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

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

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