ABCG11 is expressed in the vasculature as early as
during embryo development and then throughout plant
development They showed that the vascular pattern in
leaves of the homozygote mutant seedlings was less com-
plex than in the wild-type and discontinuous (Panikashvili
et al 2007) The present article describes a mutant that
has both cuticle and vascular system patterning defects
although it has been previously shown that mutants
altered in cuticle formation have dramatically reduced
growth and altered leaf morphology (Jenks et al 1996)
which may account for vascular defects Analysis of the
abcg11+ heterozygous seedlings that have vascular
defects but apparently no cuticle alteration suggested that
these phenotypes are probably uncoupled Because
ABCG11 dimerizes with several members of its sub-family
it seems reasonable to postulate that it is involved in the
transport of different compounds depending on its partner
Here we provide evidence that ABCG11 in addition to
forming dimers with ABCG12 (McFarlane et al 2010) in
epidermal cells is likely to dimerize with ABCG9 and
ABCG14 in phloem cells suggesting functional compart-
mentalization of these proteins and a possible role in vas-
cular system patterning in Arabidopsis
Metabolite profiling suggests that ABCG9 ABCG11 and
ABCG14 are involved in sterollipid homeostasis
We investigated in more depth the metabolic changes
induced by mutations in ABCG9 ABCG11 and ABCG14
We found that levels of metabolites of several classes
including lipids sugars and amino acids were altered in
both the heterozygotes and homozygotes and that the
profiles in the root and apical parts differed suggesting
that ABCGs may play different roles in the roots and shoot
Interestingly the modifications of amino acid and sugar
contents observed in the metabolic profiles of the abcg
mutants suggest an impaired carbonnitrogen balance that
may be directly or indirectly linked to the vascular defects
leading to possible problems with phloem loading or
unloading Saturated and unsaturated fatty acids and sterol
contents were also altered in the mutants Patterning
defects in the vascular system are often observed in
mutants altered in hormone homeostasis such as for
auxin or brassinosteroids (Kaneda et al 2011) and direct
or indirect modifications of sterol homeostasis are thought
to be one of the primary causes of these downstream
effects (Pullen et al 2010) The detected defects in sterol
homeostasis may explain the abnormal vascular develop-
ment observed in abcg9 abcg11 and abcg14 mutants
Accordingly several sterol-deficient mutants such as fac-
kelhydra (Jang et al 2000) and cpv1 (Carland et al 2002)
have also been shown to exhibit severe defects in vascular
development It has been suggested that the bushy habit
and low fertility of cpv1smt2 mutants may be due to an
increased campesterolsitosterol ratio rather than sterol
deficiency per se (Clouse 2002) This ratio is controlled by
the activity of SMT2 (STEROL METHYLTRANSFERASE2)
Interestingly strong down-regulation of SMT2 expression
was observed in all the mutant backgrounds (Figure S5)
and the metabolic data indicated a deficit in 24ndashmethylene
cholesterol (precursor of both campesterol and sitosterol)
andor sitosterol in abcg9 and abcg14 mutants respec-
tively In addition the significant decrease in sterol ester
content in the double mutant abcg9ndash2 abcg14ndash1 correlated
with down-regulation of expression of the PHOSPHOLIPID
STEROL ACYLTRANSFERASE1 (PSAT1) gene encoding the
enzyme that is mainly responsible for SE synthesis (Bou-
vier-Nave et al 2010) and is proposed to be a control step
in sterol homeostasis (Kopischke et al 2013) Altogether
these results suggest that the vascular phenotype
observed in abcg mutants may be linked to modification of
sterol homeostasis
Therefore we postulate that the various dimer combina-
tions between ABCG9 ABCG11 and ABCG14 may
participate in the transport of squalene-derived metabolites
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
820 Rozenn Le Hir et al
such as sterol andor sterol conjugates via the phloem sap
The presence of sterols in phloem sap has been observed in
various species such as barley (Hordeum vulgare Lehrer
et al 2000) and fava bean (Vicia faba Bouvaine et al
2012) and indirect proof of the presence of sterols in
phloem sap has been obtained by analysis of the phytos-
terol content in exclusively phloem-feeding aphids that are
unable to synthetize them directly (Behmer et al 2011) In
addition the evidence for long-distance transport of sterol
precursors has been supported by observations that sterol
esters accumulate in organs distal to the site of application
of exogenous precursors (Bouvier-Nave et al 2010) As
sterols have been proposed to act as signal molecules in
the control of developmental processes such as vascular
patterning (Carland et al 2002 Pullen et al 2010) modifi-
cations of sterol homeostasis may affect development of
the vascular system in the abcgmutants described here
In conclusion we propose that ABCG9 ABCG11 and
ABCG14 are important for lipidsterol homeostasis Thus
these three proteins are likely to be involved in the control
of various aspects of plant development andor physiol-
ogy including patterning of the vascular system
EXPERIMENTAL PROCEDURES
Plant material and growth conditions
Insertion lines in ABCG9 (SALK_047133 and GABI_353B01)ABCG11 (SALK_096377 and GABI_590C03) and ABCG14 (SALK_036952) genes were obtained from the European ArabidopsisStock Centre (httparabidopsisinfo) Heterozygoushomozygousplants were screened using gene-specific primers in combinationwith the specific primer for the left border of the TndashDNA insertion(Table S1)
Plants were grown in soil or in vitro as described previously(Sorin et al 2005) Arabidopsis Colndash0 cell cultures were main-tained by weekly sub-culturing in MS medium (Murashige andSkoog 1962) supplemented with 240 lg l1 24ndashdichlorophenoxy-acetic acid and 14 lg l1 kinetin and were grown under 12 h light12 h dark cycles at 25 lE m2 sec1 and with 23degC day and nighttemperatures
Morphological characterization and GUS staining
To study the venation pattern cotyledons of 7-day-old seedlingsand rosette leaves of 4-week-old plants were cleared in a buffercontaining chloral hydrate glycerol and water (831 wvv) afterwhich 200 cotyledons per genotype were analyzed and the per-centage showing particular levels of complexity or discontinuity invascularity was estimated as described by Cnops et al (2006)
Flower stem segments (1ndash2 cm long) were removed from ten3ndash4-week-old plants grown in soil embedded in 8 agarose andsectioned using a VT100S vibratome (Leica httpwwwleicacom)The cross-sections were then stained with safraninalcian blue for1ndash2 min (Sigma httpwwwsigmaaldrichcom) rinsed in waterand mounted in 50 glycerol
Histochemical GUS assays were performed as described previ-ously (Sorin et al 2005) All observations were performed usingan Axioplan light microscope equipped with an Axiocam cameraand Axiovision software (Zeiss httpwwwzeisscom)
To observe potential cuticle defects in the various mutant linesa TB test was performed by immersing in vitro-grown plantlets ina 005 wv TB solution for 2 min as described by Tanaka et al(2004)
Statistical analyses
Statistical analyses of differences between-genotype means andvariance for the studied morphological traits were performedusing Studentrsquos t test (GraphPad Prism version 50 httpwwwgraphpadcom) In the venation pattern experiment thesignificance of differences between wild-type and mutants wasassessed using R (R Development Core Team 2010) and Pearsonrsquosv2 test with Monte Carlo permutations (N = 10 000)
Plasmid constructs
The native ABCG promoters and open reading frame of ABCG11were amplified from genomic DNA or cDNA extracted from 7-day-old Arabidopsis seedlings using Phusion high-fidelity DNApolymerase (Finnzyme wwwthermoscientificbiocomfinnzymes)according to the manufacturerrsquos instructions with gene-specificprimers (Table S1) The PCR products were cloned into pENTRDndashTOPO (Invitrogen httpwwwinvitrogencom) and recombinedinto pKGWFS7 (Karimi et al 2002) to create ABCGproGUS Allrecombinations were performed using the Gateway LR Clonase
enzyme mix (Invitrogen) At each cloning step the sequenceswere confirmed by sequencing and all the constructs weretransferred into Agrobacterium strain GV3101pMP90 (C58C1)Wild-type or abcg11ndash7+ heterozygous plants were transformed byfloral dipping (Clough and Bent 1998) For ABCGGUS constructsthe expression pattern was checked in the T2 progeny of 10ndash15independent transgenic lines and one representative homozygousline was used for further characterization
Tagged protein constructs and protoplast transformation
Epitope-tagged versions of ABCG9 ABCG11 and ABCG14 proteinswere produced in pRT104ndash3xHA and pRT104ndash3xMyc plasmids(Feurouleuroop et al 2005) and pSAT6-mRFP (Citovsky et al 2006) Allthese plasmids have a 35S promoter sequence upstream of themulti-cloning site The open reading frames of ABCG11 andABCG14 and the genomic clone of ABCG9 were amplified fromcDNA or genomic DNA extracted from 7-day-old Arabidopsisseedlings using Phusion high-fidelity DNA polymerase (Finnzyme)according to the manufacturerrsquos instructions with gene-specificprimers carrying EcoRI or SalI restriction sites to facilitate subse-quent cloning (Table S1) The products obtained after PCR weredigested by EcoRI and SalI prior to ligation into appropriate plas-mids that had previously been cut open with the same restrictionenzymes Constructs were verified by sequencing Protoplastsfrom Arabidopsis cell cultures or 14-day-old Arabidopsis seedlingswere prepared and transformed as previously described (Meski-ene et al 2003 Zhai et al 2009) For co-immunoprecipitation andsubcellular localization assays 100 000 and 50 000 protoplastcells respectively were transfected with 15 and 10 ll of plasmidsrespectively (containing between 5 and 75 lg of each construct)and the resulting transformed protoplasts were processed afterincubation in the dark at room temperature for 16ndash18 and 24 hrespectively
Whole-mount immunolocalization
The HA-tagged versions of ABCG9 and ABCG14 were detected in7-day-old in vitro-grown Arabidopsis seedlings as described inMethods S1 Anti-HA antibody 12CA5 (Roche httpwwwroche
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 821
com) (1100 dilution) combined with Alexa Fluor 488-conjugatedsecondary antibody (1100 dilution) were used
Confocal laser-scanning microscopy
Images were collected using an SP2 confocal microscope (Leica)RFP and Alexa Fluor 488 were detected using laser lines at 561and 488 nm respectively according to the manufacturerrsquos set-tings RFP fluorescence emission was detected at 550ndash600 nmand Alexa Fluor 488 and GFP fluorescence emission were detectedat 495ndash545 nm For subcellular localization in protoplasts imageswere coded in cyan (GFP) magenta (RFP) and yellow (auto-fluo-rescence) resulting in purple coloration for co-localization inmerged images For the immunolocalization experiment imageswere coded in green (GFP) A water-corrected 209 objective (HCPlan-Apochromat 20907 ImmKor Leica) was used Images werecropped using Adobe Photoshop CS2 and assembled using AdobeIllustrator CS2 software (Abode httpwwwabodecom) Eachimage shown represents a single focal plan
Co-immunoprecipitation assays
Transformed protoplast proteins were extracted using 50 ll lysisbuffer containing 25 mM TrisHCl pH 78 10 mM MgCl2 75 mM
NaCl 5 mM EGTA 1 mM benzamidine 1 mM dithiothreitol 10glycerol 1 Triton Xndash100 and 19 protein inhibitor cocktail(Sigma httpsigmaaldrichcom) The transformed protoplast sus-pension was frozen in liquid nitrogen then thawed on ice andsubjected to centrifugation for 5 min at 150 g Two microliters ofanti-Myc (9E10 Covance httpwwwcovancecom) or anti-HA(16B12 Covance) antibodies were added to the extracts and themixture was incubated for 2 h at 4degC on a rotating wheel Immu-nocomplexes were captured by incubation with 10 ll Protein GndashSepharose beads for a further 2 h at 4degC on a rotating wheel afterwhich they were washed three times for 5 min each in 25 mM
NaPi 150 mM NaCl 5 glycerol and 02 Igepal CAndash630 buffer(Sigma) and then eluted using 40 ll SDS sample buffer at 37degCfor 30 min Co-immunoprecipitation of Myc- and HA-tagged pro-teins was revealed by SDSndashPAGE followed by Western blottingusing both anti-Myc (Invitrogen) and anti-HA (3F10 Roche) anti-bodies at 12000 dilution
RNA extraction and cDNA preparation
To quantify the relative amounts of the ABCG transcriptsexpressed in various organs three rosette leaves all the cau-line leaves the bottom 5 cm of the floral stem all closed flow-ers and all green siliques were harvested from each of threeindependent 4-week-old Arabidopsis plants of each genotypegrown in soil For the other experiments 10-day-old seedlingsgrown in vitro were harvested All the material was directly fro-zen in liquid nitrogen and stored at 80degC prior to RNA extrac-tion Samples were then disrupted using a MM 301 vibrationmill (Retsch GmbH httpwwwretschcom) at a frequency of25 Hz for 2 min with 3 mm tungsten carbide beads (Qiagenhttpwwwqiagencom) Total RNA was isolated from thismaterial using TRIzol (Life Technologies httplifetechnologiescom) RNA quantity and quality were measured using a Nano-Drop ND 1000 (Thermo Scientific httpwwwthermoscientificcom) All samples with 260280 nm and 260230 nm absorbanceratios lower than 18 were discarded and RNA was re-extractedDNase treatment was applied to 5 lg samples of total RNAusing a DNase I kit (Fermentas httpwwwfermentascom)Double-stranded cDNA was synthesized from 5 lg total RNAusing a SuperScript reverse transcriptase kit (Invitrogen) witholigo(dT)12 primer Finally RNase treatment was applied to the
samples using Escherichia coli RNAse H (Fermentas) The cDNAsamples obtained were diluted 30-fold (except for abcg9 linesamples which were diluted 10-fold) prior to use either toverify the absence of full-length transcripts in putative knockoutmutants or to quantify gene expression by quantitative PCR Allprimers used were designed using the Primer3 program (Rozenand Skaletsky 2000) and their sequences are listed inTable S1
QUANTITATIVE REAL-TIME PCR
Quantitative PCR amplifications were performed in 20 llmixtures containing 10 ll 59 SYBR Green I master mix
(Roche) 02 ll forward and reverse primer (30 lM each)
48 ll sterile water and 5 ll cDNA The BiondashRad CFX96
real-time PCR detection system (httpwwwbio-radcom)
was used for quantification of expression of the sterol
biosynthesis transcripts and the ABCG transcripts in the
various mutant backgrounds Finally the Roche LightCy-
cler 480 system (Roche) was used to quantify the rela-
tive abundance of ABCG transcripts in various organs
Four genes were considered as potential reference genes
[APT1 (At1g27450) TIP41 (At4g34270) EF1a (At5g60390)
and UBQ10 (At4g05320)] and their suitability was tested
using the geNorm algorithm (Vandesompele et al 2002)
to determine the most appropriate one for each experi-
ment A more detailed procedure following the Minimum
Information for Publication of Quantitative Real-Time PCR
Experiments guidelines (Bustin et al 2009) is described
in Methods S2 and the annealing temperature and
efficiencies of the primer pairs used are shown in
Table S2
VLCFA quantification
Approximately 5 mg dry weight of 10-day-old in vitro
Arabidopsis seedlings were analyzed for VLCFA quantifica-
tion as described by Li et al (2006)
Sterol extraction and GC-FID characterization
A pool of 10-day-old seedlings grown in vitro or rosettes
from 4-week-old plants grown in soil were lyophilized prior
to extraction Sterol extraction and isolation was per-
formed as described previously (Bouvier-Nave et al 2010)
Gas Chromatography-Flame Ionization Detector (GC-FID)
characterization was performed as described by Silvestro
et al (2013) To quantify the total amounts of free sterols
and sterol esters non-saponifiable lipids were extracted
using nndashhexane and sterols were derivatized as sterols ace-
tates Quantification was achieved using lupenyl-3 28ndashdi-
acetate as an internal standard
Metabolome analysis
To obtain metabolome profiles associated with wild-type
abcg11ndash7+ abcg11ndash7 abcg9ndash1 abcg9ndash2 and abcg14ndash1
genotypes 10-day-old seedlings representing each geno-
type grown in vitro as described above were harvested
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
822 Rozenn Le Hir et al
and divided into shoot and root samples In the case of
abcg9ndash2 the phenotype of the heterozygote abcg9ndash2+
was indistinguishable from the homozygote phenotype
and therefore heterozygous and homozygous seedlings
from a segregating population were pooled Hence in this
particular experiment abcg9ndash2+ refers to a mixed popula-
tion comprising two-thirds heterozygotes and one-third
homozygotes
Metabolites in five samples of shoot and root parts of
seedlings of each genotype (10 mg fresh weight per sam-
ple) were then extracted analyzed by GCMS and the
acquired data were processed as previously described
(Gullberg et al 2004 Jonsson et al 2005) All multivariate
statistical investigations (partial component analysis and
orthogonal partial least-squares discriminant analysis) for
between-genotype similarities and differences in general
metabolite profiles were performed using Simca software
(Umetrics httpwwwumetricscom)
ACKNOWLEDGEMENTS
We thank A Murphy (Plant Science and Landscape Archi-
tecture University of Maryland MD USA) for stimulating
discussions and critical reading of the manuscript I Carl-
son (Umea Plant Science Center Department of Forest
Genetics and Plant Physiology Swedish University of Agri-
cultural Sciences Umea Sweden) for technical assistance
and S Vernhettes for kindly donating seeds of the
transgenic line carrying the plasma membrane marker
GFPndashLTi6b We also thank the European Arabidopsis Stock
Centre and the GABI-Kat stock centre for providing seeds
This work was supported by allocations granted to CB by
the Swedish Research Council for Agriculture the Swedish
Foundation for Strategic Research the Swedish Research
Council for Research and Innovation for Sustainable
Growth the K amp A Wallenberg Foundation and the Carl
Trygger Foundation
SUPPORTING INFORMATION
Additional Supporting Information may be found in the onlineversion of this articleAppendix S1 Raw data quantification for compounds identified inthe metabolite profiling by GCMS
Figure S1 Relative expression of ABCG genes in various organsand mutant backgroundsFigure S2 Co-immunoprecipitation controlsFigure S3 ABCG9 and ABCG14 homo- andor heterodimerize withABCG11Figure S4 Complementary data on metabolome analysis and con-jugated sterol contentFigure S5 Relative expression of genes related to sterol homeo-stasisMethods S1 Method for whole-mount immunolocalizationMethods S2 Method for quantitative PCR
Table S1 List of primers used in this studyTable S2 Annealing temperature and efficiencies for the primerspairs used in this study
REFERENCES
Behmer ST Grebenok RJ and Douglas AE (2011) Plant sterols and host
plant suitability for a phloem-feeding insect Funct Ecol 25 484ndash491Bird D Beisson F Brigham A Shin J Greer S Jetter R Kunst L
Wu XW Yephremov A and Samuels L (2007) Characterization of Ara-
bidopsis ABCG11WBC11 an ATP binding cassette (ABC) transporter that
is required for cuticular lipid secretion Plant J 52 485ndash498Bouvaine S Behmer ST Lin GG Faure MndashL Grebenok RJ and
Douglas AE (2012) The physiology of sterol nutrition in the pea aphid
Acyrthosiphon pisum J Insect Physiol 58 1383ndash1389Bouvier-Nave P Berna A Noiriel A Compagnon V Carlsson AS
Banas A Stymne S and Schaller H (2010) Involvement of the phos-
pholipid sterol acyltransferase1 in plant sterol homeostasis and leaf
senescence Plant Physiol 152 107ndash119Bustin SA Benes V Garson JA Hellemans J Huggett J Kubista
M Mueller R Nolan T Pfaffl MW and Shipley GL (2009) The MIQE
guidelines Minimum information for publication of quantitative real--
time PCR experiments Clin Chem 55 611
Carland FM Fujioka S Takatsuto S Yoshida S and Nelson T (2002)
The identification of CVP1 reveals a role for sterols in vascular pattern-
ing Plant Cell 14 2045ndash2058Choi H Jin JY Choi S Hwang JU Kim YY Suh MC and Lee Y
(2010) An ABCGWBC-type ABC transporter is essential for transport of
sporopollenin precursors for exine formation in developing pollen Plant
J 65 181ndash193Citovsky V Lee LY Vyas S Glick E Chen MH Vainstein A Gafni
Y Gelvin SB and Tzfira T (2006) Subcellular localization of interacting
proteins by bimolecular fluorescence complementation in planta J Mol
Biol 362 1120ndash1131Clough SJ and Bent AF (1998) Floral dip a simplified method for Agro-
bacterium-mediated transformation of Arabidopsis thaliana Plant J 16
735ndash743Clouse SD (2002) Arabidopsis mutants reveal multiple roles for sterols in
plant development Plant Cell 14 1995ndash2000Cnops G Neyt P Raes J et al (2006) The TORNADO1 and TORNADO2
genes function in several patterning processes during early leaf develop-
ment in Arabidopsis thaliana Plant Cell 18 852ndash866Cutler SR Ehrhardt DW Griffitts JS and Somerville CR (2000) Random
GFPcDNA fusions enable visualization of subcellular structures in cells of
Arabidopsis at a high frequency Proc Natl Acad Sci USA 97 3718ndash3723Dou XY Yang KZ Zhang Y Wang W Liu XL Chen LQ Zhang
XQ and Ye D (2011) WBC27 an adenosine tri-phosphate-binding cas-
sette protein controls pollen wall formation and patterning in Arabidop-
sis J Integr Plant Biol 53 74ndash88Feurouleuroop K Pettko-Szandtner A Magyar Z Miskolczi P Kondorosi E
Dudits D and Bako L (2005) The Medicago CDKC1ndashCYCLINT1 kinase
complex phosphorylates the carboxy-terminal domain of RNA polymer-
ase II and promotes transcription Plant J 42 810ndash820Gullberg J Jonsson P Nordstrom A Sjostrom M and Moritz T (2004)
Design of experiments an efficient strategy to identify factors influenc-
ing extraction and derivatization of Arabidopsis thaliana samples in met-
abolomic studies with gas chromatographymass spectrometry Anal
Biochem 331 283ndash295Hertzberg M Aspeborg H Schrader J et al (2001) A transcriptional
roadmap to wood formation Proc Natl Acad Sci USA 98 14732ndash14737Jang JC Fujioka S Tasaka M Seto H Takatsuto S Ishii A Aida M
Yoshida S and Sheen J (2000) A critical role of sterols in embryonic
patterning and meristem programming revealed by the fackel mutants of
Arabidopsis thaliana Genes Dev 14 1485ndash1497Jenks MA Rashotte AM Tuttle HA and Feldmann KA (1996) Mutants
in Arabidopsis thaliana altered in epicuticular wax and leaf morphology
Plant Physiol 110 377ndash385Jonsson P Johansson AI Gullberg J Trygg J A J Grung B Markl-
und S Sjostrom M Antti H and Moritz T (2005) High-throughput
data analysis for detecting and identifying differences between samples
in GCMS-based metabolomic analyses Anal Chem 77 5635ndash5642Jungwirth H and Kuchler K (2006) Yeast ABC transporters ndash a tale of sex
stress drugs and aging FEBS Lett 580 1131ndash1138Kaneda M Schuetz M Lin B Chanis C Hamberger B Western T
Ehlting J and Samuels A (2011) ABC transporters coordinately
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 823
expressed during lignification of Arabidopsis stems include a set of AB-
CBs associated with auxin transport J Exp Bot 62 2063
Karimi M Inze D and Depicker A (2002) GATEWAYTMvectors for Agrobac-
terium-mediated plant transformation Trends Plant Sci 7 193ndash195Kneurooller A and Murphy A (2011) ABC transporters and their function at the
plasma membrane In The Plant Plasma Membrane Plant Cell Mono-
graphs vol 19 (Murphy AS Peer W and Schulz B eds) New York
NY Springer pp 353ndash377Kopischke M Westphal L Schneeberger K Clark R Ossowski S
Wewer V Fuchs R Landtag J Hause G and Deuroormann P (2013)
Impaired sterol ester synthesis alters the response of Arabidopsis thali-
ana to Phytophthora infestans Plant J 73 456ndash468Kuromori T Miyaji T Yabuuchi H Shimizu H Sugimoto E Kamiya
A Moriyama Y and Shinozaki K (2010) ABC transporter AtABCG25 is
involved in abscisic acid transport and responses Proc Natl Acad Sci
USA 107 2361ndash2366Kuromori T Itoh T Sugimoto E and Shinozaki K (2011a) Arabidopsis
mutant of AtABCG26 an ABC transporter gene is defective in pollen
maturation J Plant Physiol 168 2001ndash2005Kuromori T Sugimoto E and Shinozaki K (2011b) Arabidopsis mutants
of AtABCG22 an ABC transporter gene increase transpiration and
drought susceptibility Plant J 67 885ndash894Le Hir R Beneteau J Bellini C Vilaine F and Dinant S (2008) Gene
expression profiling keys for investigating phloem functions Trends
Plant Sci 13 273ndash280Lehrer AT Dugassa-Gobena D Vidal S and Seifert K (2000) Transport
of resistance-inducing sterols in phloem sap of barley Z Naturforsch C
55 948ndash952Li Y Beisson F Pollard M and Ohlrogge J (2006) Oil content of Arabid-
opsis seeds the influence of seed anatomy light and plant-to-plant vari-
ation Phytochemistry 67 904ndash915Luo B Xue XndashY Hu WndashL Wang LndashJ and Chen XndashY (2007) An ABC
transporter gene of Arabidopsis thaliana AtWBC11 is involved in cuticle
development and prevention of organ fusion Plant Cell Physiol 48
1790ndash1802McFarlane HE Shin JJH Bird DA and Samuels AL (2010)
Arabidopsis ABCG transporters which are required for export of diverse
cuticular lipids dimerize in different combinations Plant Cell 22 3066ndash3075
Meskiene I Baudouin E Schweighofer A Liwosz A Jonak C Rodri-
guez PL Jelinek H and Hirt H (2003) Stress-induced protein phospha-
tase 2C is a negative regulator of a mitogen-activated protein kinase
J Biol Chem 278 18945ndash18952Murashige T and Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures Physiol Plant 15 473ndash497Panikashvili D Savaldi-Goldstein S Mandel T Yifhar T Franke RB
Hofer R Schreiber L Chory J and Aharoni A (2007) The Arabidopsis
DESPERADOAtWBC11 transporter is required for cutin and wax secre-
tion Plant Physiol 145 1345ndash1360Panikashvili D Shi JX Bocobza S Franke RB Schreiber L and Aha-
roni A (2010) The Arabidopsis DSOABCG11 transporter affects cutin
metabolism in reproductive organs and suberin in roots Mol Plant 3
563ndash575Panikashvili D Shi JX Schreiber L and Aharoni A (2011) The Arabid-
opsis ABCG13 transporter is required for flower cuticle secretion and pat-
terning of the petal epidermis New Phytol 190 113ndash124Pighin JA Zheng H Balakshin LJ Goodman IP Western TL Jetter
R Kunst L and Samuels AL (2004) Plant cuticular lipid export requires
an ABC transporter Science 306 702ndash704Pullen M Clark N Zarinkamar F Topping J and Lindsey K (2010)
Analysis of vascular development in the hydra sterol biosynthetic
mutants of Arabidopsis PLoS ONE 5 e12227
Quilichini TD Friedmann MC Samuels AL and Douglas CJ (2010)
ATP-binding cassette transporter G26 is required for male fertility and
pollen exine formation in Arabidopsis Plant Physiol 154 678ndash690R Development Core Team (2010) R A Language and Environment for
Statistical Computing Vienna Austria R Foundation for Statistical
Computing
Roudier F Gissot L Beaudoin F Haslam R Michaelson L Marion J
Molino D Lima A Bach L and Morin H (2010) Very-long-chain fatty
acids are involved in polar auxin transport and developmental patterning
in Arabidopsis Plant Cell 22 364ndash375Rozen S and Skaletsky HJ (2000) Primer3 on the WWW for general users
and for biologist programmers Methods Mol Biol 132 365ndash386Schad M Lipton MS Giavalisco P Smith RD and Kehr J (2005) Eval-
uation of two-dimensional electrophoresis and liquid chromatography
tandem mass spectrometry for tissue-specific protein profiling of
laser-microdissected plant samples Electrophoresis 26 2729ndash2738Schauer N Steinhauser D Strelkov S et al (2005) GCndashMS libraries for
the rapid identification of metabolites in complex biological samples
FEBS Lett 579 1332ndash1337Schrader J Nilsson J Mellerowicz EJ Berglund A Nilsson P Hertz-
berg M and Sandberg G (2004) A high-resolution transcript profile
across the wood forming meristem of poplar identifies potential regula-
tors of cambial stem cell identity Plant Cell 16 2278ndash2292Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desi-
mone M Frommer WB Fleurougge UndashI and Kunze R (2003) ARAMEM-
NON a novel database for Arabidopsis integral membrane proteins
Plant Physiol 131 16ndash26Silvestro D Andersen TG Schaller H and Jensen PE (2013) Plant ste-
rol metabolism D7ndashsterol-C5ndashdesaturase (STE1DWARF7) D57ndashste-rol-D7ndashreductase (DWARF5) and D24ndashsterol-D24ndashreductase (DIMINUTO
DWARF1) show multiple subcellular localizations in Arabidopsis thaliana
(Heynh) L PLoS ONE 8 e56429
Sorin C Bussell JD Camus I Ljung K Kowalczyk M Geiss G McKh-
ann H Garcion C Vaucheret H and Sandberg G (2005) Auxin and
light control of adventitious rooting in Arabidopsis require ARGONA-
UTE1 Plant Cell 17 1343ndash1359Tanaka T Tanaka H Machida C Watanabe M and Machida Y (2004) A
new method for rapid visualization of defects in leaf cuticle reveals five
intrinsic patterns of surface defects in Arabidopsis Plant J 37 139ndash146Turgeon R and Wolf S (2009) Phloem transport cellular pathways and
molecular trafficking Annu Rev Plant Biol 60 207ndash221Ukitsu H Kuromori T Toyooka K et al (2007) Cytological and biochemi-
cal analysis of COF1 an Arabidopsis mutant of an ABC transporter gene
Plant Cell Physiol 48 1524ndash1533Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De
Paepe A and Speleman F (2002) Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biol 3 research00340031-00340011Verrier PJ Bird D Burla B et al (2008) Plant ABC proteins ndash a unified
nomenclature andupdated inventory Trends Plant Sci 13 151ndash159Vilaine F Palauqui JC Amselem J Kusiak C Lemoine R and Dinant
S (2003) Towards deciphering phloem a transcriptome analysis of the
phloem of Apium graveolens Plant J 36 67ndash81Zhai Z Sooksa-nguan T and Vatamaniuk OK (2009) Establishing RNA
interference as a reverse-genetic approach for gene functional analysis
in protoplasts Plant Physiol 149 642ndash652Zhang Q Blaylock LA and Harrison MJ (2010) Two Medicago truncatula
half-ABC transporters are essential for arbuscule development in
arbuscular mycorrhizal symbiosis Plant Cell 22 1483
Zhao C Craig JC Petzold HE Dickerman AW and Beers EP (2005)
The xylem and phloem transcriptomes from secondary tissues of the
Arabidopsis root-hypocotyl Plant Physiol 138 803ndash818
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
824 Rozenn Le Hir et al
shown that when the cuticle is altered toluidine blue (TB)
permeates the epidermal surface (Tanaka et al 2004)
Therefore we used TB to check for cuticle defects in the
mutants After treatment with TB no staining was
observed in the wild-type abcg11+ or the abcg9ndash2 and
abcg14ndash1 mutants (Figure 6cef) whereas abcg11ndash7
homozygous seedlings showed patches of blue staining
confirming the cuticle alteration previously described
(Figure 6d) (Ukitsu et al 2007) These observations sug-
gest that unlike mutations in ABCG11 mutations in
ABGC9 or ABCG14 do not affect cuticle formation
abcg9ndash2 and abcg14ndash1 mutants are defective in sterol
composition
To obtain an insight into the potential substrates trans-
ported by these ABCG transporters we analyzed the shoot
and root metabolome of single mutants Orthogonal partial
least-squares discriminant analysis was applied to the
GCMS data in order to classify the profiles of the geno-
types In both shoots (Figure 7andashc) and roots (Figure S4andash
c) homozygous abcg11ndash7 abcg9ndash1 abcg9ndash2 and abcg14ndash1
mutants exhibited very different metabolomic profiles from
the wild-type Interestingly the heterozygous abcg11ndash7+
and abcg9ndash2+ plants had metabolomic profiles intermedi-
ate between the wild-type and their respective homozy-
gous mutant plants (Figure 7ab and Figure S4ab) These
results suggest that a mutation in one copy of ABCG11 or
ABCG9 is sufficient to significantly affect the physiology of
the plant even if it does not dramatically affect visible
aspects of its phenotype
The metabolites showing the most significant differ-
ences were identified by comparing their retention indices
and mass spectra with entries in publicly available reten-
tion libraries (Schauer et al 2005) For all the genotypes
analyzed the metabolites identified as being significantly
different from the wild-type belonged to three main clas-
ses amino acids carbohydrates and lipidssterols The
levels of these compounds were almost all reduced in
abcg9ndash2 and abcg14ndash1 but in excess in abcg11ndash7 (Table 2
and Appendix S1)
Because our study focused on vascular development in
aerial tissues only data obtained for the apical parts are
described in detail here (Table 2) Root data are presented
in Appendix S1 Whereas many neutral amino acids were
significantly depleted in abcg9ndash2 and abcg14ndash1 aerial
Figure 4 Cotyledon venation pattern in abcg mutant lines
Venation complexity and continuity of each examined class of mutant and
wild-type seedlings Values in brackets indicate the percentage contribution
of each class Asterisks indicate statistically significant differences between
the wild-type and mutants (P lt 005 Pearsonrsquos v2 test with Monte Carlo
permutations N = 10 000)
Table 1 Distribution of vascular bundles in the floral stem
Genotype Vascular bundle number Floral stem diameter (mm) Vascular bundle density Total
Col-0 930 105 157 020 592 049 10abcg11-7+ 914 134 140 080 652 042 12abcg11-7 902 115 051 004 176 182 8abcg14-1 828 049 145 018 632 0049 1135SHA-ABCG14 abcg14-1 940 089 170 016 554 058 12abcg9-1 957 053 160 006 586 031 7abcg9-2 875 103 162 010 553 046 835SHA-ABCG9 abcg9-2 933 051 174 010 606 026 8abcg9-1 abcg14-1 871 048 154 003 565 030 8abcg9-2abcg14-1 828 049 124 015 674 084 8abcg9-2abcg14-1abcg11-7
760 054 063 005 1203 169 9
Statistically significant differences between the wild-type and mutants (P lt 005 Studentrsquos t test)
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
816 Rozenn Le Hir et al
tissues (nine and eight out of 20 neutral amino acids
respectively) only two amino acids (glutamine and gluta-
mate) associated with Arabidopsis phloem cells (Schad
et al 2005) were different in abcg11ndash7 (Table 2) Xylose
and the non-reducing sugars trehalose and raffinose were
also decreased in abcg14ndash1 (Table 2) whereas glucose
fructose galactose maltose xylose were significantly dif-
ferent in abg9ndash2 and abcg11ndash7 (reduced and increased
respectively compared to wild-type) (Table 2)
Levels of sterols (campesterol 24ndashmethylene cholesterol
and sitosterol) saturated and unsaturated fatty acids (lino-
leic acid lauric acid palmitic acid and triacontanoic acid)
glycerolipids (monoacylglycerol) and lipid metabolism
intermediates (glycerol glycerol-3ndashphosphate and inositol-
1ndashphosphate) were also significantly different from wild-
type in abcg mutants (Table 2) The abcg9ndash2 mutant
showed 30 lower levels of 24ndashmethylene cholesterol
compared to the wild-type (Appendix S1) In the abcg14ndash1
mutant 50 and 35 decreases of 24ndashmethylene cholesterol
and sitosterol levels were measured respectively (Appen-
dix S1) Finally campesterol showed a 50 increase in the
abcg11ndash7 mutant (Appendix S1) To extend this analysis
measurements of conjugated sterols [sterol ester (SE)
sterol glucoside and acylated sterol glucoside] were also
performed When seedlings were grown in vitro no differ-
ence in the sterol glucoside and acylated sterol glucoside
contents was detected between the wild-type and the vari-
ous mutant lines (Figure S4d) However there was a ten-
dency towards a reduced SE content in the double mutant
abcg9ndash2 abcg14ndash1 compared to the wild-type (Figure S4d)
This observation prompted us to examine variations in the
SE content in older plants (4-week-old plants grown in soil
in the greenhouse) At this developmental stage no differ-
ence in the free sterol (FS) content was observed between
the wild-type and the various mutant lines (Figure 7d) but
the fraction comprising FS and SE was significantly
(a) (b) (c) (d)
(e) (f) (g) (h)
(i) (j)
(k) (l) (m)
(n) (o) (p)
Figure 5 Subcellular localization and physical interactions of ABCG9 ABCG11 and ABCG14
(andashh) ABCG9 and ABCG14 co-localize to the plasma membrane when transiently expressed in Arabidopsis mesophyll protoplasts of the GFPndashLTi6b marker line
(Cutler et al 2000)
(a e) Cyan fluorescence of GFPndashLTi6b(b f) Purple fluorescence from mRFPndashABCG9 and mRFPndashABCG14 respectively
(c g) Yellow auto-fluorescence of the chloroplasts
(d h) Merged images of the three channels Scale bar = 10 lm
(i j) Confocal images of immunofluorescence staining with anti-HA antibodies in root cells of abcg9ndash2 and abcg14ndash1 mutants complemented with HA-tagged
ABCG9 (i) and HA-tagged ABCG14 (j) Scale bars = 10 lm
(kndashp) Proteins were extracted from protoplasts co-transfected with HA- or cndashMyc-tagged versions of ABCGs (lanes 1 and 4) or transfected with a single plasmid
containing the HA-tagged version (lane 2) or the c-Myc-tagged version (lane 3) In lanes 2ndash4 proteins were immunoprecipitated with the anti-c-Myc antibody
and subjected to anti-HA protein gel-blot analysis to reveal the other partner of the dimer Transient co-expression of (k) HAndashABCG9cndashMycndashABCG9 (l) HAndashABCG11cndashMycndashABCG11 (m) HAndashABCG14cndashMycndashABCG14 (n) HAndashABCG14cndashMycndashABCG11 (o) HAndashABCG14cndashMycndashABCG9 and (p) HAndashABCG11cndashMycndashABCG9
in wild-type protoplasts
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 817
reduced probably accounting for the difference in the SE
content between the wild-type and the abcg9ndash2 abcg14ndash1
double mutant (Figure 7e)
(a)
(b)
(c) (d) (e)
(f) (g)
Figure 6 Long chain and very long chain fatty acid content and cuticle
integrity in abcg9ndash2 and abcg14ndash1 mutants
(a b) Fatty acid (a) and VLCFA content (b) in abcg9ndash2 and abcg14ndash1 mutants
in apical parts of 10-day-old seedlings (three biological replicates each of
approximately 5 mg dry weight were used)
(cndashf) Toluidine blue staining patterns of abcg mutants in 7-day-old seed-
lings Scale bars = 3 cm
(a)
(b)
(c)
(d) (e)
Figure 7 Metabolome analysis and measurements of sterol content in abcg
mutants
(andashc) Results of orthogonal partial least-squares discriminant analysis of
global metabolite contents of shoots of 7-day-old seedlings from wild-type
(black squares) abcg11ndash7+ (gray triangles) abcg11ndash7 (white stars) abcg9ndash1(white hexagons) abcg9ndash2+ (gray hexagons) abcg9ndash2 (white hexagons)
and abcg14ndash1 (black circles) seedlings (five biological replicates of 10 mg
each were used for the analysis)
(d e) Free sterol (FS) and sterol ester (SE) content of wild-type (white bars)
abcg9ndash2 (light gray bars) abcg14ndash1 (dark gray bars) and abcg9ndash2 abcg14ndash1(black bars) Error bars indicate standard deviations obtained from three
independent biological replicates The asterisk indicates a statistically signif-
icant difference between the wild-type and the mutant (P lt 005 Studentrsquos
t test)
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
818 Rozenn Le Hir et al
DISCUSSION
ABCG9 and ABCG14 are two plasma membrane proteins
that interact physically in multiple homo-heterodimer
combinations
In this study we showed that like the previously described
ABCG11 (Pighin et al 2004 Bird et al 2007 Luo et al
2007 Panikashvili et al 2007 2010 Ukitsu et al 2007)
ABCG9 and ABCG14 localize to the plasma membrane
Based on co-immunoprecipitation of tagged proteins and
the bimolecular fluorescence complementation assay we
demonstrated that ABCG9 interacts with ABCG11 to form a
heterodimer and like ABCG11 (McFarlane et al 2010) can
homodimerize In contrast ABCG14 interacts only with
ABCG11 forming an obligate heterodimer This extends
the number of potential partners of ABCG11 to three as it
was previously shown to dimerize with the ABCG12CER5
protein (McFarlane et al 2010) Similarly ABCG9 under-
goes flexible dimerization to form either homo- or hetero-
dimers suggesting that ABCGs may be promiscuous
proteins with different functions depending on their inter-
acting partner andor expression profile
Mutations in ABCG genes are semi-dominant
In the last few years several Arabidopsis mutants with
altered expression of various ABCG genes such as ABCG11
(Bird et al 2007 Luo et al 2007 Panikashvili et al 2007
Ukitsu et al 2007) ABCG12CER5 (Pighin et al 2004)
ABCG13 (Panikashvili et al 2011) ABCG22 (Kuromori
et al 2011a) ABCG25 (Kuromori et al 2010) and ABCG26
(Choi et al 2010 Quilichini et al 2010 Dou et al 2011
Kuromori et al 2011b) have been characterized but even
though these half-size transporters require dimerization to
become functional all the mutations were described as
recessive Here we demonstrated that mutations in these
genes have semi-dominant effects Indeed the metabolome
analyses showed that abcg9ndash2+ and abcg11ndash7+ heterozy-
gous seedlings have a profile intermediate between that of
wild-type and homozygous seedlings The possibility of
dominant-negative effects was excluded because no
Table 2 Shoot metabolites of 10-day-old abcg9ndash2 abcg11ndash7 and abcg14ndash1 seedlings whose levels were significantly different from wild-type
Group
Metabolites that differed between mutant and wild-type
abcg9ndash2Colndash0 abcg11ndash7Colndash0 abcg14ndash1Colndash0
Amino acids Glycine Glutamate AlanineAlanine Glutamine ValineValine ThreonineThreonine GABASerine Prolinebndashalanine IsoleucineIsoleucine SerineGABA bndashalanineProline
Carbohydrates Glucose Glucose ErythritolFructose Fructose TrehaloseXylose Galactose XyloseMaltose RaffinoseSucrose
Lipids and intermediates 24ndashmethylene-cholesterol Campesterol Linoleic acidMonoacylglycerol Glycerol-3ndashphosphate Triacontanoic acidPalmitic acid Inositol-1ndashphosphate GlycerolGlycerol-3ndashphosphate Lauric acidInositol-1ndashphosphate 24ndashmethylene-cholesterol
SitosterolGlycerol-3ndashphosphate
TCA cycle and others Phosphoric acid Phosphate compound Glyceric acid24ndashdihydroxybutanoic acid Dehydroascorbic acid Fumaric acidGlyceric acid Malic acid Threonic acid-14ndashlactoneFumaric acid 2ndashoxoglutaric acid Threonic acidThreonic acid bndashhydroxy-bndashmethylglutaric acid Shikimic acidThreonic acid-14ndashlactone Gluconic acid lactone-like Succinic acidShikimic acid Phytol2ndashoxoglutaric acid Ethanolamine
Nicotinic acid
For abcg9ndash2 and abcg14ndash1 the levels of the compounds were reduced whereas they were increased in abcg11ndash7
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 819
truncated transcript was found for either abcg9ndash2 or
abcg11ndash7 mutants Hence abcg9ndash2 and abcg11ndash7 are true
null alleles These observations are consistent with the
requirement for homo- or heterodimerization of the ABCG
proteins to form functional transporters In the heterozy-
gous plants ABCG11 transcript levels were 50 lower than
in wild-type counterparts suggesting that ABCG11 protein
and thus corresponding homo- and heterodimers were
likely to be less abundant which may also explain the meta-
bolic perturbations and consequent phenotypic modifica-
tions These findings also suggest that wild-type plants are
not saturated by ABCG11 proteins as a 50 reduction in
protein content appears to be sufficient to alter the physiol-
ogy of the heterozygous mutant plants This hypothesis is
strengthened by the observation that mutants carrying the
weak abcg11ndash6 allele in which there was only a 25 reduc-
tion in ABCG11 transcript levels showed a similar pheno-
type to the heterozygous abcg11ndash7+ and abcg11ndash8+
plants Similarly the abcg9ndash1 weak allele mutant had a very
similar phenotype to the abcg9ndash2+ heterozygous seedlings
suggesting that considering the mode of action of these
proteins the semi-dominant effects that we observed are
likely to be a general feature among the ABCG genes
ABCG9 ABCG11 and ABCG14 transporters are involved in
vascular system development in Arabidopsis
We showed that the ABCG11 ABCG9 and ABCG14 promot-
ers drove expression in the vascular system of Arabidopsis
cotyledons and rosette leaves and more precisely in the
phloem of flower stems In addition mutations in ABCG9
ABCG11 and ABCG14 genes altered vascular development
in cotyledons and the floral stem Similar vascular localiza-
tion of ABCG11 has already been described by Panikashvili
et al (20072010) who showed that in addition to the epi-
dermis ABCG11 is expressed in the vasculature as early as
during embryo development and then throughout plant
development They showed that the vascular pattern in
leaves of the homozygote mutant seedlings was less com-
plex than in the wild-type and discontinuous (Panikashvili
et al 2007) The present article describes a mutant that
has both cuticle and vascular system patterning defects
although it has been previously shown that mutants
altered in cuticle formation have dramatically reduced
growth and altered leaf morphology (Jenks et al 1996)
which may account for vascular defects Analysis of the
abcg11+ heterozygous seedlings that have vascular
defects but apparently no cuticle alteration suggested that
these phenotypes are probably uncoupled Because
ABCG11 dimerizes with several members of its sub-family
it seems reasonable to postulate that it is involved in the
transport of different compounds depending on its partner
Here we provide evidence that ABCG11 in addition to
forming dimers with ABCG12 (McFarlane et al 2010) in
epidermal cells is likely to dimerize with ABCG9 and
ABCG14 in phloem cells suggesting functional compart-
mentalization of these proteins and a possible role in vas-
cular system patterning in Arabidopsis
Metabolite profiling suggests that ABCG9 ABCG11 and
ABCG14 are involved in sterollipid homeostasis
We investigated in more depth the metabolic changes
induced by mutations in ABCG9 ABCG11 and ABCG14
We found that levels of metabolites of several classes
including lipids sugars and amino acids were altered in
both the heterozygotes and homozygotes and that the
profiles in the root and apical parts differed suggesting
that ABCGs may play different roles in the roots and shoot
Interestingly the modifications of amino acid and sugar
contents observed in the metabolic profiles of the abcg
mutants suggest an impaired carbonnitrogen balance that
may be directly or indirectly linked to the vascular defects
leading to possible problems with phloem loading or
unloading Saturated and unsaturated fatty acids and sterol
contents were also altered in the mutants Patterning
defects in the vascular system are often observed in
mutants altered in hormone homeostasis such as for
auxin or brassinosteroids (Kaneda et al 2011) and direct
or indirect modifications of sterol homeostasis are thought
to be one of the primary causes of these downstream
effects (Pullen et al 2010) The detected defects in sterol
homeostasis may explain the abnormal vascular develop-
ment observed in abcg9 abcg11 and abcg14 mutants
Accordingly several sterol-deficient mutants such as fac-
kelhydra (Jang et al 2000) and cpv1 (Carland et al 2002)
have also been shown to exhibit severe defects in vascular
development It has been suggested that the bushy habit
and low fertility of cpv1smt2 mutants may be due to an
increased campesterolsitosterol ratio rather than sterol
deficiency per se (Clouse 2002) This ratio is controlled by
the activity of SMT2 (STEROL METHYLTRANSFERASE2)
Interestingly strong down-regulation of SMT2 expression
was observed in all the mutant backgrounds (Figure S5)
and the metabolic data indicated a deficit in 24ndashmethylene
cholesterol (precursor of both campesterol and sitosterol)
andor sitosterol in abcg9 and abcg14 mutants respec-
tively In addition the significant decrease in sterol ester
content in the double mutant abcg9ndash2 abcg14ndash1 correlated
with down-regulation of expression of the PHOSPHOLIPID
STEROL ACYLTRANSFERASE1 (PSAT1) gene encoding the
enzyme that is mainly responsible for SE synthesis (Bou-
vier-Nave et al 2010) and is proposed to be a control step
in sterol homeostasis (Kopischke et al 2013) Altogether
these results suggest that the vascular phenotype
observed in abcg mutants may be linked to modification of
sterol homeostasis
Therefore we postulate that the various dimer combina-
tions between ABCG9 ABCG11 and ABCG14 may
participate in the transport of squalene-derived metabolites
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
820 Rozenn Le Hir et al
such as sterol andor sterol conjugates via the phloem sap
The presence of sterols in phloem sap has been observed in
various species such as barley (Hordeum vulgare Lehrer
et al 2000) and fava bean (Vicia faba Bouvaine et al
2012) and indirect proof of the presence of sterols in
phloem sap has been obtained by analysis of the phytos-
terol content in exclusively phloem-feeding aphids that are
unable to synthetize them directly (Behmer et al 2011) In
addition the evidence for long-distance transport of sterol
precursors has been supported by observations that sterol
esters accumulate in organs distal to the site of application
of exogenous precursors (Bouvier-Nave et al 2010) As
sterols have been proposed to act as signal molecules in
the control of developmental processes such as vascular
patterning (Carland et al 2002 Pullen et al 2010) modifi-
cations of sterol homeostasis may affect development of
the vascular system in the abcgmutants described here
In conclusion we propose that ABCG9 ABCG11 and
ABCG14 are important for lipidsterol homeostasis Thus
these three proteins are likely to be involved in the control
of various aspects of plant development andor physiol-
ogy including patterning of the vascular system
EXPERIMENTAL PROCEDURES
Plant material and growth conditions
Insertion lines in ABCG9 (SALK_047133 and GABI_353B01)ABCG11 (SALK_096377 and GABI_590C03) and ABCG14 (SALK_036952) genes were obtained from the European ArabidopsisStock Centre (httparabidopsisinfo) Heterozygoushomozygousplants were screened using gene-specific primers in combinationwith the specific primer for the left border of the TndashDNA insertion(Table S1)
Plants were grown in soil or in vitro as described previously(Sorin et al 2005) Arabidopsis Colndash0 cell cultures were main-tained by weekly sub-culturing in MS medium (Murashige andSkoog 1962) supplemented with 240 lg l1 24ndashdichlorophenoxy-acetic acid and 14 lg l1 kinetin and were grown under 12 h light12 h dark cycles at 25 lE m2 sec1 and with 23degC day and nighttemperatures
Morphological characterization and GUS staining
To study the venation pattern cotyledons of 7-day-old seedlingsand rosette leaves of 4-week-old plants were cleared in a buffercontaining chloral hydrate glycerol and water (831 wvv) afterwhich 200 cotyledons per genotype were analyzed and the per-centage showing particular levels of complexity or discontinuity invascularity was estimated as described by Cnops et al (2006)
Flower stem segments (1ndash2 cm long) were removed from ten3ndash4-week-old plants grown in soil embedded in 8 agarose andsectioned using a VT100S vibratome (Leica httpwwwleicacom)The cross-sections were then stained with safraninalcian blue for1ndash2 min (Sigma httpwwwsigmaaldrichcom) rinsed in waterand mounted in 50 glycerol
Histochemical GUS assays were performed as described previ-ously (Sorin et al 2005) All observations were performed usingan Axioplan light microscope equipped with an Axiocam cameraand Axiovision software (Zeiss httpwwwzeisscom)
To observe potential cuticle defects in the various mutant linesa TB test was performed by immersing in vitro-grown plantlets ina 005 wv TB solution for 2 min as described by Tanaka et al(2004)
Statistical analyses
Statistical analyses of differences between-genotype means andvariance for the studied morphological traits were performedusing Studentrsquos t test (GraphPad Prism version 50 httpwwwgraphpadcom) In the venation pattern experiment thesignificance of differences between wild-type and mutants wasassessed using R (R Development Core Team 2010) and Pearsonrsquosv2 test with Monte Carlo permutations (N = 10 000)
Plasmid constructs
The native ABCG promoters and open reading frame of ABCG11were amplified from genomic DNA or cDNA extracted from 7-day-old Arabidopsis seedlings using Phusion high-fidelity DNApolymerase (Finnzyme wwwthermoscientificbiocomfinnzymes)according to the manufacturerrsquos instructions with gene-specificprimers (Table S1) The PCR products were cloned into pENTRDndashTOPO (Invitrogen httpwwwinvitrogencom) and recombinedinto pKGWFS7 (Karimi et al 2002) to create ABCGproGUS Allrecombinations were performed using the Gateway LR Clonase
enzyme mix (Invitrogen) At each cloning step the sequenceswere confirmed by sequencing and all the constructs weretransferred into Agrobacterium strain GV3101pMP90 (C58C1)Wild-type or abcg11ndash7+ heterozygous plants were transformed byfloral dipping (Clough and Bent 1998) For ABCGGUS constructsthe expression pattern was checked in the T2 progeny of 10ndash15independent transgenic lines and one representative homozygousline was used for further characterization
Tagged protein constructs and protoplast transformation
Epitope-tagged versions of ABCG9 ABCG11 and ABCG14 proteinswere produced in pRT104ndash3xHA and pRT104ndash3xMyc plasmids(Feurouleuroop et al 2005) and pSAT6-mRFP (Citovsky et al 2006) Allthese plasmids have a 35S promoter sequence upstream of themulti-cloning site The open reading frames of ABCG11 andABCG14 and the genomic clone of ABCG9 were amplified fromcDNA or genomic DNA extracted from 7-day-old Arabidopsisseedlings using Phusion high-fidelity DNA polymerase (Finnzyme)according to the manufacturerrsquos instructions with gene-specificprimers carrying EcoRI or SalI restriction sites to facilitate subse-quent cloning (Table S1) The products obtained after PCR weredigested by EcoRI and SalI prior to ligation into appropriate plas-mids that had previously been cut open with the same restrictionenzymes Constructs were verified by sequencing Protoplastsfrom Arabidopsis cell cultures or 14-day-old Arabidopsis seedlingswere prepared and transformed as previously described (Meski-ene et al 2003 Zhai et al 2009) For co-immunoprecipitation andsubcellular localization assays 100 000 and 50 000 protoplastcells respectively were transfected with 15 and 10 ll of plasmidsrespectively (containing between 5 and 75 lg of each construct)and the resulting transformed protoplasts were processed afterincubation in the dark at room temperature for 16ndash18 and 24 hrespectively
Whole-mount immunolocalization
The HA-tagged versions of ABCG9 and ABCG14 were detected in7-day-old in vitro-grown Arabidopsis seedlings as described inMethods S1 Anti-HA antibody 12CA5 (Roche httpwwwroche
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 821
com) (1100 dilution) combined with Alexa Fluor 488-conjugatedsecondary antibody (1100 dilution) were used
Confocal laser-scanning microscopy
Images were collected using an SP2 confocal microscope (Leica)RFP and Alexa Fluor 488 were detected using laser lines at 561and 488 nm respectively according to the manufacturerrsquos set-tings RFP fluorescence emission was detected at 550ndash600 nmand Alexa Fluor 488 and GFP fluorescence emission were detectedat 495ndash545 nm For subcellular localization in protoplasts imageswere coded in cyan (GFP) magenta (RFP) and yellow (auto-fluo-rescence) resulting in purple coloration for co-localization inmerged images For the immunolocalization experiment imageswere coded in green (GFP) A water-corrected 209 objective (HCPlan-Apochromat 20907 ImmKor Leica) was used Images werecropped using Adobe Photoshop CS2 and assembled using AdobeIllustrator CS2 software (Abode httpwwwabodecom) Eachimage shown represents a single focal plan
Co-immunoprecipitation assays
Transformed protoplast proteins were extracted using 50 ll lysisbuffer containing 25 mM TrisHCl pH 78 10 mM MgCl2 75 mM
NaCl 5 mM EGTA 1 mM benzamidine 1 mM dithiothreitol 10glycerol 1 Triton Xndash100 and 19 protein inhibitor cocktail(Sigma httpsigmaaldrichcom) The transformed protoplast sus-pension was frozen in liquid nitrogen then thawed on ice andsubjected to centrifugation for 5 min at 150 g Two microliters ofanti-Myc (9E10 Covance httpwwwcovancecom) or anti-HA(16B12 Covance) antibodies were added to the extracts and themixture was incubated for 2 h at 4degC on a rotating wheel Immu-nocomplexes were captured by incubation with 10 ll Protein GndashSepharose beads for a further 2 h at 4degC on a rotating wheel afterwhich they were washed three times for 5 min each in 25 mM
NaPi 150 mM NaCl 5 glycerol and 02 Igepal CAndash630 buffer(Sigma) and then eluted using 40 ll SDS sample buffer at 37degCfor 30 min Co-immunoprecipitation of Myc- and HA-tagged pro-teins was revealed by SDSndashPAGE followed by Western blottingusing both anti-Myc (Invitrogen) and anti-HA (3F10 Roche) anti-bodies at 12000 dilution
RNA extraction and cDNA preparation
To quantify the relative amounts of the ABCG transcriptsexpressed in various organs three rosette leaves all the cau-line leaves the bottom 5 cm of the floral stem all closed flow-ers and all green siliques were harvested from each of threeindependent 4-week-old Arabidopsis plants of each genotypegrown in soil For the other experiments 10-day-old seedlingsgrown in vitro were harvested All the material was directly fro-zen in liquid nitrogen and stored at 80degC prior to RNA extrac-tion Samples were then disrupted using a MM 301 vibrationmill (Retsch GmbH httpwwwretschcom) at a frequency of25 Hz for 2 min with 3 mm tungsten carbide beads (Qiagenhttpwwwqiagencom) Total RNA was isolated from thismaterial using TRIzol (Life Technologies httplifetechnologiescom) RNA quantity and quality were measured using a Nano-Drop ND 1000 (Thermo Scientific httpwwwthermoscientificcom) All samples with 260280 nm and 260230 nm absorbanceratios lower than 18 were discarded and RNA was re-extractedDNase treatment was applied to 5 lg samples of total RNAusing a DNase I kit (Fermentas httpwwwfermentascom)Double-stranded cDNA was synthesized from 5 lg total RNAusing a SuperScript reverse transcriptase kit (Invitrogen) witholigo(dT)12 primer Finally RNase treatment was applied to the
samples using Escherichia coli RNAse H (Fermentas) The cDNAsamples obtained were diluted 30-fold (except for abcg9 linesamples which were diluted 10-fold) prior to use either toverify the absence of full-length transcripts in putative knockoutmutants or to quantify gene expression by quantitative PCR Allprimers used were designed using the Primer3 program (Rozenand Skaletsky 2000) and their sequences are listed inTable S1
QUANTITATIVE REAL-TIME PCR
Quantitative PCR amplifications were performed in 20 llmixtures containing 10 ll 59 SYBR Green I master mix
(Roche) 02 ll forward and reverse primer (30 lM each)
48 ll sterile water and 5 ll cDNA The BiondashRad CFX96
real-time PCR detection system (httpwwwbio-radcom)
was used for quantification of expression of the sterol
biosynthesis transcripts and the ABCG transcripts in the
various mutant backgrounds Finally the Roche LightCy-
cler 480 system (Roche) was used to quantify the rela-
tive abundance of ABCG transcripts in various organs
Four genes were considered as potential reference genes
[APT1 (At1g27450) TIP41 (At4g34270) EF1a (At5g60390)
and UBQ10 (At4g05320)] and their suitability was tested
using the geNorm algorithm (Vandesompele et al 2002)
to determine the most appropriate one for each experi-
ment A more detailed procedure following the Minimum
Information for Publication of Quantitative Real-Time PCR
Experiments guidelines (Bustin et al 2009) is described
in Methods S2 and the annealing temperature and
efficiencies of the primer pairs used are shown in
Table S2
VLCFA quantification
Approximately 5 mg dry weight of 10-day-old in vitro
Arabidopsis seedlings were analyzed for VLCFA quantifica-
tion as described by Li et al (2006)
Sterol extraction and GC-FID characterization
A pool of 10-day-old seedlings grown in vitro or rosettes
from 4-week-old plants grown in soil were lyophilized prior
to extraction Sterol extraction and isolation was per-
formed as described previously (Bouvier-Nave et al 2010)
Gas Chromatography-Flame Ionization Detector (GC-FID)
characterization was performed as described by Silvestro
et al (2013) To quantify the total amounts of free sterols
and sterol esters non-saponifiable lipids were extracted
using nndashhexane and sterols were derivatized as sterols ace-
tates Quantification was achieved using lupenyl-3 28ndashdi-
acetate as an internal standard
Metabolome analysis
To obtain metabolome profiles associated with wild-type
abcg11ndash7+ abcg11ndash7 abcg9ndash1 abcg9ndash2 and abcg14ndash1
genotypes 10-day-old seedlings representing each geno-
type grown in vitro as described above were harvested
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
822 Rozenn Le Hir et al
and divided into shoot and root samples In the case of
abcg9ndash2 the phenotype of the heterozygote abcg9ndash2+
was indistinguishable from the homozygote phenotype
and therefore heterozygous and homozygous seedlings
from a segregating population were pooled Hence in this
particular experiment abcg9ndash2+ refers to a mixed popula-
tion comprising two-thirds heterozygotes and one-third
homozygotes
Metabolites in five samples of shoot and root parts of
seedlings of each genotype (10 mg fresh weight per sam-
ple) were then extracted analyzed by GCMS and the
acquired data were processed as previously described
(Gullberg et al 2004 Jonsson et al 2005) All multivariate
statistical investigations (partial component analysis and
orthogonal partial least-squares discriminant analysis) for
between-genotype similarities and differences in general
metabolite profiles were performed using Simca software
(Umetrics httpwwwumetricscom)
ACKNOWLEDGEMENTS
We thank A Murphy (Plant Science and Landscape Archi-
tecture University of Maryland MD USA) for stimulating
discussions and critical reading of the manuscript I Carl-
son (Umea Plant Science Center Department of Forest
Genetics and Plant Physiology Swedish University of Agri-
cultural Sciences Umea Sweden) for technical assistance
and S Vernhettes for kindly donating seeds of the
transgenic line carrying the plasma membrane marker
GFPndashLTi6b We also thank the European Arabidopsis Stock
Centre and the GABI-Kat stock centre for providing seeds
This work was supported by allocations granted to CB by
the Swedish Research Council for Agriculture the Swedish
Foundation for Strategic Research the Swedish Research
Council for Research and Innovation for Sustainable
Growth the K amp A Wallenberg Foundation and the Carl
Trygger Foundation
SUPPORTING INFORMATION
Additional Supporting Information may be found in the onlineversion of this articleAppendix S1 Raw data quantification for compounds identified inthe metabolite profiling by GCMS
Figure S1 Relative expression of ABCG genes in various organsand mutant backgroundsFigure S2 Co-immunoprecipitation controlsFigure S3 ABCG9 and ABCG14 homo- andor heterodimerize withABCG11Figure S4 Complementary data on metabolome analysis and con-jugated sterol contentFigure S5 Relative expression of genes related to sterol homeo-stasisMethods S1 Method for whole-mount immunolocalizationMethods S2 Method for quantitative PCR
Table S1 List of primers used in this studyTable S2 Annealing temperature and efficiencies for the primerspairs used in this study
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Douglas AE (2012) The physiology of sterol nutrition in the pea aphid
Acyrthosiphon pisum J Insect Physiol 58 1383ndash1389Bouvier-Nave P Berna A Noiriel A Compagnon V Carlsson AS
Banas A Stymne S and Schaller H (2010) Involvement of the phos-
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M Mueller R Nolan T Pfaffl MW and Shipley GL (2009) The MIQE
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Carland FM Fujioka S Takatsuto S Yoshida S and Nelson T (2002)
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J 65 181ndash193Citovsky V Lee LY Vyas S Glick E Chen MH Vainstein A Gafni
Y Gelvin SB and Tzfira T (2006) Subcellular localization of interacting
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Biol 362 1120ndash1131Clough SJ and Bent AF (1998) Floral dip a simplified method for Agro-
bacterium-mediated transformation of Arabidopsis thaliana Plant J 16
735ndash743Clouse SD (2002) Arabidopsis mutants reveal multiple roles for sterols in
plant development Plant Cell 14 1995ndash2000Cnops G Neyt P Raes J et al (2006) The TORNADO1 and TORNADO2
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GFPcDNA fusions enable visualization of subcellular structures in cells of
Arabidopsis at a high frequency Proc Natl Acad Sci USA 97 3718ndash3723Dou XY Yang KZ Zhang Y Wang W Liu XL Chen LQ Zhang
XQ and Ye D (2011) WBC27 an adenosine tri-phosphate-binding cas-
sette protein controls pollen wall formation and patterning in Arabidop-
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Dudits D and Bako L (2005) The Medicago CDKC1ndashCYCLINT1 kinase
complex phosphorylates the carboxy-terminal domain of RNA polymer-
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Design of experiments an efficient strategy to identify factors influenc-
ing extraction and derivatization of Arabidopsis thaliana samples in met-
abolomic studies with gas chromatographymass spectrometry Anal
Biochem 331 283ndash295Hertzberg M Aspeborg H Schrader J et al (2001) A transcriptional
roadmap to wood formation Proc Natl Acad Sci USA 98 14732ndash14737Jang JC Fujioka S Tasaka M Seto H Takatsuto S Ishii A Aida M
Yoshida S and Sheen J (2000) A critical role of sterols in embryonic
patterning and meristem programming revealed by the fackel mutants of
Arabidopsis thaliana Genes Dev 14 1485ndash1497Jenks MA Rashotte AM Tuttle HA and Feldmann KA (1996) Mutants
in Arabidopsis thaliana altered in epicuticular wax and leaf morphology
Plant Physiol 110 377ndash385Jonsson P Johansson AI Gullberg J Trygg J A J Grung B Markl-
und S Sjostrom M Antti H and Moritz T (2005) High-throughput
data analysis for detecting and identifying differences between samples
in GCMS-based metabolomic analyses Anal Chem 77 5635ndash5642Jungwirth H and Kuchler K (2006) Yeast ABC transporters ndash a tale of sex
stress drugs and aging FEBS Lett 580 1131ndash1138Kaneda M Schuetz M Lin B Chanis C Hamberger B Western T
Ehlting J and Samuels A (2011) ABC transporters coordinately
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ABCG proteins and vascular development 823
expressed during lignification of Arabidopsis stems include a set of AB-
CBs associated with auxin transport J Exp Bot 62 2063
Karimi M Inze D and Depicker A (2002) GATEWAYTMvectors for Agrobac-
terium-mediated plant transformation Trends Plant Sci 7 193ndash195Kneurooller A and Murphy A (2011) ABC transporters and their function at the
plasma membrane In The Plant Plasma Membrane Plant Cell Mono-
graphs vol 19 (Murphy AS Peer W and Schulz B eds) New York
NY Springer pp 353ndash377Kopischke M Westphal L Schneeberger K Clark R Ossowski S
Wewer V Fuchs R Landtag J Hause G and Deuroormann P (2013)
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ana to Phytophthora infestans Plant J 73 456ndash468Kuromori T Miyaji T Yabuuchi H Shimizu H Sugimoto E Kamiya
A Moriyama Y and Shinozaki K (2010) ABC transporter AtABCG25 is
involved in abscisic acid transport and responses Proc Natl Acad Sci
USA 107 2361ndash2366Kuromori T Itoh T Sugimoto E and Shinozaki K (2011a) Arabidopsis
mutant of AtABCG26 an ABC transporter gene is defective in pollen
maturation J Plant Physiol 168 2001ndash2005Kuromori T Sugimoto E and Shinozaki K (2011b) Arabidopsis mutants
of AtABCG22 an ABC transporter gene increase transpiration and
drought susceptibility Plant J 67 885ndash894Le Hir R Beneteau J Bellini C Vilaine F and Dinant S (2008) Gene
expression profiling keys for investigating phloem functions Trends
Plant Sci 13 273ndash280Lehrer AT Dugassa-Gobena D Vidal S and Seifert K (2000) Transport
of resistance-inducing sterols in phloem sap of barley Z Naturforsch C
55 948ndash952Li Y Beisson F Pollard M and Ohlrogge J (2006) Oil content of Arabid-
opsis seeds the influence of seed anatomy light and plant-to-plant vari-
ation Phytochemistry 67 904ndash915Luo B Xue XndashY Hu WndashL Wang LndashJ and Chen XndashY (2007) An ABC
transporter gene of Arabidopsis thaliana AtWBC11 is involved in cuticle
development and prevention of organ fusion Plant Cell Physiol 48
1790ndash1802McFarlane HE Shin JJH Bird DA and Samuels AL (2010)
Arabidopsis ABCG transporters which are required for export of diverse
cuticular lipids dimerize in different combinations Plant Cell 22 3066ndash3075
Meskiene I Baudouin E Schweighofer A Liwosz A Jonak C Rodri-
guez PL Jelinek H and Hirt H (2003) Stress-induced protein phospha-
tase 2C is a negative regulator of a mitogen-activated protein kinase
J Biol Chem 278 18945ndash18952Murashige T and Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures Physiol Plant 15 473ndash497Panikashvili D Savaldi-Goldstein S Mandel T Yifhar T Franke RB
Hofer R Schreiber L Chory J and Aharoni A (2007) The Arabidopsis
DESPERADOAtWBC11 transporter is required for cutin and wax secre-
tion Plant Physiol 145 1345ndash1360Panikashvili D Shi JX Bocobza S Franke RB Schreiber L and Aha-
roni A (2010) The Arabidopsis DSOABCG11 transporter affects cutin
metabolism in reproductive organs and suberin in roots Mol Plant 3
563ndash575Panikashvili D Shi JX Schreiber L and Aharoni A (2011) The Arabid-
opsis ABCG13 transporter is required for flower cuticle secretion and pat-
terning of the petal epidermis New Phytol 190 113ndash124Pighin JA Zheng H Balakshin LJ Goodman IP Western TL Jetter
R Kunst L and Samuels AL (2004) Plant cuticular lipid export requires
an ABC transporter Science 306 702ndash704Pullen M Clark N Zarinkamar F Topping J and Lindsey K (2010)
Analysis of vascular development in the hydra sterol biosynthetic
mutants of Arabidopsis PLoS ONE 5 e12227
Quilichini TD Friedmann MC Samuels AL and Douglas CJ (2010)
ATP-binding cassette transporter G26 is required for male fertility and
pollen exine formation in Arabidopsis Plant Physiol 154 678ndash690R Development Core Team (2010) R A Language and Environment for
Statistical Computing Vienna Austria R Foundation for Statistical
Computing
Roudier F Gissot L Beaudoin F Haslam R Michaelson L Marion J
Molino D Lima A Bach L and Morin H (2010) Very-long-chain fatty
acids are involved in polar auxin transport and developmental patterning
in Arabidopsis Plant Cell 22 364ndash375Rozen S and Skaletsky HJ (2000) Primer3 on the WWW for general users
and for biologist programmers Methods Mol Biol 132 365ndash386Schad M Lipton MS Giavalisco P Smith RD and Kehr J (2005) Eval-
uation of two-dimensional electrophoresis and liquid chromatography
tandem mass spectrometry for tissue-specific protein profiling of
laser-microdissected plant samples Electrophoresis 26 2729ndash2738Schauer N Steinhauser D Strelkov S et al (2005) GCndashMS libraries for
the rapid identification of metabolites in complex biological samples
FEBS Lett 579 1332ndash1337Schrader J Nilsson J Mellerowicz EJ Berglund A Nilsson P Hertz-
berg M and Sandberg G (2004) A high-resolution transcript profile
across the wood forming meristem of poplar identifies potential regula-
tors of cambial stem cell identity Plant Cell 16 2278ndash2292Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desi-
mone M Frommer WB Fleurougge UndashI and Kunze R (2003) ARAMEM-
NON a novel database for Arabidopsis integral membrane proteins
Plant Physiol 131 16ndash26Silvestro D Andersen TG Schaller H and Jensen PE (2013) Plant ste-
rol metabolism D7ndashsterol-C5ndashdesaturase (STE1DWARF7) D57ndashste-rol-D7ndashreductase (DWARF5) and D24ndashsterol-D24ndashreductase (DIMINUTO
DWARF1) show multiple subcellular localizations in Arabidopsis thaliana
(Heynh) L PLoS ONE 8 e56429
Sorin C Bussell JD Camus I Ljung K Kowalczyk M Geiss G McKh-
ann H Garcion C Vaucheret H and Sandberg G (2005) Auxin and
light control of adventitious rooting in Arabidopsis require ARGONA-
UTE1 Plant Cell 17 1343ndash1359Tanaka T Tanaka H Machida C Watanabe M and Machida Y (2004) A
new method for rapid visualization of defects in leaf cuticle reveals five
intrinsic patterns of surface defects in Arabidopsis Plant J 37 139ndash146Turgeon R and Wolf S (2009) Phloem transport cellular pathways and
molecular trafficking Annu Rev Plant Biol 60 207ndash221Ukitsu H Kuromori T Toyooka K et al (2007) Cytological and biochemi-
cal analysis of COF1 an Arabidopsis mutant of an ABC transporter gene
Plant Cell Physiol 48 1524ndash1533Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De
Paepe A and Speleman F (2002) Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biol 3 research00340031-00340011Verrier PJ Bird D Burla B et al (2008) Plant ABC proteins ndash a unified
nomenclature andupdated inventory Trends Plant Sci 13 151ndash159Vilaine F Palauqui JC Amselem J Kusiak C Lemoine R and Dinant
S (2003) Towards deciphering phloem a transcriptome analysis of the
phloem of Apium graveolens Plant J 36 67ndash81Zhai Z Sooksa-nguan T and Vatamaniuk OK (2009) Establishing RNA
interference as a reverse-genetic approach for gene functional analysis
in protoplasts Plant Physiol 149 642ndash652Zhang Q Blaylock LA and Harrison MJ (2010) Two Medicago truncatula
half-ABC transporters are essential for arbuscule development in
arbuscular mycorrhizal symbiosis Plant Cell 22 1483
Zhao C Craig JC Petzold HE Dickerman AW and Beers EP (2005)
The xylem and phloem transcriptomes from secondary tissues of the
Arabidopsis root-hypocotyl Plant Physiol 138 803ndash818
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
824 Rozenn Le Hir et al
tissues (nine and eight out of 20 neutral amino acids
respectively) only two amino acids (glutamine and gluta-
mate) associated with Arabidopsis phloem cells (Schad
et al 2005) were different in abcg11ndash7 (Table 2) Xylose
and the non-reducing sugars trehalose and raffinose were
also decreased in abcg14ndash1 (Table 2) whereas glucose
fructose galactose maltose xylose were significantly dif-
ferent in abg9ndash2 and abcg11ndash7 (reduced and increased
respectively compared to wild-type) (Table 2)
Levels of sterols (campesterol 24ndashmethylene cholesterol
and sitosterol) saturated and unsaturated fatty acids (lino-
leic acid lauric acid palmitic acid and triacontanoic acid)
glycerolipids (monoacylglycerol) and lipid metabolism
intermediates (glycerol glycerol-3ndashphosphate and inositol-
1ndashphosphate) were also significantly different from wild-
type in abcg mutants (Table 2) The abcg9ndash2 mutant
showed 30 lower levels of 24ndashmethylene cholesterol
compared to the wild-type (Appendix S1) In the abcg14ndash1
mutant 50 and 35 decreases of 24ndashmethylene cholesterol
and sitosterol levels were measured respectively (Appen-
dix S1) Finally campesterol showed a 50 increase in the
abcg11ndash7 mutant (Appendix S1) To extend this analysis
measurements of conjugated sterols [sterol ester (SE)
sterol glucoside and acylated sterol glucoside] were also
performed When seedlings were grown in vitro no differ-
ence in the sterol glucoside and acylated sterol glucoside
contents was detected between the wild-type and the vari-
ous mutant lines (Figure S4d) However there was a ten-
dency towards a reduced SE content in the double mutant
abcg9ndash2 abcg14ndash1 compared to the wild-type (Figure S4d)
This observation prompted us to examine variations in the
SE content in older plants (4-week-old plants grown in soil
in the greenhouse) At this developmental stage no differ-
ence in the free sterol (FS) content was observed between
the wild-type and the various mutant lines (Figure 7d) but
the fraction comprising FS and SE was significantly
(a) (b) (c) (d)
(e) (f) (g) (h)
(i) (j)
(k) (l) (m)
(n) (o) (p)
Figure 5 Subcellular localization and physical interactions of ABCG9 ABCG11 and ABCG14
(andashh) ABCG9 and ABCG14 co-localize to the plasma membrane when transiently expressed in Arabidopsis mesophyll protoplasts of the GFPndashLTi6b marker line
(Cutler et al 2000)
(a e) Cyan fluorescence of GFPndashLTi6b(b f) Purple fluorescence from mRFPndashABCG9 and mRFPndashABCG14 respectively
(c g) Yellow auto-fluorescence of the chloroplasts
(d h) Merged images of the three channels Scale bar = 10 lm
(i j) Confocal images of immunofluorescence staining with anti-HA antibodies in root cells of abcg9ndash2 and abcg14ndash1 mutants complemented with HA-tagged
ABCG9 (i) and HA-tagged ABCG14 (j) Scale bars = 10 lm
(kndashp) Proteins were extracted from protoplasts co-transfected with HA- or cndashMyc-tagged versions of ABCGs (lanes 1 and 4) or transfected with a single plasmid
containing the HA-tagged version (lane 2) or the c-Myc-tagged version (lane 3) In lanes 2ndash4 proteins were immunoprecipitated with the anti-c-Myc antibody
and subjected to anti-HA protein gel-blot analysis to reveal the other partner of the dimer Transient co-expression of (k) HAndashABCG9cndashMycndashABCG9 (l) HAndashABCG11cndashMycndashABCG11 (m) HAndashABCG14cndashMycndashABCG14 (n) HAndashABCG14cndashMycndashABCG11 (o) HAndashABCG14cndashMycndashABCG9 and (p) HAndashABCG11cndashMycndashABCG9
in wild-type protoplasts
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 817
reduced probably accounting for the difference in the SE
content between the wild-type and the abcg9ndash2 abcg14ndash1
double mutant (Figure 7e)
(a)
(b)
(c) (d) (e)
(f) (g)
Figure 6 Long chain and very long chain fatty acid content and cuticle
integrity in abcg9ndash2 and abcg14ndash1 mutants
(a b) Fatty acid (a) and VLCFA content (b) in abcg9ndash2 and abcg14ndash1 mutants
in apical parts of 10-day-old seedlings (three biological replicates each of
approximately 5 mg dry weight were used)
(cndashf) Toluidine blue staining patterns of abcg mutants in 7-day-old seed-
lings Scale bars = 3 cm
(a)
(b)
(c)
(d) (e)
Figure 7 Metabolome analysis and measurements of sterol content in abcg
mutants
(andashc) Results of orthogonal partial least-squares discriminant analysis of
global metabolite contents of shoots of 7-day-old seedlings from wild-type
(black squares) abcg11ndash7+ (gray triangles) abcg11ndash7 (white stars) abcg9ndash1(white hexagons) abcg9ndash2+ (gray hexagons) abcg9ndash2 (white hexagons)
and abcg14ndash1 (black circles) seedlings (five biological replicates of 10 mg
each were used for the analysis)
(d e) Free sterol (FS) and sterol ester (SE) content of wild-type (white bars)
abcg9ndash2 (light gray bars) abcg14ndash1 (dark gray bars) and abcg9ndash2 abcg14ndash1(black bars) Error bars indicate standard deviations obtained from three
independent biological replicates The asterisk indicates a statistically signif-
icant difference between the wild-type and the mutant (P lt 005 Studentrsquos
t test)
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
818 Rozenn Le Hir et al
DISCUSSION
ABCG9 and ABCG14 are two plasma membrane proteins
that interact physically in multiple homo-heterodimer
combinations
In this study we showed that like the previously described
ABCG11 (Pighin et al 2004 Bird et al 2007 Luo et al
2007 Panikashvili et al 2007 2010 Ukitsu et al 2007)
ABCG9 and ABCG14 localize to the plasma membrane
Based on co-immunoprecipitation of tagged proteins and
the bimolecular fluorescence complementation assay we
demonstrated that ABCG9 interacts with ABCG11 to form a
heterodimer and like ABCG11 (McFarlane et al 2010) can
homodimerize In contrast ABCG14 interacts only with
ABCG11 forming an obligate heterodimer This extends
the number of potential partners of ABCG11 to three as it
was previously shown to dimerize with the ABCG12CER5
protein (McFarlane et al 2010) Similarly ABCG9 under-
goes flexible dimerization to form either homo- or hetero-
dimers suggesting that ABCGs may be promiscuous
proteins with different functions depending on their inter-
acting partner andor expression profile
Mutations in ABCG genes are semi-dominant
In the last few years several Arabidopsis mutants with
altered expression of various ABCG genes such as ABCG11
(Bird et al 2007 Luo et al 2007 Panikashvili et al 2007
Ukitsu et al 2007) ABCG12CER5 (Pighin et al 2004)
ABCG13 (Panikashvili et al 2011) ABCG22 (Kuromori
et al 2011a) ABCG25 (Kuromori et al 2010) and ABCG26
(Choi et al 2010 Quilichini et al 2010 Dou et al 2011
Kuromori et al 2011b) have been characterized but even
though these half-size transporters require dimerization to
become functional all the mutations were described as
recessive Here we demonstrated that mutations in these
genes have semi-dominant effects Indeed the metabolome
analyses showed that abcg9ndash2+ and abcg11ndash7+ heterozy-
gous seedlings have a profile intermediate between that of
wild-type and homozygous seedlings The possibility of
dominant-negative effects was excluded because no
Table 2 Shoot metabolites of 10-day-old abcg9ndash2 abcg11ndash7 and abcg14ndash1 seedlings whose levels were significantly different from wild-type
Group
Metabolites that differed between mutant and wild-type
abcg9ndash2Colndash0 abcg11ndash7Colndash0 abcg14ndash1Colndash0
Amino acids Glycine Glutamate AlanineAlanine Glutamine ValineValine ThreonineThreonine GABASerine Prolinebndashalanine IsoleucineIsoleucine SerineGABA bndashalanineProline
Carbohydrates Glucose Glucose ErythritolFructose Fructose TrehaloseXylose Galactose XyloseMaltose RaffinoseSucrose
Lipids and intermediates 24ndashmethylene-cholesterol Campesterol Linoleic acidMonoacylglycerol Glycerol-3ndashphosphate Triacontanoic acidPalmitic acid Inositol-1ndashphosphate GlycerolGlycerol-3ndashphosphate Lauric acidInositol-1ndashphosphate 24ndashmethylene-cholesterol
SitosterolGlycerol-3ndashphosphate
TCA cycle and others Phosphoric acid Phosphate compound Glyceric acid24ndashdihydroxybutanoic acid Dehydroascorbic acid Fumaric acidGlyceric acid Malic acid Threonic acid-14ndashlactoneFumaric acid 2ndashoxoglutaric acid Threonic acidThreonic acid bndashhydroxy-bndashmethylglutaric acid Shikimic acidThreonic acid-14ndashlactone Gluconic acid lactone-like Succinic acidShikimic acid Phytol2ndashoxoglutaric acid Ethanolamine
Nicotinic acid
For abcg9ndash2 and abcg14ndash1 the levels of the compounds were reduced whereas they were increased in abcg11ndash7
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 819
truncated transcript was found for either abcg9ndash2 or
abcg11ndash7 mutants Hence abcg9ndash2 and abcg11ndash7 are true
null alleles These observations are consistent with the
requirement for homo- or heterodimerization of the ABCG
proteins to form functional transporters In the heterozy-
gous plants ABCG11 transcript levels were 50 lower than
in wild-type counterparts suggesting that ABCG11 protein
and thus corresponding homo- and heterodimers were
likely to be less abundant which may also explain the meta-
bolic perturbations and consequent phenotypic modifica-
tions These findings also suggest that wild-type plants are
not saturated by ABCG11 proteins as a 50 reduction in
protein content appears to be sufficient to alter the physiol-
ogy of the heterozygous mutant plants This hypothesis is
strengthened by the observation that mutants carrying the
weak abcg11ndash6 allele in which there was only a 25 reduc-
tion in ABCG11 transcript levels showed a similar pheno-
type to the heterozygous abcg11ndash7+ and abcg11ndash8+
plants Similarly the abcg9ndash1 weak allele mutant had a very
similar phenotype to the abcg9ndash2+ heterozygous seedlings
suggesting that considering the mode of action of these
proteins the semi-dominant effects that we observed are
likely to be a general feature among the ABCG genes
ABCG9 ABCG11 and ABCG14 transporters are involved in
vascular system development in Arabidopsis
We showed that the ABCG11 ABCG9 and ABCG14 promot-
ers drove expression in the vascular system of Arabidopsis
cotyledons and rosette leaves and more precisely in the
phloem of flower stems In addition mutations in ABCG9
ABCG11 and ABCG14 genes altered vascular development
in cotyledons and the floral stem Similar vascular localiza-
tion of ABCG11 has already been described by Panikashvili
et al (20072010) who showed that in addition to the epi-
dermis ABCG11 is expressed in the vasculature as early as
during embryo development and then throughout plant
development They showed that the vascular pattern in
leaves of the homozygote mutant seedlings was less com-
plex than in the wild-type and discontinuous (Panikashvili
et al 2007) The present article describes a mutant that
has both cuticle and vascular system patterning defects
although it has been previously shown that mutants
altered in cuticle formation have dramatically reduced
growth and altered leaf morphology (Jenks et al 1996)
which may account for vascular defects Analysis of the
abcg11+ heterozygous seedlings that have vascular
defects but apparently no cuticle alteration suggested that
these phenotypes are probably uncoupled Because
ABCG11 dimerizes with several members of its sub-family
it seems reasonable to postulate that it is involved in the
transport of different compounds depending on its partner
Here we provide evidence that ABCG11 in addition to
forming dimers with ABCG12 (McFarlane et al 2010) in
epidermal cells is likely to dimerize with ABCG9 and
ABCG14 in phloem cells suggesting functional compart-
mentalization of these proteins and a possible role in vas-
cular system patterning in Arabidopsis
Metabolite profiling suggests that ABCG9 ABCG11 and
ABCG14 are involved in sterollipid homeostasis
We investigated in more depth the metabolic changes
induced by mutations in ABCG9 ABCG11 and ABCG14
We found that levels of metabolites of several classes
including lipids sugars and amino acids were altered in
both the heterozygotes and homozygotes and that the
profiles in the root and apical parts differed suggesting
that ABCGs may play different roles in the roots and shoot
Interestingly the modifications of amino acid and sugar
contents observed in the metabolic profiles of the abcg
mutants suggest an impaired carbonnitrogen balance that
may be directly or indirectly linked to the vascular defects
leading to possible problems with phloem loading or
unloading Saturated and unsaturated fatty acids and sterol
contents were also altered in the mutants Patterning
defects in the vascular system are often observed in
mutants altered in hormone homeostasis such as for
auxin or brassinosteroids (Kaneda et al 2011) and direct
or indirect modifications of sterol homeostasis are thought
to be one of the primary causes of these downstream
effects (Pullen et al 2010) The detected defects in sterol
homeostasis may explain the abnormal vascular develop-
ment observed in abcg9 abcg11 and abcg14 mutants
Accordingly several sterol-deficient mutants such as fac-
kelhydra (Jang et al 2000) and cpv1 (Carland et al 2002)
have also been shown to exhibit severe defects in vascular
development It has been suggested that the bushy habit
and low fertility of cpv1smt2 mutants may be due to an
increased campesterolsitosterol ratio rather than sterol
deficiency per se (Clouse 2002) This ratio is controlled by
the activity of SMT2 (STEROL METHYLTRANSFERASE2)
Interestingly strong down-regulation of SMT2 expression
was observed in all the mutant backgrounds (Figure S5)
and the metabolic data indicated a deficit in 24ndashmethylene
cholesterol (precursor of both campesterol and sitosterol)
andor sitosterol in abcg9 and abcg14 mutants respec-
tively In addition the significant decrease in sterol ester
content in the double mutant abcg9ndash2 abcg14ndash1 correlated
with down-regulation of expression of the PHOSPHOLIPID
STEROL ACYLTRANSFERASE1 (PSAT1) gene encoding the
enzyme that is mainly responsible for SE synthesis (Bou-
vier-Nave et al 2010) and is proposed to be a control step
in sterol homeostasis (Kopischke et al 2013) Altogether
these results suggest that the vascular phenotype
observed in abcg mutants may be linked to modification of
sterol homeostasis
Therefore we postulate that the various dimer combina-
tions between ABCG9 ABCG11 and ABCG14 may
participate in the transport of squalene-derived metabolites
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
820 Rozenn Le Hir et al
such as sterol andor sterol conjugates via the phloem sap
The presence of sterols in phloem sap has been observed in
various species such as barley (Hordeum vulgare Lehrer
et al 2000) and fava bean (Vicia faba Bouvaine et al
2012) and indirect proof of the presence of sterols in
phloem sap has been obtained by analysis of the phytos-
terol content in exclusively phloem-feeding aphids that are
unable to synthetize them directly (Behmer et al 2011) In
addition the evidence for long-distance transport of sterol
precursors has been supported by observations that sterol
esters accumulate in organs distal to the site of application
of exogenous precursors (Bouvier-Nave et al 2010) As
sterols have been proposed to act as signal molecules in
the control of developmental processes such as vascular
patterning (Carland et al 2002 Pullen et al 2010) modifi-
cations of sterol homeostasis may affect development of
the vascular system in the abcgmutants described here
In conclusion we propose that ABCG9 ABCG11 and
ABCG14 are important for lipidsterol homeostasis Thus
these three proteins are likely to be involved in the control
of various aspects of plant development andor physiol-
ogy including patterning of the vascular system
EXPERIMENTAL PROCEDURES
Plant material and growth conditions
Insertion lines in ABCG9 (SALK_047133 and GABI_353B01)ABCG11 (SALK_096377 and GABI_590C03) and ABCG14 (SALK_036952) genes were obtained from the European ArabidopsisStock Centre (httparabidopsisinfo) Heterozygoushomozygousplants were screened using gene-specific primers in combinationwith the specific primer for the left border of the TndashDNA insertion(Table S1)
Plants were grown in soil or in vitro as described previously(Sorin et al 2005) Arabidopsis Colndash0 cell cultures were main-tained by weekly sub-culturing in MS medium (Murashige andSkoog 1962) supplemented with 240 lg l1 24ndashdichlorophenoxy-acetic acid and 14 lg l1 kinetin and were grown under 12 h light12 h dark cycles at 25 lE m2 sec1 and with 23degC day and nighttemperatures
Morphological characterization and GUS staining
To study the venation pattern cotyledons of 7-day-old seedlingsand rosette leaves of 4-week-old plants were cleared in a buffercontaining chloral hydrate glycerol and water (831 wvv) afterwhich 200 cotyledons per genotype were analyzed and the per-centage showing particular levels of complexity or discontinuity invascularity was estimated as described by Cnops et al (2006)
Flower stem segments (1ndash2 cm long) were removed from ten3ndash4-week-old plants grown in soil embedded in 8 agarose andsectioned using a VT100S vibratome (Leica httpwwwleicacom)The cross-sections were then stained with safraninalcian blue for1ndash2 min (Sigma httpwwwsigmaaldrichcom) rinsed in waterand mounted in 50 glycerol
Histochemical GUS assays were performed as described previ-ously (Sorin et al 2005) All observations were performed usingan Axioplan light microscope equipped with an Axiocam cameraand Axiovision software (Zeiss httpwwwzeisscom)
To observe potential cuticle defects in the various mutant linesa TB test was performed by immersing in vitro-grown plantlets ina 005 wv TB solution for 2 min as described by Tanaka et al(2004)
Statistical analyses
Statistical analyses of differences between-genotype means andvariance for the studied morphological traits were performedusing Studentrsquos t test (GraphPad Prism version 50 httpwwwgraphpadcom) In the venation pattern experiment thesignificance of differences between wild-type and mutants wasassessed using R (R Development Core Team 2010) and Pearsonrsquosv2 test with Monte Carlo permutations (N = 10 000)
Plasmid constructs
The native ABCG promoters and open reading frame of ABCG11were amplified from genomic DNA or cDNA extracted from 7-day-old Arabidopsis seedlings using Phusion high-fidelity DNApolymerase (Finnzyme wwwthermoscientificbiocomfinnzymes)according to the manufacturerrsquos instructions with gene-specificprimers (Table S1) The PCR products were cloned into pENTRDndashTOPO (Invitrogen httpwwwinvitrogencom) and recombinedinto pKGWFS7 (Karimi et al 2002) to create ABCGproGUS Allrecombinations were performed using the Gateway LR Clonase
enzyme mix (Invitrogen) At each cloning step the sequenceswere confirmed by sequencing and all the constructs weretransferred into Agrobacterium strain GV3101pMP90 (C58C1)Wild-type or abcg11ndash7+ heterozygous plants were transformed byfloral dipping (Clough and Bent 1998) For ABCGGUS constructsthe expression pattern was checked in the T2 progeny of 10ndash15independent transgenic lines and one representative homozygousline was used for further characterization
Tagged protein constructs and protoplast transformation
Epitope-tagged versions of ABCG9 ABCG11 and ABCG14 proteinswere produced in pRT104ndash3xHA and pRT104ndash3xMyc plasmids(Feurouleuroop et al 2005) and pSAT6-mRFP (Citovsky et al 2006) Allthese plasmids have a 35S promoter sequence upstream of themulti-cloning site The open reading frames of ABCG11 andABCG14 and the genomic clone of ABCG9 were amplified fromcDNA or genomic DNA extracted from 7-day-old Arabidopsisseedlings using Phusion high-fidelity DNA polymerase (Finnzyme)according to the manufacturerrsquos instructions with gene-specificprimers carrying EcoRI or SalI restriction sites to facilitate subse-quent cloning (Table S1) The products obtained after PCR weredigested by EcoRI and SalI prior to ligation into appropriate plas-mids that had previously been cut open with the same restrictionenzymes Constructs were verified by sequencing Protoplastsfrom Arabidopsis cell cultures or 14-day-old Arabidopsis seedlingswere prepared and transformed as previously described (Meski-ene et al 2003 Zhai et al 2009) For co-immunoprecipitation andsubcellular localization assays 100 000 and 50 000 protoplastcells respectively were transfected with 15 and 10 ll of plasmidsrespectively (containing between 5 and 75 lg of each construct)and the resulting transformed protoplasts were processed afterincubation in the dark at room temperature for 16ndash18 and 24 hrespectively
Whole-mount immunolocalization
The HA-tagged versions of ABCG9 and ABCG14 were detected in7-day-old in vitro-grown Arabidopsis seedlings as described inMethods S1 Anti-HA antibody 12CA5 (Roche httpwwwroche
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 821
com) (1100 dilution) combined with Alexa Fluor 488-conjugatedsecondary antibody (1100 dilution) were used
Confocal laser-scanning microscopy
Images were collected using an SP2 confocal microscope (Leica)RFP and Alexa Fluor 488 were detected using laser lines at 561and 488 nm respectively according to the manufacturerrsquos set-tings RFP fluorescence emission was detected at 550ndash600 nmand Alexa Fluor 488 and GFP fluorescence emission were detectedat 495ndash545 nm For subcellular localization in protoplasts imageswere coded in cyan (GFP) magenta (RFP) and yellow (auto-fluo-rescence) resulting in purple coloration for co-localization inmerged images For the immunolocalization experiment imageswere coded in green (GFP) A water-corrected 209 objective (HCPlan-Apochromat 20907 ImmKor Leica) was used Images werecropped using Adobe Photoshop CS2 and assembled using AdobeIllustrator CS2 software (Abode httpwwwabodecom) Eachimage shown represents a single focal plan
Co-immunoprecipitation assays
Transformed protoplast proteins were extracted using 50 ll lysisbuffer containing 25 mM TrisHCl pH 78 10 mM MgCl2 75 mM
NaCl 5 mM EGTA 1 mM benzamidine 1 mM dithiothreitol 10glycerol 1 Triton Xndash100 and 19 protein inhibitor cocktail(Sigma httpsigmaaldrichcom) The transformed protoplast sus-pension was frozen in liquid nitrogen then thawed on ice andsubjected to centrifugation for 5 min at 150 g Two microliters ofanti-Myc (9E10 Covance httpwwwcovancecom) or anti-HA(16B12 Covance) antibodies were added to the extracts and themixture was incubated for 2 h at 4degC on a rotating wheel Immu-nocomplexes were captured by incubation with 10 ll Protein GndashSepharose beads for a further 2 h at 4degC on a rotating wheel afterwhich they were washed three times for 5 min each in 25 mM
NaPi 150 mM NaCl 5 glycerol and 02 Igepal CAndash630 buffer(Sigma) and then eluted using 40 ll SDS sample buffer at 37degCfor 30 min Co-immunoprecipitation of Myc- and HA-tagged pro-teins was revealed by SDSndashPAGE followed by Western blottingusing both anti-Myc (Invitrogen) and anti-HA (3F10 Roche) anti-bodies at 12000 dilution
RNA extraction and cDNA preparation
To quantify the relative amounts of the ABCG transcriptsexpressed in various organs three rosette leaves all the cau-line leaves the bottom 5 cm of the floral stem all closed flow-ers and all green siliques were harvested from each of threeindependent 4-week-old Arabidopsis plants of each genotypegrown in soil For the other experiments 10-day-old seedlingsgrown in vitro were harvested All the material was directly fro-zen in liquid nitrogen and stored at 80degC prior to RNA extrac-tion Samples were then disrupted using a MM 301 vibrationmill (Retsch GmbH httpwwwretschcom) at a frequency of25 Hz for 2 min with 3 mm tungsten carbide beads (Qiagenhttpwwwqiagencom) Total RNA was isolated from thismaterial using TRIzol (Life Technologies httplifetechnologiescom) RNA quantity and quality were measured using a Nano-Drop ND 1000 (Thermo Scientific httpwwwthermoscientificcom) All samples with 260280 nm and 260230 nm absorbanceratios lower than 18 were discarded and RNA was re-extractedDNase treatment was applied to 5 lg samples of total RNAusing a DNase I kit (Fermentas httpwwwfermentascom)Double-stranded cDNA was synthesized from 5 lg total RNAusing a SuperScript reverse transcriptase kit (Invitrogen) witholigo(dT)12 primer Finally RNase treatment was applied to the
samples using Escherichia coli RNAse H (Fermentas) The cDNAsamples obtained were diluted 30-fold (except for abcg9 linesamples which were diluted 10-fold) prior to use either toverify the absence of full-length transcripts in putative knockoutmutants or to quantify gene expression by quantitative PCR Allprimers used were designed using the Primer3 program (Rozenand Skaletsky 2000) and their sequences are listed inTable S1
QUANTITATIVE REAL-TIME PCR
Quantitative PCR amplifications were performed in 20 llmixtures containing 10 ll 59 SYBR Green I master mix
(Roche) 02 ll forward and reverse primer (30 lM each)
48 ll sterile water and 5 ll cDNA The BiondashRad CFX96
real-time PCR detection system (httpwwwbio-radcom)
was used for quantification of expression of the sterol
biosynthesis transcripts and the ABCG transcripts in the
various mutant backgrounds Finally the Roche LightCy-
cler 480 system (Roche) was used to quantify the rela-
tive abundance of ABCG transcripts in various organs
Four genes were considered as potential reference genes
[APT1 (At1g27450) TIP41 (At4g34270) EF1a (At5g60390)
and UBQ10 (At4g05320)] and their suitability was tested
using the geNorm algorithm (Vandesompele et al 2002)
to determine the most appropriate one for each experi-
ment A more detailed procedure following the Minimum
Information for Publication of Quantitative Real-Time PCR
Experiments guidelines (Bustin et al 2009) is described
in Methods S2 and the annealing temperature and
efficiencies of the primer pairs used are shown in
Table S2
VLCFA quantification
Approximately 5 mg dry weight of 10-day-old in vitro
Arabidopsis seedlings were analyzed for VLCFA quantifica-
tion as described by Li et al (2006)
Sterol extraction and GC-FID characterization
A pool of 10-day-old seedlings grown in vitro or rosettes
from 4-week-old plants grown in soil were lyophilized prior
to extraction Sterol extraction and isolation was per-
formed as described previously (Bouvier-Nave et al 2010)
Gas Chromatography-Flame Ionization Detector (GC-FID)
characterization was performed as described by Silvestro
et al (2013) To quantify the total amounts of free sterols
and sterol esters non-saponifiable lipids were extracted
using nndashhexane and sterols were derivatized as sterols ace-
tates Quantification was achieved using lupenyl-3 28ndashdi-
acetate as an internal standard
Metabolome analysis
To obtain metabolome profiles associated with wild-type
abcg11ndash7+ abcg11ndash7 abcg9ndash1 abcg9ndash2 and abcg14ndash1
genotypes 10-day-old seedlings representing each geno-
type grown in vitro as described above were harvested
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
822 Rozenn Le Hir et al
and divided into shoot and root samples In the case of
abcg9ndash2 the phenotype of the heterozygote abcg9ndash2+
was indistinguishable from the homozygote phenotype
and therefore heterozygous and homozygous seedlings
from a segregating population were pooled Hence in this
particular experiment abcg9ndash2+ refers to a mixed popula-
tion comprising two-thirds heterozygotes and one-third
homozygotes
Metabolites in five samples of shoot and root parts of
seedlings of each genotype (10 mg fresh weight per sam-
ple) were then extracted analyzed by GCMS and the
acquired data were processed as previously described
(Gullberg et al 2004 Jonsson et al 2005) All multivariate
statistical investigations (partial component analysis and
orthogonal partial least-squares discriminant analysis) for
between-genotype similarities and differences in general
metabolite profiles were performed using Simca software
(Umetrics httpwwwumetricscom)
ACKNOWLEDGEMENTS
We thank A Murphy (Plant Science and Landscape Archi-
tecture University of Maryland MD USA) for stimulating
discussions and critical reading of the manuscript I Carl-
son (Umea Plant Science Center Department of Forest
Genetics and Plant Physiology Swedish University of Agri-
cultural Sciences Umea Sweden) for technical assistance
and S Vernhettes for kindly donating seeds of the
transgenic line carrying the plasma membrane marker
GFPndashLTi6b We also thank the European Arabidopsis Stock
Centre and the GABI-Kat stock centre for providing seeds
This work was supported by allocations granted to CB by
the Swedish Research Council for Agriculture the Swedish
Foundation for Strategic Research the Swedish Research
Council for Research and Innovation for Sustainable
Growth the K amp A Wallenberg Foundation and the Carl
Trygger Foundation
SUPPORTING INFORMATION
Additional Supporting Information may be found in the onlineversion of this articleAppendix S1 Raw data quantification for compounds identified inthe metabolite profiling by GCMS
Figure S1 Relative expression of ABCG genes in various organsand mutant backgroundsFigure S2 Co-immunoprecipitation controlsFigure S3 ABCG9 and ABCG14 homo- andor heterodimerize withABCG11Figure S4 Complementary data on metabolome analysis and con-jugated sterol contentFigure S5 Relative expression of genes related to sterol homeo-stasisMethods S1 Method for whole-mount immunolocalizationMethods S2 Method for quantitative PCR
Table S1 List of primers used in this studyTable S2 Annealing temperature and efficiencies for the primerspairs used in this study
REFERENCES
Behmer ST Grebenok RJ and Douglas AE (2011) Plant sterols and host
plant suitability for a phloem-feeding insect Funct Ecol 25 484ndash491Bird D Beisson F Brigham A Shin J Greer S Jetter R Kunst L
Wu XW Yephremov A and Samuels L (2007) Characterization of Ara-
bidopsis ABCG11WBC11 an ATP binding cassette (ABC) transporter that
is required for cuticular lipid secretion Plant J 52 485ndash498Bouvaine S Behmer ST Lin GG Faure MndashL Grebenok RJ and
Douglas AE (2012) The physiology of sterol nutrition in the pea aphid
Acyrthosiphon pisum J Insect Physiol 58 1383ndash1389Bouvier-Nave P Berna A Noiriel A Compagnon V Carlsson AS
Banas A Stymne S and Schaller H (2010) Involvement of the phos-
pholipid sterol acyltransferase1 in plant sterol homeostasis and leaf
senescence Plant Physiol 152 107ndash119Bustin SA Benes V Garson JA Hellemans J Huggett J Kubista
M Mueller R Nolan T Pfaffl MW and Shipley GL (2009) The MIQE
guidelines Minimum information for publication of quantitative real--
time PCR experiments Clin Chem 55 611
Carland FM Fujioka S Takatsuto S Yoshida S and Nelson T (2002)
The identification of CVP1 reveals a role for sterols in vascular pattern-
ing Plant Cell 14 2045ndash2058Choi H Jin JY Choi S Hwang JU Kim YY Suh MC and Lee Y
(2010) An ABCGWBC-type ABC transporter is essential for transport of
sporopollenin precursors for exine formation in developing pollen Plant
J 65 181ndash193Citovsky V Lee LY Vyas S Glick E Chen MH Vainstein A Gafni
Y Gelvin SB and Tzfira T (2006) Subcellular localization of interacting
proteins by bimolecular fluorescence complementation in planta J Mol
Biol 362 1120ndash1131Clough SJ and Bent AF (1998) Floral dip a simplified method for Agro-
bacterium-mediated transformation of Arabidopsis thaliana Plant J 16
735ndash743Clouse SD (2002) Arabidopsis mutants reveal multiple roles for sterols in
plant development Plant Cell 14 1995ndash2000Cnops G Neyt P Raes J et al (2006) The TORNADO1 and TORNADO2
genes function in several patterning processes during early leaf develop-
ment in Arabidopsis thaliana Plant Cell 18 852ndash866Cutler SR Ehrhardt DW Griffitts JS and Somerville CR (2000) Random
GFPcDNA fusions enable visualization of subcellular structures in cells of
Arabidopsis at a high frequency Proc Natl Acad Sci USA 97 3718ndash3723Dou XY Yang KZ Zhang Y Wang W Liu XL Chen LQ Zhang
XQ and Ye D (2011) WBC27 an adenosine tri-phosphate-binding cas-
sette protein controls pollen wall formation and patterning in Arabidop-
sis J Integr Plant Biol 53 74ndash88Feurouleuroop K Pettko-Szandtner A Magyar Z Miskolczi P Kondorosi E
Dudits D and Bako L (2005) The Medicago CDKC1ndashCYCLINT1 kinase
complex phosphorylates the carboxy-terminal domain of RNA polymer-
ase II and promotes transcription Plant J 42 810ndash820Gullberg J Jonsson P Nordstrom A Sjostrom M and Moritz T (2004)
Design of experiments an efficient strategy to identify factors influenc-
ing extraction and derivatization of Arabidopsis thaliana samples in met-
abolomic studies with gas chromatographymass spectrometry Anal
Biochem 331 283ndash295Hertzberg M Aspeborg H Schrader J et al (2001) A transcriptional
roadmap to wood formation Proc Natl Acad Sci USA 98 14732ndash14737Jang JC Fujioka S Tasaka M Seto H Takatsuto S Ishii A Aida M
Yoshida S and Sheen J (2000) A critical role of sterols in embryonic
patterning and meristem programming revealed by the fackel mutants of
Arabidopsis thaliana Genes Dev 14 1485ndash1497Jenks MA Rashotte AM Tuttle HA and Feldmann KA (1996) Mutants
in Arabidopsis thaliana altered in epicuticular wax and leaf morphology
Plant Physiol 110 377ndash385Jonsson P Johansson AI Gullberg J Trygg J A J Grung B Markl-
und S Sjostrom M Antti H and Moritz T (2005) High-throughput
data analysis for detecting and identifying differences between samples
in GCMS-based metabolomic analyses Anal Chem 77 5635ndash5642Jungwirth H and Kuchler K (2006) Yeast ABC transporters ndash a tale of sex
stress drugs and aging FEBS Lett 580 1131ndash1138Kaneda M Schuetz M Lin B Chanis C Hamberger B Western T
Ehlting J and Samuels A (2011) ABC transporters coordinately
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ABCG proteins and vascular development 823
expressed during lignification of Arabidopsis stems include a set of AB-
CBs associated with auxin transport J Exp Bot 62 2063
Karimi M Inze D and Depicker A (2002) GATEWAYTMvectors for Agrobac-
terium-mediated plant transformation Trends Plant Sci 7 193ndash195Kneurooller A and Murphy A (2011) ABC transporters and their function at the
plasma membrane In The Plant Plasma Membrane Plant Cell Mono-
graphs vol 19 (Murphy AS Peer W and Schulz B eds) New York
NY Springer pp 353ndash377Kopischke M Westphal L Schneeberger K Clark R Ossowski S
Wewer V Fuchs R Landtag J Hause G and Deuroormann P (2013)
Impaired sterol ester synthesis alters the response of Arabidopsis thali-
ana to Phytophthora infestans Plant J 73 456ndash468Kuromori T Miyaji T Yabuuchi H Shimizu H Sugimoto E Kamiya
A Moriyama Y and Shinozaki K (2010) ABC transporter AtABCG25 is
involved in abscisic acid transport and responses Proc Natl Acad Sci
USA 107 2361ndash2366Kuromori T Itoh T Sugimoto E and Shinozaki K (2011a) Arabidopsis
mutant of AtABCG26 an ABC transporter gene is defective in pollen
maturation J Plant Physiol 168 2001ndash2005Kuromori T Sugimoto E and Shinozaki K (2011b) Arabidopsis mutants
of AtABCG22 an ABC transporter gene increase transpiration and
drought susceptibility Plant J 67 885ndash894Le Hir R Beneteau J Bellini C Vilaine F and Dinant S (2008) Gene
expression profiling keys for investigating phloem functions Trends
Plant Sci 13 273ndash280Lehrer AT Dugassa-Gobena D Vidal S and Seifert K (2000) Transport
of resistance-inducing sterols in phloem sap of barley Z Naturforsch C
55 948ndash952Li Y Beisson F Pollard M and Ohlrogge J (2006) Oil content of Arabid-
opsis seeds the influence of seed anatomy light and plant-to-plant vari-
ation Phytochemistry 67 904ndash915Luo B Xue XndashY Hu WndashL Wang LndashJ and Chen XndashY (2007) An ABC
transporter gene of Arabidopsis thaliana AtWBC11 is involved in cuticle
development and prevention of organ fusion Plant Cell Physiol 48
1790ndash1802McFarlane HE Shin JJH Bird DA and Samuels AL (2010)
Arabidopsis ABCG transporters which are required for export of diverse
cuticular lipids dimerize in different combinations Plant Cell 22 3066ndash3075
Meskiene I Baudouin E Schweighofer A Liwosz A Jonak C Rodri-
guez PL Jelinek H and Hirt H (2003) Stress-induced protein phospha-
tase 2C is a negative regulator of a mitogen-activated protein kinase
J Biol Chem 278 18945ndash18952Murashige T and Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures Physiol Plant 15 473ndash497Panikashvili D Savaldi-Goldstein S Mandel T Yifhar T Franke RB
Hofer R Schreiber L Chory J and Aharoni A (2007) The Arabidopsis
DESPERADOAtWBC11 transporter is required for cutin and wax secre-
tion Plant Physiol 145 1345ndash1360Panikashvili D Shi JX Bocobza S Franke RB Schreiber L and Aha-
roni A (2010) The Arabidopsis DSOABCG11 transporter affects cutin
metabolism in reproductive organs and suberin in roots Mol Plant 3
563ndash575Panikashvili D Shi JX Schreiber L and Aharoni A (2011) The Arabid-
opsis ABCG13 transporter is required for flower cuticle secretion and pat-
terning of the petal epidermis New Phytol 190 113ndash124Pighin JA Zheng H Balakshin LJ Goodman IP Western TL Jetter
R Kunst L and Samuels AL (2004) Plant cuticular lipid export requires
an ABC transporter Science 306 702ndash704Pullen M Clark N Zarinkamar F Topping J and Lindsey K (2010)
Analysis of vascular development in the hydra sterol biosynthetic
mutants of Arabidopsis PLoS ONE 5 e12227
Quilichini TD Friedmann MC Samuels AL and Douglas CJ (2010)
ATP-binding cassette transporter G26 is required for male fertility and
pollen exine formation in Arabidopsis Plant Physiol 154 678ndash690R Development Core Team (2010) R A Language and Environment for
Statistical Computing Vienna Austria R Foundation for Statistical
Computing
Roudier F Gissot L Beaudoin F Haslam R Michaelson L Marion J
Molino D Lima A Bach L and Morin H (2010) Very-long-chain fatty
acids are involved in polar auxin transport and developmental patterning
in Arabidopsis Plant Cell 22 364ndash375Rozen S and Skaletsky HJ (2000) Primer3 on the WWW for general users
and for biologist programmers Methods Mol Biol 132 365ndash386Schad M Lipton MS Giavalisco P Smith RD and Kehr J (2005) Eval-
uation of two-dimensional electrophoresis and liquid chromatography
tandem mass spectrometry for tissue-specific protein profiling of
laser-microdissected plant samples Electrophoresis 26 2729ndash2738Schauer N Steinhauser D Strelkov S et al (2005) GCndashMS libraries for
the rapid identification of metabolites in complex biological samples
FEBS Lett 579 1332ndash1337Schrader J Nilsson J Mellerowicz EJ Berglund A Nilsson P Hertz-
berg M and Sandberg G (2004) A high-resolution transcript profile
across the wood forming meristem of poplar identifies potential regula-
tors of cambial stem cell identity Plant Cell 16 2278ndash2292Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desi-
mone M Frommer WB Fleurougge UndashI and Kunze R (2003) ARAMEM-
NON a novel database for Arabidopsis integral membrane proteins
Plant Physiol 131 16ndash26Silvestro D Andersen TG Schaller H and Jensen PE (2013) Plant ste-
rol metabolism D7ndashsterol-C5ndashdesaturase (STE1DWARF7) D57ndashste-rol-D7ndashreductase (DWARF5) and D24ndashsterol-D24ndashreductase (DIMINUTO
DWARF1) show multiple subcellular localizations in Arabidopsis thaliana
(Heynh) L PLoS ONE 8 e56429
Sorin C Bussell JD Camus I Ljung K Kowalczyk M Geiss G McKh-
ann H Garcion C Vaucheret H and Sandberg G (2005) Auxin and
light control of adventitious rooting in Arabidopsis require ARGONA-
UTE1 Plant Cell 17 1343ndash1359Tanaka T Tanaka H Machida C Watanabe M and Machida Y (2004) A
new method for rapid visualization of defects in leaf cuticle reveals five
intrinsic patterns of surface defects in Arabidopsis Plant J 37 139ndash146Turgeon R and Wolf S (2009) Phloem transport cellular pathways and
molecular trafficking Annu Rev Plant Biol 60 207ndash221Ukitsu H Kuromori T Toyooka K et al (2007) Cytological and biochemi-
cal analysis of COF1 an Arabidopsis mutant of an ABC transporter gene
Plant Cell Physiol 48 1524ndash1533Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De
Paepe A and Speleman F (2002) Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biol 3 research00340031-00340011Verrier PJ Bird D Burla B et al (2008) Plant ABC proteins ndash a unified
nomenclature andupdated inventory Trends Plant Sci 13 151ndash159Vilaine F Palauqui JC Amselem J Kusiak C Lemoine R and Dinant
S (2003) Towards deciphering phloem a transcriptome analysis of the
phloem of Apium graveolens Plant J 36 67ndash81Zhai Z Sooksa-nguan T and Vatamaniuk OK (2009) Establishing RNA
interference as a reverse-genetic approach for gene functional analysis
in protoplasts Plant Physiol 149 642ndash652Zhang Q Blaylock LA and Harrison MJ (2010) Two Medicago truncatula
half-ABC transporters are essential for arbuscule development in
arbuscular mycorrhizal symbiosis Plant Cell 22 1483
Zhao C Craig JC Petzold HE Dickerman AW and Beers EP (2005)
The xylem and phloem transcriptomes from secondary tissues of the
Arabidopsis root-hypocotyl Plant Physiol 138 803ndash818
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
824 Rozenn Le Hir et al
reduced probably accounting for the difference in the SE
content between the wild-type and the abcg9ndash2 abcg14ndash1
double mutant (Figure 7e)
(a)
(b)
(c) (d) (e)
(f) (g)
Figure 6 Long chain and very long chain fatty acid content and cuticle
integrity in abcg9ndash2 and abcg14ndash1 mutants
(a b) Fatty acid (a) and VLCFA content (b) in abcg9ndash2 and abcg14ndash1 mutants
in apical parts of 10-day-old seedlings (three biological replicates each of
approximately 5 mg dry weight were used)
(cndashf) Toluidine blue staining patterns of abcg mutants in 7-day-old seed-
lings Scale bars = 3 cm
(a)
(b)
(c)
(d) (e)
Figure 7 Metabolome analysis and measurements of sterol content in abcg
mutants
(andashc) Results of orthogonal partial least-squares discriminant analysis of
global metabolite contents of shoots of 7-day-old seedlings from wild-type
(black squares) abcg11ndash7+ (gray triangles) abcg11ndash7 (white stars) abcg9ndash1(white hexagons) abcg9ndash2+ (gray hexagons) abcg9ndash2 (white hexagons)
and abcg14ndash1 (black circles) seedlings (five biological replicates of 10 mg
each were used for the analysis)
(d e) Free sterol (FS) and sterol ester (SE) content of wild-type (white bars)
abcg9ndash2 (light gray bars) abcg14ndash1 (dark gray bars) and abcg9ndash2 abcg14ndash1(black bars) Error bars indicate standard deviations obtained from three
independent biological replicates The asterisk indicates a statistically signif-
icant difference between the wild-type and the mutant (P lt 005 Studentrsquos
t test)
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
818 Rozenn Le Hir et al
DISCUSSION
ABCG9 and ABCG14 are two plasma membrane proteins
that interact physically in multiple homo-heterodimer
combinations
In this study we showed that like the previously described
ABCG11 (Pighin et al 2004 Bird et al 2007 Luo et al
2007 Panikashvili et al 2007 2010 Ukitsu et al 2007)
ABCG9 and ABCG14 localize to the plasma membrane
Based on co-immunoprecipitation of tagged proteins and
the bimolecular fluorescence complementation assay we
demonstrated that ABCG9 interacts with ABCG11 to form a
heterodimer and like ABCG11 (McFarlane et al 2010) can
homodimerize In contrast ABCG14 interacts only with
ABCG11 forming an obligate heterodimer This extends
the number of potential partners of ABCG11 to three as it
was previously shown to dimerize with the ABCG12CER5
protein (McFarlane et al 2010) Similarly ABCG9 under-
goes flexible dimerization to form either homo- or hetero-
dimers suggesting that ABCGs may be promiscuous
proteins with different functions depending on their inter-
acting partner andor expression profile
Mutations in ABCG genes are semi-dominant
In the last few years several Arabidopsis mutants with
altered expression of various ABCG genes such as ABCG11
(Bird et al 2007 Luo et al 2007 Panikashvili et al 2007
Ukitsu et al 2007) ABCG12CER5 (Pighin et al 2004)
ABCG13 (Panikashvili et al 2011) ABCG22 (Kuromori
et al 2011a) ABCG25 (Kuromori et al 2010) and ABCG26
(Choi et al 2010 Quilichini et al 2010 Dou et al 2011
Kuromori et al 2011b) have been characterized but even
though these half-size transporters require dimerization to
become functional all the mutations were described as
recessive Here we demonstrated that mutations in these
genes have semi-dominant effects Indeed the metabolome
analyses showed that abcg9ndash2+ and abcg11ndash7+ heterozy-
gous seedlings have a profile intermediate between that of
wild-type and homozygous seedlings The possibility of
dominant-negative effects was excluded because no
Table 2 Shoot metabolites of 10-day-old abcg9ndash2 abcg11ndash7 and abcg14ndash1 seedlings whose levels were significantly different from wild-type
Group
Metabolites that differed between mutant and wild-type
abcg9ndash2Colndash0 abcg11ndash7Colndash0 abcg14ndash1Colndash0
Amino acids Glycine Glutamate AlanineAlanine Glutamine ValineValine ThreonineThreonine GABASerine Prolinebndashalanine IsoleucineIsoleucine SerineGABA bndashalanineProline
Carbohydrates Glucose Glucose ErythritolFructose Fructose TrehaloseXylose Galactose XyloseMaltose RaffinoseSucrose
Lipids and intermediates 24ndashmethylene-cholesterol Campesterol Linoleic acidMonoacylglycerol Glycerol-3ndashphosphate Triacontanoic acidPalmitic acid Inositol-1ndashphosphate GlycerolGlycerol-3ndashphosphate Lauric acidInositol-1ndashphosphate 24ndashmethylene-cholesterol
SitosterolGlycerol-3ndashphosphate
TCA cycle and others Phosphoric acid Phosphate compound Glyceric acid24ndashdihydroxybutanoic acid Dehydroascorbic acid Fumaric acidGlyceric acid Malic acid Threonic acid-14ndashlactoneFumaric acid 2ndashoxoglutaric acid Threonic acidThreonic acid bndashhydroxy-bndashmethylglutaric acid Shikimic acidThreonic acid-14ndashlactone Gluconic acid lactone-like Succinic acidShikimic acid Phytol2ndashoxoglutaric acid Ethanolamine
Nicotinic acid
For abcg9ndash2 and abcg14ndash1 the levels of the compounds were reduced whereas they were increased in abcg11ndash7
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 819
truncated transcript was found for either abcg9ndash2 or
abcg11ndash7 mutants Hence abcg9ndash2 and abcg11ndash7 are true
null alleles These observations are consistent with the
requirement for homo- or heterodimerization of the ABCG
proteins to form functional transporters In the heterozy-
gous plants ABCG11 transcript levels were 50 lower than
in wild-type counterparts suggesting that ABCG11 protein
and thus corresponding homo- and heterodimers were
likely to be less abundant which may also explain the meta-
bolic perturbations and consequent phenotypic modifica-
tions These findings also suggest that wild-type plants are
not saturated by ABCG11 proteins as a 50 reduction in
protein content appears to be sufficient to alter the physiol-
ogy of the heterozygous mutant plants This hypothesis is
strengthened by the observation that mutants carrying the
weak abcg11ndash6 allele in which there was only a 25 reduc-
tion in ABCG11 transcript levels showed a similar pheno-
type to the heterozygous abcg11ndash7+ and abcg11ndash8+
plants Similarly the abcg9ndash1 weak allele mutant had a very
similar phenotype to the abcg9ndash2+ heterozygous seedlings
suggesting that considering the mode of action of these
proteins the semi-dominant effects that we observed are
likely to be a general feature among the ABCG genes
ABCG9 ABCG11 and ABCG14 transporters are involved in
vascular system development in Arabidopsis
We showed that the ABCG11 ABCG9 and ABCG14 promot-
ers drove expression in the vascular system of Arabidopsis
cotyledons and rosette leaves and more precisely in the
phloem of flower stems In addition mutations in ABCG9
ABCG11 and ABCG14 genes altered vascular development
in cotyledons and the floral stem Similar vascular localiza-
tion of ABCG11 has already been described by Panikashvili
et al (20072010) who showed that in addition to the epi-
dermis ABCG11 is expressed in the vasculature as early as
during embryo development and then throughout plant
development They showed that the vascular pattern in
leaves of the homozygote mutant seedlings was less com-
plex than in the wild-type and discontinuous (Panikashvili
et al 2007) The present article describes a mutant that
has both cuticle and vascular system patterning defects
although it has been previously shown that mutants
altered in cuticle formation have dramatically reduced
growth and altered leaf morphology (Jenks et al 1996)
which may account for vascular defects Analysis of the
abcg11+ heterozygous seedlings that have vascular
defects but apparently no cuticle alteration suggested that
these phenotypes are probably uncoupled Because
ABCG11 dimerizes with several members of its sub-family
it seems reasonable to postulate that it is involved in the
transport of different compounds depending on its partner
Here we provide evidence that ABCG11 in addition to
forming dimers with ABCG12 (McFarlane et al 2010) in
epidermal cells is likely to dimerize with ABCG9 and
ABCG14 in phloem cells suggesting functional compart-
mentalization of these proteins and a possible role in vas-
cular system patterning in Arabidopsis
Metabolite profiling suggests that ABCG9 ABCG11 and
ABCG14 are involved in sterollipid homeostasis
We investigated in more depth the metabolic changes
induced by mutations in ABCG9 ABCG11 and ABCG14
We found that levels of metabolites of several classes
including lipids sugars and amino acids were altered in
both the heterozygotes and homozygotes and that the
profiles in the root and apical parts differed suggesting
that ABCGs may play different roles in the roots and shoot
Interestingly the modifications of amino acid and sugar
contents observed in the metabolic profiles of the abcg
mutants suggest an impaired carbonnitrogen balance that
may be directly or indirectly linked to the vascular defects
leading to possible problems with phloem loading or
unloading Saturated and unsaturated fatty acids and sterol
contents were also altered in the mutants Patterning
defects in the vascular system are often observed in
mutants altered in hormone homeostasis such as for
auxin or brassinosteroids (Kaneda et al 2011) and direct
or indirect modifications of sterol homeostasis are thought
to be one of the primary causes of these downstream
effects (Pullen et al 2010) The detected defects in sterol
homeostasis may explain the abnormal vascular develop-
ment observed in abcg9 abcg11 and abcg14 mutants
Accordingly several sterol-deficient mutants such as fac-
kelhydra (Jang et al 2000) and cpv1 (Carland et al 2002)
have also been shown to exhibit severe defects in vascular
development It has been suggested that the bushy habit
and low fertility of cpv1smt2 mutants may be due to an
increased campesterolsitosterol ratio rather than sterol
deficiency per se (Clouse 2002) This ratio is controlled by
the activity of SMT2 (STEROL METHYLTRANSFERASE2)
Interestingly strong down-regulation of SMT2 expression
was observed in all the mutant backgrounds (Figure S5)
and the metabolic data indicated a deficit in 24ndashmethylene
cholesterol (precursor of both campesterol and sitosterol)
andor sitosterol in abcg9 and abcg14 mutants respec-
tively In addition the significant decrease in sterol ester
content in the double mutant abcg9ndash2 abcg14ndash1 correlated
with down-regulation of expression of the PHOSPHOLIPID
STEROL ACYLTRANSFERASE1 (PSAT1) gene encoding the
enzyme that is mainly responsible for SE synthesis (Bou-
vier-Nave et al 2010) and is proposed to be a control step
in sterol homeostasis (Kopischke et al 2013) Altogether
these results suggest that the vascular phenotype
observed in abcg mutants may be linked to modification of
sterol homeostasis
Therefore we postulate that the various dimer combina-
tions between ABCG9 ABCG11 and ABCG14 may
participate in the transport of squalene-derived metabolites
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
820 Rozenn Le Hir et al
such as sterol andor sterol conjugates via the phloem sap
The presence of sterols in phloem sap has been observed in
various species such as barley (Hordeum vulgare Lehrer
et al 2000) and fava bean (Vicia faba Bouvaine et al
2012) and indirect proof of the presence of sterols in
phloem sap has been obtained by analysis of the phytos-
terol content in exclusively phloem-feeding aphids that are
unable to synthetize them directly (Behmer et al 2011) In
addition the evidence for long-distance transport of sterol
precursors has been supported by observations that sterol
esters accumulate in organs distal to the site of application
of exogenous precursors (Bouvier-Nave et al 2010) As
sterols have been proposed to act as signal molecules in
the control of developmental processes such as vascular
patterning (Carland et al 2002 Pullen et al 2010) modifi-
cations of sterol homeostasis may affect development of
the vascular system in the abcgmutants described here
In conclusion we propose that ABCG9 ABCG11 and
ABCG14 are important for lipidsterol homeostasis Thus
these three proteins are likely to be involved in the control
of various aspects of plant development andor physiol-
ogy including patterning of the vascular system
EXPERIMENTAL PROCEDURES
Plant material and growth conditions
Insertion lines in ABCG9 (SALK_047133 and GABI_353B01)ABCG11 (SALK_096377 and GABI_590C03) and ABCG14 (SALK_036952) genes were obtained from the European ArabidopsisStock Centre (httparabidopsisinfo) Heterozygoushomozygousplants were screened using gene-specific primers in combinationwith the specific primer for the left border of the TndashDNA insertion(Table S1)
Plants were grown in soil or in vitro as described previously(Sorin et al 2005) Arabidopsis Colndash0 cell cultures were main-tained by weekly sub-culturing in MS medium (Murashige andSkoog 1962) supplemented with 240 lg l1 24ndashdichlorophenoxy-acetic acid and 14 lg l1 kinetin and were grown under 12 h light12 h dark cycles at 25 lE m2 sec1 and with 23degC day and nighttemperatures
Morphological characterization and GUS staining
To study the venation pattern cotyledons of 7-day-old seedlingsand rosette leaves of 4-week-old plants were cleared in a buffercontaining chloral hydrate glycerol and water (831 wvv) afterwhich 200 cotyledons per genotype were analyzed and the per-centage showing particular levels of complexity or discontinuity invascularity was estimated as described by Cnops et al (2006)
Flower stem segments (1ndash2 cm long) were removed from ten3ndash4-week-old plants grown in soil embedded in 8 agarose andsectioned using a VT100S vibratome (Leica httpwwwleicacom)The cross-sections were then stained with safraninalcian blue for1ndash2 min (Sigma httpwwwsigmaaldrichcom) rinsed in waterand mounted in 50 glycerol
Histochemical GUS assays were performed as described previ-ously (Sorin et al 2005) All observations were performed usingan Axioplan light microscope equipped with an Axiocam cameraand Axiovision software (Zeiss httpwwwzeisscom)
To observe potential cuticle defects in the various mutant linesa TB test was performed by immersing in vitro-grown plantlets ina 005 wv TB solution for 2 min as described by Tanaka et al(2004)
Statistical analyses
Statistical analyses of differences between-genotype means andvariance for the studied morphological traits were performedusing Studentrsquos t test (GraphPad Prism version 50 httpwwwgraphpadcom) In the venation pattern experiment thesignificance of differences between wild-type and mutants wasassessed using R (R Development Core Team 2010) and Pearsonrsquosv2 test with Monte Carlo permutations (N = 10 000)
Plasmid constructs
The native ABCG promoters and open reading frame of ABCG11were amplified from genomic DNA or cDNA extracted from 7-day-old Arabidopsis seedlings using Phusion high-fidelity DNApolymerase (Finnzyme wwwthermoscientificbiocomfinnzymes)according to the manufacturerrsquos instructions with gene-specificprimers (Table S1) The PCR products were cloned into pENTRDndashTOPO (Invitrogen httpwwwinvitrogencom) and recombinedinto pKGWFS7 (Karimi et al 2002) to create ABCGproGUS Allrecombinations were performed using the Gateway LR Clonase
enzyme mix (Invitrogen) At each cloning step the sequenceswere confirmed by sequencing and all the constructs weretransferred into Agrobacterium strain GV3101pMP90 (C58C1)Wild-type or abcg11ndash7+ heterozygous plants were transformed byfloral dipping (Clough and Bent 1998) For ABCGGUS constructsthe expression pattern was checked in the T2 progeny of 10ndash15independent transgenic lines and one representative homozygousline was used for further characterization
Tagged protein constructs and protoplast transformation
Epitope-tagged versions of ABCG9 ABCG11 and ABCG14 proteinswere produced in pRT104ndash3xHA and pRT104ndash3xMyc plasmids(Feurouleuroop et al 2005) and pSAT6-mRFP (Citovsky et al 2006) Allthese plasmids have a 35S promoter sequence upstream of themulti-cloning site The open reading frames of ABCG11 andABCG14 and the genomic clone of ABCG9 were amplified fromcDNA or genomic DNA extracted from 7-day-old Arabidopsisseedlings using Phusion high-fidelity DNA polymerase (Finnzyme)according to the manufacturerrsquos instructions with gene-specificprimers carrying EcoRI or SalI restriction sites to facilitate subse-quent cloning (Table S1) The products obtained after PCR weredigested by EcoRI and SalI prior to ligation into appropriate plas-mids that had previously been cut open with the same restrictionenzymes Constructs were verified by sequencing Protoplastsfrom Arabidopsis cell cultures or 14-day-old Arabidopsis seedlingswere prepared and transformed as previously described (Meski-ene et al 2003 Zhai et al 2009) For co-immunoprecipitation andsubcellular localization assays 100 000 and 50 000 protoplastcells respectively were transfected with 15 and 10 ll of plasmidsrespectively (containing between 5 and 75 lg of each construct)and the resulting transformed protoplasts were processed afterincubation in the dark at room temperature for 16ndash18 and 24 hrespectively
Whole-mount immunolocalization
The HA-tagged versions of ABCG9 and ABCG14 were detected in7-day-old in vitro-grown Arabidopsis seedlings as described inMethods S1 Anti-HA antibody 12CA5 (Roche httpwwwroche
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 821
com) (1100 dilution) combined with Alexa Fluor 488-conjugatedsecondary antibody (1100 dilution) were used
Confocal laser-scanning microscopy
Images were collected using an SP2 confocal microscope (Leica)RFP and Alexa Fluor 488 were detected using laser lines at 561and 488 nm respectively according to the manufacturerrsquos set-tings RFP fluorescence emission was detected at 550ndash600 nmand Alexa Fluor 488 and GFP fluorescence emission were detectedat 495ndash545 nm For subcellular localization in protoplasts imageswere coded in cyan (GFP) magenta (RFP) and yellow (auto-fluo-rescence) resulting in purple coloration for co-localization inmerged images For the immunolocalization experiment imageswere coded in green (GFP) A water-corrected 209 objective (HCPlan-Apochromat 20907 ImmKor Leica) was used Images werecropped using Adobe Photoshop CS2 and assembled using AdobeIllustrator CS2 software (Abode httpwwwabodecom) Eachimage shown represents a single focal plan
Co-immunoprecipitation assays
Transformed protoplast proteins were extracted using 50 ll lysisbuffer containing 25 mM TrisHCl pH 78 10 mM MgCl2 75 mM
NaCl 5 mM EGTA 1 mM benzamidine 1 mM dithiothreitol 10glycerol 1 Triton Xndash100 and 19 protein inhibitor cocktail(Sigma httpsigmaaldrichcom) The transformed protoplast sus-pension was frozen in liquid nitrogen then thawed on ice andsubjected to centrifugation for 5 min at 150 g Two microliters ofanti-Myc (9E10 Covance httpwwwcovancecom) or anti-HA(16B12 Covance) antibodies were added to the extracts and themixture was incubated for 2 h at 4degC on a rotating wheel Immu-nocomplexes were captured by incubation with 10 ll Protein GndashSepharose beads for a further 2 h at 4degC on a rotating wheel afterwhich they were washed three times for 5 min each in 25 mM
NaPi 150 mM NaCl 5 glycerol and 02 Igepal CAndash630 buffer(Sigma) and then eluted using 40 ll SDS sample buffer at 37degCfor 30 min Co-immunoprecipitation of Myc- and HA-tagged pro-teins was revealed by SDSndashPAGE followed by Western blottingusing both anti-Myc (Invitrogen) and anti-HA (3F10 Roche) anti-bodies at 12000 dilution
RNA extraction and cDNA preparation
To quantify the relative amounts of the ABCG transcriptsexpressed in various organs three rosette leaves all the cau-line leaves the bottom 5 cm of the floral stem all closed flow-ers and all green siliques were harvested from each of threeindependent 4-week-old Arabidopsis plants of each genotypegrown in soil For the other experiments 10-day-old seedlingsgrown in vitro were harvested All the material was directly fro-zen in liquid nitrogen and stored at 80degC prior to RNA extrac-tion Samples were then disrupted using a MM 301 vibrationmill (Retsch GmbH httpwwwretschcom) at a frequency of25 Hz for 2 min with 3 mm tungsten carbide beads (Qiagenhttpwwwqiagencom) Total RNA was isolated from thismaterial using TRIzol (Life Technologies httplifetechnologiescom) RNA quantity and quality were measured using a Nano-Drop ND 1000 (Thermo Scientific httpwwwthermoscientificcom) All samples with 260280 nm and 260230 nm absorbanceratios lower than 18 were discarded and RNA was re-extractedDNase treatment was applied to 5 lg samples of total RNAusing a DNase I kit (Fermentas httpwwwfermentascom)Double-stranded cDNA was synthesized from 5 lg total RNAusing a SuperScript reverse transcriptase kit (Invitrogen) witholigo(dT)12 primer Finally RNase treatment was applied to the
samples using Escherichia coli RNAse H (Fermentas) The cDNAsamples obtained were diluted 30-fold (except for abcg9 linesamples which were diluted 10-fold) prior to use either toverify the absence of full-length transcripts in putative knockoutmutants or to quantify gene expression by quantitative PCR Allprimers used were designed using the Primer3 program (Rozenand Skaletsky 2000) and their sequences are listed inTable S1
QUANTITATIVE REAL-TIME PCR
Quantitative PCR amplifications were performed in 20 llmixtures containing 10 ll 59 SYBR Green I master mix
(Roche) 02 ll forward and reverse primer (30 lM each)
48 ll sterile water and 5 ll cDNA The BiondashRad CFX96
real-time PCR detection system (httpwwwbio-radcom)
was used for quantification of expression of the sterol
biosynthesis transcripts and the ABCG transcripts in the
various mutant backgrounds Finally the Roche LightCy-
cler 480 system (Roche) was used to quantify the rela-
tive abundance of ABCG transcripts in various organs
Four genes were considered as potential reference genes
[APT1 (At1g27450) TIP41 (At4g34270) EF1a (At5g60390)
and UBQ10 (At4g05320)] and their suitability was tested
using the geNorm algorithm (Vandesompele et al 2002)
to determine the most appropriate one for each experi-
ment A more detailed procedure following the Minimum
Information for Publication of Quantitative Real-Time PCR
Experiments guidelines (Bustin et al 2009) is described
in Methods S2 and the annealing temperature and
efficiencies of the primer pairs used are shown in
Table S2
VLCFA quantification
Approximately 5 mg dry weight of 10-day-old in vitro
Arabidopsis seedlings were analyzed for VLCFA quantifica-
tion as described by Li et al (2006)
Sterol extraction and GC-FID characterization
A pool of 10-day-old seedlings grown in vitro or rosettes
from 4-week-old plants grown in soil were lyophilized prior
to extraction Sterol extraction and isolation was per-
formed as described previously (Bouvier-Nave et al 2010)
Gas Chromatography-Flame Ionization Detector (GC-FID)
characterization was performed as described by Silvestro
et al (2013) To quantify the total amounts of free sterols
and sterol esters non-saponifiable lipids were extracted
using nndashhexane and sterols were derivatized as sterols ace-
tates Quantification was achieved using lupenyl-3 28ndashdi-
acetate as an internal standard
Metabolome analysis
To obtain metabolome profiles associated with wild-type
abcg11ndash7+ abcg11ndash7 abcg9ndash1 abcg9ndash2 and abcg14ndash1
genotypes 10-day-old seedlings representing each geno-
type grown in vitro as described above were harvested
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
822 Rozenn Le Hir et al
and divided into shoot and root samples In the case of
abcg9ndash2 the phenotype of the heterozygote abcg9ndash2+
was indistinguishable from the homozygote phenotype
and therefore heterozygous and homozygous seedlings
from a segregating population were pooled Hence in this
particular experiment abcg9ndash2+ refers to a mixed popula-
tion comprising two-thirds heterozygotes and one-third
homozygotes
Metabolites in five samples of shoot and root parts of
seedlings of each genotype (10 mg fresh weight per sam-
ple) were then extracted analyzed by GCMS and the
acquired data were processed as previously described
(Gullberg et al 2004 Jonsson et al 2005) All multivariate
statistical investigations (partial component analysis and
orthogonal partial least-squares discriminant analysis) for
between-genotype similarities and differences in general
metabolite profiles were performed using Simca software
(Umetrics httpwwwumetricscom)
ACKNOWLEDGEMENTS
We thank A Murphy (Plant Science and Landscape Archi-
tecture University of Maryland MD USA) for stimulating
discussions and critical reading of the manuscript I Carl-
son (Umea Plant Science Center Department of Forest
Genetics and Plant Physiology Swedish University of Agri-
cultural Sciences Umea Sweden) for technical assistance
and S Vernhettes for kindly donating seeds of the
transgenic line carrying the plasma membrane marker
GFPndashLTi6b We also thank the European Arabidopsis Stock
Centre and the GABI-Kat stock centre for providing seeds
This work was supported by allocations granted to CB by
the Swedish Research Council for Agriculture the Swedish
Foundation for Strategic Research the Swedish Research
Council for Research and Innovation for Sustainable
Growth the K amp A Wallenberg Foundation and the Carl
Trygger Foundation
SUPPORTING INFORMATION
Additional Supporting Information may be found in the onlineversion of this articleAppendix S1 Raw data quantification for compounds identified inthe metabolite profiling by GCMS
Figure S1 Relative expression of ABCG genes in various organsand mutant backgroundsFigure S2 Co-immunoprecipitation controlsFigure S3 ABCG9 and ABCG14 homo- andor heterodimerize withABCG11Figure S4 Complementary data on metabolome analysis and con-jugated sterol contentFigure S5 Relative expression of genes related to sterol homeo-stasisMethods S1 Method for whole-mount immunolocalizationMethods S2 Method for quantitative PCR
Table S1 List of primers used in this studyTable S2 Annealing temperature and efficiencies for the primerspairs used in this study
REFERENCES
Behmer ST Grebenok RJ and Douglas AE (2011) Plant sterols and host
plant suitability for a phloem-feeding insect Funct Ecol 25 484ndash491Bird D Beisson F Brigham A Shin J Greer S Jetter R Kunst L
Wu XW Yephremov A and Samuels L (2007) Characterization of Ara-
bidopsis ABCG11WBC11 an ATP binding cassette (ABC) transporter that
is required for cuticular lipid secretion Plant J 52 485ndash498Bouvaine S Behmer ST Lin GG Faure MndashL Grebenok RJ and
Douglas AE (2012) The physiology of sterol nutrition in the pea aphid
Acyrthosiphon pisum J Insect Physiol 58 1383ndash1389Bouvier-Nave P Berna A Noiriel A Compagnon V Carlsson AS
Banas A Stymne S and Schaller H (2010) Involvement of the phos-
pholipid sterol acyltransferase1 in plant sterol homeostasis and leaf
senescence Plant Physiol 152 107ndash119Bustin SA Benes V Garson JA Hellemans J Huggett J Kubista
M Mueller R Nolan T Pfaffl MW and Shipley GL (2009) The MIQE
guidelines Minimum information for publication of quantitative real--
time PCR experiments Clin Chem 55 611
Carland FM Fujioka S Takatsuto S Yoshida S and Nelson T (2002)
The identification of CVP1 reveals a role for sterols in vascular pattern-
ing Plant Cell 14 2045ndash2058Choi H Jin JY Choi S Hwang JU Kim YY Suh MC and Lee Y
(2010) An ABCGWBC-type ABC transporter is essential for transport of
sporopollenin precursors for exine formation in developing pollen Plant
J 65 181ndash193Citovsky V Lee LY Vyas S Glick E Chen MH Vainstein A Gafni
Y Gelvin SB and Tzfira T (2006) Subcellular localization of interacting
proteins by bimolecular fluorescence complementation in planta J Mol
Biol 362 1120ndash1131Clough SJ and Bent AF (1998) Floral dip a simplified method for Agro-
bacterium-mediated transformation of Arabidopsis thaliana Plant J 16
735ndash743Clouse SD (2002) Arabidopsis mutants reveal multiple roles for sterols in
plant development Plant Cell 14 1995ndash2000Cnops G Neyt P Raes J et al (2006) The TORNADO1 and TORNADO2
genes function in several patterning processes during early leaf develop-
ment in Arabidopsis thaliana Plant Cell 18 852ndash866Cutler SR Ehrhardt DW Griffitts JS and Somerville CR (2000) Random
GFPcDNA fusions enable visualization of subcellular structures in cells of
Arabidopsis at a high frequency Proc Natl Acad Sci USA 97 3718ndash3723Dou XY Yang KZ Zhang Y Wang W Liu XL Chen LQ Zhang
XQ and Ye D (2011) WBC27 an adenosine tri-phosphate-binding cas-
sette protein controls pollen wall formation and patterning in Arabidop-
sis J Integr Plant Biol 53 74ndash88Feurouleuroop K Pettko-Szandtner A Magyar Z Miskolczi P Kondorosi E
Dudits D and Bako L (2005) The Medicago CDKC1ndashCYCLINT1 kinase
complex phosphorylates the carboxy-terminal domain of RNA polymer-
ase II and promotes transcription Plant J 42 810ndash820Gullberg J Jonsson P Nordstrom A Sjostrom M and Moritz T (2004)
Design of experiments an efficient strategy to identify factors influenc-
ing extraction and derivatization of Arabidopsis thaliana samples in met-
abolomic studies with gas chromatographymass spectrometry Anal
Biochem 331 283ndash295Hertzberg M Aspeborg H Schrader J et al (2001) A transcriptional
roadmap to wood formation Proc Natl Acad Sci USA 98 14732ndash14737Jang JC Fujioka S Tasaka M Seto H Takatsuto S Ishii A Aida M
Yoshida S and Sheen J (2000) A critical role of sterols in embryonic
patterning and meristem programming revealed by the fackel mutants of
Arabidopsis thaliana Genes Dev 14 1485ndash1497Jenks MA Rashotte AM Tuttle HA and Feldmann KA (1996) Mutants
in Arabidopsis thaliana altered in epicuticular wax and leaf morphology
Plant Physiol 110 377ndash385Jonsson P Johansson AI Gullberg J Trygg J A J Grung B Markl-
und S Sjostrom M Antti H and Moritz T (2005) High-throughput
data analysis for detecting and identifying differences between samples
in GCMS-based metabolomic analyses Anal Chem 77 5635ndash5642Jungwirth H and Kuchler K (2006) Yeast ABC transporters ndash a tale of sex
stress drugs and aging FEBS Lett 580 1131ndash1138Kaneda M Schuetz M Lin B Chanis C Hamberger B Western T
Ehlting J and Samuels A (2011) ABC transporters coordinately
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ABCG proteins and vascular development 823
expressed during lignification of Arabidopsis stems include a set of AB-
CBs associated with auxin transport J Exp Bot 62 2063
Karimi M Inze D and Depicker A (2002) GATEWAYTMvectors for Agrobac-
terium-mediated plant transformation Trends Plant Sci 7 193ndash195Kneurooller A and Murphy A (2011) ABC transporters and their function at the
plasma membrane In The Plant Plasma Membrane Plant Cell Mono-
graphs vol 19 (Murphy AS Peer W and Schulz B eds) New York
NY Springer pp 353ndash377Kopischke M Westphal L Schneeberger K Clark R Ossowski S
Wewer V Fuchs R Landtag J Hause G and Deuroormann P (2013)
Impaired sterol ester synthesis alters the response of Arabidopsis thali-
ana to Phytophthora infestans Plant J 73 456ndash468Kuromori T Miyaji T Yabuuchi H Shimizu H Sugimoto E Kamiya
A Moriyama Y and Shinozaki K (2010) ABC transporter AtABCG25 is
involved in abscisic acid transport and responses Proc Natl Acad Sci
USA 107 2361ndash2366Kuromori T Itoh T Sugimoto E and Shinozaki K (2011a) Arabidopsis
mutant of AtABCG26 an ABC transporter gene is defective in pollen
maturation J Plant Physiol 168 2001ndash2005Kuromori T Sugimoto E and Shinozaki K (2011b) Arabidopsis mutants
of AtABCG22 an ABC transporter gene increase transpiration and
drought susceptibility Plant J 67 885ndash894Le Hir R Beneteau J Bellini C Vilaine F and Dinant S (2008) Gene
expression profiling keys for investigating phloem functions Trends
Plant Sci 13 273ndash280Lehrer AT Dugassa-Gobena D Vidal S and Seifert K (2000) Transport
of resistance-inducing sterols in phloem sap of barley Z Naturforsch C
55 948ndash952Li Y Beisson F Pollard M and Ohlrogge J (2006) Oil content of Arabid-
opsis seeds the influence of seed anatomy light and plant-to-plant vari-
ation Phytochemistry 67 904ndash915Luo B Xue XndashY Hu WndashL Wang LndashJ and Chen XndashY (2007) An ABC
transporter gene of Arabidopsis thaliana AtWBC11 is involved in cuticle
development and prevention of organ fusion Plant Cell Physiol 48
1790ndash1802McFarlane HE Shin JJH Bird DA and Samuels AL (2010)
Arabidopsis ABCG transporters which are required for export of diverse
cuticular lipids dimerize in different combinations Plant Cell 22 3066ndash3075
Meskiene I Baudouin E Schweighofer A Liwosz A Jonak C Rodri-
guez PL Jelinek H and Hirt H (2003) Stress-induced protein phospha-
tase 2C is a negative regulator of a mitogen-activated protein kinase
J Biol Chem 278 18945ndash18952Murashige T and Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures Physiol Plant 15 473ndash497Panikashvili D Savaldi-Goldstein S Mandel T Yifhar T Franke RB
Hofer R Schreiber L Chory J and Aharoni A (2007) The Arabidopsis
DESPERADOAtWBC11 transporter is required for cutin and wax secre-
tion Plant Physiol 145 1345ndash1360Panikashvili D Shi JX Bocobza S Franke RB Schreiber L and Aha-
roni A (2010) The Arabidopsis DSOABCG11 transporter affects cutin
metabolism in reproductive organs and suberin in roots Mol Plant 3
563ndash575Panikashvili D Shi JX Schreiber L and Aharoni A (2011) The Arabid-
opsis ABCG13 transporter is required for flower cuticle secretion and pat-
terning of the petal epidermis New Phytol 190 113ndash124Pighin JA Zheng H Balakshin LJ Goodman IP Western TL Jetter
R Kunst L and Samuels AL (2004) Plant cuticular lipid export requires
an ABC transporter Science 306 702ndash704Pullen M Clark N Zarinkamar F Topping J and Lindsey K (2010)
Analysis of vascular development in the hydra sterol biosynthetic
mutants of Arabidopsis PLoS ONE 5 e12227
Quilichini TD Friedmann MC Samuels AL and Douglas CJ (2010)
ATP-binding cassette transporter G26 is required for male fertility and
pollen exine formation in Arabidopsis Plant Physiol 154 678ndash690R Development Core Team (2010) R A Language and Environment for
Statistical Computing Vienna Austria R Foundation for Statistical
Computing
Roudier F Gissot L Beaudoin F Haslam R Michaelson L Marion J
Molino D Lima A Bach L and Morin H (2010) Very-long-chain fatty
acids are involved in polar auxin transport and developmental patterning
in Arabidopsis Plant Cell 22 364ndash375Rozen S and Skaletsky HJ (2000) Primer3 on the WWW for general users
and for biologist programmers Methods Mol Biol 132 365ndash386Schad M Lipton MS Giavalisco P Smith RD and Kehr J (2005) Eval-
uation of two-dimensional electrophoresis and liquid chromatography
tandem mass spectrometry for tissue-specific protein profiling of
laser-microdissected plant samples Electrophoresis 26 2729ndash2738Schauer N Steinhauser D Strelkov S et al (2005) GCndashMS libraries for
the rapid identification of metabolites in complex biological samples
FEBS Lett 579 1332ndash1337Schrader J Nilsson J Mellerowicz EJ Berglund A Nilsson P Hertz-
berg M and Sandberg G (2004) A high-resolution transcript profile
across the wood forming meristem of poplar identifies potential regula-
tors of cambial stem cell identity Plant Cell 16 2278ndash2292Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desi-
mone M Frommer WB Fleurougge UndashI and Kunze R (2003) ARAMEM-
NON a novel database for Arabidopsis integral membrane proteins
Plant Physiol 131 16ndash26Silvestro D Andersen TG Schaller H and Jensen PE (2013) Plant ste-
rol metabolism D7ndashsterol-C5ndashdesaturase (STE1DWARF7) D57ndashste-rol-D7ndashreductase (DWARF5) and D24ndashsterol-D24ndashreductase (DIMINUTO
DWARF1) show multiple subcellular localizations in Arabidopsis thaliana
(Heynh) L PLoS ONE 8 e56429
Sorin C Bussell JD Camus I Ljung K Kowalczyk M Geiss G McKh-
ann H Garcion C Vaucheret H and Sandberg G (2005) Auxin and
light control of adventitious rooting in Arabidopsis require ARGONA-
UTE1 Plant Cell 17 1343ndash1359Tanaka T Tanaka H Machida C Watanabe M and Machida Y (2004) A
new method for rapid visualization of defects in leaf cuticle reveals five
intrinsic patterns of surface defects in Arabidopsis Plant J 37 139ndash146Turgeon R and Wolf S (2009) Phloem transport cellular pathways and
molecular trafficking Annu Rev Plant Biol 60 207ndash221Ukitsu H Kuromori T Toyooka K et al (2007) Cytological and biochemi-
cal analysis of COF1 an Arabidopsis mutant of an ABC transporter gene
Plant Cell Physiol 48 1524ndash1533Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De
Paepe A and Speleman F (2002) Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biol 3 research00340031-00340011Verrier PJ Bird D Burla B et al (2008) Plant ABC proteins ndash a unified
nomenclature andupdated inventory Trends Plant Sci 13 151ndash159Vilaine F Palauqui JC Amselem J Kusiak C Lemoine R and Dinant
S (2003) Towards deciphering phloem a transcriptome analysis of the
phloem of Apium graveolens Plant J 36 67ndash81Zhai Z Sooksa-nguan T and Vatamaniuk OK (2009) Establishing RNA
interference as a reverse-genetic approach for gene functional analysis
in protoplasts Plant Physiol 149 642ndash652Zhang Q Blaylock LA and Harrison MJ (2010) Two Medicago truncatula
half-ABC transporters are essential for arbuscule development in
arbuscular mycorrhizal symbiosis Plant Cell 22 1483
Zhao C Craig JC Petzold HE Dickerman AW and Beers EP (2005)
The xylem and phloem transcriptomes from secondary tissues of the
Arabidopsis root-hypocotyl Plant Physiol 138 803ndash818
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
824 Rozenn Le Hir et al
DISCUSSION
ABCG9 and ABCG14 are two plasma membrane proteins
that interact physically in multiple homo-heterodimer
combinations
In this study we showed that like the previously described
ABCG11 (Pighin et al 2004 Bird et al 2007 Luo et al
2007 Panikashvili et al 2007 2010 Ukitsu et al 2007)
ABCG9 and ABCG14 localize to the plasma membrane
Based on co-immunoprecipitation of tagged proteins and
the bimolecular fluorescence complementation assay we
demonstrated that ABCG9 interacts with ABCG11 to form a
heterodimer and like ABCG11 (McFarlane et al 2010) can
homodimerize In contrast ABCG14 interacts only with
ABCG11 forming an obligate heterodimer This extends
the number of potential partners of ABCG11 to three as it
was previously shown to dimerize with the ABCG12CER5
protein (McFarlane et al 2010) Similarly ABCG9 under-
goes flexible dimerization to form either homo- or hetero-
dimers suggesting that ABCGs may be promiscuous
proteins with different functions depending on their inter-
acting partner andor expression profile
Mutations in ABCG genes are semi-dominant
In the last few years several Arabidopsis mutants with
altered expression of various ABCG genes such as ABCG11
(Bird et al 2007 Luo et al 2007 Panikashvili et al 2007
Ukitsu et al 2007) ABCG12CER5 (Pighin et al 2004)
ABCG13 (Panikashvili et al 2011) ABCG22 (Kuromori
et al 2011a) ABCG25 (Kuromori et al 2010) and ABCG26
(Choi et al 2010 Quilichini et al 2010 Dou et al 2011
Kuromori et al 2011b) have been characterized but even
though these half-size transporters require dimerization to
become functional all the mutations were described as
recessive Here we demonstrated that mutations in these
genes have semi-dominant effects Indeed the metabolome
analyses showed that abcg9ndash2+ and abcg11ndash7+ heterozy-
gous seedlings have a profile intermediate between that of
wild-type and homozygous seedlings The possibility of
dominant-negative effects was excluded because no
Table 2 Shoot metabolites of 10-day-old abcg9ndash2 abcg11ndash7 and abcg14ndash1 seedlings whose levels were significantly different from wild-type
Group
Metabolites that differed between mutant and wild-type
abcg9ndash2Colndash0 abcg11ndash7Colndash0 abcg14ndash1Colndash0
Amino acids Glycine Glutamate AlanineAlanine Glutamine ValineValine ThreonineThreonine GABASerine Prolinebndashalanine IsoleucineIsoleucine SerineGABA bndashalanineProline
Carbohydrates Glucose Glucose ErythritolFructose Fructose TrehaloseXylose Galactose XyloseMaltose RaffinoseSucrose
Lipids and intermediates 24ndashmethylene-cholesterol Campesterol Linoleic acidMonoacylglycerol Glycerol-3ndashphosphate Triacontanoic acidPalmitic acid Inositol-1ndashphosphate GlycerolGlycerol-3ndashphosphate Lauric acidInositol-1ndashphosphate 24ndashmethylene-cholesterol
SitosterolGlycerol-3ndashphosphate
TCA cycle and others Phosphoric acid Phosphate compound Glyceric acid24ndashdihydroxybutanoic acid Dehydroascorbic acid Fumaric acidGlyceric acid Malic acid Threonic acid-14ndashlactoneFumaric acid 2ndashoxoglutaric acid Threonic acidThreonic acid bndashhydroxy-bndashmethylglutaric acid Shikimic acidThreonic acid-14ndashlactone Gluconic acid lactone-like Succinic acidShikimic acid Phytol2ndashoxoglutaric acid Ethanolamine
Nicotinic acid
For abcg9ndash2 and abcg14ndash1 the levels of the compounds were reduced whereas they were increased in abcg11ndash7
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 819
truncated transcript was found for either abcg9ndash2 or
abcg11ndash7 mutants Hence abcg9ndash2 and abcg11ndash7 are true
null alleles These observations are consistent with the
requirement for homo- or heterodimerization of the ABCG
proteins to form functional transporters In the heterozy-
gous plants ABCG11 transcript levels were 50 lower than
in wild-type counterparts suggesting that ABCG11 protein
and thus corresponding homo- and heterodimers were
likely to be less abundant which may also explain the meta-
bolic perturbations and consequent phenotypic modifica-
tions These findings also suggest that wild-type plants are
not saturated by ABCG11 proteins as a 50 reduction in
protein content appears to be sufficient to alter the physiol-
ogy of the heterozygous mutant plants This hypothesis is
strengthened by the observation that mutants carrying the
weak abcg11ndash6 allele in which there was only a 25 reduc-
tion in ABCG11 transcript levels showed a similar pheno-
type to the heterozygous abcg11ndash7+ and abcg11ndash8+
plants Similarly the abcg9ndash1 weak allele mutant had a very
similar phenotype to the abcg9ndash2+ heterozygous seedlings
suggesting that considering the mode of action of these
proteins the semi-dominant effects that we observed are
likely to be a general feature among the ABCG genes
ABCG9 ABCG11 and ABCG14 transporters are involved in
vascular system development in Arabidopsis
We showed that the ABCG11 ABCG9 and ABCG14 promot-
ers drove expression in the vascular system of Arabidopsis
cotyledons and rosette leaves and more precisely in the
phloem of flower stems In addition mutations in ABCG9
ABCG11 and ABCG14 genes altered vascular development
in cotyledons and the floral stem Similar vascular localiza-
tion of ABCG11 has already been described by Panikashvili
et al (20072010) who showed that in addition to the epi-
dermis ABCG11 is expressed in the vasculature as early as
during embryo development and then throughout plant
development They showed that the vascular pattern in
leaves of the homozygote mutant seedlings was less com-
plex than in the wild-type and discontinuous (Panikashvili
et al 2007) The present article describes a mutant that
has both cuticle and vascular system patterning defects
although it has been previously shown that mutants
altered in cuticle formation have dramatically reduced
growth and altered leaf morphology (Jenks et al 1996)
which may account for vascular defects Analysis of the
abcg11+ heterozygous seedlings that have vascular
defects but apparently no cuticle alteration suggested that
these phenotypes are probably uncoupled Because
ABCG11 dimerizes with several members of its sub-family
it seems reasonable to postulate that it is involved in the
transport of different compounds depending on its partner
Here we provide evidence that ABCG11 in addition to
forming dimers with ABCG12 (McFarlane et al 2010) in
epidermal cells is likely to dimerize with ABCG9 and
ABCG14 in phloem cells suggesting functional compart-
mentalization of these proteins and a possible role in vas-
cular system patterning in Arabidopsis
Metabolite profiling suggests that ABCG9 ABCG11 and
ABCG14 are involved in sterollipid homeostasis
We investigated in more depth the metabolic changes
induced by mutations in ABCG9 ABCG11 and ABCG14
We found that levels of metabolites of several classes
including lipids sugars and amino acids were altered in
both the heterozygotes and homozygotes and that the
profiles in the root and apical parts differed suggesting
that ABCGs may play different roles in the roots and shoot
Interestingly the modifications of amino acid and sugar
contents observed in the metabolic profiles of the abcg
mutants suggest an impaired carbonnitrogen balance that
may be directly or indirectly linked to the vascular defects
leading to possible problems with phloem loading or
unloading Saturated and unsaturated fatty acids and sterol
contents were also altered in the mutants Patterning
defects in the vascular system are often observed in
mutants altered in hormone homeostasis such as for
auxin or brassinosteroids (Kaneda et al 2011) and direct
or indirect modifications of sterol homeostasis are thought
to be one of the primary causes of these downstream
effects (Pullen et al 2010) The detected defects in sterol
homeostasis may explain the abnormal vascular develop-
ment observed in abcg9 abcg11 and abcg14 mutants
Accordingly several sterol-deficient mutants such as fac-
kelhydra (Jang et al 2000) and cpv1 (Carland et al 2002)
have also been shown to exhibit severe defects in vascular
development It has been suggested that the bushy habit
and low fertility of cpv1smt2 mutants may be due to an
increased campesterolsitosterol ratio rather than sterol
deficiency per se (Clouse 2002) This ratio is controlled by
the activity of SMT2 (STEROL METHYLTRANSFERASE2)
Interestingly strong down-regulation of SMT2 expression
was observed in all the mutant backgrounds (Figure S5)
and the metabolic data indicated a deficit in 24ndashmethylene
cholesterol (precursor of both campesterol and sitosterol)
andor sitosterol in abcg9 and abcg14 mutants respec-
tively In addition the significant decrease in sterol ester
content in the double mutant abcg9ndash2 abcg14ndash1 correlated
with down-regulation of expression of the PHOSPHOLIPID
STEROL ACYLTRANSFERASE1 (PSAT1) gene encoding the
enzyme that is mainly responsible for SE synthesis (Bou-
vier-Nave et al 2010) and is proposed to be a control step
in sterol homeostasis (Kopischke et al 2013) Altogether
these results suggest that the vascular phenotype
observed in abcg mutants may be linked to modification of
sterol homeostasis
Therefore we postulate that the various dimer combina-
tions between ABCG9 ABCG11 and ABCG14 may
participate in the transport of squalene-derived metabolites
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
820 Rozenn Le Hir et al
such as sterol andor sterol conjugates via the phloem sap
The presence of sterols in phloem sap has been observed in
various species such as barley (Hordeum vulgare Lehrer
et al 2000) and fava bean (Vicia faba Bouvaine et al
2012) and indirect proof of the presence of sterols in
phloem sap has been obtained by analysis of the phytos-
terol content in exclusively phloem-feeding aphids that are
unable to synthetize them directly (Behmer et al 2011) In
addition the evidence for long-distance transport of sterol
precursors has been supported by observations that sterol
esters accumulate in organs distal to the site of application
of exogenous precursors (Bouvier-Nave et al 2010) As
sterols have been proposed to act as signal molecules in
the control of developmental processes such as vascular
patterning (Carland et al 2002 Pullen et al 2010) modifi-
cations of sterol homeostasis may affect development of
the vascular system in the abcgmutants described here
In conclusion we propose that ABCG9 ABCG11 and
ABCG14 are important for lipidsterol homeostasis Thus
these three proteins are likely to be involved in the control
of various aspects of plant development andor physiol-
ogy including patterning of the vascular system
EXPERIMENTAL PROCEDURES
Plant material and growth conditions
Insertion lines in ABCG9 (SALK_047133 and GABI_353B01)ABCG11 (SALK_096377 and GABI_590C03) and ABCG14 (SALK_036952) genes were obtained from the European ArabidopsisStock Centre (httparabidopsisinfo) Heterozygoushomozygousplants were screened using gene-specific primers in combinationwith the specific primer for the left border of the TndashDNA insertion(Table S1)
Plants were grown in soil or in vitro as described previously(Sorin et al 2005) Arabidopsis Colndash0 cell cultures were main-tained by weekly sub-culturing in MS medium (Murashige andSkoog 1962) supplemented with 240 lg l1 24ndashdichlorophenoxy-acetic acid and 14 lg l1 kinetin and were grown under 12 h light12 h dark cycles at 25 lE m2 sec1 and with 23degC day and nighttemperatures
Morphological characterization and GUS staining
To study the venation pattern cotyledons of 7-day-old seedlingsand rosette leaves of 4-week-old plants were cleared in a buffercontaining chloral hydrate glycerol and water (831 wvv) afterwhich 200 cotyledons per genotype were analyzed and the per-centage showing particular levels of complexity or discontinuity invascularity was estimated as described by Cnops et al (2006)
Flower stem segments (1ndash2 cm long) were removed from ten3ndash4-week-old plants grown in soil embedded in 8 agarose andsectioned using a VT100S vibratome (Leica httpwwwleicacom)The cross-sections were then stained with safraninalcian blue for1ndash2 min (Sigma httpwwwsigmaaldrichcom) rinsed in waterand mounted in 50 glycerol
Histochemical GUS assays were performed as described previ-ously (Sorin et al 2005) All observations were performed usingan Axioplan light microscope equipped with an Axiocam cameraand Axiovision software (Zeiss httpwwwzeisscom)
To observe potential cuticle defects in the various mutant linesa TB test was performed by immersing in vitro-grown plantlets ina 005 wv TB solution for 2 min as described by Tanaka et al(2004)
Statistical analyses
Statistical analyses of differences between-genotype means andvariance for the studied morphological traits were performedusing Studentrsquos t test (GraphPad Prism version 50 httpwwwgraphpadcom) In the venation pattern experiment thesignificance of differences between wild-type and mutants wasassessed using R (R Development Core Team 2010) and Pearsonrsquosv2 test with Monte Carlo permutations (N = 10 000)
Plasmid constructs
The native ABCG promoters and open reading frame of ABCG11were amplified from genomic DNA or cDNA extracted from 7-day-old Arabidopsis seedlings using Phusion high-fidelity DNApolymerase (Finnzyme wwwthermoscientificbiocomfinnzymes)according to the manufacturerrsquos instructions with gene-specificprimers (Table S1) The PCR products were cloned into pENTRDndashTOPO (Invitrogen httpwwwinvitrogencom) and recombinedinto pKGWFS7 (Karimi et al 2002) to create ABCGproGUS Allrecombinations were performed using the Gateway LR Clonase
enzyme mix (Invitrogen) At each cloning step the sequenceswere confirmed by sequencing and all the constructs weretransferred into Agrobacterium strain GV3101pMP90 (C58C1)Wild-type or abcg11ndash7+ heterozygous plants were transformed byfloral dipping (Clough and Bent 1998) For ABCGGUS constructsthe expression pattern was checked in the T2 progeny of 10ndash15independent transgenic lines and one representative homozygousline was used for further characterization
Tagged protein constructs and protoplast transformation
Epitope-tagged versions of ABCG9 ABCG11 and ABCG14 proteinswere produced in pRT104ndash3xHA and pRT104ndash3xMyc plasmids(Feurouleuroop et al 2005) and pSAT6-mRFP (Citovsky et al 2006) Allthese plasmids have a 35S promoter sequence upstream of themulti-cloning site The open reading frames of ABCG11 andABCG14 and the genomic clone of ABCG9 were amplified fromcDNA or genomic DNA extracted from 7-day-old Arabidopsisseedlings using Phusion high-fidelity DNA polymerase (Finnzyme)according to the manufacturerrsquos instructions with gene-specificprimers carrying EcoRI or SalI restriction sites to facilitate subse-quent cloning (Table S1) The products obtained after PCR weredigested by EcoRI and SalI prior to ligation into appropriate plas-mids that had previously been cut open with the same restrictionenzymes Constructs were verified by sequencing Protoplastsfrom Arabidopsis cell cultures or 14-day-old Arabidopsis seedlingswere prepared and transformed as previously described (Meski-ene et al 2003 Zhai et al 2009) For co-immunoprecipitation andsubcellular localization assays 100 000 and 50 000 protoplastcells respectively were transfected with 15 and 10 ll of plasmidsrespectively (containing between 5 and 75 lg of each construct)and the resulting transformed protoplasts were processed afterincubation in the dark at room temperature for 16ndash18 and 24 hrespectively
Whole-mount immunolocalization
The HA-tagged versions of ABCG9 and ABCG14 were detected in7-day-old in vitro-grown Arabidopsis seedlings as described inMethods S1 Anti-HA antibody 12CA5 (Roche httpwwwroche
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 821
com) (1100 dilution) combined with Alexa Fluor 488-conjugatedsecondary antibody (1100 dilution) were used
Confocal laser-scanning microscopy
Images were collected using an SP2 confocal microscope (Leica)RFP and Alexa Fluor 488 were detected using laser lines at 561and 488 nm respectively according to the manufacturerrsquos set-tings RFP fluorescence emission was detected at 550ndash600 nmand Alexa Fluor 488 and GFP fluorescence emission were detectedat 495ndash545 nm For subcellular localization in protoplasts imageswere coded in cyan (GFP) magenta (RFP) and yellow (auto-fluo-rescence) resulting in purple coloration for co-localization inmerged images For the immunolocalization experiment imageswere coded in green (GFP) A water-corrected 209 objective (HCPlan-Apochromat 20907 ImmKor Leica) was used Images werecropped using Adobe Photoshop CS2 and assembled using AdobeIllustrator CS2 software (Abode httpwwwabodecom) Eachimage shown represents a single focal plan
Co-immunoprecipitation assays
Transformed protoplast proteins were extracted using 50 ll lysisbuffer containing 25 mM TrisHCl pH 78 10 mM MgCl2 75 mM
NaCl 5 mM EGTA 1 mM benzamidine 1 mM dithiothreitol 10glycerol 1 Triton Xndash100 and 19 protein inhibitor cocktail(Sigma httpsigmaaldrichcom) The transformed protoplast sus-pension was frozen in liquid nitrogen then thawed on ice andsubjected to centrifugation for 5 min at 150 g Two microliters ofanti-Myc (9E10 Covance httpwwwcovancecom) or anti-HA(16B12 Covance) antibodies were added to the extracts and themixture was incubated for 2 h at 4degC on a rotating wheel Immu-nocomplexes were captured by incubation with 10 ll Protein GndashSepharose beads for a further 2 h at 4degC on a rotating wheel afterwhich they were washed three times for 5 min each in 25 mM
NaPi 150 mM NaCl 5 glycerol and 02 Igepal CAndash630 buffer(Sigma) and then eluted using 40 ll SDS sample buffer at 37degCfor 30 min Co-immunoprecipitation of Myc- and HA-tagged pro-teins was revealed by SDSndashPAGE followed by Western blottingusing both anti-Myc (Invitrogen) and anti-HA (3F10 Roche) anti-bodies at 12000 dilution
RNA extraction and cDNA preparation
To quantify the relative amounts of the ABCG transcriptsexpressed in various organs three rosette leaves all the cau-line leaves the bottom 5 cm of the floral stem all closed flow-ers and all green siliques were harvested from each of threeindependent 4-week-old Arabidopsis plants of each genotypegrown in soil For the other experiments 10-day-old seedlingsgrown in vitro were harvested All the material was directly fro-zen in liquid nitrogen and stored at 80degC prior to RNA extrac-tion Samples were then disrupted using a MM 301 vibrationmill (Retsch GmbH httpwwwretschcom) at a frequency of25 Hz for 2 min with 3 mm tungsten carbide beads (Qiagenhttpwwwqiagencom) Total RNA was isolated from thismaterial using TRIzol (Life Technologies httplifetechnologiescom) RNA quantity and quality were measured using a Nano-Drop ND 1000 (Thermo Scientific httpwwwthermoscientificcom) All samples with 260280 nm and 260230 nm absorbanceratios lower than 18 were discarded and RNA was re-extractedDNase treatment was applied to 5 lg samples of total RNAusing a DNase I kit (Fermentas httpwwwfermentascom)Double-stranded cDNA was synthesized from 5 lg total RNAusing a SuperScript reverse transcriptase kit (Invitrogen) witholigo(dT)12 primer Finally RNase treatment was applied to the
samples using Escherichia coli RNAse H (Fermentas) The cDNAsamples obtained were diluted 30-fold (except for abcg9 linesamples which were diluted 10-fold) prior to use either toverify the absence of full-length transcripts in putative knockoutmutants or to quantify gene expression by quantitative PCR Allprimers used were designed using the Primer3 program (Rozenand Skaletsky 2000) and their sequences are listed inTable S1
QUANTITATIVE REAL-TIME PCR
Quantitative PCR amplifications were performed in 20 llmixtures containing 10 ll 59 SYBR Green I master mix
(Roche) 02 ll forward and reverse primer (30 lM each)
48 ll sterile water and 5 ll cDNA The BiondashRad CFX96
real-time PCR detection system (httpwwwbio-radcom)
was used for quantification of expression of the sterol
biosynthesis transcripts and the ABCG transcripts in the
various mutant backgrounds Finally the Roche LightCy-
cler 480 system (Roche) was used to quantify the rela-
tive abundance of ABCG transcripts in various organs
Four genes were considered as potential reference genes
[APT1 (At1g27450) TIP41 (At4g34270) EF1a (At5g60390)
and UBQ10 (At4g05320)] and their suitability was tested
using the geNorm algorithm (Vandesompele et al 2002)
to determine the most appropriate one for each experi-
ment A more detailed procedure following the Minimum
Information for Publication of Quantitative Real-Time PCR
Experiments guidelines (Bustin et al 2009) is described
in Methods S2 and the annealing temperature and
efficiencies of the primer pairs used are shown in
Table S2
VLCFA quantification
Approximately 5 mg dry weight of 10-day-old in vitro
Arabidopsis seedlings were analyzed for VLCFA quantifica-
tion as described by Li et al (2006)
Sterol extraction and GC-FID characterization
A pool of 10-day-old seedlings grown in vitro or rosettes
from 4-week-old plants grown in soil were lyophilized prior
to extraction Sterol extraction and isolation was per-
formed as described previously (Bouvier-Nave et al 2010)
Gas Chromatography-Flame Ionization Detector (GC-FID)
characterization was performed as described by Silvestro
et al (2013) To quantify the total amounts of free sterols
and sterol esters non-saponifiable lipids were extracted
using nndashhexane and sterols were derivatized as sterols ace-
tates Quantification was achieved using lupenyl-3 28ndashdi-
acetate as an internal standard
Metabolome analysis
To obtain metabolome profiles associated with wild-type
abcg11ndash7+ abcg11ndash7 abcg9ndash1 abcg9ndash2 and abcg14ndash1
genotypes 10-day-old seedlings representing each geno-
type grown in vitro as described above were harvested
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
822 Rozenn Le Hir et al
and divided into shoot and root samples In the case of
abcg9ndash2 the phenotype of the heterozygote abcg9ndash2+
was indistinguishable from the homozygote phenotype
and therefore heterozygous and homozygous seedlings
from a segregating population were pooled Hence in this
particular experiment abcg9ndash2+ refers to a mixed popula-
tion comprising two-thirds heterozygotes and one-third
homozygotes
Metabolites in five samples of shoot and root parts of
seedlings of each genotype (10 mg fresh weight per sam-
ple) were then extracted analyzed by GCMS and the
acquired data were processed as previously described
(Gullberg et al 2004 Jonsson et al 2005) All multivariate
statistical investigations (partial component analysis and
orthogonal partial least-squares discriminant analysis) for
between-genotype similarities and differences in general
metabolite profiles were performed using Simca software
(Umetrics httpwwwumetricscom)
ACKNOWLEDGEMENTS
We thank A Murphy (Plant Science and Landscape Archi-
tecture University of Maryland MD USA) for stimulating
discussions and critical reading of the manuscript I Carl-
son (Umea Plant Science Center Department of Forest
Genetics and Plant Physiology Swedish University of Agri-
cultural Sciences Umea Sweden) for technical assistance
and S Vernhettes for kindly donating seeds of the
transgenic line carrying the plasma membrane marker
GFPndashLTi6b We also thank the European Arabidopsis Stock
Centre and the GABI-Kat stock centre for providing seeds
This work was supported by allocations granted to CB by
the Swedish Research Council for Agriculture the Swedish
Foundation for Strategic Research the Swedish Research
Council for Research and Innovation for Sustainable
Growth the K amp A Wallenberg Foundation and the Carl
Trygger Foundation
SUPPORTING INFORMATION
Additional Supporting Information may be found in the onlineversion of this articleAppendix S1 Raw data quantification for compounds identified inthe metabolite profiling by GCMS
Figure S1 Relative expression of ABCG genes in various organsand mutant backgroundsFigure S2 Co-immunoprecipitation controlsFigure S3 ABCG9 and ABCG14 homo- andor heterodimerize withABCG11Figure S4 Complementary data on metabolome analysis and con-jugated sterol contentFigure S5 Relative expression of genes related to sterol homeo-stasisMethods S1 Method for whole-mount immunolocalizationMethods S2 Method for quantitative PCR
Table S1 List of primers used in this studyTable S2 Annealing temperature and efficiencies for the primerspairs used in this study
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Behmer ST Grebenok RJ and Douglas AE (2011) Plant sterols and host
plant suitability for a phloem-feeding insect Funct Ecol 25 484ndash491Bird D Beisson F Brigham A Shin J Greer S Jetter R Kunst L
Wu XW Yephremov A and Samuels L (2007) Characterization of Ara-
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is required for cuticular lipid secretion Plant J 52 485ndash498Bouvaine S Behmer ST Lin GG Faure MndashL Grebenok RJ and
Douglas AE (2012) The physiology of sterol nutrition in the pea aphid
Acyrthosiphon pisum J Insect Physiol 58 1383ndash1389Bouvier-Nave P Berna A Noiriel A Compagnon V Carlsson AS
Banas A Stymne S and Schaller H (2010) Involvement of the phos-
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M Mueller R Nolan T Pfaffl MW and Shipley GL (2009) The MIQE
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Carland FM Fujioka S Takatsuto S Yoshida S and Nelson T (2002)
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J 65 181ndash193Citovsky V Lee LY Vyas S Glick E Chen MH Vainstein A Gafni
Y Gelvin SB and Tzfira T (2006) Subcellular localization of interacting
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Biol 362 1120ndash1131Clough SJ and Bent AF (1998) Floral dip a simplified method for Agro-
bacterium-mediated transformation of Arabidopsis thaliana Plant J 16
735ndash743Clouse SD (2002) Arabidopsis mutants reveal multiple roles for sterols in
plant development Plant Cell 14 1995ndash2000Cnops G Neyt P Raes J et al (2006) The TORNADO1 and TORNADO2
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GFPcDNA fusions enable visualization of subcellular structures in cells of
Arabidopsis at a high frequency Proc Natl Acad Sci USA 97 3718ndash3723Dou XY Yang KZ Zhang Y Wang W Liu XL Chen LQ Zhang
XQ and Ye D (2011) WBC27 an adenosine tri-phosphate-binding cas-
sette protein controls pollen wall formation and patterning in Arabidop-
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Dudits D and Bako L (2005) The Medicago CDKC1ndashCYCLINT1 kinase
complex phosphorylates the carboxy-terminal domain of RNA polymer-
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Design of experiments an efficient strategy to identify factors influenc-
ing extraction and derivatization of Arabidopsis thaliana samples in met-
abolomic studies with gas chromatographymass spectrometry Anal
Biochem 331 283ndash295Hertzberg M Aspeborg H Schrader J et al (2001) A transcriptional
roadmap to wood formation Proc Natl Acad Sci USA 98 14732ndash14737Jang JC Fujioka S Tasaka M Seto H Takatsuto S Ishii A Aida M
Yoshida S and Sheen J (2000) A critical role of sterols in embryonic
patterning and meristem programming revealed by the fackel mutants of
Arabidopsis thaliana Genes Dev 14 1485ndash1497Jenks MA Rashotte AM Tuttle HA and Feldmann KA (1996) Mutants
in Arabidopsis thaliana altered in epicuticular wax and leaf morphology
Plant Physiol 110 377ndash385Jonsson P Johansson AI Gullberg J Trygg J A J Grung B Markl-
und S Sjostrom M Antti H and Moritz T (2005) High-throughput
data analysis for detecting and identifying differences between samples
in GCMS-based metabolomic analyses Anal Chem 77 5635ndash5642Jungwirth H and Kuchler K (2006) Yeast ABC transporters ndash a tale of sex
stress drugs and aging FEBS Lett 580 1131ndash1138Kaneda M Schuetz M Lin B Chanis C Hamberger B Western T
Ehlting J and Samuels A (2011) ABC transporters coordinately
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ABCG proteins and vascular development 823
expressed during lignification of Arabidopsis stems include a set of AB-
CBs associated with auxin transport J Exp Bot 62 2063
Karimi M Inze D and Depicker A (2002) GATEWAYTMvectors for Agrobac-
terium-mediated plant transformation Trends Plant Sci 7 193ndash195Kneurooller A and Murphy A (2011) ABC transporters and their function at the
plasma membrane In The Plant Plasma Membrane Plant Cell Mono-
graphs vol 19 (Murphy AS Peer W and Schulz B eds) New York
NY Springer pp 353ndash377Kopischke M Westphal L Schneeberger K Clark R Ossowski S
Wewer V Fuchs R Landtag J Hause G and Deuroormann P (2013)
Impaired sterol ester synthesis alters the response of Arabidopsis thali-
ana to Phytophthora infestans Plant J 73 456ndash468Kuromori T Miyaji T Yabuuchi H Shimizu H Sugimoto E Kamiya
A Moriyama Y and Shinozaki K (2010) ABC transporter AtABCG25 is
involved in abscisic acid transport and responses Proc Natl Acad Sci
USA 107 2361ndash2366Kuromori T Itoh T Sugimoto E and Shinozaki K (2011a) Arabidopsis
mutant of AtABCG26 an ABC transporter gene is defective in pollen
maturation J Plant Physiol 168 2001ndash2005Kuromori T Sugimoto E and Shinozaki K (2011b) Arabidopsis mutants
of AtABCG22 an ABC transporter gene increase transpiration and
drought susceptibility Plant J 67 885ndash894Le Hir R Beneteau J Bellini C Vilaine F and Dinant S (2008) Gene
expression profiling keys for investigating phloem functions Trends
Plant Sci 13 273ndash280Lehrer AT Dugassa-Gobena D Vidal S and Seifert K (2000) Transport
of resistance-inducing sterols in phloem sap of barley Z Naturforsch C
55 948ndash952Li Y Beisson F Pollard M and Ohlrogge J (2006) Oil content of Arabid-
opsis seeds the influence of seed anatomy light and plant-to-plant vari-
ation Phytochemistry 67 904ndash915Luo B Xue XndashY Hu WndashL Wang LndashJ and Chen XndashY (2007) An ABC
transporter gene of Arabidopsis thaliana AtWBC11 is involved in cuticle
development and prevention of organ fusion Plant Cell Physiol 48
1790ndash1802McFarlane HE Shin JJH Bird DA and Samuels AL (2010)
Arabidopsis ABCG transporters which are required for export of diverse
cuticular lipids dimerize in different combinations Plant Cell 22 3066ndash3075
Meskiene I Baudouin E Schweighofer A Liwosz A Jonak C Rodri-
guez PL Jelinek H and Hirt H (2003) Stress-induced protein phospha-
tase 2C is a negative regulator of a mitogen-activated protein kinase
J Biol Chem 278 18945ndash18952Murashige T and Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures Physiol Plant 15 473ndash497Panikashvili D Savaldi-Goldstein S Mandel T Yifhar T Franke RB
Hofer R Schreiber L Chory J and Aharoni A (2007) The Arabidopsis
DESPERADOAtWBC11 transporter is required for cutin and wax secre-
tion Plant Physiol 145 1345ndash1360Panikashvili D Shi JX Bocobza S Franke RB Schreiber L and Aha-
roni A (2010) The Arabidopsis DSOABCG11 transporter affects cutin
metabolism in reproductive organs and suberin in roots Mol Plant 3
563ndash575Panikashvili D Shi JX Schreiber L and Aharoni A (2011) The Arabid-
opsis ABCG13 transporter is required for flower cuticle secretion and pat-
terning of the petal epidermis New Phytol 190 113ndash124Pighin JA Zheng H Balakshin LJ Goodman IP Western TL Jetter
R Kunst L and Samuels AL (2004) Plant cuticular lipid export requires
an ABC transporter Science 306 702ndash704Pullen M Clark N Zarinkamar F Topping J and Lindsey K (2010)
Analysis of vascular development in the hydra sterol biosynthetic
mutants of Arabidopsis PLoS ONE 5 e12227
Quilichini TD Friedmann MC Samuels AL and Douglas CJ (2010)
ATP-binding cassette transporter G26 is required for male fertility and
pollen exine formation in Arabidopsis Plant Physiol 154 678ndash690R Development Core Team (2010) R A Language and Environment for
Statistical Computing Vienna Austria R Foundation for Statistical
Computing
Roudier F Gissot L Beaudoin F Haslam R Michaelson L Marion J
Molino D Lima A Bach L and Morin H (2010) Very-long-chain fatty
acids are involved in polar auxin transport and developmental patterning
in Arabidopsis Plant Cell 22 364ndash375Rozen S and Skaletsky HJ (2000) Primer3 on the WWW for general users
and for biologist programmers Methods Mol Biol 132 365ndash386Schad M Lipton MS Giavalisco P Smith RD and Kehr J (2005) Eval-
uation of two-dimensional electrophoresis and liquid chromatography
tandem mass spectrometry for tissue-specific protein profiling of
laser-microdissected plant samples Electrophoresis 26 2729ndash2738Schauer N Steinhauser D Strelkov S et al (2005) GCndashMS libraries for
the rapid identification of metabolites in complex biological samples
FEBS Lett 579 1332ndash1337Schrader J Nilsson J Mellerowicz EJ Berglund A Nilsson P Hertz-
berg M and Sandberg G (2004) A high-resolution transcript profile
across the wood forming meristem of poplar identifies potential regula-
tors of cambial stem cell identity Plant Cell 16 2278ndash2292Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desi-
mone M Frommer WB Fleurougge UndashI and Kunze R (2003) ARAMEM-
NON a novel database for Arabidopsis integral membrane proteins
Plant Physiol 131 16ndash26Silvestro D Andersen TG Schaller H and Jensen PE (2013) Plant ste-
rol metabolism D7ndashsterol-C5ndashdesaturase (STE1DWARF7) D57ndashste-rol-D7ndashreductase (DWARF5) and D24ndashsterol-D24ndashreductase (DIMINUTO
DWARF1) show multiple subcellular localizations in Arabidopsis thaliana
(Heynh) L PLoS ONE 8 e56429
Sorin C Bussell JD Camus I Ljung K Kowalczyk M Geiss G McKh-
ann H Garcion C Vaucheret H and Sandberg G (2005) Auxin and
light control of adventitious rooting in Arabidopsis require ARGONA-
UTE1 Plant Cell 17 1343ndash1359Tanaka T Tanaka H Machida C Watanabe M and Machida Y (2004) A
new method for rapid visualization of defects in leaf cuticle reveals five
intrinsic patterns of surface defects in Arabidopsis Plant J 37 139ndash146Turgeon R and Wolf S (2009) Phloem transport cellular pathways and
molecular trafficking Annu Rev Plant Biol 60 207ndash221Ukitsu H Kuromori T Toyooka K et al (2007) Cytological and biochemi-
cal analysis of COF1 an Arabidopsis mutant of an ABC transporter gene
Plant Cell Physiol 48 1524ndash1533Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De
Paepe A and Speleman F (2002) Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biol 3 research00340031-00340011Verrier PJ Bird D Burla B et al (2008) Plant ABC proteins ndash a unified
nomenclature andupdated inventory Trends Plant Sci 13 151ndash159Vilaine F Palauqui JC Amselem J Kusiak C Lemoine R and Dinant
S (2003) Towards deciphering phloem a transcriptome analysis of the
phloem of Apium graveolens Plant J 36 67ndash81Zhai Z Sooksa-nguan T and Vatamaniuk OK (2009) Establishing RNA
interference as a reverse-genetic approach for gene functional analysis
in protoplasts Plant Physiol 149 642ndash652Zhang Q Blaylock LA and Harrison MJ (2010) Two Medicago truncatula
half-ABC transporters are essential for arbuscule development in
arbuscular mycorrhizal symbiosis Plant Cell 22 1483
Zhao C Craig JC Petzold HE Dickerman AW and Beers EP (2005)
The xylem and phloem transcriptomes from secondary tissues of the
Arabidopsis root-hypocotyl Plant Physiol 138 803ndash818
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
824 Rozenn Le Hir et al
truncated transcript was found for either abcg9ndash2 or
abcg11ndash7 mutants Hence abcg9ndash2 and abcg11ndash7 are true
null alleles These observations are consistent with the
requirement for homo- or heterodimerization of the ABCG
proteins to form functional transporters In the heterozy-
gous plants ABCG11 transcript levels were 50 lower than
in wild-type counterparts suggesting that ABCG11 protein
and thus corresponding homo- and heterodimers were
likely to be less abundant which may also explain the meta-
bolic perturbations and consequent phenotypic modifica-
tions These findings also suggest that wild-type plants are
not saturated by ABCG11 proteins as a 50 reduction in
protein content appears to be sufficient to alter the physiol-
ogy of the heterozygous mutant plants This hypothesis is
strengthened by the observation that mutants carrying the
weak abcg11ndash6 allele in which there was only a 25 reduc-
tion in ABCG11 transcript levels showed a similar pheno-
type to the heterozygous abcg11ndash7+ and abcg11ndash8+
plants Similarly the abcg9ndash1 weak allele mutant had a very
similar phenotype to the abcg9ndash2+ heterozygous seedlings
suggesting that considering the mode of action of these
proteins the semi-dominant effects that we observed are
likely to be a general feature among the ABCG genes
ABCG9 ABCG11 and ABCG14 transporters are involved in
vascular system development in Arabidopsis
We showed that the ABCG11 ABCG9 and ABCG14 promot-
ers drove expression in the vascular system of Arabidopsis
cotyledons and rosette leaves and more precisely in the
phloem of flower stems In addition mutations in ABCG9
ABCG11 and ABCG14 genes altered vascular development
in cotyledons and the floral stem Similar vascular localiza-
tion of ABCG11 has already been described by Panikashvili
et al (20072010) who showed that in addition to the epi-
dermis ABCG11 is expressed in the vasculature as early as
during embryo development and then throughout plant
development They showed that the vascular pattern in
leaves of the homozygote mutant seedlings was less com-
plex than in the wild-type and discontinuous (Panikashvili
et al 2007) The present article describes a mutant that
has both cuticle and vascular system patterning defects
although it has been previously shown that mutants
altered in cuticle formation have dramatically reduced
growth and altered leaf morphology (Jenks et al 1996)
which may account for vascular defects Analysis of the
abcg11+ heterozygous seedlings that have vascular
defects but apparently no cuticle alteration suggested that
these phenotypes are probably uncoupled Because
ABCG11 dimerizes with several members of its sub-family
it seems reasonable to postulate that it is involved in the
transport of different compounds depending on its partner
Here we provide evidence that ABCG11 in addition to
forming dimers with ABCG12 (McFarlane et al 2010) in
epidermal cells is likely to dimerize with ABCG9 and
ABCG14 in phloem cells suggesting functional compart-
mentalization of these proteins and a possible role in vas-
cular system patterning in Arabidopsis
Metabolite profiling suggests that ABCG9 ABCG11 and
ABCG14 are involved in sterollipid homeostasis
We investigated in more depth the metabolic changes
induced by mutations in ABCG9 ABCG11 and ABCG14
We found that levels of metabolites of several classes
including lipids sugars and amino acids were altered in
both the heterozygotes and homozygotes and that the
profiles in the root and apical parts differed suggesting
that ABCGs may play different roles in the roots and shoot
Interestingly the modifications of amino acid and sugar
contents observed in the metabolic profiles of the abcg
mutants suggest an impaired carbonnitrogen balance that
may be directly or indirectly linked to the vascular defects
leading to possible problems with phloem loading or
unloading Saturated and unsaturated fatty acids and sterol
contents were also altered in the mutants Patterning
defects in the vascular system are often observed in
mutants altered in hormone homeostasis such as for
auxin or brassinosteroids (Kaneda et al 2011) and direct
or indirect modifications of sterol homeostasis are thought
to be one of the primary causes of these downstream
effects (Pullen et al 2010) The detected defects in sterol
homeostasis may explain the abnormal vascular develop-
ment observed in abcg9 abcg11 and abcg14 mutants
Accordingly several sterol-deficient mutants such as fac-
kelhydra (Jang et al 2000) and cpv1 (Carland et al 2002)
have also been shown to exhibit severe defects in vascular
development It has been suggested that the bushy habit
and low fertility of cpv1smt2 mutants may be due to an
increased campesterolsitosterol ratio rather than sterol
deficiency per se (Clouse 2002) This ratio is controlled by
the activity of SMT2 (STEROL METHYLTRANSFERASE2)
Interestingly strong down-regulation of SMT2 expression
was observed in all the mutant backgrounds (Figure S5)
and the metabolic data indicated a deficit in 24ndashmethylene
cholesterol (precursor of both campesterol and sitosterol)
andor sitosterol in abcg9 and abcg14 mutants respec-
tively In addition the significant decrease in sterol ester
content in the double mutant abcg9ndash2 abcg14ndash1 correlated
with down-regulation of expression of the PHOSPHOLIPID
STEROL ACYLTRANSFERASE1 (PSAT1) gene encoding the
enzyme that is mainly responsible for SE synthesis (Bou-
vier-Nave et al 2010) and is proposed to be a control step
in sterol homeostasis (Kopischke et al 2013) Altogether
these results suggest that the vascular phenotype
observed in abcg mutants may be linked to modification of
sterol homeostasis
Therefore we postulate that the various dimer combina-
tions between ABCG9 ABCG11 and ABCG14 may
participate in the transport of squalene-derived metabolites
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
820 Rozenn Le Hir et al
such as sterol andor sterol conjugates via the phloem sap
The presence of sterols in phloem sap has been observed in
various species such as barley (Hordeum vulgare Lehrer
et al 2000) and fava bean (Vicia faba Bouvaine et al
2012) and indirect proof of the presence of sterols in
phloem sap has been obtained by analysis of the phytos-
terol content in exclusively phloem-feeding aphids that are
unable to synthetize them directly (Behmer et al 2011) In
addition the evidence for long-distance transport of sterol
precursors has been supported by observations that sterol
esters accumulate in organs distal to the site of application
of exogenous precursors (Bouvier-Nave et al 2010) As
sterols have been proposed to act as signal molecules in
the control of developmental processes such as vascular
patterning (Carland et al 2002 Pullen et al 2010) modifi-
cations of sterol homeostasis may affect development of
the vascular system in the abcgmutants described here
In conclusion we propose that ABCG9 ABCG11 and
ABCG14 are important for lipidsterol homeostasis Thus
these three proteins are likely to be involved in the control
of various aspects of plant development andor physiol-
ogy including patterning of the vascular system
EXPERIMENTAL PROCEDURES
Plant material and growth conditions
Insertion lines in ABCG9 (SALK_047133 and GABI_353B01)ABCG11 (SALK_096377 and GABI_590C03) and ABCG14 (SALK_036952) genes were obtained from the European ArabidopsisStock Centre (httparabidopsisinfo) Heterozygoushomozygousplants were screened using gene-specific primers in combinationwith the specific primer for the left border of the TndashDNA insertion(Table S1)
Plants were grown in soil or in vitro as described previously(Sorin et al 2005) Arabidopsis Colndash0 cell cultures were main-tained by weekly sub-culturing in MS medium (Murashige andSkoog 1962) supplemented with 240 lg l1 24ndashdichlorophenoxy-acetic acid and 14 lg l1 kinetin and were grown under 12 h light12 h dark cycles at 25 lE m2 sec1 and with 23degC day and nighttemperatures
Morphological characterization and GUS staining
To study the venation pattern cotyledons of 7-day-old seedlingsand rosette leaves of 4-week-old plants were cleared in a buffercontaining chloral hydrate glycerol and water (831 wvv) afterwhich 200 cotyledons per genotype were analyzed and the per-centage showing particular levels of complexity or discontinuity invascularity was estimated as described by Cnops et al (2006)
Flower stem segments (1ndash2 cm long) were removed from ten3ndash4-week-old plants grown in soil embedded in 8 agarose andsectioned using a VT100S vibratome (Leica httpwwwleicacom)The cross-sections were then stained with safraninalcian blue for1ndash2 min (Sigma httpwwwsigmaaldrichcom) rinsed in waterand mounted in 50 glycerol
Histochemical GUS assays were performed as described previ-ously (Sorin et al 2005) All observations were performed usingan Axioplan light microscope equipped with an Axiocam cameraand Axiovision software (Zeiss httpwwwzeisscom)
To observe potential cuticle defects in the various mutant linesa TB test was performed by immersing in vitro-grown plantlets ina 005 wv TB solution for 2 min as described by Tanaka et al(2004)
Statistical analyses
Statistical analyses of differences between-genotype means andvariance for the studied morphological traits were performedusing Studentrsquos t test (GraphPad Prism version 50 httpwwwgraphpadcom) In the venation pattern experiment thesignificance of differences between wild-type and mutants wasassessed using R (R Development Core Team 2010) and Pearsonrsquosv2 test with Monte Carlo permutations (N = 10 000)
Plasmid constructs
The native ABCG promoters and open reading frame of ABCG11were amplified from genomic DNA or cDNA extracted from 7-day-old Arabidopsis seedlings using Phusion high-fidelity DNApolymerase (Finnzyme wwwthermoscientificbiocomfinnzymes)according to the manufacturerrsquos instructions with gene-specificprimers (Table S1) The PCR products were cloned into pENTRDndashTOPO (Invitrogen httpwwwinvitrogencom) and recombinedinto pKGWFS7 (Karimi et al 2002) to create ABCGproGUS Allrecombinations were performed using the Gateway LR Clonase
enzyme mix (Invitrogen) At each cloning step the sequenceswere confirmed by sequencing and all the constructs weretransferred into Agrobacterium strain GV3101pMP90 (C58C1)Wild-type or abcg11ndash7+ heterozygous plants were transformed byfloral dipping (Clough and Bent 1998) For ABCGGUS constructsthe expression pattern was checked in the T2 progeny of 10ndash15independent transgenic lines and one representative homozygousline was used for further characterization
Tagged protein constructs and protoplast transformation
Epitope-tagged versions of ABCG9 ABCG11 and ABCG14 proteinswere produced in pRT104ndash3xHA and pRT104ndash3xMyc plasmids(Feurouleuroop et al 2005) and pSAT6-mRFP (Citovsky et al 2006) Allthese plasmids have a 35S promoter sequence upstream of themulti-cloning site The open reading frames of ABCG11 andABCG14 and the genomic clone of ABCG9 were amplified fromcDNA or genomic DNA extracted from 7-day-old Arabidopsisseedlings using Phusion high-fidelity DNA polymerase (Finnzyme)according to the manufacturerrsquos instructions with gene-specificprimers carrying EcoRI or SalI restriction sites to facilitate subse-quent cloning (Table S1) The products obtained after PCR weredigested by EcoRI and SalI prior to ligation into appropriate plas-mids that had previously been cut open with the same restrictionenzymes Constructs were verified by sequencing Protoplastsfrom Arabidopsis cell cultures or 14-day-old Arabidopsis seedlingswere prepared and transformed as previously described (Meski-ene et al 2003 Zhai et al 2009) For co-immunoprecipitation andsubcellular localization assays 100 000 and 50 000 protoplastcells respectively were transfected with 15 and 10 ll of plasmidsrespectively (containing between 5 and 75 lg of each construct)and the resulting transformed protoplasts were processed afterincubation in the dark at room temperature for 16ndash18 and 24 hrespectively
Whole-mount immunolocalization
The HA-tagged versions of ABCG9 and ABCG14 were detected in7-day-old in vitro-grown Arabidopsis seedlings as described inMethods S1 Anti-HA antibody 12CA5 (Roche httpwwwroche
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 821
com) (1100 dilution) combined with Alexa Fluor 488-conjugatedsecondary antibody (1100 dilution) were used
Confocal laser-scanning microscopy
Images were collected using an SP2 confocal microscope (Leica)RFP and Alexa Fluor 488 were detected using laser lines at 561and 488 nm respectively according to the manufacturerrsquos set-tings RFP fluorescence emission was detected at 550ndash600 nmand Alexa Fluor 488 and GFP fluorescence emission were detectedat 495ndash545 nm For subcellular localization in protoplasts imageswere coded in cyan (GFP) magenta (RFP) and yellow (auto-fluo-rescence) resulting in purple coloration for co-localization inmerged images For the immunolocalization experiment imageswere coded in green (GFP) A water-corrected 209 objective (HCPlan-Apochromat 20907 ImmKor Leica) was used Images werecropped using Adobe Photoshop CS2 and assembled using AdobeIllustrator CS2 software (Abode httpwwwabodecom) Eachimage shown represents a single focal plan
Co-immunoprecipitation assays
Transformed protoplast proteins were extracted using 50 ll lysisbuffer containing 25 mM TrisHCl pH 78 10 mM MgCl2 75 mM
NaCl 5 mM EGTA 1 mM benzamidine 1 mM dithiothreitol 10glycerol 1 Triton Xndash100 and 19 protein inhibitor cocktail(Sigma httpsigmaaldrichcom) The transformed protoplast sus-pension was frozen in liquid nitrogen then thawed on ice andsubjected to centrifugation for 5 min at 150 g Two microliters ofanti-Myc (9E10 Covance httpwwwcovancecom) or anti-HA(16B12 Covance) antibodies were added to the extracts and themixture was incubated for 2 h at 4degC on a rotating wheel Immu-nocomplexes were captured by incubation with 10 ll Protein GndashSepharose beads for a further 2 h at 4degC on a rotating wheel afterwhich they were washed three times for 5 min each in 25 mM
NaPi 150 mM NaCl 5 glycerol and 02 Igepal CAndash630 buffer(Sigma) and then eluted using 40 ll SDS sample buffer at 37degCfor 30 min Co-immunoprecipitation of Myc- and HA-tagged pro-teins was revealed by SDSndashPAGE followed by Western blottingusing both anti-Myc (Invitrogen) and anti-HA (3F10 Roche) anti-bodies at 12000 dilution
RNA extraction and cDNA preparation
To quantify the relative amounts of the ABCG transcriptsexpressed in various organs three rosette leaves all the cau-line leaves the bottom 5 cm of the floral stem all closed flow-ers and all green siliques were harvested from each of threeindependent 4-week-old Arabidopsis plants of each genotypegrown in soil For the other experiments 10-day-old seedlingsgrown in vitro were harvested All the material was directly fro-zen in liquid nitrogen and stored at 80degC prior to RNA extrac-tion Samples were then disrupted using a MM 301 vibrationmill (Retsch GmbH httpwwwretschcom) at a frequency of25 Hz for 2 min with 3 mm tungsten carbide beads (Qiagenhttpwwwqiagencom) Total RNA was isolated from thismaterial using TRIzol (Life Technologies httplifetechnologiescom) RNA quantity and quality were measured using a Nano-Drop ND 1000 (Thermo Scientific httpwwwthermoscientificcom) All samples with 260280 nm and 260230 nm absorbanceratios lower than 18 were discarded and RNA was re-extractedDNase treatment was applied to 5 lg samples of total RNAusing a DNase I kit (Fermentas httpwwwfermentascom)Double-stranded cDNA was synthesized from 5 lg total RNAusing a SuperScript reverse transcriptase kit (Invitrogen) witholigo(dT)12 primer Finally RNase treatment was applied to the
samples using Escherichia coli RNAse H (Fermentas) The cDNAsamples obtained were diluted 30-fold (except for abcg9 linesamples which were diluted 10-fold) prior to use either toverify the absence of full-length transcripts in putative knockoutmutants or to quantify gene expression by quantitative PCR Allprimers used were designed using the Primer3 program (Rozenand Skaletsky 2000) and their sequences are listed inTable S1
QUANTITATIVE REAL-TIME PCR
Quantitative PCR amplifications were performed in 20 llmixtures containing 10 ll 59 SYBR Green I master mix
(Roche) 02 ll forward and reverse primer (30 lM each)
48 ll sterile water and 5 ll cDNA The BiondashRad CFX96
real-time PCR detection system (httpwwwbio-radcom)
was used for quantification of expression of the sterol
biosynthesis transcripts and the ABCG transcripts in the
various mutant backgrounds Finally the Roche LightCy-
cler 480 system (Roche) was used to quantify the rela-
tive abundance of ABCG transcripts in various organs
Four genes were considered as potential reference genes
[APT1 (At1g27450) TIP41 (At4g34270) EF1a (At5g60390)
and UBQ10 (At4g05320)] and their suitability was tested
using the geNorm algorithm (Vandesompele et al 2002)
to determine the most appropriate one for each experi-
ment A more detailed procedure following the Minimum
Information for Publication of Quantitative Real-Time PCR
Experiments guidelines (Bustin et al 2009) is described
in Methods S2 and the annealing temperature and
efficiencies of the primer pairs used are shown in
Table S2
VLCFA quantification
Approximately 5 mg dry weight of 10-day-old in vitro
Arabidopsis seedlings were analyzed for VLCFA quantifica-
tion as described by Li et al (2006)
Sterol extraction and GC-FID characterization
A pool of 10-day-old seedlings grown in vitro or rosettes
from 4-week-old plants grown in soil were lyophilized prior
to extraction Sterol extraction and isolation was per-
formed as described previously (Bouvier-Nave et al 2010)
Gas Chromatography-Flame Ionization Detector (GC-FID)
characterization was performed as described by Silvestro
et al (2013) To quantify the total amounts of free sterols
and sterol esters non-saponifiable lipids were extracted
using nndashhexane and sterols were derivatized as sterols ace-
tates Quantification was achieved using lupenyl-3 28ndashdi-
acetate as an internal standard
Metabolome analysis
To obtain metabolome profiles associated with wild-type
abcg11ndash7+ abcg11ndash7 abcg9ndash1 abcg9ndash2 and abcg14ndash1
genotypes 10-day-old seedlings representing each geno-
type grown in vitro as described above were harvested
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
822 Rozenn Le Hir et al
and divided into shoot and root samples In the case of
abcg9ndash2 the phenotype of the heterozygote abcg9ndash2+
was indistinguishable from the homozygote phenotype
and therefore heterozygous and homozygous seedlings
from a segregating population were pooled Hence in this
particular experiment abcg9ndash2+ refers to a mixed popula-
tion comprising two-thirds heterozygotes and one-third
homozygotes
Metabolites in five samples of shoot and root parts of
seedlings of each genotype (10 mg fresh weight per sam-
ple) were then extracted analyzed by GCMS and the
acquired data were processed as previously described
(Gullberg et al 2004 Jonsson et al 2005) All multivariate
statistical investigations (partial component analysis and
orthogonal partial least-squares discriminant analysis) for
between-genotype similarities and differences in general
metabolite profiles were performed using Simca software
(Umetrics httpwwwumetricscom)
ACKNOWLEDGEMENTS
We thank A Murphy (Plant Science and Landscape Archi-
tecture University of Maryland MD USA) for stimulating
discussions and critical reading of the manuscript I Carl-
son (Umea Plant Science Center Department of Forest
Genetics and Plant Physiology Swedish University of Agri-
cultural Sciences Umea Sweden) for technical assistance
and S Vernhettes for kindly donating seeds of the
transgenic line carrying the plasma membrane marker
GFPndashLTi6b We also thank the European Arabidopsis Stock
Centre and the GABI-Kat stock centre for providing seeds
This work was supported by allocations granted to CB by
the Swedish Research Council for Agriculture the Swedish
Foundation for Strategic Research the Swedish Research
Council for Research and Innovation for Sustainable
Growth the K amp A Wallenberg Foundation and the Carl
Trygger Foundation
SUPPORTING INFORMATION
Additional Supporting Information may be found in the onlineversion of this articleAppendix S1 Raw data quantification for compounds identified inthe metabolite profiling by GCMS
Figure S1 Relative expression of ABCG genes in various organsand mutant backgroundsFigure S2 Co-immunoprecipitation controlsFigure S3 ABCG9 and ABCG14 homo- andor heterodimerize withABCG11Figure S4 Complementary data on metabolome analysis and con-jugated sterol contentFigure S5 Relative expression of genes related to sterol homeo-stasisMethods S1 Method for whole-mount immunolocalizationMethods S2 Method for quantitative PCR
Table S1 List of primers used in this studyTable S2 Annealing temperature and efficiencies for the primerspairs used in this study
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Wu XW Yephremov A and Samuels L (2007) Characterization of Ara-
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is required for cuticular lipid secretion Plant J 52 485ndash498Bouvaine S Behmer ST Lin GG Faure MndashL Grebenok RJ and
Douglas AE (2012) The physiology of sterol nutrition in the pea aphid
Acyrthosiphon pisum J Insect Physiol 58 1383ndash1389Bouvier-Nave P Berna A Noiriel A Compagnon V Carlsson AS
Banas A Stymne S and Schaller H (2010) Involvement of the phos-
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M Mueller R Nolan T Pfaffl MW and Shipley GL (2009) The MIQE
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Carland FM Fujioka S Takatsuto S Yoshida S and Nelson T (2002)
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(2010) An ABCGWBC-type ABC transporter is essential for transport of
sporopollenin precursors for exine formation in developing pollen Plant
J 65 181ndash193Citovsky V Lee LY Vyas S Glick E Chen MH Vainstein A Gafni
Y Gelvin SB and Tzfira T (2006) Subcellular localization of interacting
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Biol 362 1120ndash1131Clough SJ and Bent AF (1998) Floral dip a simplified method for Agro-
bacterium-mediated transformation of Arabidopsis thaliana Plant J 16
735ndash743Clouse SD (2002) Arabidopsis mutants reveal multiple roles for sterols in
plant development Plant Cell 14 1995ndash2000Cnops G Neyt P Raes J et al (2006) The TORNADO1 and TORNADO2
genes function in several patterning processes during early leaf develop-
ment in Arabidopsis thaliana Plant Cell 18 852ndash866Cutler SR Ehrhardt DW Griffitts JS and Somerville CR (2000) Random
GFPcDNA fusions enable visualization of subcellular structures in cells of
Arabidopsis at a high frequency Proc Natl Acad Sci USA 97 3718ndash3723Dou XY Yang KZ Zhang Y Wang W Liu XL Chen LQ Zhang
XQ and Ye D (2011) WBC27 an adenosine tri-phosphate-binding cas-
sette protein controls pollen wall formation and patterning in Arabidop-
sis J Integr Plant Biol 53 74ndash88Feurouleuroop K Pettko-Szandtner A Magyar Z Miskolczi P Kondorosi E
Dudits D and Bako L (2005) The Medicago CDKC1ndashCYCLINT1 kinase
complex phosphorylates the carboxy-terminal domain of RNA polymer-
ase II and promotes transcription Plant J 42 810ndash820Gullberg J Jonsson P Nordstrom A Sjostrom M and Moritz T (2004)
Design of experiments an efficient strategy to identify factors influenc-
ing extraction and derivatization of Arabidopsis thaliana samples in met-
abolomic studies with gas chromatographymass spectrometry Anal
Biochem 331 283ndash295Hertzberg M Aspeborg H Schrader J et al (2001) A transcriptional
roadmap to wood formation Proc Natl Acad Sci USA 98 14732ndash14737Jang JC Fujioka S Tasaka M Seto H Takatsuto S Ishii A Aida M
Yoshida S and Sheen J (2000) A critical role of sterols in embryonic
patterning and meristem programming revealed by the fackel mutants of
Arabidopsis thaliana Genes Dev 14 1485ndash1497Jenks MA Rashotte AM Tuttle HA and Feldmann KA (1996) Mutants
in Arabidopsis thaliana altered in epicuticular wax and leaf morphology
Plant Physiol 110 377ndash385Jonsson P Johansson AI Gullberg J Trygg J A J Grung B Markl-
und S Sjostrom M Antti H and Moritz T (2005) High-throughput
data analysis for detecting and identifying differences between samples
in GCMS-based metabolomic analyses Anal Chem 77 5635ndash5642Jungwirth H and Kuchler K (2006) Yeast ABC transporters ndash a tale of sex
stress drugs and aging FEBS Lett 580 1131ndash1138Kaneda M Schuetz M Lin B Chanis C Hamberger B Western T
Ehlting J and Samuels A (2011) ABC transporters coordinately
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ABCG proteins and vascular development 823
expressed during lignification of Arabidopsis stems include a set of AB-
CBs associated with auxin transport J Exp Bot 62 2063
Karimi M Inze D and Depicker A (2002) GATEWAYTMvectors for Agrobac-
terium-mediated plant transformation Trends Plant Sci 7 193ndash195Kneurooller A and Murphy A (2011) ABC transporters and their function at the
plasma membrane In The Plant Plasma Membrane Plant Cell Mono-
graphs vol 19 (Murphy AS Peer W and Schulz B eds) New York
NY Springer pp 353ndash377Kopischke M Westphal L Schneeberger K Clark R Ossowski S
Wewer V Fuchs R Landtag J Hause G and Deuroormann P (2013)
Impaired sterol ester synthesis alters the response of Arabidopsis thali-
ana to Phytophthora infestans Plant J 73 456ndash468Kuromori T Miyaji T Yabuuchi H Shimizu H Sugimoto E Kamiya
A Moriyama Y and Shinozaki K (2010) ABC transporter AtABCG25 is
involved in abscisic acid transport and responses Proc Natl Acad Sci
USA 107 2361ndash2366Kuromori T Itoh T Sugimoto E and Shinozaki K (2011a) Arabidopsis
mutant of AtABCG26 an ABC transporter gene is defective in pollen
maturation J Plant Physiol 168 2001ndash2005Kuromori T Sugimoto E and Shinozaki K (2011b) Arabidopsis mutants
of AtABCG22 an ABC transporter gene increase transpiration and
drought susceptibility Plant J 67 885ndash894Le Hir R Beneteau J Bellini C Vilaine F and Dinant S (2008) Gene
expression profiling keys for investigating phloem functions Trends
Plant Sci 13 273ndash280Lehrer AT Dugassa-Gobena D Vidal S and Seifert K (2000) Transport
of resistance-inducing sterols in phloem sap of barley Z Naturforsch C
55 948ndash952Li Y Beisson F Pollard M and Ohlrogge J (2006) Oil content of Arabid-
opsis seeds the influence of seed anatomy light and plant-to-plant vari-
ation Phytochemistry 67 904ndash915Luo B Xue XndashY Hu WndashL Wang LndashJ and Chen XndashY (2007) An ABC
transporter gene of Arabidopsis thaliana AtWBC11 is involved in cuticle
development and prevention of organ fusion Plant Cell Physiol 48
1790ndash1802McFarlane HE Shin JJH Bird DA and Samuels AL (2010)
Arabidopsis ABCG transporters which are required for export of diverse
cuticular lipids dimerize in different combinations Plant Cell 22 3066ndash3075
Meskiene I Baudouin E Schweighofer A Liwosz A Jonak C Rodri-
guez PL Jelinek H and Hirt H (2003) Stress-induced protein phospha-
tase 2C is a negative regulator of a mitogen-activated protein kinase
J Biol Chem 278 18945ndash18952Murashige T and Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures Physiol Plant 15 473ndash497Panikashvili D Savaldi-Goldstein S Mandel T Yifhar T Franke RB
Hofer R Schreiber L Chory J and Aharoni A (2007) The Arabidopsis
DESPERADOAtWBC11 transporter is required for cutin and wax secre-
tion Plant Physiol 145 1345ndash1360Panikashvili D Shi JX Bocobza S Franke RB Schreiber L and Aha-
roni A (2010) The Arabidopsis DSOABCG11 transporter affects cutin
metabolism in reproductive organs and suberin in roots Mol Plant 3
563ndash575Panikashvili D Shi JX Schreiber L and Aharoni A (2011) The Arabid-
opsis ABCG13 transporter is required for flower cuticle secretion and pat-
terning of the petal epidermis New Phytol 190 113ndash124Pighin JA Zheng H Balakshin LJ Goodman IP Western TL Jetter
R Kunst L and Samuels AL (2004) Plant cuticular lipid export requires
an ABC transporter Science 306 702ndash704Pullen M Clark N Zarinkamar F Topping J and Lindsey K (2010)
Analysis of vascular development in the hydra sterol biosynthetic
mutants of Arabidopsis PLoS ONE 5 e12227
Quilichini TD Friedmann MC Samuels AL and Douglas CJ (2010)
ATP-binding cassette transporter G26 is required for male fertility and
pollen exine formation in Arabidopsis Plant Physiol 154 678ndash690R Development Core Team (2010) R A Language and Environment for
Statistical Computing Vienna Austria R Foundation for Statistical
Computing
Roudier F Gissot L Beaudoin F Haslam R Michaelson L Marion J
Molino D Lima A Bach L and Morin H (2010) Very-long-chain fatty
acids are involved in polar auxin transport and developmental patterning
in Arabidopsis Plant Cell 22 364ndash375Rozen S and Skaletsky HJ (2000) Primer3 on the WWW for general users
and for biologist programmers Methods Mol Biol 132 365ndash386Schad M Lipton MS Giavalisco P Smith RD and Kehr J (2005) Eval-
uation of two-dimensional electrophoresis and liquid chromatography
tandem mass spectrometry for tissue-specific protein profiling of
laser-microdissected plant samples Electrophoresis 26 2729ndash2738Schauer N Steinhauser D Strelkov S et al (2005) GCndashMS libraries for
the rapid identification of metabolites in complex biological samples
FEBS Lett 579 1332ndash1337Schrader J Nilsson J Mellerowicz EJ Berglund A Nilsson P Hertz-
berg M and Sandberg G (2004) A high-resolution transcript profile
across the wood forming meristem of poplar identifies potential regula-
tors of cambial stem cell identity Plant Cell 16 2278ndash2292Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desi-
mone M Frommer WB Fleurougge UndashI and Kunze R (2003) ARAMEM-
NON a novel database for Arabidopsis integral membrane proteins
Plant Physiol 131 16ndash26Silvestro D Andersen TG Schaller H and Jensen PE (2013) Plant ste-
rol metabolism D7ndashsterol-C5ndashdesaturase (STE1DWARF7) D57ndashste-rol-D7ndashreductase (DWARF5) and D24ndashsterol-D24ndashreductase (DIMINUTO
DWARF1) show multiple subcellular localizations in Arabidopsis thaliana
(Heynh) L PLoS ONE 8 e56429
Sorin C Bussell JD Camus I Ljung K Kowalczyk M Geiss G McKh-
ann H Garcion C Vaucheret H and Sandberg G (2005) Auxin and
light control of adventitious rooting in Arabidopsis require ARGONA-
UTE1 Plant Cell 17 1343ndash1359Tanaka T Tanaka H Machida C Watanabe M and Machida Y (2004) A
new method for rapid visualization of defects in leaf cuticle reveals five
intrinsic patterns of surface defects in Arabidopsis Plant J 37 139ndash146Turgeon R and Wolf S (2009) Phloem transport cellular pathways and
molecular trafficking Annu Rev Plant Biol 60 207ndash221Ukitsu H Kuromori T Toyooka K et al (2007) Cytological and biochemi-
cal analysis of COF1 an Arabidopsis mutant of an ABC transporter gene
Plant Cell Physiol 48 1524ndash1533Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De
Paepe A and Speleman F (2002) Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biol 3 research00340031-00340011Verrier PJ Bird D Burla B et al (2008) Plant ABC proteins ndash a unified
nomenclature andupdated inventory Trends Plant Sci 13 151ndash159Vilaine F Palauqui JC Amselem J Kusiak C Lemoine R and Dinant
S (2003) Towards deciphering phloem a transcriptome analysis of the
phloem of Apium graveolens Plant J 36 67ndash81Zhai Z Sooksa-nguan T and Vatamaniuk OK (2009) Establishing RNA
interference as a reverse-genetic approach for gene functional analysis
in protoplasts Plant Physiol 149 642ndash652Zhang Q Blaylock LA and Harrison MJ (2010) Two Medicago truncatula
half-ABC transporters are essential for arbuscule development in
arbuscular mycorrhizal symbiosis Plant Cell 22 1483
Zhao C Craig JC Petzold HE Dickerman AW and Beers EP (2005)
The xylem and phloem transcriptomes from secondary tissues of the
Arabidopsis root-hypocotyl Plant Physiol 138 803ndash818
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
824 Rozenn Le Hir et al
such as sterol andor sterol conjugates via the phloem sap
The presence of sterols in phloem sap has been observed in
various species such as barley (Hordeum vulgare Lehrer
et al 2000) and fava bean (Vicia faba Bouvaine et al
2012) and indirect proof of the presence of sterols in
phloem sap has been obtained by analysis of the phytos-
terol content in exclusively phloem-feeding aphids that are
unable to synthetize them directly (Behmer et al 2011) In
addition the evidence for long-distance transport of sterol
precursors has been supported by observations that sterol
esters accumulate in organs distal to the site of application
of exogenous precursors (Bouvier-Nave et al 2010) As
sterols have been proposed to act as signal molecules in
the control of developmental processes such as vascular
patterning (Carland et al 2002 Pullen et al 2010) modifi-
cations of sterol homeostasis may affect development of
the vascular system in the abcgmutants described here
In conclusion we propose that ABCG9 ABCG11 and
ABCG14 are important for lipidsterol homeostasis Thus
these three proteins are likely to be involved in the control
of various aspects of plant development andor physiol-
ogy including patterning of the vascular system
EXPERIMENTAL PROCEDURES
Plant material and growth conditions
Insertion lines in ABCG9 (SALK_047133 and GABI_353B01)ABCG11 (SALK_096377 and GABI_590C03) and ABCG14 (SALK_036952) genes were obtained from the European ArabidopsisStock Centre (httparabidopsisinfo) Heterozygoushomozygousplants were screened using gene-specific primers in combinationwith the specific primer for the left border of the TndashDNA insertion(Table S1)
Plants were grown in soil or in vitro as described previously(Sorin et al 2005) Arabidopsis Colndash0 cell cultures were main-tained by weekly sub-culturing in MS medium (Murashige andSkoog 1962) supplemented with 240 lg l1 24ndashdichlorophenoxy-acetic acid and 14 lg l1 kinetin and were grown under 12 h light12 h dark cycles at 25 lE m2 sec1 and with 23degC day and nighttemperatures
Morphological characterization and GUS staining
To study the venation pattern cotyledons of 7-day-old seedlingsand rosette leaves of 4-week-old plants were cleared in a buffercontaining chloral hydrate glycerol and water (831 wvv) afterwhich 200 cotyledons per genotype were analyzed and the per-centage showing particular levels of complexity or discontinuity invascularity was estimated as described by Cnops et al (2006)
Flower stem segments (1ndash2 cm long) were removed from ten3ndash4-week-old plants grown in soil embedded in 8 agarose andsectioned using a VT100S vibratome (Leica httpwwwleicacom)The cross-sections were then stained with safraninalcian blue for1ndash2 min (Sigma httpwwwsigmaaldrichcom) rinsed in waterand mounted in 50 glycerol
Histochemical GUS assays were performed as described previ-ously (Sorin et al 2005) All observations were performed usingan Axioplan light microscope equipped with an Axiocam cameraand Axiovision software (Zeiss httpwwwzeisscom)
To observe potential cuticle defects in the various mutant linesa TB test was performed by immersing in vitro-grown plantlets ina 005 wv TB solution for 2 min as described by Tanaka et al(2004)
Statistical analyses
Statistical analyses of differences between-genotype means andvariance for the studied morphological traits were performedusing Studentrsquos t test (GraphPad Prism version 50 httpwwwgraphpadcom) In the venation pattern experiment thesignificance of differences between wild-type and mutants wasassessed using R (R Development Core Team 2010) and Pearsonrsquosv2 test with Monte Carlo permutations (N = 10 000)
Plasmid constructs
The native ABCG promoters and open reading frame of ABCG11were amplified from genomic DNA or cDNA extracted from 7-day-old Arabidopsis seedlings using Phusion high-fidelity DNApolymerase (Finnzyme wwwthermoscientificbiocomfinnzymes)according to the manufacturerrsquos instructions with gene-specificprimers (Table S1) The PCR products were cloned into pENTRDndashTOPO (Invitrogen httpwwwinvitrogencom) and recombinedinto pKGWFS7 (Karimi et al 2002) to create ABCGproGUS Allrecombinations were performed using the Gateway LR Clonase
enzyme mix (Invitrogen) At each cloning step the sequenceswere confirmed by sequencing and all the constructs weretransferred into Agrobacterium strain GV3101pMP90 (C58C1)Wild-type or abcg11ndash7+ heterozygous plants were transformed byfloral dipping (Clough and Bent 1998) For ABCGGUS constructsthe expression pattern was checked in the T2 progeny of 10ndash15independent transgenic lines and one representative homozygousline was used for further characterization
Tagged protein constructs and protoplast transformation
Epitope-tagged versions of ABCG9 ABCG11 and ABCG14 proteinswere produced in pRT104ndash3xHA and pRT104ndash3xMyc plasmids(Feurouleuroop et al 2005) and pSAT6-mRFP (Citovsky et al 2006) Allthese plasmids have a 35S promoter sequence upstream of themulti-cloning site The open reading frames of ABCG11 andABCG14 and the genomic clone of ABCG9 were amplified fromcDNA or genomic DNA extracted from 7-day-old Arabidopsisseedlings using Phusion high-fidelity DNA polymerase (Finnzyme)according to the manufacturerrsquos instructions with gene-specificprimers carrying EcoRI or SalI restriction sites to facilitate subse-quent cloning (Table S1) The products obtained after PCR weredigested by EcoRI and SalI prior to ligation into appropriate plas-mids that had previously been cut open with the same restrictionenzymes Constructs were verified by sequencing Protoplastsfrom Arabidopsis cell cultures or 14-day-old Arabidopsis seedlingswere prepared and transformed as previously described (Meski-ene et al 2003 Zhai et al 2009) For co-immunoprecipitation andsubcellular localization assays 100 000 and 50 000 protoplastcells respectively were transfected with 15 and 10 ll of plasmidsrespectively (containing between 5 and 75 lg of each construct)and the resulting transformed protoplasts were processed afterincubation in the dark at room temperature for 16ndash18 and 24 hrespectively
Whole-mount immunolocalization
The HA-tagged versions of ABCG9 and ABCG14 were detected in7-day-old in vitro-grown Arabidopsis seedlings as described inMethods S1 Anti-HA antibody 12CA5 (Roche httpwwwroche
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 821
com) (1100 dilution) combined with Alexa Fluor 488-conjugatedsecondary antibody (1100 dilution) were used
Confocal laser-scanning microscopy
Images were collected using an SP2 confocal microscope (Leica)RFP and Alexa Fluor 488 were detected using laser lines at 561and 488 nm respectively according to the manufacturerrsquos set-tings RFP fluorescence emission was detected at 550ndash600 nmand Alexa Fluor 488 and GFP fluorescence emission were detectedat 495ndash545 nm For subcellular localization in protoplasts imageswere coded in cyan (GFP) magenta (RFP) and yellow (auto-fluo-rescence) resulting in purple coloration for co-localization inmerged images For the immunolocalization experiment imageswere coded in green (GFP) A water-corrected 209 objective (HCPlan-Apochromat 20907 ImmKor Leica) was used Images werecropped using Adobe Photoshop CS2 and assembled using AdobeIllustrator CS2 software (Abode httpwwwabodecom) Eachimage shown represents a single focal plan
Co-immunoprecipitation assays
Transformed protoplast proteins were extracted using 50 ll lysisbuffer containing 25 mM TrisHCl pH 78 10 mM MgCl2 75 mM
NaCl 5 mM EGTA 1 mM benzamidine 1 mM dithiothreitol 10glycerol 1 Triton Xndash100 and 19 protein inhibitor cocktail(Sigma httpsigmaaldrichcom) The transformed protoplast sus-pension was frozen in liquid nitrogen then thawed on ice andsubjected to centrifugation for 5 min at 150 g Two microliters ofanti-Myc (9E10 Covance httpwwwcovancecom) or anti-HA(16B12 Covance) antibodies were added to the extracts and themixture was incubated for 2 h at 4degC on a rotating wheel Immu-nocomplexes were captured by incubation with 10 ll Protein GndashSepharose beads for a further 2 h at 4degC on a rotating wheel afterwhich they were washed three times for 5 min each in 25 mM
NaPi 150 mM NaCl 5 glycerol and 02 Igepal CAndash630 buffer(Sigma) and then eluted using 40 ll SDS sample buffer at 37degCfor 30 min Co-immunoprecipitation of Myc- and HA-tagged pro-teins was revealed by SDSndashPAGE followed by Western blottingusing both anti-Myc (Invitrogen) and anti-HA (3F10 Roche) anti-bodies at 12000 dilution
RNA extraction and cDNA preparation
To quantify the relative amounts of the ABCG transcriptsexpressed in various organs three rosette leaves all the cau-line leaves the bottom 5 cm of the floral stem all closed flow-ers and all green siliques were harvested from each of threeindependent 4-week-old Arabidopsis plants of each genotypegrown in soil For the other experiments 10-day-old seedlingsgrown in vitro were harvested All the material was directly fro-zen in liquid nitrogen and stored at 80degC prior to RNA extrac-tion Samples were then disrupted using a MM 301 vibrationmill (Retsch GmbH httpwwwretschcom) at a frequency of25 Hz for 2 min with 3 mm tungsten carbide beads (Qiagenhttpwwwqiagencom) Total RNA was isolated from thismaterial using TRIzol (Life Technologies httplifetechnologiescom) RNA quantity and quality were measured using a Nano-Drop ND 1000 (Thermo Scientific httpwwwthermoscientificcom) All samples with 260280 nm and 260230 nm absorbanceratios lower than 18 were discarded and RNA was re-extractedDNase treatment was applied to 5 lg samples of total RNAusing a DNase I kit (Fermentas httpwwwfermentascom)Double-stranded cDNA was synthesized from 5 lg total RNAusing a SuperScript reverse transcriptase kit (Invitrogen) witholigo(dT)12 primer Finally RNase treatment was applied to the
samples using Escherichia coli RNAse H (Fermentas) The cDNAsamples obtained were diluted 30-fold (except for abcg9 linesamples which were diluted 10-fold) prior to use either toverify the absence of full-length transcripts in putative knockoutmutants or to quantify gene expression by quantitative PCR Allprimers used were designed using the Primer3 program (Rozenand Skaletsky 2000) and their sequences are listed inTable S1
QUANTITATIVE REAL-TIME PCR
Quantitative PCR amplifications were performed in 20 llmixtures containing 10 ll 59 SYBR Green I master mix
(Roche) 02 ll forward and reverse primer (30 lM each)
48 ll sterile water and 5 ll cDNA The BiondashRad CFX96
real-time PCR detection system (httpwwwbio-radcom)
was used for quantification of expression of the sterol
biosynthesis transcripts and the ABCG transcripts in the
various mutant backgrounds Finally the Roche LightCy-
cler 480 system (Roche) was used to quantify the rela-
tive abundance of ABCG transcripts in various organs
Four genes were considered as potential reference genes
[APT1 (At1g27450) TIP41 (At4g34270) EF1a (At5g60390)
and UBQ10 (At4g05320)] and their suitability was tested
using the geNorm algorithm (Vandesompele et al 2002)
to determine the most appropriate one for each experi-
ment A more detailed procedure following the Minimum
Information for Publication of Quantitative Real-Time PCR
Experiments guidelines (Bustin et al 2009) is described
in Methods S2 and the annealing temperature and
efficiencies of the primer pairs used are shown in
Table S2
VLCFA quantification
Approximately 5 mg dry weight of 10-day-old in vitro
Arabidopsis seedlings were analyzed for VLCFA quantifica-
tion as described by Li et al (2006)
Sterol extraction and GC-FID characterization
A pool of 10-day-old seedlings grown in vitro or rosettes
from 4-week-old plants grown in soil were lyophilized prior
to extraction Sterol extraction and isolation was per-
formed as described previously (Bouvier-Nave et al 2010)
Gas Chromatography-Flame Ionization Detector (GC-FID)
characterization was performed as described by Silvestro
et al (2013) To quantify the total amounts of free sterols
and sterol esters non-saponifiable lipids were extracted
using nndashhexane and sterols were derivatized as sterols ace-
tates Quantification was achieved using lupenyl-3 28ndashdi-
acetate as an internal standard
Metabolome analysis
To obtain metabolome profiles associated with wild-type
abcg11ndash7+ abcg11ndash7 abcg9ndash1 abcg9ndash2 and abcg14ndash1
genotypes 10-day-old seedlings representing each geno-
type grown in vitro as described above were harvested
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
822 Rozenn Le Hir et al
and divided into shoot and root samples In the case of
abcg9ndash2 the phenotype of the heterozygote abcg9ndash2+
was indistinguishable from the homozygote phenotype
and therefore heterozygous and homozygous seedlings
from a segregating population were pooled Hence in this
particular experiment abcg9ndash2+ refers to a mixed popula-
tion comprising two-thirds heterozygotes and one-third
homozygotes
Metabolites in five samples of shoot and root parts of
seedlings of each genotype (10 mg fresh weight per sam-
ple) were then extracted analyzed by GCMS and the
acquired data were processed as previously described
(Gullberg et al 2004 Jonsson et al 2005) All multivariate
statistical investigations (partial component analysis and
orthogonal partial least-squares discriminant analysis) for
between-genotype similarities and differences in general
metabolite profiles were performed using Simca software
(Umetrics httpwwwumetricscom)
ACKNOWLEDGEMENTS
We thank A Murphy (Plant Science and Landscape Archi-
tecture University of Maryland MD USA) for stimulating
discussions and critical reading of the manuscript I Carl-
son (Umea Plant Science Center Department of Forest
Genetics and Plant Physiology Swedish University of Agri-
cultural Sciences Umea Sweden) for technical assistance
and S Vernhettes for kindly donating seeds of the
transgenic line carrying the plasma membrane marker
GFPndashLTi6b We also thank the European Arabidopsis Stock
Centre and the GABI-Kat stock centre for providing seeds
This work was supported by allocations granted to CB by
the Swedish Research Council for Agriculture the Swedish
Foundation for Strategic Research the Swedish Research
Council for Research and Innovation for Sustainable
Growth the K amp A Wallenberg Foundation and the Carl
Trygger Foundation
SUPPORTING INFORMATION
Additional Supporting Information may be found in the onlineversion of this articleAppendix S1 Raw data quantification for compounds identified inthe metabolite profiling by GCMS
Figure S1 Relative expression of ABCG genes in various organsand mutant backgroundsFigure S2 Co-immunoprecipitation controlsFigure S3 ABCG9 and ABCG14 homo- andor heterodimerize withABCG11Figure S4 Complementary data on metabolome analysis and con-jugated sterol contentFigure S5 Relative expression of genes related to sterol homeo-stasisMethods S1 Method for whole-mount immunolocalizationMethods S2 Method for quantitative PCR
Table S1 List of primers used in this studyTable S2 Annealing temperature and efficiencies for the primerspairs used in this study
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Behmer ST Grebenok RJ and Douglas AE (2011) Plant sterols and host
plant suitability for a phloem-feeding insect Funct Ecol 25 484ndash491Bird D Beisson F Brigham A Shin J Greer S Jetter R Kunst L
Wu XW Yephremov A and Samuels L (2007) Characterization of Ara-
bidopsis ABCG11WBC11 an ATP binding cassette (ABC) transporter that
is required for cuticular lipid secretion Plant J 52 485ndash498Bouvaine S Behmer ST Lin GG Faure MndashL Grebenok RJ and
Douglas AE (2012) The physiology of sterol nutrition in the pea aphid
Acyrthosiphon pisum J Insect Physiol 58 1383ndash1389Bouvier-Nave P Berna A Noiriel A Compagnon V Carlsson AS
Banas A Stymne S and Schaller H (2010) Involvement of the phos-
pholipid sterol acyltransferase1 in plant sterol homeostasis and leaf
senescence Plant Physiol 152 107ndash119Bustin SA Benes V Garson JA Hellemans J Huggett J Kubista
M Mueller R Nolan T Pfaffl MW and Shipley GL (2009) The MIQE
guidelines Minimum information for publication of quantitative real--
time PCR experiments Clin Chem 55 611
Carland FM Fujioka S Takatsuto S Yoshida S and Nelson T (2002)
The identification of CVP1 reveals a role for sterols in vascular pattern-
ing Plant Cell 14 2045ndash2058Choi H Jin JY Choi S Hwang JU Kim YY Suh MC and Lee Y
(2010) An ABCGWBC-type ABC transporter is essential for transport of
sporopollenin precursors for exine formation in developing pollen Plant
J 65 181ndash193Citovsky V Lee LY Vyas S Glick E Chen MH Vainstein A Gafni
Y Gelvin SB and Tzfira T (2006) Subcellular localization of interacting
proteins by bimolecular fluorescence complementation in planta J Mol
Biol 362 1120ndash1131Clough SJ and Bent AF (1998) Floral dip a simplified method for Agro-
bacterium-mediated transformation of Arabidopsis thaliana Plant J 16
735ndash743Clouse SD (2002) Arabidopsis mutants reveal multiple roles for sterols in
plant development Plant Cell 14 1995ndash2000Cnops G Neyt P Raes J et al (2006) The TORNADO1 and TORNADO2
genes function in several patterning processes during early leaf develop-
ment in Arabidopsis thaliana Plant Cell 18 852ndash866Cutler SR Ehrhardt DW Griffitts JS and Somerville CR (2000) Random
GFPcDNA fusions enable visualization of subcellular structures in cells of
Arabidopsis at a high frequency Proc Natl Acad Sci USA 97 3718ndash3723Dou XY Yang KZ Zhang Y Wang W Liu XL Chen LQ Zhang
XQ and Ye D (2011) WBC27 an adenosine tri-phosphate-binding cas-
sette protein controls pollen wall formation and patterning in Arabidop-
sis J Integr Plant Biol 53 74ndash88Feurouleuroop K Pettko-Szandtner A Magyar Z Miskolczi P Kondorosi E
Dudits D and Bako L (2005) The Medicago CDKC1ndashCYCLINT1 kinase
complex phosphorylates the carboxy-terminal domain of RNA polymer-
ase II and promotes transcription Plant J 42 810ndash820Gullberg J Jonsson P Nordstrom A Sjostrom M and Moritz T (2004)
Design of experiments an efficient strategy to identify factors influenc-
ing extraction and derivatization of Arabidopsis thaliana samples in met-
abolomic studies with gas chromatographymass spectrometry Anal
Biochem 331 283ndash295Hertzberg M Aspeborg H Schrader J et al (2001) A transcriptional
roadmap to wood formation Proc Natl Acad Sci USA 98 14732ndash14737Jang JC Fujioka S Tasaka M Seto H Takatsuto S Ishii A Aida M
Yoshida S and Sheen J (2000) A critical role of sterols in embryonic
patterning and meristem programming revealed by the fackel mutants of
Arabidopsis thaliana Genes Dev 14 1485ndash1497Jenks MA Rashotte AM Tuttle HA and Feldmann KA (1996) Mutants
in Arabidopsis thaliana altered in epicuticular wax and leaf morphology
Plant Physiol 110 377ndash385Jonsson P Johansson AI Gullberg J Trygg J A J Grung B Markl-
und S Sjostrom M Antti H and Moritz T (2005) High-throughput
data analysis for detecting and identifying differences between samples
in GCMS-based metabolomic analyses Anal Chem 77 5635ndash5642Jungwirth H and Kuchler K (2006) Yeast ABC transporters ndash a tale of sex
stress drugs and aging FEBS Lett 580 1131ndash1138Kaneda M Schuetz M Lin B Chanis C Hamberger B Western T
Ehlting J and Samuels A (2011) ABC transporters coordinately
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ABCG proteins and vascular development 823
expressed during lignification of Arabidopsis stems include a set of AB-
CBs associated with auxin transport J Exp Bot 62 2063
Karimi M Inze D and Depicker A (2002) GATEWAYTMvectors for Agrobac-
terium-mediated plant transformation Trends Plant Sci 7 193ndash195Kneurooller A and Murphy A (2011) ABC transporters and their function at the
plasma membrane In The Plant Plasma Membrane Plant Cell Mono-
graphs vol 19 (Murphy AS Peer W and Schulz B eds) New York
NY Springer pp 353ndash377Kopischke M Westphal L Schneeberger K Clark R Ossowski S
Wewer V Fuchs R Landtag J Hause G and Deuroormann P (2013)
Impaired sterol ester synthesis alters the response of Arabidopsis thali-
ana to Phytophthora infestans Plant J 73 456ndash468Kuromori T Miyaji T Yabuuchi H Shimizu H Sugimoto E Kamiya
A Moriyama Y and Shinozaki K (2010) ABC transporter AtABCG25 is
involved in abscisic acid transport and responses Proc Natl Acad Sci
USA 107 2361ndash2366Kuromori T Itoh T Sugimoto E and Shinozaki K (2011a) Arabidopsis
mutant of AtABCG26 an ABC transporter gene is defective in pollen
maturation J Plant Physiol 168 2001ndash2005Kuromori T Sugimoto E and Shinozaki K (2011b) Arabidopsis mutants
of AtABCG22 an ABC transporter gene increase transpiration and
drought susceptibility Plant J 67 885ndash894Le Hir R Beneteau J Bellini C Vilaine F and Dinant S (2008) Gene
expression profiling keys for investigating phloem functions Trends
Plant Sci 13 273ndash280Lehrer AT Dugassa-Gobena D Vidal S and Seifert K (2000) Transport
of resistance-inducing sterols in phloem sap of barley Z Naturforsch C
55 948ndash952Li Y Beisson F Pollard M and Ohlrogge J (2006) Oil content of Arabid-
opsis seeds the influence of seed anatomy light and plant-to-plant vari-
ation Phytochemistry 67 904ndash915Luo B Xue XndashY Hu WndashL Wang LndashJ and Chen XndashY (2007) An ABC
transporter gene of Arabidopsis thaliana AtWBC11 is involved in cuticle
development and prevention of organ fusion Plant Cell Physiol 48
1790ndash1802McFarlane HE Shin JJH Bird DA and Samuels AL (2010)
Arabidopsis ABCG transporters which are required for export of diverse
cuticular lipids dimerize in different combinations Plant Cell 22 3066ndash3075
Meskiene I Baudouin E Schweighofer A Liwosz A Jonak C Rodri-
guez PL Jelinek H and Hirt H (2003) Stress-induced protein phospha-
tase 2C is a negative regulator of a mitogen-activated protein kinase
J Biol Chem 278 18945ndash18952Murashige T and Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures Physiol Plant 15 473ndash497Panikashvili D Savaldi-Goldstein S Mandel T Yifhar T Franke RB
Hofer R Schreiber L Chory J and Aharoni A (2007) The Arabidopsis
DESPERADOAtWBC11 transporter is required for cutin and wax secre-
tion Plant Physiol 145 1345ndash1360Panikashvili D Shi JX Bocobza S Franke RB Schreiber L and Aha-
roni A (2010) The Arabidopsis DSOABCG11 transporter affects cutin
metabolism in reproductive organs and suberin in roots Mol Plant 3
563ndash575Panikashvili D Shi JX Schreiber L and Aharoni A (2011) The Arabid-
opsis ABCG13 transporter is required for flower cuticle secretion and pat-
terning of the petal epidermis New Phytol 190 113ndash124Pighin JA Zheng H Balakshin LJ Goodman IP Western TL Jetter
R Kunst L and Samuels AL (2004) Plant cuticular lipid export requires
an ABC transporter Science 306 702ndash704Pullen M Clark N Zarinkamar F Topping J and Lindsey K (2010)
Analysis of vascular development in the hydra sterol biosynthetic
mutants of Arabidopsis PLoS ONE 5 e12227
Quilichini TD Friedmann MC Samuels AL and Douglas CJ (2010)
ATP-binding cassette transporter G26 is required for male fertility and
pollen exine formation in Arabidopsis Plant Physiol 154 678ndash690R Development Core Team (2010) R A Language and Environment for
Statistical Computing Vienna Austria R Foundation for Statistical
Computing
Roudier F Gissot L Beaudoin F Haslam R Michaelson L Marion J
Molino D Lima A Bach L and Morin H (2010) Very-long-chain fatty
acids are involved in polar auxin transport and developmental patterning
in Arabidopsis Plant Cell 22 364ndash375Rozen S and Skaletsky HJ (2000) Primer3 on the WWW for general users
and for biologist programmers Methods Mol Biol 132 365ndash386Schad M Lipton MS Giavalisco P Smith RD and Kehr J (2005) Eval-
uation of two-dimensional electrophoresis and liquid chromatography
tandem mass spectrometry for tissue-specific protein profiling of
laser-microdissected plant samples Electrophoresis 26 2729ndash2738Schauer N Steinhauser D Strelkov S et al (2005) GCndashMS libraries for
the rapid identification of metabolites in complex biological samples
FEBS Lett 579 1332ndash1337Schrader J Nilsson J Mellerowicz EJ Berglund A Nilsson P Hertz-
berg M and Sandberg G (2004) A high-resolution transcript profile
across the wood forming meristem of poplar identifies potential regula-
tors of cambial stem cell identity Plant Cell 16 2278ndash2292Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desi-
mone M Frommer WB Fleurougge UndashI and Kunze R (2003) ARAMEM-
NON a novel database for Arabidopsis integral membrane proteins
Plant Physiol 131 16ndash26Silvestro D Andersen TG Schaller H and Jensen PE (2013) Plant ste-
rol metabolism D7ndashsterol-C5ndashdesaturase (STE1DWARF7) D57ndashste-rol-D7ndashreductase (DWARF5) and D24ndashsterol-D24ndashreductase (DIMINUTO
DWARF1) show multiple subcellular localizations in Arabidopsis thaliana
(Heynh) L PLoS ONE 8 e56429
Sorin C Bussell JD Camus I Ljung K Kowalczyk M Geiss G McKh-
ann H Garcion C Vaucheret H and Sandberg G (2005) Auxin and
light control of adventitious rooting in Arabidopsis require ARGONA-
UTE1 Plant Cell 17 1343ndash1359Tanaka T Tanaka H Machida C Watanabe M and Machida Y (2004) A
new method for rapid visualization of defects in leaf cuticle reveals five
intrinsic patterns of surface defects in Arabidopsis Plant J 37 139ndash146Turgeon R and Wolf S (2009) Phloem transport cellular pathways and
molecular trafficking Annu Rev Plant Biol 60 207ndash221Ukitsu H Kuromori T Toyooka K et al (2007) Cytological and biochemi-
cal analysis of COF1 an Arabidopsis mutant of an ABC transporter gene
Plant Cell Physiol 48 1524ndash1533Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De
Paepe A and Speleman F (2002) Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biol 3 research00340031-00340011Verrier PJ Bird D Burla B et al (2008) Plant ABC proteins ndash a unified
nomenclature andupdated inventory Trends Plant Sci 13 151ndash159Vilaine F Palauqui JC Amselem J Kusiak C Lemoine R and Dinant
S (2003) Towards deciphering phloem a transcriptome analysis of the
phloem of Apium graveolens Plant J 36 67ndash81Zhai Z Sooksa-nguan T and Vatamaniuk OK (2009) Establishing RNA
interference as a reverse-genetic approach for gene functional analysis
in protoplasts Plant Physiol 149 642ndash652Zhang Q Blaylock LA and Harrison MJ (2010) Two Medicago truncatula
half-ABC transporters are essential for arbuscule development in
arbuscular mycorrhizal symbiosis Plant Cell 22 1483
Zhao C Craig JC Petzold HE Dickerman AW and Beers EP (2005)
The xylem and phloem transcriptomes from secondary tissues of the
Arabidopsis root-hypocotyl Plant Physiol 138 803ndash818
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
824 Rozenn Le Hir et al
com) (1100 dilution) combined with Alexa Fluor 488-conjugatedsecondary antibody (1100 dilution) were used
Confocal laser-scanning microscopy
Images were collected using an SP2 confocal microscope (Leica)RFP and Alexa Fluor 488 were detected using laser lines at 561and 488 nm respectively according to the manufacturerrsquos set-tings RFP fluorescence emission was detected at 550ndash600 nmand Alexa Fluor 488 and GFP fluorescence emission were detectedat 495ndash545 nm For subcellular localization in protoplasts imageswere coded in cyan (GFP) magenta (RFP) and yellow (auto-fluo-rescence) resulting in purple coloration for co-localization inmerged images For the immunolocalization experiment imageswere coded in green (GFP) A water-corrected 209 objective (HCPlan-Apochromat 20907 ImmKor Leica) was used Images werecropped using Adobe Photoshop CS2 and assembled using AdobeIllustrator CS2 software (Abode httpwwwabodecom) Eachimage shown represents a single focal plan
Co-immunoprecipitation assays
Transformed protoplast proteins were extracted using 50 ll lysisbuffer containing 25 mM TrisHCl pH 78 10 mM MgCl2 75 mM
NaCl 5 mM EGTA 1 mM benzamidine 1 mM dithiothreitol 10glycerol 1 Triton Xndash100 and 19 protein inhibitor cocktail(Sigma httpsigmaaldrichcom) The transformed protoplast sus-pension was frozen in liquid nitrogen then thawed on ice andsubjected to centrifugation for 5 min at 150 g Two microliters ofanti-Myc (9E10 Covance httpwwwcovancecom) or anti-HA(16B12 Covance) antibodies were added to the extracts and themixture was incubated for 2 h at 4degC on a rotating wheel Immu-nocomplexes were captured by incubation with 10 ll Protein GndashSepharose beads for a further 2 h at 4degC on a rotating wheel afterwhich they were washed three times for 5 min each in 25 mM
NaPi 150 mM NaCl 5 glycerol and 02 Igepal CAndash630 buffer(Sigma) and then eluted using 40 ll SDS sample buffer at 37degCfor 30 min Co-immunoprecipitation of Myc- and HA-tagged pro-teins was revealed by SDSndashPAGE followed by Western blottingusing both anti-Myc (Invitrogen) and anti-HA (3F10 Roche) anti-bodies at 12000 dilution
RNA extraction and cDNA preparation
To quantify the relative amounts of the ABCG transcriptsexpressed in various organs three rosette leaves all the cau-line leaves the bottom 5 cm of the floral stem all closed flow-ers and all green siliques were harvested from each of threeindependent 4-week-old Arabidopsis plants of each genotypegrown in soil For the other experiments 10-day-old seedlingsgrown in vitro were harvested All the material was directly fro-zen in liquid nitrogen and stored at 80degC prior to RNA extrac-tion Samples were then disrupted using a MM 301 vibrationmill (Retsch GmbH httpwwwretschcom) at a frequency of25 Hz for 2 min with 3 mm tungsten carbide beads (Qiagenhttpwwwqiagencom) Total RNA was isolated from thismaterial using TRIzol (Life Technologies httplifetechnologiescom) RNA quantity and quality were measured using a Nano-Drop ND 1000 (Thermo Scientific httpwwwthermoscientificcom) All samples with 260280 nm and 260230 nm absorbanceratios lower than 18 were discarded and RNA was re-extractedDNase treatment was applied to 5 lg samples of total RNAusing a DNase I kit (Fermentas httpwwwfermentascom)Double-stranded cDNA was synthesized from 5 lg total RNAusing a SuperScript reverse transcriptase kit (Invitrogen) witholigo(dT)12 primer Finally RNase treatment was applied to the
samples using Escherichia coli RNAse H (Fermentas) The cDNAsamples obtained were diluted 30-fold (except for abcg9 linesamples which were diluted 10-fold) prior to use either toverify the absence of full-length transcripts in putative knockoutmutants or to quantify gene expression by quantitative PCR Allprimers used were designed using the Primer3 program (Rozenand Skaletsky 2000) and their sequences are listed inTable S1
QUANTITATIVE REAL-TIME PCR
Quantitative PCR amplifications were performed in 20 llmixtures containing 10 ll 59 SYBR Green I master mix
(Roche) 02 ll forward and reverse primer (30 lM each)
48 ll sterile water and 5 ll cDNA The BiondashRad CFX96
real-time PCR detection system (httpwwwbio-radcom)
was used for quantification of expression of the sterol
biosynthesis transcripts and the ABCG transcripts in the
various mutant backgrounds Finally the Roche LightCy-
cler 480 system (Roche) was used to quantify the rela-
tive abundance of ABCG transcripts in various organs
Four genes were considered as potential reference genes
[APT1 (At1g27450) TIP41 (At4g34270) EF1a (At5g60390)
and UBQ10 (At4g05320)] and their suitability was tested
using the geNorm algorithm (Vandesompele et al 2002)
to determine the most appropriate one for each experi-
ment A more detailed procedure following the Minimum
Information for Publication of Quantitative Real-Time PCR
Experiments guidelines (Bustin et al 2009) is described
in Methods S2 and the annealing temperature and
efficiencies of the primer pairs used are shown in
Table S2
VLCFA quantification
Approximately 5 mg dry weight of 10-day-old in vitro
Arabidopsis seedlings were analyzed for VLCFA quantifica-
tion as described by Li et al (2006)
Sterol extraction and GC-FID characterization
A pool of 10-day-old seedlings grown in vitro or rosettes
from 4-week-old plants grown in soil were lyophilized prior
to extraction Sterol extraction and isolation was per-
formed as described previously (Bouvier-Nave et al 2010)
Gas Chromatography-Flame Ionization Detector (GC-FID)
characterization was performed as described by Silvestro
et al (2013) To quantify the total amounts of free sterols
and sterol esters non-saponifiable lipids were extracted
using nndashhexane and sterols were derivatized as sterols ace-
tates Quantification was achieved using lupenyl-3 28ndashdi-
acetate as an internal standard
Metabolome analysis
To obtain metabolome profiles associated with wild-type
abcg11ndash7+ abcg11ndash7 abcg9ndash1 abcg9ndash2 and abcg14ndash1
genotypes 10-day-old seedlings representing each geno-
type grown in vitro as described above were harvested
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
822 Rozenn Le Hir et al
and divided into shoot and root samples In the case of
abcg9ndash2 the phenotype of the heterozygote abcg9ndash2+
was indistinguishable from the homozygote phenotype
and therefore heterozygous and homozygous seedlings
from a segregating population were pooled Hence in this
particular experiment abcg9ndash2+ refers to a mixed popula-
tion comprising two-thirds heterozygotes and one-third
homozygotes
Metabolites in five samples of shoot and root parts of
seedlings of each genotype (10 mg fresh weight per sam-
ple) were then extracted analyzed by GCMS and the
acquired data were processed as previously described
(Gullberg et al 2004 Jonsson et al 2005) All multivariate
statistical investigations (partial component analysis and
orthogonal partial least-squares discriminant analysis) for
between-genotype similarities and differences in general
metabolite profiles were performed using Simca software
(Umetrics httpwwwumetricscom)
ACKNOWLEDGEMENTS
We thank A Murphy (Plant Science and Landscape Archi-
tecture University of Maryland MD USA) for stimulating
discussions and critical reading of the manuscript I Carl-
son (Umea Plant Science Center Department of Forest
Genetics and Plant Physiology Swedish University of Agri-
cultural Sciences Umea Sweden) for technical assistance
and S Vernhettes for kindly donating seeds of the
transgenic line carrying the plasma membrane marker
GFPndashLTi6b We also thank the European Arabidopsis Stock
Centre and the GABI-Kat stock centre for providing seeds
This work was supported by allocations granted to CB by
the Swedish Research Council for Agriculture the Swedish
Foundation for Strategic Research the Swedish Research
Council for Research and Innovation for Sustainable
Growth the K amp A Wallenberg Foundation and the Carl
Trygger Foundation
SUPPORTING INFORMATION
Additional Supporting Information may be found in the onlineversion of this articleAppendix S1 Raw data quantification for compounds identified inthe metabolite profiling by GCMS
Figure S1 Relative expression of ABCG genes in various organsand mutant backgroundsFigure S2 Co-immunoprecipitation controlsFigure S3 ABCG9 and ABCG14 homo- andor heterodimerize withABCG11Figure S4 Complementary data on metabolome analysis and con-jugated sterol contentFigure S5 Relative expression of genes related to sterol homeo-stasisMethods S1 Method for whole-mount immunolocalizationMethods S2 Method for quantitative PCR
Table S1 List of primers used in this studyTable S2 Annealing temperature and efficiencies for the primerspairs used in this study
REFERENCES
Behmer ST Grebenok RJ and Douglas AE (2011) Plant sterols and host
plant suitability for a phloem-feeding insect Funct Ecol 25 484ndash491Bird D Beisson F Brigham A Shin J Greer S Jetter R Kunst L
Wu XW Yephremov A and Samuels L (2007) Characterization of Ara-
bidopsis ABCG11WBC11 an ATP binding cassette (ABC) transporter that
is required for cuticular lipid secretion Plant J 52 485ndash498Bouvaine S Behmer ST Lin GG Faure MndashL Grebenok RJ and
Douglas AE (2012) The physiology of sterol nutrition in the pea aphid
Acyrthosiphon pisum J Insect Physiol 58 1383ndash1389Bouvier-Nave P Berna A Noiriel A Compagnon V Carlsson AS
Banas A Stymne S and Schaller H (2010) Involvement of the phos-
pholipid sterol acyltransferase1 in plant sterol homeostasis and leaf
senescence Plant Physiol 152 107ndash119Bustin SA Benes V Garson JA Hellemans J Huggett J Kubista
M Mueller R Nolan T Pfaffl MW and Shipley GL (2009) The MIQE
guidelines Minimum information for publication of quantitative real--
time PCR experiments Clin Chem 55 611
Carland FM Fujioka S Takatsuto S Yoshida S and Nelson T (2002)
The identification of CVP1 reveals a role for sterols in vascular pattern-
ing Plant Cell 14 2045ndash2058Choi H Jin JY Choi S Hwang JU Kim YY Suh MC and Lee Y
(2010) An ABCGWBC-type ABC transporter is essential for transport of
sporopollenin precursors for exine formation in developing pollen Plant
J 65 181ndash193Citovsky V Lee LY Vyas S Glick E Chen MH Vainstein A Gafni
Y Gelvin SB and Tzfira T (2006) Subcellular localization of interacting
proteins by bimolecular fluorescence complementation in planta J Mol
Biol 362 1120ndash1131Clough SJ and Bent AF (1998) Floral dip a simplified method for Agro-
bacterium-mediated transformation of Arabidopsis thaliana Plant J 16
735ndash743Clouse SD (2002) Arabidopsis mutants reveal multiple roles for sterols in
plant development Plant Cell 14 1995ndash2000Cnops G Neyt P Raes J et al (2006) The TORNADO1 and TORNADO2
genes function in several patterning processes during early leaf develop-
ment in Arabidopsis thaliana Plant Cell 18 852ndash866Cutler SR Ehrhardt DW Griffitts JS and Somerville CR (2000) Random
GFPcDNA fusions enable visualization of subcellular structures in cells of
Arabidopsis at a high frequency Proc Natl Acad Sci USA 97 3718ndash3723Dou XY Yang KZ Zhang Y Wang W Liu XL Chen LQ Zhang
XQ and Ye D (2011) WBC27 an adenosine tri-phosphate-binding cas-
sette protein controls pollen wall formation and patterning in Arabidop-
sis J Integr Plant Biol 53 74ndash88Feurouleuroop K Pettko-Szandtner A Magyar Z Miskolczi P Kondorosi E
Dudits D and Bako L (2005) The Medicago CDKC1ndashCYCLINT1 kinase
complex phosphorylates the carboxy-terminal domain of RNA polymer-
ase II and promotes transcription Plant J 42 810ndash820Gullberg J Jonsson P Nordstrom A Sjostrom M and Moritz T (2004)
Design of experiments an efficient strategy to identify factors influenc-
ing extraction and derivatization of Arabidopsis thaliana samples in met-
abolomic studies with gas chromatographymass spectrometry Anal
Biochem 331 283ndash295Hertzberg M Aspeborg H Schrader J et al (2001) A transcriptional
roadmap to wood formation Proc Natl Acad Sci USA 98 14732ndash14737Jang JC Fujioka S Tasaka M Seto H Takatsuto S Ishii A Aida M
Yoshida S and Sheen J (2000) A critical role of sterols in embryonic
patterning and meristem programming revealed by the fackel mutants of
Arabidopsis thaliana Genes Dev 14 1485ndash1497Jenks MA Rashotte AM Tuttle HA and Feldmann KA (1996) Mutants
in Arabidopsis thaliana altered in epicuticular wax and leaf morphology
Plant Physiol 110 377ndash385Jonsson P Johansson AI Gullberg J Trygg J A J Grung B Markl-
und S Sjostrom M Antti H and Moritz T (2005) High-throughput
data analysis for detecting and identifying differences between samples
in GCMS-based metabolomic analyses Anal Chem 77 5635ndash5642Jungwirth H and Kuchler K (2006) Yeast ABC transporters ndash a tale of sex
stress drugs and aging FEBS Lett 580 1131ndash1138Kaneda M Schuetz M Lin B Chanis C Hamberger B Western T
Ehlting J and Samuels A (2011) ABC transporters coordinately
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 823
expressed during lignification of Arabidopsis stems include a set of AB-
CBs associated with auxin transport J Exp Bot 62 2063
Karimi M Inze D and Depicker A (2002) GATEWAYTMvectors for Agrobac-
terium-mediated plant transformation Trends Plant Sci 7 193ndash195Kneurooller A and Murphy A (2011) ABC transporters and their function at the
plasma membrane In The Plant Plasma Membrane Plant Cell Mono-
graphs vol 19 (Murphy AS Peer W and Schulz B eds) New York
NY Springer pp 353ndash377Kopischke M Westphal L Schneeberger K Clark R Ossowski S
Wewer V Fuchs R Landtag J Hause G and Deuroormann P (2013)
Impaired sterol ester synthesis alters the response of Arabidopsis thali-
ana to Phytophthora infestans Plant J 73 456ndash468Kuromori T Miyaji T Yabuuchi H Shimizu H Sugimoto E Kamiya
A Moriyama Y and Shinozaki K (2010) ABC transporter AtABCG25 is
involved in abscisic acid transport and responses Proc Natl Acad Sci
USA 107 2361ndash2366Kuromori T Itoh T Sugimoto E and Shinozaki K (2011a) Arabidopsis
mutant of AtABCG26 an ABC transporter gene is defective in pollen
maturation J Plant Physiol 168 2001ndash2005Kuromori T Sugimoto E and Shinozaki K (2011b) Arabidopsis mutants
of AtABCG22 an ABC transporter gene increase transpiration and
drought susceptibility Plant J 67 885ndash894Le Hir R Beneteau J Bellini C Vilaine F and Dinant S (2008) Gene
expression profiling keys for investigating phloem functions Trends
Plant Sci 13 273ndash280Lehrer AT Dugassa-Gobena D Vidal S and Seifert K (2000) Transport
of resistance-inducing sterols in phloem sap of barley Z Naturforsch C
55 948ndash952Li Y Beisson F Pollard M and Ohlrogge J (2006) Oil content of Arabid-
opsis seeds the influence of seed anatomy light and plant-to-plant vari-
ation Phytochemistry 67 904ndash915Luo B Xue XndashY Hu WndashL Wang LndashJ and Chen XndashY (2007) An ABC
transporter gene of Arabidopsis thaliana AtWBC11 is involved in cuticle
development and prevention of organ fusion Plant Cell Physiol 48
1790ndash1802McFarlane HE Shin JJH Bird DA and Samuels AL (2010)
Arabidopsis ABCG transporters which are required for export of diverse
cuticular lipids dimerize in different combinations Plant Cell 22 3066ndash3075
Meskiene I Baudouin E Schweighofer A Liwosz A Jonak C Rodri-
guez PL Jelinek H and Hirt H (2003) Stress-induced protein phospha-
tase 2C is a negative regulator of a mitogen-activated protein kinase
J Biol Chem 278 18945ndash18952Murashige T and Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures Physiol Plant 15 473ndash497Panikashvili D Savaldi-Goldstein S Mandel T Yifhar T Franke RB
Hofer R Schreiber L Chory J and Aharoni A (2007) The Arabidopsis
DESPERADOAtWBC11 transporter is required for cutin and wax secre-
tion Plant Physiol 145 1345ndash1360Panikashvili D Shi JX Bocobza S Franke RB Schreiber L and Aha-
roni A (2010) The Arabidopsis DSOABCG11 transporter affects cutin
metabolism in reproductive organs and suberin in roots Mol Plant 3
563ndash575Panikashvili D Shi JX Schreiber L and Aharoni A (2011) The Arabid-
opsis ABCG13 transporter is required for flower cuticle secretion and pat-
terning of the petal epidermis New Phytol 190 113ndash124Pighin JA Zheng H Balakshin LJ Goodman IP Western TL Jetter
R Kunst L and Samuels AL (2004) Plant cuticular lipid export requires
an ABC transporter Science 306 702ndash704Pullen M Clark N Zarinkamar F Topping J and Lindsey K (2010)
Analysis of vascular development in the hydra sterol biosynthetic
mutants of Arabidopsis PLoS ONE 5 e12227
Quilichini TD Friedmann MC Samuels AL and Douglas CJ (2010)
ATP-binding cassette transporter G26 is required for male fertility and
pollen exine formation in Arabidopsis Plant Physiol 154 678ndash690R Development Core Team (2010) R A Language and Environment for
Statistical Computing Vienna Austria R Foundation for Statistical
Computing
Roudier F Gissot L Beaudoin F Haslam R Michaelson L Marion J
Molino D Lima A Bach L and Morin H (2010) Very-long-chain fatty
acids are involved in polar auxin transport and developmental patterning
in Arabidopsis Plant Cell 22 364ndash375Rozen S and Skaletsky HJ (2000) Primer3 on the WWW for general users
and for biologist programmers Methods Mol Biol 132 365ndash386Schad M Lipton MS Giavalisco P Smith RD and Kehr J (2005) Eval-
uation of two-dimensional electrophoresis and liquid chromatography
tandem mass spectrometry for tissue-specific protein profiling of
laser-microdissected plant samples Electrophoresis 26 2729ndash2738Schauer N Steinhauser D Strelkov S et al (2005) GCndashMS libraries for
the rapid identification of metabolites in complex biological samples
FEBS Lett 579 1332ndash1337Schrader J Nilsson J Mellerowicz EJ Berglund A Nilsson P Hertz-
berg M and Sandberg G (2004) A high-resolution transcript profile
across the wood forming meristem of poplar identifies potential regula-
tors of cambial stem cell identity Plant Cell 16 2278ndash2292Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desi-
mone M Frommer WB Fleurougge UndashI and Kunze R (2003) ARAMEM-
NON a novel database for Arabidopsis integral membrane proteins
Plant Physiol 131 16ndash26Silvestro D Andersen TG Schaller H and Jensen PE (2013) Plant ste-
rol metabolism D7ndashsterol-C5ndashdesaturase (STE1DWARF7) D57ndashste-rol-D7ndashreductase (DWARF5) and D24ndashsterol-D24ndashreductase (DIMINUTO
DWARF1) show multiple subcellular localizations in Arabidopsis thaliana
(Heynh) L PLoS ONE 8 e56429
Sorin C Bussell JD Camus I Ljung K Kowalczyk M Geiss G McKh-
ann H Garcion C Vaucheret H and Sandberg G (2005) Auxin and
light control of adventitious rooting in Arabidopsis require ARGONA-
UTE1 Plant Cell 17 1343ndash1359Tanaka T Tanaka H Machida C Watanabe M and Machida Y (2004) A
new method for rapid visualization of defects in leaf cuticle reveals five
intrinsic patterns of surface defects in Arabidopsis Plant J 37 139ndash146Turgeon R and Wolf S (2009) Phloem transport cellular pathways and
molecular trafficking Annu Rev Plant Biol 60 207ndash221Ukitsu H Kuromori T Toyooka K et al (2007) Cytological and biochemi-
cal analysis of COF1 an Arabidopsis mutant of an ABC transporter gene
Plant Cell Physiol 48 1524ndash1533Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De
Paepe A and Speleman F (2002) Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biol 3 research00340031-00340011Verrier PJ Bird D Burla B et al (2008) Plant ABC proteins ndash a unified
nomenclature andupdated inventory Trends Plant Sci 13 151ndash159Vilaine F Palauqui JC Amselem J Kusiak C Lemoine R and Dinant
S (2003) Towards deciphering phloem a transcriptome analysis of the
phloem of Apium graveolens Plant J 36 67ndash81Zhai Z Sooksa-nguan T and Vatamaniuk OK (2009) Establishing RNA
interference as a reverse-genetic approach for gene functional analysis
in protoplasts Plant Physiol 149 642ndash652Zhang Q Blaylock LA and Harrison MJ (2010) Two Medicago truncatula
half-ABC transporters are essential for arbuscule development in
arbuscular mycorrhizal symbiosis Plant Cell 22 1483
Zhao C Craig JC Petzold HE Dickerman AW and Beers EP (2005)
The xylem and phloem transcriptomes from secondary tissues of the
Arabidopsis root-hypocotyl Plant Physiol 138 803ndash818
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
824 Rozenn Le Hir et al
and divided into shoot and root samples In the case of
abcg9ndash2 the phenotype of the heterozygote abcg9ndash2+
was indistinguishable from the homozygote phenotype
and therefore heterozygous and homozygous seedlings
from a segregating population were pooled Hence in this
particular experiment abcg9ndash2+ refers to a mixed popula-
tion comprising two-thirds heterozygotes and one-third
homozygotes
Metabolites in five samples of shoot and root parts of
seedlings of each genotype (10 mg fresh weight per sam-
ple) were then extracted analyzed by GCMS and the
acquired data were processed as previously described
(Gullberg et al 2004 Jonsson et al 2005) All multivariate
statistical investigations (partial component analysis and
orthogonal partial least-squares discriminant analysis) for
between-genotype similarities and differences in general
metabolite profiles were performed using Simca software
(Umetrics httpwwwumetricscom)
ACKNOWLEDGEMENTS
We thank A Murphy (Plant Science and Landscape Archi-
tecture University of Maryland MD USA) for stimulating
discussions and critical reading of the manuscript I Carl-
son (Umea Plant Science Center Department of Forest
Genetics and Plant Physiology Swedish University of Agri-
cultural Sciences Umea Sweden) for technical assistance
and S Vernhettes for kindly donating seeds of the
transgenic line carrying the plasma membrane marker
GFPndashLTi6b We also thank the European Arabidopsis Stock
Centre and the GABI-Kat stock centre for providing seeds
This work was supported by allocations granted to CB by
the Swedish Research Council for Agriculture the Swedish
Foundation for Strategic Research the Swedish Research
Council for Research and Innovation for Sustainable
Growth the K amp A Wallenberg Foundation and the Carl
Trygger Foundation
SUPPORTING INFORMATION
Additional Supporting Information may be found in the onlineversion of this articleAppendix S1 Raw data quantification for compounds identified inthe metabolite profiling by GCMS
Figure S1 Relative expression of ABCG genes in various organsand mutant backgroundsFigure S2 Co-immunoprecipitation controlsFigure S3 ABCG9 and ABCG14 homo- andor heterodimerize withABCG11Figure S4 Complementary data on metabolome analysis and con-jugated sterol contentFigure S5 Relative expression of genes related to sterol homeo-stasisMethods S1 Method for whole-mount immunolocalizationMethods S2 Method for quantitative PCR
Table S1 List of primers used in this studyTable S2 Annealing temperature and efficiencies for the primerspairs used in this study
REFERENCES
Behmer ST Grebenok RJ and Douglas AE (2011) Plant sterols and host
plant suitability for a phloem-feeding insect Funct Ecol 25 484ndash491Bird D Beisson F Brigham A Shin J Greer S Jetter R Kunst L
Wu XW Yephremov A and Samuels L (2007) Characterization of Ara-
bidopsis ABCG11WBC11 an ATP binding cassette (ABC) transporter that
is required for cuticular lipid secretion Plant J 52 485ndash498Bouvaine S Behmer ST Lin GG Faure MndashL Grebenok RJ and
Douglas AE (2012) The physiology of sterol nutrition in the pea aphid
Acyrthosiphon pisum J Insect Physiol 58 1383ndash1389Bouvier-Nave P Berna A Noiriel A Compagnon V Carlsson AS
Banas A Stymne S and Schaller H (2010) Involvement of the phos-
pholipid sterol acyltransferase1 in plant sterol homeostasis and leaf
senescence Plant Physiol 152 107ndash119Bustin SA Benes V Garson JA Hellemans J Huggett J Kubista
M Mueller R Nolan T Pfaffl MW and Shipley GL (2009) The MIQE
guidelines Minimum information for publication of quantitative real--
time PCR experiments Clin Chem 55 611
Carland FM Fujioka S Takatsuto S Yoshida S and Nelson T (2002)
The identification of CVP1 reveals a role for sterols in vascular pattern-
ing Plant Cell 14 2045ndash2058Choi H Jin JY Choi S Hwang JU Kim YY Suh MC and Lee Y
(2010) An ABCGWBC-type ABC transporter is essential for transport of
sporopollenin precursors for exine formation in developing pollen Plant
J 65 181ndash193Citovsky V Lee LY Vyas S Glick E Chen MH Vainstein A Gafni
Y Gelvin SB and Tzfira T (2006) Subcellular localization of interacting
proteins by bimolecular fluorescence complementation in planta J Mol
Biol 362 1120ndash1131Clough SJ and Bent AF (1998) Floral dip a simplified method for Agro-
bacterium-mediated transformation of Arabidopsis thaliana Plant J 16
735ndash743Clouse SD (2002) Arabidopsis mutants reveal multiple roles for sterols in
plant development Plant Cell 14 1995ndash2000Cnops G Neyt P Raes J et al (2006) The TORNADO1 and TORNADO2
genes function in several patterning processes during early leaf develop-
ment in Arabidopsis thaliana Plant Cell 18 852ndash866Cutler SR Ehrhardt DW Griffitts JS and Somerville CR (2000) Random
GFPcDNA fusions enable visualization of subcellular structures in cells of
Arabidopsis at a high frequency Proc Natl Acad Sci USA 97 3718ndash3723Dou XY Yang KZ Zhang Y Wang W Liu XL Chen LQ Zhang
XQ and Ye D (2011) WBC27 an adenosine tri-phosphate-binding cas-
sette protein controls pollen wall formation and patterning in Arabidop-
sis J Integr Plant Biol 53 74ndash88Feurouleuroop K Pettko-Szandtner A Magyar Z Miskolczi P Kondorosi E
Dudits D and Bako L (2005) The Medicago CDKC1ndashCYCLINT1 kinase
complex phosphorylates the carboxy-terminal domain of RNA polymer-
ase II and promotes transcription Plant J 42 810ndash820Gullberg J Jonsson P Nordstrom A Sjostrom M and Moritz T (2004)
Design of experiments an efficient strategy to identify factors influenc-
ing extraction and derivatization of Arabidopsis thaliana samples in met-
abolomic studies with gas chromatographymass spectrometry Anal
Biochem 331 283ndash295Hertzberg M Aspeborg H Schrader J et al (2001) A transcriptional
roadmap to wood formation Proc Natl Acad Sci USA 98 14732ndash14737Jang JC Fujioka S Tasaka M Seto H Takatsuto S Ishii A Aida M
Yoshida S and Sheen J (2000) A critical role of sterols in embryonic
patterning and meristem programming revealed by the fackel mutants of
Arabidopsis thaliana Genes Dev 14 1485ndash1497Jenks MA Rashotte AM Tuttle HA and Feldmann KA (1996) Mutants
in Arabidopsis thaliana altered in epicuticular wax and leaf morphology
Plant Physiol 110 377ndash385Jonsson P Johansson AI Gullberg J Trygg J A J Grung B Markl-
und S Sjostrom M Antti H and Moritz T (2005) High-throughput
data analysis for detecting and identifying differences between samples
in GCMS-based metabolomic analyses Anal Chem 77 5635ndash5642Jungwirth H and Kuchler K (2006) Yeast ABC transporters ndash a tale of sex
stress drugs and aging FEBS Lett 580 1131ndash1138Kaneda M Schuetz M Lin B Chanis C Hamberger B Western T
Ehlting J and Samuels A (2011) ABC transporters coordinately
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
ABCG proteins and vascular development 823
expressed during lignification of Arabidopsis stems include a set of AB-
CBs associated with auxin transport J Exp Bot 62 2063
Karimi M Inze D and Depicker A (2002) GATEWAYTMvectors for Agrobac-
terium-mediated plant transformation Trends Plant Sci 7 193ndash195Kneurooller A and Murphy A (2011) ABC transporters and their function at the
plasma membrane In The Plant Plasma Membrane Plant Cell Mono-
graphs vol 19 (Murphy AS Peer W and Schulz B eds) New York
NY Springer pp 353ndash377Kopischke M Westphal L Schneeberger K Clark R Ossowski S
Wewer V Fuchs R Landtag J Hause G and Deuroormann P (2013)
Impaired sterol ester synthesis alters the response of Arabidopsis thali-
ana to Phytophthora infestans Plant J 73 456ndash468Kuromori T Miyaji T Yabuuchi H Shimizu H Sugimoto E Kamiya
A Moriyama Y and Shinozaki K (2010) ABC transporter AtABCG25 is
involved in abscisic acid transport and responses Proc Natl Acad Sci
USA 107 2361ndash2366Kuromori T Itoh T Sugimoto E and Shinozaki K (2011a) Arabidopsis
mutant of AtABCG26 an ABC transporter gene is defective in pollen
maturation J Plant Physiol 168 2001ndash2005Kuromori T Sugimoto E and Shinozaki K (2011b) Arabidopsis mutants
of AtABCG22 an ABC transporter gene increase transpiration and
drought susceptibility Plant J 67 885ndash894Le Hir R Beneteau J Bellini C Vilaine F and Dinant S (2008) Gene
expression profiling keys for investigating phloem functions Trends
Plant Sci 13 273ndash280Lehrer AT Dugassa-Gobena D Vidal S and Seifert K (2000) Transport
of resistance-inducing sterols in phloem sap of barley Z Naturforsch C
55 948ndash952Li Y Beisson F Pollard M and Ohlrogge J (2006) Oil content of Arabid-
opsis seeds the influence of seed anatomy light and plant-to-plant vari-
ation Phytochemistry 67 904ndash915Luo B Xue XndashY Hu WndashL Wang LndashJ and Chen XndashY (2007) An ABC
transporter gene of Arabidopsis thaliana AtWBC11 is involved in cuticle
development and prevention of organ fusion Plant Cell Physiol 48
1790ndash1802McFarlane HE Shin JJH Bird DA and Samuels AL (2010)
Arabidopsis ABCG transporters which are required for export of diverse
cuticular lipids dimerize in different combinations Plant Cell 22 3066ndash3075
Meskiene I Baudouin E Schweighofer A Liwosz A Jonak C Rodri-
guez PL Jelinek H and Hirt H (2003) Stress-induced protein phospha-
tase 2C is a negative regulator of a mitogen-activated protein kinase
J Biol Chem 278 18945ndash18952Murashige T and Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures Physiol Plant 15 473ndash497Panikashvili D Savaldi-Goldstein S Mandel T Yifhar T Franke RB
Hofer R Schreiber L Chory J and Aharoni A (2007) The Arabidopsis
DESPERADOAtWBC11 transporter is required for cutin and wax secre-
tion Plant Physiol 145 1345ndash1360Panikashvili D Shi JX Bocobza S Franke RB Schreiber L and Aha-
roni A (2010) The Arabidopsis DSOABCG11 transporter affects cutin
metabolism in reproductive organs and suberin in roots Mol Plant 3
563ndash575Panikashvili D Shi JX Schreiber L and Aharoni A (2011) The Arabid-
opsis ABCG13 transporter is required for flower cuticle secretion and pat-
terning of the petal epidermis New Phytol 190 113ndash124Pighin JA Zheng H Balakshin LJ Goodman IP Western TL Jetter
R Kunst L and Samuels AL (2004) Plant cuticular lipid export requires
an ABC transporter Science 306 702ndash704Pullen M Clark N Zarinkamar F Topping J and Lindsey K (2010)
Analysis of vascular development in the hydra sterol biosynthetic
mutants of Arabidopsis PLoS ONE 5 e12227
Quilichini TD Friedmann MC Samuels AL and Douglas CJ (2010)
ATP-binding cassette transporter G26 is required for male fertility and
pollen exine formation in Arabidopsis Plant Physiol 154 678ndash690R Development Core Team (2010) R A Language and Environment for
Statistical Computing Vienna Austria R Foundation for Statistical
Computing
Roudier F Gissot L Beaudoin F Haslam R Michaelson L Marion J
Molino D Lima A Bach L and Morin H (2010) Very-long-chain fatty
acids are involved in polar auxin transport and developmental patterning
in Arabidopsis Plant Cell 22 364ndash375Rozen S and Skaletsky HJ (2000) Primer3 on the WWW for general users
and for biologist programmers Methods Mol Biol 132 365ndash386Schad M Lipton MS Giavalisco P Smith RD and Kehr J (2005) Eval-
uation of two-dimensional electrophoresis and liquid chromatography
tandem mass spectrometry for tissue-specific protein profiling of
laser-microdissected plant samples Electrophoresis 26 2729ndash2738Schauer N Steinhauser D Strelkov S et al (2005) GCndashMS libraries for
the rapid identification of metabolites in complex biological samples
FEBS Lett 579 1332ndash1337Schrader J Nilsson J Mellerowicz EJ Berglund A Nilsson P Hertz-
berg M and Sandberg G (2004) A high-resolution transcript profile
across the wood forming meristem of poplar identifies potential regula-
tors of cambial stem cell identity Plant Cell 16 2278ndash2292Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desi-
mone M Frommer WB Fleurougge UndashI and Kunze R (2003) ARAMEM-
NON a novel database for Arabidopsis integral membrane proteins
Plant Physiol 131 16ndash26Silvestro D Andersen TG Schaller H and Jensen PE (2013) Plant ste-
rol metabolism D7ndashsterol-C5ndashdesaturase (STE1DWARF7) D57ndashste-rol-D7ndashreductase (DWARF5) and D24ndashsterol-D24ndashreductase (DIMINUTO
DWARF1) show multiple subcellular localizations in Arabidopsis thaliana
(Heynh) L PLoS ONE 8 e56429
Sorin C Bussell JD Camus I Ljung K Kowalczyk M Geiss G McKh-
ann H Garcion C Vaucheret H and Sandberg G (2005) Auxin and
light control of adventitious rooting in Arabidopsis require ARGONA-
UTE1 Plant Cell 17 1343ndash1359Tanaka T Tanaka H Machida C Watanabe M and Machida Y (2004) A
new method for rapid visualization of defects in leaf cuticle reveals five
intrinsic patterns of surface defects in Arabidopsis Plant J 37 139ndash146Turgeon R and Wolf S (2009) Phloem transport cellular pathways and
molecular trafficking Annu Rev Plant Biol 60 207ndash221Ukitsu H Kuromori T Toyooka K et al (2007) Cytological and biochemi-
cal analysis of COF1 an Arabidopsis mutant of an ABC transporter gene
Plant Cell Physiol 48 1524ndash1533Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De
Paepe A and Speleman F (2002) Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biol 3 research00340031-00340011Verrier PJ Bird D Burla B et al (2008) Plant ABC proteins ndash a unified
nomenclature andupdated inventory Trends Plant Sci 13 151ndash159Vilaine F Palauqui JC Amselem J Kusiak C Lemoine R and Dinant
S (2003) Towards deciphering phloem a transcriptome analysis of the
phloem of Apium graveolens Plant J 36 67ndash81Zhai Z Sooksa-nguan T and Vatamaniuk OK (2009) Establishing RNA
interference as a reverse-genetic approach for gene functional analysis
in protoplasts Plant Physiol 149 642ndash652Zhang Q Blaylock LA and Harrison MJ (2010) Two Medicago truncatula
half-ABC transporters are essential for arbuscule development in
arbuscular mycorrhizal symbiosis Plant Cell 22 1483
Zhao C Craig JC Petzold HE Dickerman AW and Beers EP (2005)
The xylem and phloem transcriptomes from secondary tissues of the
Arabidopsis root-hypocotyl Plant Physiol 138 803ndash818
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
824 Rozenn Le Hir et al
expressed during lignification of Arabidopsis stems include a set of AB-
CBs associated with auxin transport J Exp Bot 62 2063
Karimi M Inze D and Depicker A (2002) GATEWAYTMvectors for Agrobac-
terium-mediated plant transformation Trends Plant Sci 7 193ndash195Kneurooller A and Murphy A (2011) ABC transporters and their function at the
plasma membrane In The Plant Plasma Membrane Plant Cell Mono-
graphs vol 19 (Murphy AS Peer W and Schulz B eds) New York
NY Springer pp 353ndash377Kopischke M Westphal L Schneeberger K Clark R Ossowski S
Wewer V Fuchs R Landtag J Hause G and Deuroormann P (2013)
Impaired sterol ester synthesis alters the response of Arabidopsis thali-
ana to Phytophthora infestans Plant J 73 456ndash468Kuromori T Miyaji T Yabuuchi H Shimizu H Sugimoto E Kamiya
A Moriyama Y and Shinozaki K (2010) ABC transporter AtABCG25 is
involved in abscisic acid transport and responses Proc Natl Acad Sci
USA 107 2361ndash2366Kuromori T Itoh T Sugimoto E and Shinozaki K (2011a) Arabidopsis
mutant of AtABCG26 an ABC transporter gene is defective in pollen
maturation J Plant Physiol 168 2001ndash2005Kuromori T Sugimoto E and Shinozaki K (2011b) Arabidopsis mutants
of AtABCG22 an ABC transporter gene increase transpiration and
drought susceptibility Plant J 67 885ndash894Le Hir R Beneteau J Bellini C Vilaine F and Dinant S (2008) Gene
expression profiling keys for investigating phloem functions Trends
Plant Sci 13 273ndash280Lehrer AT Dugassa-Gobena D Vidal S and Seifert K (2000) Transport
of resistance-inducing sterols in phloem sap of barley Z Naturforsch C
55 948ndash952Li Y Beisson F Pollard M and Ohlrogge J (2006) Oil content of Arabid-
opsis seeds the influence of seed anatomy light and plant-to-plant vari-
ation Phytochemistry 67 904ndash915Luo B Xue XndashY Hu WndashL Wang LndashJ and Chen XndashY (2007) An ABC
transporter gene of Arabidopsis thaliana AtWBC11 is involved in cuticle
development and prevention of organ fusion Plant Cell Physiol 48
1790ndash1802McFarlane HE Shin JJH Bird DA and Samuels AL (2010)
Arabidopsis ABCG transporters which are required for export of diverse
cuticular lipids dimerize in different combinations Plant Cell 22 3066ndash3075
Meskiene I Baudouin E Schweighofer A Liwosz A Jonak C Rodri-
guez PL Jelinek H and Hirt H (2003) Stress-induced protein phospha-
tase 2C is a negative regulator of a mitogen-activated protein kinase
J Biol Chem 278 18945ndash18952Murashige T and Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures Physiol Plant 15 473ndash497Panikashvili D Savaldi-Goldstein S Mandel T Yifhar T Franke RB
Hofer R Schreiber L Chory J and Aharoni A (2007) The Arabidopsis
DESPERADOAtWBC11 transporter is required for cutin and wax secre-
tion Plant Physiol 145 1345ndash1360Panikashvili D Shi JX Bocobza S Franke RB Schreiber L and Aha-
roni A (2010) The Arabidopsis DSOABCG11 transporter affects cutin
metabolism in reproductive organs and suberin in roots Mol Plant 3
563ndash575Panikashvili D Shi JX Schreiber L and Aharoni A (2011) The Arabid-
opsis ABCG13 transporter is required for flower cuticle secretion and pat-
terning of the petal epidermis New Phytol 190 113ndash124Pighin JA Zheng H Balakshin LJ Goodman IP Western TL Jetter
R Kunst L and Samuels AL (2004) Plant cuticular lipid export requires
an ABC transporter Science 306 702ndash704Pullen M Clark N Zarinkamar F Topping J and Lindsey K (2010)
Analysis of vascular development in the hydra sterol biosynthetic
mutants of Arabidopsis PLoS ONE 5 e12227
Quilichini TD Friedmann MC Samuels AL and Douglas CJ (2010)
ATP-binding cassette transporter G26 is required for male fertility and
pollen exine formation in Arabidopsis Plant Physiol 154 678ndash690R Development Core Team (2010) R A Language and Environment for
Statistical Computing Vienna Austria R Foundation for Statistical
Computing
Roudier F Gissot L Beaudoin F Haslam R Michaelson L Marion J
Molino D Lima A Bach L and Morin H (2010) Very-long-chain fatty
acids are involved in polar auxin transport and developmental patterning
in Arabidopsis Plant Cell 22 364ndash375Rozen S and Skaletsky HJ (2000) Primer3 on the WWW for general users
and for biologist programmers Methods Mol Biol 132 365ndash386Schad M Lipton MS Giavalisco P Smith RD and Kehr J (2005) Eval-
uation of two-dimensional electrophoresis and liquid chromatography
tandem mass spectrometry for tissue-specific protein profiling of
laser-microdissected plant samples Electrophoresis 26 2729ndash2738Schauer N Steinhauser D Strelkov S et al (2005) GCndashMS libraries for
the rapid identification of metabolites in complex biological samples
FEBS Lett 579 1332ndash1337Schrader J Nilsson J Mellerowicz EJ Berglund A Nilsson P Hertz-
berg M and Sandberg G (2004) A high-resolution transcript profile
across the wood forming meristem of poplar identifies potential regula-
tors of cambial stem cell identity Plant Cell 16 2278ndash2292Schwacke R Schneider A van der Graaff E Fischer K Catoni E Desi-
mone M Frommer WB Fleurougge UndashI and Kunze R (2003) ARAMEM-
NON a novel database for Arabidopsis integral membrane proteins
Plant Physiol 131 16ndash26Silvestro D Andersen TG Schaller H and Jensen PE (2013) Plant ste-
rol metabolism D7ndashsterol-C5ndashdesaturase (STE1DWARF7) D57ndashste-rol-D7ndashreductase (DWARF5) and D24ndashsterol-D24ndashreductase (DIMINUTO
DWARF1) show multiple subcellular localizations in Arabidopsis thaliana
(Heynh) L PLoS ONE 8 e56429
Sorin C Bussell JD Camus I Ljung K Kowalczyk M Geiss G McKh-
ann H Garcion C Vaucheret H and Sandberg G (2005) Auxin and
light control of adventitious rooting in Arabidopsis require ARGONA-
UTE1 Plant Cell 17 1343ndash1359Tanaka T Tanaka H Machida C Watanabe M and Machida Y (2004) A
new method for rapid visualization of defects in leaf cuticle reveals five
intrinsic patterns of surface defects in Arabidopsis Plant J 37 139ndash146Turgeon R and Wolf S (2009) Phloem transport cellular pathways and
molecular trafficking Annu Rev Plant Biol 60 207ndash221Ukitsu H Kuromori T Toyooka K et al (2007) Cytological and biochemi-
cal analysis of COF1 an Arabidopsis mutant of an ABC transporter gene
Plant Cell Physiol 48 1524ndash1533Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De
Paepe A and Speleman F (2002) Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multiple internal
control genes Genome Biol 3 research00340031-00340011Verrier PJ Bird D Burla B et al (2008) Plant ABC proteins ndash a unified
nomenclature andupdated inventory Trends Plant Sci 13 151ndash159Vilaine F Palauqui JC Amselem J Kusiak C Lemoine R and Dinant
S (2003) Towards deciphering phloem a transcriptome analysis of the
phloem of Apium graveolens Plant J 36 67ndash81Zhai Z Sooksa-nguan T and Vatamaniuk OK (2009) Establishing RNA
interference as a reverse-genetic approach for gene functional analysis
in protoplasts Plant Physiol 149 642ndash652Zhang Q Blaylock LA and Harrison MJ (2010) Two Medicago truncatula
half-ABC transporters are essential for arbuscule development in
arbuscular mycorrhizal symbiosis Plant Cell 22 1483
Zhao C Craig JC Petzold HE Dickerman AW and Beers EP (2005)
The xylem and phloem transcriptomes from secondary tissues of the
Arabidopsis root-hypocotyl Plant Physiol 138 803ndash818
copy 2013 The AuthorsThe Plant Journal copy 2013 John Wiley amp Sons Ltd The Plant Journal (2013) 76 811ndash824
824 Rozenn Le Hir et al
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