Transcriptional regulation of aquaporins in accessionsof Arabidopsis in response to drought stress
Erik Alexandersson1,†,‡, Jonas A.H. Danielson1,‡, Johan Rade2, Vamsi K. Moparthi1, Magnus Fontes2, Per Kjellbom1 and
Urban Johanson1,*
1Department of Biochemistry, Center for Molecular Protein Science, Lund University, PO Box 124, SE-221 00, Lund, Sweden, and2Centre for Mathematical Sciences, Lund University, SE-221 00 Lund, Sweden
Received 28 April 2009; revised 28 October 2009; accepted 12 November 2009; published online 4 January 2010.*For correspondence (fax +46 462224116; e-mail [email protected]).†Present address: Institute for Wine Biotechnology, Faculty of AgriSciences Stellenbosch University, Private Bag X1, 7602 Matieland, South Africa.‡These authors contributed equally to this work.
SUMMARY
Aquaporins facilitate water transport over cellular membranes, and are therefore believed to play an important
role in water homeostasis. In higher plants aquaporin-like proteins, also called major intrinsic proteins (MIPs),
are divided into five subfamilies. We have previously shown that MIP transcription in Arabidopsis thaliana is
generally downregulated in leaves upon drought stress, apart from two members of the plasma membrane
intrinsic protein (PIP) subfamily, AtPIP1;4 and AtPIP2;5, which are upregulated. In order to assess whether this
regulation is general or accession-specific we monitored the gene expression of all PIPs in five Arabidopsis
accessions. The overall drought regulation of PIPs was well conserved for all five accessions tested, suggesting
a general and fundamental physiological role of this drought response. In addition, significant differences
among accessions were identified for transcripts of three PIP genes. Principal component analysis showed that
most of the PIP transcriptional variation during drought stress could be explained by one variable linked to leaf
water content. Promoter-GUS constructs of AtPIP1;4, AtPIP2;5 and also AtPIP2;6, which is unresponsive to
drought stress, had distinct expression patterns concentrated in the base of the leaf petioles and parts of the
flowers. The presence of drought stress response elements within the 1.6-kb promoter regions of AtPIP1;4 and
AtPIP2;5 was demonstrated by comparing transcription of the promoter reporter construct and the
endogenous gene upon drought stress. Analysis by ATTED-II and other web-based bioinformatical tools
showed that several of the MIPs downregulated upon drought are strongly co-expressed, whereas AtPIP1;4,
AtPIP2;5 and AtPIP2;6 are not co-expressed.
Keywords: aquaporins, major intrinsic proteins, drought stress response, natural variation, ecotypes,
Arabidopsis.
INTRODUCTION
In plants, aquaporin (AQP) homologs, also called major
intrinsic proteins (MIPs), form a large protein family, with
35 identified members in Arabidopsis (Johanson et al.,
2001), and 33 in maize (Zea mays) and rice (Oryza sativa)
(Chaumont et al., 2001; Sakurai et al., 2005). Based on
sequence comparison, higher plant MIPs are divided into
five subfamilies: plasma membrane intrinsic proteins
(PIPs), tonoplast intrinsic proteins (TIPs), NOD26-like
intrinsic proteins (NIPs), small basic intrinsic proteins
(SIPs) and the recently identified uncharacterized intrinsic
proteins (XIPs) (Danielson and Johanson, 2008). Members
of the PIP and TIP subfamilies, which are abundant in the
plasma membrane and in the vacuolar membrane, where
they reside as tetramers, have been shown to be active
water channels in Xenopus oocytes (e.g. Maurel et al.,
1993; Johansson et al., 1998). The PIP subfamily can be
further divided into the PIP1 and PIP2 groups, based on
sequence similarity. Several PIP2 isoforms have been
reported to function as water channels when expressed in
Xenopus oocytes (AtPIP2;1, AtPIP2;2, AtPIP2;3, AtPIP2;7;
Daniels et al., 1994; Kammerloher et al., 1994; Weig et al.,
1997), and because of the high degree of sequence
650 ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd
The Plant Journal (2010) 61, 650–660 doi: 10.1111/j.1365-313X.2009.04087.x
conservation (‡84% identity) it is reasonable to assume
that all the AtPIP2s have the same specificity. PIP1 iso-
forms generally have much lower or no water channel
activity (Kammerloher et al., 1994; Yamada et al., 1995;
Biela et al., 1999; Chaumont et al., 2000; Moshelion et al.,
2002). However, Fetter et al. (2004) showed that
co-expression of ZmPIP1;2 and different maize PIP2
isoforms led to increased water permeability, dependent on
the quantity of ZmPIP1;2 cRNA injected into Xenopus
oocytes. Further transient co-expression of PIP genes in
maize protoplasts demonstrated that the localization of
PIP1s to the plasma membrane seems to be dependent on
the simultaneous expression of PIP2 isoforms (Zelazny
et al., 2007). This suggests that PIP1 isoforms have a role in
water permeability, possibly interacting with PIP2 isoforms
either in heterotetramers or between homotetramers.
Aquaporins (AQPs) have been shown to affect plant water
homeostasis. For example, downregulation of certain PIP
isoforms through antisense RNA leads to a decrease in
osmotic water permeability of protoplasts, an increased size
of the root system, and/or a higher susceptibility to drought
and osmotic stress (Kaldenhoff et al., 1998; Martre et al.,
2002; Siefritz et al., 2002). Accordingly, overexpression of
PIP isoforms has, among other effects, been shown to give
an increase in root osmotic hydraulic conductivity, transpi-
ration and shoot to root ratio (Aharon et al., 2003; Katsuhara
et al., 2003; Lian et al., 2004). Still, the benefits of AQP
overexpression in stress conditions are uncertain, and some
studies have shown a decreased resistance to salt or
drought stress in comparison with wild-type plants (Aharon
et al., 2003; Katsuhara et al., 2003).
A key question in the study of the MIP family in plants is
why the plant requires so many MIP genes. One explanation
is that although many PIP2 and several TIP isoforms have
been shown to be permeable to water, MIPs can also be
channels for a number of other small solutes. Consequently,
the substrate specificity can differ between individual
isoforms. PIP1 members have been proposed to be perme-
able to e.g. glycerol (Biela et al., 1999; Moshelion et al.,
2002), boric acid (Dordas et al., 2000), urea (Gaspar et al.,
2003) and carbon dioxide (Uehlein et al., 2003), even though
the physiological importance of MIPs as facilitators of some
of these solutes remains unclear. A number of TIP isoforms
have been shown to be permeable to glycerol, urea and
hydrogen peroxide (Gerbeau et al., 1999; Klebl et al., 2003;
Liu et al., 2003; Bienert et al., 2007), and in wheat
(Triticum aestivum) and Arabidopsis, TIP isoforms have
been recognized to be permeable to NH4+/NH3 and urea
(Jahn et al., 2004; Loque et al., 2005).
Another reason for the redundancy of MIPs is the
specific spatial expression pattern, limited to certain
organs or cell types, or developmental stages, as shown
for some of the MIPs (e.g. see review by Fraysse et al.,
2005; Maurel et al., 2002). We previously monitored the
expression of MIPs in roots, leaves and flowers (Alexan-
dersson et al., 2005). The transcription of MIPs are also
affected by environmental cues, e.g. in drought-stressed
plants grown on soil the expression of MIPs are generally
downregulated, with the exception of AtPIP1;4 and
AtPIP2;5, which are instead upregulated, and AtPIP2;6,
which is unaffected (Alexandersson et al., 2005). There
have been previous reports of upregulation of MIPs upon
osmotic stress in plants (Jang et al., 2004; Lian et al.,
2004; Guo et al., 2006). Upon drought the PIP1, Trg-31,
was shown to be rapidly upregulated in pea (Pisum
sativum) (Guerrero et al., 1990), and, like AtPIP1;4, Trg-31
was shown to be weakly expressed in roots (Guerrero and
Crossland, 1993).
Lately, several studies have estimated the variation in
transcription between different Arabidopsis accessions
(Chen et al., 2005; Vuylsteke et al., 2005; Kliebenstein
et al., 2006). These studies show that phenotypic variation
between accessions can be assigned to transcriptional
differences, and that there is an over-representation of
differently regulated genes involved in signaling and
stress responses (Chen et al., 2005), and also in transport
(Kliebenstein et al., 2006). Furthermore, differences in
transcriptional regulation of MIPs between plant varieties
have been shown to occur. Lian et al. (2004, 2006)
showed, for example, that upon osmotic stress OsPIP1;3
was upregulated in highland rice that has better drought
avoidance in comparison with lowland rice, in which
OsPIP1;3 did not show a change in expression, leading to
the conclusion that OsPIP1;3 must have a role in the
difference in drought avoidance between the two rice
varieties. Recently, it has also been shown that VvPIP1;1 in
grapevine (Vitis vinifera) roots are expressed in a cultivar-
specific manner in response to drought stress, reflecting
the differences in hydraulic conductivity observed
(Vandeleur et al., 2009).
In this study, we further investigate PIP expression and
regulation upon drought stress in five Arabidopsis acces-
sions. Importantly, the use of several accessions with
varying water use efficiency (WUE) and different geograph-
ical origin (Nienhuis et al., 1994) enabled us to show that the
PIP expression generally conforms to the pattern previously
observed in the Col-0 background, at the same time it
allowed us to detect some minor but significant differences
in the expression of three PIP genes.
In addition, we generated promoter-GUS constructs for
the AtPIP1;4, AtPIP2;5 and AtPIP2;6 isoforms to monitor
expression patterns. With the help of the Arabidopsis
thaliana trans-factor and cis-element prediction database
(ATTED-II), and other publicly available microarray data
and bioinformatics tools, we show that several of the
MIPs downregulated upon drought stress are strongly
co-expressed, whereas AtPIP1;4, AtPIP2;5 and AtPIP2;6 do
not co-express with this group.
Conserved drought regulation of aquaporin genes 651
ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 650–660
RESULTS
PIP expression in different accessions upon drought stress
and analysis of variation in expression
In Figure 1 the expression ratio (log2 scale) of control and
drought-stressed plants of all 13 Arabidopsis PIP genes
for the five accessions tested can been seen as mean
values of the three biological replicates. The relatively
large standard deviations observed for some genes are
mainly the result of variation between biological repli-
cates, rather than between technical replicates (Figure S1).
In general the expression pattern of PIPs was conserved
between accessions. AtPIP1;4 and AtPIP2;5 were upregu-
lated for all five accessions, whereas the remaining PIP
genes, with the exception of AtPIP2;6, showed a general
downregulation. For three genes, small but significant
differences in drought responses among accessions were
recorded. In the accession Edi-0, AtPIP2;4 and AtPIP2;6
were upregulated compared with Lip-0, and for AtPIP2;4,
also relative to Kas-1. The upregulation of AtPIP2;5 was
significantly higher, about four-fold, in Kas-1 compared
with Col-0, Ler-0 and Lip-0. There were also differences in
the normalized unstressed expression levels of specific
PIP transcripts between accessions (Table S2). Most
notably, the expression of AtPIP2;8 appears to be seven-
fold higher in Lip-0 compared with Col-0, whereas differ-
ences in expression for the other PIP transcripts were
within a twofold change, and no major differences in
expression intensity between accessions can be seen for
AtPIP1;4, AtPIP2;5 and AtPIP2;6.
As differences in expression ratio can arise from differ-
ences in the level of drought stress between accessions and/
or experimental replicates, drought stress was assessed by
measuring the water content in sampled leaf tissue. Further
examination employing principle component analysis (PCA)
of the regulation of PIP expression during drought showed
that 90% of the total variation in the dataset is described by
the first principle component (PC1; Figure 2a). The contri-
bution of the individual variables (PIP genes) to PC1 and PC2
is depicted in Figure 2b. There is no clear subclustering of
the different ecotypes in the PCA plots (data not shown).
However, when the samples are colored for leaf water
content, a correlation of leaf water content and variation in
PIP expression can be seen (Figure 2a). In Figure 3, PC1 is
shown as a function of leaf water content, displaying a weak
linear correlation (r2 = 0.7).
To extend the expression analysis of PIP genes to also
include other conditions than drought stress, as well as
other genes, expression profiles were investigated in silico.
Figure 4 shows a gene co-expression network of all AtPIPs
and the most highly expressed AtTIP genes based on data
retrieved from ATTED-II and visualized in CYTOSCAPE 2.2
(http://www.cytoscape.org). In ATTED-II the correlation coef-
ficients were calculated from Affymetrix data corresponding
to 58 different experimental conditions, which covered
various stress treatments and developmental stages. Many
AtPIPs and AtTIPs are highly co-expressed, whereas
AtPIP1;4, AtPIP2;4, AtPIP2;5 and AtPIP2;6 are co-expressed
with other AtPIPs and AtTIPs to a much lesser extent. A list of
the most highly co-expressed genes to AtPIP1;4, AtPIP2;5
and PIP2;6 reveals a correlation of AtPIP2;6 with photosyn-
thetic genes (Table S3). The cluster of co-expressing AtPIPs
and AtTIPs is also confirmed by co-correlation plots using
Arabidopsis co-expression tool (ACT; University of Leeds,
UK; Figure S2). In particular, the gene pairs AtPIP1;2 and
AtPIP2;1, AtTIP1;2 and AtTIP2;1, and AtPIP1;3 and AtPIP2;7/
2;8, which are among the most co-expressed MIPs (Fig-
ure 4), show very similar patterns of correlations with
the expression of other genes (Figure S2). These high
co-correlations (r2 ‡ 0.85) are contrasted by a comparison
of PIP1;2 and PIP1;4 (r2 = 0.30), for example, where the latter
is not part of the cluster in Figure 4.
Figure 1. Drought regulation of AtPIP expres-
sion in five Arabidopsis accessions.
Ratios (log2) of mRNA levels in drought-stressed
and control plants for all 13 plasma membrane
intrinsic protein (PIP) genes in five accessions of
Arabidopsis, as measured with RT-qPCR. The
bars show means � SDs (error bars) from three
replicates of the drought stress experiment.
Accession-specific differences in mRNA regula-
tion, as measured by ANOVA (P < 0.05), were
found for PIP2;4, PIP2;5 and PIP2;6. Dashed lines
show which accessions the difference was found
between (ad hoc test with non-overlapping 95%
confidence intervals).
652 Erik Alexandersson et al.
ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 650–660
Expression analysis of AtPIP1;4, AtPIP2;5 and AtPIP2;6
promoter-GUS constructs
The GUS expression patterns regulated by the 1.6-kb long
AtPIP1;4, AtPIP2;5 and AtPIP2;6 promoters of the respective
gene showed a concentration in the base of the petioles for
all three constructs (Figure 5). No major differences were
seen in leaf expression patterns between plants grown on
soil and MS agar. GUS expression was also seen in petiole
vascular tissues, and to a somewhat lesser extent in leaf
vascular tissues (Figure 5a–c, e–g). There was also a differ-
ence in the expression between individual leaves in single
plants, with the highest expression located in younger
leaves. This expression difference was not as clear for GUS
regulated by the 1.6-kb AtPIP2;6 promoter, which had a
distinct expression pattern restricted to the mid-nerve and
hydathodes in leaves (Figure 5i–k). All three constructs were
expressed in flower tissue, centered to the sepal and petal
abscission zone, and just below the stigma (Figure 5d,h
and l). However, only the AtPIP2;6 promoter-GUS construct
showed expression in stamens. In roots, GUS expression
regulated by the AtPIP1;4 promoter was restricted to the root
tips, whereas GUS expression regulated by the AtPIP2;5
promoter was foremost detected in lateral roots, but not in
root tips (not shown). GUS expression regulated by the
AtPIP2;6 promoter showed weak expression in the middle
of the main root (not shown).
Comparison of GUS transcription regulated by MIP
promoters and transcription of endogenous MIP genes
in response to drought stress
In order to assess if all necessary drought-responsive ele-
ments were present within the respective 1.6-kb promoter-
GUS construct, lines carrying the AtPIP1;4, AtPIP2;5 or
AtPIP2;6 promoter-GUS construct were drought stressed on
soil. Drought regulation of uid (GUS) and the endogenous
AtPIP1;4, AtPIP2;5 or AtPIP2;6 genes, under control of their
complete regulatory region, were then analyzed in leaves of
each line. In Figure 6 the ratios of the drought regulation of
GUS transcripts and the corresponding endogenous gene in
the same line are compared for three independent lines for
each construct. For AtPIP1;4, AtPIP2;5 and AtPIP2;6 there is
Figure 2. Principle component analysis (PCA) of AtPIP expression in drought-
stressed accessions.
PCA was performed with in-house computer software. The PIP expression
regulation of Figure 1 was used as input data for the PCA.
(a) Projections of the 15 samples (five accessions and three drought stress
experiments) along PC1 and PC2. The samples are colored for leaf water
content (red, low leaf water content; green, high leaf water content).
(b) The loading of the 13 variables (PIP gene expression ratios) used in the
analysis. The positions of the 13 variables reflect their relative contribution to
the variables PC1 and PC2. The axes PC1 and PC2 show the directions of the
main variation, accounting for 90 and 4%, respectively, of the total variation.
The lengths of the axes represent one arbitrary unit. Note that the relative
positions of the variables and samples have a well-defined interpretation, i.e.
the rightmost samples have the highest values of the rightmost variables,
and the lowest values of the leftmost variables, and vice versa. For example,
for the samples to the left, the expression ratios of PIP2;2 and PIP1;5 are high,
whereas the expression ratios of PIP2;5 and PIP1;4 are low.
Figure 3. Principal component 1 (PC1) and leaf water content for all 15
drought stress experiments.
The values of PC1 (from the principle component analysis, PCA) plotted
against leaf water content for samples from 15 drought stress experiments
(five accessions and three replicates). The control sample point is the mean of
the leaf water content of all 15 controls.
Conserved drought regulation of aquaporin genes 653
ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 650–660
no significant difference in the regulation of the GUS con-
struct compared with the endogenous gene, as log2(GUS
response/PIP response) is close to zero in at least two out of
three independent lines, suggesting that the main drought
regulatory cis-elements are contained within the 1.6 kb
upstream of the translational start codon. However, for
Figure 4. Co-expression analysis of all AtPIPs
and four AtTIPs.
Co-expressed gene network generated in CYTO-
SCAPE 2.2 by Pearson’s coefficients of all plasma
membrane intrinsic proteins (PIPs) and the four
most highly expressed tonoplast intrinsic pro-
teins (TIPs) retrieved from the Arabidopsis thali-
ana trans-factor and cis-element prediction
database (ATTED-II). Correlation coefficients
below 0.6 are colored gray for visibility reasons.
Note that there are no specific probes for
AtPIP2;2 and AtPIP2;3, and for AtPIP2;7 and
AtPIP2;8, on the Affymetrix ATH1 chip, because
of the high level of sequence identity between
these two PIP pairs.
Figure 5. Expression pattern from 1.6-kb pro-
moter::GUS constructs of AtPIP1;4, AtPIP2;5
and AtPIP2;6.
Plants transformed by the AtPIP1;4 promoter-
GUS construct grown for 2 weeks on agar plates
(a), 2 weeks on soil (b) and 3 weeks on soil (c),
and flowers (d). Plants transformed by the
AtPIP2;5 promoter-GUS construct grown for
2 weeks on agar plates (e), 2 weeks on soil (f)
and 3 weeks on soil (g), and flower (h). Plants
transformed by the AtPIP2;6 promoter-GUS con-
struct grown for 2 weeks on agar plates (i),
2 weeks on soil (j) and 3 weeks on soil (k), and
flowers (l).
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ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 650–660
PIP2;5, the marginally but consistently lower drought
induction of the GUS transcript might indicate that the 1.6-kb
promoter-GUS construct is not sufficient to completely
mimic the drought regulation of AtPIP2;5.
DISCUSSION
Regulation of PIP expression upon drought in different
accessions
To monitor possible differences in the regulation of PIP
expression during drought stress, five different accessions
were chosen based on geographical origin and WUE, as
determined by Nienhuis et al. (1994). By testing the variation
of WUE, defined as accumulated biomass per volume of
water used, in 31 Arabidopsis accessions, Nienhuis et al.
(1994) found that Lip-0 had the highest WUE and Edi-0 had
the the lowest (2.40 and 1.86 mg plant dry weight/g H2O,
respectively). In addition, Col-0 (2.24 mg plant dry weight/g
H2O; Nienhuis et al., 1994) and Ler-0, which are among the
most commonly used accessions, were included together
with Kas-1, as it originates from central Asia, and is thus
likely to be genetically more divergent from the other four
accessions. The WUE of the latter two accessions was not
tested by Nienhuis et al. (1994).
The overall pattern of expression regulation of PIPs was
found to be conserved between the five accessions during
drought stress (Figure 1). Importantly, the upregulation of
AtPIP1;4 and AtPIP2;5, and the downregulation of most PIPs,
as we previously showed only in the Col-0 accession
(Alexandersson et al., 2005), seem to be a general pattern
of PIP regulation during drought stress. Although the
regulation of AtPIP1;4 is consistent with the finding of an
upregulated PIP1 in response to drought in pea (Guerrero
et al., 1990), it could only be speculated whether this pattern
could also be expected in other plants. It is possible that the
drought regulation of PIPs represents an adaptation to the
habitat and lifestyle specific for Arabidopsis. A general
downregulation of AQPs might be a way for the plant to
minimize water loss and uphold turgor in leaves. The
upregulation of a few individual PIPs could be a way for
the plant to direct water flow to specific organs or cells that
are crucial for plant survival during drought or necessary for
a fast recovery upon rehydration of the plant, as we have
previously proposed (Alexandersson et al., 2005). Even
though the general regulation pattern is well conserved
among the accessions, there are significant minor differ-
ences in PIP regulation between some of these for AtPIP2;4,
AtPIP2;5 and AtPIP2;6. Interestingly, Lip-0 and Edi-0, the two
accessions with the highest difference in WUE are found
among these accessions. However, the variation in basal
expression levels among accessions complicates the inter-
pretation of the regulatory differences, and the relatively
large variation between biological replicates could mask
more subtle differences that might exist between, for
example, Col-0 (that has a slightly lower WUE than Lip-0)
and Edi-0. Whether AtPIP2;4 and AtPIP2;6 contribute to the
differences in WUE, and what mechanisms might lie behind
this, remains to be investigated.
Principal component analysis is a very powerful method
to identify and visualize the major variation in multivariate
data sets without the requirement of specifying any partic-
ular hypothesis regarding potential correlations. In PCA,
new variables – the so-called principal components (PCs) –
are constructed by a linear combination of the original
variables, so that the major variation is captured by the first
principal component, followed by the second and so on. In
this way the main variation among multidimensional sam-
ples may be projected onto a plane defined by PC1 and PC2.
Although we do not find any accession-specific patterns, the
variation introduced by analyzing all the different accessions
together revealed that: (i) for each sample the regulation of
PIP expression in the drought experiments is closely asso-
ciated with PC1 (Figure 2a); (ii) PC1 explains 90% of the
variation, and accordingly the regulation of most Arabidop-
sis PIP genes is closely linked to PC1 (Figure 2b); and (iii) PC1
seems to be correlated to leaf water content (Figures 2a and
3). Thus, during drought stress PIP expression is strongly
reflected by PC1, which in its turn seems to be dependent on
the leaf water content. Even though the exact nature of this
dependency is unclear because of the limited volume of
data, it is tempting to hypothesize that there is an associa-
tion between leaf water content and the variation in PIP
expression, resulting from a common regulatory mecha-
nism of PIP expression. Possible scenarios as the leaf water
content decreases ranges from a simple induction of tran-
scription factors, acting directly as activators or repressors
Figure 6. Correlation between GUS and PIP transcriptions.
The bars show the ratio of uid (encoding GUS) transcriptional regulation
(regulated by the AtPIP1;4, AtPIP2;5 or AtPIP2;6 promoter) and the transcrip-
tional regulation of the endogenous AtPIP1;4, AtPIP2;5 or AtPIP2;6 gene in
response to drought stress. Three independent lines are shown for each
construct (transformed by promoter AtPIP1;4 (1.6 kb), AtPIP2;5 (1.6 kb) or
AtPIP2;6 (1.6 kb) GUS). Ratios are shown in log2 scale (0 means that the ratio
is 1, e.g. the GUS and PIP transcripts are regulated exactly the same upon
drought stress). The error bars represent � SDs from three replicates of the
drought stress experiment. Note that for one of the PIP2;6 promoter construct
lines, only data from one drought stress experiment was available, and hence
no error bars are shown for that line.
Conserved drought regulation of aquaporin genes 655
ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 650–660
on the up- and downregulated groups of PIPs, to a more
complex regulatory network, where the PIPs are indirectly
regulated via a cascade of transcription factors. The
co-expression of MIPs is further discussed below in connec-
tion with the ATTED-II analysis.
Co-variation of MIP expression
Gene co-expression of MIPs as a family has been reported
before. In Arabidopsis, Schmid et al. (2005) found that 33
members of the MIP family had a higher level of
co-expression than could be expected by chance, when
examining the expression in 79 diverse samples covering
different developmental stages. Using several internet-
based databases and tools, which enable the discovery and
investigation of co-expressed genes (ACT, ATTED-II and
csb.db) we thoroughly analyzed the co-expression of PIPs
and TIPs. In Figure 4 the gene co-expression network con-
structed by co-expression coefficients retrieved from
ATTED-II shows that many AtPIP and AtTIP genes that
are downregulated upon drought stress are strongly
co-expressed. In contrast, AtPIP1;4 and AtPIP2;5, which were
upregulated, and AtPIP2;6, which was stably expressed
upon drought, do not fall into this group or co-express with
each other (r < 0.6). We obtained similar results by per-
forming expression co-correlation plots with the help of ACT
(Figure S2). As it is possible to retrieve genes with nega-
tively correlated expression in ACT, we surveyed ACT but
did not find any clear negative correlations (r < )0.5) among
PIPs and TIPs (not shown).
The Arabidopsis genome has extensive syntenic regions,
indicative of several rounds of genome-wide duplications
(reviewed in Henry et al., 2006). Several of the closely related
PIP pairs in the co-expressed cluster (e.g. PIP1;1 and PIP1;2,
and PIP2;1 and PIP2;2/2;3) are located in duplicated regions,
and hence a positive co-correlation could be expected
because the promoter regions were most likely duplicated
together with the gene. Interestingly, the three gene pairs
with strongest co-correlations are not closely related: two of
them are PIP1 and PIP2 pairs, whereas the third is a TIP1 and
TIP2 pair. This points to a functional link between these
MIPs, as transcripts in a pair are both needed at the same
time under most conditions, leading to a very similar
transcriptional relation to other genes (Figure S2A–C).
Whether this reflects a direct physical interaction, as has
been suggested for PIP1s and PIP2s (Zelazny et al., 2007), a
functional complement in subcellular localization, or sub-
strate specificity, awaits further investigation.
The expressional regulation of AtPIP2;5 shows similarity
to genes connected to the establishment of shoot meristem
formation, and/or leaf shape and growth (GIF1, PINHEAD/
ZWILLE, MYB17; Kim and Kende, 2004; Newman et al., 2002;
Schmitz and Theres, 2005). This co-expression might reflect
the localization of AtPIP2;5 to the base of leaf petioles, and
possibly the shoot meristem, seen by AtPIP2;5 promoter-
GUS constructs (see below). The co-expression of AtPIP2;5
with other MIP genes is low, and only AtPIP1;4 is among the
300 most co-expressed genes according to ATTED-II
(r = 0.47). In comparison with AtPIP1;4 and AtPIP2;5,
AtPIP2;6 shows a higher degree of co-expression with many
genes, and interestingly 30 out of the 50 most co-expressed
genes are annotated to be associated with photosynthesis
(ATTED-II). The co-expression of AtPIP2;6 with several genes
encoding chloroplast proteins is also seen in the ACT and
csb.db databases (not shown). This co-expression is consis-
tent with our earlier results showing that AtPIP2;6 is the
major MIP transcript in leaves (Alexandersson et al., 2005).
However, although the promoter-GUS fusion (see below)
confirms that AtPIP2;6 is strongly expressed in leaves, the
GUS staining is not found in mesophyll cells where the
chloroplasts are most abundant. This would argue against a
localization of PIP2;6 to the chloroplast, as suggested for the
PIP1 isoform NtAQP1 in tobacco (Uehlein et al., 2008).
Tissue-specific expression of AtPIP1;4, AtPIP2;5 and
AtPIP2;6, and promoter element analysis
Promoter-GUS constructs with 1.6-kb-long AtPIP1;4,
AtPIP2;5 and AtPIP2;6 promoter sequences were con-
structed in order to monitor the spatial expression pattern in
the plant, and to determine if the cis-element(s) regulating
expression upon drought was present in these promoter
regions (Figures 5 and 6).
In unstressed tissue GUS expression shows a concentra-
tion at the base of the petioles, as well as in the vascular
tissues of petioles and leaves, in some developmental
stages for all three constructs. Notably, AtPIP1;4 is
expressed in emerging and young leaves. AtPIP2;6 displays
a distinct expression pattern in leaves, restricted to the mid-
nerve, and was also the only one of the three genes with an
expression in hydathodes. In flower tissue all show a similar
expression pattern, except that AtPIP2;6 is also expressed in
stamens. In accordance with earlier transcript expression
studies (Jang et al., 2004; Alexandersson et al., 2005), the
expression of the three genes is low in roots. AtPIP1;4 seems
to be exclusively expressed in root tips, and AtPIP2;5 in
lateral roots (not shown). Thus, the expression of AtPIP1;4
and AtPIP2;5 seem to be spatially divided in roots. The
expression patterns, level of expression (AtPIP1;4 comprises
a much higher portion of the total expression of PIP isoforms
in rosette leaves than AtPIP2;5; Table S2) and lack of
correlation of expression (Figure 5) points to AtPIP1;4 and
AtPIP2;5 having different roles in drought stress, and in
stress response in general.
To investigate whether the promoter regions contained
the necessary cis-element(s) for regulating expression upon
drought, a promoter-GUS-transformed Arabidopsis lines
were exposed to drought stress on soil, as previously
performed for the accessions. Regulation of the expression
of GUS transcripts under the control of AtPIP1;4, AtPIP2;5 or
656 Erik Alexandersson et al.
ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 650–660
AtPIP2;6 promoters was compared with the endogenous
AtPIP1;4, AtPIP2;5 or AtPIP2;6 transcripts. This showed that
GUS and endogenous AtPIP1;4, AtPIP2;5 and AtPIP2;6
transcription were not significantly different in two out of
three lines (Figure 6), suggesting that the 1.6-kb AtPIP1;4
and AtPIP2;5 promoter regions include the drought-respon-
sive cis-element(s), leading to an upregulation during
drought stress. Supporting this, we were also able to
visually detect increased GUS activity in lines transformed
by AtPIP1;4-GUS or AtPIP2;5-GUS promoter constructs
drought stressed on agar plates. For lines transformed by
AtPIP1;4-GUS promoter constructs, GUS activity was also
spatially increased in leaves, whereas the higher expression
of AtPIP2;5 seems instead to relate to an increased expres-
sion intensity at the base of the petioles upon drought stress
(Figure S3). As drought induction of the endogenous
AtPIP1;4 gene and the AtPIP1;4-GUS promoter constructs
are similar, it is likely that the extended expression pattern
observed for the promoter-GUS construct is also represen-
tative for endogenous AtPIP1;4 expression. However, the
physiological relevance of the increased expression of
AtPIP1;4 and AtPIP2;5 is unclear.
For AtPIP2;6, the results indicate that the cloned region is
sufficient to mimic the drought-insensitive transcription of
the endogenous gene. The observed difference between
individual lines carrying the same construct may result from
positional effects caused by the random insertion of the
promoter-GUS construct in the genome. For AtPIP2;5, the
GUS transcript was slightly less induced by drought in all
three lines, possibly because of missing regulatory elements
outside of the cloned 1.6-kb promoter region. Our results
demonstrate that the relative expression levels of a pro-
moter–reporter gene and the corresponding endogenous
gene can be used to map promoter cis-elements. In addition
to validating that the cloned promoter contains all the
response elements of interest, this method provides a large
advantage to an absolute comparison in drought experi-
ments, as it is virtually impossible to achieve exactly the
same level of drought stress in different plants. Using this
technique, promoter regions may be further examined by
deletion studies to better define the cis-elements.
CONCLUSIONS
Here, we show that the general pattern of drought regulation
of PIPs is conserved among five accessions of Arabidopsis. In
our studies, AtPIP1;4 and AtPIP2;5 are the only AQP tran-
scripts that show distinct upregulation upon drought stress,
whereas most AtPIPs are downregulated. At the same time
there are significant differences in the expression of certain
PIPs between accessions that could have importance for
accession-specific traits, e.g. WUE. A bioinformatics analysis
of microarray data reveals that most of the downregulated
PIPs have correlated expression, with specific pairs of PIP1s
and PIP2s having the highest correlations. In contrast,
AtPIP1;4 and AtPIP2;5 expression are not correlated in gen-
eral, even though both are upregulated by drought stress.
Differences in expression patterns visualized by GUS staining
also suggest that these genes have distinct functional roles.
Future investigations should further clarify the physiological
role of the general drought stress expression pattern of AQPs
in plants, as well as the relevance of the identified natural
variation in PIP expression among accessions.
EXPERIMENTAL PROCEDURES
Plant material and treatments
Accessions Lip-0, Edi-0 and Kas-1, with stock codes N1337, N1123and N1265, were obtained from the European Arabidopsis StockCentre (http://arabidopsis.info). For drought stress experimentsArabidopsis seeds were planted on soil and vernalized at 8�C 1 weekbefore trays were placed in growth chambers (100 lE, 14-h lightperiod) at 20�C with 70–80% humidity. Plants were grown for4–4.5 weeks before drought stress was initiated by ceasing watering.Control plants were grown next to stress-treated plants, and werecontinuously watered during the experiments. In order to monitorthe soil water loss of the stressed plants and maintain constant soilwater content of control plants, the trays were weighed throughoutthe experiment. Drought stress was carried out for 8–11 days beforesampling to achieve a similar degree of stress based on soil watercontent. In each experimental repetition all accessions were har-vested at the same time to avoid developmental differences. Rosetteleaves of between five and seven plants (12–15 for plants trans-formed by promoter-GUS constructs, see below) of drought-stres-sed and control plants were harvested at every sampling point, aswell as leaves of between four and six plants to determine dry weightand leaf water content, which was determined by comparing thefresh weight with the dry weight after drying plant material at 80�Cfor 24 h. Harvesting was always carried out 4 h after the onset oflight. Plants with promoter-GUS constructs were grown both on soiland on MS agar plates (half-strength MS + MS vitamins; Murashigeand Skoog, 1962) in order to monitor the expression of GUS.
RNA isolation and RT-qPCR
RNA was isolated with the Plant Total RNA Miniprep System(Viogene, http://www.viogene.com) following the manufacturer’sinstructions, and was then treated with RNase-free DNaseI (Pro-mega, http://www.promega.com) to degrade contaminating geno-mic DNA. qPCR-primers (MWG, http://www.eurofinsdna.com) weredesigned according to specific criteria, which included a predictedmelting temperature of 60 � 2�C, a primer length of 18–24 nucleo-tides, a product size of 60–150 bp and a GC content of 45–60%.Primer sequences are available in Table S1. cDNA from approxi-mately 500 ng total RNA (determined by spectrophotometry, A260)was synthesized with the use of oligo(dT23) primers (ProtoScript kit;New England Biolabs, http://www.neb.com) following themanufacturer’s instructions. qPCR was carried out with a Rotor-Gene 3000 (Corbett, http://www.corbettlifescience.com) usingPlatinum qPCR SYBR�green supermix (Invitrogen, http://www.invitrogen.com). Melting curve analyses were performed for allsamples by elevating the temperature from 55 to 99�C at a rate of0.5�C s)1, and products were routinely checked by 2% agarose gelelectrophoresis. To account for possible differences between sam-ples in original RNA quantity and efficiency of cDNA transcription,samples were normalized by actin2, a reference gene that wasshown to be only weakly affected by drought stress in previousmicroarray experiments (Alexandersson et al., 2005). This was also
Conserved drought regulation of aquaporin genes 657
ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 650–660
confirmed by Genevestigator (Zimmermann et al., 2004), andCzechowski et al. (2005) showed that actin2 expression changed byless than 50% upon drought. In addition, the conserved overallexpression pattern of PIPs among the accessions supported thechoice of the reference gene. The comparison of expression wasperformed by the function ‘Comparative Quantification’ included inROTOR-GENE 6 (Corbett).
Differences in gene regulation during drought for the accessionswere analyzed in a two-way fashion. First, one-way ANOVA was usedto find accession-specific deviations from a common mean. Then apost-hoc analysis (‘multcompare’, as implemented in MAT-
LAB R2006b; The MathWorks Inc., http://www.mathworks.com)was performed on genes with a significant deviation (as indicatedby ANOVA) in order to find out which accessions differed signifi-cantly from each other.
Promoter-GUS construction and plant transformation
Approximately 1.6-kb-long sequences upstream of the ATG startcodon of AtPIP1;4, AtPIP2;5 and AtPIP2;6 were amplified fromgenomic DNA isolated from Arabidopsis Col-0 plants by the fol-lowing primer pairs (numbers denote the position of the primer inrelation to the start codon, set as position +1; all forward primershave CACC added in the 5¢ end, in order to allow directionalrecombination of the PCR product into the pENTR D-TOPO-vectorusing the Gateway� system). Promoter AtPIP1;4, forward,CACC)1659GGCAACGAAGCATGTGAGAT)1639; promoter AtPIP1;4,reverse,)1CAAAAGAGGGAGAGGGAGAGAA)22;promoterAtPIP2;5,forward, CACC)1692TTTTGGTGCTGACGAGAGTG)1672; promoterAtPIP2;5, reverse )1AAGTTTGGTGAAGAATAAGAAGACA)25; pro-moter AtPIP2;6, forward, CACC)1622ATGGTCGTCGCTCGATTA-TT)1602; promoter AtPIP2;6, reverse, )1CGTGAAGGCTAAGTCTGAAAGA)22.
In the following step, the promoter regions were recombined intothe pKGWFS7 destination vector, which contains the uid genecoding for GUS (Plant Systems Biology, http://www.psb.ugent.be),using the LR recombination mix (Invitrogen, http://www.invitro-gen.com/).
The constructs were transformed into competent Agrobacteriumtumefaciens GV3101, and were introduced into Arabidopsis Col-0by floral dip as described previously (Clough and Bent, 1998). The400-ml cultures of A. tumefaciens containing the promoter-GUSconstructs were grown overnight at 28�C. The bacteria were pelletedand resuspended in 400 ml 5% sucrose containing 200 ll of thedetergent Silwet L-77 (Lehle Seeds, http://www.arabidopsis.com).Bolting Arabidopsis plants were dipped into the bacterial suspen-sion for 3 min, followed by coverage with plastic foil for 2 days. Thedipping procedure was repeated after 1 week to increase thetransformation frequency. Seeds were germinated on agar plateswith kanamycin (half-strength MS + MS vitamins, 50 lg ml)1 kana-mycin) to select for transformed plants. The surviving plants werethen transplanted to soil.
GUS staining
The GUS expression patterns were visualized by dispensing theplant tissue to substrate solution [1 mM X-gluc, 100 mM potassiumphosphate buffer, pH 7.0, 10 mM EDTA, pH 8.0, 1 mM potassiumferricyanide, 0.1% (v/v) Triton X-100] and incubating at 37�C over-night. The plant tissues were then kept in 95% ethanol at 4–8�C. Plantstransformed by GUS constructs were grown on agar plates withoutkanamycin (half-strength MS + MS vitamins) for 2 weeks before theGUS expression pattern was visualized in intact seedlings. Flowers,leaves and siliques (containing seeds) for GUS staining were takenfrom plants grown for 6–10 weeks. Twenty of the 30 promoter
AtPIP1;4 GUS lines examined showed GUS expression. The corre-sponding numbers for the lines transformed with the AtPIP2;5 andAtPIP2;6 promoter-GUS constructs were eight of 11 and 12 of 28,respectively. All plants studied were either T2 or T3 generations. Nopropagation of homozygous lines was performed.
Database search and PCA analysis
Principal component analysis stands for a spectral decompositionof a given data covariance or correlation matrix, consisting ofmeasurements of variables for a set of given samples. It finds un-correlated directions (the PCs) in variable space ordered by thevariance that the samples have in the corresponding direction.Thus, for example, the first principal component is the direction invariable space in which the samples have the highest variance. PCAwas introduced by K. Pearson (1901), and is now widely used inexploratory data analysis. In this work an uncentered version of PCAwas used. This makes it possible to visualize and estimate both theintervariability in the dataset as well as the overall behavior. ThePCA of variation of leaf water content and PIP gene expression wascalculated from expression ratios (drought/control), and was visu-alized by QLUCORE OMICS EXPLORER (Qlucore Omics Explorer 2009;Qlucore, http://www.qlucore.com). Analysis of the co-variation ofPIP and TIP transcripts was carried out with ACT (http://www.arabidopsis.leeds.ac.uk/act/; Jen et al., 2006), ATTED-II (http://atted.jp/; Obayashi et al., 2004) and csb.db (http://csbdb.mpimp-golm.mpg.de/; Rautengarten et al., 2005). ATTED-II expression datacomprised 58 different experimental conditions from a total of 1388Affymetrix (ATH1) arrays, and ACT expression data consisted of 322Affymetrix (ATH1) arrays covering various stress treatments anddevelopmental stages. Data was retrieved in August 2006 (ATTED-IIand csb.db) and February 2009 (ACT).
ACKNOWLEDGEMENTS
We would like to thank Adine Karlsson for help with maintainingand growing the plants. Funding by the Swedish Research Council,Formas, Carl Tryggers Stiftelse for Vetenskaplig Forskning and theResearch School of Pharmaceutical Sciences (FLAK) is gratefullyacknowledged.
SUPPORTING INFORMATION
The following Supporting Information is available for this articleonline:Figure S1. Ratios (log2) of mRNA levels of drought-stressed andcontrol plants for the 13 plasma membrane intrinsic protein (PIP)transcripts in five different accessions of Arabidopsis.Figure S2. Co-correlation scatter plots generated by ACT of PIPs andTIPs with Pearson’s correlation coefficients higher than or equal to0.6 in the ATTED analysis.Figure S3. Expression patterns with and without drought stress ofrepresentative lines carrying the 1.6-kb promoter::GUS constructsof AtPIP1;4 (A, B) and AtPIP2;5 (C, D).Table S1 Primers used for qPCR.Table S2 Level of plasma membrane intrinsic protein (PIP) expres-sion in comparison with actin2 in Col-0, Ler-0, Lip-0, Edi-0 and Kas-1.Table S3 The genes most highly co-expressed with the non-drought-inhibited major intrinsic proteins (MIPs; AtPIP1;4, AtPIP2;5and AtPIP2;6), according to data retrieved from ATTED-II.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.
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