ORIGINAL PAPER

Modulation of transcription factor and metabolic pathwaygenes in response to water-deficit stress in rice

Swatismita Ray & Prasant K. Dansana & Jitender Giri & Priyanka Deveshwar &

Rita Arora & Pinky Agarwal & Jitendra P. Khurana & Sanjay Kapoor &

Akhilesh K. Tyagi

Received: 3 April 2010 /Revised: 10 August 2010 /Accepted: 16 August 2010 /Published online: 7 September 2010# Springer-Verlag 2010

Abstract Water-deficit stress is detrimental for rice growth,development, and yield. Transcriptome analysis of 1-week-old rice (Oryza sativa L. var. IR64) seedling under water-deficit stress condition using Affymetrix 57 K GeneChip®has revealed 1,563 and 1,746 genes to be up- anddownregulated, respectively. In an effort to amalgamatedata across laboratories, we identified 5,611 differentiallyexpressing genes under varying extrinsic water-deficitstress conditions in six vegetative and one reproductivestage of development in rice. Transcription factors (TFs)involved in ABA-dependent and ABA-independent path-ways have been found to be upregulated during water-deficit stress. Members of zinc-finger TFs namely, C2H2,C2C2, C3H, LIM, PHD, WRKY, ZF-HD, and ZIM, alongwith TF families like GeBP, jumonji, MBF1 and ULTexpress differentially under water-deficit conditions. NAC(NAM, ATAF and CUC) TF family emerges to be apotential key regulator of multiple abiotic stresses. Amongthe 12 TF genes that are co-upregulated under water-deficit,salt and cold stress conditions, five belong to the NAC TFfamily. We identified water-deficit stress-responsive genes

encoding key enzymes involved in biosynthesis of osmo-protectants like polyols and sugars; amino acid andquaternary ammonium compounds; cell wall looseningand structural components; cholesterol and very long chainfatty acid; cytokinin and secondary metabolites. Compari-son of genes responsive to water-deficit stress conditionswith genes preferentially expressed during panicle and seeddevelopment revealed a significant overlap of transcriptomealteration and pathways.

Keywords Oryza sativa L. var. IR64 .Microarray .

Transcriptome analysis .Water-deficit stress .

Metabolic pathways . Transcription factors

Introduction

Rice is a moisture-loving plant and, thus, its production isadversely affected by drought, which alone causes loss ofapproximately 18 million metric tons of rice productionworld-wide (Widawsky and O’Toole 1990; Evenson 1996).Moreover, global climate change is increasing the threat ofwater stress in near future (Tuba and Lichtenthaler 2007).Hence, identification of key genes, involved in regulation ofthe complex trait of drought tolerance and their manipulationby molecular breeding and/or genetic engineering fordevelopment of drought tolerant varieties, have becomestrategically important. Several quantitative trait loci associ-ated with drought tolerance have been identified throughphenotyping and marker-assisted selection (Tuberosa andSalvi 2006). Generation of ESTs from rice tissues subjectedto drought has also been an effective approach foridentifying genes associated with drought stress (Babu etal. 2002; Reddy et al. 2002; Markandeya et al. 2005;Gorantla et al. 2007; Varshney et al. 2009). The completion

Electronic supplementary material The online version of this article(doi:10.1007/s10142-010-0187-y) contains supplementary material,which is available to authorized users.

S. Ray : P. K. Dansana : J. Giri : P. Deveshwar : R. Arora :P. Agarwal : J. P. Khurana : S. Kapoor :A. K. Tyagi (*)Interdisciplinary Centre for Plant Genomics and Department ofPlant Molecular Biology, University of Delhi South Campus,New Delhi 110021, Indiae-mail: akhilesh@genomeindia.org

Present Address:A. K. TyagiNational Institute of Plant Genome Research,Aruna Asaf Ali Marg,New Delhi 110067, India

Funct Integr Genomics (2011) 11:157–178DOI 10.1007/s10142-010-0187-y

of genome sequences from several plant species, likeArabidopsis, rice, poplar, grape, papaya, Medicago, lotus,tomato, sorghum, and maize, and advances in the high-throughput techniques have revolutionized the gene discov-ery process followed by global gene expression (Vij andTyagi 2007; Paterson et al. 2009). Expression Microarrayplatform has been widely used in identifying genes involvedin abiotic stress tolerance (Rensink and Buell 2005).

In 2001, Seki et al. identified 44 cDNAs expressingdifferentially in Arabidopsis under drought and cold stressusing a microarray of ~1,300 full-length cDNAs. Subse-quently, Seki et al. (2002b) identified 277 drought-induciblegenes from Arabidopsis. Other, more extensive microarraystudies in Arabidopsis have revealed that initial perception ofstress might not be very specific to individual stress but laterthey attain specificity by involving transcription factors,signaling components and metabolic pathways (Kreps et al.2002; Oono et al. 2003; Kilian et al. 2007).

Abscisic acid (ABA) is known to have important role indrought stress response. Seki et al. (2002a) identified 299ABA responsive genes, 155 of which were upregulatedunder drought stress condition as well. Effect of ABAtreatment on guard cells revealed 64 differentially expressinggenes (Leonhardt et al. 2004). A comparative study of water-deficit stress-induced genes from microarray experiments ofSeki et al. (2002a, b), Kreps et al. (2002), and Kawaguchi etal. (2004) helped identify 27 genes that were induced underall three conditions, falling in six functional categories, e.g.,metabolism, transport, signaling, transcription, hydrophilicproteins and unknown proteins (Bray 2004).

Transcriptome analysis of rice under water-deficit stresshas been carried out earlier (Cooper et al. 2003; Rabbani etal. 2003; Hazen et al. 2005; Lan et al. 2005; Wang et al.2007; Zhou et al. 2007). Lan et al. (2004) identified that ofthe 253 cDNAs involved in pollination/fertilization in rice,136 genes (Lan et al. 2005) coding for regulatory proteinsinvolved in signal transduction and gene expression werealso involved in dehydration. Under drought stress condi-tion, the regulation of transcriptome of two divergentcultivars with high- and low-osmotic adjustment capacitywas found to be remarkably distinct. Genes encoding forsucrose synthase, a pore protein, a heat shock protein, and aLEA protein, were found exclusively upregulated in high-osmotic adjustment variety which could be contributing tothe tolerance trait (Hazen et al. 2005). In another study,much higher number of genes was found to be upregulatedin sensitive variety of rice than tolerant variety underdrought stress condition. Interestingly, ribosomal andplastid protein synthesis genes were downregulated insensitive variety but not in tolerant variety; whereas, genesfor protein degradation, amino acid degradation, lipiddegradation were triggered in sensitive variety but not intolerant variety. Moreover, genes encoding for cytochrome

P450 were significantly upregulated in the tolerant varietybut not in sensitive variety. All these factors cumulativelycould be contributing to drought tolerance (Degenkolbe etal. 2009). Transcriptome analysis under water-deficit stresshas also been studied in various other species like chickpea,sunflower, barley, tobacco, Physcomitrella patens, Pinustaeda, Populus trichocarpa, Populus euphratica, Sorghumbicolor, Zea mays, Thellungiella salsuginea, and Vitis vinifera(Oztur et al. 2002; Rizhsky et al. 2002; Watkinson et al.2003; Yu and Setter 2003; Zheng et al. 2004; Brosche et al.2005; Buchanan et al. 2005; Andjelkovic and Thompson2006; Street et al. 2006; Wong et al. 2006; Cramer et al.2007; Cuming et al. 2007; Mantri et al. 2007; Roche et al.2007; Talame et al. 2007; Tattersall et al. 2007; Varshney etal. 2009). However, a comprehensive analysis of stress-responsive genes during various stages of plant life cycle isnot yet available.

In this study, an attempt has been made to identify andenlist the water-deficit stress-responsive genes from micro-array gene expression profiling of 1-week-old rice seedlingunder water-deficit stress condition. To achieve a consensuson water-deficit stress-responsive genes, a cumulativeanalysis has been performed considering various otherhigh-throughput studies across the globe. The water-deficit stress-responsive genes have thus been analyzed inseven developmental (vegetative and reproductive) stages,although water-deficit conditions varied widely. Here, wealso report a repertoire of genes commonly triggered bywater-deficit, cold, and salt stress conditions. Moreover, anoverlap between genes differentially expressed duringvarious stages of panicle and seed development and thoseresponsive to water-deficit stress condition has beenestablished.

Materials and methods

Plant material, growth condition, and stress treatment

The seeds of indica rice (Oryza sativa L. var. IR64,IET9671), after disinfection with 0.1% HgCl2 and thoroughwashing with reverse-osmosis (RO) water, were soakedovernight in RO water. Next day, seeds were spread on ameshed float and grown hydroponically at 28±1°C inculture room with a daily photoperiodic cycle of 14 h lightand 10 h dark. After 1 week of growth, the seedlings weregiven different stress treatments (Mukhopadhyay et al.2004). One-week-old seedling stage was chosen foranalysis to emphasize on transcriptome regulation atseedling survival in early stage of development underwater-deficit stress condition. Specifically, for cold stress,the seedlings were kept at 4±1°C in RO water, for saltstress the seedlings were transferred into a beaker contain-

158 Funct Integr Genomics (2011) 11:157–178

ing 200 mM NaCl solution and for water-deficit stress theywere air-dried on a Whatman 3 mm sheet at 28±1°C, andall stresses were given for 3 h. For control, 1-week-oldseedlings were maintained in water in 100 ml beaker for3 h. Different developmental stages of rice panicle wereobtained from field-grown rice (O. sativa ssp. indica var.IR64). The young panicles were taken out from the sheathand measured to be categorized in six groups (P1, 0–3 cm;P2, 3–5 cm; P3, 5–10 cm; P4, 10–15 cm; P5, 15–22 cm,and P6, 22–30 cm) based on length of the panicle and thelandmark developmental events (Itoh et al. 2005), andfrozen in liquid nitrogen. The rice seed were tagged fromthe day of pollination (DAP), and developing seeds werecollected on each DAP from 0 to 30 DAP. These werepooled into S1, S2, S3, S4, and S5, representing, 0–2, 3–4,5–10, 11–20, and 21–29 DAP, respectively. Mature leaveswere harvested from the same plants.

Affymetrix GeneChip hybridization and data collection

Total RNAwas isolated from vegetative tissue of 1-week-oldrice seedlings (root and shoot tissue), the stress samples,mature leaf and panicle of rice and quality of the RNA wasascertained as described previously (Jain et al. 2006). Forisolating total RNA from rice seed, RNA isolation methodfrom carbohydrate-rich seeds was followed (Sharma et al.2003). The microarray analysis using Affymetrix GeneChip®Rice Genome Array was carried out according to Affymetrixmanual for one-cycle target labeling and control reagents(Affymetrix, Santa Clara, CA) using 5 μg of RNA as startingmaterial. Target preparation, hybridization to arrays, washing,staining, and scanning were carried out as described earlier(Jain et al. 2007; Ray et al. 2007). The cell intensity data files(*.cel) generated by the Gene Chip Operating Software(GCOS 1.2) (Schadt et al. 2001) were imported in ArrayAssist® software (Strandgenomics, Bangalore, India) forsubsequent data processing. The data from 12 chips werenormalised by using GeneChip robust multi-array average(GCRMA) algorithm (Wu et al. 2004). The correlationbetween the biological replicates were assessed usingPearson’s correlation coefficient (R) on the signal intensitiesand the R values between the three replicates were≥0.95 forfour stress experimental stages. For further data analysis, thethree replicates under each stress condition (water-deficit,cold, or salt) and control tissue were normalized as individualexperimental pairs by using GCRMA algorithm. The finaldataset after normalization contained 57,381 probesets fromwhich hybridization controls, TE-related and redundantprobesets, were removed after an extensive manual curation.The final number of unique probeset was determined to be37,927 (mentioned as genes in this study). The unique probesets include gene loci identified by TIGR (The Institute forGenomic Research; http://www.tigr.org/) and the KOME

(Knowledge-based Oryza Molecular biological Encyclopedia;http://red.dna.affrc.go.jp/cDNA/) cDNAs which were notpresent in the sequenced genome. The normalized data werelog2 transformated, and differential expression analysis wereperformed, using paired t test method. A gene was designatedas up- or downregulated if the signal ratios were ≥2 at pvalue <0.005 with respect to 1-week-old unstressed seedlingfor stress samples and mature leaf was considered as controlfor the reproductive developmental stages. To avoid lowexpressing genes under water-deficit, cold, and salt stresscondition, from the differentially up- and downregulatedgenes list, genes having average normalized intensity value≥50 in stress sample and control sample, respectively, wereconsidered for further analysis. The respective log trans-formed intensity values were used for hierarchical clusteringby using Euclidean distance matrix and K-Means. Forcomparative study of genes regulated by extrinsic andintrinsic (developmental) water-deficit stress, cell intensitydata files for mature leaf, six panicle stages (P1, P2, P3, P4,P5, and P6), and five seed stages (S1, S2, S3, S4, and S5)were used to make a project along with the cell intensity datafiles of stressed and unstressed 1-week-old seedlings.GCRMA normalized data was analyzed for differentialexpression and genes showing twofold changes at a p value≥0.05 were called as differentially expressed genes. Benja-mini–Hoschberg correction was applied for all t testsperformed. Co-regulation of water-deficit stress-responsivegenes with seed preferential expression was identified asthose genes that were at least two-fold upregulated in any ofthe seed stage with respect to any of the panicle stage suchthat the maximum signal intensity amongst the five seeddevelopment stages is higher than the maximum signalintensity amongst the six panicle development stages.Furthermore, they were filtered for those which had at leasttwo-fold upregulation in any of the seed stages with respectto mature leaf, which served as the vegetative control.Similarly, co-regulation with panicle preferential genes wasidentified where expression of panicle was up in comparisonto seed and mature leaf in water-deficit-induced genes.Further analyses were carried out in Microsoft Excel. Micro-array data from this article have been deposited in the GeneExpression Omnibus database at the National Center forBiotechnology Information under the series accession numb-ers GSE6893 and GSE6901.

Literature database analysis

Literature search was made to compile a list of alreadyknown water-deficit stress-related genes from differentcultivars, tissue, time points, level of stress, and platformsstudied. Only those studies were taken into considerationfor which data was available in retrievable form (Cooper etal. 2003; Rabbani et al. 2003; Wang et al. 2007; Zhou et al.

Funct Integr Genomics (2011) 11:157–178 159

2007). Gene IDs provided in these papers were mapped togene loci of TIGR version 5. Corresponding Affymetrixprobeset IDs for genes retrieved from published sourceswere searched from Rice Multi-platform Microarray Search(http://www.ricearray.org/matrix.search.shtml). Probe IDscorresponding to the 37,927 unique probe set were usedfor further analysis. The up- and downregulation of thesegenes were determined after comparing the data providedfor treated tissue and control tissue. Few genes we report tobe up- and downregulated in same tissue as we arereporting a concise data of all time course experiments.

Functional classification

Gene Ontology-based functional analysis

Genes responsive to water-deficit stress condition wereclassified according to the function of the protein theyencode according to Gene Ontology (GO) database (http://www.geneontology.org/). The underlying fact of the GOclassification is that every gene could be part of all threemain classification types i.e., molecular function, cellularprocess, and biological process. However, these numberswould change depending on the current state of ourunderstanding about that particular gene or protein.

Metabolic pathway analysis

From RiceCyc in GRAMENE (Jaiswal et al. 2006), wedownloaded metabolic pathway-associated genes whose IDwas clustering on metabolic pathways. Pathways werereconstructed using Adobe Illustrator® software.

Results

Identification and analysis of genes responsive to extrinsicwater-deficit stress during rice development

IR64 variety of indica rice was chosen for transcriptomeanalysis under water-deficit stress condition using RiceGenome Array. IR64, a semi-dwarf lowland variety withgood yield potential, is widely grown in irrigated area intropical Asia (Khush 1995; Narciso and Hossain 2002).Sampling for microarray gene expression study was doneafter 3 h of water-deficit stress to 1-week-old rice seedlingwhen relative water content (Barr and Weatherley 1962)was approximately 31–37%. Most of the previouslyidentified water-deficit stress-responsive genes, includingLEA protein (LOC_Os01g12580), dehydrin-Rab16B(LOC_ Os11g26780), Rab21 (LOC_Os11g26790),COR410 (LOC_Os02g44870), aquaporin-TIP3.1(LOC_Os10g35050), and DREB1 (LOC_Os04g55520)

showed increased transcript accumulation in our microarrayexperiment. The microarray data for abiotic stress-responsive genes of selected gene families have beenalready validated in our previous studies by quantitativePCR analysis (Agarwal et al. 2007; Arora et al. 2007; Jainet al. 2007; Ray et al. 2007; Nijhawan et al. 2008; Vij et al.2008). Under water-deficit stress condition, 1,563 and1,746 genes were differentially up- and downregulated,respectively. Initiative for identification of water-deficitstress-responsive genes in different cultivars of rice (japon-ica and indica), at varied developmental stages (2-, 4-, and6-week-old, 1-week-before-heading, and 4-tiller stage), andexperimental conditions has been taken-up across laborato-ries (Cooper et al. 2003; Rabbani et al. 2003; Wang et al.2007; Zhou et al. 2007) as listed in Table 1.

A total of 5,901 unique genes were thus identified afterremoving the redundant ones from the cumulative list of 7,222differentially regulated genes (Table 1). Among the 5,901unique genes, 5,611 were found to be represented in the37,927 subset of unique genes on Rice Genome Array whichhas been used for further analysis (Electronic supplementaryTable S1). Expression of genes under water-deficit stressresponse in seven developmental stages, including sixvegetative (1-, 2-, 4-, 6-week, 1-week-before-heading, and4-tiller stage) and one reproductive (panicle: 1-week-before-heading) stage, was found to be spatially and temporallyregulated. Under water-deficit stress condition, 2,505 and2,925 genes were found to be up- and downregulated,respectively, wherein, 181 genes were found to be up- aswell as downregulated in different studies implying that theirdifferential regulation in response to water-deficit stress maybe developmental stage-specific (Electronic supplementaryTables S2, S3, and S4). Maximum number of genes (1,968and 2,631 up- and downregulated, respectively) was found tobe expressing differentially under individual experimentalcondition, followed by those that showed differentialexpression under two or more experimental conditions(Fig. 1). A set of 24 genes (22 and 2 genes in four andfive developmental stages, respectively) were found to beinduced at many developmental stages under water-deficitstress. They were also involved in regulation (transcriptionfactors), signaling (kinase), and metabolism (CTP synthase,epimearse), although few genes remain to be assignedspecific function (Fig. 1).

Transcription factors expressing differentiallyunder water-deficit condition

The rice genome has been found to code for 2,314 TFsbelonging to 68 TF families (unpublished data). Out of 68TF families, at least one member of each of the 58 TFfamilies showed differential expression under water-deficitstress condition (Electronic supplementary Table S5).

160 Funct Integr Genomics (2011) 11:157–178

Members of the same TF family (33 families) showedvaried response indicating that TFs work individually inresponse to stress (Fig. 2a). Under water-deficit condition,more than ten genes encoding for members of AP2, MYB,bHLH, NAC, bZIP, C2H2, Homeobox, WRKY, and MADs TFfamily were upregulated, however, >10 members of MYB

and C2H2 family also showed downregulation under stress.Ten interesting families of TFs, about which not much isknown for their involvement in water-deficit stress, had one(jumonji (LOC_Os10g42690), Multiprotein bridging factor 1(MBF1; LOC_Os06g39240), PBF-2-like (whirly;LOC_Os06g05350), SHI-related sequence (SRS;LOC_Os01g72490), and ULTRAPETALA1 (ULT;LOC_Os01g57240)) or two (cell-shape-control protein phos-phatase (CPP; LOC_Os01g55580; LOC_Os07g07974),ethylene-insensitive3-like (EIL; LOC_Os07g48630;LOC_Os09g31400), GL1 enhancer binding protein (GeBP;LOC_Os01g14720; LOC_Os03g50110), tr ihel ix(LOC_Os04g51320; LOC_Os04g45750) and tubby(LOC_Os12g06630; LOC_Os05g36190)) member(s) eachthat were upregulated, whereas, none of the members of thesegene families were downregulated under stress (Electronicsupplementary Table S5).

Genes encoding for 58 TFs were found to express inmore than one developmental stage under water-deficitstress condition (Fig. 2b). Precisely, 14 stress-responsivegenes identified from Zhou et al. (2007) were found to beupregulated in panicle tissue under water-deficit stresswhich encoded for TFs belonging to AP2, bromo domain,GeBP, FHA, MADs, MYB, NAC, PBF-2-like (whirly),TCP and C2H2 family (Electronic supplementary Table S2).Under water-deficit stress, TF encoding genes, bHLH(LOC_Os03g56950), WRKY (LOC_Os05g03900), C2H2

(LOC_Os12g39220), and LSD1 (LOC_Os08g06280)showed decrease in transcript accumulation in threedevelopmental stages (Fig. 2c). Interestingly, 20 TFs under

248

42

4

403

110

22

20

0 500 1000 1500 2000 2500 3000

1

2

3

4

5

Number of genes

Upregulated

Downregulated

Num

ber

of s

tage

s

19682631

Fig. 1 Developmental stage-wise distribution of differentiallyexpressing genes in rice under water-deficit stress condition. Hori-zontal bars represent total number of differentially expressed genes inthe number of rice developmental stages (six vegetative; 1-, 2-, 4-, 6-week-old, and 1-week-before-heading and 4-tiller stage and onereproductive (1-week-before-heading) stage) analyzed (for detailsrefer Table 1). No gene was found to be differentially regulated inmore than five stages

Data source Total genes inmicroarray analysis

Microarrayplatform

Plant material Regulation No. ofgenes

Laboratorydata

57,381 Affymetrix 1-week-old seedling Up 1,563

Down 1,746

Cooper et al.(2003)

~21,000 Affymetrix 6-week-old seedling Up 73

Down 28

Rabbani etal. (2003)

1,718 cDNA array 2-week-old seedling Up 59

Wang et al.(2007)

1,991 cDNA array 4-week-old seedlingUpland Rice

Up 65

4-week-old seedlingLowland Rice

Up 74

Zhou et al.(2007)

41,754 Oligonucleotidearray

1-week-before-heading Panicle

Up 449

Down 935

1-week-before-heading Flag leaf

Up 465

Down 615

4-tiller-stage Shoot Up 813

Down 337

Total 7,222

Unique 5,901

Represented in 37,927a probesets 5,611

Table 1 Number of genesidentified by microarrayanalyses with altered regulationin response to five differentwater-deficit stress experimentalsystems in seven developmentalstages of rice

a Subset of 37,927 genes withunique probe IDs on Rice GenomeArray; for details, refer to “Mate-rials and methods”

Funct Integr Genomics (2011) 11:157–178 161

water-deficit stress were up/downregulated in a develop-mental stage-dependent manner (Electronic supplementaryFig. S1). Ten genes encoding for TFs belonging to CO-like(LOC_Os08g15050), MADs (LOC_Os12g10540), MYB(LOC_Os10g33810) , NAC (LOC_Os03g60080;LOC_Os05g34830), PLATZ (LOC_Os10g42410), ZIM(LOC_Os10g25230), WRKY (LOC_Os05g39720), C2H2

(LOC_Os01g62460), and Dof (LOC_Os04g58190) familywere downregulated in panicle, but upregulated in vegeta-tive tissues. Even few TF families showed strict regulationof expression among vegetative developmental stages,where members of CO-like, MYB and Dof family wereupregulated in shoot tissue of 4-tiller stage, wherein, other

members of CO-like and MYB family were downregulatedin flag leaf tissue; moreover, another Dof family memberwas also found to be downregulated in 1-week-oldseedling. A gene coding for PLATZ TF was found to beupregulated in flag leaf but another gene of the same TFfamily was found to be downregulated in 1-week-oldseedling (Electronic supplementary Fig. S1).

Shared response to water-deficit, cold, and salt stress

Co-regulation of water-deficit stress-responsive genes withcold and salt stress is shown in Fig. 3a. A higher percentageof water-deficit stress regulated genes (27%) are also

3310 15

Repressed48 TF family

Induced43 TF family

Stress responsive58 TF family

2

3

Num

ber

of s

tage

s

Dof

WRKY

WRKY

C2H2, LSD1

C2H2, MYB, MADs

bHLH, LIM, NAC, PHD

bHLH

Homeobox, Pseudo ARR_B

Pseudo ARR_B, MYB, HSF

week(s) 1-week before heading

4-tiller

1 2 6 PANICLE FLAG SHOOT

3

2

4

Num

ber

of s

tage

s

1 2 4 6

week(s)

PANICLE FLAG

1-week before heading

4-tiller

SHOOT

bZIP

bZIP

C2H2

NAC

HMG

bZip, MADs

bHLH

AP2, HSF

Homeobox, MYB, C3H

G2-like

Aux_IAA, Lim, WRKY, C2H2

AP2, bZIP, Homeobox, HSF, NAC, PHD

AP2, Homeobox, C3H, GRAS, HSF, Jumonji, MYB, NAC, Pseudo ARR_B, SBP, ZIM, Tubby, WRKY, C2H2, PHD

a

b

c

Fig. 2 Transcription factor fam-ilies responsive to water-deficitstress condition in rice. a Venndiagram showing total numberof TF families in rice whosemembers were induced and re-pressed under water-deficitstress. Few members of 33 TFfamilies were up- and somewere downregulated underwater-deficit stress. b Stage-specific upregulation of TFfamily members in seven stagesof rice development. c Stage-specific downregulation of TFfamily members in seven stagesof rice development. x-axis rep-resents seven developmentalstages of rice, and y-axis repre-sents number of stages in whichdifferential expression is found.The different shades of the hor-izontal bars signify the numberof developmental stages inwhich the members of the TFfamilies are expressing

162 Funct Integr Genomics (2011) 11:157–178

COLD SALTWATER-DEFICIT

338

159

2505

693

958

233

97

2925

437

675

145

2715

76

34

181

Up

Up/down

Down

Con

trol

see

dlin

g

Wat

er-d

efic

it

Col

dS

alt

Stress

Upr

egul

ated

Dow

nreg

ulat

ed

5%

14%7%

17%

1%

8%

39%

9%

6%

29%

12%9%

6%

9%

29%

SIGNAL TRANSDUCTION

TRANSLATION REGULATOR

TRANSCRIPTION REGULATOR

TRANSPORTER ACTIVITYBINDING PROTEIN

OTHER MOLECULAR FUNCTION

CATALYTIC ACTIVITYUNIDENTIFIED

Expression values in log2

a

b

Fig. 3 Relationship between thegenes that are up/downregulatedunder water-deficit, salt and coldstress along with expressionpattern of genes having sharedregulation pattern. a Blockarrows showing co-regulatedgenes between water-deficit,cold, or salt stress. Numbers indark circles represents co-regulated genes under water-deficit, cold and salt stress con-dition. b K-Means clustering ofthe co-up- (76) and down- (34)regulated genes. Pie chart rep-resents the Gene Ontology-based functional categorizationof co-regulated genes. Colorscale for average signal intensityvalues is given in log2

Funct Integr Genomics (2011) 11:157–178 163

affected by salt stress, however, only 6.3% genes werefound to be co-regulated between water-deficit and coldstress dataset. It was also observed that a homeobox-leucinezipper protein (LOC_Os02g43330), water stress-inducibleprotein Rab21 (LOC_Os11g26790) and seed maturationprotein (LOC_Os08g23870) encoding genes are among thefive genes showing highest differential regulation in 1-week-old rice seedling under water-deficit and salt stress(Electronic supplementary Table S6). Furthermore, amongthe genes co-regulated by both salt and cold stress, aMKKK2 (LOC_Os01g50420) and expressed protein(LOC_Os06g46140) genes exhibit greater degree of differ-ential expression under cold stress condition. Of the 181genes, which showed both up/downregulation of transcriptunder water-deficit stress in a tissue and developmentalstage-dependent manner, 14 and 5 genes, respectively, wereup- and downregulated under cold stress condition, where-as, more genes were co-regulated under salt stress from thiscategory of genes.

Of the 693 co-regulated genes under water-deficit and saltstress conditions, 76 were also upregulated under cold stress(Fig. 3a). These 76 genes code for proteins involved in signaltransduction, transcription and translation regulation, trans-porters and catalytic activity; function to a few proteins is yetto be assigned (Fig. 3b). The signal transduction componentgenes include proteins belonging to calcium-regulated cascade(namely calmodulin, EF-hand family protein and ATPases),kinases, phosphatases, heat shock proteins, transporters, andhormone action. More genes encoding for TFs were co-upregulated (17%) than co-downregulated (6%). One or twomembers of TF families namely, bHLH (LOC_Os08g42470),CPP (LOC_Os07g07974), C2H2 (LOC_Os03g60570;LOC_Os03g60560), MYB (LOC_Os04g43680), WRKY(LOC_Os06g44010), and AP2 (LOC_Os01g58420), wereamong the co-regulated genes; additionally, five genesencoded for NAC TF family (LOC_Os01g60020;LOC_Os11g03370; LOC_Os01g15640; LOC_Os01g66120;LOC_Os11g03300). Moreover, among these five NAC TFs,one gene (LOC_Os01g66120) was upregulated in uplanddrought tolerant variety of rice under water-deficit stress(Wang et al. 2007). These co-regulated TFs might be keyplayers in downstream responses induced by different kind ofabiotic stresses. Water-deficiency limits photosynthesis, salin-ity leads to ion toxicity and low-temperature directly affectscellular functioning, thus, collectively, they affect normalmetabolic processes of plant which is reflected in the declineof transcript accumulation of genes having catalytic activity.Among these downregulated genes, cytochrome p450 86A2(LOC_Os03g04530) , phosphoe thano lamine N-methyltransferase (LOC_Os05g47540), two expressed pro-teins (LOC_Os04g11120 and LOC_Os04g11060) andCRK10 (LOC_Os07g43570) encoding genes showed maxi-mum decline in transcript accumulation under water-deficit

stress in 1-week-old seedling (Electronic supplementary TableS6). More genes encoding for proteins having catalyticactivity were downregulated (29%) than upregulated (14%).

Functional categorization and pathway determinationof the water-deficit responsive genes

Gene Ontology-based analysis showed that 2,408 differen-tially regulated genes were involved in biological processesand molecular function could be assigned to 1,382 and1,611 up- and downregulated genes, respectively; however,a large number of differentially regulated genes (1,857)remains to be annotated (Electronic supplementary Fig. S2).

Ricecyc (http://www.gramene.org/pathway) databasewas further used for metabolic profiling of the up anddownregulated genes under water-deficit condition. Carbo-hydrate, energy, lipid, amino acid, nucleotide, cofactor,vitamin, and secondary metabolite metabolism were alteredunder water-deficit stress condition. Moreover, processeslike transcription, translation, replication and repair, fold-ing/sorting/degradation, transport and signaling were alsofound to be affected. While many functional categorieswere similarly represented in the up- and downregulatedgroups, not every regulated gene has a role in water-deficitstress tolerance and the change in expression in some ofthem may simply be the result of damages caused by stress(Bray 1997; Chaves et al. 2003).

The detailed list of the pathways affected under water-deficit stress condition is provided as Electronic supplemen-tary Tables S7, S8, and S9. A large range of osmolytes havebeen implicated in preventing damage to proteins caused bywater-deficit stress. Among the seven upregulated genesencoding for enzymes involved in trehalose biosynthesis,three of them represented isomers of trehalose-6-phosphatesynthase (TPS; LOC_Os02g54820; LOC_Os08g34580;LOC_Os09g23350) and two for isomers of trehalosesynthase (LOC_Os01g53000; LOC_Os01g54560). Thegenes encoding for trehalose-phosphate phosphatase (TPP;LOC_Os10g40550) and trehalase (LOC_Os10g37660) werealso upregulated (Fig. 4; Electronic supplementary Fig. S3A).In an alternative trehalose biosynthesis pathway, where glyco-gen is converted to trehalose, gene encoding for isoamylase-type starch debranching enzyme (LOC_Os05g32710) wasupregulated by fourfold in 1-week-old seedling under water-deficit stress, however, hydrolase was downregulated in thepanicle (Zhou et al. 2007), suggesting that under water-deficitstress condition this alternative pathway might be cell-typespecific.

Sucrose synthesis utilizes glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, and sugar nucleotide UDP-D-glucose as hexose phosphate pool (Hoekstra et al. 2001).Under water-deficit stress, gene encoding for sucrose synthase1 (LOC_Os03g22120) was upregulated which mediated

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conversion of UDP-D-glucose to sucrose. However, regulationof other isoform of sucrose synthase 1 and 2 gene(LOC_Os03g28330; LOC_Os06g09450) was found to bestage specific. Moreover, genes encoding for sucrose-phosphate synthase (LOC_Os01g69030, LOC_Os01g27880),which mediates synthesis of sucrose utilizing fructose-6-phosphate, were downregulated. This would cause increasein the hexose phosphate pool (Fig. 4; Electronic supplementaryFig. S3B). Transcript level of gene encoding for ß-fructofur-anosidase (LOC_Os02g01590), involved in biosynthesis offructan, a polysaccharide functioning as storage carbohydrate,was found to have strict regulation depending on develop-mental stage and organ, i.e. sixfold upregulated in 1-week-oldseedling, whereas it was downregulated in panicle and flagleaf (Fig. 4; Electronic supplementary Fig. S3B). Transcriptlevel of stachyose synthase enzyme encoding gene(LOC_Os01g07530) involved in biosynthesis of anotheroligosaccharide, stachyose, which is used by plants as storagematerial and known to act as protective agent during seedmaturation and cold stress (Bentsink et al. 2000; Gilmour etal. 2000), was found to be upregulated by 11-fold in 1-week-old seedling under water-deficit stress condition (Fig. 4;Electronic supplementary Fig. S3C). In mannitol biosynthesispathway, isomerization of fructose-6-phosphate to mannose-6-phosphate is mediated by mannose-6-phosphate isomerase(LOC_Os01g03710), whose transcript level increased byeightfold in 1-week-old seedling under water-deficit stresscondition (Fig. 4; Electronic supplementary Fig. S4A)establishing a positive correlation between accumulation ofmannitol and water-deficit stress.

Genes encoding for enzymes involved in biosynthesis ofprimary cell wall components were upregulated under water-deficit stress condition. Synthesis of GDP-D-rhamnose andGDP-L-fucose were favored under stress condition. Moreover,transcript level of hexokinase gene (LOC_Os06g45980) thatmediates conversion of GDP-D-mannose to mannose-6-phosphate in GDP-mannose metabolism was sixfold down-regulated in 1-week-old seedling, which in turn could facilitatechanneling of more GDP-D-mannose into synthesis of GDP-D-rhamnose and GDP-L-fucose (Fig. 4; Electronic supplemen-tary Fig. S4A). UDP-galacturonate and UDP-xylose are twoother cell wall components derived from UDP-glucuronate,which is synthesized via inositol oxidation pathway (Fig. 4;Electronic supplementary Fig. S4B). Increased accumulationof transcript of myo-inositol oxygenase encoding gene(LOC_Os06g3656) occurred under stress condition whichwould convert myo-inositol to glucuronate.Moreover, inositol-3-phosphate synthase gene (LOC_Os03g09250) involved inmyo-inositol biosynthesis pathway showed six fold increase inits transcript levels (Fig. 4; Electronic supplementary Fig. S4B).Interestingly, it was also observed that the synthesis of myo-inositol from α-D-glucose-6-phosphate was favored oversynthesis of UDP-D-glucose under stress condition. 3-deoxy-

D-manno-octulosonate (KDO) is a component of rhamnoga-lacturonanII pectin fraction of the primary cell wall. The genecoding for 3-deoxy-manno-octulosonate-cytidylyltransferase(CKS; LOC_Os05g48750), which activates KDO by couplingit to CMP (cytidine monophosphote), was found to beupregulated under stress condition (Fig. 4; Electronic supple-mentary Fig. S4C).

Genes encoding for enzymes involved in amino acidmetabolism pathways were found to be differentially regulat-ed under water-deficit stress condition (Electronic supple-mentary Tables S7, S8, and S9). Genes involved inbiosynthesis of ß-alanine, histidine, and serine were prefer-entially upregulated under water-deficit stress. Inproline biosynthesis, genes encoding for enzymes δ-1-pyrroline-5-carboxylate synthase (LOC_Os05g38150;LOC_Os01g62900), oxidoreductase (LOC_Os01g12710),aldehyde dehydrogenase (LOC_Os09g26880), and NADP-dependent glyceraldehydes-3-phosphate dehydrogenase(LOC_Os08g34210) were upregulated under stress condition(Fig. 4; Electronic supplementary Fig. S5). Transcriptaccumulation of six enzymes (LOC_Os11g39220;LOC_Os06g23870; LOC_Os06g24704; LOC_Os05g03480;LOC_Os05g07090; LOC_Os05g46480) involved in synthe-sis of β-alanine from propionate was also found to beincreased (Fig. 4; Electronic supplementary Fig. S6). Thepathways related to biosynthesis of cholesterol, very longchain fatty acid, flavonol and isoflavonol derivatives werealso stimulated under water-deficit stress condition (Fig. 4).

Analysis of commonly regulated genes by extrinsic(environmental) and intrinsic (developmental) water-deficitconditions

The reproductive developmental stages of rice were categorizedinto six stages of panicle development followed by five stagesof seed development, which have been described in detail inmaterials and methods section. Among the 2,686 water-deficitstress-induced genes, 490 genes were found to be inducedduring panicle development (Electronic supplementary TableS10) and 400 genes (Electronic supplementary Table S11)showed upregulation during seed development (Fig. 5a).

Fig. 5 Correlation between extrinsic and intrinsic water-deficit stressresponse during six stages of panicle (P1–P6) and five stages of seeddevelopment (S1–S5). a Venn diagram showing relationship amongwater-deficit stress-responsive genes having panicle and seed devel-opmental stage-specific regulation. b K-Means clustering of 490panicle preferential genes under water-deficit stress condition. Clusterswith similar trend of expression during panicle and seed developmenthave been grouped together into six groups. c K-Means clustering of400 seed preferential genes under water-deficit stress condition.Clusters with similar trend of expression during panicle and seeddevelopment have been grouped together into five groups. Color scalefor average log ratio values is given in log2

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Overlap of gene expression between water-deficit stresscondition and panicle development

Zhou et al. (2007) identified 408 genes to be inducible inpanicle under water-deficit stress condition. Ninety of thesegenes were also found to be differentially regulated duringnormal course of panicle development, indicating towards anunderlying connection in panicle development and water-deficit stress response. In this study, 490 water-deficit stress-responsive genes with preferential expression in panicle werecategorized into six major groups using K-means clustering(Group 1–6; Fig. 5b). Genes in group 3a (48) showed hightranscript accumulation uniformly from P1 to P6 stages ofpanicle development with decrease in expression duringseed maturation. Among these, only seven genes(LOC_Os01g54080, LOC_Os04g47520, LOC_Os07g01600,LOC_Os07g37850, LOC_Os09g31031, LOC_Os08g35710,and LOC_Os06g06780) showed upregulation in panicle tissueunder water-deficit stress (Zhou et al. 2007), whereas, othercluster members showed transcript accumulation at variousvegetative developmental stages under water-deficit stress.The P5 and P6 stages of panicle development includematuration of stamen, carpel, pollen, and anther dehiscence,which involves natural dehydration process. Two clusters (6aand 6b) of group 6 include genes expressing in the later stages(P5 and P6) of panicle development. Interestingly, a largefraction of (41.38% and 31.37%) genes belonging to cluster6a and b, respectively, showed upregulation in panicle underwater-deficit stress (Zhou et al. 2007) and the remaininggenes were responsive to external water-deficit stress atvarious vegetative stages. Taken together, among the genesshowing specific upregulation during various stages ofpanicle development, 490 genes showed correlation withthe stress upregulated genes and these may participate innatural desiccation process related to panicle development.

Overlap of gene expression between water-deficit stressand seed development

The ten K-means derived clusters, representing 400 seedpreferential genes upregulated under water-deficit stress, couldbe grouped in five distinct groups (Fig. 5c). Group 4 genesshowed higher level of transcript accumulation in seedcompared to panicle development. Group 4c genesmostly encoding for LEA proteins (LOC_Os06g23350;LOC_Os01g12580), dehydrin (LOC_Os11g26750), em-bryonic protein (LOC_Os05g28210; LOC_Os04g52110;LOC_Os11g26570), aquaporin (LOC_Os10g35050),phosphatase (LOC_Os06g04790), l ipid- t ransferprotein (LOC_Os03g02050), calcium-sensory protein(LOC_Os10g09850), and NAC transcription factor(LOC_Os03g21060) were sharply upregulated after S1 stageof seed development. By 5th day after anthesis (S3), the

embryo sac is filled with endosperm cells and its gradualmaturation continues till S5 stage of seed development. Genesbelonging to cluster (4b) were found to be encoding for proteinsmostly involved in synthesis of storage proteins such asglobulin (LOC_Os05g41970), glutelin (LOC_Os02g16830;LOC_Os02g15169; LOC_Os02g25640), prolamine(LOC_Os11g33000; LOC_Os06g31070) along with oleosin(LOC_Os03g49190), and patatin (LOC_Os01g67310), as wellas aquaporin (LOC_Os04g44570), NAC transcription factor(LOC_Os02g12310) and LEA protein (LOC_Os12g43140).Endoplasmic reticulum (ER) stress-related genes were foundclustered in group 4a, namely heat shock 70 kDa protein 1(LOC_ Os03g11910), heat shock 22 kDa protein(LOC_Os02g52150), 17.4 kDa class I heat shock protein 2(LOC_Os03g16020), heat shock cognate 70 kDa protein(LOC_Os05g38530), and heat shock protein 101(LOC_Os05g44340), which started accumulating from P6stage of panicle development till last stage of seed maturation.In group 5a cluster, transcript accumulation is maximum in S1–S2 stages and then decreases with seed maturity, which is adiagonally opposite expression profile to the group 4 genes.MYB (LOC_Os05g37060) and C2H2 (LOC_Os03g60560)transcription factors, ABC-transporter (LOC_Os01g03144),ATPase-3 (LOC_Os03g58790), GA inactivating enzyme(gibberellin 2-beta-dioxygenase; LOC_Os01g55240)and defense related proteins, acidic endochitinase(LOC_Os01g47070) and immediate-early fungal elicitorprotein CMPG1 (LOC_Os02g50460) encoding genes arerepresented in group 5 cluster. Therefore, it is apparent fromthis analysis that the genes known traditionally to be involvedin reproductive development could also be potential players inabiotic stress response.

Overlap in metabolic pathways between water-deficit stressand reproductive development

Metabolic pathways regulated under water-deficit stress andvarious stages of panicle/seed development are given inElectronic supplementary Tables S12 and S13. Genesencoding for proteins involved in secondary metabolitebiosynthesis pathways namely, flavonol, maackiain, medi-carpin, phenylpropanoid, salicylate, divinyl ether, coumarinbiosynthesis were upregulated during panicle developmentand under water-deficit stress. However, stachyose synthaseenzyme encoding gene (LOC_Os01g07530), genes encodingfour enzymes (metabolite transport protein-csbC, ADP-glucose pyrophosphorylase large subunit 3, starch synthaseand 1,4-α-glucan branching enzyme) involved in starchbiosynthesis pathway and transketolase (LOC_Os04g19740)enzyme involved in pentose phosphate pathway, glucosefermentation to lactate II, Calvin cycle and xylulose-monophosphate cycle, were preferentially upregulated dur-ing seed development as well as under water-deficit stress.

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Discussion

Our study primarily focused on the identification andanalysis of water-deficit stress-responsive genes from ricecultivars at different developmental stages including sixvegetative (1-, 2-, 4-, 6-week, 1-week-before-heading, and4-tiller stage) and one reproductive (1-week-before-head-ing) stages with the aim to identify important componentsof water-deficit stress response. Data across laboratorieswas used for this analyses as it were thought to enrich thelist of water-deficit stress-responsive genes under variedexperimental conditions (Bray 2004). While preparing themanuscript, another substantial study on the effect of long-term drought stress on rice cultivars was published byDegenkolbe et al. (2009); most of the differentiallyexpressed genes identified in this study were found to berepresented in the comprehensive non-redundant gene listprepared in this study.

Transcription factors regulating water-deficit stressresponse

Previous studies have shown that transcription factors areimportant regulators involved in plant response to environmen-tal stress (Chinnusamy et al. 2004; Mahajan and Tuteja 2005;Yamaguchi-Shinozaki and Shinozaki 2006; Shinozaki andYamaguchi-Shinozaki 2007; Nakashima et al. 2009). Both,ABA-independent and ABA-dependent regulatory pathwaysexist for water-deficit stress-responsive genes in plants. It wasobserved that under water-deficit stress condition, members ofTF families involved in both ABA-independent (AP2/ERF,bHLH and NAC) and ABA-dependent (MYB, bZIP, bHLH(MYC), NAC and homeodomain) pathways are upregulatedin rice. However, members of a gene family are differentiallyup or downregulated indicating that TFs work individually inresponse to stress. TFs belonging to these families have beenknown to interact with specific cis-elements and/or proteinsand their overexpression conferred stress tolerance in heterol-ogous systems (Abe et al. 1997, 2003; Jaglo-Ottosen et al.1998; Liu et al. 1998; Kasuga et al. 1999; Kang et al. 2002;Fujita et al. 2004, 2005; Tran et al. 2004; Furihata et al. 2006;Hu et al. 2006; Ito et al. 2006; Sakuma et al. 2006; Chen et al.2007; Dai et al. 2007; Jung et al. 2008; Zhou et al. 2009).Expression of TFs in an organ-specific manner was noted byZhou et al. (2007). In our study also, the regulation of TFswas found to be very precise in terms of spatial and temporaldistribution, as detailed in Fig. 2 and Electronic supplemen-tary Fig. S1.

Certain TF gene families (NAC, zinc-finger) have beenshown to play important role during stress (Ciftci-Yilmazand Mittler 2008; Nakashima et al. 2009). Involvement ofNAC TF in biotic and abiotic stress response is well known(Olsen et al. 2005; Hu et al. 2006; Nakashima et al. 2009;

Seo and Park 2010). Transgenic rice overexpressingOsNAC10, OsNAC6, and ONAC045 showed improveddrought and salt tolerance (Zheng et al. 2004; Nakashimaet al. 2009; Jeong et al. 2010). SNAC1 gene was found tobe involved in stomatal closure leading to drought tolerance(Hu et al. 2006). Fang et al. (2008) reported 20 rice NACTF genes to be drought-inducible, however, our studyrevealed 22 NAC TF genes to be upregulated under water-deficit stress (Electronic supplementary Table S2). Five ofthe NAC TF genes shared upregulation under water-deficit,cold and salt stress condition. Moreover, two of these NACTF genes (LOC_Os11g03370; LOC_Os11g03300) showedpanicle preferential regulation; however, they were notinducible by water-deficit stress condition in panicle tissue(Zhou et al. 2007). Six other water-deficit stress-inducibleNAC TF genes (LOC_Os02g36880; LOC_Os07g37920;LOC_Os06g46270; LOC_Os03g21060; LOC_Os02g56600;LOC_Os02g12310) showed seed preferential expression.Involvement of NAC TF in development as well as in stresstolerance has been reported in earlier studies (Sablowski andMeyerowitz 1998; Guo and Gan 2006; Peng et al. 2009).Arabidopsis NAC TF gene ANAC092 demonstrated anintricate overlap of ANAC092-mediated gene regulatorynetworks during salt-promoted senescence and seed matura-tion (Balazadeh et al. 2010). Hence, these studies highlightinterplay of pathways regulated by NAC TFs during stressand developmental stages.

Out of zinc-finger motif containing TFs, C2H2, C2C2-Dof, C3H, PHD, WRKY and ZIM are particularly repre-sented in water-deficit stress-responsive transcriptome.Genes belonging to these categories have been shown toconfer stress tolerance on overexpression in transgenicsystems (Chen et al. 1996; Bowman 2000; Eliasson et al.2000; Rao et al. 2000; Rizhsky et al. 2002; Kim et al. 2004;Narusaka et al. 2004; Reyes et al. 2004; Rizhsky et al.2004; Sakamoto et al. 2004; Yanagisawa 2004; Davletovaet al. 2005; Zhang and Wang 2005; Jiang and Deyholos2006; Major and Constabel 2006; Agarwal et al. 2007;Huang et al. 2007; Park et al. 2007; Sun et al. 2007; VanHolme et al. 2007; Ciftci-Yilmaz and Mittler 2008).

Members of ten TF families were only induced (none of themembers were repressed) under water-deficit stress implyingthat they might play special role in stress tolerance. Amongthese families, members of SRS, CPP, EIL, Tubby and trihelixfamily have been earlier reported to be induced in rice roottissue under osmotic stress condition (Ma et al. 2009). Amongthe other TF families, PBF-2-like (whirly) proteins are mostlyknown to play role in defense response and could alsofunction in the chloroplast as well as the nucleus (Desveaux etal. 2005). Members of the remaining four TF families(jumonji, MBF1, ULT, and GeBP), which are conventionallynot known to be stress-responsive and are primarily involvedin developmental processes and phytohormone responses

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(Curaba et al. 2003; Noh et al. 2004; Tsuda et al. 2004; Carleset al. 2005; Chevalier et al. 2008; Yu et al. 2008) wereupregulated under water-deficit stress condition. The preciserole of these gene products in water-deficit stress responserequires more detailed investigations.

Modulation of metabolic pathways under water-deficitstress condition

Osmoprotectants

One of the most important class of molecules known toprotect plant cells from dehydration represents osmopro-tectants (Ramanjulu and Bartels 2002). Osmoprotectantsfall in several groups―polyols and sugars (mannitol,trehalose, sucrose, and fructan), amino acids (e.g., proline)and quaternary ammonium compounds (glycine betaine).Trehalose functions in the stabilization of biologicalstructures under abiotic stress in bacteria, fungi andinvertebrates (Ramanjulu and Bartels 2002). We observedthat biosynthesis of trehalose is favored under water-deficitstress condition by more accumulation of transcripts ofseven genes coding for trehalase, TPS, trehalose synthaseand TPP enzymes (trehalase: LOC_Os10g37660;TPS: LOC_Os02g54820, LOC_Os08g34580 andLOC_Os09g23350; trehalose synthase: LOC_Os01g53000and LOC_Os01g54560; TPP: LOC_Os10g40550). Abioticstress tolerance was successfully achieved in rice byoverexpression of Escherichia coli trehalose biosyntheticgenes, otsA and otsB, as a fusion gene (encoding for TPSand TPP, respectively; Garg et al. 2002). TPS1 is alsorequired for normal vegetative development and floraltransition in Arabidopsis (Ramon and Rolland 2007). Thus,trehalose, along with its protective role in stabilizingproteins, might also be helping plants in sustaining normalvegetative and reproductive growth by maintaining normalcell division, cellular differentiation and associated tran-scriptional changes under water-deficit stress condition.The alternative pathway of trehalose showed tissue-specificregulation. This kind of cell type-specific regulation hasbeen reported earlier, however, a possible physiological rolefor such tissue-specific accumulation is unclear (Leyman etal. 2001). However, the alternative trehalose pathway is notyet characterized in rice, but it is well characterized inRhizobium sp. M-11 (Iturriaga et al. 2009). The genesrelated to biosynthesis of sucrose, fructan and mannitol arealso stimulated under water-deficit stress; for example,sucrose synthase (SUS) gets upregulated. SUS expressionwas reported earlier to be induced by cold, dehydration, andosmotic stress (Hesse and Willmitzer 1996; Dejardin et al.1999; Kleines et al. 1999). The transcript level of AtSUS3from Arabidopsis was found to increase under droughtstress and mannitol treatment, as well as during seed

maturation (Baud et al. 2004). Enhanced sucrose biosyn-thesis under temperature shock and cold acclimation inArabidopsis has been reported (Kaplan et al. 2004, 2007).Resurrection plants have been shown to accumulate sucroseand trehalose when dehydrating (Whittaker et al. 2001;Moore et al. 2007). Recently, it has also been found thatglucose and sucrose accumulate in specific locations inresurrection plant tissue during dehydration from desicca-tion (Martinelli 2008). Fructans are known to prevent lipidcondensation during the phase transition and are believed toprotect biological membranes under stress (Hincha et al.2002; Vereyken et al. 2003). Sugars, which play versatilerole in plant development, could also trigger an oxidativeburst in tissues under abiotic stress conditions (reviewed byVan den Ende and Valluru 2009). Under osmotic stress,accumulation of proline helps in stabilizing proteins,membranes and subcellular structures; it also protectscellular metabolism by scavenging reactive oxygen species(Ramanjulu and Bartels 2002). One of the genes upregu-lated during water-deficit stress was δ-1-pyrroline-5-car-boxylate synthetase. It has been reported earlier thatPetunia plants expressing δ-1-pyrroline-5-carboxylate syn-thetase genes (AtP5CS from Arabidopsis or OsP5CS fromrice) accumulated proline and the transgenic plants couldtolerate 14 days of drought stress (Yamada et al. 2005).

Synthesis of non-protein amino acid β-alanine may beenhanced, as seven genes encoding for enzymes involved inbiosynthesis of β-alanine from propionate were upregulatedunder water-def ic i t s t ress (LOC_Os02g17390,LOC_Os05g46480, LOC_ Os05g07090, LOC_Os05g03480,LOC_Os06g24704, LOC_Os06g23870, LOC_Os11g 39220;Electronic supplementary Fig. S6). β-alanine in turn isconverted to β-alanine betaine, which acts as an osmopro-tectant in most members of the highly stress tolerant plantfamily Plumbaginaceae (Rathinasabapathi et al. 2001), medi-ated by N-methyltransferase. Two isomeric genes encodingfor N-methyltransferase were up (LOC_Os06g06560;LOC_Os07g42280) and downregulated (LOC_Os07g49300;LOC_Os09g29710), respectively, under water-deficit stresscondition.

Cell wall components

A number of transcripts encoding for enzymes involved insynthesis of primary cell wall component and enzymesresponsible for cell wall loosening like xyloglucan endo-transglycosylase (XET: LOC_Os08g13920) and six expansins(LOC_Os02g16730, LOC_Os10g39110, LOC_Os02g44108,LOC_Os10g39640, LOC_Os10g40710, LOC_Os06g50400)were upregulated under water-deficit stress condition. It hasbeen shown in earlier studies that cell wall plays crucial role incell enlargement, which is indispensable part of plant growthand development (Cosgrove 2001). Moreover, it has been

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found that the tensile property of cell wall helps inameliorating the shearing force generated during droughtstress in grasses (Balsamo et al. 2006). Hence, loosening ofcell wall and synthesis of structural constituents together couldhelp in coping with the water-deficit stress.

Lipid metabolism

The gene coding for squalene synthase (LOC_Os03g59040),involved in cholesterol biosynthesis, was found to be upregu-lated in the present study. In parallel to this observation,squalene synthase ESTwere found to be overrepresented undera variety of abiotic stresses (Houde et al. 2006). Recent studieshave shown that sterols are required for secretory vesicletargeting which might be facilitating stress adaptation process(Carter et al. 2004). Plant sterols have been shown to cyclebetween plasma membrane and endosomes in an actin-dependent manner (Grebe et al. 2003). Abiotic stress causessignificant intracellular restructuring in plants which leads totransportation, removal and compartmentalization of certainmolecule via vesicle trafficking. Very long chain fatty acidbiosynthesis-related gene (long-chain-3-hydroxyacyl-CoA de-hydrogenase; LOC_Os02g17390) was also upregulated underwater-deficit stress. VLCAFs are known to mainly function asprecursors for components of the cuticle, such as cutin andepicuticular waxes (Post-Beittenmiller 1996), which areknown to prevent water loss at leaf surface.

Secondary metabolite

Accumulation of anthocyanin pigments in vegetative tissueis hallmark of plant stress (Winkel-Shirley 2002). Increasein transcript accumulation of phenylalanine ammonia-lyase(PAL; LOC_Os02g41670) gene was observed, whichmediates conversion of L-phenylalanine to trans-cinnamate,favoring salicylate, flavonoid, coumarin and phenylpropa-noid biosynthesis (Fig. 4; Electronic supplementary Fig.S7). Induction of PAL genes under salt, drought, cold stress(Guo and Wang 2009), wounding, and fungal elicitortreatment (Zhu et al. 1995) have been observed in variousplant species. Flavonol synthase (FLS; LOC_Os02g52840),a key enzyme in flavonoid biosynthetic pathway, wasupregulated in the present study. Flavonoids are known tohave role in providing UV protection (Bharti and Khurana1997; Ryan et al. 2001, 2002), conferring resistance toaluminium toxicity in maize (Kidd et al. 2001) and haveantimicrobial as well as antifungal properties (Dixon andSteele 1999). Isoforms of FLS gene in Arabidopsis showtissue-specific expression as well as differential expressionin response to different environmental conditions (Owens etal. 2008). Recently, a R2R3-MYB transcription factor,MYB12, from Arabidopsis, has been found to be thetranscriptional regulator of flavonol synthase (Mehrtens et

al. 2005). AtMYB60, another member of R2R3-MYBfamily has been assigned the role of transcriptionalmodulator of physiological responses in guard cells whichcould help plants survive desiccation (Cominelli et al.2005). Interestingly, the overexpression of a rice R2R3-MYB transcription factor, OsMYB3R-2, in Arabidopsisconferred tolerance to cold, drought and salt stress (Dai etal. 2007). It has also been reported that overexpression ofCpMYB10 from resurrection plant, Craterostigma planta-gineum, in Arabidopsis led to desiccation and salt toleranceof transgenic lines by altering ABA and Glc signaling(Villalobos et al. 2004). Thus, among the 25 MYB TFsupregulated under water-deficit stress condition in ourstudy, some could be regulating secondary metabolitebiosynthesis helpful in stress response.

Hormone metabolism

Genes coding for enzymes involved in cytokinin biosyn-thesis showed increased transcript accumulation underwater-deficit stress condition (Fig. 4). However, cytokinindegradation-related genes were also found to be upregu-lated under water-deficit stress. Cytokinin is essential fornormal functioning of plants as well as mediating stressresponse by stimulating accumulation of anthocyanin,proline and ethylene as reviewed by Hare et al. (1997).Studies showed that overexpression of isopentenyltransfer-ase (IPT) gene, involved in cytokinin synthesis, on theonset of senescence resulted in suppression of drought-induced leaf senescence leading to drought tolerance oftransgenic tobacco plants (Rivero et al. 2007), however, ithas also been found that overexpression of IPT gene intobacco under light-inducible promoter resulted in elevatedaccumulation of cytokinin level which induced wiltingsymptom as observed during salinity stress (Thomas et al.1995). In Arabidopsis, cytokinin receptor histidine kinases,AHK2, AHK3, and CRE1, have been found to act asnegative regulators in stress responses in a cytokinin-dependent manner (Tran et al. 2007). Thus, it is apparentthat cytokinin mediated regulation might be dependent onthe nature of target cells and precise developmental stages.

Resistant variety (upland rice), on exposure to stress, hasalso been found to trigger biosynthesis of osmoprotectants,cell wall strengthening components, oxidation protectionmolecules, secondary metabolite, and ion transport compo-nents to ensure normal cellular functioning under stresscondition (Chao et al. 2005; Walia et al. 2005; Wang et al.2007). We noted in metabolic pathway analysis that genesinvolved in proline biosynthesis (LOC_Os01g12710;LOC_Os09g26880), sucrose (LOC_Os03g22120), and cellwall component (LOC_Os06g36560; LOC_Os07g04690)were upregulated in upland rice variety (Wang et al. 2007).This observation strengthens the fact that along with such

Funct Integr Genomics (2011) 11:157–178 171

genes, other upregulated genes involved in these pathways might be responsible for conferring stress tolerance.Our study also revealed upregulation of metallothionein(LOC_Os12g38051), methionine sulfoxide reductase(LOC_Os03g24600) , respi ra tory burs t oxidase(LOC_Os11g33120; LOC_Os05g45210), calcium transportingATPase (LOC_Os05g02940; LOC_Os04g51610) and ninecalmodulin binding proteins (Electronic supplementary TableS1). Genes belonging to same groups were upregulated in rootof upland rice variety (Prata Ligeiro) and not in lowlandvariety (IRAT20) after drought stress at anthesis stage(Rabello et al. 2008).

Physiological parameters like root development andstomatal movement have utmost importance in water-deficit stress tolerance. Development of root in bothlowland and upland variety of rice is intricately related todrought tolerance (Fukai and Cooper 1995). Two genes(LOC_Os12g01550 (upregulated in 1-week-old seedling)and LOC_Os03g45750 (downregulated in panicle)) codingfor LOB domain proteins, which are essential for adventi-tious root formation in rice (Liu et al. 2005), weredifferentially regulated under water-deficit stress condition.Involvement of the vacuolar Ca2+-activated channel TPC1and protein phosphatase 2C, are already known in stomatalmovement (Peiter et al. 2005; Pandey et al. 2007). Ourstudy identified more genes coding for homologues of calciumchannel protein TPC1 (LOC_Os01g48680), protein phospha-tase 2C ABI1 (LOC_Os05g49730; LOC_Os01g46760), pro-tein phosphatase 2C ABI2 (LOC_Os05g46040;LOC_Os05g51510; LOC_Os01g40094), and 11 more phos-phatase 2C to be upregulated under water-deficit stress(Electronic supplementary Table S1). These genes,which could be essential for root development andstomatal movement, are still not assigned any pathway,hence, further study is needed for their functionalcharacterization.

Regulation of genes in relation to oxidativeand water-deficit stress

On comparing our 5,611 differentially regulated genes with1,062 oxidative stress-responsive genes from rice, we foundthat 4% of water-deficit stress-responsive genes are alsoresponsive to oxidative stress (Liu et al. 2010). However, weadd more genes coding for enzymes with antioxidant proper-ties, namely, ascorbate peroxidase (LOC_Os04g14680), andsuperoxide dismutase (LOC_Os07g46990). Developmentalstage-specific expression of antioxidant genes under water-deficit stress has also been observed (Electronic supplementaryTables S2 and S3). Jain et al. (2010) reported sevenglutathione-S-transferases (GST) genes to be upregulatedunder water-deficit stress in 1-week old seedling. Weidentified another isoform of GST gene (LOC_Os01g70770)

to be upregulated only in shoot tissue under water-deficit stress,emphasizing on tissue-specific regulation of oxidative stress-related gene expression. Hence, we suggest that specificpathways are operational under water-deficit stress to counter-act oxidative stress in a developmental stage-specific manner.

Downregulation of genes

In higher plants, foliar photosynthetic rate is known todecrease with low relative water content (Lawlor 2002).Stomatal limitation, decrease in ATP content and CO2

concentration, limited metabolic processes, and loss ofrubisco are considered to be the determinant of reducedphotosynthesis under drought condition (Cornic et al. 2000;Lawlor 2002; Parry et al. 2002; Vu and Allen 2009).Present study also showed downregulation of transcriptsrelated to photosynthesis (i.e., photosynthesis-antennaprotein, porphyrin, and chlorophyll metabolism; Electronicsupplementary Table S14) under water-deficit stress. More-over, genes encoding for proteins related to normal geneticfunctioning like, components of RNA polymerase,aminoacyl-tRNA biosynthesis, DNA replication and repairwere also found widely downregulated under stress. Similarobservation was made by Seki et al. (2002b), where genesrelated to photosynthesis were downregulated underdrought stress. They also reported that transcripts involvedin DNA damage repair showed decrease in accumulationunder drought. The downregulation of these genes could infact mean switching between the productivity and thesustenance mode.

Members of TF families, zf_DHHC (four members),SNF2 (three members), SET (two members), and sigma70(two members) were downregulated under water-deficitstress but none were upregulated (Electronic supplementaryTable S15). SET, SNF2 and zf_DHHC TFs are also knownto be involved in development of reproductive organs(Farrona et al. 2004; Thorstensen et al. 2008; Verdier et al.2008). Moreover, six of the 17 members of CO-like TFfamily were found to be downregulated under water-deficitstress (Electronic supplementary Table S3), however, threeother members (LOC_Os06g19444; LOC_Os02g49230;LOC_Os08g15050) were found to have tissue-specific up/downregulation, indicating their involvement in tissuedependent stress response in rice (Electronic supplementaryFig. S1). CONSTANS (CO) gene is known to be involved inthe photoperiodic regulation of flowering (Imaizumi andKay 2006; Kobayashi and Weigel 2007; Kim et al. 2008).However, in earlier studies interplay between stress- andcircadian-regulated gene expression have also been shown(Kreps et al. 2002; Hannah et al. 2005; Achard et al. 2007).Hence, we suggest that the signaling cascade or pathways,which are downregulated under stress, could be targeted byregulated overexpression of these downregulated genes,

172 Funct Integr Genomics (2011) 11:157–178

which might help in sustaining normal functioning understress.

Correlation between water-deficit, cold, and salt stress

Among the genes which are responsive to water-deficit,cold, and salt stress, more overlap was found between saltand water-deficit stress-responsive genes, whereas, water-deficit and cold stress showed least number of commonlyregulated genes. Similar trend of regulation was also notedby Rabbani et al. (2003). Drought and salt stress ultimatelyresult in dehydration of the cell causing osmotic imbalance,which leads to de-regulation of almost every aspect ofcellular physiology and metabolism. Therefore, prominentcross-talk of components regulated by these two stressconditions is not surprising. Even cold stress induces severemembrane damage due to dehydration associated withfreezing, which might be responsible for regulation ofcommon genes under these two stress conditions. The geneexpression data indicate that the major abiotic stresses,water-deficit, low temperature, and salinity, are complexstimuli, because they possess common, yet differentattributes, resulting in signaling cascades that are uniqueto each stress condition and/or have complex networking(Xiong and Zhu 2002; Chinnusamy et al. 2004;Yamaguchi-Shinozaki and Shinozaki 2006; Nakashima etal. 2009).

Genetic overlap in development and water-deficit stress

Complex genetic network functions during development oforgan in plants and it has been found that substantialoverlaps exist between the developmental pathways and thestress-response pathways (Cooper et al. 2003). Some of thegenes are expressed under stress as well as in embryoniccells or during seed desiccation (Sivamani et al. 2000;Medina et al. 2001). Lan et al. (2005) showed that a largefraction of genes regulated by dehydration is also upregu-lated by pollination/fertilization. Similar conclusions weredrawn from analyses of promoter-GUS fusions of cold-inducible RD29A, COR15A, KIN1, and COR6.6 genes inArabidopsis which were regulated during plant develop-ment under both stressed (cold) and unstressed conditions(Yamaguchi-Shinozaki and Shinozaki 1993; Baker et al.1994; Wang and Cutler 1995).

In cereals, it has been seen that water-deficit stressduring flower induction and inflorescence developmentleads to a delay or complete inhibition in flowering (Winkelet al. 1997). Interestingly, TFs belonging to AP2, MYB andPBF2-like (whirly) family were found to be involved inpanicle development as well as water-deficit stress re-sponse, implying that they may represent the cross-talkcomponents between development and stress.

Acknowledgement This work is supported by the Department ofBiotechnology, Government of India, the Council of Scientific andIndustrial Research (research fellowship to P.D.), and the UniversityGrants Commission (research fellowship to P.K.D.).

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