Hypocotyl Transcriptome Reveals Auxin Regulation of
Growth-Promoting Genes through GA-Dependent and
Elisabeth J. Chapman1.¤, Kathleen Greenham1., Cristina Castillejo1, Ryan Sartor1, Agniezska Bialy1, Tai-
ping Sun2, Mark Estelle3*
1Section of Cell and Developmental Biology, Division of Biological Sciences, University of California San Diego, La Jolla, California, United States of America, 2Biology
Department, Duke University, Durham, North Carolina, United States of America, 3Howard Hughes Medical Institute, University of California San Diego, La Jolla, California,
United States of America
Many processes critical to plant growth and development are regulated by the hormone auxin. Auxin responses are
initiated through activation of a transcriptional response mediated by the TIR1/AFB family of F-box protein auxin receptors
as well as the AUX/IAA and ARF families of transcriptional regulators. However, there is little information on how auxin
regulates a specific cellular response. To begin to address this question, we have focused on auxin regulation of cell
expansion in the Arabidopsis hypocotyl. We show that auxin-mediated hypocotyl elongation is dependent upon the TIR1/
AFB family of auxin receptors and degradation of AUX/IAA repressors. We also use microarray studies of elongating
hypocotyls to show that a number of growth-associated processes are activated by auxin including gibberellin biosynthesis,
cell wall reorganization and biogenesis, and others. Our studies indicate that GA biosynthesis is required for normal
response to auxin in the hypocotyl but that the overall transcriptional auxin output consists of PIF-dependent and -
independent genes. We propose that auxin acts independently from and interdependently with PIF and GA pathways to
regulate expression of growth-associated genes in cell expansion.
Citation: Chapman EJ, Greenham K, Castillejo C, Sartor R, Bialy A, et al. (2012) Hypocotyl Transcriptome Reveals Auxin Regulation of Growth-Promoting Genes
through GA-Dependent and -Independent Pathways. PLoS ONE 7(5): e36210. doi:10.1371/journal.pone.0036210
Editor: Miguel A. Blazquez, Instituto de Biologı ´a Molecular y Celular de Plantas, Spain
Received March 23, 2012; Accepted March 28, 2012; Published May 9, 2012
Copyright: ? 2012 Chapman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Institute of Health Institute of General Medicine (GM 43644) and the Department of Energy (De-FG02-
02ER15312). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funders had no role
in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
¤ Current address: Forage Genetics International, West Salem, Wisconsin, United States of America
The plant hormone auxin has diverse roles in plant growth and
development including, but not limited to, embryogenesis, cell
division and expansion, root initiation, tropic responses, apical
dominance, flowering, and fruit and seed development . A
major challenge in the field of auxin biology is to understand how
a small molecule can specify such distinct changes in morphogen-
esis and growth throughout the life cycle of a plant. Current
models suggest that auxin levels are highly regulated through
changes in auxin biosynthesis, conjugation and storage, degrada-
tion, and polar transport. Auxin level is then interpreted by the
auxin perception machinery resulting in tissue- and cell type-
specific changes in gene expression [2,3,4].
Auxin regulation of transcription involves a large family (23 in
Arabidopsis) of DNA-binding transcription factors called the
AUXIN RESPONSE FACTORs (ARF) [5,6]. ARFs bind to
promoters of auxin-responsive genes at cis-elements referred to as
auxin response elements (AuxREs) [7,8]. A TGTCTC sequence
motif first identified in the auxin-responsive GH3 promoter from
soybean was shown to recruit multiple members of the Arabidopsis
ARF family, with TGTC being absolutely required for ARF-DNA
binding . However, the TGTCTC element is not found in all
auxin-responsive promoters. In some cases tandem repeats of the
TGTC portion of the AuxRE are sufficient for auxin induction
[10,11]. ARF proteins are characterized by a B3-like DNA
binding domain, a middle region associated with transcriptional
repression or activation, and a C-terminal domain (CTD) involved
in homo- and hetero-dimerization [2,7,8]. The CTD region is
similar to the C-terminal domains III and IV of the Aux/IAA
transcriptional regulators .
The Aux/IAAs are a 29 member family of small nuclear
proteins in Arabidopsis that are involved in repressing auxin-
regulated transcription . Aux/IAA proteins contain four
conserved domains (I–IV), of which domains I, II and IV contain
nuclear localization motifs. Domain III contains a sequence that is
related to the baa DNA binding domain that is required for Aux/
IAA homo- and hetero-dimerization. However, there is currently
no evidence that Aux/IAA proteins bind DNA directly [14,15].
Rather, Aux/IAAs are recruited to promoters through interactions
with ARF proteins that are mediated by domains III and IV of the
two proteins. Domain II of Aux/IAAs is highly conserved and
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contains a degron motif that is important for degradation by the
SCFTIR1E3 ubiquitin ligase complex [12,16]. Mutations in this
degron result in stabilization of the protein and reduced auxin
response, causing various defects in growth and development
Functional redundancies within the ARF and Aux/IAA gene
families make assigning specific roles of each protein a challenge.
However, genetic studies have revealed ARF and Aux/IAA
combinations that are essential for certain processes. BDL/IAA12
and MP/ARF5 specify apical-basal polarity during embryogenesis
, SLR/IAA14 and NPH4/ARF7 are required for lateral root
initiation, and MSG2/IAA19 and NPH4/ARF7 are involved in
tropic hypocotyl growth . ARF2, ARF8, and ARF19 are
involved in root and hypocotyl growth and development, although
Aux/IAA partners in these processes are not clear [20,21,22].
Recently, the apical-basal polarity determinant TOPLESS (TPL)
was shown to act as a transcriptional co-repressor with IAA12/
BDL to repress ARF5/MP transcriptional activity . It has yet
to be seen whether all the Aux/IAAs interact with TPL to repress
the auxin response in specific developmental pathways.
Auxin exerts changes in gene expression by interacting with the
TIR1/AFB family of auxin receptors. These proteins are the F-
box protein subunits of SCF (Skp1/Cullin/F-box) complexes that
target the Aux/IAAs for proteasome-mediated degradation
[24,25,26]. The Arabidopsis genome encodes 5 proteins related to
TIR1, Auxin Signaling F-Box (AFB) proteins AFB1, 2, 3, 4 and 5.
Previous work has shown that, like TIR1, AFB1–5 function as
auxin receptors that interact with Aux/IAA repressors in an auxin-
dependent manner [26,27]. Mutant analysis reveals overlapping
functions of TIR1/AFB1–3. The most severely affected tir1 afb1
afb2 afb3 quadruple mutants arrest shortly after germination .
The AFB4 clade of receptors, including AFB4 and AFB5, display a
unique affinity for the synthetic auxin picloram. The afb5-5 single
mutant shows almost complete resistance to picloram-induced
hypocotyl growth .
In order to develop successful models for auxin regulation of
growth and development, it will be important to identify the gene
targets of the TIR1/AFB pathway(s) and understand their function
in cell growth. Several studies of auxin-responsive transcriptomes
have identified large numbers of candidate auxin targets. The
results of supporting genetic studies ascribe developmental roles to
a small number of these . A potential barrier to identification
of distinct auxin pathways from such studies lies in the complexity
of the tissue sampled for the experiment. Auxin mediates distinct
responses in different tissue types, for example inhibiting primary
root elongation while stimulating lateral root initiation and
outgrowth . Therefore, auxin-responsive transcriptomes in
entire plants are too complex to facilitate separation of distinct
In this study we focus on the role of auxin signaling in cell
expansion. We chose the hypocotyl, which grows entirely by cell
expansion, as a model tissue for this study . The hypocotyl
elongates in plants overexpressing auxin biosynthetic genes 
and in response to high temperature , due to elevated auxin
levels. Hypocotyl elongation is tightly regulated and many
signaling pathways overlap to regulate uniform, as well as
directional, hypocotyl cell expansion. Light is a major repressor
of hypocotyl growth and as a consequence, mutations in the
phytochrome light receptors result in seedlings with long hypocotyl
phenotypes . Light-activated forms of the phytochromes
interact with members of the phytochrome-interacting factor
(PIF) family of bHLH transcription factors, signaling rapid phyA-
and phyB-mediated degradation of PIF3,4 and 5 in the light
[34,35,36]. PIFs have also recently been shown to function in GA
signaling . The PIFs appear to be the major positive regulators
of hypocotyl growth, as they are required for growth responses to
time of day, direction of light source, nutrients, high temperature
and other stimuli [38,39,40,41]. PIF mRNA and protein levels are
controlled by the circadian clock, light, and GA signaling, such
that PIF activities and hypocotyl growth are repressed during the
day [39,42,37]. Within the PIF family, several PIF and PIF-LIKE
(PIL) genes are implicated in germination and early seedling
growth . PIF4 and PIF5 seem to be particularly important for
hypocotyl growth as expression of these factors is circadian
regulated and correlates with hypocotyl growth [39,42]. In
addition the pif4pif5 double mutant has a short hypocotyl
Here we identify auxin signaling components required for
auxin-responsive hypocotyl elongation. In addition we character-
ize the auxin transcriptome specifically in elongating hypocotyl
tissue. Our findings indicate that auxin-induced hypocotyl
elongation is associated with regulation of a suite of growth-
associated genes and involves GA biosynthesis. Importantly, we
also show that auxin works in part through pathways independent
of GA and PIF activities.
Results and Discussion
Auxin Promotes Elongation of Arabidopsis Hypocotyls
To explore the function of auxin in plant growth, we have
elected to focus on the Arabidopsis hypocotyl. Our first task was to
develop a robust assay for auxin response in this system. Seedlings
were grown at 22C for 5 days in various day-night cycles, exposed
to auxin, and measured after different treatment times. Initially,
we treated seedlings with the synthetic auxin picloram because
earlier studies showed that this compound promoted hypocotyl
elongation while the natural auxin indole acetic acid (IAA),
generally inhibited elongation [27,44,45]. However, under our
conditions we found that both picloram and IAA promoted
hypocotyl growth in continuous light (LL), long days (LD), or short
days (SD) (Fig. 1A). Unless otherwise stated, LD conditions were
used for additional experiments designed to characterize auxin-
responsive hypocotyl growth (Fig. 1B). Importantly, in our
conditions the auxin dose-response curve for hypocotyl growth
of wild-type seedlings is bell-shaped (Fig. 2). This differentiates our
growth conditions from those in which auxin treatment or
[22,44,46]. Interestingly, the bell-shaped response curve is similar
to auxin dose response in root system growth modeled previously
, suggesting that an auxin signaling level optimal for eliciting a
growth response may be a common feature among auxin-
Auxin-mediated Hypocotyl Elongation Requires
Transcriptional Auxin Signaling
To confirm that the auxin-dependent elongation response
requires activation of transcriptional auxin signaling pathways,
we measured the response in a series of Aux/IAA gain-of-function
mutants in which auxin-regulated transcription is repressed
[48,49,50,51,52]. In slr-1/iaa14, axr2-1/iaa7 and axr5-1/iaa1, the
auxin response was significantly reduced compared to wild-type
plants (Fig. 1C). Interestingly, the response of msg2-1/iaa19
seedlings was similar to that of wild type, even though this mutant
is deficient in tropic growth in the hypocotyl. This suggests that
different auxin signaling pathways have specific roles in hypocotyl
growth. This has been shown previously for apical-basal polarity
determination  and lateral root initiation .
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We explored the possibility of functional specialization among
the TIR1/AFB auxin receptors in hypocotyl elongation by
analyzing the phenotypes of various tir1/afb mutants. We
observed slight auxin resistance or hypersensitivity in single tir1/
afb receptor mutants (Fig. 2A) with the exception of afb5-5 and
afb4-2 afb5-5, which are highly resistant to picloram . The
basis for auxin hypersensitivity in afb1-3 and afb3-4 mutants is
unclear, however, double mutant combinations among afb1-3,
afb2-3, and afb3-4 eliminated this hypersensitivity (Fig. 2B)
suggesting that increased growth response may be due to
enhanced activity of other TIR1/AFB family members that is
lost in the higher order mutants. The afb3-5 mutant overexpresses
AFB1 and AFB2 due to alterations in small RNA-mediated
regulation, and afb2-3 overexpresses AFB1 and AFB3 . Thus,
TIR1/AFB single mutants may not display predictable loss-of-
function phenotypes. Future analysis of the expression patterns of
the receptors in the single and double mutant backgrounds will be
necessary to determine whether elevated receptor activity in
TIR1/AFB mutants could explain the hypersensitivity we
Double and triple mutants carrying tir1-1 each displayed
increased auxin resistance when compared to the tir1-1 mutant
(Fig. 2C). The triple mutant tir1-1 afb2-3 afb3-4 displays an
incompletely penetrant phenotype in which a significant percent-
age of individuals fail to develop basal structures such as roots and
hypocotyls . In tir1-1 afb2-3 afb3-4 individuals with developed
basal structures, hypocotyls were shorter than those of wild-type
plants and displayed the highest degree of resistance to IAA-
Figure 1. Auxin promotes hypocotyl elongation in light-grown
seedlings. Auxin promotes hypocotyl elongation in a range of day-
length conditions. Average hypocotyl length of wild-type seedlings
grown in short days (SD; 8/16), long days (LD; 16/8) or constant light
(LL) and treated with 5 mM picloram was determined following 24, 48,
or 72 hours of auxin treatment. Hypocotyl length on auxin is shown as a
percentage of length on control medium. Error bars indicate standard
error. (B) Auxin response in seedlings increases with auxin concentra-
tion. Images of aerial portions of individual 7 day-old seedlings were
captured following 48 hours of IAA treatment at the indicated
concentrations. (C) Hypocotyl auxin response requires auxin signaling.
Average hypocotyl length of wild-type or aux/iaa mutant seedlings
treated with 5 mM picloram (red bars) or IAA (blue bars) was measured
following 48 hours of auxin treatment. Hypocotyl length on auxin
relative to the untreated control is shown as in (a). Error bars indicate
standard error. Statistical significance was determined using a Tukey
HSD post hoc comparison among the means on the analysis of variance
using type III sums of squares (p,0.05).
Figure 2. Hypocotyl auxin response requires TIR1/AFB auxin
receptors. (A–C) Hypocotyl length of wild-type or tir1/afb single or
multiple mutant seedlings grown in short days and treated with IAA at
the indicated concentrations was measured following 48 hours of auxin
treatment. Asterisks represent mutants showing a significantly different
response to hormone treatment compared to wildtype. A general linear
model (glm performed in R using the car package ) was used to
determine significance and main effects for genotype were confirmed
using ANOVA type III sums of squares. All assumptions for GLM were
fulfilled. Error bars indicate standard error.
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mediated elongation of all tir1/afb receptor mutants (Fig. 2C). The
reliance of the elongation response on the TIR1/AFB auxin
receptors and degradation of Aux/IAA proteins confirms that
auxin mediated growth requires transcriptional auxin signaling
Identification of Auxin-responsive Cell Expansion-
associated Genes in Elongating Hypocotyls
Based on our finding that auxin-mediated hypocotyl elongation
requires the TIR1/AFB pathway, we hypothesized that elongation
is preceded by changes in expression of a suite of auxin-responsive
genes. To identify such genes, we profiled auxin-responsive
transcription in hypocotyls in a series of microarray experiments.
We incorporated several parameters into our microarray design to
maximize the likelihood of identifying auxin-regulated genes
associated with anisotropic cell expansion. To enrich our dataset
for cell expansion genes that may not be identified in whole
seedling experiments, we sampled hypocotyl tissue dissected from
auxin- or control-treated whole seedlings. To minimize time-of-
day and circadian effects and avoid mis-identification of auxin-
responsive genes, we treated experimental and control seedlings at
the same time of day and limited the dissection time to 30 minutes.
To maximize the amplitude of the transcriptional auxin response,
we treated seedlings two hours after subjective dawn, when
hypocotyl growth is minimal in the absence of exogenous auxin
. Finally, we used the synthetic auxin picloram and included
the afb5-5 mutant in our microarray design, as this mutant is
picloram-resistant but does not otherwise display obvious growth
defects [27,55]. We theorized that cell expansion-associated genes
differentially expressed in wild-type hypocotyls elongating in
response to picloram might not be responsive in afb5-5 hypocotyls,
which fail to elongate in response to picloram.
For microarray experiment ‘‘a’’, we sampled hypocotyls from
wild-type plants treated for 30 minutes or 2 hours with picloram or
a solvent-only control. For experiment ‘‘b’’, we sampled hypocot-
yls from wild-type or afb5-5 plants treated for 2 hours with
picloram or a solvent-only control (Fig. 3A, Table S1). Following
auxin or control treatment of seedlings, hypocotyls were individ-
ually dissected and frozen for subsequent RNA isolation.
To identify genes differentially expressed among the treatments,
we used a moderated linear model  and an FDR cutoff of
,0.05 to filter data from each microarray experiment. From this
initial analysis we identified 65 genes differentially expressed
following the 30-minute auxin treatment (Table S2), and 3544
(experiment ‘‘a’’) or 804 (experiment ‘‘b’’) genes differentially
expressed following a 2-hour auxin treatment (Fig. 3A). Consistent
with the picloram-resistant phenotype of afb5-5, no differential
expression was detected in afb5-5 following picloram treatment
using the analysis method described. Interestingly, we were also
unable to identify genes differentially expressed between wild-type
and afb5-5 untreated samples (Fig. 3A). So far, picloram
perception and regulation of picloram-responsive transcription is
the only known function of the AFB5 auxin receptor. The
identification of additional functions for AFB5 will require
alternative experimental approaches. Analysis of genes differen-
tially expressed in wild-type hypocotyls following 30 minutes of
picloram treatment indicated that SAUR genes, AUX/IAA genes,
GH3 genes and others shown elsewhere to be early auxin-
responsive  were induced by picloram and were the
predominant genes to be regulated at this time-point (Table S2).
For additional insight into gene expression associated with auxin
response, we focused on data from the 2-hour time-point samples.
Comparison of gene lists from the 2-hour auxin treatment in
experiments ‘‘a’’ and ‘‘b’’ identified 267 genes differentially
Figure 3. The afb5 mutant fails to respond to picloram. The afb5-
5 mutant fails to regulate transcription in response to picloram.
Differential gene expression between hypocotyl samples of solvent-
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expressed in both experiments. This modest overlap may be due to
experimental variables such as differences in RNA extraction
methods and microarray hybridization parameters, or perhaps
more importantly, to ‘lab-effects’ such as those previously shown to
serve as a source of variability among microarray experiments
performed on the same platform at different laboratories  (see
Methods). To increase the validity and statistical strength of the
comparison we used the RankProd package in R that accepts pre-
processed data generated from different laboratories and platforms
. This package is an extension of the rank product method that
implements a non-parametric statistic to compare the expression-
based rankings of genes across samples . From this analysis, we
identified 1193 genes differentially expressed between control and
2 hour auxin-treated samples; 740 of these genes are induced, and
453 are repressed by picloram (Table S3). The mean expression
levels of these two gene sets in microarray ‘‘b’’ are not affected by
picloram treatment of afb5-5 mutant plants (Fig. 3B), suggesting
that these are indeed downstream targets of picloram-stimulated
transcriptional auxin signaling. We focused on the set of 1193
candidate auxin-responsive cell expansion-associated genes for all
Picloram and IAA Regulate a Common Set of Target
The synthetic auxin picloram induces a hypocotyl elongation
response similar to that observed with IAA, suggesting that the
downstream targets of picloram- and IAA-stimulated auxin
signaling are common between these two auxin pathways. The
failure of afb5-5 to regulate this set of genes or to elongate in
response to picloram is consistent with a model in which the genes
are targets of auxin signaling and involved in the elongation
response. To confirm this, we performed a comprehensive
comparison between our auxin-responsive gene set and publicly
available microarray data. Our first comparison was done using
the MASTA package available from the BAR website (http://bar.
utoronto.ca/welcome.htm) that probes differentially expressed
genes against a database of 600 contrasts obtained from publicly
available microarray datasets. Of the 740 genes upregulated by
picloram in our dataset, 219 were identified as auxin-upregulated
in IAA treatment arrays; of 453 genes downregulated by picloram
in our dataset, 121 genes were identified as auxin-downregulated
in IAA arrays (data not shown). These overlaps are statistically
significant (p.value ,0.001) and confirm that picloram affects
known IAA-responsive genes. We also performed independent
comparisons with the Nemhauser et al.  and Stepanova et al.
 auxin treatment datasets (see Methods for details of
comparison). In both cases, more than 50% of the IAA-induced
genes were induced by picloram in our experiments (Fig. S1A).
The Stepanova et al.  dataset was obtained from experiments
using root tissue suggesting that many of the genes involved in
hypocotyl growth are common to root tissue. We would expect
these genes to be specifically involved in cell elongation during
Importantly, our analysis identified many genes that are not
presented in other auxin transcriptome datasets (Table S4)
[61,62]. Of the 740 induced genes, 521 were not described in
the Nemhauser et al. (62) and Stepanova et al. (63) datasets or the 7
IAA treatment arrays found in the MASTA database. Eighty-one
of these are not represented on ATH1 chips and not well
characterized as auxin responsive. We expect that many of the
remaining 440 genes are specifically auxin regulated in the
elongating hypocotyl and were not identified in other studies
because of the relative complexity of the auxin response in
seedlings and roots. Similarly, 332 of the 453 repressed genes had
not been described in these other datasets, 68 of which are on the
Nimblegen chip but not ATH1.
To further validate the effects of picloram on auxin-responsive
genes, we confirmed that a set of auxin ‘‘marker’’ genes, proposed
to serve as hallmarks of auxin activity , were identified as
picloram-responsive in our microarray data analysis. Overall,
expression of the marker genes was responsive to picloram in wild-
type hypocotyls, but not in hypocotyls from afb5-5 mutant plants
(Fig. S1B). We further validated the picloram response of several of
these genes, GH3.3, GH3.5, HAT2, IAA5, IAA19 and SAUR15, by
quantitative RT-PCR using wild-type and afb5-5 hypocotyls.
Expression of each gene was induced in wild-type hypocotyls by
picloram treatment, and induction was dependent upon AFB5
(Fig. S1C). This indicates that picloram and IAA regulate an
overlapping set of target genes, although the picloram signal is
uniquely transduced by AFB5.
Finally, we analyzed our picloram-responsive gene set for
association with auxin Gene Ontology terms and overrepresenta-
tion of AuxRE-containing promoter elements. GO terms associated
with auxin response and hormone signaling are enriched in the
annotations of our auxin-responsive gene set (Table S5), and we
identified several overrepresented AuxRE-containing promoter
elements in the promoter gene set (Fig. S2). From these results
we conclude that picloram regulates the same downstream
transcriptional targets as IAA, and therefore promotes hypocotyl
elongation through the same transcriptional pathways as IAA. For
the remaining experiments, we used picloram and IAA inter-
changeably or in parallel, and we did not observe qualitative
differences in responses to these two auxins.
A Profile of the Transcriptional Auxin Response Preceding
Further examination of GO terms associated with our auxin-
responsivegene set revealed
involved in cell wall maintenance, cell expansion, growth and
hormone signaling (Fig. 4A, Table S5, Fig. S3). Enriched GO
terms associated with the auxin-induced gene set included cell
wall metabolism and gibberellin biosynthesis. Terms associated
metabolism and plastoquinone assembly (Fig. 4A). Representa-
tion of these GO processes in our auxin-responsive gene set is
consistent with a role for auxin in transcriptional regulation of
cell expansion-associated genes. Cell expansion in the hypocotyl,
as well as in other growing plant tissues, is gated by the
circadian clock and shows non-uniform patterns across a 24-
hour period [63,54,39]. This is likely due in part to circadian
patterns of expression of many genes involved in auxin
signaling, biosynthesis and transport, and varying sensitivity to
auxin at different times of day . We theorized that genes we
found to be auxin-responsive in elongating hypocotyls may
treated wild-type (Col-0) or afb5-5 seedlings (afb5) or seedlings treated
with picloram (+ pic) for 30 minutes (30) or 120 minutes (120), as
determined by analysis of microarray data. The number of genes
differentially expressed between samples is shown in lines connecting
each sample pair. Data from microarray experiments (a) and (b) were
combined for identification of 1193 picloram-responsive genes. (B)
Average expression values of 740 auxin-induced (upper panel) or 453
auxin-repressed (lower panel) genes are not different in hypocotyls
from control seedlings (Col-0), control-treated afb5-5 mutant seedlings
(control afb5) or picloram-treated afb5-5 mutant seedlings (pic afb5).
Differentially expressed genes identified using the RankProd package
were selected and average expression values for microarray ‘b’ are
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follow circadian expression patterns. To determine whether
circadian-regulated genes are overrepresented in our auxin-
responsive gene set, we generated a gene subset consisting of the
top 400 auxin-induced genes according to statistical significance,
and analyzed this subset using the Phaser tool (http://phaser.
cgrb.oregonstate.edu/) . We observed significant enrichment
of genes showing peak expression during phases 0–2 and 22–23
in LD conditions, during which hypocotyl growth is active
(Fig. 4B) . We further explored our auxin-induced gene set
for additional determinants of expression profile by analyzing
the corresponding promoter set for overrepresented regulatory
elements. Interestingly, the predicted MYC/MYB binding site
‘CACATG’ was the most highly overrepresented element
identified in this analysis (data not shown). The ‘CACATG’
element was previously identified as the Hormone Up at Dawn
(HUD) element enriched in promoters of genes responsive to
phytohormones and showing peak expression levels during
periods of growth . Together, these findings suggest that
auxin promotes hypocotyl growth by regulating expression of
cell expansion-associated genes whose expression levels are
controlled by the circadian clock. This is consistent with auxin
gating by the clock to maintain the diurnal pattern of hypocotyl
elongation under normal growth conditions .
Auxin-mediated Hypocotyl Elongation Requires GA
A number of studies have shown that auxin and GA interact to
[65,66,67,68,69]. For example, in pea stem and Arabidopsis
seedlings, auxin regulates the expression of a number of GA
metabolic genes including members of the GA20OX and GA3OX
gene families, involved in synthesis of active GAs, as well as
GA2OX genes, involved in GA inactivation [66,70]. In addition,
Frigerio et al  showed that the long hypocotyl phenotype
conferred by overexpression of YUCCA1 is suppressed by the GA
biosynthesis inhibitor paclobutrazol, indicating that GA synthesis
is required for auxin-dependent hypocotyl growth. We found that
GA20OX1, GA20OX2, GA2OX8, and GA3OX1 are auxin-regulated
in the hypocotyl (Table S2, Table S3). To expand on the role of
GA biosynthesis in auxin-mediated hypocotyl elongation, we
tested the effect of adding paclobutrazol to auxin treatment assays.
Paclobutrazol inhibited the effects of exogenous auxin in our
system, as co-treatment with paclobutrazol attenuated, but did not
abolish, the hypocotyl elongation promoted by picloram (Fig. 5A)
or IAA (Fig. 5B). This suggests that active GA biosynthesis is
required for optimal hypocotyl auxin response, consistent with
earlier reports .
These results are consistent with a model in which increased
auxin levels promote an increase in GA levels, and this GA
increase is required for the elongation response. Auxin and GA
are known to be involved in the effects of elevated temperature
on hypocotyl elongation. Temperature-mediated
depends upon auxin biosynthesis [32,71,72]. Stavang et al.
 showed that the temperature response also requires GA
and that both GA20ox1 and GA3ox1 are up-regulated at higher
temperature. They concluded that GA and auxin act indepen-
dently, based on the behavior of pentuple della mutants .
However, it has also been shown that co-treatment of seedlings
with GA and NPA attenuates the GA response . These
results suggest that auxin and GA act interdependently to
regulate hypocotyl length.
We further examined our microarray data for overlap between
the auxin/GA pathway we present here and the temperature-
response pathway mediating hypocotyl elongation. Interestingly,
we find that our hypocotyl auxin-regulated gene set is quite distinct
from the gene set responding to elevated temperature in seedlings.
Of the 113 temperature up-regulated genes presented by Stavang
et al., only 13 are also induced by auxin in the hypocotyl .
These findings suggest that most transcriptional changes associated
with temperature are not related to auxin, or may occur
predominantly in non-hypocotyl seedling tissues.
GA acts by stimulating the degradation of growth repressing
proteins called the DELLAs . Previous work has shown that
auxin promotes degradation of the DELLA proteins in the root
and that this degradation is required for GA regulated root growth
. However, how auxin regulates DELLA levels is not clear.
One possibility is that loss of the DELLAs is caused by an auxin-
dependent increase in GA levels. To determine if this might be
happening in the hypocotyl, we tested the effects of exogenous
auxin on stability of the DELLA protein RGA. Treatment of
seedlings expressing RGA-GFP with IAA or GA resulted in loss of
RGA protein from hypocotyl cells within 2 hours (Fig. 5C). This
auxin effect was abolished by co-treatment with paclobutrazol
(Fig. 5C). While it is possible that the observed loss of RGA protein
in auxin-treated seedlings is due to an effect of auxin on
transcription of RGA, we think this is unlikely as we did not
identify RGA as an auxin-downregulated gene in our microarray
experiments (although we did identify RGA-LIKE1 (AT1G66350)
and RGA-LIKE3 (AT5G17490) as auxin-upregulated genes, see
Table S3). DELLA protein abundance is also affected by circadian
regulation of GA signaling . However, it is unlikely that
circadian regulation fully explains the effects we observed on
RGA-GFP levels, as DELLA protein levels increase during the day
 where we observed a decrease. A more likely possibility is that
auxin regulation of GA levels results in degradation of RGA-GFP
protein in the seedlings. We did observe that the RGA-GFP signal
decreased in the hypocotyl throughout the course of the
experiment, and therefore that the signal in control seedlings
was weaker at the 24-hour time point than at time zero. This is
also unlikely to be due to circadian patterns, which follow a 24-
hour cycle. It is possible that the overall abundance of RGA-GFP
in hypocotyls changes with growth dynamics, and that more
sensitive imaging methods could be used to visualize the protein in
We further explored the requirement for GA biosynthesis and
signaling in auxin response by examining the behavior of the
ga20ox1 ga20ox2 double mutant in the hypocotyl elongation assay.
Plants compromised in endogenous GA levels due to mutations in
GA20OX1 and GA20OX2 showed partial auxin resistance (Fig. 5D).
These data suggest that auxin and GA act interdependently in
hypocotyl cell expansion.
We noted, however, that in our hormone treatment assays,
paclobutrazol did not completely abolish the auxin effect (Fig. 5A,
5B). While this inhibitor may not fully suppress GA accumulation
in the seedlings, our results suggest that the elongation-promoting
effects of auxin may not be limited to regulation of GA
metabolism. We further explored the auxin-GA interaction by
testing the ability of GA to restore the short hypocotyl phenotypes
of several gain-of-function Aux/IAA mutants. We found that GA
did not significantly affect the hypocotyl length of the axr2 mutant,
and that the hypocotyl phenotypes of these mutants overall were
only partially restored by treatment with GA (Fig. S4A). These
data indicate that auxin signaling is required for a growth program
independent of the regulation of GA metabolism, and that
constitutive repression of auxin signaling in the Aux/IAA mutants
represses this program. We propose that auxin promotes hypocotyl
growth in part through GA and in part through an unknown
independent pathway(s). A mechanism by which auxin can induce
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hypocotyl growth independently of GA synthesis may be
important for rapid growth responses.
It is important to note that while the results of our paclobutrazol
experiments are consistent with a model in which auxin stimulates
synthesis of GA, which then contributes to the elongation
response, this may be an oversimplification. GA levels are under
negative feedback regulation in which expression of GA biosyn-
thesis genes is repressed as GA levels increase . Auxin
biosynthesis in turn is regulated in part by PIF4, which is indirectly
activated by GA. Dynamic regulation between these two hormone
pathways is likely to be important for hormone-mediated cell
Auxin Promotes Cell Expansion Independent of Time of
Day in Part through Regulation of PIF-independent
As previously mentioned, several signaling pathways are
important for controlling hypocotyl growth, including light
signaling and the circadian clock, as well as hormone signaling
[37,39,42]. Many of the growth-associated downstream genes in
these pathways are regulated by PIF transcription factors
[37,39,42], recently shown to be required for activation of
transcription downstream of GA signaling [37,39,42]. PIF4 and
PIF5 are two members of the PIF family that are circadian
Figure 4. Auxin regulates a suite of growth-associated genes preceding hypocotyl elongation. Gene Ontology (GO) term enrichment in
the hypocotyl datasets is similar to an IAA-responsive dataset but includes novel categories. Venn diagrams indicating the number of enriched GO
terms in the auxin-induced or -repressed hypocotyl datasets or IAA datasets from the AtGenExpress project  are shown. The top-ranked GO terms
unique to the hypocotyl dataset are shown in the lower set of Venn diagrams. (B) Picloram induced genes are circadian regulated. The top 400
statistically significant picloram induced genes were analyzed using the Phaser tool (http://phaser.cgrb.oregonstate.edu/). Bars represent z-scores for
the enrichment of cycling genes within our gene list compared to all the genes shown to cycle under long day conditions at a given phase of the day.
Phase 0 signifies the start of the day. Asterisks indicate significant enrichment with a p,0.05.
Transcriptional Basis for Auxin-Mediated Growth
PLoS ONE | www.plosone.org7 May 2012 | Volume 7 | Issue 5 | e36210
regulated and for which expression level is correlated with
hypocotyl growth [39,42,77]. A recent study by Nozue et al. 
suggests that PIF5 is a modulator of auxin signaling and that PIF4
and PIF5 regulate auxin sensitivity to control hypocotyl growth.
There are several possible mechanisms by which transcriptional
auxin signaling may promote growth either by feeding into a
PIF4/5-mediated pathway or acting independently. First, auxin
might promote PIF4/5 activity by inducing PIF4/5 transcription
during the day; second, auxin might indirectly promote PIF4/5
activity by stimulating GA synthesis consequently degrading the
DELLA repressors of the PIFs; third, auxin might act indepen-
dently of PIF4/5 and regulate transcription of PIF4/5 targets
during the day; last, auxin might act independently of the PIFs and
regulate PIF4/5-independent growth genes. We addressed the first
Figure 5. Gibberellin biosynthesis is required for hypocotyl auxin response. (A,B,C) Asterisk represents mutants showing a significantly
different response to hormone treatment compared to wildtype or control treatment. A general linear model (glm performed in R using the car
package ) was used to determine significance and main effects for genotype were confirmed using ANOVA type III sums of squares. All
assumptions for GLM were fulfilled. (A)_Paclobutrazol inhibits hypocotyl auxin response. Hypocotyl length of wild-type seedlings grown in short-day
conditions and treated with paclobutrazol at the indicated concentrations (black line) or paclobutrazol plus 5 mM picloram (red line) was measured
following 48 hours of treatment. Error bars indicate standard error. (B) Paclobutrazol-mediated inhibition of hypocotyl elongation is not overcome by
higher auxin concentration. Hypocotyl length of wild-type seedlings treated with IAA (blue line) or picloram (red line) at the indicated concentrations
or IAA and 2.5 mM paclobutrazol (PAC; green line) was measured following 48 hours of treatment. Error bars indicate standard error. (C) RGA protein
degrades in response to auxin treatment in hypocotyls of auxin-treated seedlings. Abundance of RGA-GFP protein in hypocotyl tissues was analyzed
by epifluorescence microscopy over a 0–24 hour time course. Three day-old seedlings were treated for 2, 4, or 24 hours with 50 mM GA3, 5 mM IAA,
5 mM IAA +2.5 mM paclobutrazol, or a solvent control, prior to imaging. (D) A GA biosynthesis mutant is partially auxin-resistant. Hypocotyl length of
wild-type seedlings (Col-0) or the ga20ox1/ox2 mutant  treated with IAA at the indicated concentrations was measured following 48 hours of
auxin treatment. Hypocotyl length on auxin is shown as a percentage of length on control medium. Error bars indicate standard error.
Transcriptional Basis for Auxin-Mediated Growth
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possibility by analyzing our microarray data. We did not detect a
transcriptional response to auxin for the PIF4/5 genes, suggesting
that auxin either enhances residual PIF activity that may be
present during the day, or acts in parallel to promote elongation
independently of these proteins during the day.
We asked whether auxin, GA, and PIF4/5 are required for the
initial growth response to a pulse of auxin using a time course
elongation assay done during the day. A 2-hour auxin treatment
led to an increase in hypocotyl length in wild-type seedlings within
2 hours (Fig. 6A). The response of pif4pif5 mutant seedlings was
indistinguishable from that of wild type, suggesting that this initial
growth response does not require PIF4/5 protein. This result is
not surprising given that PIF4 and PIF5 are rapidly degraded by a
phyb-dependent mechanism and transcriptionally inhibited by the
DELLAs during the day, and so are unlikely to be required for
daytime growth . In contrast, both the ga20ox2 double mutant
and the axr2-1 are completely resistant to auxin in this assay (Fig.
S4B). These results suggest that both auxin and GA are required
for the initial growth response.
To address whether the transcriptional targets of auxin signaling
are also PIF targets, we performed an extensive comparison of our
auxin-responsive cell expansion data set with existing growth-
related microarray datasets. Nozue et al.  describe a series of
global expression analyses in the pif4pif5 mutant to classify sets of
‘‘growth’’ and ‘‘stationary’’ phase genes that are PIF4/5-depen-
dent or -independent. Using the resulting gene lists as well as
datasets obtained using various light conditions in wild type and a
pif1 pif3 pif4 pif5 PIF quadruple mutant (pifq) , we compared
our gene lists to the growth-regulated genes identified in these
selected arrays. For a description of the arrays selected and the
method of comparison see Methods, Table 1 and Table S6. We
compared our auxin-induced and auxin-repressed gene lists to
each array dataset and identified 490 auxin-induced genes and
270 auxin-repressed genes also presented in these growth datasets.
We converted these results into a matrix in which each row
represents an auxin-responsive gene from our list, and each
column represents a microarray condition (Table S6). We then
used hierarchical clustering to generate maps of each matrix. We
divided each map into ‘growth’ and ‘stationary’ sections to reflect
the conditions with which regulation of each gene is associated, as
described by Nozue et al.  (Fig. 6B). We also included a
column of genes associated with cell wall reorganization, ‘CW’
A pattern that emerges from our matrix maps is that many
picloram-induced genes are co-regulated by conditions where
growth is occurring. We found that 46% of our auxin-induced
genes are induced in wild type 2-day-old seedlings grown in the
dark when compared to light-grown seedlings (Fig. 6B column 1),
and 21% are repressed by a 2-day red light treatment that inhibits
hypocotyl elongation (Fig. 6B column 3). Similarly, the overlap
between stationary phase genes and auxin-repressed genes is
greater (the sum of values in columns 9–15 is 348 for 269 genes)
than between stationary phase genes and auxin-induced genes (the
sum of values in columns 9–15 is 191 for 490 genes) (Fig. 6B, left
and right maps, columns 11,12,14,15). Therefore, our auxin-
induced gene list consists at least in part of genes that are
associated with growth, such as ARGOS (AT3G59900) and
(AT5G15580) and LNG2 (AT3G02170)  and several EX-
PANSIN and EXPANSIN-LIKE genes (Table S6) [83,84,85].
The matrix maps highlight a significant overlap between PIF-
regulated genes and auxin targets in elongating hypocotyls (Fig. 6B
columns 3–6). This is consistent with previous results from Nozue et
al.  that show that auxin-regulated genes are overrepresented
among genes differentially expressed between pif4pif5 double
mutant and wildtypeplants. Notsurprisingly, genesinthiscategory
include genes associated with GA pathways including gibberellin
biosynthesis genes GA3OX1 and GA2OX8, the GA repressor RGL1,
PIF3-LIKE2 (AT3G62090) and SOMNUS (AT1G03790), a germi-
nation gene downstream of PIL5 (AT2G20180). Of the 81 genes
defined by Nozue et al.  as upregulated by growth and PIF4- or
PIF5-dependent, 38 are also classified in that study as auxin
regulated. Of these 38, 35 are in our auxin-induced list. Our auxin-
induced list also includes an additional 17 PIF4/5-dependent genes
not classified by Nozue et al. as auxin-responsive .
These findings raise the question of whether auxin regulates PIF
target genes through induction of GA biosynthesis and consequent
PIF activation, through a GA-independent PIF process, or
through a PIF-independent mechanism. We predicted that a set
of PIF4/5-dependent growth-associated genes might be auxin-
regulated in the absence of PIF activity, since the hypocotyl growth
response to the transient auxin treatment during the day did not
require PIF4/5 (Fig. 6A). We tested the response of a subset of
growth-associated genes, including SAUR23 (AT5G18060), IAA2
(AT3G23030) and ARGOS, to auxin using qRT-PCR. We found
that each of these three genes was induced by a 2 hr IAA
treatment in pif4pif5 double mutant seedlings (Fig. 6C). This
suggests that these genes are directly regulated by auxin. This has
been confirmed for IAA2, which is rapidly induced by auxin in the
presence of cyclohexamide . The fact that these three genes
are PIF-dependent in growth promoting-conditions can be
explained by the recent discovery that PIF4/5 directly regulates
auxin biosynthesis at elevated temperature [39,42,77,87,88].
Genes in this category may be direct auxin targets whose
expression in growth-promoting conditions, such as elevated
temperature, is dependent upon PIF regulation of auxin biosyn-
thesis. However, we do not rule out the possibility that such genes
may also be directly regulated by the PIF family in some
conditions. Thus, our results support a growth model in which a
number of important cell expansion-associated genes are common
targets of multiple growth-promoting pathways.
Finally, our analysis also revealed overlap between auxin-
responsive genes and growth-upregulated genes that are PIF4/5-
independent. More than 200 of our auxin-induced genes are in
this category. While this group predictably includes auxin
AT3G23050; IAA5, AT1G15580), genes in the GA pathway
(GAI, AT1G14920; GA20OX2, AT5G51810), ethylene pathway
(ETHYLENE RESPONSE 2, AT3G23150; ETHYLENE RE-
SPONSE SENSOR 1, AT2G40940; ERS2, AT1G04310), and
AT4G36780; BRASSINAZOLE-RESISTANT 1, AT1G75080) are
also present. Additionally, several genes with roles in cell wall
metabolism are present, including XYLOGLUCAN ENDOTRANS-
(AT1G65310) and XTH8 (AT1G11545), CELLULOSE SYN-
THASE-LIKE D3 (AT3G03050), and CELLULOSE SYNTHASE-
INTERACTIVE PROTEIN 1 (AT2G22125). Together, these genes
are candidate direct auxin targets involved in growth and in cross-
talk with other signaling pathways.
Auxin Regulates Additional Candidate Cell Expansion
In ourauxin-responsive genelist, 81genes weidentified asauxin-
induced and 70 genes we identified as auxin-repressed are
interrogated by the NimbleChip but not by the Affymetrix ATH1
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Affymetrix/). These genes are presented in Table S4. Several genes
among these have predicted functions in cell expansion, including
growth , KIDARI (AT1G26945), which promotes shoot
elongation downstream of GA , and PAR2 (AT3G58850), a
transcription factor induced during the shade avoidance response
. Due to a lack of available microarray data, we did not further
we confirmed auxin-responsiveness of PAR2 as well as of CTR1
(AT5G03730) and BRIL (AT1G55610) (new candidate growth
genes that are not represented on the ATH1 chip) inseedlings using
qRT-PCR (Fig. S5).
Our hypocotyl sampling approach enabled us to detect auxin-
responsive growth-associated genes that have not been detected
Figure 6. Auxin promotes hypocotyl elongation through PIF-dependent and –independent pathways. PIF4 and PIF5 are not required for
transient auxin response. Average hypocotyl length of wild-type (Col-0) or pif4pif5 mutant seedlings treated with 5 mM IAA for two hours was
measured each hour for 7 hours. Error bars indicate standard error. Hypocotyl length at each time point is shown as a percentage of length at time 0.
(B) Auxin-responsive genes are associated with growth conditions. Picloram-induced genes are also induced in the dark (column 1, up in WTD) and
during growth (2, upG; 6, upG PIF4/5), and repressed by light (3, down in WTRc; 7, DL) and in the pifq mutant (4, down in pifqR1; 5, down in pifqD).
Picloram-repressed genes are also repressed in the dark (column 9, down in WTD) and upregulated by light (10, upL; 15 up in WTRc), during
stationary phase (11, upS; 13 Nozue upS PIF4/5), and in the pifq mutant (12, up in pifqR1; 14, up in pifqD) (see Methods, Table 1 and Table S6 for
complete description of array conditions shown). CW indicates genes associated with cell wall metabolism (column 8). (C) PIF4/5-dependent genes
are regulated by auxin in seedlings. Wild-type (Col-0) or pif4pif5 mutant 5-day-old seedlings were treated with 5 mM IAA or a solvent control for 2
hours and used for RNA isolation. Expression value of each gene shown, relative to a control gene, was determined by qRT-PCR.
Transcriptional Basis for Auxin-Mediated Growth
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in many whole seedling arrays. It is possible that the large
number of genes in our auxin-responsive lists that were not found
in the MASTA analysis or the comparison with the Nemhauser et
al.  and Stepanova et al.  datasets represent genes that
are auxin-responsive in a specific spatio-temporal pattern that is
masked in experimental designs using diverse tissue homogenates.
Results from this study emphasize the value of tissue-specific
analyses when addressing a particular developmental question.
We have uncovered a large set of auxin-regulated genes that are
expressed inelongating hypocotyls,
biosynthesis enzymes (Fig. 7). Our results suggest that auxin
regulates GA biosynthesis to release DELLA-dependent growth
repression . Genetic analyses confirmed the importance of
auxin-GA cross-talk for a complete hypocotyl growth response, a
process that has also been reported in pea . However, we
also demonstrated that regulation of GA is not the only
mechanism for auxin-stimulated hypocotyl growth and an
independent pathway is required for optimal response. Interest-
ingly, auxin-GA interplay is also involved in tropic hypocotyl
growth, although in these processes GA is required to attenuate
growth through repression of auxin signaling . It will be
important for a complete understanding of hormone-regulated
growth to assign downstream growth genes to specific hormone
pathways or identify mechanisms and conditions in which these
genes are downstream of multiple signaling pathways, as we have
proposed for IAA2, ARGOS, and other genes.
Under normal growth conditions, the circadian clock main-
tains diurnal hypocotyl growth by gating auxin response
primarily through PIF4 and PIF5 [39,41,42]. However, various
stress conditions cause plants to stimulate rapid changes in
growth during the day in order to survive. For example, rapid
flooding causes changes in hormone levels within 1 h of
submergence. Studies in Rumex palustris have revealed the
importance of ethylene, IAA and GA in stimulating stem
elongation following submergence due to rapid flooding .
Our hypocotyl transcriptome analysis was performed under
conditions that mimic a rapid increase in auxin levels during the
day leading to a hypocotyl elongation response. We have
identified many cell elongation genes that are known to be
growth-associated but have not been previously described as
auxin-responsive (Fig. 7). A subset of these genes is described as
being PIF4/5-dependent and we would expect this regulation to
be active during normal hypocotyl growth conditions during the
night when the PIFs are present [39,42]. However, our results
suggest that auxin activates these genes in the absence of PIF4/5
suggesting that auxin promotes hypocotyl growth by an
independent pathway during the day. It will be interesting to
determine how the activities and expression patterns of these
genes are important for the extent and timing of hypocotyl
growth. Among our auxin-responsive gene list are genes involved
in cell wall biogenesis and secretory pathways known to be
important for cell elongation. Using the hypocotyl tissue-specific
approach and the NimbleChip, we have also uncovered
additional hypocotyl growth genes that may also be important
for other cell expansion dependent processes such as petiole
growth. With this study we present a transcriptional framework
for rapid stimulation of hypocotyl elongation during the day,
independent of PIF4 and PIF5, and we provide a genomic basis
for the model that auxin, GA, and the PIFs have overlapping
roles in regulating growth.
Materials and Methods
Arabidopsis thaliana mutants and transgenic lines used in this
study were all in the Columbia (Col-0) ecotype. Mutants msg2-1
[48,96], slr-1 , and pif4-101/pif5-1  were described
previously. tir1-1, afb1-3, afb2-3, afb3-4, afb4-2, afb5-5 and
higher-order combinations among these mutants were described
previously [27,97]. RGA::GFP-RGA (CS16360) was obtained from
the Arabidopsis Biological Resource Center at The Ohio State
University, and the ga20ox1ox2 mutant  was a generous gift
from Peter Hedden. For hormone treatment assays and RNA
isolations, seeds were plated on K6 Murashige-Skoog medium
containing 1% sucrose and 1% agar, and stratified 2–4 days in the
dark at 4uC.
Hypocotyl Growth Assays and Imaging
Seedlings were grown under long day photoperiods (16 h light/
8 h dark) at 23uC unless otherwise indicated, with white light
intensity of ,80 mmol/m2/s. For treatment assays and RNA
isolations, 5-day-old seedlings were transferred to plates containing
the chemical being tested or the solvent control (DMSO was used
for picloram; ethanol was used for IAA, GA3, paclobutrazol and
NPA) for an additional 48 hours unless otherwise stated.
Hypocotyl images were taken using a Nikon SMZ1500 dissecting
scope and all measurements were done using ImageJ software.
Data shown represent an average of at least 10 seedlings per
treatment; error bars represent standard error.
Table 1. Microarray data selected for comparison to auxin-regulated gene sets.
ReferenceGEO ID Conditions analyzed
Leivar et al. 2009GSE17159 wild-type vs. pifq (pif1 pif2 pif3 pif4 pif5 mutant) 2 day dark
wild-type vs. pifq 2 day dark plus 1 hr red light
wild-type 2 day dark treatment vs. wild-type 2 day red light
wild-type seed vs. wild-type 2 day dark
Nozue et al. 2011 GSE21684 UpG PIF4/5 - Genes up in growth phase and PIF4/5 Dependent
UpG - Genes up in stationary phase and PIF4/5 independent
upS PIF4/5 - Genes up in stationary phase and PIF4/5 dependent
upS - Genes up in stationary phase and PIF4/5 independent
Ma et al. 2005 GSE14648 6 day old light grown hypocotyls
6 day old dark grown hypocotyls
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Seedlings used for visualization of RGA-GFP were grown in
long day conditions, and RGA-GFP fluorescence at time zero was
analyzed four hours after subjective dawn in two day-old seedlings.
Seedlings were then submerged in liquid medium (K6 Mura-
shige-Skoog medium containing 1% sucrose) containing the
chemical being tested or the solvent control (ethanol) for an
additional 2–24 hours, and a subset of seedlings was removed from
treatment and imaged at the time points indicated. GFP
fluorescence in pRGA:RGA-GFP was visualized using a Nikon
SMZ1500 dissecting scope.
overlaid with sterilized nylon mesh (110 micron pore size; www.
smallparts.com). Two hours after chamber lights came on, mesh
rafts containing 5-day-old seedlings were transferred to medium
containing 5 mM picloram or an equivalent volume of DMSO for
30 min or two hours. Hypocotyls were dissected over a 30-minute
period and frozen in liquid nitrogen. Tissue samples were collected
over several days and pooled into biological replicates containing
at least 400 hypocotyls. RNA extractions were done using Trizol
reagent (Sigma) followed by additional phenol extraction and
ethanol precipitation steps. mRNA was amplified using the
MessageAmp II aRNA Amplification kit (Ambion) and the
manufacturer’s protocol. Labeled cDNA was prepared from
aRNA using the Superscript ds cDNA synthesis kit (Invitrogen),
Cy3- and Cy5-labeled random nonomers (TriLink) and Klenow
fragment (Promega). Samples representing three biological
replicates were selected for hybridization to the 4-plex NimbleGen
chip at the Center for Genomics and Bioinformatics at Indiana
University. Experiment ‘a’ was hybridized to the NimbleGen 4-
plex chip using dual-color labeling.
Seedlings were grown and treated as
described for experiment ‘a’; however, roughly 700 hypocotyls
were included in each biological replicate to avoid RNA
amplification. RNA extractions were performed using the
Invitrogen PureLink RNA mini Kit. Three biological replicates
were sampled and used for cDNA synthesis and hybridization to
the 12-plex NimbleGen chip according to manufacturer’s instruc-
tions. Experiment ‘b’ was hybridized to the NimbleGen 12-plex
Stratified seeds were plated on medium
chip using single-color labeling. Microarray ‘b’ was carried out at
the GeneChipTMMicroarray Core facility at the University of
California San Diego.
All microarray analysis was done using R (R Development Core
Team (2011), http://www.R-project.org/) and Bioconductor .
annotated based on TAIR10. Annotation packages were built with
pdInfoBuilder using raw data files (.xys) along with a Nimblegen
using oligo in R with this annotation package. Normalized data for
array ‘a’ and ‘b’ were analyzed independently using a linear model
method  performed in the LIMMA package in R. Differentially
expressed genes were chosen based on an Empirical Bayes method
of differentially expressed genes from microarray ‘a’ and ‘b’, Rank
Product method was used due to the difficulty comparing datasets
derived from independent experiments . As shown in Vert et al.
, this method [60,99] often outperforms the linear model when
comparing microarray experiments derived from different labora-
of pre-processed data, eliminating the requirement for normalizing
heterogeneous data together that will often retain ‘lab-effects’ .
The Rank Product method includes fewer assumptions under the
are transformed into ranks allowing for the integration of datasets
from a variety of platforms [59,60]. Genes or splice forms that were
not present on both chips were removed from the analysis.
Upregulated and downregulated gene lists from RankProd were
used for the comparisons described below. Microarray data have
been deposited inNCBI’s Gene Expression Omnibus and are
The MASTA package available from the BAR website (www.
bar.utoronto.ca) was used to compare RankProd-generated lists
with the 7 IAA wild-type treatment arrays included in the MASTA
Figure 7. Model for the transcriptional auxin response preceding hypocotyl elongation. Auxin levels in hypocotyl tissue elevate in
response to growth-promoting conditions or exogenous auxin, which activates transcriptional auxin signaling. Early auxin-responsive genes include
those encoding GA oxidases, cell wall modifying enzymes, and other factors that may contribute directly to cell elongation or regulate expression of
additional growth-promoting genes. These pathways may be reinforced by activity of PIF4 and PIF5, which are liberated from DELLA repression due
to auxin-mediated modulation of GA levels. In growth-promoting conditions, auxin-responsive genes may be PIF-dependent due to PIF regulation of
auxin biosynthesis [39,42,77,87].
Transcriptional Basis for Auxin-Mediated Growth
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package. The IAA root treatment data from Stepanova et al. 
was downloaded from the Gene Expression Omnibus (GEO)
database (www.ncbi.nlm.nih.gov/geo/). CEL files were RMA
normalized using the Affymetrix package and input into
RankProd. Differentially expressed genes with an FDR less than
0.05 were selected and compared to the picloram-responsive gene
lists. For the Nemhauser et al.  comparisons the genes defined
by the authors as auxin-responsive were used.
Microarrays selected for the growth gene comparisons are listed
in Table 1, Tables S6 and S7. CEL files were not available for all
of the arrays selected; picloram-responsive genes identified in this
study were compared with genes defined as differentially expressed
according to the publication associated with the data in GEO.
Matrices were generated with the picloram-induced and –
repressed genes in which each row represents an auxin-responsive
gene and each column represents a treatment condition from the
array being compared. Genes were assigned a value of 1 if defined
as differentially expressed in the associated publication, or a value
of 0 if absent from the data set. The resulting matrix was used to
generate a hierarchical clustered based map in R. Columns were
manually arranged based on conditions where growth is occurring
(growth phase) or inhibited (stationary phase). The middle column
in each map (cw) includes genes that were defined by Jamet et al.
 as being involved in cell wall biogenesis or secretory pathways
likely important for cell wall expansion.
RNA samples collected from hypocotyl and whole seedling
tissue were obtained from tissue frozen in liquid N2 using the
INVITROGEN PureLink RNA minikit. RNA yield and quality
was quantified using the Thermo Scientific NanoDrop 2000.
Equal amounts of RNA were pre-treated with DNase using the
DNA-free Kit (Ambion) according to manufacturer’s instructions
and used to generate cDNA with SuperScript III First-Strand
Synthesis (Invitrogen) with 20-mer oligo(dT) primers. Quantita-
tive RT-PCR was done with SyBR green and the primers listed
in Table S8. Primer pairs were evaluated for specificity and
efficiency using three serial dilutions of cDNA using the
CFX96TMReal-Time PCR Detection System (Biorad). Data
were normalized to the reference gene PP2AA3  according
to the DDCt method . Primers were designed using
QuantPrime . Experiments with hypocotyl or seedling
tissue were done with two biological replicates and three
targets. (A) Microarray analysis of picloram-regulated genes in
hypocotyls and IAA-regulated genes in seedlings or roots identified
common target genes. Venn diagrams of auxin-upregulated genes
in IAA-treated materials (IAA-up) and hypocotyls of picloram-
treated seedlings (Pic-up) are shown. Numbers of genes identified
in common are shown in the overlap sections of each diagram. (B)
Auxin marker genes are picloram-responsive in hypocotyls from
wild-type, but not afb5-5 mutant, seedlings. Hierarchical clustering
result of IAA marker gene expression in hypocotyls of picloram-
treated or control wild-type (Col-0) or afb5-5 (afb5) seedlings, as
determined by analysis of microarray data using ArrayStar, is
shown. (C) IAA marker genes are regulated by picloram in
hypocotyls of picloram-treated seedlings. Wild-type (Col-0) or
afb5-5 mutant seedlings were treated with picloram or a solvent
control for 2 hours and used for hypocotyl dissection and RNA
Picloram and IAA share transcriptional
isolation. Expression value of each gene shown, relative to a
control gene, was determined by qRT-PCR.
in picloram-responsive promoters. Statistical significance of
overrepresentation of each AuxRE-containing sequence element
(p-value) is plotted on the x-axis; the number of promoters
containing the element is plotted on the y-axis. Overrepresented
sequences were identified using ELEMENT .
Auxin response elements are overrepresented
responsive transcription. Overrepresented GO terms and
enrichment scores were identified using GOMiner . Only
GO terms not overrepresented in the AtGenExpress datasets 
GO terms newly associated with auxin-
pendently to regulate hypocotyl elongation. (A) Auxin
signaling mutants are partially restored by treatment with GA3.
Average hypocotyl length of wild-type seedlings or the indicated
mutants grown in long days and treated with 50 mM GA3was
determined following 48 hours of treatment. Statistical significance
was determined using a Tukey HSD post hoc comparison among
the means on the analysis of variance using type III sums of
squares (p,0.05). Error bars indicate standard error. (b)The axr2-1
and ga20ox2 double mutant are deficient in transient auxin
response. Average hypocotyl length of wild-type and mutant
seedlings treated with 5 mM IAA for two hours was measured each
hour for 7 hours. Hypocotyl length at each time point is shown as
a percentage of length at time 0. Error bars indicate standard
GA and auxin act independently and interde-
seedlings. Wild-type seedlings were treated with IAA or a solvent
control for 2 hours and used for RNA isolation. Expression value
of each gene shown, relative to a control gene, was determined by
PIF4/5-independent genes are regulated by auxin in
Microarray experimental design.
Genes auxin-responsive at 30 minutes.
Genes auxin-responsive at 120 minutes.
Newly identified auxin-responsive genes.
GO terms associated with auxin-responsive gene lists.
Genes and microarray datasets presented in Figure 7B,
Genes and microarray datasets presented in Figure 7B,
Primer sequences used for quantitative RT-PCR.
Transcriptional Basis for Auxin-Mediated Growth
PLoS ONE | www.plosone.org13 May 2012 | Volume 7 | Issue 5 | e36210
Colleen Doherty assisted with microarray analysis. Mon-Ray Shao,
Amanda Budiman, Tatiana Manchenkov and Britta Baynes assisted with
plant propagation and provided technical assistance. Stuart Grande and
Zak Gezon assisted with the statistical analysis. Jason Reed, members of the
Estelle lab particularly Luz Irina A. Caldero ´n Villalobos, and anonymous
reviewers provided helpful comments on early drafts of the manuscript.
Conceived and designed the experiments: EJC KG CC ME. Performed the
experiments: EJC KG CC RS AB. Analyzed the data: EJC KG CC RS
ME. Contributed reagents/materials/analysis tools: T-PS. Wrote the
paper: EJC KG CC ME.
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