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The Plant Cell, Vol. 28: 345–366, February 2016, www.plantcell.org ã 2016 American Society of Plant Biologists.

All rights reserved.

LARGE-SCALE BIOLOGY ARTICLE

Time-Series Transcriptomics Reveals That AGAMOUS-LIKE22


Affects Primary Metabolism and Developmental Processes in
Drought-Stressed Arabidopsis OPEN

Ulrike Bechtold,a,1 Christopher A. Penfold,b,2 Dafyd J. Jenkins,b Roxane Legaie,b,3 Jonathan D. Moore,b
Tracy Lawson,a Jack S.A. Matthews,a Silvere R.M. Vialet-Chabrand,a Laura Baxter,b Sunitha Subramaniam,a
Richard Hickman,b,4 Hannah Florance,c,5 Christine Sambles,c Deborah L. Salmon,c Regina Feil,d Laura Bowden,e,6
Claire Hill,e Neil R. Baker,a John E. Lunn,d Bärbel Finkenstädt,f Andrew Mead,e,7 Vicky Buchanan-Wollaston,b,e
Jim Beynon,b,e David A. Rand,b David L. Wild,b Katherine J. Denby,b,e Sascha Ott,b Nicholas Smirnoff,c
and Philip M. Mullineauxa
a School of Biological Sciences, University of Essex, Colchester CO4 3SQ, United Kingdom
b Systems Biology Centre, University of Warwick, Coventry CV4 7AL, United Kingdom
c College of Life and Environmental Sciences, University of Exeter, Exeter EX4 4QD, United Kingdom
d Max Planck Institute of Molecular Plant Physiology, 14476 Potsdam-Golm, Germany
e School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom
f Department of Statistics, University of Warwick, Coventry CV4 7AL, United Kingdom

ORCID IDs: 0000-0003-2320-3890 (U.B.); 0000-0002-5486-0407 (J.D.M.); 0000-0002-7282-8929 (J.S.A.M.); 0000-0003-3287-7547 (L.B.);
0000-0003-3712-1589 (H.F.); 0000-0002-7219-0398 (C.S.); 0000-0002-2575-8278 (D.L.S.); 0000-0002-2198-696X (N.R.B.); 0000-0002-4909-
8235 (A.M.); 0000-0002-2217-3274 (D.A.R.); 0000-0002-7857-6814 (K.J.D.); 0000-0001-5630-5602 (N.S.); 0000-0002-1998-3540 (P.M.M.)

In Arabidopsis thaliana, changes in metabolism and gene expression drive increased drought tolerance and initiate diverse drought
avoidance and escape responses. To address regulatory processes that link these responses, we set out to identify genes that
govern early responses to drought. To do this, a high-resolution time series transcriptomics data set was produced, coupled with
detailed physiological and metabolic analyses of plants subjected to a slow transition from well-watered to drought conditions. A
total of 1815 drought-responsive differentially expressed genes were identified. The early changes in gene expression coincided with
a drop in carbon assimilation, and only in the late stages with an increase in foliar abscisic acid content. To identify gene regulatory
networks (GRNs) mediating the transition between the early and late stages of drought, we used Bayesian network modeling of
differentially expressed transcription factor (TF) genes. This approach identified AGAMOUS-LIKE22 (AGL22), as key hub gene in a TF
GRN. It has previously been shown that AGL22 is involved in the transition from vegetative state to flowering but here we show that
AGL22 expression influences steady state photosynthetic rates and lifetime water use. This suggests that AGL22 uniquely regulates
a transcriptional network during drought stress, linking changes in primary metabolism and the initiation of stress responses.

INTRODUCTION change (Easterling et al., 2000; Christensen et al., 2007; Seager et al.,
2007; Famiglietti and Rodell, 2013). Reduced water availability leads
Water limitation in agriculture is poised to intensify in the coming to drought stress, which is a major constraint on the physiology,
decades due to urbanization, industrialization, depletion of aquifers, growth, development, and productivity of plants (Boyer, 1970, 1982;
and increasingly erratic rainfall patterns exacerbated by climate Lobell and Field, 2007; Roberts and Schlenker, 2009; Skirycz et al.,
1 Address correspondence to ubech@essex.ac.uk. 2010; Lobell et al., 2011; Verelst et al., 2013). Therefore, un-
2 Current address: Wellcome Trust/Cancer Research UK, Gurdon In- derstanding the mechanisms of drought response in plants is es-
stitute, Tennis Court Road, Cambridge CB2 1QN, UK. sential for the improvement of plant performance under water-limiting
3 Current address: QFAB Bioinformatics, The University of Queensland,
conditions and has been the subject of many investigations over the
St. Lucia QLD 4072, Australia.
4 Current address: Institute of Environmental Biology, University of years (Shinozaki and Yamaguchi-Shinozaki, 1997, 2007; Chaves
Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands. et al., 2009; Nakashima et al., 2009; Pinheiro and Chaves, 2011).
5 Current address: Systems and Synthetic Biology, CH Waddington
Water deficit responses are complex and require stress sensing and
Building, Max Born Crescent, Edinburgh EH9 3BF, UK. signaling to adjust plant growth, maintain water status through os-
6 Current address: Science and Advice for Scottish Agriculture, Rodding-

law Road, Edinburgh EH12 9FJ, UK. moregulation, prevent water loss through decreases in stomatal
7 Current address: Computational and Systems Biology, Rothamsted conductance, and activate detoxification processes (Passioura,
Research, Harpenden, Hertfordshire AL5 2JQ, UK. 1996; Chaves et al., 2003; Pinheiro and Chaves, 2011). An important
The author responsible for distribution of materials integral to the findings consideration is that even a slight reduction in water availability can
presented in this article in accordance with the policy described in the Instruc-
tions for Authors (www.plantcell.org) is: Ulrike Bechtold (ubech@essex.ac.uk).
elicit stomatal closure and a reduction in CO2 assimilation and in
OPEN
Articles can be viewed online without a subscription. combination with the diversion of resources toward drought defense
www.plantcell.org/cgi/doi/10.1105/tpc.15.00910 mechanisms will affect plant productivity (Chaves et al., 2003).
346 The Plant Cell

Plants have adopted different strategies to respond to water use of a single or a small number of time points and different types of
limitation, such as drought escape through early flowering and re- experimental conditions lead to very different outcomes. One
ducing the size of plants to increase water use efficiency or drought consequence of this is that very little is known about the early events
avoidance through enhanced soil moisture capture or reduced in the perception of drought stress signals (Ueguchi et al., 2001;
transpiration (Ludlow, 1989; Blum, 2005; Aguirrezabal et al., 2006; Wohlbach et al., 2008; Pinheiro and Chaves, 2011).
Franks, 2011). In this context, the influence of drought on plant To address the above issues, we set out to gain detailed in-
development and growth through its effects on developmental formation on the processes that occur during the transition from well-
processes such as germination, seedling growth, and leaf de- watered to drought conditions, in which the intensity of the stress
velopment has been studied extensively in the past decade (van der becomes gradually greater. We monitored the physiological and
Weele et al., 2000; Finkelstein et al., 2002; Xiong et al., 2006; Yaish metabolic status of plants through a progressive drought experiment
et al., 2011). Optimal timing of flowering and inflorescence de- and mapped onto these data the temporal responses of the tran-
velopment are important traits essential in determining plant yield, scriptome. Our intention was to use the highly resolved transcrip-
and these can vary greatly in response to water limitation (Eckhart tional profiling data to construct gene regulatory networks (GRNs)
et al., 2004; Franke et al., 2006; Su et al., 2013; Ma et al., 2014). using dynamic Bayesian network modeling (Beal et al., 2005; Breeze
At the cellular level, plants respond to drought with changes in et al., 2011; Penfold and Wild, 2011) with the aim of identifying
gene expression and protein and metabolite abundances (Charlton regulatory genes functional during drought perception and signaling.
et al., 2008; Harb et al., 2010; Wilkins et al., 2010; Baerenfaller et al., The goal was to link early physiological and metabolic drought
2012), which are part of defense mechanisms and detoxification avoidance responses with later drought escape and/or tolerance
processes (Shinozaki and Yamaguchi-Shinozaki, 2007; Begcy responses (Claeys and Inzé, 2013). This initially required testing of
et al., 2011; Ozfidan et al., 2012). Recent progress in genomics, the network modeling to evaluate the capability of these approaches
transcriptomics, and bioinformatics has paved the way for to identify genes important in the regulation of drought responses.
dissecting drought-response mechanisms and has enabled the This was achieved by selecting a highly connected candidate gene,
targeted manipulation of drought-responsive genes in plants. For AGAMOUS-LIKE22 (AGL22; also known as SHORT VEGETATIVE
example, the overexpression of a number of genes that code for PROTEIN), from the GRNs. AGL22 has an established function in
transcription factors (TFs) leads to drought resistance (Sakuma plant development (Gregis et al., 2013; Méndez-Vigo et al., 2013), but
et al., 2006; Nelson et al., 2007; Chen et al., 2008; Quan et al., 2010; in this study, it was shown to play a thus far undiscovered role in the
Tang et al., 2012). critical early stages of the plant’s response to drought. These results
In many studies to identify genes important in the regulation of demonstrated the potential value of experimental strategies that
drought responses, the effects of water limitation at the tran- combine time-series transcriptomics data with dynamic modeling as
scriptional level have been analyzed by exposing plants to severe a means of identifying stress-responsive genes.
dehydration. This involves treatments such as cutting and air
drying leaves and/or roots or induction of osmotic shock through
RESULTS
the application of highly concentrated osmotica such as poly-
ethylene glycol or mannitol (Kreps et al., 2002; Seki et al., 2002; Time-series experiments were performed analyzing physiological,
Kawaguchi et al., 2004; Kilian et al., 2007; Weston et al., 2008; metabolic, and transcriptional changes in Arabidopsis thaliana to
Fujita et al., 2009; Abdeen et al., 2010; Deyholos, 2010; Mizoguchi reveal the chronology of plant responses to drought stress. A pro-
et al., 2010). These experiments have substantially increased our gressive slow-drying experiment starting at 95% relative gravimetric
knowledge of molecular responses under severe drought stress, soil water content (rSWC) and drying down to 17% rSWC was per-
but they do not always reflect physiological conditions experi- formed on 5-week-old Arabidopsis plants (Figure 1). To determine the
enced by drought-stressed soil-grown plants (Bechtold et al., severity of the stress, daily measurements of relative leaf water content
2010, 2013; Harb et al., 2010; Wilkins et al., 2010; Lawlor, 2013; (RWC; Figure 1A) and leaf water potential were also performed (Figure
Zhang et al., 2014). Physiological responses such as stomatal 1B). During the experiment, the average rSWC loss was ;10% per
conductance, photosynthetic performance, and metabolic day, but RWC was maintained throughout the progressive drying
changes are usually not measured during the progression of the period until the point of wilting at 17% rSWC (Figure 1A).
drought stress, and the varied nature of the stress induction
treatments makes comparative analysis between experiments Maximum Photosynthetic Capacity Responds Similarly in
problematic. Slow developing soil water deficits have different Well-Watered and Drought-Stressed Plants during
physiological consequences than those induced by rapid tissue a Progressive Drought Experiment
dehydration and therefore possibly utilize different gene networks
(Chaves et al., 2003, 2009; Pinheiro and Chaves, 2011). Stomatal conductance (gs) and photosynthetic carbon assimilation
This inconsistency among experiments was first noted in a meta- (A) were measured daily on well-watered and drought-stressed
analysis of microarray experiments comparing air drying, soil drying, plants through the progressive drought treatment (Figures 1C and
and mannitol treatments (Bray, 2004). This analysis found very few 1D). Stomatal conductance declined at ;60% rSWC (day 5; Figure
differentially expressed genes (DEGs) common to all treatments 1C), which was followed by a decline in carbon assimilation at ;45%
(Bray, 2004). Consequently, recent experiments have focused on rSWC (day 7), indicating that stomatal diffusional limitations affected
soil-grown plants (Harb et al., 2010; Wilkins et al., 2010; Zhang et al., carbon assimilation (Figure 1D). Plant growth evaluated as rosette
2014). From these studies, an overall integrative picture of the fresh weight and rosette area ceased at ;40% rSWC (Supplemental
temporal responses to drought is emerging slowly, and it is clear that Figures 1A to 1C).
Temporal Drought Dynamics in Arabidopsis 347

Figure 1. Plant Responses during a Progressive Drought Experiment.

(A) RWC (open triangles) and rSWC (closed triangles) during a 13-d drying period. The data represent the mean (n = 6; 6SE).
(B) Leaf water potential in well-watered (open circles) and drought-stressed (closed circles) plants during a 13-d drying period. The data represent the mean
(n = 5; 6SE).
(C) Stomatal conductance of well-watered (open circles) and drought-stressed (closed circles) plants, measured at the prevailing growth conditions (see
Methods). The data represent the mean (n = 6; 6SE).
(D) Carbon assimilation of well-watered (open circles) and drought-stressed (closed circles) plants, measured at the prevailing growth conditions (see
Methods). The data represent the mean (n = 6; 6SE).

The light and CO2-saturated maximum photosynthetic rate (Amax), samples harvested at early (day 2, ;80% rSWC), mid (day 7, ;45%
maximum rate of carboxylation (VCmax), and the rate of ribulose-1,5- rSWC), and late (day 13, 17% rSWC) stages of the drought stress.
bisphosphate (RuBP) regeneration (Jmax) showed no difference This analysis showed that the majority of the metabolome was
between well-watered and drought-stressed plants (Figure 2A). In unchanged throughout most of the drought treatment, and distinct
addition,maximum andoperatingefficienciesofphotosystemII(Fv/Fm, clustering between well-watered and drought-stressed samples
Fv9/Fm9, and Fq9/Fm9; Baker, 2008) showed no change during the emerged only by the final day of drought stress (17% rSWC) (Figure
drought period (Figure 2B; Supplemental Figure 1D), suggesting 3A). Leaf development was a major factor for sample separation, with
that the overall primary metabolic capacity was maintained as days clustered more closely together than treatments (Figure 3A).
drought conditions progressed. The sequential changes in photo- Targeted metabolite analysis was performed to determine the
synthetic physiology, relative water content, and leaf water poten- foliar levels of 102 stress-associated compounds (Supplemental
tial suggest that these conditions allowed us to capture the transition Data Sets 1 to 3), which also revealed a mainly late response for
between early physiological changes and later stress responses. many of these stress-associated metabolites (Supplemental Data
Sets 1 to 3). Often these changes were limited to the last two to
Metabolite Profiling Indicates the Stable Nature of Primary three time points (between ;30 and 17% rSWC; Supplemental
Metabolism during Drought Stress Figures 2 and 3). For example, metabolites indicative of drought
stress increased only during the late stages of the dehydration
The decline in stomatal conductance and carbon assimilation led us period (Figures 3B and 3C). There was a significant increase in
to perform metabolite analysis to evaluate changes in primary and abscisic acid (ABA) levels during the last four time points (Xiong
secondary metabolism (Figure 3). Untargeted liquid chromatography- et al., 2002; Figure 3B), while proline, a drought stress-responsive
mass spectrometry metabolite profiling was performed on compatible solute in vascular plants (Sperdouli and Moustakas,
348 The Plant Cell

Figure 2. Potential Photosynthesis in Response to Drought.

(A) A/Ci curves were performed under saturating light conditions of 1000 µmol m22 s21 at six selected time points throughout the drying period, and potential
photosynthesis was calculated, including light and [CO2]-saturated net CO2 assimilation (Amax), maximum rate of RuBP regeneration (Jmax), and maximum
rate of carboxylation (VCmax).
(B) Maximum and operating quantum efficiencies of photosystem II (Fv/Fm, Fq9/Fm9, and Fv9/Fm9). The data represent the mean (n = 3; 6SE).

2012), accumulated to significant levels only during the last two associated with drought stress (Taji et al., 2002; Xiong et al., 2002;
time points (Figure 3B). Additionally, the accumulation of sec- Sperdouli and Moustakas, 2012) only became evident during the
ondary metabolites commonly associated with stress responses last three to four time points.
such as anthocyanins and flavonols were altered during the late
stages of the drought response (Sperdouli and Moustakas, 2012; Transcriptomics Analysis on a Single Leaf Identifies 1815
Supplemental Data Sets 1 and 3 and Supplemental Figure 3). DEGs during Progressive Drought Stress
Oligosaccharides/disaccharides associated with osmotic pro-
tection during drought and osmotic stresses (galactinol and raf- Transcriptome profiling was performed on leaf 7 to integrate the
finose; Taji et al., 2002) significantly accumulated during the last 4 complex physiological and metabolic responses with changes at
days of the drought response (Figure 3C; Supplemental Data the gene expression level. Leaf 7 was fully expanded at the time of
Set 1). In conclusion, leaf metabolism remained largely stable the experiment (Supplemental Figure 1C) and was chosen because
during the first 9 d of the experiment, while changes previously a detailed temporal transcriptome analysis of leaf development was
Temporal Drought Dynamics in Arabidopsis 349

Figure 3. Metabolite Levels during Progressive Drought.


(A) Liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry metabolite profiling of Arabidopsis leaves under well-
watered (W) and progressive soil drought (D) conditions. Leaf extracts were analyzed in negative and positive ionization modes. The heat maps show the
normalized abundances of all detected chemical features. Samples and chemical features were clustered using a Pearson distance measure and the Ward
clustering algorithm (Supplemental Data Set 18).
(B) Relative concentrations of ABA (black bars) and proline (gray bars).
(C) Relative concentrations of galactinol (black bars) and raffinose (gray bars). The data represent the mean of the ratio (n = 4; 6SE; *P < 0.05 or **P < 0.01).

available (Breeze et al., 2011). Single leaf 7 samples for transcriptome each gene (Supplemental Data Set 6; Figure 4A). In our experi-
analysis were taken each day at the midpoint of the light period. RNA ment, leaf water potential correlated significantly with rSWC
from four leaf samples per treatment and time point was hybridized (Figure 4B) as well as the number of DEGs (Figure 4C) and showed
on CATMA v4 arrays (Sclep et al., 2007; see Methods). An adapted a weaker correlation with carbon assimilation (Figure 4D). The
MAANOVA (microarray analysis of variance) method was used to biggest drop in leaf water potential occurred between 40% (day 8)
analyze the data for each comparison (Wu et al., 2003; Churchill, and ;30% (day 9) rSWC, which coincided with the biggest in-
2004; Breeze et al., 2011; Windram et al., 2012). This generated crease in DEGs (Figures 4A and 4C), potentially indicating a shift
a single normalized expression value for each gene. A Gaussian from mild to severe drought stress.
process two-sample test (GP2S; Stegle et al., 2010) was used to To evaluate the transcriptome data set in the context of other
identify DEGs. Choosing a Bayes factor value (likelihood of drought experiments, we also compared our data set to two soil-
differential expression) of >6 resulted in a total of 1815 DEGs based drought studies in Arabidopsis. The first experiment per-
(Supplemental Data Set 4). The upregulated group of genes showed formed by Harb et al. (2010) comprised a microarray comparison of
on overrepresentation of Gene Ontology (GO) terms related to leaf samples under moderate drought stress (maintaining soil water
carbohydrate biosynthesis, flavonoid, and secondary metabolic content at 30% of field capacity) and progressive drought stress at
processes, while downregulated genes were enriched in protein the prewilting (;15% field capacity) and wilting (;10% field ca-
translation, cell wall-associated processes, pigment biosynthesis, pacity) stages. In the progressive drought stress treatment, 3005
and chloroplast associated processes (Supplemental Data Set 5). genes responded >2- and <0.5-fold, in comparison to 441 genes for
GO terms related to stress, dehydration, and hormonal regu- the moderate drought treatments (Supplemental Data Set 7). The
lation, including ABA, were not enriched in the complete data set. second study analyzed samples at a loss of 25% soil water of field
This result suggested that the overall progressive drought ex- capacity measured at 6-h intervals across a 24-h period (Wilkins
periment was not a severe dehydration stress response, as in- et al., 2010) and identified 570 genes that responded across a 24-h
dicated by the maintenance of primary metabolic capacity (Figure interval (hereafter called diel; Supplemental Data Set 7). A general
2) as well as the late responses of stress-associated metabolites overview of overlapping genes between the different experiments
(Figure 3). Leaf water potential has been used as a measure of the showed that only 30 genes were common to all 4 treatments (Figure
progression and effect of drought stress on plants (Zhang et al., 5A). Among the overlapping genes were known stress-responsive,
2014). We estimated the cumulative number of DEGs at each time ABA-responsive, and secondary metabolism genes, which pre-
point by determining the time of first differential expression for dominantly responded during the latter half of the drought
350 The Plant Cell

Figure 4. The Relationship between Stress Severity and Differential Gene Expression.

(A) Number of genes for which the first differential expression was observed at each time point, indicating a late transcriptional response.
(B) Correlation between leaf water potential and rSWC.
(C) Correlation between leaf water potential and number of differentially expressed genes.
(D) Correlation between carbon assimilation and leaf water potential. Line represents the linear regression; r2 and P values are given.

experiment (Supplemental Data Set 8). While there were common including CHALCONE SYNTHASE, FLAVONOL SYNTHASE1,
elements in all four treatments, 63% of the DEGs in our data set LEUCOANTHOCYANIDIN DIOXYGENASE, PRODUCTION
were unique to the time series (Figure 5A) and were potentially OF ANTHOCYANIN PIGMENT1, and ANTHOCYANINLESS2
related to the adjustments to early and moderate drought stress. (Supplemental Figure 5 and Supplemental Data Set 9). This co-
incided with increased accumulation of flavonol and anthocyanin
Slow Soil Drying Induces a Senescence Response in Leaf 7 (from day 11; Supplemental Data Set 1 and Supplemental Figure 3)
and suggested that the plants had entered a severe stress phase
To assess developmental changes in leaf 7 during drought stress, we (Vanderauwera et al., 2005). Therefore, it was concluded that slow
compared the drought time series to an Arabidopsis leaf 7 senescence soil drying induces senescence in leaf 7 but only at the point of severe
time-series data set (Breeze et al., 2011; Supplemental Data Set 7). In drought stress. By contrast, early responses to soil drying (days 1 to
all, 842 genes overlapped with the senescence data set (p-hyper, 7) were mostly unique to the drought time series. Importantly for later
4.4E-135;Figure 5B), of which 83% respondedbetween days 8 and13 considerations, the induction of leaf senescence in response to
(40 to 17% rSWC; Supplemental Data Set 9). The overlap contained drought did not affect flowering time (Supplemental Figure 4B).
genes associated with oxidation/reduction-related processes, pig-
ment biosynthesis, and primary metabolism, which were pre- Temporal Clustering Reveals Coregulated Groups of Genes,
dominantly downregulated during drought stress (Figures 5C and 5D; but Does Not Reveal Specific Regulatory Mechanisms
Supplemental Data Set 10). The induction of senescence-related
processes during drought stress is a known phenomenon (Munné- The cumulative number of DEGs at each time point (Supplemental
Bosch and Alegre, 2004) and was further confirmed by a significant Data Set 6) confirmed that major gene expression changes oc-
overlapof genes between the publisheddrought andsenescencedata curred late during the drought experiment as the number of genes
sets (Supplemental Data Set 7 and Supplemental Figure 4A). that showed first differential expression at each time point was
In addition, changes in the expression of secondary metabolism highest between days 8 and 11 (Figure 4A). A total of 336 genes
genes were observed in the drought time series (Figure 5E), responded during the first half of the experiment, while the majority
Temporal Drought Dynamics in Arabidopsis 351

Figure 5. Comparative Meta-Analysis with Publicly Available Data Sets and MapMan Analysis (Thimm et al., 2004) of Primary and Secondary Metabolism
Pathways.

(A) Comparative meta-analysis of the 1815 DEGs with publicly available drought data sets. The Venn diagram shows the overlap of time series DEGs with
those responsive to moderate (mDr; Harb et al., 2010) or progressive drought (pDr; Harb et al., 2010) and a moderate drought at different times of day (diel;
Wilkins et al., 2010).
(B) Comparative meta-analysis of the 1815 DEGs with a publicly available leaf 7 senescence time-series data set (Breeze et al., 2011). The Venn diagram
shows the overlap of drought and senescence DEGs.
(C) Overview of antioxidant, photosynthesis. and photorespiration-related gene expression at two different time points (95% rSWC and 17% rSWC).
(D) Overview of oxidation/reduction-related gene expression at two different time points (95% rSWC and 17% rSWC).
(E) Overview of secondary metabolism-related gene expression at two different time points (95% rSWC and 17% rSWC). All MapMan diagrams show gene
expression data in leaf 7, where blue indicates increased and yellow indicates decreased gene expression according to the scale. Each square represents
a single gene within the pathways.

of genes (1479) showed first differential expression during the responding at different times throughout the progressive drought.
latter half of the experiment. A Euclidean distance matrix of the Early upregulated genes were associated with carbohydrate and
average expression values of the four biological replicates for each glycoside biosynthetic processes, general carbohydrate metabolic
data set was generated and used in hierarchical cluster analysis. processes, and inorganic cation transporter activities (Table 1). The
The resulting dendrogram showed that samples clustered in re- late upregulated genes encompassed flavonoid and secondary
lation to treatments and within the drought treatment into early and metabolite biosynthesis, while the late downregulated genes were
late stage responses (Figure 6A). Dividing the data set into early involved in translation, pigment biosynthesis, photosynthesis-
(days 1 to 7, ;95 to 45% rSWC) and late responses (days 8 to 13, related processes, and oxidation/reduction processes (Table 1). To
;40 to 17% rSWC) also revealed functional groups of genes gain insight into the molecular factors underlying this temporal
352 The Plant Cell

separation of samples, hierarchical cluster analysis of the DEGs was many significant edges. The M-VBSSM consensus model indicated
performed using SplineCluster (Heard et al., 2005) on the basis of that developmental genes played an important role in the regulation
gene expression patterns in the drought stressed leaf only. Using of drought, as four out of the top 10 highly connected TFs were
a prior precision value of 0.01, the 1815 genes were divided into 28 associated with the regulation of plant development (Supplemental
clusters (Figures 6B and 7; Supplemental Data Set 11). The first 14 Data Set 15). In addition, two of the top 10 TFs were among the group
clusters showed an overall upregulation and contained 1149 genes, of early responding TFs (Supplemental Data Sets 14 and 15). While
while the last 14 downregulated clusters contained 667 genes the consensus model was useful for the initial ranking of genes, it did
(Figure 7; Supplemental Data Set 11). The 28 clusters were hy- not represent a causal model, instead representing a type of aver-
pothesized to represent groups of genes that are coregulated during aging of many different network models. For this reason, we opted to
the drought experiment. To explore potential regulatory mecha- model a smaller selection of genes, which was advantageous for two
nisms of genes clustered in specific temporal expression profiles, we reasons. First, the final model was smaller and sparser, and therefore
analyzed each individual cluster for over- and underrepresentation of more interpretable, and second, the resultant interactions could be
GO terms in the Biological Process and Molecular Function and for interpreted causally.
overrepresentation of known TF binding motifs in promoters Therefore, the top 10 “hub” TFs with the highest frequency of
(Supplemental Data Sets 12 and 13 and Supplemental Figure 6). A occurrence and 90 random transcription factors were chosen for
few clusters showed enrichment of expected GO terms in response analysis with the VBSSM package (Beal et al., 2005; Supplemental
to drought, such as flavonoid biosynthesis, photosynthesis, pigment Data Set 16). As part of this selection, we also chose early and late
biosynthesis, and response to stress (Supplemental Data Set 12; responding TFs. In total, 19 early responding TFs (days 1 to 7) were
Figure 7). However, most clusters did not show any enrichment or included in the model to establish a potential transcriptional link
underrepresentation of GO terms (Supplemental Data Set 12; Figure between early and late responses (Supplemental Data Set 16).
7), and only two clusters (cluster 1 and 9) contained the ABRE binding The resultant model placed the early-responding TF gene
motif, known to perceive ABA-mediated drought and osmotic stress AGL22 at the center of a 25-TF gene network (Figure 8A). AGL22
signals (Supplemental Figure 6 and Supplemental Data Set 13; Kim was differentially expressed in the drought experiment beginning
et al., 2011). at day 5 (Supplemental Figure 7A). Fifteen TF genes that were part
of the GRN were initially analyzed by qPCR to check for differential
Bayesian State Space Modeling Identifies Genes That Link expression under drought in wild-type plants (20% rSWC). All
Drought Responses to Plant Development through genes were differentially expressed in line with the levels observed
Regulating Transcriptional Networks in the microarray experiment, except for PACLOBUTRAZOL
RESISTANCE1, BASIC HELIX-LOOP-HELIX038, and AUXIN
The data presented so far did not allow us to draw conclusions about RESPONSE FACTOR1 (Figure 8B; Supplemental Figure 7B).
a specific regulatory mechanism across the whole time series but AGL22 is known to affect flowering time and plant development
suggested that early and late responses to a decline in soil water (Supplemental Figure 7C; Gregis et al., 2013; Méndez-Vigo et al.,
content are regulated differently. Large-scale transcriptional 2013); however, it was not regulated during leaf senescence
reprogramming and metabolic adjustment did not play a dominant (Supplemental Data Set 9), suggesting that AGL22 uniquely
role during the early phases of the dehydration response. Never- regulated a transcriptional network during drought stress. This
theless, among the 337 DEGs responding between 95 and 40% was further explored by performing VBSSM using the control time-
rSWC were 33 TF genes (Supplemental Data Set 14), of which series data set of the same transcription factor genes. If AGL22
25% had a functional annotation of development (GO:0032502; was a hub gene in the control time series, it would suggest a role in
Supplemental Data Set 14). This suggested that a reprogramming of developmental reprogramming over the 13-d experimental period
developmental processes during drought stress may have occurred regardless of drought stress. The Bayesian modeling resulted in
in response to the observed early physiological changes (closure of a number of fragmented connections of a small number of genes
stomata and reduction in leaf water potential). We reasoned that the (Supplemental Figure 8A), suggesting these genes were not part of
expression of early TF genes must therefore play a role in orches- a gene regulatory network under well-watered conditions. The
trating this acclimation to drought stress. highly connected genes in the drought model, AGL22 and
Metropolis Variational Bayesian State Space Modeling RAP2.12, did not feature, not even as peripheral genes
(M-VBSSM; Penfold, University of Warwick; Supplemental (Supplemental Figure 8A).
Methods) was initially performed on 176 differentially expressed Therefore, the early responding TF AGL22 was chosen for
TFs in the data set (Supplemental Data Set 14). This approach further analysis to establish how far an unbiased modeling ap-
selects subsets of TFs to generate a network, which is continually proach can be used to identify genes capable of influencing plant
updated by probabilistic replacement of TFs to generate a series drought phenotypes and downstream network connections. Two
of networks and provides a consensus model based on the independent T-DNA insertion lines were isolated (see Methods),
marginal likelihood (Supplemental Methods). From the consensus both of which were confirmed knockout mutants for AGL22
model, we could calculate the occurrence of each TF (the number (Figure 8C; Supplemental Figure 8B). The mutants were sub-
of times a particular TF appeared over all models) and a count of sequently analyzed for their effect on the AGL22-centered net-
the number of downstream connections each TF had across all work interactions after drought stress. Eight out of 15 TF genes
models at a particular z-score, in this case indicating a 95% differentially expressed under drought conditions exhibited al-
confidence threshold (Supplemental Data Set 15). TFs that scored tered gene expression in at least one of the agl22 mutants
well in both rankings were deemed highly connected hubs with compared with the wild type (Figure 8D). This implied that ;50% of
Temporal Drought Dynamics in Arabidopsis 353

Figure 6. Temporal Clustering of 1815 Differentially Expressed Genes.


(A) Dendrogram of the hierarchical clustering using a Euclidian distance divides the data set into early and late responses for both the control (white circles)
and drought (gray circles) samples.
(B) Heat map of the SplineCluster analysis of the 1815 DEGs on differentially expressed genes (drought samples only) across the time series using
normalized and averaged data (Supplemental Data Set 11). The heat map demonstrates expression profiles for genes in each cluster with red representing
high expression and green representing low expression.

the network connections were regulated at least partially through plant chambers at 90, 74, and 25% rSWC (see Methods). The in-
AGL22, but also suggested the possibility of redundancy within creased water loss was primarily driven by a greater rosette area
the network (Figures 8A and 8D). Four late TFs (WRKY20, GIS, (Figure 9A), despite a significant reduction in stomatal conductance
DREB1A, and FBH3) were substantially downregulated in both (Figure 9C). Accordingly, light saturated carbon assimilation (Asat) was
agl22 mutants after drought, suggesting that these TFs were significantly reduced throughout the drying period already under well-
primarily regulated through AGL22 (Figure 8D). watered conditions (Figure 9D), leading to a significant reduction in
total aboveground biomass (Supplemental Figure 10A). Flowering
Both agl22 Mutants Had Early-Flowering, Fast-Drying time remained constant between well-watered and drought treat-
Drought Escape Phenotypes ments in both agl22 mutants, indicating that drought stress con-
ditions did not affect flowering time in the agl22 mutants (Figure 9E).
It is important to note that due to the early-flowering phenotype of
the agl22 mutants (Supplemental Figure 7C), drought stress was
begun at day 22 after sowing, when there was no visible differ- DISCUSSION
ences in rosette leaf number (Supplemental Figures 9A to 9C), but
with a significant increase in rosette area (Figure 9A). Chronology of the Drought Response Suggests Early
We observed an increased drying rate in both agl22 mutants, Adjustments in Stomatal Conductance and Carbon
suggesting increased water use (Figure 9B). To determine if this Assimilation Are Followed by Changes in ABA and
was due to developmental or metabolic changes, we performed Transcriptional Reprogramming
light response curve measurements of photosynthesis at specific
times throughout the drying period (Supplemental Figure 9D). Due to Responses to drought are complex and depend on the type and
the small leaf and rosette size, light curves were measured in whole strength of the drought stress imposed (Harb et al., 2010; Wilkins
354 The Plant Cell

Table 1. Functional Categorization of Early (95 to 45% rSWC) and Late (40 to 17% rSWC) Responsive Genes

Category GO Term Biological Process/Molecular Function Fold P Value

Early upregulated GO:0034637 Cellular carbohydrate biosynthetic process 6.9 0.003


Early upregulated GO:0006812 Cation transport 4.3 0.014
Early upregulated GO:0044262 Cellular carbohydrate metabolic process 3.7 0.018
Early upregulated GO:0022890 Inorganic cation transmembrane transporter activity 5.1 0.0432
Late upregulated GO:0009812 Flavonoid metabolic process 7.1 1.74E-05
Late upregulated GO:0009813 Flavonoid biosynthetic process 7.1 5.82E-05
Late upregulated GO:0019748 Secondary metabolic process 2.3 0.004
Late upregulated GO:0009699 Phenylpropanoid biosynthetic process 3.8 0.006
Late upregulated GO:0016051 Carbohydrate biosynthetic process 2.7 0.011
Late upregulated GO:0006519 Cellular amino acid and derivative metabolic process 2.0 0.011
Late upregulated GO:0019438 Aromatic compound biosynthetic process 2.9 0.021
Late upregulated GO:0042398 Cellular amino acid derivative biosynthetic process 2.9 0.044
Late downregulated GO:0006412 Translation 2.1 4.96E-05
Late downregulated GO:0015995 Chlorophyll biosynthetic process 11.4 6.50E-04
Late downregulated GO:0044085 Cellular component biogenesis 2.4 0.002
Late downregulated GO:0042254 Ribosome biogenesis 3.6 0.003
Late downregulated GO:0006334 Nucleosome assembly 6.9 0.003
Late downregulated GO:0015979 Photosynthesis 3.9 0.011
Late downregulated GO:0033014 Tetrapyrrole biosynthetic process 7.7 0.014
Late downregulated GO:0055114 Oxidation reduction 1.8 0.02
P value was adjusted using the Benjamini-Hochberg method (Benjamini and Hochberg, 1995). “Category” indicates the timing and direction of change
in gene expression across the time series. “Fold” indicates the fold enrichment.

et al., 2010; Zhang et al., 2014). The slow steady drought experiment predominantly adjustments to stomatal conductance leading to
performed in this study allowed us to investigate the full range of restricted CO2 diffusion for photosynthetic carbon assimilation
temporal physiological, transcriptional, and metabolic responses in (Boyer, 1970; Passioura, 1996; Figure 1C) with some associated
a single fully expanded Arabidopsis leaf. Additionally, by measuring transcriptional changes accounting for 17% of the 1815 DEGs
leaf water potential (Figure 1B) and RWC (Figure 1A), we were able to (Supplemental Data Sets 1 to 12; Table 1). By contrast, late re-
monitor the progression and degree of drought stress in relation to sponses (from 40% rSWC) encompassed hormonal (ABA), tran-
the physiological, transcriptome, and metabolome changes. The scriptional, and major metabolic changes associated with
decline in carbon assimilation at ;45% rSWC was primarily driven by senescence (Supplemental Data Sets 1 to 12; Table 1). These later
reduced stomatal conductance limiting CO2 diffusion. There were no responses corresponded with the many different phenological
underlying metabolic constraints, as photosynthetic capacity was and physiological changes observed in other studies, including
unaffected by the drought treatment (Figure 2; Supplemental Figure impaired photosynthesis, increased solute accumulation, and
1D), and metabolite profiles remained unchanged throughout the ma- growth arrest (Boyer, 1970; Passioura, 1996). At the cellular level,
jority of the drying period (Figure 3A; Supplemental Figures 2 and 3), soluble sugars, oligosaccharides, antioxidants, and proline ac-
suggesting that Arabidopsis Col-0 is a drought-tolerant ecotype. cumulation are known to enhance the tolerance to drought stress
Stomatal limitation as the primary factor in reducing photosyn- by acting as osmolytes or as reactive oxygen species scavengers,
thesis under mild drought conditions has been observed in other especially hydroxyl radicals (Smirnoff and Cumbes, 1989; Cuin
studies; however, severe dehydration stress is believed to lead to and Shabala, 2007). The accumulation of these compounds
metabolic constraints, associated with RuBP availability (Flexas and during the latter stages of the drought period (Figures 3B and 3C),
Medrano, 2002). We did not observe a reduction in the maximum together with the increase in secondary metabolites, such as
capacity for carbon assimilation (Amax), Rubisco carboxylation flavonoids (Supplemental Data Set 1 and Supplemental Figures 3
(VCmax), and RuBP regeneration (Jmax; Figure 2A). In addition, max- and 4), suggests a role in the defense against severe drought
imum and operating efficiencies of photosystem II photochemistry stress (Tattini et al., 2004; Lei et al., 2006; Xiao et al., 2007; Xu et al.,
(Figure 2B), photochemical and nonphotochemical quenching 2008; Harb et al., 2010; Fini et al., 2011; Page et al., 2012). In
(Supplemental Figure 1D), were maintained throughout the drying conclusion, this time series covers all phases during a progressive
period despite a decline in carbon assimilation (Figures 1D), in- drought stress and therefore provides the opportunity to study
dicating very little stress on photosystem II. This suggested that an different stages of stress responses in greater detail than has been
alternative electron sink, most likely photorespiration (reviewed in previously possible (Supplemental Figure 10B).
Chaves et al., 2003; Lawson et al., 2014), must have been operating
under drought stress, and increased gene expression in the pho- Transcriptional Regulation of Drought Stress Responses
torespiratory pathway supports this notion (Figure 5C).
In general, two distinct phases in response to progressive soil At the gene expression level, a slowly developing soil water deficit
drying could be discerned (Figures 6 and 7). Early responses were is different from rapid tissue dehydration. From this study and in
Temporal Drought Dynamics in Arabidopsis 355

Figure 7. Descriptions of the 28 Clusters Derived from SplineCluster.

The blue line indicates the mean expression profile for each of the 28 clusters. Individual genes present in each cluster are available in Supplemental Data Set
11. The red line indicates the switch from early (95 to 45% rSW; days 1 to 7) to late (40 to 17% rSWC; days 8 to 13). Selected enriched GO terms (Supplemental
Data Set 12) are indicated on each cluster.

comparison with two other drought experiments (Harb et al., 2010; Previous studies have often focused on the identification of
Wilkins et al., 2010), it is clear that different genes respond depending genes coding for TF classes responding to terminal or severe
on the nature of the drought stress applied (Figure 5A). Only few drought stress, including BASIC LEUCINE ZIPPER (bZIPs, e.g.,
genes overlapped, with the majority of genes, responding in the final ABA-responsive element binding protein/ABRE binding factor),
4 d of the experiment (Supplemental Data Set 8). AP2/EREBP (e.g., DREB/CBF), NAC transcription factors (NAM,
356 The Plant Cell

Figure 8. Constructing and Evaluating a TF Regulatory Network.

(A) Gene regulatory network generated using VBSSM with the drought time-series data (threshold z-score = 1.65). The nodes highlighted in red were
upregulated during drought stress including the central hub gene, AGL22. Nodes highlighted in green were genes downregulated during drought stress.
Blue nodes signify genes that were not regulated by AGL22 as predicted from the model (see [D]). All red, green, and blue nodes were selected for evaluation
after drought stress;
(B) Relative gene expression of selected genes under drought conditions (17% rSWC). Gene expression was analyzed by qPCR. The numbers are expressed
as fold changes of drought over control (n = 5 6 SE). Significance of the fold changes are indicated by either *P < 0.05 or **P < 0.01. For gene and primer list, see
Supplemental Data Set 16 and Supplemental Table 1.
(C) Relative expression levels of AGL22 in two knockout lines, agl22-3 and agl22-4, compared with the wild type determined by qPCR. Significance of the
fold changes are indicated **P < 0.01.
(D) Relative gene expression profiles of 16 genes predicted to be regulated by AGL22 under drought stress in agl22-3 (black bars) and agl22-4 (gray bars)
compared with the wild type. The data represent the mean (n = 7; 6SE), and significance of the fold changes are indicated by either *P < 0.05 or **P < 0.01.
Asterisks located centrally indicate both mutants are significantly different to the wild type, while asterisks located over one mutant indicate significance for
the specific mutant.

ATAF1-2, CUP-SHAPED COTYLEDON2), CCAAT binding (e.g., responses (Supplemental Data Set 14). Similar classes of TF
NUCLEAR FACTOR Y), and ZINC-FINGER (e.g., C2H2 zinc finger genes have also been shown to respond to drought stress in
protein) families (Umezawa et al., 2004; Bartels and Sukar, 2005; Medicago truncatula where 8% of the responding genes coding
Karaba et al., 2007; Li et al., 2008; Licausi et al., 2010; Jensen et al., for TFs responded late throughout the drying period (Zhang et al.,
2013). The majority of TF genes in our study also responded 2014). By contrast, among the early-responding TF genes (95 to
relatively late in the drought period (from 40% rSWC), especially 45% rSWC; Supplemental Data Set 14), ;25% were linked to
those associated with ABA, dehydration, and oxidative stress plant development, indicating that early physiological changes
Temporal Drought Dynamics in Arabidopsis 357

Figure 9. Stress and Plant Growth Phenotypes of agl22 Mutants.


Due to the early-flowering phenotype of both agl22 mutant alleles, drought stress was begun at 22 d after sowing.
(A) Rosette area (cm2) of Col-0 (light gray), agl22-3 (black), and agl22-4 (dark gray) plants at different soil water contents (n = 5). The asterisk indicates
significant difference compared with the wild type at P < 0.05.
(B) Rate of water loss in agl22-3 and agl22-4 plants compared with the wild type averaged over 13 d of water withdrawal (n = 10). The asterisk indicates
significant difference compared with the wild type at P < 0.05.
(C) Stomatal conductance (Gs) at different soil water contents in Co-0 (light gray), agl22-3 (black), and agl22-4 (dark gray) (n = 5). The asterisk indicates
significant difference compared with the wild type at P < 0.05.
(D) Light-saturated carbon assimilation (Asat) at different soil water contents in Col-0 (light gray), agl22-3 (black), and agl22-4 (dark gray; n = 5). The asterisk
indicates significant difference compared with the wild type at P < 0.05.
(E) Days to flowering in well-watered (light gray) and drought-stressed (black) plants (n = 10). Plants were grown under short-day conditions as described in
Methods. At 5 weeks, plants were subjected to progressive drought stress. When 17% rSWC was reached, plants were rewatered and flowering time was
recorded as days after sowing. Control plants were maintained well watered. The asterisk indicates significant difference compared with the wild type at P <
0.05.
358 The Plant Cell

may influence lifetime traits before the initiation of acute stress quantitative trait locus mapping, VBSSM also managed to establish
defense and senescence responses, highlighting the balancing some valid network interactions from time-series transcriptomics
act between the need to grow and to induce effective stress data, potentially allowing for a temporal reconstruction of events and
tolerance mechanisms (Claeys and Inzé, 2013). biological processes occurring during progressive drought stress.

Dynamic Bayesian Network Modeling Identifies Genes That A Flowering Time Gene Influences Water Use and
Regulate Plant Development Photosynthesis under Well-Watered and Drought
Stress Conditions
Analysis of promoter binding sites (Supplemental Data Set 13 and
Supplemental Figure 6) did not indicate specific regulatory net- Both agl22 mutants exhibited elevated water loss and rapid de-
works or mechanisms during the early events. We therefore as- velopment already under nonstress conditions (Figures 9A and
sessed the use of a high-throughput gene expression approach 9B). This could imply a trade-off between drought avoidance and
coupled with dynamic Bayesian network modeling to identify escape in environments where drought shortens the growing
genes associated with the regulation of early drought responses. season (Franks, 2011). Selecting for early flowering may be
To make sense of large high-throughput data sets, network in- beneficial for plant survival but not necessarily for achieving high
ference algorithms were developed, which are capable of es- biomass (Supplemental Figure 10A), which suggests that drought
tablishing regulatory interactions among genes (Bansal et al., survival and the ability to maintain biomass under sustained water-
2007). A number of different inference algorithms were used to limiting conditions depend on different mechanisms (Skirycz et al.,
successfully reconstruct known gene regulatory networks to 2011).
validate these approaches (Cantone et al., 2009; Penfold and Wild, The agl22 mutants exhibited 36 and 46% reductions in the
2011). VBSSM is such an algorithm developed specifically for steady state light-saturated photosynthetic rate under well-
highly resolved temporal gene expression data sets, with the aim watered conditions (Figure 9D), which appeared to be partly as-
of identifying genes that are the key regulators in a given system sociated with reduced stomatal conductance (Figure 9C). However,
(Beal et al., 2005). A recent comparison of modeling algorithms during drought stress, the photosynthetic rate in both agl22
used to infer GRNs has shown that VBSSM is competitive with mutants was reduced by only 11 and 13%, suggesting that both
network reconstructions based on experimental data (Penfold and agl22 mutants were able to maintain substantial photosynthetic
Wild, 2011; Windram et al., 2014; Penfold and Buchanan-Wollaston, rates (Figure 9D). This is supported by the fact that agl22 mutants
2014). Due to the limited number of experimental observations also maintained rosette growth throughout the drying period in
compared with the much greater number of differentially expressed comparison to wild-type plants (Figure 9A), and although total
genes, the system is inherently underdetermined (Penfold and aboveground biomass was significantly reduced in both mutant
Buchanan-Wollaston, 2014), and previous experience suggested alleles (Supplemental Figure 10A), biomass distribution shifted
that for these kinds of data sets VBSSM can model around 100 from vegetative growth to reproductive growth (Supplemental
genes (Breeze et al., 2011). However, this type of approach can Figure 10C). The complex links between plant growth, primary
introduce a bias during the process of gene selection, while the metabolism, and flowering time in Arabidopsis are highlighted in
M-VBSSM approach (see Methods) generally avoids this gene a recent article where increased plant growth was positively as-
selection bias (Supplemental Methods). We therefore opted to use sociated with early-flowering phenotypes (El-Lithy et al., 2010),
both M-VBSSM and VBSSM to identify key drought-regulatory which may explain the larger rosette area observed prior to
genes and drought phenotypes associated with those genes. flowering in 30-d-old agl22 mutant plants compared with the wild
The initial emergence of several development-associated type (Figure 9A; Supplemental Figure 9B). In addition, starch/
transcription factors from the M-VBSSM approach (Supplemental carbohydrate status and metabolite levels have been linked to
Data Set 15) and the subsequent selection of developmental and rosette growth (Meyer et al., 2007; Sulpice et al., 2009), as well as
nondevelopmental TFs for VBSSM (Supplemental Data Set 16) development and flowering time (Zhou et al., 1998; Moore et al.,
confirmed the flowering time regulator AGL22 as a hub gene in the 2003; Funck et al., 2012). Both photosynthesis and flowering are
drought response. Importantly, we did not observe any in- regulated by the light environment and are clearly linked via the
volvement of AGL22 in leaf senescence (Supplemental Data Set 9 carbohydrate status (Zhou et al., 1998; Moore et al., 2003; Funck
and Supplemental Figure 8A), suggesting that the gene plays et al., 2012), connecting primary metabolism with plant growth and
a unique role in drought stress responses. development. However, little is known about the link between
The connection between flowering time and drought in Arabi- photosynthetic performance and flowering time especially in
dopsis has been established independently in a number of studies, in flowering time mutants, and at this point, it is unclear why the agl22
which carbon isotope discrimination (Farquhar et al., 1982, 1989) mutants exhibited a substantial reduction in photosynthesis al-
quantitative trait loci colocated with known developmental/flowering ready under well-watered conditions.
time loci (Hausmann et al., 2005; Masle et al., 2005; Juenger et al., Furthermore, the observations regarding AGL22 reinforces that
2005; McKay et al., 2008). The identification of a flowering time gene water use in relation to overall plant productivity requires a balance
and subsequent verification of its influence on plant water use, of developmental and physiological processes to successfully
photosynthesis, and phenology (discussed below) suggest that this complete a lifecycle in the prevailing climatic conditions. AGL22
type of dynamic modeling can provide an important means of was identified from moderately drought-stressed plants, which
discovering genes that will produce phenotypes associated with also suggests that not all drought-responsive genes may work in
lifetime water use and plant development. However, unlike all water deficit scenarios. This may especially be the case for
Temporal Drought Dynamics in Arabidopsis 359

those genes that have been selected under terminal or severe was determined as the slope of the decline in relative soil water content,
drought conditions (Hu et al., 2006; Nelson et al., 2007; Xiao et al., measured daily throughout the drying period.
2009). It is important to note that none of the TF genes selected as
key regulators of the drought response in earlier studies (Hu et al., RNA Extractions, Labeling, Microarray Hybridization, and Analysis
2006; Nelson et al., 2007; Xiao et al., 2009) was a hub in the TF GRN
Total RNA was extracted, labeled, and hybridized to CATMA v4 arrays
(Figure 8A), although many of these TF genes were differentially (Sclep et al., 2007) as previously described (Breeze et al., 2011; Windram
expressed during drought stress (Supplemental Data Set 14). This et al., 2012). The experimental design for the drought time-series hy-
is supported by the notion that many genes identified with a role in bridization is shown in Supplemental Figure 11.
stress tolerance under severe stress conditions seem to have little Arrays were hybridized and washed as described (Windram et al., 2012).
effect on plant growth in mild drought conditions (Skirycz et al., Arrays were scanned on a 428 Affymetrix scanner at wavelengths of 532 nm
2011). for Cy3 and 635 nm for Cy5. Cy3 and Cy5 scans for each slide were
Interestingly, two targets of AGL22, DREB1A and FBH3 (Figure combined and processed in ImaGene version 8.0 (BioDiscovery) to extract
8D), have previously been shown to be involved in the regulation of raw intensity and background corrected data values for each spot on the
array. The data have been deposited in the Gene Expression Omnibus
abiotic stress responses. Overexpression of DREB1A leads to
under accession number GSE65046.
drought, salt, and freezing tolerance (Kasuga et al., 1999, 2004),
An adaptation of the MAANOVA package (Wu et al., 2003) was used to
while FBH3 has been shown to regulate stomatal opening analyze the extracted microarray data as described by Breeze et al. (2011)
(Takahashi et al., 2013) and functions in ABA signaling in response and Windram et al. (2012), using a mixed-model analysis. The MAANOVA
to osmotic stress (Yoshida et al., 2015). Both genes were late- fitted model considered dye and array slide as random variables, and time
responding targets with few network connections (Figure 8A; point, treatment, and biological replicate as fixed variables. The model
Supplemental Data Set 15). The model therefore may allow us to allowed assessment of the main effect of treatment, the main effect of time
predict the role of AGL22 during drought stress and provide point, the interaction between these factors, and the nested effect of bi-
a potential link between mild/moderate and severe drought re- ological replicate. Predicted means were calculated for each gene in each
sponses. of the 112 combinations of treatment, time point, and biological replicate
and for each of the 28 combinations of treatment and time point, averaged
This study demonstrates that network inference incorporating
across biological replicates.
highly resolved time-series transcriptomics data is able to predict
TF networks and identify genes with regulatory importance during
drought stress. Moreover, by focusing on the transition from early Differential Gene Expression Analysis
physiological changes to drought stress responses, we were able Genes were ranked based on their Gaussian process two-sample (GP2S)
to identify AGL22 as a gene associated with lifetime water use. Bayes factor differentially expressed score, a cutoff of $6 gave 2496
Consequently, VBSSM as a gene discovery tool promotes the differentially expressed genes. Genes identified in the F-test as being
selection of unknown, yet highly connected genes for further differentially expressed that were not in the GP2S list of 2496 genes were
phenotypic evaluation. added manually. The expression profiles of the genes ranked 1800 to 3150
were then plotted and assigned visually as differentially expressed or not.
This resulted in a false positive rate of 23.3% for this group, and a final cutoff
METHODS of 6 for the GP2S was chosen, duplicates were removed, and a list of 1934
differentially expressed genes was produced. Removal of probes and
Plant Material, Plant Growth, and Drought Stress genes with no annotation in TAIR9 left a list of 1815 unique differentially
expressed genes. The time at which genes first became differentially
Arabidopsis thaliana plants (Col-0, agl22-3 [SALK_141674], and agl22-4 expressed (TOFDE) was subsequently determined using the GP2S time-
[SAIL_583_C08]) were obtained from the European Arabidopsis Stock local method (Stegle et al., 2010).
Centre and were grown under a 8:16-h light:dark cycle at 23°C, 60%
relative humidity, and light intensity of 150 µmol m22 s21, using a mixture of
Promoter Analysis
cold and warm white fluorescent tubes. Arabidopsis seed was stratified for
3 d in 0.1% agarose at 4°C before individual seeds were sown onto a soil Publically available position-specific scoring matrices (PSSMs) were
mix (Scotts Levington’s F2+S compost:fine grade vermiculite in a ratio of collected from the PLACE and JASPAR databases (Higo et al., 1999;
6:1). For the drought time-course experiment, pots (7 3 7 3 9 cm) were filled Sandelin et al., 2004). PSSMs were clustered by similarity, and a repre-
with the same amount of soil mix. Control pots, to determine 100 and 0% sentative of each cluster was chosen for screening. Promoter regions
soil water content, were set up at the same time. Plants were transferred corresponding to 200 bp upstream of the transcription start site were
into individual pots 2 weeks after the sowing date and were kept well retrieved from the Ensembl Plants sequence database (release 50). For any
watered until the beginning of the drying episode at 5 weeks after sowing. given PSSM and promoter, we scanned the sequence and computed
Half the plants were maintained under well-watered conditions, while for a matrix similarity score (Kel et al., 2003) at each position on both strands. P
the remaining half, water was withdrawn and pot weight was determined values for each score were computed from a score distribution obtained by
daily. Relative soil water content was calculated for each day and pots were applying the PSSM to randomly generated sequences. We took the top k
left to dry until 17% rSWC was reached. Five-week-old plants were sat- nonoverlapping hits and performed the binomial test (pbinom function in R
urated in water to reach 95% rSWC, and watering was stopped in the Stats package) for the occurrence of k sites with observed P values within
treatment plants until ;17% rSWC was reached. The control plants were a sequence of length 200 bp. The parameter k is optimized within the range
maintained under well-watered conditions at ;95% rSWC. Due to the 1 to 5 for minimum binomial P value to allow detection of binding sites
early-flowering phenotype of both agl22 mutant alleles, drought experi- without a fixed threshold per binding site. To determine the presence or
ments were performed on 22-d-old plants to ensure the experiments were absence of a PSSM in a promoter, in each case the promoters were sorted
performed at similar rosette developmental stages and prior to the onset of by binomial P value, and we applied a cutoff to select the top 2000. For each
flowering, as indicated in Supplemental Figures 9B and 9C. The drying rate PSSM, its frequency in promoters of each cluster was compared with its
360 The Plant Cell

occurrence in all promoters in the genome. Motif enrichment was calcu- A/Ci Curves (Maximum Photosynthetic Rates)
lated using the hypergeometric distribution (see statistical analysis). For
motif enrichment analyses P values # 1e-5 were considered significant, to Five weeks after emergence, (A) and (gs) were measured on leaf 7, using an
allow for multiple testing. infrared gas exchange system (PP Systems). The response of A to changes in
the intercellular CO2 concentration (Ci) was measured under a saturating PFD,
provided by a combination of red and white LEDs (PP Systems). In addition,
RT-PCR and qPCR
the response of A to changes in PFD from saturating to subsaturating levels
Leaves were harvested and frozen in liquid nitrogen. Total RNA was was measured using the same light source at the current atmospheric CO2
extracted from a pool of three plants using Tri-reagent (Sigma-Aldrich) concentration (390 µmol mol21). All gas analysis was made at a leaf tem-
according to the manufacturer’s instructions. A minimum of five replicates perature of 20 (61)°C and a vapor pressure deficit of 1 (60.2) kPa. Plants were
of whole plants from separate experiments was performed for mutant sampled between 1 and 4 h after the beginning of the photoperiod. For each
analysis and drought treatments. For cDNA synthesis for real-time qPCR leaf, steady state rates of A and gs at current atmospheric [CO2] were recorded
and RT-PCR, 1 µg total RNA was treated with RNase-free DNase (Ambion) at the beginning of each measurement. The A/Ci parameters, VCmax (maximum
according to the manufacturer’s instructions and reverse transcribed as RuBP-saturated rate of carboxylation in vivo), Amax (light and CO2 saturated
previously described (Ball et al., 2004). qPCR-PCR was performed using rate of carbon assimilation in vivo), and Jmax (maximum in vivo rate of electron
a SYBR green fluorescence-based assay as described previously transport contributing to RuBP regeneration) were calculated by fitting
(Bechtold et al., 2010, 2013). Gene-specific cDNA amounts were calcu- equations described by Farquhar et al. (1980) with subsequent modifications
lated from threshold cycle (Ct) values and expressed relative to controls described by McMurtrie and Wang (1993).
and normalized with respect to ACTIN and CYCLOPHILIN cDNA according
to Gruber et al. (2001). RT-PCR was performed to amplify the full-length
AGL22 gene on both agl22 mutant alleles. The primers used for qPCR and
Chlorophyll Fluorescence Imaging
RT-PCR are given in Supplemental Table 1.
Plants were analyzed at various stages of the progressive drought stress
Physiological Measurements using the dark (Fv/Fm)- and light (Fv9/Fm9, Fq9/Fm9, and Fq9/Fv9)-adapted
chlorophyll a fluorescence parameters, using a chlorophyll fluorescence
Relative Water Content imaging instrument (Fluorimager; Barbagallo et al., 2003).
Whole rosettes of five plants were harvested each day throughout the
drying period. The RWC of the leaf was calculated using the formula: rLWC
(%) = (FW 2 DW)/(SW 2 DW) 3 100, where FW is the actual rosette weight Light Response Curves Using Whole-Plant Chambers
at the day of harvest, SW is the fully saturated rosette weight, and DW is the
dry weight of the rosette. A/Q response curves were measured using whole-plant gas exchange system
developed at the University of Essex, with a heliospectra LED light source
(Heliospectra). Input air was maintained at a relative humidity of 50 to 60%, air
temperature of 22°C, and CO2 concentration of 400 mmol mol21, matching
Leaf Water Potential that of the growth conditions. Plants were initially stabilized for 30 min at
saturating irradiance 800 µmol m22 s21, after which PPFD was reduced in nine
The leaf water potential was measured via the Scholander pressure bomb
steps (Supplemental Figure 9D), with assimilation (A) and stomatal conduc-
technique (Scholander et al., 1964) using a SKPM1400 plant moisture
tance (gs) being recorded at each new PPFD level.
system (Skye Instruments). Leaf water potential was measured daily
throughout the drying period on both control and drought-stressed plants
according to the manufacturer’s instructions. Metabolite and Hormone Analysis

During the experimental period, two leaves per plant were harvested every
day, for a total of six plants per treatment. Samples were frozen in liquid ni-
Plant Development and Biomass Measurements trogen, freeze-dried overnight, and stored at room temperature in darkness
until extraction. Primary metabolites were extracted from frozen tissue with
Arabidopsis development was assessed using the scale developed by
chloroform-methanol as described by Lunn et al. (2006). T6P, other phos-
Boyes et al. (2001). Once the final flower had opened, watering was ceased
phorylated intermediates, and organic acids were measured by high-
and plants were bagged and left to dry out before harvesting. At harvesting,
performance anion-exchange chromatography coupled to tandem mass
rosettes, stalks, and seeds were separated. The seed weight and dry
spectrometry as described by Lunn et al. (2006). Trehalose was measured
weight of rosettes and stalks/pods were determined (Bechtold et al., 2010).
enzymatically with fluorometric detection as described by Carillo et al. (2013).
At least 10 plants per line and watering regime were measured.
For sugars, amino acids, hormones, and secondary metabolites, freeze-
dried leaf powder (10 mg) was extracted in 0.8 mL methanol containing 1%
Photosynthesis Measurements
acetic acid. After centrifugation (10 min at 16,100g, 4°C), the samples were
Photosynthetic Rate (Snapshot Measurements) filtered through a 0.2-µm PVDF syringe filter (Chromacol). For nontargeted
Liquid chromatography quadrupole time-of-flight mass spectrometry
Instantaneous measurements of net CO2 uptake rate (A) and stomatal metabolite profiling, 5 mL extract was injected onto a Zorbax StableBond
conductance to water (gs) were made on leaf 7, using an open gas ex- C18 1.8 mm, 2.1 3 100 mm (QToF) reversed-phase analytical column
change system (PP Systems). Leaves were placed in the cuvette at ambient (Agilent Technologies). Chromatography and mass spectrometry con-
CO2 concentration (Ca) of 400 µmol mol21, leaf temperature was main- ditions were described by Page et al. (2012). Peaks were extracted and
tained at 22 6 2°C, vapor pressure deficit was ;1 kPa, and irradiance was aligned using XCMS (Smith et al., 2006), and statistical analysis and data
set to growth conditions (150 µmol m22 s21). A reading was recorded every visualization were performed with MetaboAnalyst 2.0 (Xia et al., 2015). For
3 min when the IRGA conditions had stabilized (;1.5 min), but before the liquid chromatography-tandem mass spectrometry analysis of hormones,
leaf had a response to the new environment (Parsons et al., 1997). 10 mg freeze-dried leaf powder was extracted in 0.8 mL 10% methanol + 1%
Temporal Drought Dynamics in Arabidopsis 361

acetic acid containing deuterated standards (Forcat et al., 2008). VBSSM and M-VBSSM
Secondary metabolites and hormones were analyzed with an Agilent
Significant numbers of genes can be differentially expressed in response to
6420B triple quadrupole (QQQ) mass spectrometer (Agilent Technologies)
environmental stress, which, given the limited number of experimental
coupled to a 1200 series Rapid Resolution HPLC system. Two microliters
measurements, means that network models are often unidentifiable (Penfold
of sample extract was loaded onto a Zorbax Eclipse Plus C18 (3.5 mm,
and Buchanan-Wollaston,2014;Windram et al., 2014, and references therein).
2.1 3 150 mm) for amino acids, sugars, and hormones, respectively. The
Furthermore, the interpretation of large, densely connected networks can
following gradient was used: 0 min to 0% B; 1 min to 0% B; 5 min to 20% B;
often be difficult, and any hypothesis we extract from them can therefore be
20 min to 100% B; 25 min to 100% B; 27 min to 0% B; 7 min post-time. QQQ
ambiguous. One solution is to select a more limited number of genes to model,
source conditions were as follows: gas temperature 350°C, drying gas flow
either based upon prior knowledge, heuristic approaches, or random se-
rate 9 liters min21, nebulizer pressure 35 psig, and capillary voltage 4 kV.
lection. The reduced number of genes means network inference approaches
The polarity, fragmentor voltage, and collision energies were optimized for
can be applied such as the VBSSM of Beal et al. (2005). Within the VBSSM the
each compound. The multiple reaction monitoring modes used for com-
expression of the genes can be written in the form:
pound identification are shown in Supplemental Data Set 17, and data are
reported as peak areas. Flavonoid identification was based on previous yt f½BC þ Dyt 2 1 ;
tandem mass spectrometry identification of flavonoids in Arabidopsis
(Tohge et al., 2005; Stobiecki et al., 2006). For sugar and polyol analysis, where the term ½BC þ Dij captures all information about how gene j reg-
5 mL of sample extract was loaded onto an XBridge amide HILIC column ulates gene i. Rather than infer a point estimate for each interaction
(particle size 3.5 µm, 2.1 mm i.d. 3150mm; Waters)with a constantflow rate of ½BC þ Dij , Beal et al. (2005) infer a posterior distribution and use standard
0.3 mL min21 and a column temperature of 35°C for the duration. Mobile Z-statistics to assess the statistical significance. However, the pre-
phases comprised water:acetonitrile with 0.1% ammonia (mobile phase A selection of genes described above may result in some bias. Here, we
was 90% acetonitrile, and B was 10% acetonitrile with 5 mM ammonium chose to additionally build a network model around a particular gene of
formate). Sugars (5 µL) were separated using the following gradient: 0 to 17
interest, using random selection via a Metropolis algorithm. At each step in
min, 0 to 54% B; 17 to 19 min, 54% B; 19 to 20 min, 54 to 0% B, with a 10 min
the Metropolis algorithm, a Bayesian state space model is fitted to the time-
reequilibration time. The QQQ was operated in negative ion mode. Electro-
series gene expression profiles for the selected genes, and the marginal
spray ionization source conditions were as follows: gas temperature, 350°C;
drying gas flow rate, 9 liters min21; nebulizer pressure, 35 psig; and capillary likelihood or “model evidence” used as the selection criteria. In this way, we
voltage, 4 kV. Data were acquired in selected ion monitoring mode with a dwell can infer small network models around each gene that we are interested in
time of 50 ms. The fragmentor voltage was 50 V for all sugars. The sugars were (for full details, see Supplemental Methods and Supplemental Figure 12).
quantified by reference to standards (Supplemental Data Set 17). Amino acids For the drought data, a total of 176 transcription factors were differ-
were separated with a ZIC-HILIC column (150 3 2.1 cm, 3.5-µm particle size; entially expressed (Supplemental Data Set 14). We therefore used the
Merck SeQuant). Sample (2 µL) was injected into the column with a flow rate of Metropolis model selection to systematically build a network of 88 genes
0.25 mL min21. Mobile phases comprised of water:acetonitrile with 0.1% around each of the 176 genes in turn. Within the Metropolis selection, each
formic acid (mobile phase A was 95% acetonitrile and B was 5% acetonitrile of the 176 network models was run for 2000 iterations in the MCMC chain,
with 5 mM ammonium acetate). Compounds were separated using the fol- by which point the marginal likelihood was seen to be plateau and the
lowing gradient: 0 to 10 min, 5 to 50% B, 10 to 15 min, 50-90% B, 15 to 20 min, algorithm was terminated. The 176 networks at step 2000 were then
90% B, 20 to 25 min, 90 to 5% B, with an 11-min reequilibration time. The QQQ combined to create a meta-network, which was used to compile summary
was operated in positive ion mode and electrospray ionization source con- statistics, such as the number of times a particular gene was found in each of
ditions were as follows: gas temperature, 350°C; drying gas flow rate, 9 liters those 176 network models or the number of downstream connections
min21; nebulizer pressure, 35 psig; and capillary voltage, 4 kV. Data were a particular gene had over those 176 models. Ranked lists of genes can be
acquired in multiple reaction monitoring mode with a dwell time of 50 ms. found in Supplemental Data Set 13. Of the top 10 genes with the highest
Amino acids were quantified by multiple reaction monitoring (Supplemental number of downstream connections, four were annotated as being de-
Data Set 17) and data are reported as peak areas (Supplemental Data Set 18). velopmental in nature, suggesting a link between drought response and
developmental programs. A selection of 99 random differentially expressed
transcription factors including the top 10 highly connected genes were se-
Analysis of Publicly Available Microarray Data Sets
lected from the larger pool of 176 TFs that were DE during the drought stress
Publicly available microarray data sets from different experiments in which (Supplemental Data Set 16). Both the control and drought time series for these
Arabidopsis was subjected to drought and senescence (Harb et al., 2010; genes were normalized to have zero-mean and unit variance and sub-
Wilkins et al., 2010; Breeze et al., 2011) were located in supplementary files sequently modeled using the VBSSM of Beal et al. (2005) using a z-score of
of already published articles and were compared with the drought time- 1.65 to select the control and drought-specific networks, and a final VBSSM
series data sets using VENNY (Oliveros, 2007). Hypergeometric dis- model (Beal et al., 2005) was fitted to the gene expression for AGL22.
tributions were calculated for different overlaps using the phyper function in
R version 3.0.2. A cutoff of -p(log) of 5 was chosen as highly a significant Statistical Analysis
overlap between two or more data sets.
Statistical analyses were performed using SPSS version 19.0. Parameter
differences between the wild type and agl22 mutants were determined
Analysis of GO
using one-way ANOVA with appropriate post-hoc analysis. Tukey’s HSD
GO annotation analysis was performed using DAVID version 6.7 (Huang test was used if variances of means were homogenous, and Games Howell
et al., 2009), BINGO (Maere et al., 2005), and Agrigo (Du et al., 2010) with the test, if variances were not homogenous. The SE of the calculated ratios of
GO_Biological_Process category, as described by Ashburner et al. (2000). fold differences for metabolite and gene expression data and errors of in-
Overrepresented GO_Biological_Process and GO_Molecular_Function dividual means were combined “in quadrature” as the final ratio was
categories were identified using a hypergeometric test with a significance a combination of the error of the two different means of the control and drought
threshold of 0.05 after Benjamini-Hochberg correction (Benjamini and stress samples. Correlations were estimated among drying rate and flowering
Hochberg, 1995) or Bonferroni correction (Holm, 1979) with the whole time as the standard Pearson product-moment correlation between the
annotated genome as the reference set. genotype means. Hypergeometric distributions were analyzed using the
362 The Plant Cell

phyper function in the R stats package. Generally, P values # 1e-5 were Supplemental Data Set 10. Gene Ontology analysis of genes over-
considered significant to allow for multiple testing. The metabolite data were lapping in the drought and senescence time series.
analyzed by between subjects two-way ANOVA for time series data and
Supplemental Data Set 11. Hierarchical clustering divides the 1815
probability values with false discovery rate multiple testing correction are
genes into 28 clusters.
tabulated. For secondary compounds, amino acids, and sugars, compounds
not detected in >50% of samples were discarded and remaining missing data Supplemental Data Set 12. Gene Ontology analysis: over- and
imputed using KNN (Supplemental Data Set 3). Abundance data were nor- underrepresentation of biological process and molecular function of
malized to total signal in each sample, log2 transformed, mean-centered, and individual clusters.
divided by SD of each variable using Metabolonalyst 3.0 (Xia et al., 2015). Supplemental Data Set 13. Analysis of TF binding sites.
Supplemental Data Set 14. Differentially expressed transcription
Accession Numbers
regulators divided into transcription factor families.
Sequence data from this article can be found in the Gene Expression
Supplemental Data Set 15. Output of the TF Metropolis VBSSM
Omnibus data library under accession number GSE65046.
(M-VBSSM).

Supplemental Data Supplemental Data Set 16. Selected TFs for VBSSM modeling.

Supplemental Figure 1. Plant growth and chlorophyll fluorescence Supplemental Data Set 17. MRMs used for compound identification.
during progressive drought stress. Supplemental Data Set 18. Output from XCMS alignment of peaks
Supplemental Figure 2. Targeted metabolite analyses of secondary from LC-QToF metabolite profiling.
metabolites (except flavonoids), sugars, and amino acids.
Supplemental Figure 3. Targeted metabolite analyses of flavonoids
and anthocyanins. ACKNOWLEDGMENTS
Supplemental Figure 4. The effect of drought stress on plant
The authors acknowledge the support of the UK Biotechnology and Biological
development.
Science Research Council (BBSRC; Grant BB/F005806/1). S.S. was sup-
Supplemental Figure 5. Temporal expression patterns of five se- ported by a University of Essex PhD studentship. J.S.A.M. is supported by an
lected flavonol biosynthesis genes. NERC-CASE award (ENV-EATR-DTP: NE/L002582/1), and S.R.M.V-C. is
Supplemental Figure 6. Analysis of TF binding sites. supported by the BBSRC (BB/1001187_1). R.F. and J.E.L. are supported
by the Max Planck Society. N.S. and H.F. are supported by Exeter Science
Supplemental Figure 7. Gene expression of selected drought- Strategy funding, and C.S. is supported by the BBSRC and the Department for
responsive genes and growth analysis of agl22 mutants. Environment, Food, and Rural Affairs through the NORNEX project. D.L.S
Supplemental Figure 8. Validation of the knockout phenotype in agl22 was supported by a Wellcome Trust Institutional Strategic Support Fund.
insertion mutants and the specific role of AGL22 during drought stress. We thank Susan Corbett and Phillip A. Davey for help with the drought
experiments and gas exchange measurements.
Supplemental Figure 9. Growth and photosynthetic phenotype of
Col-0 and agl22 mutants during drought stress.
Supplemental Figure 10. Biomass production in agl22 mutants AUTHOR CONTRIBUTIONS
compared with the wild type and timeline of events.
U.B. designed, carried out, and analyzed the drought experiments. T.L.
Supplemental Figure 11. Schematic overview of array hybridizations
designed the gas exchange and fluorescence measurements. J.S.A.M.
across the 13 time points and two different treatments.
and S.R.M.V.-C. performed whole-chamber gas exchange experiments on
Supplemental Figure 12. Validation of the M-VBSSM approach. gene mutants. S.S. isolated hub gene mutants. R.F. and J.E.L. measured
Supplemental Table 1. Primers for qPCR, mutant screen, and RT- primary metabolites. H.F., C.S., D.L.S., and N.S. measured and analyzed
PCR analyses. targeted and untargeted metabolites. C.A.P., D.J.J., L.B., L.B., R.H., S.O.,
R.L., C.H., and J.D.M. were responsible for data analysis. K.J.D., N.R.B.,
Supplemental Methods. Model comparison via a Metropolis search. P.M.M., N.S., A.M., D.L.W., B.F., D.R., J.B., S.O., V.B.-W., and U.B. all had
Supplemental Data Set 1. LC-MS of sugars and LC-MS/MS analysis input into the design of the experiments and analysis. U.B. wrote the article
of secondary metabolites and amino acids. with contributions from all authors.
Supplemental Data Set 2. LC-MS/MS analysis of sugar phosphates,
other phosphorylated compounds, and organic acids.
Received October 27, 2015; revised January 14, 2016; accepted February
Supplemental Data Set 3. ANOVA of metabolites. 2, 2016; published February 3, 2016.
Supplemental Data Set 4. List of differentially expressed genes.
Supplemental Data Set 5. Functional categorization of 1815 DEGs.
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Time-Series Transcriptomics Reveals That AGAMOUS-LIKE22 Affects Primary Metabolism and
Developmental Processes in Drought-Stressed Arabidopsis
Ulrike Bechtold, Christopher A. Penfold, Dafyd J. Jenkins, Roxane Legaie, Jonathan D. Moore, Tracy
Lawson, Jack S.A. Matthews, Silvere R.M. Vialet-Chabrand, Laura Baxter, Sunitha Subramaniam,
Richard Hickman, Hannah Florance, Christine Sambles, Deborah L. Salmon, Regina Feil, Laura
Bowden, Claire Hill, Neil R. Baker, John E. Lunn, Bärbel Finkenstädt, Andrew Mead, Vicky
Buchanan-Wollaston, Jim Beynon, David A. Rand, David L. Wild, Katherine J. Denby, Sascha Ott,
Nicholas Smirnoff and Philip M. Mullineaux
Plant Cell 2016;28;345-366; originally published online February 3, 2016;
DOI 10.1105/tpc.15.00910
This information is current as of December 15, 2016

Supplemental Data http://www.plantcell.org/content/suppl/2016/02/03/tpc.15.00910.DC1.html


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