Proteomic Analysis of Wheat Seed in Response to Drought Stress

Available online at www. sciencedirect.comJournal of Integrative AgricultureScienceDirect2014, 13(5): 919-925May 2014RESEARCH ARTICLEProteomic Analysis of Wheat Seed in Response to Drought StressZHANG Yu-feng, HUANG Xiu-wen, WANG Li-li, WEI Liu, WU Zhi-hui, YOU Ming shan and LI Bao-yunBeijing Key Laboratory of Crop Genetic Improvement/Key Laboratory of Crop Heterosis & Utilization, Ministry of Education/College ofAgronomy and Biotechnology, China Agricultural University, Beijing 100193, P.R.ChinaAbstractDrought stress is one of the major factors affecting in wheat yield and grain quality. In order to investigate how droughtstress might influence wheat quality during grain flling, three wheat cultivars Gaocheng 8901, Jagger and Nongda 3406 weresubjected to drought stress during the grain flling stage. Neither globulin and glutenin, nor the relative percentage of amylosesignificantly changed following drought treatments, whereas albumin and gliadin concentrations did. The SDS-sedimentation,which has a strong linear correlation with wheat baking quality was markedly decreased following drought stress. Theseresults indicated that drought had an adverse efect on wheat quality. In order to investigate the protein complexes in thewheat flour, the data from native PAGE and SDS-PAGE were combined and a total of 14 spots were successfully identified,and of these eight protein types were determined to be potential complex forming proteins.Key words: wheat seed, drought stress, two-dimensional electrophoresis (2-DE)decreased under drought stress, Yang et al. (2011)INTRODUCTIONfound that 0- and γ-gliadin decreased in response todrought stress, and Fabian et al. (201 1) found that theIt has long been known that drought stress significant-A-type starch increased, whereas the B-type starch ac-ly affects common wheat (Triticum aestivum L) yield tually decreased.and quality, especially the composition of seed storageProteomics is an effective method to investigateproteins that form during the grain filling stage. In changes in wheat seed protein concentration in .fact, the baking quality of spring wheat flour can beresponse to drought stress. Vensel et al. (2005)negatively affected by drought stress during the de-analyzed the differences in wheat seed proteinsvelopment period (Day and Barmore 1971). Under between 10-36 DAP (days after pollination). In thisdrought stress, the protein content, SDS-sedimenta-survey, 250 differentially expressed proteins weretion value and wet gluten of winter wheat have been found, and were involved in 13 different biologicaldemonstrated to increase by 18.1, 16.5 and 21.9% fromprocesses. Analysis of protein content revealed thatgermination to maturation, whereas they increased most endosperm proteins were related to carbohydrateonly by 8.3, 8.7 and 10.8% respectively during grainmetabolism, transcription, translation, proteinfilling (Ozturk and Aydin 2004). However, Gooding synthesis at 10 DAP, and carbohydrate metabolismet al. (2003) found that the SDS-sedimentation valueand protein synthesis at 36 DAP, but stress, defenseReceived 7 March, 2013 Acepted 2 July, 2013中国煤化工Correspondence LI Ba-yun, Tel: +86- 10-6273 1064, Fax: +86-10-62732568, E-mail: baoyunli@cau. edu.cnYHCNM HG◎2014, CAAS. All rights reserved. Published by Elsevier Ltd.doi: 10.1016/S2095-3119(13)60601-292ZHANG Yu-feng e1 al.and storage proteins predominated (e.g, heat shock, separated into single subunits, with most functionalgranule-bound starch synthase, β-amylase, glucose-1-proteins containing multiple subunits. The individualphosphate adenyltransferase, etc.).proteins, which were separated by SDS-PAGE, are of-The protein expression profiles of heat-sensitive (cv.ten associated with each other, forming temporary, orWyuna) and heat-tolerant (cv. Fang) wheat cultivars stable larger protein complexes. Thus it is dificult tohave been compared following heat shock (Skylasascertain which proteins form a complex through IEF-et al.2002). In this study, 48 proteins were differen-SDS- PAGE alone. In the present study, proteins weretially expressed, and of these 17 were identified as heat separated by native PAGE, which does not break apartshock proteins. This is consistent with that found byprotein complexes in the first dimension (Fig. 1). Fol-Majoul et al. (2003, 2004) in which 25 proteins werelowed by the second dimension, protein complexes aredifferentially expressed proteins following heat shock,separated by SDS-PAGE, in which some flour proteinamong which 24 were up-regulated, yet only onecomplexes could be identified, and thus provided awas down-regulated. Some of these were enzymesmore in-depth understanding of flour protein interac-involved in different plant metabolic pathways, such tions.as granule-bound starch synthase and glucose- 1-phos-phate adenyltransferase (both involved in the starchAsynthesis), β-amylase, (involved in carbohydrate|o8[&)8\σmetabolism) and the ATP synthase β-chain that wasrelated to four heat-decreased proteins. Non-prolamin竺proteins of Durum wheat were affected by heat stress|2during grain flling, and 132 differentially expressedspots were revealed. Of these 47 were HSPs, and|/7|proteins involved in glycolysis, carbohydrate metab-c8|olism and stress-related proteins were analyzed byMALDI-TOF and MALDI-TOF-TOF MS. Sanchoet al. (2008) examined the influence of genotype andFig. 1 Protein complexes electrophoresis diagram. A, first, native-environment on the accumulation of soluble wheatPAGE. B, second, SDS-PAGE.dough liquor protein (Gliadin) under hot/dry, and cool/wet regimes using 2-DE gel technology. In such, theRESULTS7S globulin (vicilin-like), R-globulin families, someprotective proteins including members of the serpin,Changes in protein composition and starchand chitinase families were differentially expressed.The combination of water deficit and high-temperaturecontent in response to drought stressresulted in differential expression of primary metabo-lism, storage, and stress response related proteins (e.g. Four seed storage proteins (e.g. albumin, globulin,late embryogenesis abundant proteins, peroxiredoxin,gliadin, and glutenin) were determined in the cultivara.-amylase inhibitors, and 14-3-3 proteins, etc., YangGaocheng 8901 to assess if their content was affectedet al. 2011). In addition, the effect of different nitro-by drought stress. To analyze the accumulation ofgen and sulfur levels on tris-soluble, and glutenin pro-different storage protein classes, seed protein wastein fractions have been determined via use of theseextracted from mature grains and separated into2-DE gels (Grove et al. 2009). In such, 20 tris-soluble albumin, globulin, gliadin, and glutenin. The albuminand 16 types of glutenin were differentially expressed.and gliadin concentrations increased significantlyAlthough this study demonstrated that a sulfur defi- under drought stress, whereas globulin and gluteninciency during grain flling might result in changes inconcentrations did not (Fig. 2). .protein composition, this was not evident in Dupont'sThe relative percentage. of amylose in Gaochenget al. (2006) investigation. This study used a degen- 8901 flour under中国煤化工al growtheration condition in which albumin and globulin wereconditions were (C N M H Gpercentage◎2014, CAAS. All rights reserved. Published by Elsevier Ltd.Proteomic Analysis of Wheat Seed in Response to Drought Stress21口Albumin日 Globulin 8 Gliadin■ Glutenin6]4]0十10 |0.DroughtNormal0-Fig. 3 The percentage of amylose content in flour of Gaocheng0+8901 grown under drought and normal conditons. No significantdifference was detected.Fig. 2 Albumin, globulin, gliadin, and glutenin under drought; (Jagger, Nongda 3406 and Gaocheng 8901) werestress in Gaocheng 8901 flour." , P<0.05," ， P<0.01. Data aremeans+SD. The same as below.subjected to SDS-sedimentation. Results of the SDS-sedimentation at5, 10, 15, and 20 min are summarizedof amylose content was 11.4 and 13.0% within seedsin Fig. 4. The SDS-sedimentation values of these threeof drought stressed wheat and normally grown wheat,cultivars under drought stess were significantly lowerrespectively (Fig. 3). Though no statistically signifi- than that observed for those under normal conditions.cant differences were observed between the two treat-The results suggested that drought stress may have aments, the quality of wheat flour may be influenced by negative effect on the final baking quality of wheat.the slight decrease within drought-stressed seeds.Ptotein profiling of wheat flour in response toSDS-sedimentation of wheat flourdrought stressTo evaluate the effect of drought stress on bakingFist dimensional native PAGE can be used to separatequality, flour of three drought-stressed wheat cultivars protein complexes, while a second dimensional SDSagger25Nongda 340630..25 t20 -15-昌1010-5-0Drot| Norm| Drot| Norm| Drot| Norm| Drot| Norm)1020Time (min)35Gaocheng 890130 +25 -20 t5」Drot| Norm| Drot| Norm Drot| Norm| Drot| NormFig. 4 SDS-sedime-' droughtstress and normal co中国煤化工uIght andnormal, respectively.MYHCNM HG◎2014. CAAS. All rights reserved. Published by Elsevier Ltd.92ZHANG Yu-feng e1 al.Native PAGENative-PAGE一CI2D1F2一 一C2Fig. 6 Enlargement of the native PAGE-SDS PAGE images.NommalDroughtFig. 5 Native PAGE-SDS PAGE images in drought sample andBnormal sample.PAGE is used to further resolve subunits of proteincomplexes, which may fail to separate in the firstFig. 7 Enlargement of the native PAGE-SDS PAGE images.dimension. Albumin and globulin were extracted byKCl solution buffer (Fig. 5).The β-amylase was composed of subunits (A1 andA2). There may be interaction between β-amylaseI一and UDP-glucose pyrophosphorylase (H1 and H2).The interaction between UDP-glucose pyrophos-phorylase and oligopeptidase A-like (C1 and C2)may also exist. Both D1 and D2 were 14-3-3 protein(Fig. 6 and Table).Some subunits (B1, B2) of serpin, a supposedlystress-related protein might have interaction with cyto-Fig. 8 Enlargement of the native PAGE-SDS PAGE images.solic 3-phosphoglycerate kinase (B2, Fig. 7 and Table).As illustrated in Fig. 8, E2 was identified as a 27-a-amylase inhibitor may consist of subunits, andkDa protein, and both F1 and F2 were identified as these subunits combine to form the final functionala.-amylase inhibitors. These results suggest thatcomplex.Table Proteins identified by MS-MS"SpcProteinSpeciesAcession no.MW (kDa)coreAB-AmylaseTriticum aestivumgi324007643087228β-Amylase237Serpingil57345064328425Cytosolic 3-phosphoglycerate kinasegil2817291131315Triticum destivumngil22458927043113gi|573450643 284Oligopeptidase A-likeBrachypodium distachyongil35714406787143UDP-glucose pyrophosphorylaseOryza sativagil1 14848914516741514-3-3-like protein GF14-6Zea maysgil26235993529 66214-3-3 proteingil4078 16052927443a-Amylase inhibitorTriticuom aestivuomgil22504219621Triticum aestiwumgil22504326977Triticum ae stivumgil3240076402F2gi1154805710L”Protein complexes, which were failed to separate following the second dimension SDS-PAGE were excised, anFourteen proteinswere sccessfully identified. Those identified included metabolic enzymes (B-amylase, UDP-glucose pyrophos中国煤化工1433 porein,.and stress related proteins (a-amylase inhibitor and serpin).YHCNM HG◎2014, CAAS. All rights reserved. Published by EIsevier Ltd.Proteomic Analysis of Wheat Seed in Response to Drought Stress23DISCUSSIONMATERIALS AND METHODSWheat quality is susceptible to prevailing environ-Plant materials and drought treatmentsmental conditions, of which drought stress is one suchfactor. During grain filling, drought stress affectsWheat cultivars Gaocheng 8901, Jagger and Nongda 3406were grown in 50 cm (L)x30 cm (H)x40 cm (W) plastic boxesgrain carbohydrates, storage protein synthesis andat 25°C and a soil water content of 20 25%, and moved intoaccumulation, subsequently negatively affecting yieldphytotron chambers until anthesis. The growing condition wasand quality (Jiang and Yu 2007). Previous studies .at 20°C (13 h day)/12°C (11 h night). Soil water content wasindicated that the protein content, SDS-sedimentation cotolled as in Apile (2009) in which normal was consideredvalue, and wet gluten of winter wheat were increasedto 20-25%, and drought was 7- 12%, the normal condition wasby 18.1, 16.5 and 21.9%, respectively, under droughtat 20-25%. Soil water was controlled as described by Aprileet al. (2009), the soil water content in drought condition was atstress during germination to maturation, and protein7-12%, the normal condition was at 20-25%.content, sedimentation value and wet gluten contentwere increased by 8.3, 8.7 and 10.8%, respectivelyProtein and amylose content determinationduring grain flling (Ozturk and Aydin 2004). Theseresults are similar to those using Gaocheng 8901.Albumin, globulin, gliadin, and gluten were extracted asAlbumin and gliadin contents increased underpreviously described (Tian 2006). Briefly, mature grain wasmilled with a Brabender D-4100, and the resulting flour (100drought stress, whereas globulin and glutenin didmg) suspended in I mL distlled H2O. These suspensionsnot change appreciably. We did find that all proteinwere incubated at 50°C for 30 min with intermittentcontentrations increased in seed of plants exposed to mixing, and then centrifuged at 4000 r min' for 20 mindrought stress. For example, drought stress resultsat 4°C, repeated three times. This process resulted in thein a significant increase in total seed protein, andalbumin being retained in the soluble fraction. Remainingpellets were suspended in 1 mL 10% NaCl, and incubated,grain glutenin/gliadin protein ratio (Lan et al. 2004;Dai et al. 2006). Although the total protein contentresulting in globulin retained in the soluble fraction. Theincreases under drought stress, protein production remaining pellets were again then suspended in 1 mL 75%EtOH, treated and incubated, mixed and centrifuged at 4000 rdecreases due to reduced grain production (Fan et al.min' for 20 min at 4°C, and any gliadin was retained in2004). Protein complexes research is available forthe soluble fraction. Lastly, the remaining pellets werestudying adversity stress in wheat.suspended in 1 mL 0.2% NaOH, and incubated, mixedand centrifuged 4000 r min' for 20 min at 4°C, and anyremaining gluten retained in the soluble fraction. ProteinCONCLUSIONcontent was determined by the Bradford method. Amylosewas extracted and determined by suspending 100 mg of flourThe present study indicated that protein concentrationsin 1 mL EtOH, to which was added 9 mL NaOH(1 mol L).is changed under drought stress. The albumin andSuspensions were subsequently incubated in boiling(100°C) H2O for 10 min, and cooled in ice water. Aliquotsgliadin concentrations increased significantly, butof the each suspension (20 mL) were transferred to 50 mLglobulin and glutenin concentrations did not change.centrifuge tubes and incubated with 10 mL petroleum etherBesides, amylose content are not observed staistically for 10 min, heated at 25°C for 15 min, repeated thre times.significant differences between the two treatments.The upper fraction contained amylose solution, and theSDS-sedimentation suggest that drought stress haveamylose content was determined using the iodine-potassiumiodide method. The upper fraction was amylase solution,a negative effect on the final baking quality of wheat.and amylose content was determined by iodine-potassiumA total of 14 spots were successfully identified byiodide method. The experimental results were analyzed bycombining native PAGE and SDS-PAGE, which Excel 2010 sofware.revealed many proteins were composed of a fewSDS sedimentation analysisfunctional complexes in wheat flour. Therefore, .protein complexes research may lead to innovativeWheat flour sampl中国煤化工;0.7 mL H2Ostrategies for studying adversity stress in plant.(containing 100incubatedYHCNMH G .◎2014. CAAS. All rights reserved. Published by Elsevier Ltd.924ZHANG Yu-feng etal.at room temperature (≈25°C) for 5 min; 16.7 mL of SDSto terminal drought stress reveals differential responsessolution (2% SDS, 0.012 mol L' lactic acid) was then addedalong the wheat genome. BMC Genomics, 10, 279.to each sample, and each was again incubated at roomDai Y B, Zhao H, Jing Q, Jiang D, Cao w x. 2006. Effectstemperature for an additional 5 min. Sedimentation volumesof high temperature and water stress during grain flligwere recorded every 5 min for a total of 20 min.on grain protein and starch formation in winter wheat.Acta Ecologica Sinica, 26, 3670-3676.Protein electrophoresisDay A D, Barmore M A.1971. Effects of soil moisturestress on the yield and quality of flour from wheatThe frst-dimensional electrophoresis was conducted on a 10%(Triticum aestivum L.). Agronomy Journal, 63, 115-116.native PAGE, at 300 V per gel, and 16°C for 3-4 h until theDupont'sF M, Hurkmana W J, Vensel W H, Chanadye front exited the gel. Gel strips containing protein wereR, Lopezb R, Tanakaa C K, Altenbacha s B. 2006.cut from the gel and equilibrated in 0.0125 mol IDifferential accumulation of sulfur-rich andi sulfurpH 6.8, containing 4% w/v SDS, 0.02% w/v bromophenolpoor wheat four proteins is affected by temperature andblue, and 0.9% w/v beta meraptoethanol for 20 min. Formineral nutrition during grain development. Joural ofthe second-dimensional electrophoresis, gel strips wereCereal Science, 44, 101-112.transferred to 12.5% SDS-PAGE, at 300 V per gel and 16°CFan x M, Jiang D, DaiY B, Jing Q, Cao w D. 2004. ffeetsfor 3-4 h until the dye front exited the gel. Proteins wereof post-anthesis and waterlogging on the quality of grainthen visualized by silver staining as PlusOneTM Staining Kitformation in different wheat varieties. Acta Ecologica .(GE Healthcare, USA).Sinica, 26, 680-685.Fabian A, Jager K, Rakszcegi M, Barmabis B. 2011. Embryo .Protein identification by MALDI-TOF-TOFand endosperm development in wheat (Triticumaestivum L.) kernels subjected to drought stress. PlantCell Reports, 30, 551-563.Proteins of interest were manually excised from the silver-stained gels, and subjected to in-gel trypsin digestion.Gooding M J, Ellis R H, Shewry P R, Schofiel J D.2003.Following digestion, peptides were extracted twice withEffects of restricted water availability and increased0.1% rifiutemperature on the grain flling, drying and quality oftifuracetic acid (TFA) in 50% acetonitrile. Extractswere pooled, dried completely in a SpeedVac, and mixtureswinter wheat. JouJournal of Cereal Science, 37, 295-309.re-dissolved in 0.1% trifuoracetic acid. Peptide solutionsGrove H, Hollung K, Moldestad A, Fergestad E M, Uhlen(0.8 μL) were mixed with 0.4 μL of matrix (a-cyano-4-A K. 2009. Proteome changes in wheat subjected to .hydroxycinnamic acid (CHCA) in 30% ACN, 0.1% TFA)different nitrogen and sulfur fertilizations. Journal ofprior to soting on the target plate.Agricultural and Food Chemistry, 57, 4250-4258.Proteins were identified on an AB SCIEX MALDIJiang D Y, Yu Z W.2007. Effects of soil water on yield andTOF-TOFTM 5800 analyzer (AB SCIEX, Foster City, CA)grain quality of wheat. Journal of Nuclear Agriculuraeequipped with a neodymium: ytrium-aluminum-garnet laserSciences, 21, 641-645.(349 nm). The TOF/TOF calibration mixtures (AB SCIEX)Lan T,Jiang D, XieZJ, Dai Y B, Jing Q, Cao W x.2004.were used to calibrate the spectrum to a mass tolerance ofEffects of post-anthesis drought and waterlogging onwithin 150 ppm. The raw MS and MS/MS spectra data weregrain quality traits in different specialty wheat varieties.processed using GPS ExplorerTM software. Proteins withJournal of Soil and Water Conservation, 18, 193- 196.score confidence intervals (C.I.) above 95% (protein scoreMajoul T, Bancel E, Triboi E, Ben Hamida J, Branlard G.>66) were considered confident identifications. Identified2003. Proteomic analysis of the efct of heat stressproteins were then matched to specific biological processes,on hexaploid wheat grain: Characterization of heat-or molecular functions using the Gene Ontology databaseresponsive proteins from total endosperm. Proteomics, 3,(http://www.geneontology.org/).Acknowledgements2004. Proteomic analysis of the efct of heat stressThis work was supported by the National Natural Scienceresponsive proteins from non-prolamins fraction.Foundation of China (31071412), the High-Tech R&DProteomis, 4, 505-513.Program of China (2012AA101105) and the Key BasicOzturk A, Aydin F. 2004. Effeet of water stress at variousResearch Program of China (2009CB1 18300).growth stages on some quality characteristics of winterwheat. Jourmal of Agronomy and Crop Science, 190, 93-99.ReferencesSancho A I, Gillabert M, Tapp H, Shewry P R, Skeggs PK,Aprile A, Mastrangelo A M, de Leonardis A M, Galiba G,Mills E N. 2008. Effect of environmental stress duringRoncaglia E, Ferrari F, de Bellis L, Turchi L, Giuliano G,grain flling on(Triticum，Cativelli L. 2009. Transcriptional profiling in responseaestivum) doug中国煤化工re and FoodYHCNMH G .0 2014. CAAS. AlI ights reserved. Pullshed by Elsevier LId.Proteomic Analysis of Wheat Seed in Response to Drought Stress25Chemistry, 56, 5386- 5393.B B, Hurkman W J. 2005. Developmental changes inSkylas D J, Cordwell S J, Hains P G, Larsen M R, Bassealthe metabolic protein profiles of wheat endosperm.D J, Walsh B J, Blumenthal C, Rathmell W, Copeland L,Proteomics, 5, 1594-1611.Wrigley C W. 2002. Heat shock of wheat during grainYang F, Jorgensen A D, Li H W, Sondergaard I, Finniefilling: Proteins associated with heat-tolerance. JournalC, Svensson B, Jiang D, Wollenweber B, Jacobsen B.of Cereal Science, 35, 175-188.201 1. Implications of high-temperature events and waterTianJ C, Chen J S, Hu R B.2006. Grain Quality Testingdeficits on protein profiles in wheat (Triticum aestivum L.Theory and Method. Science Press, Beijing. (in Chinese)cv. Vinjett) grain. Proteomics, 11, 1684-1695.Vensel W H, TanakaC K, Cai N, Wong J H, Buchanan(Managing editor WANG Ning)中国煤化工MHCNM HG◎2014. CAAS. All rights reserved. Published by Elsevier Ltd.