Identification of genes induced upon water-deficit stress in a drought-tolerant rice cultivar

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Journal of Plant Physiology 163 (2006) 577—584

KEYWORDcDNA-AFLPDrought stGene exprOryza sati

0176-1617/$ - sdoi:10.1016/j.

AbbreviationCDK; cyclin-detein kinases; Gscription factoPTGRP; prolinefamily of recepfragments�Correspond

fax: +53 7 33 17E-mail addr

(O. Borras-Hida

www.elsevier.de/jplph

Identification of genes induced upon water-deficitstress in a drought-tolerant rice cultivar

Mayra Rodrıguez, Eduardo Canales, Carlos J. Borroto, Elva Carmona,Junior Lopez, Merardo Pujol, Orlando Borras-Hidalgo�

Laboratory of Plant Functional Genomics, Head of the Plant Functional Genomic Department, Plant Division, Centerfor Genetic Engineering and Biotechnology (C.I.G.B.), P.O. Box 6162, La Habana 10600, Cuba

Received 4 May 2005; accepted 5 July 2005

S;ress;ession;va L.

ee front matter & 200jplph.2005.07.005

: AFLP; amplified fragpendent kinase; CDPKRP; glycine-rich proters; MAPKs; mitogen-ac–threonine–glycine-ritor protein kinases; T

ing author. Tel.: +53 779.ess: orlando.borras@clgo).

SummaryAmong the abiotic stresses, the availability of water is the most important factor thatlimits the productive potential of higher plants. The identification of novel genes,determination of their expression patterns, and the understanding of their functionsin stress adaptation is essential to improve stress tolerance. Amplified fragmentlength polymorphism analysis of cDNA was used to identify rice genes differentiallyexpressed in a tolerant rice variety upon water-deficit stress. In total, 103 transcript-derived fragments corresponding to differentially induced genes were identified. Theresults of the sequence comparison in BLAST database revealed that severaldifferentially expressed TDFs were significantly homologous to stress regulatedgenes/proteins isolated from rice or other plant species. Most of the transcriptsidentified here were genes related to metabolism, energy, protein biosynthesis, celldefence, signal transduction, and transport. New genes involved in the response towater-deficit stress in a tolerant rice variety are reported here.& 2005 Elsevier GmbH. All rights reserved.

5 Elsevier GmbH. All rights rese

ment length polymorphism;s; calcium-dependent pro-in; HSF; heat-shock tran-tivated protein kinases;ch protein; SRKs; S-locusDFs; transcript-derived

271 6022x3151;

igb.edu.cu

Introduction

Rice (Oryza sativa L. subsp. Indica) is the staplefood for more than half of the world’s population.With limited water resources, future increases inrice production will largely rely on rainfed produc-tion. Upland rice, which relies strictly on rainfall asa source of water, is often exposed to droughtstress and has developed drought-resistant traits(Yadav et al., 1997). Abiotic stress is the primary

rved.

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cause of crop loss worldwide, reducing averageyields for most major crop plants by more than 50%.In Cuba, 1 580 996 ha of soils are affected bydrought and desertification being 14% of total soil(Naciones Unidas, 2000).

Among the abiotic factors that have shaped andcontinue shaping plant evolution, water availabilityis the most important (Bray et al., 2000). Thecomplex plant response to abiotic stress involvesmany genes and biochemical–molecular mechan-isms. Changes in gene expression are induced by acomplex of signal transduction events that have notbeen clearly delineated. Various genes respond todrought stress in several species, and functions oftheir gene products have been predicted fromsequence homology with known proteins. Manydrought-inducible genes are also induced by saltstress and low temperature, which suggests theexistence of similar mechanisms of stress responses(Zhu, 2001).

Genes induced during drought-stress conditionsare thought to function not only in protecting cellsfrom water deficit by the production of importantmetabolic proteins but also in the regulation ofgenes for signal transduction in the drought-stressresponse (Shinozaki et al., 2003). Thus, these geneproducts are classified into three major groups. (1)those that encode products that directly protectplant cells against stresses such as heat stressproteins or chaperones, LEA proteins, osmoprotec-tants, antifreeze proteins, detoxification enzymesand free-radical scavengers (Bray et al., 2000); (2)those that are involved in signalling cascades and intranscriptional control, such as mitogen-activatedprotein kinases (MAPKs), calcium-dependent pro-tein kinases (CDPKs) (Ludwig et al., 2004) and SOSkinase (Zhu, 2001), phospholipases (Frank et al.,2000), and transcriptional factors (Shinozaki andYamaguchi-Shinozaki, 2000); (3) those that areinvolved in water and ion uptake and transportsuch as aquaporins and ion transporters (Blumwald,2000).

Stress-inducible genes have been used to im-prove the stress tolerance of plants by genetransfer. It is important to analyze the functionsof stress-inducible genes not only to understand themolecular mechanisms of stress tolerance and theresponses of higher plants, but also to improve thestress tolerance of crops by gene manipulation(Seki et al., 2003).

Expression profiling has become an importanttool to investigate how an organism responds toenvironmental changes. Sometimes these tran-scriptional changes are successful adaptationsleading to tolerance while in other instances theplant ultimately fails to adapt to the new environ-

ment and is labeled as sensitive to that condition.Expression profiling can define both tolerant andsensitive responses. These profiles of plant re-sponse to environmental extremes are expected tolead to regulators that will be useful in biotechno-logical approaches to improve stress tolerance aswell as to new tools for studying regulatory geneticcircuitry (Hazen et al., 2003). cDNA-amplifiedfragment length polymorphism (AFLP) is an effi-cient, sensitive and reproducible technology thatoffers several advantages over other methods ofgene expression analysis (Bachem et al., 1996).

The aim of this work is to identify genes that areactivated and associated with tolerance in riceupon water-deficit stress. This is pursued byisolation of differentially expressed transcription-derived fragments that are induced upon water-deficit stress in a tolerant rice variety, based on acDNA-AFLP approach.

Materials and methods

Plant materials and water-deficit treatment

Seeds from two varieties of rice (seed providedby the Rice Researches Institute, Cuba) withcontrasting drought tolerance (O. sativa L. subsp.Indica cv. ‘‘LC8866’’, tolerant and O. sativa L.subsp. Indica cv. ‘‘IACuba-27’’, sensitive) weregerminated in 6-in pots containing normal soilunder controlled conditions in growth chambers at23 1C. Seedlings were watered every day. Thirty-day old seedlings from both varieties were droughtstressed by withholding water for a period of 3weeks. Phenotypic analysis was developed at 1, 2and 3 weeks after drought conditions.

RNA isolation and cDNA-AFLP

Leaf samples from ‘‘LC8866’’ variety wereharvested at 1, 2 and 3 weeks after droughtconditions. Also, leaf samples from unstressed‘‘LC8866’’ variety were harvested at 1, 2 and 3weeks. The samples were immediately frozen inliquid nitrogen and stored at �70 1C for further RNAextraction. Leaf samples from ‘‘LC8866’’ variety at1, 2 and 3 weeks of drought conditions were pooledbefore RNA extraction. A leaf samples pool fromunstressed ‘‘LC8866’’ variety at 1, 2 and 3 weekswere used as control. Total RNA (10 mg) wasextracted using a commercially available kit (SVtotal RNA Isolation System, Promega, Madison, WI)according to the manufacturer’s instructions. Theintegrity of the RNA was checked on a denaturing

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Identification of rice stress tolerant genes 579

agarose gel. Finally, double-stranded cDNA wassynthesized (cDNA Synthesis System, Promega,Madison, WI). cDNA prepared from ‘‘LC8866’’variety under drought condition and unstressed‘‘LC8866’’ variety were used in cDNA-AFLP analysis.The cDNA-AFLP procedure described by Bachem etal. (1996) was utilized with a few modifications.The EcoRI/MseI enzyme combination was employedand a total of 9 primer pairs (9 with EcoRI+1/MseI+1combination and 9 with EcoRI+2/MseI+2 combina-tions) were used to generate PCR amplificationproducts. The amplification products were sepa-rated on a 6% polyacrylamide gel containing urea(8.0M) at 110W until the bromophenol bluereached the bottom of the gel. The cDNA bandswere stained with silver nitrate, according to theprotocol DNA Sequencing System kit (Promega,Madison, USA).

Sequence determination of differentiallyexpressed TDFs

Differentially expressed (those that are ex-pressed in the stressed tolerant variety but not inunstressed tolerant variety) transcript-derivedfragment (TDFs) were marked, cut out and incu-bated in 150 mL TE (10mM Tris, pH 7.5, and 1mMEDTA, pH 8.0) overnight at 37 1C. Extracted targetbands were used as template for re-amplificationusing PCR. The sequences were determined with anautomated sequencer (Perkin Elmer ABI PRISM DyeTerminator Cycle sequencing kit and ABI Model 377DNA sequencer) using an EcoRI+TA and MseI+GTprimer pair. Nucleotide sequences as well astranslated sequences were compared with nucleo-tide and protein sequences of the GenBank non-redundant databases and sequences of theexpressed sequence tag databases by using theBLAST sequence alignment program (Altschul et al.,1997).

RNA blot analysis

In a separate experiment, total RNA from‘‘LC8866’’ variety (tolerant) and ‘‘IACuba-27’’variety (sensitive) rice varieties at 0, 1, 2 and 3weeks under drought conditions were size-fractio-nated on a 1.2% agarose/0.4M formaldehyde RNAgel and transferred to Hybond N+ Nylon membrane(Amersham-Pharmacia, Buckinghamshire, UK).Probes were generated from PCR-amplified frag-ments of selected TDFs with higher E value usingthe ReadyPrime random primed DNA labeling kit(Amersham-Pharmacia, Buckinghamshire, UK) with[32P] (ICN Biomedicals, Irvine, CA, USA). Blots were

hybridized and washed according to standardprocedures (Sambrook et al., 1989).

Results

The IACuba-27 and LC8866 rice varieties are usedin the production and as parental into rice breedingprogram in Cuba. The IACuba-27 is a short-cyclevariety with high yield and resistance to maindisease, but very sensitive to water-stress condi-tion. This variety was obtained after mutagenesisprocess of O. sativa L. subsp. Indica cv. ‘‘J104’’. Onthe other hand, ‘‘LC8866’’ variety was introducedfrom Vietnam and the origin is unknown. Thisvariety showed high tolerance to water-stresscondition in Cuban soils (Alvarez et al., 1999).Phenotypic changes in the sensitive variety wereobserved 2 weeks upon water-deficit stress. Plantsfrom sensitive variety were significantly affectedafter water-deficit stress, while tolerant varietyremained green. However, stress symptoms wereobserved after 3 weeks upon water-deficit stress inboth varieties. These symptoms were higher in thesensitive variety (Fig. 1).

Rice genes whose expression was regulated bydrought stress were identified by a cDNA-AFLPtechnique. cDNA-AFLP analysis can reveal alteredexpression of any gene provided that it carries therestriction sites that have been chosen for analysis.Highly reproducible banding patterns were ob-tained with the EcoRI+TA and Mse I+GT primer pair.Using this primer pair, a total of 103 bands wereobtained to be differentially expressed in tolerantvariety (LC8866) upon water-deficit stress, com-pared to unstressed ‘‘LC8866’’ variety. The origin ofthese 103 TDFs was from leaf samples pool ofstressed tolerant variety at 1, 2 and 3 weeks underdrought conditions. In addition, all differentiallyexpressed TDFs were up-regulated and sequenced.

Following sequencing, the TDFs were organizedinto several categories according to their putativefunction. The majority of the TDFs were groupedinto defense/stress, signal transduction, transpor-ters, metabolism, protein synthesis, energy andunknown function categories (Fig. 2). The genes ofknown function were sorted into the 6 primaryfunctional categories. The largest set of genes(20%) was assigned to the defense/stress category,while genes involved in transporter and proteinsynthesis constituted the smallest groups, compris-ing 12%, respectively. Genes with unknown functionconstituted 4% of the TDFs collection.

The results of the sequence comparison in BLASTdatabase revealed that several differentially

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Figure 1. Behavior of tolerant (LC8866) and sensitive (IACuba-27) rice varieties after 0 (A), 1 (B), 2 (C) and 3 (D) weeksupon water-deficit stress.

Figure 2. Classification of identified TDFs according tothe putative function. All clones with scores below 10�5

for the E value were considered as significant andincluded. Genes were grouped using the same functionalclassification used for Arabidopsis thaliana MIPS (http://www.mips.biochem.mpg.de).

M. Rodrıguez et al.580

expressed TDFs were significantly homologous tostress regulated genes/proteins isolated from riceor other plant species. TDFs showing homology torust resistance-like protein, glycine-rich protein(GRP), calcium-dependent protein kinase, S-recep-

tor kinase, cyclin-dependent kinase (CDK) C,carbamoyl phosphate synthase and ethylene-re-sponsive protein kinase were found (Table 1).

Differential gene expression patterns were com-pared between drought-stress tolerant variety anddrought-stress sensitive variety by RNA blot analy-sis. RNA from sensitive variety for RNA-blot analysiswas only isolated at 0, 1 and 2 weeks after stresscondition, because the quality of the leaves wasnot good after 3 weeks under stress. The analysiswas performed on a selection of most interestingdifferentially expressed TDFs according to E value.The RNA blot analysis indicated that expressionlevels of the transcripts in the tolerant variety werevery high compared to the sensitive variety forwhich transcript levels were either very low orundetectable (Fig. 3). It is possible that lack ofhybridization reflects differences in sequencebetween the two cultivars. For that reason, wehybridized some of the probes to genomic DNA fromthe sensitive cultivar and verified that the geneswere present and sequences conserved betweenthe two cultivars (data non shown).

The ‘‘GBAS12’’ transcript which exhibited se-quence homology with rust resistance-like proteinwas up-regulated in the first week under drought

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Figure 3. RNA blot analysis for transcription-derived fragments isolated from tolerant rice variety upon water-deficitstress. RNA blots were prepared using total RNA (10 mg each lane) isolated from tolerant variety (LC8866) or sensitivevariety (IACuba-27), after 0, 1, 2 and 3 weeks under stress, respectively. Ribosomal RNAs were stained with ethidiumbromide (EtdBr).

Table 1. Homology of transcription-derived fragment (TDF) sequences.

AFLP fragment Accession number Sequence homology/match E value

GBAS02 Q9LXP8 Hypothetical 26.4 kDa protein 7e�12GBAS08 Q9HXK5 2-isopropylmalate synthase 7e�07GBAS12 AAM0301 Rust resistance-like protein RP1-3 [Zea mays] 3e�31GBAS14 BM929246 Rice root nitrate-induced subtracted library [Oryza sativa] 1e�05GBAS19 P22198 Serine/threonine protein phosphatase [Zea mays] 4e�22GBAS23 S20846 Glycine-rich protein [Zea mays] 2e�29GBAS30 AAK38343.1 Seven transmembrane protein Mlo7 [Zea mays] 3e�12GBAS36 BAA13232.1 Calcium-dependent protein kinase [Zea mays] 5e�34GBAS37 T02053 S-receptor kinase (EC 2.7.1.-) KIK1 precursor [Zea mays] 1e�52GBAS39 CAC85725.1 Putative carbamoyl phosphate synthase small subunit [Nicotiana tabacum] 3e�34GBAS57 BG451891 Drought Medicago truncatula cDNA, mRNAsequence 4e�18GBAS60 AB049715 Pisum sativum ssa-4 mRNA for putative senescence-associated protein 4e�07GBAS77 BE363768 Water-stressed 1 (WS1) Sorghum bicolor cDNA, mRNA sequence. 5e�19GBAS80 CAC51391.1 Cyclin dependent kinase C [Lycopersicon esculentum] 2e�76GBAS81 AF096250.1 Ethylene-responsive protein kinase TCTR1 [Lycopersicon esculentum] 2e�23

Identification of rice stress tolerant genes 581

stress in tolerant variety and afterwards it appearsto be reduced. TDF that had sequence homologywith GRP (GBAS23) was activated after the secondweek of drought treatment, while it was reduced atthe third week in tolerant variety. The ‘‘GBAS37’’homologous to a precursor of S-receptor kinase and‘‘GBAS80’’ with homology to CDK C showed the

same behavior in tolerant variety, respectively.Meanwhile, ‘‘GBAS37’’ transcript was also up-regulated in sensitive variety; however, the ex-pression was not high. The ‘‘GBAS36’’ and‘‘GBAS39’’ transcripts that showed homology toCDPK and a carbamoyl phosphate synthase wereinduced in the first week and remained expressed

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during all time points evaluated in tolerant variety.Finally, the homolog of the ethylene-responsiveprotein kinase (GBAS81) was induced during 1 and 2weeks upon water-deficit stress in tolerant varietyand repressed after 3 weeks, respectively. Never-theless, this transcript was only expressed 2weeks upon water-deficit stress in sensitive variety(Fig. 3).

Discussion

When plants are subjected to drought stress, anumber of physiological responses have beenobserved. Osmotic adjustment, as a process ofactive accumulation of compatible osmolytes inplant cells exposed to water deficit, may enable (1)a continuation of leaf elongation, though atreduced rates; (2) stomatal and photosyntheticadjustments; (3) maintained root development andsoil moisture extraction; (4) delayed leaf senes-cence; (5) better dry matter accumulation andyield production for crops in stressful environments(Boyer, 1982).

cDNA-AFLP analysis was used to identify differ-entially expressed genes in tolerant variety uponwater-deficit stress. Genes induced by droughtstress has been allocated to at least 10 differentfunctional categories: metabolism; energy; tran-scription; protein destination; transport; cell com-munication/signal transduction; cell rescue,defence, death and ageing; cellular organiza-tion; cell growth, cell division and DNA syn-thesis and cellular transport (Bray, 2002). Mostof the transcripts identified here were genesrelated to metabolism, energy, protein biosynth-esis, cell defence, signal transduction, and trans-port (Fig. 2).

We found that ‘‘GBAS12’’ exhibited sequencehomology with rust resistance-like protein reveal-ing that stress response is a complex regulatorynetwork of signals that allows plants to respondoptimally to their changing environment. However,no report about the relation of this protein withdrought stress in plants has been found.

In addition, the ‘‘GBAS36’’ transcript showedhomology to CDPK. It has been reported that cold,drought and salinity induce transient Ca2+ influxinto the cell cytoplasm. Channels responsible forthis Ca2+ influx represent one type of sensor forthese stress signals. CDPKs are implicated asimportant sensors of Ca2+ influx in plants inresponse to these stresses. CDPKs are encoded bymultigene families, and expression levels of thesegenes are spatially and temporally controlled

throughout development. In addition, a subset ofCDPK genes responds to external stimuli. Biochem-ical evidence supports the idea that CDPKs areinvolved in signal transduction during stress condi-tions. Furthermore, loss-of-function and gain-of-function studies revealed that signaling pathwaysleading to cold, salt, drought or pathogen resis-tance are mediated by specific CDPK isoforms(Ludwig et al., 2004).

On the other hand, ‘‘GBAS23’’ had sequencehomology with GRP. Similar GRP from normalizedlibrary of drought-stressed rice seedlings wasreported in O. sativa (Reddy et al., 2002). GRPscontaining more than 60% glycine have been foundin different tissues from many eukaryotic species.Some of these proteins are components of the cellwalls of many higher plants. In most cases, it hasbeen shown that they are accumulated in thevascular tissues and that their synthesis is part ofthe plant’s defence mechanism. Other distincttypes of GRPs are characterized by having struc-tures and functions similar to animal cytokeratinsor by a domain with typical RNA-binding motifs(Mousavi and Hotta, 2005). Report on wild tomatospecies (Lycopersicon chilense) revealed that aproline-, threonine-, and GRP is down-regulated bydrought stress. The level of the mRNA in leaves andstems of 8-d drought-stressed plants decreased 5-to 10-fold compared with that in regularly wateredplants. Proline–threonine–glycine-rich protein(PTGRP) was the first drought-regulated proteinthat has been precisely localized in the cell wall(Harrak et al., 1999). In our research, the RNA-blotanalysis showed that ‘‘GBAS23’’ appeared to be up-regulated at the second week of drought stress;however, at the third week a very weak hybridiza-tion signal was observed. This fact is pointing outthat its expression was suppressed after 2 weeks.Currently, the role of GRPs in abotic stress in plantsis still unknown.

According to the sequence analysis, ‘‘GBAS37’’seems to be related to the S-locus family ofreceptor protein kinases (SRKs) because its homol-ogy to a precursor of S-receptor kinase. A similarfinding was recently reported by Bassett et al.(2005) who isolated a gene induced by water-deficittreatment from peach.

The carbamoyl phosphate synthase has beenreported in mammalian as a part of carbamoyl-phosphate synthetase–aspartate carbamoyltrans-ferase-dihydroorotase, a multienzymatic proteinrequired for the de novo synthesis of pyrimidinenucleotides and cell growth carbamoylphosphate isa common intermediate in the metabolic pathwaysleading to the biosynthesis of arginine and pyrimi-dines (Hewagama et al., 1999). Studies carried out

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by Graves et al. (2000) indicated its direct linkbetween activation of the MAPK cascade and denovo biosynthesis of pyrimidine nucleotides. MAPKsplay a key role in plant responses to stress andpathogens. In the last few years, MAPK cascadeshave been proposed as major pathways for signaltransduction into intracellular response. MAPKsidentified from different plant species are inducedin response to wounding, pathogen attack, drought,temperature and osmotic stress (Kumar et al.,2003). It should be noted that ‘‘GBAS39’’ had a verystrong hybridization signal during the water stresstreatment and it showed homology to carbamoylphosphate synthase. We could suggest that carba-moyl phosphate synthase play an important roleduring drought stress in rice.

Additionally, ‘‘GBAS80’’ transcript showedhomology to CDK. CDKs control cell cycle progres-sion through timely coordinated phosphorylationevents (Vanstraelen et al., 2004). In human cells, aswell as in Arabidopsis, phosphorylation of heat-shock transcription factors (HSFs) through variouskinases may integrate growth signals. As yet, it isunknown whether CDKs are involved in HSF phos-phorylation in animals or whether MAPKs play a rolein HSF regulation in plants (Schoffl et al., 1998).

Stress gene induction occurs primarily at thelevel of transcription, and regulating the temporaland spatial expression patterns of specific stressgenes is an important part of the plant stressresponse (Karam et al., 2002). Induction of geneexpression does not necessarily imply that a genewill play an adaptive role. Depending upon theconditions to which the plant was subjected, someof genes that are expressed may indicate that theplant has been subjected to a secondary stress.

It is uncertainly whether these genes are induceddirectly by cellular water deficit or by resultingsecondary stress. Techniques such as silencing orover-expression of certain signaling componentsmay confirm their role in particular pathways. Inconclusion, further characterization and functionalanalysis of the genes that are identified in thisstudy can lead to a more comprehensive under-standing of stress tolerance to drought in rice.

Acknowledgments

We are grateful to Rice Researches Institute,Cuba for providing the seeds for the experiments.Also, we are grateful to MACROGENE for sequencingservice. The work was supported by the CubanState Council.

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