高温诱导表达基因筛选

Harsh Chauhan •Neetika Khurana •

Akhilesh K.Tyagi •Jitendra P.Khurana •

Paramjit Khurana

Received:25November 2009/Accepted:30September 2010/Published online:23October 2010ÓSpringer Science+Business Media B.V.2010

Abstract To elucidate the effect of high temperature,wheat plants (Triticum aestivum cv.CPAN 1676)were given heat shock at 37and 42°C for 2h,and responsive genes were identified through PCR-Select Subtraction technology.Four subtractive cDNA libraries,including three forward and one reverse subtraction,were con-structed from three different developmental stages.A total of 5,500ESTs were generated and 3,516high quality ESTs submitted to Genbank.More than one-third of the ESTs generated fall in unknown/no hit category upon homology search through BLAST analysis.Differential expression was confirmed by macroarray and by northern/RT-PCR analysis.Expression analysis of wheat plants sub-jected to high temperature stress,after 1and 4days of recovery,showed fast recovery in seedling tissue.How-ever,even after 4days,recovery was negligible in the developing seed tissue after 2h of heat stress.Ten selected genes were analyzed in further detail including one unknown protein and a new heat shock factor,by quanti-tative real-time PCR in an array of 35different wheat tis-sues representing major developmental stages as well as

different abiotic stresses.Tissue specificity was examined along with cross talk with other abiotic stresses and puta-tive signalling molecules.

Keywords Heat stress ÁHSF ÁHSPs Á

Subtractive hybridization ÁTranscriptome ÁWheat

Introduction

Climate change and abiotic stress affect agriculture and crop production adversely.Of the various climatic factors affecting agriculture,temperature is one of the most important as higher temperatures adversely affect plant growth and yield (Kurek et al.2007).Global mean surface temperature increased by ^0.5°C in the twentieth century and is projected to rise in a range from 1.8to 4.5°C by 2100(Working group I report,IPCC 2007).An in depth analysis carried out by Lobell and Field (2007),involving effect of global warming on six major food crops from the year 1982–2002,revealed combined yield loss of around 40million tonnes for wheat,corn and barley per year,where wheat alone accounts for almost half of the yield loss (19mt year -1).

At the global level,the demand for wheat is growing at approximately 2%per year (Rosegrant and Cline 2003).However,genetic gains in yield potential of wheat stand at less than 1%(Sayre et al.1997).Consequently,an yield plateau has been observed in the last decade and has been attributed to many factors among which high occurrence of terminal heat stress appears to be the most prominent (Nagarajan 2005).A significant proportion of wheat grown in South Asia experiences heat stress out of which a major portion is present in India (Joshi et al.2007).Wheat is grown in India under sub-tropical environment during mild

Electronic supplementary material The online version of this article (doi:10.1007/s11103-010-9702-8)contains supplementary material,which is available to authorized users.

H.Chauhan ÁN.Khurana ÁA.K.Tyagi ÁJ.P.Khurana ÁP.Khurana (&)

Department of Plant Molecular Biology,University of Delhi,South Campus,Benito Juarez Road,Dhaula Kuan,New Delhi 110021,India

e-mail:param@genomeindia.org

Present Address:A.K.Tyagi

National Institute of Plant Genome Research,Aruna Asaf Ali Marg,New Delhi 110067,India

Plant Mol Biol (2011)75:35–51DOI 10.1007/s11103-010-9702-8

Tolerance to heat stress is a complex phenomenon and controlled by multiple genes imparting a number of phys-iological and biochemical changes such as alteration in membrane structures and function,tissue water content, composition of protein,lipids,primary and secondary metabolites(Shinozaki and Dennis2003;Zhang et al. 2005;Kotak et al.2007;Barnabas et al.2008;Huang and Xu2008)and no single trait fully explains why some wheat varieties are able to give better yield even when they experience heat stress.A significant increase in our understanding of molecular basis of plant stress tolerance will allow breeders to address the problem of built-in resistance against high temperature stress.Hence,there is an urgent need to elucidate molecular and genetic basis of heat tolerance in wheat,to identify beneficial genes and alleles,and to utilize them in molecular breeding pro-grammes targeted to produce superior wheat cultivars in the future.Recent advances in genome sequencing and global gene expression analysis has led to the identification of hundreds of plant genes governing abiotic stress response(Fowler and Thomashow2002;Kreps et al. 2002).

In wheat,although the effect of high temperature on physiology and biochemistry has been well investigated, there are only a few studies devoted to transcript profiling of wheat during high temperature stress(Altenbach and Kothari2004;Altenbach et al.2007,2008;Qin et al.2008). Moreover,almost all of these studies concentrate on the effect of high temperature during the grain growth period however,studies focusing on the effect of elevated tem-perature on transcriptome of seedlings andflowering stages are rare(Nagarajan2005).

Subtractive hybridization coupled with Suppression Subtractive Hybridization(SSH)is a powerful tool for cloning differentially expressed genes and has been applied in many studies to identify abiotic stress regulated gene transcripts in plants(Boominathan et al.2004;Liu et al. 2008).Keeping this in mind,we carried out a detailed transcriptome analysis through suppression subtractive hybridization of heat stressed and non-stressed tissues of wheat at three different growth stages,viz.young seedling, pre-pollinatedflower and developing grains.Heat tolerance related gene transcripts were identified based on their putative functions and validated by cDNA macroarray and northern/RT-PCR analysis,with the aim to unravel the complexity associated with heat stress response in wheat. Materials and methods

Plant material,growth conditions and stress treatment

Indian bread wheat(Triticum aestivum)cv CPAN1676, which is an intermediate type of variety for heat tolerance was used in this study and plants were grown in a growth chamber(Conviron,Canada)maintained at20°C day:night temperature in a16:8h photoperiod.Plants were also grown in the departmental garden during crop season in potted soil.To reduce the environmental variation,the same experiment was conducted twice with two separate growth chambers switched for control and heat stress treatments and the plant tissues from two experiments were pooled together.The plants were arranged in a randomized complete block design within each growth chamber.For seedlings,heat stress was given by transferring10-day-old plants to a growth chamber maintained at37°C for2h and then at42°C for another2h.After heat stress treatment, control and stressed seedling shoot tissues were sampled,flash frozen in liquid nitrogen and stored at-80°C until RNA isolation.Remaining seedlings were transferred back to the control growth chamber for recovery and seedling shoot tissue was again sampled after1and4days of recovery.For unfertilizedflower library,heat stress was given to the potted plants after spikes were fully extended from theflag leaf sheath but prior to anthesis.Sampling offlower tissue was done by removing lemma and palea from the spike of stressed and control plants,remainingflower organs(anther and ovary)were stored at-80°C until RNA isolation.For developing grains,potted plants grown in departmental garden were transferred for2h to the growth chamber maintained at37and42°C at7,15,21and 30days after anthesis followed by recovery period as above.Sampling of developing grains was done from spikes of control,stressed and recovery plants and devel-oping seeds were stored at-80°C until RNA isolation. Similarly,for northern analysis heat stress was given at 37°C as well as at42°C for all the tissues.

For other stress and hormone treatments,10-day-old seedlings were used and salt stress was given by immersing seedlings in150mM NaCl solution,drought stress by2% mannitol solution for1day.Similarly,ABA(10l M, Sigma,USA),brassinosteroid(1l M epibrassinolide, Sigma),salicylic Acid(100l M)and CaCl2(10mM) treatments were also given for1day.After treatment, sampling of seedling shoot tissue was done and tissue frozen and stored at-80°C.

Isolation of total RNA,mRNA and construction

of subtracted cDNA libraries

Total RNA from seedling shoot andflower tissues(anthers and ovaries)were isolated by Trizole Reagent(Invitrogen) as per the manufacturer protocol.Total RNA from devel-oping wheat seeds were isolated(stages as mentioned above)as per the method described by Singh et al.(2003). mRNA was isolated from total RNA by PolyATract mRNA isolation system(Promega,USA)as per the manufacturer’s protocol.Subtracted cDNA library was constructed by using CLONTECH PCR-Select cDNA subtraction kit (CLONTECH Laboratories,USA)following manufac-turer’s protocol.In brief,tester(stressed)and driver(con-trol)double stranded cDNAs were prepared from2l g mRNA.Tester and Driver cDNAs were separately digested with Rsa I to obtain shorter blunt ended molecules.Two tester populations were created by ligating two aliquots of diluted tester cDNA with two different adaptors(adaptors1 and2R),separately.First hybridization was performed by the following procedure.Each tester population was mixed with an excess of digested driver cDNA.The samples were heat denatured and allowed to anneal at68°C for8h.The two samples from thefirst hybridization reaction were mixed together,and more denatured driver cDNA was added for further overnight hybridization to enrich differ-entially expressed sequences.Differentially expressed cDNAs,with different adaptor sequences at two ends,were selectively amplified by PCR and a second PCR was done with nested primers to further reduce the background.The subtracted and enriched DNA fragments were directly cloned into pGEMTeasy T/A cloning vector(Promega, USA).Electro competent cells of Escherichia coli DH5a (Invitrogen)were transformed with the ligation mix and plated on LB-agar(Pronadisa,Spain)plates containing ampicillin,isopropylthio-b galactoside,and X-gal for blue-white selection(Sambrook and Russel2001).For seedling and unfertilizedflower library,subtraction was done in forward direction,while for developing seed library,sub-traction was done in both forward and reverse direction. Thus,a total of four different subtracted libraries were constructed.

Sequencing and EST analysis

For high-throughput sequencing,clones from all libraries were cultured in96well deepwell plates containing 1.25ml LB broth(Pronadisa,Spain)supplemented with 100l g ml-1ampicillin for22h at37°C with shaking at 200rpm.Plasmid DNA was prepared using alkaline lysis preparation protocol(Genesis Workstation200,TECAN, Switzerland).All sequencing reactions were conducted with the modified M13reverse primer(50-AGC GGA TAA CAA TTT CAC ACA GG-30).Sequencing was performed in an ABI3700capillary sequencer.The tracefiles obtained from the ABI sequencer were then entered into a pipeline that entailed a semiautomatic process to perform the base calling using Phred(http://www.phrap.org).Each sequence was screened for overall base quality and contaminating vector,mitochondrial,ribosomal,and E.coli sequences were removed.All sequences of the assembled EST data-base,singlets and contigs,were examined for homology to the NCBI nr database by BLASTX analysis(Altschul et al. 1997).For Gene Ontology(GO)term identification BLASTX was done against TIGR rice genome database to identify Locus IDs of rice homologs.

Amplification of cDNA inserts and preparation

of cDNA macroarrays

Individual clones of the subtracted cDNA libraries were amplified in a96-well PCR reaction plate using adapter primer1and2R in a50-l l reaction.PCR was conducted with the following program using Taq DNA Polymerase (Roche,Germany):initial denaturation at94°C for5min, followed with94°C for30s,60°C for30s,72°C for1min with30cycles.The products were analyzed in agarose gel to confirm the insert size,quality,and quantity by running with GeneRuler TM DNA size markers(Fermentas,Lithu-ania).PCR products were denatured by adding an equal volume of0.6M sodium hydroxide.Equal volume of the denatured PCR product(about100ng)of C250bp of size was spotted on two Hybond N membranes(Amersham Pharmacia Biotech,UK)to make two identical arrays usingdot-blot apparatus(Amersham Pharmacia Biotech,UK)in 96-well format.In addition,a PCR product of wheat actin cDNA(GenBank accession no.AY663392)using primer sequences(50-ATG GCT GAC GGT GAG GAC ATC-30 and50-AGG TGC CAC ACG GAG CTC ATT G-30)was spotted as internal control to normalize the signals of two different blots corresponding to stressed and unstressed samples.A PCR product of neomycin phosphotransferase (NPTII)gene from the vector pCAMBIA2301(GenBank accession no.AF234316)using primer sequences(50-TTT TCT CCC AAT CAG GCT TG-30and50-TCA GGC TCT TTC ACT CCA TC-30)was also spotted as a negative control to subtract the background noise.The membranes were neutralized with neutralization buffer(0.5M Tris–HCl,pH7.5,1.5M NaCl)for2min,washed with29SSC, and cross linked using UV cross linker(Amersham Phar-macia Biotech,UK).

Probe preparation,hybridization,and data analysis Control and stress mRNAs were labeled with a32P-dATP (BRIT,India)byfirst-strand reverse transcription using SuperScriptIII First Strand cDNA Synthesis Kit(Life Technologies,USA).Two micrograms of mRNA was labeled in a20-l l reaction volume containing19reaction buffer,1l L of oligo dT(20)primer(50l M), 2.5mM dCTP,dTTP,dGTP,0.02mM dATP,5l L of a32P-dATP (10mCi/ml;3,000Ci/mmol),and200units of reverse transcriptase SuperscriptIII.After incubation at45°C for 1h,RNA was removed by incubating with RNase H at 37°C for20min.Radio labeled cDNAs were purified by Sephadex G-25column(Amersham-Pharmacia Biotech, UK)and suspended in ExpressHyb hybridization buffer (CLONTECH Laboratories,USA).Nylon membranes were prehybridized with the same buffer with10l g/ml of denatured herring sperm DNA for4h at65°C and hybridized with denatured control and experimental cDNA probes at the same condition for24h.The membranes were washed three timesfirst with2xSSC,0.05%SDS for 10min and then once with0.19SSC,0.1%SDS,for 10min,at65°C.Membranes were than exposed to BioMax MSfilms(Kodak,USA)with intensifying screens and stored at-80°C for2–3days.Images of the membranes were scanned in Typhoon(Amersham-Pharmacia Biotech, U.K.)and signal intensities were analyzed.The program allows normalization of the signal against background. Fold change was calculated as suggested by Boominathan et al.(2004).

Fold change¼

Effective signal intensity in heat

Effective signal intensity in control

Ä

Intensity of actin in heat

Intensity of actin in control

Heatmap was constructed using Mayday 2.0software

(http://www.zbit.uni-tuebingen.de/pas/mayday/).The val-

ues obtained from fold change were used as input data,and

four maps were constructed.

Northern hybridization

Twenty micrograms of total RNA from control and treated

samples were resolved in 1.2%agarose gel containing

formaldehyde and transferred to Hybond N membrane

(Amersham Pharmacia Biotech,UK).PCR-amplified

individual cDNA fragment(with primers corresponding to

adaptor1and2R)was purified from agarose gel.Probes

were labeled with a32P-dATP using Megaprime DNA

labelling system(Amersham Pharmacia Biotech,UK)and

purified through Probquant G-25column(Amersham

Pharmacia Biotech,UK).Northern hybridization was per-

formed and band intensity calculated following the proce-

dure described above.

Semi-quantitative RT-PCR

For expression study of wheat heat stress-related genes

from unfertilizedflower tissue under high temperature,a

two-step RT-PCR was used in place of northern analysis

because of the limited amount of the tissue available.2l g

of mRNA from each sample was used to synthesize thefirst

strand cDNA using the SuperScriptIII First Strand cDNA

Synthesis Kit(Life Technologies,USA)in a20l l reaction

volume as described above for cDNA labelling.After

RNase H treatment,80l L MQ was added to each tube.

1l l of this diluted cDNA template was added to each PCR

reaction with wheat actin gene primers and gene-specific

primers.Wheat gene-specific primers were designed by

using GENERUNNER programme(www.generunner.com)

(Supplementary Table1).PCR was conducted with the

following program using Taq DNA Polymerase(Roche,

Germany):initial denaturation at94°C for5min,followed

with94°C for30s,60°C for30s,72°C for1min with

30cycles.The PCR products were checked on1.5%aga-

rose gel in19TAE buffer with EtBr.All gene-specific

primers amplified cDNA fragments with corresponding

sizes predicted by the designed primer.The expression

level of genes in each sample were compared using actin as

an internal control.

Real time expression analysis

Total RNA was isolated using the RNeasy Plant mini kit

(Qiagen,Germany)according to the manufacturer’s

instructions,including on-column DNase I treatment to

remove genomic DNA contamination.2l g of the totalRNA was used as template to synthesize cDNA employing the High Capacity cDNA Archive kit(Applied Biosystems, USA)and mixed with200nM of each primer and SYBR Green PCR Master Mix(Applied Biosystems)for real-time PCR analysis,using the ABI Prism7000Sequence Detection System and Software(PE Applied Biosystems) according to the manufacturer’s protocol.The primer pairs were designed by using Primer Express2.0software(PE Applied Biosystems)and were checked by the BLAST program in wheat sequences available in the NCBI data-base to ensure that the primers amplify a desired unique cDNA segment(Supplementary Table2).The specificity of the reactions was verified by melting curve analysis.The relative mRNA levels for the genes studied in RNA iso-lated from various tissue samples were quantified with respect to the internal control,Actin.At least two inde-pendent RNA isolations(biological replicates)were used for cDNA synthesis,and each cDNA sample was subjected to real-time PCR analysis in triplicate.The Ct(threshold cycles)values were averaged for three replicates and the values normalized with the Ct values of internal control Actin.

Results and discussion

Temperatures above optimum levels are known to impair dry matter accumulation,resulting in reduced grain weight and size in all major cereal crops,including wheat,maize, barley and rice.Studies concerning physiological and molecular effects of elevated temperatures due to global warming are thus inevitable(Yamakawa et al.2007).We generated wheat heat stress ESTs representing three dif-ferent tissues and two subtractive hybridizations and sub-mitted a total of3516ESTs to public database.Data obtained were further validated by northern and dot blot analyses and some significantfindings of these analyses are discussed below.

Identification and characterization of heat stress related ESTs

Four subtracted cDNA libraries,represented by a total of 5,500clones,were constructed(Table1);three forward subtracted libraries(FSH)contained transcripts putatively differential/up-regulated by heat stress(total4,000clones) and the reverse subtracted library of developing grains representing transcripts putatively down-regulated by heat stress(total1,500clones).The insert size in the four libraries ranged between250bp to2kb.High throughput sequencing was done for all5,500clones.These ESTs were assembled into995contigs and666singlets by PHRAP including CROSS-MATCH for screening vector sequences.Table1gives the details of contigs and singlets present in each library.These ESTs were submitted to GenBank dbEST(www.ncbi.nlm.nih.gov/dbEST),with accession number from GD186391to GD1906.In order to annotate these contigs/singlets from clustered ESTs, protein homologies were searched in public database (NCBI and TIGR)using BLASTX and putative functions assigned.There were a number of redundant clones,indi-cating their abundance in the heat stressed samples,how-ever,the largest category was of no match or proteins with unknown functions(Table1).The group‘No hits found’constitutes around40%of all contigs and singlets taken together.

Contigs and singlets included in all the libraries were classified separately by GO(Gene Ontology)annotations of rice.For identification of GO terms of corresponding rice homologues,all contigs and singlet reads were

Table1Description of different libraries

Library name Tissue Total EST

sequenced No.of

contigs

No.of

singlets

No Hit/

unknown

ESTs checked by

macroarray

ESTs submitted to

GENBANK database

(Accession number)

Taz10days seedling(FSH)1,500201170145262846

(GD1061-GD1906) Tau Unfertilizedflower(FSH)1,00020990165127546

(GD188515-GD1060) Taw Developing seed(RSH)1,5003002302121481,146

(GD187369-GD188514) Tax Developing seed(FSH)1,50028517614995978

(GD186391-GD1873680) Total5,5009956666716323,516

FSH forward subtractive hybridization,RSH reverse subtractive hybridizationsubjected to BLAST against rice genome database (www.tigr.org)and locus ID retrieved.These locus IDs were used to assign GO terms under three main categories viz.biological process,cellular component and molecular function(http://www.tigr.org/tdb/e2k1/osa1/GO.retrieval. shtml)(Fig.1a–c).Functional characterization through GO based Molecular Function category showed great diversity among the libraries(Fig.1a).For seedling and flower forward subtracted libraries,more than one-third transcripts could not be assigned any known GO term, possibly due to a large number of unknown/novel genes identified in the present study.Catalytic activity makes the second largest subcategory.Transporter activity also makes a significant part forflower library and reverse subtracted developing seed library.The reverse subtracted developing seed library showed most complex picture and was sub-divided into16subcategories,with transcription regula-tion,hydrolase activity and catalytic activity being the largest subcategories.However,in the forward subtracted developing seed library,transcription factor is the largest subcategory followed by no GO ID.In the biological process category,one-third of the transcripts account for stress-related subcategory in all the three forward sub-tracted libraries,while it was only13%of the reverse subtracted developing seed library showing a2-threefold increase in the number of stress-related genes by high temperature(Fig.1b).Transcription and metabolic process makes the second and third largest subcategories,respec-tively,in forward subtractions.Metabolic process is the largest subcategory in the reverse subtracted developing seed library followed by transcription and carbohydrate metabolism,as expected,because developing wheat seed is a reservoir of endospermic carbohydrate(Fig.1b).All the libraries were essentially similar for cellular component category,with nucleus,mitochondria and plastids accounting for more than60%of total transcripts.How-ever,the components representing cytoskeleton and endo-plasmic reticulum appeared in both the developing grain

libraries suggesting a more elaborated role in this tissue (Fig.1c).

To gain further insight into putative functions of ESTs from all four libraries,protein sequences of all corre-sponding rice unknown/hypothetical proteins based on BLASTX homology search were retrieved.These protein sequences were again searched for presence of putative conserved domain using SMART database.It was found that a majority of the proteins have one or more domains related to stress-associated functions,such as Aha1_N SNARE_assoc,FKBP_C and TPR,STI1and UBA,SUI1,AAA,DnaJ,Inos-1-P-synth,zinc finger zf-DHHC and zf-C2HC,Usp,STI1and TBR,along with many other conserved domains of unknown function.Supplementary Table 3lists the library contigs,corresponding rice homolog accession numbers as well as details of the con-served domains along with their Pfam accession numbers.A combination of approaches were employed to assess differential gene expression analysis in response to heat stress as well as to gain some insight into its regulation and signal transduction by plant hormones,and to check for cross talk with other abiotic stresses.The relative fold change was calculated by reverse northern cDNA macro-array.Figure 2shows Heatmap generated by MAYDAY software of all the genes regulated by heat stress as well as by at least one inducer in seedling library,along with effect of heat stress in other three libraries.Of these,178tran-scripts showed more than fivefold increase after 2h of treatment while more than 95%transcripts showed more than twofold induction (Fig.2).The complete list of all 632transcripts considered for reverse northern analysis is given in the Supplementary Tables 4to 7,along with respective fold change and Genbank Accession Numbers.Seedling response to heat stress and hormonal regulation

One-day and four-day recovery tissues along with control tissues treated with CaCl 2,ABA,BR,SA and NaCl were subjected to reverse northern analysis for a total of 262transcripts of the seedling library.After 1-day recovery,only 46transcripts showed more than twofold induction,indicating a fast recovery in the seedling tissue,while,after 4days of recovery,52transcripts showed more than two-fold induction,possibly due to reappearance of some genes which are important for acclimation and transcriptional reprogramming for normal growth and development (Supplementary Table 4).

Fig.2Heat map representing expression analysis of ESTs in different libraries and treatments.The order of the genes is the same as given in respective Supplementary table 4–7

c H S 1 D a y R e c o v e r y 4 D a y R e c o v e r y C a C l 2

A B A N a C l S A B

R

H

S

H S 1 D a y R e c o v e r y 4 D a y R e c o v e r

y

H

S

-14.24+21.26

Abiotic stresses cause rapid elevation in the cytosolic calcium ion concentration and this level remains consid-erably high even after long recovery(Knight et al.1998; Gong et al.1998).This change in calcium level affects the binding activity of heat stress transcription factors(HSF)to the heat shock elements(HSE)(Mosser et al.1990).Liu et al.(2003)proposed a putative Ca2?mediated signal transduction pathway for heat stress.The heat stress signals are perceived by an unidentified receptor,which upon activation increases the concentration of calcium ion through opening of calcium channels.This elevated cal-cium directly activates calmodulin which in turn promotes the DNA binding activity of heat shock factors,thus ini-tiating synthesis of heat stress-related proteins such as HSPs.Very recently,Saidi et al.(2009)have found a Ca2? permeable channel in the plasma membrane that is acti-vated by high temperature and modulate the intensity of heat shock response.In the present study,we found that 16%of seedling subtractive library ESTs are also up-reg-ulated by a2-h treatment with10mM CaCl2.Mainly HSPs and many calcium binding protein like TaCAM3-1,calci-neurin and calnexin along with other unidentified calcium binding domain containing proteins,are induced,as revealed by the analysis of all the three forward subtractive libraries.Previous studies have shown that level of cal-modulin is increased in heat stress response in maize(Gong et al.1997)and wheat(Liu et al.2003).

ABA is the key hormone involved in drought stress signal transduction and most of the genes that respond to drought also respond to exogenous ABA treatment(Seki et al.2001,2002).Since drought and heat stress frequently occur simultaneously in thefield conditions in many parts of the world,ABA induction is an important regulator of thermotolerance(Maestri et al.2002).Gong et al.(1998) found that as far as thermotolerance is concerned,ABA works synergistically with calcium.In the present investi-gation,we found that ABA is regulating49ESTs and most of them fall in the unknown category.Brassinosteroids protect plants from a variety of environmental stresses such as high and low temperatures,drought,salinity and herbi-cide(Khripach et al.2000;Krishna2003;Kagale et al. 2007).Daubhadel et al.(1999,2002)found that exogenous application of24-epibrassinolide increases the basic ther-motolerance of Brassica napus and tomato seedlings.We found that among the ESTs generated from the seedling library,the largest number of ESTs(total86)are up-reg-ulated after a2-h treatment with100nM epibrassinolide. Among the other genes,ESTs related to HSPs and detox-ifiers were highly up-regulated by brassinosteroid(Sup-plementary Table4).Although salicylic acid(SA)is known to take part primarily in biotic stress signalling (Heil and Bostock2002),levels of SA were found to increase within5min of heat stress onset suggesting its role in heat stress signalling as well(Kaplan et al.2004). Similarly,in the present study,SA treatment stimulated the expression of83out of262ESTs checked through cDNA macroarray.Several studies have shown that exogenous application of SA helps to confer thermotolerance in plants (Dat et al.1998;Clarke et al.2004).It is well documented that several steps in heat stress response pathway overlap with those involved in the response to various other forms of abiotic stresses such as drought and cold(Zhu2001; Rizhsky et al.2004;Yamaguchi-Shinozaki and Shinozaki 2006;Swindell et al.2007).The present data suggest that salinity is also not an exception in this respect.Thus,it is evident that the heat stress signal transduction is quite complex.There is a possibility of many parallel heat stress transduction pathways being governed by specific hor-mones or there could be a single pathway and different hormones play specific roles in the network.

We also checked36gene transcripts from the seedling library by northern analysis for their response at37and 42°C(Fig.3).These genes were selected from different functional categories,such as molecular chaperons and HSPs,transporters,protein modifiers,signalling molecules, stress-related and unknown functions.Figure3shows that most of these genes are highly inducible by high tempera-ture and remain stable at both temperature regimes.How-ever,there are a few which have greater expression at42°C. Two of such genes encode unknown proteins and may be of special interest as they are induced at42°C only,suggesting their specific role during severe heat stress.Another such gene is Rubisco Activase B(RcaB),which is induced at 37°C,but its transcript is relatively reduced at42°C.

Transcripts of many genes,such as HSPs,lipid transfer protein,L-myo -inositol-1-phosphate synthase (MIPS),cpn60,calcium binding proteins and membrane binding proteins were observed to be induced and stable at 37and 42°C.Calcineurin is a Ca 2?and calmodulin dependent serine/threonine phosphatase and its expression is observed during various abiotic stresses in Arabidopsis (Kudla et al.1999).Nonspecific lipid transfer proteins have also been seen to increase by both biotic and abiotic stresses as they protect plants by formation of a hydrophobic layer (Trevino and Ma 1998).L-myo -inositol-1-phosphate synthase (MIPS)is known to protect plants from salt and drought stress (Boominathan et al.2004;Majee et al.2004),and under transgenic conditions provide higher photosynthetic com-petence and better growth during salinity stress.

Expression profiles of ten selected genes from four libraries in 35different wheat tissues sampled on the basis of various growth stages and stress treatments were also monitored by real time PCR (Fig.4).Among these genes,one unknown putative membrane protein encoding gene (chosen from seedling library)was found to be seedling specific and besides heat stress,was also regulated by other stresses,brassinosteroids and ABA.This contig (Taz Con 95)harbours a full-length EST having an ORF coding for 71amino acids.The deduced amino acid sequence indicated the protein to be highly hydrophobic with one uncharacterized transmembrane domain UPF0057.Sub-stantial transcript amount was present even after 4days of heat stress recovery,suggesting its role in stress amelio-ration and recovery (Fig.4a).We chose two heat shock proteins for detailed analysis by real time PCR,i.e.HSP 80and a chloroplastic small HSP 26.Both these HSPs were present in seedling,flower and in developing seed libraries and induced by heat stress as well as by other stresses in different tissues but not present after 4days of recovery in root and shoot tissues (Fig.4b,c).Interestingly,HSP 26transcript was found in very high amount in the heat-stressed flower and developing seed tissues.

A number of ESTs generated in the present study repre-sent the proteins related to protein synthesis,such as initia-tion and elongation factors,protein disulphide isomerases,ADP-ribosylation factor,ribosomal proteins,Clp protease and proteosome subunits,nucleic acid binding proteins,signal transduction molecules and transcription factors,and proteins involved in various metabolic pathways like for-mate dehydrogenase,beta amylase and triose phosphate isomerase.Heat stress is known to result in misfolding of newly synthesized proteins and the denaturation of existing proteins.Chaperon functions,the prevention of denaturation by misfolding or the refolding of already denatured proteins

MIPS (GD1122)

Hypothetical protein (GD1691)PIP2Putative transmembrane protein (GD1191)Unknown (GD1761)

Lipid transfer protein (GD1881)mRNA binding protein (GD1727)

Transmembrane protein Precursor (GD1144)Unknown (GD1204)Glutathione transferase (GD1321)HSP100 (GD1439)Unknown (GD1309)HSP16.9 (GD1366)Rubisco activase (GD1104) Unknown (GD1371)Ta WIN1 (GD1859)Chaperonin 60 (GD1195)Calmodulin (GD188788)C 37o C 42o C

HSP70 (GD1256)STi like (GD1073)HSP 80 (GD1345)

Peptydylprolyl isomerase (GD1773)Calmodulin binding protein (GD1073)HSP17.3 (GD1311)HSP26.6 (GD1297)Cinnamoyl Co A reductase (GD1084)Unknown (GD1185)

Ribosomal protein L34 (GD1184)Calcineurin (GD1399)

HSP 18 (GD1311)C 37o C 42o C

Stress responsive protein (GD1397)HSP23.5 (GD1560)Unknown (GD1217)Expressed protein (GD1097)Unknown (GD1087)Unknown (GD1114)

is one of the major functions of HSPs(Klueva et al.2001).In our study,HSPs make the single largest contribution in seedling heat stress subtractive library,accounting for nearly 20%of total ESTs and we found that almost all the HSPs are highly represented in developing seed forward subtractive library also.Although in vivo function of HSPs is largely unknown,in vitro experimentation suggests that cytosolic small HSPs function as molecular chaperones by preventing thermal aggregation of proteins and facilitate their reacti-vation after stress(Lee et al.1995,1997).Majoul et al. (2004)while working on proteomics of wheat seed devel-opment under heat stress found the constitutive accumula-tion of small HSPs,but a clear increase in their relative protein abundance upon stress treatment.Small HSPs are known to protect developing embryo from heat stress as it is not responsive to heat stress on its own(DeRocher and Vierling1994).

Rubisco is the enzyme that converts CO2into plant biomass.Its activation in light is regulated by stromal enzyme Rubisco Activase(Salvucci and Ogren1996). High temperature is well known to inhibit photosynthetic CO2fixation in many plants,due to temperature induced decrease in the activation state of Rubisco(Law and Crafts-Brandner1999;Salvucci and Crafts-Brandner2004a,b). We found that in wheat,at least one isoform,RCAb,is heat induced(Fig.3)as revealed by dot blot as well as by northern analysis.Recently,an association has been reported between heat-induced chloroplastal cpn60and rubisco activase(Salvucci2008)and a positive significant correlation was found between the expression of a wheat RCA and yield under heat stress environment(Ristic et al. 2009).Kurek et al.(2007)produced thermotolerant trans-genic Arabidopsis plants by over-expressing a heat stress tolerant RCA,thereby validating the role of RCA in acquired thermotolerance.

Flower response to heat stress

In theflower forward-subtracted(FSH)library,all the127 transcripts showed rapid induction after2h of heat stress (Supplementary Table.5).Highest induction was shown by an unknown protein gene(21-fold induction)followed by a wheat calmodulin(TaCAM3-1),C3HC4type zincfinger protein,14-3-3protein,chitinase and wheat HSP82(wheat HSP90homolog)(Supplementary Table5).Additionally, these127genes were also tested for anther or ovary specificity through the macroarray probed with anther or ovary heat stressed RNA.We found that majority of these 127genes have common induction in anther and ovary,but there are some genes which have differential/altered induction pattern(Supplementary Table5).Figure5shows genes selected from unfertilizedflower subtracted library. We have checked a total of19genes through RT-PCR analysis for their response towards heat stress and their specificity,if any for anther and ovary tissues.Most of these genes are present in both the tissues,however,a putative translation factor is specific to ovary and a lipid transfer protein(LTP-2)is more abundant in anther tissue. Recently,a lipid transfer protein of Arabidopsis thaliana (AtLTP5)has been functionally characterized and pollen-targeted overexpression of Wild-Type LTP5or ltp5-1resulted in adverse effects on pollen tube tip growth and seed set(Chae et al.2009).Transcription factors,such as b-ZIP and zincfinger proteins,along with a calmodulin (TaCAM3-1)are highly expressed upon high temperature stress(Supplementary Table5).We have also checked the relative transcript abundance of this wheat calmodulin by real time PCR analysis and found that apart from different flower tissues,a significant amount of its transcript was also present in drought-stressed root and shoot tissue but was totally absent from various developing seed tissues (Fig.4d).Another heat inducible gene is a14-3-3protein, which is more inducible in ovary tissue than the anthers (Fig.5).There are several reports to show that14-3-3 proteins are involved in plants response to environmental stress such as stress induced senescence and increased drought tolerance(Yan et al.2004)and salt and cold stress (Chen et al.2006).Another such gene is a C3HC4RING finger protein,which is heat inducible in anther tissues and

C 37o C C 37o C C 37o Crelatively constitutive in ovary tissue(Fig.5).These pro-teins have been studied in both Arabidopsis and rice(Stone et al.2005;Ma et al.2009)and found to be responsive to abiotic stresses.Another transcription factor identified fromflower library is a bZIP protein,which is inducible by heat stress(Fig.5).In plants,bZIP transcription factors control many processes such as ABA signalling,seed and flower development(Jakoby et al.2002)and are involved in imparting stress tolerance(Fujita et al.2005;Zhang et al.2008).

Floral abnormalities induced by heat stress leading to spikelet sterility are known in rice(Takeoka et al.1991). Saini and Aspinall(1982)reported that temperature above 30°C duringfloret formation causes complete sterility in wheat.Sinclair and Jamieson(2006)also highlighted that grain number in wheat is highly dependent on environ-mental conditions prior to and duringflower formation. Comparative studies have shown that high temperatures cause more damage to anthers as compared to ovaries,and pollen formation is one of the most heat sensitive devel-opmental stage in cereals in general and in wheat in par-ticular(Saini and Aspinall1982;Saini et al.1984).In barley,Abiko et al.(2005)have shown through serial analysis of gene expression that high temperature results in the transcriptional inhibition of genes that are active in anther under normal temperature conditions.We have observed that148ESTs are affected by heat stress treat-ment in unfertilized wheatflower and confirmed their expression by dot blot and RT-PCR analysis.Apart from many unknown proteins,we also found a large number of transcription factors up-regulated by high temperature, which are being further characterized in our laboratory. Developing seed and heat stress

Forward Subtraction

The macroarray representing developing seed forward subtracted library with95transcripts,showed highest induction by high temperature stress among all the libraries made in this study.These transcripts correspond to many stress-related proteins,such as helicase-like protein,ala-nine amino transferase,myo-inositol1-P-synthase(MIPS), stress-induced protein Sti-1,activator of HSP90,peptydyl prolyl isomerase,heat shock factor(HSF),along with all types of HSPs.Supplementary Table6provides details of fold induction of these genes during heat stress and after recovery of1-day and4-day.It is clear from Supplemen-tary Table6and Fig.2that,in the case of developing seeds,recovery is not as fast as in the seedling,and almost all the95genes show more than twofold induction even after4days of recovery period.Contrary to this,only20%of seedling ESTs showed more than twofold increase in the transcript levels after1-day of recovery.

For developing seed forward subtractive library,50 genes were checked by northern analysis(Fig.6).As expected,most of the HSPs have a basal constitutive expression in developing seed tissue,but transcript levels of almost all are enhanced at37and42°C treatment given for2h.Along with many unknown proteins,a novel seed specific heat shock factor(HSF)and a putative helicase inducible by heat stress could be identified in developing seed tissue.This heat shock factor from developing seed forward subtractive library was also checked by real time analysis and found to be induced by calcium and heat stress,particularly in variousflower and seed tissues,and significant amounts were also present in4day recovery tissue and20DAA control seed,suggesting its dual role in stress amelioration and as well as in seed maturation (Fig.4e).This seed-specific HSF is being further charac-terized.Peptidyl prolyl cis-trans isomerases(FKBP)are known to protect plants from abiotic stresses and a heat inducible FKBP77is known to express in wheat roots (Dwivedi et al.2003;Geisler and Bailly2007).We thus monitored the expression of wheat FKBP77in different wheat tissues and found its expression induced in shoot and root tissue by heat as well as by drought stress.However, FKBP77transcript is severely reduced inflower tissue upon heat stress but,its expression was induced again during early stages of seed development(7and10DAA). At20DAA stage,the expression was almost same for control and heat stressed developing seeds and a significant amount was also present in the mature seed(Fig.4f).

While studying the effect of high temperature in rice grain development,Yamakawa et al.(2007)reported on an average%reduction in the activities of starch biosyn-thesis enzymes and translocaters,while there is consider-able increase in the transcripts of HSPs and starch consuming a-amylase coding gene.High temperature not only advance but also compress the normal seed develop-ment in wheat.Studies have shown that high temperature enhances apoptosis in developing wheat grains and hastens the activity of various seed specific genes(Hurkman et al. 2003)and early deposition of various gluten proteins, reducing the overall weight and size of wheat grains (Altenbach et al.2003).Employing a proteomic approach, Hurkman et al.(2009)analyzed the effect of heat stress in developing wheat grains.They also found early and higher accumulation of proteins related to stress and plant defense pathways while proteins related to biosynthesis and metabolism decreased under heat stress.In the present study we alsofind that even2h heat treatment during grain development caused irreparable damage and accumulation of stress associated transcripts during the recovery phase. Additionally,we report involvement of a large number of

novel uncharacterized transcripts in wheat heat stress response in three different tissues.One such gene is a putative HSF,which is highly induced in developing seed by heat stress.The Arabidopsis genome has 21members of HSFs belonging to 3classes and 14groups (Nover et al.2001).Out of 24,000genes,11%genes of Arabidopsis showed regulation during heat stress and many of these genes proved to be controlled by HSF1a/1b,as determined by global gene expression analysis (Busch et al.2005).However,whether the HSF reported in this study has similar functions to play in wheat is yet to be determined.Reverse subtraction

A total of 148transcripts of developing seed reverse sub-tracted library were checked for down-regulation by heat stress.As expected,severely affected genes are those involved in carbohydrate metabolism,encoding components like sucrose synthase,amylase inhibitor,triose phosphate isomerase and soluble starch synthase.We found a large number of genes encoding seed storage proteins (gliadins and glutanins)affected by high temperature (Supplementary Table 7).As seen in other libraries too,there are a large number of unknown genes which may be involved in seed development and down-regulated by heat stress.

We also checked many seed storage as well as starch synthesis associated genes from reverse subtracted devel-oping wheat grain library by northern analysis (Fig.7).Most of the storage protein genes selected for northern analysis showed decrease in transcript abundance at 37and 42°C (for 2h),however,the expression of a few genes encoding glutenin subunits is relatively stable during heat stress.Protein disulfide isomerases are necessary cellular proteins for sorting and trafficking and three wheat protein disulfide isomerase encoding genes are negatively affected by high temperature (Supplementary Table 7).For all the

Unknown (GD186823)

ClpB heat shock proein (GD187042)ABC transporter homolog (GD187018)Putative RNA binding protein (GD187020)Putative placental protein (GD187033)Putative 60s ribosomal protein (GD187123)HSP 16.9 (GD187358)

Unknown (GD186997)

mRNA for ribosomal protein (GD186957)

Vacuolar membrane proton translocater (GD186918)Putative zinc finger protein (GD1837)

Putative vacuolar targeting receptor (GD186397)Nhp2HSP 80Unknown protein (GD1801)DnaJ GRP94 homologue (GD186794)hsp17.3 (GD188732)stress No significant match (GD186540)Unknown (GD186506)Serpin(WSZ1c) (GD186525)Myb transcription factor (GD1884)Putative Se binding protein (GD186542)Endopeptidase NP1 precursor (GD186550)F-Putative B2 protein (GD186592)Proteosome family protein (GD187211)Chloroplast HSP (GD186670)Peptydylprolyl isomerase (GD186615)Alanine aminotransferase (GD186946)Hypothetical protein (GD186715)Activator of HSP 90 (GD186533)Luminal BiP (GD1880) Sui 1 homolog (GD186633)5a2 protein (GD18)MIPS (GD1869)HSP70 (GD186603)

HSP100 (GD186625)Ribosomal protein L7 (GD187068)

Putative senescence associated protein (GD187293)RAN GTPase activating protein (GD1865)Helicase like protein (GD1869)Putative SNARE protein (GD186516)

Leucine rich repeat family protein (GD186523)HSF (GD186916)

Unknown protein (GD187065)C 37C 42C

C 37C 42C

starch synthesis enzymes checked in the present study,heat stress response showed a decrease in transcript abundance at 37and 42°C,while the transcripts of starch branching enzyme IIb and sucrose synthase type II were compara-tively stable at 37°C.One MADS box protein identified in this study from developing seed reverse subtraction was found to be down-regulated by heat stress in flower and developing seed tissues.Its expression was particularly reduced in the anther tissue upon heat stress,however,in the ovary it was rather stable under heat stress (Fig.4g).Foldases are important for the final dough making quality of wheat flour (Johnson et al.2001).We have studied expression of three genes encoding protein disulphide isomerases (PDI)along with one peptidyl prolyl cis-trans isomerase (FKBP77).All the three PDI encoding genes studied behaved almost similarly in all the tissues and their expression was significantly reduced in developing seed upon heat stress (Fig.4h–j).Interestingly,all these three PDI genes were highly up-regulated by drought stress in root tissue.

In rice,Yamakawa et al.(2007)reported 68%decrease in the transcript levels of protein disulfide isomerase,an enzyme necessary for precise sorting of storage proteins.We have identified three wheat protein disulfide isomerase negatively affected by high temperature (Fig.4h–j).Additionally,the transcript levels corresponding to all the major enzymes responsible for starch biosynthesis were found to be decreased under heat stress,as also reported earlier in wheat (Hurkman et al.2003).

Conclusions

The demand of wheat is increasing at a rapid pace and by the year 2017additional 200million tonnes/year of wheat

and corn amounting to additional 6million ha of corn and 4million ha of wheat,would be required to meet the demand (Edgerton 2009).Despite the importance of wheat,current information of its genome sequence is not sufficient for functional genomics analysis (Gill et al.2004).The com-plete sequencing of the wheat genome is challenging because of its large genome size (16,000Mb;Bennett and Leitch 2005).Nevertheless,mapping and characterizing ESTs offers a manageable approach to the complex architecture and functioning of the wheat transcriptome and helps in unravelling the genetics of stress response (Ramalingam et al.2006;Barnabas et al.2008).Through SSH libraries we have generated a collection of heat stress responsive genes critical for various growth stages in wheat.This study will act as a foundation for further work in the field of wheat genomics for overcoming high tem-perature stress.Currently,the ESTs of many of the heat responsive genes identified in this study are being com-pleted using 50and 30RACE,with the aim to functionally validate them through transgenic technology.

Acknowledgments This work was financially supported by Department of Biotechnology,Government of India,and partially by Indo-Swiss Collaboration in Biotechnology (ISCB).HC thanks Council for Scientific and Industrial Research for Junior and Senior Research Fellowships.

References

Abiko M,Akibayashi K,Sakata T,Kimura M,Kihara M,Itoh K,

Asamizu E,Sato S,Takahashi H,Higashitani A (2005)High-temperature induction of male sterility during barley (Hordeum vulgare L.)anther development is mediated by transcriptional inhibition.Sexual Plant Rep 18:91–100

Altenbach SB,Kothari KM (2004)Transcript profiling of genes

expressed in endosperm tissue are altered by high temperature during wheat grain development.J Cer Sci 40:115–126

LMW glutenin GluD3-1(GD187473) Protein di sulfide isomerase (187938)G1-Soluble starch synthase (GD188087)LMW glutenin L4-36 (GD187474)HMW glutenin Glu-D1-2b (GD187453) LMW glutenin GluD3-6 (GD187379)HMW glutenin 1By15 (GD187772)Glutenin subunit LMW.S6 (GD187442)Sucrose phosphate synthase (GD188200)

Sucrose 6 phosphate phosphotransferase (GD187169)Sucrose synthase type 2 (GD188175)Starch branching enzyme IIb (GD188500)ADP glucose pyrophosphorylase (GD187843)ADP-ATP carrier (GD187457)

C 37C 42C

C 37C 42C

Altenbach SB,Kothari KM,Tanaka CK,Hurkman WJ(2007)Genes encoding the PR-4protein wheatwin are developmentally regulated in wheat grains and respond to high temperature during grainfill.Plant Sci173:135–143

Altenbach SB,Kothari KM,Tanaka CK,Hurkman WJ(2008) Expression of9-kDa non-specific lipid transfer protein genes in developing wheat grain is enhanced by high temperatures but not by post-anthesis fertilizer.J Cer Sci47:201–213

Altschul SF,Madden TL,Schaffer AA,Zhang J,Zhang Z,Miller W, Lipman DJ(1997)Gapped BLAST and PSI-BLAST:a new generation of protein database search programs.Nucleic Acids Res25:33–3402

Barnabas B,Jager K,Feher A(2008)The effect of drought and heat stress on reproductive processes in cereals.Plant Cell Environ 31:11–38

Bennett MD,Leitch IJ(2005)Nuclear DNA amounts in angiosperms: progress,problems and prospects.Ann Bot95:45–90 Boominathan P,Shukla R,Kumar A,Manna D,Negi D,Verma PK, Chattopadhyay D(2004)Long term transcript accumulation during the development of dehydration adaptation in Cicer arietinum.Plant Physiol135:1608–1620

Busch W,Wunderlich M,SchofflF(2005)Identification of novel heat shock factor-dependent genes and biochemical pathways in Arabidopsis thaliana.Plant J41:1–14

Chae K,Kieslich CA,Morikis D,Kim SC,Lord EM(2009)A gain-of-function mutation of Arabidopsis Lipid Transfer Protein5 disturbs pollen tube tip growth and fertilization.Plant Cell 21:3902–3914

Chen F,Li Q,Sun L,He Z(2006)The rice14–3-3gene family and its involvement in responses to biotic and abiotic stress.DNA Res 13:53–63

Clarke SM,Mur LA,Wood JE,Scott IM(2004)Salicylic acid dependent signalling promotes basal thermotolerance but is not essential for acquired thermotolerance in Arabidopsis thaliana.

Plant J38:432–447

Dat JF,Foyer CH,Scott IM(1998)Changes in salicylic acid and antioxidants during induced thermotolerance in mustard seed-lings.Plant Physiol118:1455–1461

DeRocher AE,Vierling E(1994)Developmental control of small heat shock protein expression during pea seed maturation.Plant J 5:93–102

Dhaubhadel S,Chaudhary S,Dobinson KF,Krishna P(1999) Treatment with24-epibrassinolide,a brassinosteroid,increases the basic thermotolerance of Brassica napus and tomato seedlings.Plant Mol Biol40:333–342

Dhaubhadel S,Browning KS,Gallie DR,Krishna P(2002)Brassi-nosteroid functions to protect the translational machinery and heat-shock protein synthesis following thermal stress.Plant J 29:681–691

DuPont FM,Hurkman WJ,Vensel WH,Tanaka CK,Kothari KM, Chung OK,Altenbach SB(2006)Protein accumulation and composition in wheat grains:effects of mineral nutrients and high temperature.Europian J Agronomy25:96–107

Dwivedi RS,Breiman A,Herman EM(2003)Differential distribution of the cognate and heat-stress-induced isoforms of high Mr cis-trans prolyl peptidyl isomerase(FKBP)in the cytoplasm and nucleoplasm.J Exp Bot54:2679–26

Edgerton MD(2009)Increasing crop productivity to meet global needs for feed,food,and fuel.Plant Physiol149:7–13Fowler S,Thomashow MF(2002)Arabidopsis transcriptome profil-ing indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway.Plant Cell14:1675–1690

Fujita Y,Fujita M,Satoh R,Maruyama K,Parvez MM,Seki M, Hiratsu K,Ohme-Takagi M,Shinozaki K,Yamaguchi-Shinozaki K(2005)AREB1Is a transcription activator of novel ABRE-dependent ABA signaling that enhances drought stress tolerance in Arabidopsis.Plant Cell17:3470–3488

Geisler M,Bailly A(2007)Tete-a-tete:the function of FKBPs in plant development.Trends Plant Sci12:465–473

Gill BS,Appels R,Botha-Oberholster AM,Buell CR,Bennetzen JL, Chalhoub B,Chumley F,Dvorak J,Iwanaga M,Keller B,Li W, McCombie WR,Ogihara Y,Quetier F,Sasaki T(2004)A workshop report on wheat genome sequencing:International Genome Research on Wheat Consortium.Genetics168:1087–1096

Gong M,Li YJ,Dai X,Tian M,Li ZG(1997)Involvement of calcium and calmodulin in the acquisition of heat-shock induced thermotolerance in maize.J Plant Physiol150:615–621

Gong M,ver dan Luit AH,Knight MR,Trewavas AJ(1998)Heat-shock-induced changes in intracellular Ca2?level in tobacco seedlings in relation to thermotolerance.Plant Physiol116:429–437

Heil M,Bostock RM(2002)Induced systemic resistance(ISR) against pathogens in the context of induced plant defences.Ann Bot:503–512

Huang B,Xu C(2008)Identification and characterization of proteins associated with plant tolerance to heat stress.J Int Plant Biol 50:1230–1237

Hurkman WJ,McCue KF,Altenbach SB,Korn A,Tanaka CK, Kothari KM,Johnson EL,Bechtel DB,Wilson JD,Anderson OD,DuPont FM(2003)Effect of temperature on expression of genes encoding enzymes for starch biosynthesis in developing wheat endosperm.Plant Sci1:873–881

Hurkman WJ,Vensel WH,Tanaka CK,Whiteland L,Altenbach SB (2009)Effect of high temperature on albumin and globulin accumulation in the endosperm proteome of developing wheat grain.J Cer Sci49:12–23

Hussain SS,Mudasser M(2007)Prospects for wheat production under changing climate in mountain areas of Pakistan-an econometric analysis.Agriculture Systems94:494–501 Jakoby M,Weisshaar B,Droge-Laser W,Vicente-Carbajosa J, Tiedemann J,Kroj T,Parcy F(2002)bZIP transcription factors in Arabidopsis.Trends Plant Sci7:106–111

Johnson JC,Clarke BC,Bhave M(2001)Isolation and characterisa-tion of cDNAs encoding protein disulphide isomerases and cyclophilins in wheat.J Cer Sci34:159–171

Joshi AK,Mishra B,Chatrath R,Ferrara GO,Singh RP(2007)Wheat improvement in India:present status,emerging challenges and future prospects.Euphytica157:431–446

Kagale S,Divi UK,Krochko JE,Keller WA,Krishna P(2007) Brassinosteroid confers tolerance in Arabidopsis thaliana and Brassica napus to a range of abiotic stresses.Planta225:353–3 Kaplan F,Kopka J,Haskell DW,Zhao W,Schiller KC,Gatzke N, Sung DY,Guy CL(2004)Exploring the temperature-stress metabolome of Arabidopsis.Plant Physiol136:4159–4168 Khripach V,Zhabinskii V,de Groot A(2000)Twenty years of brassinosteroids:steroidal plant hormones warrant better crops for the XXI century.Ann Bot86:441–447

Klueva NY,Maestri E,Marmiroli N,Nguyen HT(2001)Mechanisms of thermotolerance in crops.Food Products Press,Binghamton Knight H,Brandt S,Knight MR(1998)A history of stress alters drought calcium signalling pathways in Arabidopsis.Plant J 16:681–687

Kotak S,Larkindale J,Lee U,von Koskull-Doring P,Vierling E, Scharf K-D(2007)Complexity of heat stress response in plants.

Curr Opin Plant Biol10:310–316Kreps JA,Wu Y,Chang HS,Zhu T,Wang X,Harper JF(2002) Transcriptome changes for Arabidopsis in response to salt, osmotic,and cold stress.Plant Physiol130:2129–2141 Krishna P(2003)Brassinosteroid-mediated stress responses.J Plant Growth Regul22:2–297

Kudla J,Xu Q,Harter K,Gruissem W,Luan S(1999)Genes for calcineurin B-like proteins in Arabidopsis are differentially regulated by stress signals.Proc Natl Acad Sci USA96: 4718–4723

Kurek I,Chang TK,Bertain SM,Madrigal A,Liu L,Lassner MW, Zhu G(2007)Enhanced thermostability of Arabidopsis Rubisco activase improves photosynthesis and growth rates under moderate heat stress.Plant Cell19:3230–3241

Law RD,Crafts-Brandner SJ(1999)Inhibition and acclimation of photosynthesis to heat stress is closely correlated with activation of ribulose-1,5-bisphosphate Carboxylase/Oxygenase.Plant Physiol120:173–182

Lee GJ,Pokala N,Vierling E(1995)Structure and in vitro molecular chaperone activity of cytosolic small heat shock proteins from pea.J Biol Chem270:10432–10438

Lee GJ,Roseman AM,Saibil HR,Vierling E(1997)A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state.EMBO J 16:659–671

Liu HT,Li B,Shang ZL,Li XZ,Mu RL,Sun DY,Zhou RG(2003) Calmodulin is involved in heat shock signal transduction in wheat.Plant Physiol132:1186–1195

Liu L,Zhou Y,Zhou G,Ye R,Zhao L,Li X,Lin Y(2008) Identification of early senescence-associated genes in riceflag leaves.Plant Mol Biol67:37–55

Lobell DB,Field CB(2007)Global scale climate-crop yield relationships and the impacts of recent warming.Environment Res Lett2:014002(7pp)

Ma K,Xiao J,Li X,Zhang Q,Lian X(2009)Sequence and expression analysis of the C3HC4-type RINGfinger gene family in rice.Gene444:33–45

Maestri E,Klueva N,Perrotta C,Gulli M,Nguyen HT,Marmiroli N (2002)Molecular genetics of heat tolerance and heat shock proteins in cereals.Plant Mol Biol48:667–681

Majee M,Maitra S,Dastidar KG,Pattnaik S,Chatterjee A,Hait NC, Das KP,Majumder AL(2004)A novel salt-tolerant L-myo-inositol-1-phosphate synthase from Porteresia coarctata(Roxb.) Tateoka,a halophytic wild rice:molecular cloning,bacterial overexpression,characterization,and functional introgression into tobacco-conferring salt tolerance phenotype.J Biol Chem 279:28539–28552

Majoul T,Bancel E,Triboi E,Ben Hamida J,Branlard G(2004) Proteomic analysis of the effect of heat stress on hexaploid wheat grain:characterization of heat-responsive proteins from non-prolamins fraction.Proteomics4:505–513

McDonald GK,Suttan BG,Ellison FW(1983)The effect of time of sowing on the grain yield of irrigated wheat in the Namoi Valley, New South Wales.Australian J Agri Res34:229–240

Mosser DD,Kotzbauer PT,Sarge KD,Morimoto RI(1990)In vitro activation of heat shock transcription factor DNA-binding by calcium and biochemical conditions that affect protein confor-mation.Proc Natl Acad Sci USA87:3748–3752

Nagarajan S(2005)Can India produce enough wheat even by2020?

Curr Sci:1467–1471

Nover L,Bharti K,Doring P,Mishra SK,Ganguli A,Scharf KD (2001)Arabidopsis and the heat stress transcription factor world: how many heat stress transcription factors do we need?Cell Stress Chaperones6:177–1

Qin D,Wu H,Peng H,Yao Y,Ni Z,Li Z,Zhou C,Sun Q(2008)Heat stress-responsive transcriptome analysis in heat susceptible and

tolerant wheat(Triticum aestivum L.)by using Wheat Genome Array.BMC Genomics9:432

Ramalingam J,Pathan MS,Feril O,Ross K,Ma XF,Mahmoud AA, Layton J,Rodriguez-Milla MA,Chikmawati T,Valliyodan B, Skinner R,Matthews DE,Gustafson JP,Nguyen HT(2006) Structural and functional analyses of the wheat genomes based on expressed sequence tags(ESTs)related to abiotic stresses.

Genome49:1324–1340

Rane J,Chauhan H(2002)Rate of grain growth in advanced wheat (Triticum aestivum)accession under late-sown environment.

Indian J Agric Sci72:581–585

Ristic Z,Momcilovic I,Bukovnik U,Prasad PV,Fu J,Deridder BP, Elthon TE,Mladenov N(2009)Rubisco activase and wheat productivity under heat-stress conditions.J Exp Bot60: 4003–4014

Rizhsky L,Liang H,Shuman J,Shulaev V,Davletova S,Mittler R (2004)When defense pathways collide.The response of Arabidopsis to a combination of drought and heat stress.Plant Physiol134:1683–1696

Rosegrant MW,Cline SA(2003)Global food security:challenges and policies.Science302:1917–1919

Saidi Y,Finka A,Muriset M,Bromberg Z,Weiss YG,Maathuis FJM, Goloubinoff P(2009)The heat shock response in moss plants is regulated by specific calcium-permeable channels in the plasma membrane.Plant Cell21:2829–2843

Saini HS,Aspinall D(1982)Abnormal sporogenesis in wheat (Triticum aestivum L.)induced by short periods of high temperature.Ann Bot49:835–846

Saini HS,Sedgley M,Aspinall D(1984)Developmental anatomy in wheat of male sterility induced by heat stress,water deficit or abscisic acid.Aust J Plant Physiol11:243–253

Salvucci ME(2008)Association of Rubisco activase with chapero-nin-60beta:a possible mechanism for protecting photosynthesis during heat stress.J Exp Bot59:1923–1933

Salvucci ME,Crafts-Brandner SJ(2004a)Inhibition of photosynthe-sis by heat stress:the activation state of Rubisco as a limiting factor in photosynthesis.Physiol Plant120:179–186

Salvucci ME,Crafts-Brandner SJ(2004b)Relationship between the heat tolerance of photosynthesis and the thermal stability of Rubisco activase in plants from contrasting thermal environ-ments.Plant Physiol134:1460–1470

Salvucci ME,Ogren WL(1996)The mechanism of Rubisco activase: insights from studies of the properties and structure of the enzyme.Photosynth Res47:1–11

Sambrook J,Russel DW(2001)Molecular cloning:a laboratory manual.Cold Spring Harbour Press,New York

Sayre KD,Rajaram S,Fischer RA(1997)Yield potential progress in short bread wheats in northwest Mexico.Crop Sci37:36–42 Seki M,Narusaka M,Abe H,Kasuga M,Yamaguchi-Shinozaki K, Carninci P,Hayashizaki Y,Shinozaki K(2001)Monitoring the expression pattern of1,300Arabidopsis genes under drought and cold stresses by using a full-length cDNA microarray.Plant Cell 13:61–72

Seki M,Ishida J,Narusaka M,Fujita M,Nanjo T,Umezawa T, Kamiya A,Nakajima M,Enju A,Sakurai T,Satou M,Akiyama K,Yamaguchi-Shinozaki K,Carninci P,Kawai J,Hayashizaki Y,Shinozaki K(2002)Monitoring the expression pattern of around7,000Arabidopsis genes under ABA treatments using

a full-length cDNA microarray.Funct Integr Genomics2:

282–291

Shinozaki K,Dennis ES(2003)Cell signalling and gene regulation: global analyses of signal transduction and gene expression profiles.Curr Opin Plant Biol6:405–409

Sinclair TR,Jamieson PD(2006)Grain number,wheat yield and bottling beer:an analysis.Field Crops Res98:60–67Singh G,Kumar S,Singh P(2003)A quick method to isolate RNA from wheat and other carbohydrate-rich seeds.Plant Mol Biol Rep21:93a–93f

Stone PJ(2001)The effect of heat stress on cereal yield and quality.

Food Products Press,Binghamton

Stone SL,Hauksdottir H,Troy A,Herschleb J,Kraft E,Callis J (2005)Functional analysis of the RING-type ubiquitin ligase family of Arabidopsis.Plant Physiol137:13–30

Swindell WR,Huebner M,Weber AP(2007)Transcriptional profiling of Arabidopsis heat shock proteins and transcription factors reveals extensive overlap between heat and non-heat stress response pathways.BMC Genomics8:125

Takeoka Y,Hirori K,Kitano H,Wada T(1991)Pistil hyperplasia in rice spikelets as affected by heat-stress.Sexual Plant Rep4: 39–43

Trevino MB,Ma OC(1998)Three drought-responsive members of the nonspecific lipid-transfer protein gene family in Lycopers-icon pennellii show different developmental patterns of expres-sion.Plant Physiol116:1461–1468

Wardlaw IF,Blumenthal CS,Larroque O,Wrigley CW(2002) Contrasting effect of chronic heat stress and heat shock on kernel weight andflour quality in wheat.Funct Plant Biol29:25–34Yamaguchi-Shinozaki K,Shinozaki K(2006)Transcriptional regu-latory networks in cellular responses and tolerance to dehydra-tion and cold stresses.Annu Rev Plant Biol57:781–803 Yamakawa H,Hirose T,Kuroda M,Yamaguchi T(2007)Compre-hensive expression profiling of rice grainfilling-related genes under high temperature using DNA microarray.Plant Physiol 144:258–277

Yan J,He C,Wang J,Mao Z,Holaday SA,Allen RD,Zhang H(2004) Overexpression of the Arabidopsis14–3-3protein GF14lambda in cotton leads to a‘‘stay-green’’phenotype and improves stress tolerance under moderate drought conditions.Plant Cell Physiol 45:1007–1014

Zhang Y,Mian MA,Chekhovskiy K,So S,Kupfer D,Lai H,Roe BA (2005)Differential gene expression in Festuca under heat stress conditions.J Exp Bot56:7–907

Zhang X,Wollenweber B,Jiang D,Liu F,Zhao J(2008)Water deficits and heat shock effects on photosynthesis of a transgenic Arabidopsis thaliana constitutively expressing ABP9,a bZIP transcription factor.J Exp Bot59:839–848

Zhu JK(2001)Cell signaling under salt,water and cold stresses.Curr Opin Plant Biol4:401–406