Research Article Identification of Differential Expression...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 576189 11 pageshttpdxdoiorg1011552013576189

Research ArticleIdentification of Differential Expression Genes inLeaves of Rice (Oryza sativa L) in Response to Heat Stress bycDNA-AFLP Analysis

Yunying Cao1 Qian Zhang1 Yanhong Chen1 Hua Zhao1 Youzhong Lang2

Chunmei Yu1 and Jianchang Yang2

1 College of Life Science Nantong University Nantong Jiangsu 226019 China2 Key Laboratory of Crop Genetics and Physiology of Jiangsu Province Yangzhou University Yangzhou Jiangsu 225009 China

Correspondence should be addressed to Chunmei Yu ychmeintueducn and Jianchang Yang jcyangyzueducn

Received 2 August 2012 Revised 14 December 2012 Accepted 7 January 2013

Academic Editor Juan Francisco Jimenez Bremont

Copyright copy 2013 Yunying Cao et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

High temperature impedes the growth and productivity of various crop species To date rice (Oryza sativa L) has not beenexploited to understand the molecular basis of its abnormally high level of temperature tolerance To identify transcripts inducedby heat stress twenty-day-old rice seedlings of different rice cultivars suffering from heat stress were treated at different timesand differential gene expression analyses in leaves were performed by cDNA-AFLP and further verified by real-time RT-PCR Inaggregate more than three thousand different fragments were indentified and 49 fragments were selected for the sequence anddifferential expressed genes were classified functionally into different groups 6 of 49 fragments were measured by real-time RT-PCR In addition the variations of three different polyamine contents in response to heat stress through high-performance liquidchromatography (HPLC) analysis were also performedThe results and their direct and indirect relationships to heat stress tolerancemechanism were discussed

1 Introduction

With the development of industrialization the impact ofongoing climate change on the natural environment dete-rioration was more and more obviously shown Due toan increase in mean global temperature heat stress hasbecome amajor disastrous factor that severely affects the cropcultivation and productivity Heat stress-induced decreaseof the duration of developmental phases leading to fewerorgans smaller organs reduced light perception over theshortened life cycle and perturbation of the processes relatedto carbohydrate metabolism (transpiration photosynthesisand respiration) is most significant for losses in cereal yields[1]

Being a staple food and a principal calorie source forpeople living inAsia [2] rice (Oryza sativaL) production andits factors has attracted more and more attention Howeverrice is also a prime example of cereal crops whose growth

and reproduction could be impaired by heat stress It hasbeen indicated that rice production will be severely affectedby the aberrant change of temperature [3] Comprehend-ing the mechanisms of which rice response to heat stresswould facilitate the development of heat-tolerant cultivarswith enhanced productivity in a warmer future climateThe researches that underlie molecular responses both ingenomics and proteomics level of plants to heat stress havetherefore attracted a great deal of attention [4ndash6]

Previous proteomic analysis with the employment ofvarious advanced techniques such as two-dimensional elec-trophoresis provides a cogent approach to investigate themolecular mechanisms of rice response to abiotic stresses[7 8] Relative proteomic studies about rice in response toabiotic stresses have been deep into subcellular proteomeand posttranslational modifications [7] However it is wellknown that the expression of proteins is regulated by the genetranscription and translation processes although proteomic

2 BioMed Research International

study about rice in response to heat stress is significant theassessment of gene level regulation of such type of temper-ature variability on rice is also unneglectful In additionrice may confront with different high-temperature stressesduring its lifespan and different growth phases may havedifferent responses to the stresses which will be inevitablyregulated by the variation of the expression of genes Previousproteomic studies revealed that seedlings-phased rice couldalso be affected by the heat stress environment [9] Althoughgenomewide expression analysis showed that transcriptionlevel of heat shock proteins (Hsps) and heat shock factors(Hsfs) are induced under heat stress at young seedlingstages [10 11] further investigations to uncover the regulativemechanisms corresponding to heat tolerance at genomicslevel are still necessary

Among techniques employed in gene identificationmanytechniques such as differential display reverse transcription-polymerase chain reaction (DDRT-PCR) representationaldifference analysis (RDA) serial analysis of gene expression(SAGE) suppression subtractive hybridization (SSH) andcDNAmicroarray provide effective approaches for transcrip-tion analysis [12] As a high-efficient less labor-intensivemRNA fingerprinting method for isolation of differentiallyexpressed genes [13] cDNA-amplified fragment length poly-morphism (cDNA-AFLP) which has been employed in thestudy is a robust genomewide expression tool [12] for genediscovery without a prerequisite of the prior knowledge ofthe sequences [14] In addition due to its high detectivesensitivity of cDNA-AFLP analysis some rare transcriptscould also be detected by this method [15] This techniquehas been further ameliorated to avoid the possibility ofseveral transcript-derived fragments (TDFs) from a singlegenecDNA [16]

Currently several rice varieties defined by temperaturethresholds [17] have been identified with genotype variationin spikelet sterility at high temperature in indica and japonica[18 19] In our study forty-nine critical genes differentiallyexpressed in rice seedling in response to heat stress usingcDNA-AFLP technique have been identified To validate theirexpression patterns six gene fragments involving differentphysiological activitieswere analyzed through real-timePCRIn addition due to direct interactions of polyamines withother metabolic pathways during the stress response [20] andtheir regulatory functions in plant abiotic stress tolerance[21] the variations of three different polyamine contentsin response to heat stress through high-performance liquidchromatography (HPLC) analysiswere also performedTheseoutputs may lay the fundamental basis of the rice varietybreeding in the future

2 Materials and Methods

21 Plant Material Seeds of two indica rice cultivars (Shu-anggui 1 heat sensitive Huanghuazhan heat tolerant) wereprovided by the Rice Research Institute of GuangdongAcademy of Agricultural Sciences (Guangzhou China)The seedlings were grown in Kimura B complete nutri-ent solution under greenhouse conditions (28∘C day22∘C

night relative humidity 60ndash80 the light intensity 600ndash1000 120583molmminus2 sminus1 and photoperiod of 14 h day10 h night)with 50 seedlings per pot Then twenty-day old seedlingswere heat-stressed (at 39∘C day30∘C night) for two differenttreatment times 24 h or 48 h while control seedlings weregrown under the conditions mentioned above Each treat-ment had 3 pots as replications Samples from rice seedlingleaves were harvested and were immediately frozen in liquidnitrogen and stored at minus80∘C until use Heights of riceseedlings of Shuanggui 1 at 28∘C control condition or 39∘Cstress condition were determined with a ruler from the landsurface to the top of the seedling leaves with means of threereplications (50 plants per replication)

22 RNA Extraction Total RNA was extracted using RNeasyPlant Mini Kit (QIAGEN) according to the manufacturerrsquosinstructionsThe integrity of the RNAwas checked by agarosegel electrophoresis and the concentration was determined at260 nm by spectrophotometer

23 cDNA-AFLP Analysis An cDNA-AFLP method wasadapted from Bachem et al [13 22] with a few modificationsSynthesis of double-stranded cDNA was performed with M-MLVRTase cDNA Synthesis kit (TaKaRa China) and refinedusing phenolchloroform extraction 5 120583L was checked usingagarose gel electrophoresis in order to observe an expectedsmear between 100 bp and 1000 bp The rest of cDNA wasdigested using the restriction enzymes EcoR I (Fermentas2 h at 37∘C) and Mse I (Tru1I Fermentas 2 h at 65∘C)The digested products were ligated to adaptors (EcoR I5 120583mol Lminus1 forward primer 51015840-CTCGTAGACTGCGATCC-31015840 reverse primer 51015840-AATTGGTACGCAGTCTAC-31015840Mse I50120583mol Lminus1 forward primer 51015840-GACGATGAGTCCTGAG-31015840 reverse primer 51015840-TACTCAGGACTCAT-31015840) with T4-DNA ligase (Fermentas) for 13 h at 16∘C The products ofligation were amplified with the corresponding preamplifica-tion primers (EcoR I 51015840-GACTGCGTACCAATTC-31015840Mse I51015840-GATGAGTCCTGAGTAA-31015840) Preamplification was initi-ated at 94∘C for 3min and followed by 30 cycles at 94∘C for30 s 56∘C for 30 s 72∘C for 1min and terminated at 72∘C for5min The products of the preamplification were checked byagarose gel electrophoresis (expected smear between 100 bpand 1000 bp) From a 20-fold dilution of the pre-amplifiedsamples a 5 120583L sample was used for the final selective ampli-fications using the primers 51015840-GACTGCGTACCAATTCNN-31015840 (EcoR I NN represents AC AG GA and GT) and51015840-GATGAGTCCTGAGTAAMM-31015840 (Mse I MM representsTC TG CA and CT) After initial denaturation (94∘C for3min) 12 cycles were performed with touchdown annealing(94∘C for 30 s 65∘C to 56∘C in 07∘C steps for 30 s 72∘C for1min) followed by 24 cycles (94∘C for 30 s 56∘C for 30 s 72∘Cfor 1min) and final elongation (72∘C for 5min) Altogether 64primer combinations of two base extensions (denoted as NNand MM) were used

24 Polyacrylamide Gel Electrophoresis The selective ampli-fication products were separated on a sequencing polyacry-lamide gel (6 polyacrylamide 8mol Lminus1 urea 1timesTBE) at a

BioMed Research International 3

constant current mode in a vertical slab gel electrophoresisapparatus (Hoefer Pharmacia Biotech Inc CA USA) Theglass plates were treated by Repel-Silane and Binding-Silanerespectively (Dingguo China) following the manufacturerrsquosinstructions The system of electrophoresis was prerun in1timesTBE buffer about 30 minutes for 40∘Cndash45∘C of the gel sur-face temperature at 1500 v Then the samples were separatedabout 4 h of migration until the bromophenol blue reachedthe bottom of the gel The cDNA bands were visualized bysilver staining according to Bassam et al [23]

25 Isolation Cloning and Sequencing of Differential Frag-ments Fragments of interest were eluted from silver-stainedgels using the procedure of Frost and Guggenheim [24]with modifications The band was eluted in 50 120583L of steriledouble-distilled water initially at 95∘C for 15min and thenhydrated overnight at 4∘C Amplifications were performedwith appropriate primers with 5120583L of this solution PCRreactions were initiated at 94∘C for 3min followed by 35cycles at 94∘C for 30 s 56∘C for 30 s 72∘C for 1min andterminated at 72∘C for 10min The PCR products wereresolved in a 2 1timesTBE agarose gel and each single bandwas isolated and eluted using the DNA Fragment QuickPurification Kit (Dingguo China) Cloning was performedfrom fresh PCR products with the pGEM-T Easy vector(Promega) according to the manufacturerrsquos instructions andusing chemical transformation of one shot E coli (DH5120572)competent cells using Ampicillin as the selecting agentAfter plasmid purification using a small-scale plasmid DNApurification kit (QIAGEN) the insert size was checked byPCR amplification using the corresponding selective ampli-fication primer and the clones were sequenced by Invitrogenbiotechnology service company (Shanghai China)

26 Real-Time PCRAnalysis RNA extraction was performedaccording to the above mention Total RNA was thenpurified with DNase I Kit (Invitrogen China) accordingto the manufacturerrsquos instructions First-stranded cDNAswere synthesized using PrimeScript RT Master Mix PerfectReal Time Kit (TaKaRa China) These cDNAs were usedfor PCR experiments using gene-specific primers designedwith DNAMAN software Forward and reverse primerswere used for producing a single amplification for the fol-lowing genes Os04g0429600 (51015840-AGGTGCCGCAGAGTT-TCTAC-31015840 and 51015840-TACAGTGGAAGCAACCCGTTC-31015840)Os06g0124900 (51015840-CCCTCTTCGTCGTCTCCAAT-31015840 and51015840-TAAGCCTCTCACCTTCACGG-31015840) SRP14 (51015840-CGT-TTCTGAGCGAGTTGACG-31015840 and 51015840-GTTCTTCTTGCC-ATCGGTGG-31015840) Os02g0611200 (51015840-TCGGCTACAGCA-TTGAGGAC-31015840 and 51015840-GGAAGAAAGGAAGCAGGA-CTGA-31015840) Os02g0788800 (51015840-TCGGAGTTTAGAGGA-GTTGCG-31015840 and 51015840-TAAGAGCCATCACGAGACCG-31015840)qRT-PCR experiments were performed inABI 7500 real-timePCR system and 7500 Software v 204 All PCR reactionswere mixed as follows 2 120583L of diluted cDNA 10 120583L of2timesSYBR Premix Ex Taq II (TaKaRa China) 04 120583L 50timesROX Reference Dye II and 400 nmolL of primers in afinal volume of 20 120583L PCRs All reactions were repeated

four times As an internal standard a fragment of rice actingene (51015840-CTTCATAGGAATGGAAGCTGCGGGTA-31015840 and51015840-CGACCACCTTGATCTTCATGCTGCTA-31015840) was usedAll the PCRs were performed under the following program30 s at 95∘C followed by 40 cycles of 5 s at 95∘C and 34 s at60∘C in 96-well optical reaction plates (Applied BiosystemsUSA)

27 High-Performance Liquid Chromatography (HPLC) Anal-ysis of Free Polyamines Rice leaves (10000 g) were weighedand crushed to homogenate using precooled marble pestleand mortar with 4mL 5 perchloric acid The homogenatewas then suspended at 4∘C for 1 h and centrifuged at 14000 gfor 30min Supernatant was collected for the followingHPLCseparation and identification All standards and polyaminesextracts of spermidine spermine and putrescine were ana-lyzed on an Agilent 1200 HPLC system (Agilent Corp USA)Chromatographic separation and collection of extracts wereachieved using an Eclipse Plus C18 reversed-phase column(250mm times 46mm 5mm Agilent)The condition for HPLCseparation was determined to be a mobile phase composedof methanol and water (75 25 vv) and a flow-rate of 1mLper min The volume of sample injected was 10120583L for thequalitative evaluation the detection wavelength and the col-umn temperature were set at 254 nm and 30∘C respectivelyQuantification of spermidine spermine and putrescine inthe product was determined by comparison with externalstandards

28 Analysis of Sequences and Data Analysis The sequenceswere analyzed for their homology against the publicly avail-able nonredundant genesESTsTranscripts in the database(httpwwwncbinlmnihgovBLAST) using the BLASTNand BLASTX algorithms [25] The data was analyzed forvariance using the SASSTAT statistical analysis package(version 612 SAS Institute USA) Means were tested byDuncanrsquos test at the 119875

005level

3 Results

31 Morphological Responses of Rice Seedlings to High-Temperature Treatment 20-day-old seedlings were heat-stressed at 39∘C day30∘C night for two days After 48 hthe morphologies of the leaves were compared among theseseedlings treated at 39∘Cwith those treated at 28∘C As shownin Figure 1 at 28∘C leaves developed normally and showeddeep green (Figure 1(a)) However conspicuous symptomhasbeen appeared under high temperature low-growing leaveswith yellow color characters have been appeared in the heatstress treatment (Figure 1(b)) The average height of plantwith 28∘C treatment was 218 plusmn 125 cm while 39∘C heat-stressed plants showed 182 plusmn 095 cm high which was muchlower than those of 28∘C-treated rice leaves (Figure 1(c))

32 Identification of High-Temperature-Regulated TranscriptsIn order to further understand the response of rice seedlingsto heat stress cDNA-AFLP gels of gene bands from rice leavesafter treatment for 48 h at 39∘C treatment were compared


(a) (b)

0

5

10

15

20

25

28∘C 39∘C

Plan

thei

ght (

cm)

Treatment

a

b

(c)

Figure 1 Behavior of rice seedlings (Shuanggui 1) (a) 28∘C day22∘C night treatment (b) 39∘C day30∘C night treatment (c)The comparisonof plant height after 48 h at 39∘C Data from plant heights are means of three replications (50 plants per replication) plusmn SE Dissimilar lettersabove bars differ significantly at 119875 le 005

with those reduce extra space at 28∘C treatment Accurategene expression profiles were determined by visual observa-tion and analysis of band intensities and subtle differencesin transcriptional activity were revealed After cDNA-AFLPaverage 724 TDFs per pair of primers could be reproduciblydetected mainly at 28∘C while a total of average 505 TDFsper pair of primers changed in abundance in response to39∘C (Table 1) Therefore on average 61 bands (TDFs) wereproducedwith each primer combination which yieldedmorethan seven thousand TDFs from seedling leaves both undercontrol (28∘C) and heat stress (39∘C) treatments A total of145 TDFs were isolated from the silver-stained cDNA-AFLPgels based on their presenceabsence (qualitative variants) ordifference in the levels of expression (quantitative variants)(see Figure S1 in Supplementary Material available online atdoi httpdxdoiorg1011552013576189)

Table 1 Summary of PAGE showing bands after 64 pairs of primersamplification (per pair of primes)

Treatment Average number ofamplified gene bands

Average number ofupregulated and nascent

fragments28∘C 724 37339∘C 505 15928∘C 28∘C (day)22∘C (night) 39∘C 39∘C (day)30∘C (night)

Compared with the genomics profile at 28∘C average159 TDFs per pair of primers were upregulated under 39∘Ctreatment However compared with the genomics profileat 39∘C an average of 373 TDFs per pair of primers wereupregulated under 28∘C condition (Table 1 Figure S1) and


more than three thousand differential expressing fragmentswere indentified Thus the quantity of both upregulated ordownregulated cDNA fragments increased with increasedtemperature This implied that the higher the temperaturethe more rice life processes are affected

33 Changes of Rice Leaf Genomics Profile Under High-Temperature Stress With the 64 primer combinations tested65 reproducible (ie same expression profile in 2009 and 2010seasons) TDFs were identified and 49 of these were clonedand sequenced 49 differential expressed gene sequences wereperformed according to BLASTN (score ge 50) and BLASTX(E le 10minus5) analyses in NCBI and Gene Ontology databasesAccording to MIPS (httpmipshelmholtz-muenchendeprojfuncatDBsearch main framehtml) database differen-tial expressed genes in leaves suffered from heat stress wereclassified functionally asmetabolism (including carbohydratemetabolism protein metabolism polyamine metabolismamino acid metabolism ribonucleotide metabolism andcellulose synthesis) material transport stress response cellcycle and fate signal transduction and unclear functionalprotein The sequence comparison against the databaserevealed that most of them could be sorted into the followingten functional groups (Table 2)

The most abundant groups were related to carbohydratemetabolism Fourteen TDFs response to the heat stress wereidentified as related to carbohydrate metabolism Amongthem four TDFs (H51-4 H53-1 -2 and H57-3 Figure S1and Table 2) corresponding to photosynthesis II-relatedprotein NADH dehydrogenase subunit 2 chloroplast ATPsynthase a chain precursor and chloroplast ATP synthasea chain precursor respectively were upregulated by heatstress treatment (Figure S1 and Table 2) while two TDFs(N21-1 N58-3) one of them related to photorespiration pro-teins (NAD-dependent epimerasedehydratase family pro-tein) and the other corresponding to phospho-2-dehydro-3-deoxyheptonate aldolase 1 were downregulated (Table 2)Two TDFs (H1-5 H51-5) corresponding to ubiquitin familyprotein and 60S ribosomal protein which were sorted intoprotein metabolism group were also upregulated by the heatstress (Table 2) One identified TDF (H57-5) correspondingto S-adenosylmethionine decarboxylase which has beenclassified into polyamine metabolism was also affected byheat stress treatment To further validate the function of S-adenosylmethionine decarboxylase in rice seedling leaveswe used HPLC to investigate the effect of heat stress onthe concentration of spermidine spermine and putrescinein rice seedling leaves As shown in Figure 3 spermidinespermine and putrescine contents were increased dramat-ically at 39∘C compared with those at 28∘C In additionone TDF (H51-1) corresponding to glutamate decarboxylasewhich could be sorted into amino acid metabolism has alsoshown increasing trend under heat stress treatment Twoupregulated heat stress-induced differential fragments (H3-2-8 H46-3) which are corresponding to CTP synthase andcellulose synthesis family protein were classified into ribonu-cleotide metabolism and cellulose metabolism respectivelyThere are five heat-stressed induced genes identified and

classified into material transport group 2 (H51-3 H59-4)of 3 corresponding to phophate translocator and aminoacidpolyamine transporter II were upregulated at 39∘C(Table 2) Other two TDFs (H53-8 N60-4) correspondingto thioredoxin h isoform 1 and heat shock protein (HSP)DnaJ respectively could be sorted into stress response groupOne upregulated heat stress-induced differential fragment(H4-1) and the other downregulated fragment (N6-1) whichare corresponding to S-phase-specific ribosomal protein andputative senescence-associated protein were classified intocell cycle and fate group (Table 2) Other gene fragmentsregulated with 39∘C treatment included sixteen gene bandscorresponding to hypothetical proteins (H3-2-1 H51-2 N60-5 etc) whose functions were unknown or unclear (Table 2and Figure S1)

34 Differential Response of Gene Fragments to Heat StressTo validate the results of cDNA-AFLP experiment and quan-titatively assess the relative abundance of the transcripts inrice seedling leaves under the heat stress five upregulatedTDFs (H46-3 Os04g0429600 H51-2 Os06g0124900 H51-6 SRP14H57-5 Os02g0611200 and H59-4 Os02g0788800)and one downregulated TDF (N60-4 Os07g0620200) wereselected for RNA expression analysis Initially a semiquan-titative RT-PCR was performed to analyze the changesin transcripts of all six TDFs in response to the heatstress (data not shown) Obtained RT-PCR results weresubstantiated with quantitative real-time PCR (qRT-PCR)(Figure 2) Rice actin gene was selected as internal control fornormalization

The transcript of all six genes fragments (Os04g0429600Os06g0124900 SRP14 Os02g0611200 Os02g0788800 andOs07g0620200) responded to heat stress (Figure 2)Themostpronounced effect was observed in the case of Os06g0124900gene which increased more than twice after 48 h at 39∘Ctreatment both in heat-sensitive ldquoShuanggui 1rdquo and heat-tolerant ldquoHuanghuazhanrdquo and more increasing trend couldbe observed in heat-tolerant cultivar though there wasno obvious change after 24 h with 28∘C treatment Fasttranscript accumulation was observed in case of SRP14 andOs07g0620200 with heat stress SRP14 showed significantincrease in transcript accumulation only after 24 h at 39∘Ccompared with that with same time at 28∘C and relativeexpression of this gene presented more increasing trendin heat-tolerant cultivar compared to that in heat-sensitivecultivar Fast downregulated trend has been observed inOs07g0620200 gene which has also happened only after24 h heat stress treatment in heat-sensitive cultivar andconsiderable decreasing presented has been shown after48 h under heat stress condition However fast effect ofheat stress did not take place in heat-tolerant cultivarwith no considerable variation after 24 h treatment both at28∘C or 39∘C while obvious transcript increase could beobserved after 48 h of heat stress treatment The transcriptsof Os04g0429600 Os02g0611200 and Os02g0788800 alsoshowed up-regulation with no more than twofold due toheat stress with no significant change after 24 h and gradualincrease after 48 h In general five TDFs (Os04g0429600


Table 2Thenucleotide-homology of the transcript-derived fragments (TDFs)with known gene sequences in the database using the BLASTNand BLASTX algorithms along with their expression patterns

TDF no TDF size (bp) GenBankgene Corresponding or relatedprotein Score (bits) Identities 119864 value

Carbohydrate metabolism

N21-1 130 ABF960131NAD-dependentepimerasedehydratase familyprotein

732 (178) 3233 (97) 2119890 minus 14lowast

H50-1 195 PsaB PSI P700 apoprotein A2 200 (108) 108108 (100) 3119890 minus 48

H51-4 198 YP 654233 PS II protein H 578 (138) 2627 (96) 2119890 minus 09lowast

H51-8 138 ABI347571Ribulose-15-bisphosphatecarboxylaseoxygenase largesubunit (Pentameris aspera)

805 (197) 3435 (97) 3119890 minus 17lowast

H53-1 495 NP 0394321 NADH dehydrogenasesubunit 2 205 (522) 139171 (81) 1119890 minus 60

lowast

H53-2 389 Os10g0527100 Chloroplast ATP synthase achain precursor 217 (117) 119120 (99) 6119890 minus 53

H53-3 299 Os08g0472600 Alpha-1 3-fucosyltransferase 152 (82) 8789 (98) 1119890 minus 33


H53-7 220 Os07g0662900 4-Alpha-glucanotransferase 768 (41) 4648 (96) 6119890 minus 11

H56-3 229 AAA845881 atpB gene product 145 (366) 7176 (93) 1119890 minus 39lowast



H57-6 195 Os01g0881600 Photosystem II reaction centerJ protein 110 (59) 6162 (98) 5119890 minus 21

N58-3 270 Os08g0484500Phospho-2-dehydro-3-deoxyheptonate aldolase 1chloroplast precursor

207 (112) 116118 (98) 3119890 minus 50

Protein metabolism

H1-5 227 Os03g0131300 Ubiquitin domain containingprotein 283 (153) 159162 (98) 3119890 minus 73

H51-5 185 AF093630 60S ribosomal protein L21(RPL21) 298 (161) 168171 (98) 1119890 minus 77

N53-6 294 Os07g0555200 Eukaryotic translationinitiation factor 4G 147 (79) 8283 (99) 6119890 minus 32

Polyamine metabolism

H57-5 260 Os02g0611200S-adenosylmethioninedecarboxylase proenzyme(AdoMetDC) (SamDC)

169 (91) 9698 (98) 1119890 minus 38

Amino acid metabolismH51-10 118 GAD Glutamate decarboxylase 172 (93) 103107 (96) 3119890 minus 40

Ribonucleotide metabolismH3-2-8 102 Os12g0556600 CTP synthase family protein 143 (77) 7980 (99) 2119890 minus 31

Cellulose synthesis

H46-3 193 Os04g0429600 Cellulose synthase-likeprotein H1 124 (67) 100115 (87) 2119890 minus 25

Material transport

N51-17 181 Os12g0166000 Peptidase S59 nucleoporinfamily protein 189 (102) 111115 (97) 6119890 minus 45


Table 2 Continued


H51-3 238 Os01g0239200 Phophate translocator 399 (216) 218219 (99) 3119890 minus 108

N53-1 372 Os08g0517200 Ca2+-ATPase isoform 9 580 (314) 316317 (99) 2119890 minus 162

H57-2 304 Os02g0176700Potentialcalcium-transportingATPase 9 plasma membranetype

505 (273) 282286 (99) 9119890 minus 140

H59-4 276 Os02g0788800 Amino acidpolyaminetransporter II family protein 466 (252) 252252 (100) 4119890 minus 128

Stress responseH53-8 191 Os07g0186000 Thioredoxin h isoform 1 189 (102) 104105 (99) 6119890 minus 45

H53-9 186 Os07g0186000 Thioredoxin h isoform 1 187 (101) 104105 (99) 2119890 minus 44

N60-4 307 Os07g0620200Heat shock protein DnaJN-terminal domaincontaining protein

403 (218) 218218 (100) 3119890 minus 109

Signal transduction

H51-6 175 SRP14 Signal recognition particleSubunit 14 279 (151) 156158 (99) 3119890 minus 72


Cell cycle and fate

H4-1 357 RSPSP94 S-phase-specific ribosomalprotein 604 (327) 329330 (99) 1119890 minus 169

N6-1 296 AY4367731Putative senescence-associated protein (Pyruscommunis)

503 (272) 277279 (99) 3119890 minus 139

Unclear functional proteinsH3-2-1 200 Os05g0156800 Hypothetical protein 263 (142) 144145 (99) 4119890 minus 67

N19-2 369 BAH800651 Putative retrotransposonprotein 173 (438) 97100 (97) 4119890 minus 48

lowast

N51-2 436 Os12g0597400 Hypothetical protein 758 (410) 414416 (99) 00N51-3 411 Os12g0444500 Hypothetical protein 479 (259) 270275 (98) 8119890 minus 132

N51-4 399 BAD685981 Hypothetical protein 466 (109) 2959 (49) 5119890 minus 04lowast

H51-2 250 Os06g0124900 Hypothetical protein 265 (143) 179194 (92) 1119890 minus 67


N53-5 298 AAQ565701 Polyprotein 145 (367) 79125 (63) 1119890 minus 41lowast

H53-11 143 Os04g0599650 Tetratricopeptide-like helicaldomain containing protein 230 (124) 139145 (96) 3119890 minus 57






N60-5 303 EEC845891 Hypothetical proteinOsI 31400 909 (224) 4548 (94) 5119890 minus 19

lowast

N62-8 305 AAT441711 Hypothetical protein 732 (178) 3536 (97) 1119890 minus 15lowast

The ldquoHrdquo before the numbers of the TDFs represents upregulated expression in high-temperature treated plants while ldquoNrdquo represents upregulated expression innormal temperature treated plants (downregulated gene under high temperature) All are BLASTN scores except for thosemarked with ldquolowastrdquo which are BLASTXscores


0

05

1

15

2

25

Shuanggui 1 Huanghuazhan

Relat

ivee

xpre

ssio

n

Cultivar

Os04g0429600 a

c

cc

b

ccc

(a)

005

115

225

335

445

5

Os06g0124900


Relat

ivee

xpre

ssio

n

Cultivar

a

ccc

b

ccc

(b)

0

05

1

15

2

25

3

SRP14 a

b

dd

b

c

dd


Relat

ivee

xpre

ssio

n

Cultivar

(c)

0

1

2

3

4

5Os02g0611200

a

b

ccc c

b

c


Relat

ivee

xpre

ssio

n

Cultivar

(d)


Relat

ivee

xpre

ssio

n

Cultivar

28∘39∘

28∘

39∘

Os02g0788800

a

c

b

c

a

cc

c

0

05

1

15

2

25

C 24 hC 24 h

C 48 hC 48 h

(e)

0

05

1

15

2

25

3

Os07g0620200a

bbbb b

c

d


Relat

ivee

xpre

ssio

n

Cultivar

28∘39∘

28∘

39∘C 24 hC 24 h

C 48 hC 48 h

(f)

Figure 2 Relative expression ofOs04g0429600Os06g0124900 SRP14Os02g0611200Os02g0788800 andOs07g0620200 compared to that ofactin detected by real-time quantitative RT-PCR in plants at 2822∘Cor plants at 3930∘C for 24 hours and 48 hours in heat-sensitive Shuanggui1 and heat-tolerant Huanghuazhan Values indicate relative expression levels against those of the same genes at 2822∘C in three biologicalreplications from cDNA prepared from leaves Os04g0429600 cellulose synthase-like protein H1 Os06g0124900 hypothetical proteinSRP14 signal recognition particle subunit 14 Os02g0611200 S-adenosylmethionine decarboxylase proenzyme (AdoMetDC) (SamDC)Os02g0788800 amino acidpolyamine transporter II Os07g0620200 heat shock protein DnaJ N-terminal domain containing protein


0

100

200

300

400

Putrescine Spermidine Sperimine

Poly

amin

esco

nten

t (nm

ol gminus1FW

)

28∘C39∘C

a

b

c

ef

d

Figure 3 Variation of levels of free spermidine spermine andputrescine in rice seedlings under 39∘C and 28∘C treatments withHPLC analysis Data from levels of free spermidine spermine andputrescine in rice seedlings are means of three replications plusmn SEDissimilar letters above bars differ significantly at 119875 le 005

Os06g0124900 SRP14 Os02g0611200 and Os02g0788800)expressions were increased under heat stress with oneTDF (Os07g0620200) shown downregulated under the samecondition

4 Discussion

Previous genomic and proteomic studies on rice responses toheat stress have provided some valuable results Genes [26]and proteins [27] of rice grain under heat stress conditionshave been identified Changes of proteomic profiles in riceseedling leaves at high temperature [4] have also beenreported In this study responses of rice seedlings to heatstress environment (39∘C day30∘C night) were investigatedAmong TDFs which were responsive to 39∘C 49 of themhave been identified The corresponding proteins or relativenucleotides of these identified TDFs were related to energy(photosynthesis and photorespiration) transportation tran-scription or translation and other biological functionsrespectively Furthermore unlike previous transcriptomic[26] and proteomic [4 9 27] studies in our study 49 TDFswere identified as high-temperature-responsive genes Theidentified genes provided valuable information by which thetolerance mechanisms of rice exposed to heat stress can beunveiled in detail

41 Effect of Heat Stress on the Biological Process of theRice Seedling In this study there are fourteen differen-tial expression fragments which were classified into car-bohydrate metabolism group The upregulated TDF (H51-4 Table 2) in response to heat stress is the chloroplastgene (psbH) which encodes a 9-10 kDa thylakoid membrane

protein (PSII-H) PSII protein H is associated with photo-systhem II and is subject to light-dependent phosphoryla-tion at a threonine residue located on the stromal side ofthe membrane [28] Thus the up-regulation of psbH genecould protect the photosynthetic machinery in the high-temperature-stressed rice seedlings Other two differentialexpression genes (N21-1 H53-1 Table 2) which are involvedin carbohydrate metabolism are also induced by the heatstress TDF (N21-1 Table 2) corresponding to NAD depen-dent epimerasedehydratase family protein is downregulatedunder 39∘C treatment TDF (H53-1 Table 2) correspond-ing to NADH dehydrogenase subunit 2 is upregulated bythe stress treatment NAD-dependent epimerasedehydratasefamily protein takes part in the carbohydrate metabolicbiological process which includes the formation of car-bohydrate derivatives by the addition of a carbohydrateresidue to another molecule Hence downregulated TDF(N21-1 Table 2) suggested that heat stress may inhibit thecarbohydrate derivatives formation in the seedling stage ofseedling leaves NADH dehydrogenase which is respon-sible for the oxidative phosphorylation [29] is so-calledentry-enzyme of the mitochondrial electron transport chainThe up-regulation of NADH dehydrogenase indicated thatprotective mechanism involved in electron transportationprocess may be stimulated in the heat-stressed rice seedlingIn spite of down-regulation of NAD-dependent epimeraseinvolved in carbohydrate derivatives formation the moresignificant electronic transportation process regulated byNADH dehydrogenase showed upregulated trend indicatingthe protective capability of rice seedling confronted with heatstress

In cellulose metabolism category TDF (H46-3 Table 2and Figure S1) which encodes cellulose synthase familyprotein was also upregulated by the heat stress stimulationThe transcript expression of this enzyme in cDNA-AFLPwas also correlated with the results in real-time RT-PCR(Figure 2) Earlier work concluded that cellulose synthaseis the key enzyme involved in cellulose synthesis in cellwall [30] The up-regulation of cellulose synthase during theheat stress indicated that high-temperature may improve theformation of cell wall and hence assumed the protectivefunction against the damage generated by the temperature forthe rice seedling

Besides the most abundant groups mentioned abovetherewere still otherTDFswhichwere identified as functionsinvolved in material transportation cell structure and cyclepolyaminemetabolism and signal transduction and so forthIn spite of fewer TDFs found in these groups the accordanceof the semiquantitative RT-PCR results (data not shown)and the real-time PCR results (Figure 2) with the cDNA-AFLP analysis (Figure S1) indicated that these fragmentswith corresponding functional groups were also affectedseverely by the increase of the temperature Therefore theidentification of the TDFs will be a solid foundation for thefuture researches

Five genes were which involved in material transporta-tion were increasely expressed under heat stress whichimplied a temperature-related activation of this processIn this study TDF (H59-4 Table 2) identified as amino


acid permease which could import serine and generatesphingoid bases during heat stress [31] was significantlyupregulated at 39∘C treatment The activity change of TDF(H59-4) agreeing well with the change of mRNA expres-sion of amino acid permease (Figure 2) confirmed thatpermease-regulated amino acid uptake could be increasedby the stimulation of the heat stress In addition anotherTDF (H51-3 Table 2 and Figure S1) corresponding to triosephosphate translocator protein could also be included intomaterial transporters group Triose phosphate translocatorprotein is a membrane protein responsible for exchanging allcarbohydrate products produced in photosynthesis in plantsand therefore could mediate the export of fixed carbon inthe form of triose phosphates and 3-phosphoglycerate fromthe chloroplasts into the cytosol [32] So the up-regulation ofTDFs (H51-3) indicated that the increase of the temperaturecould promote the carbohydrate products transportationproduced in photosynthesis and hence provide the protec-tive mechanism for the rice seedling leaves during the heatstress

There are other two upregulated TDFs (H51-6 Figure S1and Table 2 H57-5 Figure S1 and Table 2) which encode sig-nal recognition particle subunit and S-adenosylmethioninedecarboxylase classified into two newly functional groupssignal transduction and polyamine metabolism The up-regulation of TDF (H51-6 Figure S1 and Table 2) whichencodes signal recognition particle subunit suggested thathigh temperature could also improve the signal transduc-tion process during the heat stress S-Adenosylmethioninedecarboxylase is an enzyme whose capability is to catalyzethe conversion of S-adenosyl methionine to S-adenosylme-thioninamine S-adenosylmethionine decarboxylase plays anessential regulatory role in the polyamine biosynthetic path-way by producing the n-propylamine residue required for thesynthesis of spermidine and spermine from putrescine [3334] Spermidine and spermine synthase (SPDS EC 25116and SPMS EC 25122) could synthesize higher spermidineand spermine by the successive addition of aminopropylgroups to putrescine [35] Our result has also proved thatthe upregulated expression of S-adenosylmethionine decar-boxylase gene agreed well with the contents of spermidineand spermine (Figures 2 and 3) In addition one TDF (N60-4 Table 2 Figure S1) corresponding to heat shock protein(hsp) has been classified into stress response group Its down-regulation function which was agreeing well with the real-time RT-PCR result (Figure 2) indicated that the gene wasaffected obviously under heat stress condition

In conclusion this study reported 49 identified differ-ential expression fragments in rice seedlings in respons tothe heat stress Meanwhile ten functional groups classifica-tion containing 49 TDFs indicating different strategies wereemployed by rice seedlings exposed to different tempera-tures the higher the temperature the more effects could beobserved Our future research could be focused to revealfunctions of the genes indentified under heat stress

Authorsrsquo Contribution

Y Cao and Q Zhang contributed equally to this work

Acknowledgments

The authors gratefully acknowledge the Grants from theNational Natural Science Foundation of China (3067122530771274) the Natural Science Foundation of JiangsuProvince (BK2009005 BK2007071) Science and TechnologyProgram of Nantong (AS2010017 HL2012028) Open Foun-dation of Key Laboratory of Crop Genetics and Physiology ofJiangsu Province (027388003K10008) the Doctor InitiationFoundation (12B006) and the Natural Science Foundation ofNantong University (11ZY011 03080503 03041068)

References

[1] P Stone ldquoThe effects of heat stress on cereal yield and qualityrdquoin Crop Responses and Adaptations To Temperature Stress pp243ndash291 Binghamton New York NY USA 2001

[2] G S Khush ldquoGreen revolution the way forwardrdquo NatureReviews Genetics vol 2 no 10 pp 815ndash822 2001

[3] A Grover A Chandramouli S Agarwal S Katiyar-AgarwalM Agarwal and C Sahi ldquoTransgenic rice for tolerance againstabiotic stressesrdquo in Rice Improvement in the Genomic Era pp237ndash267 Hawarth Press 2009

[4] D G Lee N Ahsan S H Lee et al ldquoA proteomic approach inanalyzing heat-responsive proteins in rice leavesrdquo Proteomicsvol 7 no 18 pp 3369ndash3383 2007

[5] A Singh U Singh D Mittal and A Grover ldquoGenome-wideanalysis of rice ClpBHSP100 ClpC and ClpD genesrdquo BMCGenomics vol 11 no 1 article 95 2010

[6] K Kosova P Vıtamvas I T Prasil and J Renaut ldquoPlantproteome changes under abiotic stress-Contribution of pro-teomics studies to understanding plantstress responserdquo Journalof Proteomics vol 74 no 8 pp 1301ndash1322 2011

[7] J V Jorrın-Novo A M Maldonado S Echevarrıa-Zomeno etal ldquoPlant proteomics update (2007-2008) second-generationproteomic techniques an appropriate experimental designand data analysis to fulfill MIAPE standards increase plantproteome coverage and expand biological knowledgerdquo Journalof Proteomics vol 72 no 3 pp 285ndash314 2009

[8] G K Agrawal N S Jwa and R Rakwal ldquoRice proteomicsending phase i and the beginning of phase IIrdquo Proteomics vol9 no 4 pp 935ndash963 2009

[9] F Han H Chen X J Li M F Yang G S Liu and S H Shen ldquoAcomparative proteomic analysis of rice seedlings under varioushigh-temperature stressesrdquo Biochimica et Biophysica Acta vol1794 no 11 pp 1625ndash1634 2009

[10] W Hu G Hu and B Han ldquoGenome-wide survey and expres-sion profiling of heat shock proteins and heat shock factorsrevealed overlapped and stress specific response under abioticstresses in ricerdquo Plant Science vol 176 no 4 pp 583ndash590 2009

[11] A Goswami R Banerjee and S Raha ldquoMechanisms of plantadaptationmemory in rice seedlings under arsenic and heatstress expression of heat-shock protein gene HSP70rdquo AoBPlants vol 2010 Article ID plq023 2010

[12] P Breyne and M Zabeau ldquoGenome-wide expression analysisof plant cell cycle modulated genesrdquo Current Opinion in PlantBiology vol 4 no 2 pp 136ndash142 2001

[13] C W B Bachem R S van der Hoeven S M de Bruijn DVreugdenhil M Zabeau and R G F Visser ldquoVisualizationof differential gene expression using a novel method of RNAfingerprinting based on AFLP analysis of gene expression


during potato tuber developmentrdquoThe Plant Journal vol 9 no5 pp 745ndash753 1996

[14] R F Ditt E W Nester and L Comai ldquoPlant gene expressionresponse to Agrobacterium tumefaciensrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 98 no 19 pp 10954ndash10959 2001

[15] R FukumuraH Takahashi T Saito et al ldquoA sensitive transcrip-tome analysis method that can detect unknown transcriptsrdquoNucleic Acids Research vol 31 no 16 p e94 2003

[16] P Breyne R Dreesen B Cannoot et al ldquoQuantitative cDNA-AFLP analysis for genome-wide expression studiesrdquo MolecularGenetics and Genomics vol 269 no 2 pp 173ndash179 2003

[17] HNakagawa THorie andTMatsui ldquoEffects of climate changeon rice production and adaptive technologiesrdquo in Rice ScienceInnovations and Impact For Livelihood pp 635ndash658 BeijingChina 2003 Proceedings of the International Rice ResearchConference

[18] P V V Prasad K J Boote L H Allen Jr J E Sheehy andJ M G Thomas ldquoSpecies ecotype and cultivar differences inspikelet fertility and harvest index of rice in response to hightemperature stressrdquo Field Crops Research vol 95 no 2-3 pp398ndash411 2006

[19] T Matsui K Omasa and T Horie ldquoThe difference in sterilitydue to high temperatures during the flowering period amongJaponica-rice varietiesrdquo Plant Production Science vol 4 no 2pp 90ndash93 2001

[20] M Bitrian X Zarza T Altabella A F Tiburcio and R AlcazarldquoPolyamines under abiotic stress metabolic crossroads andhormonal crosstalks in plantsrdquoMetabolites vol 2 no 3 pp 516ndash528 2012

[21] R Alcazar T Altabella F Marco et al ldquoPolyamines moleculeswith regulatory functions in plant abiotic stress tolerancerdquoPlanta vol 231 no 6 pp 1237ndash1249 2010

[22] C W B Bachem R J F J Oomen and R G F VisserldquoTranscript Imaging with cDNA-AFLP a step-by-step proto-colrdquo Plant Molecular Biology Reporter vol 16 no 2 pp 157ndash1731998

[23] B J Bassam G Caetano-Anolles and P M Gresshoff ldquoFastand sensitive silver staining of DNA in polyacrylamide gelsrdquoAnalytical Biochemistry vol 196 no 1 pp 80ndash83 1991

[24] M R Frost and J A Guggenheim ldquoPrevention of depurinationduring elution facilitates the reamplification of DNA fromdifferential display gelsrdquo Nucleic Acids Research vol 27 no 15p e6 1999

[25] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[26] H Yamakawa T Hirose M Kuroda and T YamaguchildquoComprehensive expression profiling of rice grain filling-relatedgenes under high temperature using DNA microarrayrdquo PlantPhysiology vol 144 no 1 pp 258ndash277 2007

[27] S K Lin M C Chang Y G Tsai and H S Lur ldquoProteomicanalysis of the expression of proteins related to rice quality dur-ing caryopsis development and the effect of high temperatureon expressionrdquo Proteomics vol 5 no 8 pp 2140ndash2156 2005

[28] H E OrsquoConnor S V Ruffle A J Cain et al ldquoThe 9-kDaphosphoprotein of photosystem II Generation and character-isation of Chlamydomonas mutants lacking PSII-H and a site-directedmutant lacking the phosphorylation siterdquoBiochimica etBiophysica Acta vol 1364 no 1 pp 63ndash72 1998

[29] E Nakamaru-Ogiso H Han A Matsuno-Yagi et al ldquoThe ND2subunit is labeled by a photoaffinity analogue of asimicin apotent complex I inhibitorrdquo FEBS Letters vol 584 no 5 pp883ndash888 2010

[30] R A Burton N J Shirley B J King A J Harvey and G BFincher ldquoThe CesA gene family of barley Quantitative analysisof transcripts reveals two groups of co-expressed genesrdquo PlantPhysiology vol 134 no 1 pp 224ndash236 2004

[31] L A Cowart and Y A Hannun ldquoSelective substrate supplyin the regulation of yeast de novo sphingolipid synthesisrdquo TheJournal of Biological Chemistry vol 282 no 16 pp 12330ndash123402007

[32] U I Flugge ldquoPhosphate translocators in plastidsrdquo AnnualReview of Plant Physiology and Plant Molecular Biology vol 50no 1 pp 27ndash45 1999

[33] P D van Poelje and E E Snell ldquoPyruvoyl-dependent enzymesrdquoAnnual Review of Biochemistry vol 59 pp 29ndash59 1990

[34] A E Pegg H Xiong D J Feith and L M Shantz ldquoS-adenosylmethionine decarboxylase structure function andregulation by polyaminesrdquo Biochemical Society Transactionsvol 26 no 4 pp 580ndash586 1998

[35] J F Jimenez-Bremont O A Ruiz and M Rodrıguez-KesslerldquoModulation of spermidine and spermine levels in maizeseedlings subjected to long-term salt stressrdquo Plant Physiologyand Biochemistry vol 45 no 10-11 pp 812ndash821 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


study about rice in response to heat stress is significant theassessment of gene level regulation of such type of temper-ature variability on rice is also unneglectful In additionrice may confront with different high-temperature stressesduring its lifespan and different growth phases may havedifferent responses to the stresses which will be inevitablyregulated by the variation of the expression of genes Previousproteomic studies revealed that seedlings-phased rice couldalso be affected by the heat stress environment [9] Althoughgenomewide expression analysis showed that transcriptionlevel of heat shock proteins (Hsps) and heat shock factors(Hsfs) are induced under heat stress at young seedlingstages [10 11] further investigations to uncover the regulativemechanisms corresponding to heat tolerance at genomicslevel are still necessary

Among techniques employed in gene identificationmanytechniques such as differential display reverse transcription-polymerase chain reaction (DDRT-PCR) representationaldifference analysis (RDA) serial analysis of gene expression(SAGE) suppression subtractive hybridization (SSH) andcDNAmicroarray provide effective approaches for transcrip-tion analysis [12] As a high-efficient less labor-intensivemRNA fingerprinting method for isolation of differentiallyexpressed genes [13] cDNA-amplified fragment length poly-morphism (cDNA-AFLP) which has been employed in thestudy is a robust genomewide expression tool [12] for genediscovery without a prerequisite of the prior knowledge ofthe sequences [14] In addition due to its high detectivesensitivity of cDNA-AFLP analysis some rare transcriptscould also be detected by this method [15] This techniquehas been further ameliorated to avoid the possibility ofseveral transcript-derived fragments (TDFs) from a singlegenecDNA [16]

Currently several rice varieties defined by temperaturethresholds [17] have been identified with genotype variationin spikelet sterility at high temperature in indica and japonica[18 19] In our study forty-nine critical genes differentiallyexpressed in rice seedling in response to heat stress usingcDNA-AFLP technique have been identified To validate theirexpression patterns six gene fragments involving differentphysiological activitieswere analyzed through real-timePCRIn addition due to direct interactions of polyamines withother metabolic pathways during the stress response [20] andtheir regulatory functions in plant abiotic stress tolerance[21] the variations of three different polyamine contentsin response to heat stress through high-performance liquidchromatography (HPLC) analysiswere also performedTheseoutputs may lay the fundamental basis of the rice varietybreeding in the future

2 Materials and Methods

21 Plant Material Seeds of two indica rice cultivars (Shu-anggui 1 heat sensitive Huanghuazhan heat tolerant) wereprovided by the Rice Research Institute of GuangdongAcademy of Agricultural Sciences (Guangzhou China)The seedlings were grown in Kimura B complete nutri-ent solution under greenhouse conditions (28∘C day22∘C

night relative humidity 60ndash80 the light intensity 600ndash1000 120583molmminus2 sminus1 and photoperiod of 14 h day10 h night)with 50 seedlings per pot Then twenty-day old seedlingswere heat-stressed (at 39∘C day30∘C night) for two differenttreatment times 24 h or 48 h while control seedlings weregrown under the conditions mentioned above Each treat-ment had 3 pots as replications Samples from rice seedlingleaves were harvested and were immediately frozen in liquidnitrogen and stored at minus80∘C until use Heights of riceseedlings of Shuanggui 1 at 28∘C control condition or 39∘Cstress condition were determined with a ruler from the landsurface to the top of the seedling leaves with means of threereplications (50 plants per replication)

22 RNA Extraction Total RNA was extracted using RNeasyPlant Mini Kit (QIAGEN) according to the manufacturerrsquosinstructionsThe integrity of the RNAwas checked by agarosegel electrophoresis and the concentration was determined at260 nm by spectrophotometer

23 cDNA-AFLP Analysis An cDNA-AFLP method wasadapted from Bachem et al [13 22] with a few modificationsSynthesis of double-stranded cDNA was performed with M-MLVRTase cDNA Synthesis kit (TaKaRa China) and refinedusing phenolchloroform extraction 5 120583L was checked usingagarose gel electrophoresis in order to observe an expectedsmear between 100 bp and 1000 bp The rest of cDNA wasdigested using the restriction enzymes EcoR I (Fermentas2 h at 37∘C) and Mse I (Tru1I Fermentas 2 h at 65∘C)The digested products were ligated to adaptors (EcoR I5 120583mol Lminus1 forward primer 51015840-CTCGTAGACTGCGATCC-31015840 reverse primer 51015840-AATTGGTACGCAGTCTAC-31015840Mse I50120583mol Lminus1 forward primer 51015840-GACGATGAGTCCTGAG-31015840 reverse primer 51015840-TACTCAGGACTCAT-31015840) with T4-DNA ligase (Fermentas) for 13 h at 16∘C The products ofligation were amplified with the corresponding preamplifica-tion primers (EcoR I 51015840-GACTGCGTACCAATTC-31015840Mse I51015840-GATGAGTCCTGAGTAA-31015840) Preamplification was initi-ated at 94∘C for 3min and followed by 30 cycles at 94∘C for30 s 56∘C for 30 s 72∘C for 1min and terminated at 72∘C for5min The products of the preamplification were checked byagarose gel electrophoresis (expected smear between 100 bpand 1000 bp) From a 20-fold dilution of the pre-amplifiedsamples a 5 120583L sample was used for the final selective ampli-fications using the primers 51015840-GACTGCGTACCAATTCNN-31015840 (EcoR I NN represents AC AG GA and GT) and51015840-GATGAGTCCTGAGTAAMM-31015840 (Mse I MM representsTC TG CA and CT) After initial denaturation (94∘C for3min) 12 cycles were performed with touchdown annealing(94∘C for 30 s 65∘C to 56∘C in 07∘C steps for 30 s 72∘C for1min) followed by 24 cycles (94∘C for 30 s 56∘C for 30 s 72∘Cfor 1min) and final elongation (72∘C for 5min) Altogether 64primer combinations of two base extensions (denoted as NNand MM) were used

24 Polyacrylamide Gel Electrophoresis The selective ampli-fication products were separated on a sequencing polyacry-lamide gel (6 polyacrylamide 8mol Lminus1 urea 1timesTBE) at a








005level

3 Results




(a) (b)

0

5

10

15

20

25

28∘C 39∘C

Plan

thei

ght (

cm)

Treatment

a

b

(c)




















732 (178) 3233 (97) 2119890 minus 14lowast




805 (197) 3435 (97) 3119890 minus 17lowast


lowast










207 (112) 116118 (98) 3119890 minus 50

Protein metabolism






169 (91) 9698 (98) 1119890 minus 38



Cellulose synthesis


Material transport



Table 2 Continued





505 (273) 282286 (99) 9119890 minus 140





403 (218) 218218 (100) 3119890 minus 109

Signal transduction



Cell cycle and fate



503 (272) 277279 (99) 3119890 minus 139



lowast













lowast




0

05

1

15

2

25


Relat

ivee

xpre

ssio

n

Cultivar

Os04g0429600 a

c

cc

b

ccc

(a)

005

115

225

335

445

5

Os06g0124900


Relat

ivee

xpre

ssio

n

Cultivar

a

ccc

b

ccc

(b)

0

05

1

15

2

25

3

SRP14 a

b

dd

b

c

dd


Relat

ivee

xpre

ssio

n

Cultivar

(c)

0

1

2

3

4

5Os02g0611200

a

b

ccc c

b

c


Relat

ivee

xpre

ssio

n

Cultivar

(d)


Relat

ivee

xpre

ssio

n

Cultivar

28∘39∘

28∘

39∘

Os02g0788800

a

c

b

c

a

cc

c

0

05

1

15

2

25

C 24 hC 24 h

C 48 hC 48 h

(e)

0

05

1

15

2

25

3

Os07g0620200a

bbbb b

c

d


Relat

ivee

xpre

ssio

n

Cultivar

28∘39∘

28∘

39∘C 24 hC 24 h

C 48 hC 48 h

(f)



0

100

200

300

400


Poly

amin

esco

nten

t (nm

ol gminus1FW

)

28∘C39∘C

a

b

c

ef

d



4 Discussion













Acknowledgments


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








005level

3 Results




(a) (b)

0

5

10

15

20

25

28∘C 39∘C

Plan

thei

ght (

cm)

Treatment

a

b

(c)




















732 (178) 3233 (97) 2119890 minus 14lowast




805 (197) 3435 (97) 3119890 minus 17lowast


lowast










207 (112) 116118 (98) 3119890 minus 50

Protein metabolism






169 (91) 9698 (98) 1119890 minus 38



Cellulose synthesis


Material transport



Table 2 Continued





505 (273) 282286 (99) 9119890 minus 140





403 (218) 218218 (100) 3119890 minus 109

Signal transduction



Cell cycle and fate



503 (272) 277279 (99) 3119890 minus 139



lowast













lowast




0

05

1

15

2

25


Relat

ivee

xpre

ssio

n

Cultivar

Os04g0429600 a

c

cc

b

ccc

(a)

005

115

225

335

445

5

Os06g0124900


Relat

ivee

xpre

ssio

n

Cultivar

a

ccc

b

ccc

(b)

0

05

1

15

2

25

3

SRP14 a

b

dd

b

c

dd


Relat

ivee

xpre

ssio

n

Cultivar

(c)

0

1

2

3

4

5Os02g0611200

a

b

ccc c

b

c


Relat

ivee

xpre

ssio

n

Cultivar

(d)


Relat

ivee

xpre

ssio

n

Cultivar

28∘39∘

28∘

39∘

Os02g0788800

a

c

b

c

a

cc

c

0

05

1

15

2

25

C 24 hC 24 h

C 48 hC 48 h

(e)

0

05

1

15

2

25

3

Os07g0620200a

bbbb b

c

d


Relat

ivee

xpre

ssio

n

Cultivar

28∘39∘

28∘

39∘C 24 hC 24 h

C 48 hC 48 h

(f)



0

100

200

300

400


Poly

amin

esco

nten

t (nm

ol gminus1FW

)

28∘C39∘C

a

b

c

ef

d



4 Discussion













Acknowledgments


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

0

5

10

15

20

25

28∘C 39∘C

Plan

thei

ght (

cm)

Treatment

a

b

(c)




















732 (178) 3233 (97) 2119890 minus 14lowast




805 (197) 3435 (97) 3119890 minus 17lowast


lowast










207 (112) 116118 (98) 3119890 minus 50

Protein metabolism






169 (91) 9698 (98) 1119890 minus 38



Cellulose synthesis


Material transport



Table 2 Continued





505 (273) 282286 (99) 9119890 minus 140





403 (218) 218218 (100) 3119890 minus 109

Signal transduction



Cell cycle and fate



503 (272) 277279 (99) 3119890 minus 139



lowast













lowast




0

05

1

15

2

25


Relat

ivee

xpre

ssio

n

Cultivar

Os04g0429600 a

c

cc

b

ccc

(a)

005

115

225

335

445

5

Os06g0124900


Relat

ivee

xpre

ssio

n

Cultivar

a

ccc

b

ccc

(b)

0

05

1

15

2

25

3

SRP14 a

b

dd

b

c

dd


Relat

ivee

xpre

ssio

n

Cultivar

(c)

0

1

2

3

4

5Os02g0611200

a

b

ccc c

b

c


Relat

ivee

xpre

ssio

n

Cultivar

(d)


Relat

ivee

xpre

ssio

n

Cultivar

28∘39∘

28∘

39∘

Os02g0788800

a

c

b

c

a

cc

c

0

05

1

15

2

25

C 24 hC 24 h

C 48 hC 48 h

(e)

0

05

1

15

2

25

3

Os07g0620200a

bbbb b

c

d


Relat

ivee

xpre

ssio

n

Cultivar

28∘39∘

28∘

39∘C 24 hC 24 h

C 48 hC 48 h

(f)



0

100

200

300

400


Poly

amin

esco

nten

t (nm

ol gminus1FW

)

28∘C39∘C

a

b

c

ef

d



4 Discussion













Acknowledgments


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













732 (178) 3233 (97) 2119890 minus 14lowast




805 (197) 3435 (97) 3119890 minus 17lowast


lowast










207 (112) 116118 (98) 3119890 minus 50

Protein metabolism






169 (91) 9698 (98) 1119890 minus 38



Cellulose synthesis


Material transport



Table 2 Continued





505 (273) 282286 (99) 9119890 minus 140





403 (218) 218218 (100) 3119890 minus 109

Signal transduction



Cell cycle and fate



503 (272) 277279 (99) 3119890 minus 139



lowast













lowast




0

05

1

15

2

25


Relat

ivee

xpre

ssio

n

Cultivar

Os04g0429600 a

c

cc

b

ccc

(a)

005

115

225

335

445

5

Os06g0124900


Relat

ivee

xpre

ssio

n

Cultivar

a

ccc

b

ccc

(b)

0

05

1

15

2

25

3

SRP14 a

b

dd

b

c

dd


Relat

ivee

xpre

ssio

n

Cultivar

(c)

0

1

2

3

4

5Os02g0611200

a

b

ccc c

b

c


Relat

ivee

xpre

ssio

n

Cultivar

(d)


Relat

ivee

xpre

ssio

n

Cultivar

28∘39∘

28∘

39∘

Os02g0788800

a

c

b

c

a

cc

c

0

05

1

15

2

25

C 24 hC 24 h

C 48 hC 48 h

(e)

0

05

1

15

2

25

3

Os07g0620200a

bbbb b

c

d


Relat

ivee

xpre

ssio

n

Cultivar

28∘39∘

28∘

39∘C 24 hC 24 h

C 48 hC 48 h

(f)



0

100

200

300

400


Poly

amin

esco

nten

t (nm

ol gminus1FW

)

28∘C39∘C

a

b

c

ef

d



4 Discussion













Acknowledgments


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 2 Continued





505 (273) 282286 (99) 9119890 minus 140





403 (218) 218218 (100) 3119890 minus 109

Signal transduction



Cell cycle and fate



503 (272) 277279 (99) 3119890 minus 139



lowast













lowast




0

05

1

15

2

25


Relat

ivee

xpre

ssio

n

Cultivar

Os04g0429600 a

c

cc

b

ccc

(a)

005

115

225

335

445

5

Os06g0124900


Relat

ivee

xpre

ssio

n

Cultivar

a

ccc

b

ccc

(b)

0

05

1

15

2

25

3

SRP14 a

b

dd

b

c

dd


Relat

ivee

xpre

ssio

n

Cultivar

(c)

0

1

2

3

4

5Os02g0611200

a

b

ccc c

b

c


Relat

ivee

xpre

ssio

n

Cultivar

(d)


Relat

ivee

xpre

ssio

n

Cultivar

28∘39∘

28∘

39∘

Os02g0788800

a

c

b

c

a

cc

c

0

05

1

15

2

25

C 24 hC 24 h

C 48 hC 48 h

(e)

0

05

1

15

2

25

3

Os07g0620200a

bbbb b

c

d


Relat

ivee

xpre

ssio

n

Cultivar

28∘39∘

28∘

39∘C 24 hC 24 h

C 48 hC 48 h

(f)



0

100

200

300

400


Poly

amin

esco

nten

t (nm

ol gminus1FW

)

28∘C39∘C

a

b

c

ef

d



4 Discussion













Acknowledgments


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

05

1

15

2

25


Relat

ivee

xpre

ssio

n

Cultivar

Os04g0429600 a

c

cc

b

ccc

(a)

005

115

225

335

445

5

Os06g0124900


Relat

ivee

xpre

ssio

n

Cultivar

a

ccc

b

ccc

(b)

0

05

1

15

2

25

3

SRP14 a

b

dd

b

c

dd


Relat

ivee

xpre

ssio

n

Cultivar

(c)

0

1

2

3

4

5Os02g0611200

a

b

ccc c

b

c


Relat

ivee

xpre

ssio

n

Cultivar

(d)


Relat

ivee

xpre

ssio

n

Cultivar

28∘39∘

28∘

39∘

Os02g0788800

a

c

b

c

a

cc

c

0

05

1

15

2

25

C 24 hC 24 h

C 48 hC 48 h

(e)

0

05

1

15

2

25

3

Os07g0620200a

bbbb b

c

d


Relat

ivee

xpre

ssio

n

Cultivar

28∘39∘

28∘

39∘C 24 hC 24 h

C 48 hC 48 h

(f)



0

100

200

300

400


Poly

amin

esco

nten

t (nm

ol gminus1FW

)

28∘C39∘C

a

b

c

ef

d



4 Discussion













Acknowledgments


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

100

200

300

400


Poly

amin

esco

nten

t (nm

ol gminus1FW

)

28∘C39∘C

a

b

c

ef

d



4 Discussion













Acknowledgments


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







Acknowledgments


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Identification of Differential Expression...

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