Yufeng Zhang,Hongyao Lou,Dandan Guo,Ruiqi Zhang,Meng Su,Zhenghong Hou,Haiying Zhou,Rongqi Liang,Chaojie Xie,Mingshan You,Baoyun Li*

Beijing Key Laboratory of Crop Genetic Improvement/Key Laboratory of Crop Heterosis&Utilization,College of Agronomy and Biotechnology,China Agricultural University,Beijing 100193,China

Keywords:Winter wheat Heat stress GO enrichment KEGG pathway enrichment Interaction networks

ABSTRACT Wheat(Triticum aestivum L.)is one of the three major global food crops.Hightemperature stress can affect its yield and quality.Studies of the effect of hightemperature stress on wheat kernel development are important because they can reveal the stability of wheat quality and lead to the genetic improvement of wheat quality traits.In this study,the isobaric tags for relative and absolute quantitation(iTRAQ)method was adopted to analyze changes in the protein expression profile of wheat cultivars under high temperature stress.The protein content of wheat grain increased under heat stress,while the SDS-sedimentation value and starch content decreased.Grain filling was deficient under high temperature stress,which reduced thousandkernel weight but did not affect wheat kernel length.The 207 differentially expressed proteins identified in Gaocheng 8901 under heat stress were associated with energy metabolism,growth and development,and stress response.Gene Ontology enrichment analysis showed that the annotated proteins that were differentially expressed in Gaocheng 8901 under heat stress were involved mainly in stimulus response,abiotic stress response,stress response,and plasma membrane.A set of 78 differentially expressed proteins were assigned to 83 KEGG signaling/metabolic pathways.KEGG pathway enrichment analysis showed that this set of proteins was significantly enriched in members of 51 pathways,and the proteins participated mainly in protein synthesis in the endoplasmic reticulum,starch and sucrose metabolism,and reaction on ribosomes.Five differentially expressed proteins were involved in protein–protein interaction networks that may greatly influence the yield and quality of wheat grain.In wheat,high-temperature stress leads to a variety of effects on protein expression and may ultimately cause changes in yield and quality.

1.Introduction

Wheat is a major crop that is grown in many different environments worldwide and is an important source of calories for human nutrition.Rising average global temperatures are predicted to reduce the yield and quality of staple food crops such as wheat[1,2].Short episodes of extreme heat are expected to become more frequent during the grain-filling stage of wheat in the main wheat production areas[3,4].Heat stress is reported to lead to severe crop-yield losses and reduced milling quality,which is predicted to result in food crises in the future[3,4].The relationships between the environment and the yield and quality of wheat are complex[5].Heat stress affects the synthesis of kernel proteins,cellular metabolism,carbohydrate metabolism,and the activities of critical enzymes in transcription and translation,ultimately disturbing normal kernel development[6–8].

Proteomics,especially by two-dimensional electrophoresis(2-DE),has become a popular method of identifying a large number of proteins in an organism[9–11].Many wheat proteins differentially expressed under high-temperature stress have been identified by 2-DE[12–19].Many of the identified differential proteins were subsequently studied and analyzed.Protein expression differences observed when hightemperature treatments have been applied at different stages of wheat kernel development have been inconsistent.The abundance of some proteins increased up to 8.5-fold[14],while that of others increased as much as 19-fold when high temperature was applied throughout grain development[17].Changes in expression were generally＜2.5-fold when hightemperature treatment was applied early in grain development[15–19].It may not be surprising that most wheat proteins differentially expressed under heat stress are classified as stress/defensive proteins.Approximately 30 proteins that respond to high temperature have been identified,including embryogenesis-rich(LEA)proteins,xylanase inhibitors,1-cys peroxiredoxin,glyoxal,tetrameric alpha-amylase inhibitor WTAI-CM17,several chitinases,sulfhydryl reductase,9 kDa lipid transporter(LTP),monomeric and dimeric alpha-amylase inhibitors(WMAI,WDAI),tetrasodium αamylase inhibitor WTAI-CM3, wheat alpha-amylase/subtilisin inhibitor(WASI),and multiple serpins[12–19].

Approximately 20 proteins involved in carbohydrate metabolism also showed a response to heat stress[12–19].For example,β-amylase,glucose and ribitol dehydrogenase are involved in glucose degradation,whereas glyceraldehyde-3-phosphate dehydrogenase(GADPH)and triosephosphate isomerase are involved in glycolysis and granule-bound starch synthase(GBSS)is involved in starch synthesis.Heat shock proteins are chaperones involved in protein folding and assembly that undergo obviously changes in response to high temperature.Many heat shock proteins have been identified as responsive to heat stress[16,17].Skylas et al.[18]found that expression of some heat shock proteins within the molecular weight range of 15–30 kDa immediately increased 15–17 days post anthesis(DPA)under heattreatment.Heatshockprotein70(HSP70)showedatransient increase in expression during grain filling[14]and under heat stress[15,16,19].Protein disulfide isomerase,involved in protein folding,showed differential expression under heat stress[14,20].

Some proteins not previously thought to be involved in stress response,such as the storage protein globulin,have been shown to be differentially expressed under heat stress.Wheat gluten is a complex group of abundant proteins and is a majordeterminantofwheatflourquality.Glutens consisting of α/β-, γ-,and ω-gliadins,as well as those consisting of three α-gliadins,were also identified as responsive to heat stress.During kernel filling,the abundance of low-molecular-weight glutenin subunits(LMW-GSs)was reduced,while that of HMW-GSs and α-gliadins was increased[12,13,16].Proteins that are up-or downregulated under heat stress,such as guanine nucleotide binding protein,the 20S proteasome alpha subunit,the 26S proteasome,poly(A)binding protein,embryo specific protein,cell division control protein 4B,14-3-3 protein,and puroindoline,may also play a role in high-temperature stress[12–19].

Two-dimensional gel electrophoresis (2-DE)00 and MassSpectrometry(MS)arecommonlyusedtoidentify changes in the proteome.Previous studies have identified differentially expressed proteins in response to high temperature,but few studies have used the isobaric tags for relative and absolute quantitation(iTRAQ)to analyze changes in protein abundance under heat stress.In comparison with 2-DE,iTRAQ has greater sensitivity,which allows detection of low-abundance proteins.iTRAQ can be used to distinguish many protein types,including high-molecular-weight proteins,acidic and basic proteins,and membrane-bound and insoluble proteins.iTRAQ can be used to analyze eight samples simultaneously and can process multiple time points or processed proteins simultaneously.In addition,iTRAQ can be performed with a high degree of automation.In this study,iTRAQ was used to identify wheat proteins differentially expressed under high-temperature stress with the goal of understanding the high-temperature adaptation mechanisms used by wheat.Further analysis of the molecular functions of these differentially expressed proteins should yield important information about wheat breeding,yield,and quality stability.

2.Materials and methods

2.1.Plant material and growth conditions

The winter wheat cultivar Gaocheng 8901 was grown under natural conditions in 50 cm×30 cm×40 cm plastic boxes.The plants were moved to an artificial climate chamber after flowering.The growing conditions for the control group were as follows:25 °C for 12 h(day)/18 °C for 12 h(night)with relative humidity of 20%–25%.For the treatment group,the temperature of the chamber was raised to 40°C for 2 h(12:00–14:00)from flowering to maturity.The treatment and control groups each contained nine pots.Ten plants were planted in each pot,with three biological replicates.

2.2.SDS-sedimentation analysis

Wheat flour samples(2 g)were suspended in 16.7 mL H2O containing 0.01%bromophenol blue and incubated at room temperature(approximately 25°C)for 5 min,after which 16.7 mL of SDS solution(2%SDS,0.012 mol L-1lactic acid)was added to each sample,and the samples were incubated at room temperature for an additional 5 min.Sedimentation volumes were recorded every 5 min for 20 min.Each experiment included three biological replicates.

2.3.Protein and starch content determination

The total protein content of the samples was determined using the Kjeldahl method with some modifications[21].Albumin,globulin,gliadin,and gluten were extracted as previously described[22].Briefly,mature grain was milled with a Brabender D-4100 mill(Brabender Technologie,Germany)and the resulting flour(100 mg)was suspended in 1 mL of distilled H2O.These suspensions were incubated at 50°C for 30 min with intermittent mixing and centrifuged at 4000 r min-1for 20 min at 4°C.The incubation/suspension process was repeated three times,after which albumin was retained in the soluble fraction.The remaining pellets were suspended in 1 mL of 10%NaCl,mixed,and centrifuged at 4000 r min-1for 20 min at 4°C,after which globulin was retained in the soluble fraction.The remaining pellets were suspended in 1 mL of 75%EtOH,mixed,and centrifuged at 4000 r min-1for 20 min at 4°C,after which gliadin was retained in the soluble fraction.Finally,the remaining pellets were suspended in 1 mL of 0.2%NaOH,mixed,and centrifuged 4000 r min-1for 20 min at 4°C,after which any remaining gluten was retained in the soluble fraction.Protein content was determined by the Bradford method.Amylose was extracted and determined by suspending 100 mg of flour in 1 mL EtOH,to which was added 9 mL NaOH(1 mol L-1).The suspensions were then incubated in boiling(100°C)H2O for 10 min and cooled in ice water.Aliquots(20 mL)of each suspension were transferred to 50-mL centrifuge tubes,incubated with 10 mL petroleum ether for 10 min,and heated at 25°C for 15 min.This incubation/heating process was repeated three times.The upper fraction contained the amylose solution,and the amylose content was determined using the iodine–potassium iodide method.The experimental results were analyzed using Excel 2010 software(Microsoft Co.,Redmond,WA,USA).

The starch content of wheat grain was determined using a DA7200 near-infrared spectrometer(Perten Company,Switzerland).The near-infrared calibration curve for wheat was modeled and corrected by the Chinese Ministry of Agriculture's Grain Quality Supervision and Testing Center(Tai'an,Shandong,China),and the results were automatically analyzed by the DA7200.All processes were performed in triplicate and each experiment consisted of three independent biological replicates.

2.4.RNA extraction and qRT-PCR

Normal and heat-treated wheat kernels were collected at 5,10,15,and 20 DPA.Total RNA was extracted using an RNA Pure Plant Kit(Tiangen,China).RNA extraction and qRT-PCR were performed following Zhang et al.[23].The 2–ΔΔCtmethod was used to calculate the expression levels of target genes.All reactions were performed in triplicate,and each experiment consisted of three independent biological replicates.

2.5.Protein extraction

Grain samples were ground into fine powder in liquid nitrogen using a mortar and pestle,and 2-g samples were extracted for 2 h with 3 mL of extraction buffer(50 mmol L-1Tris-HCl,pH 8.0,0.1 mol L-1KCl,5 mmol L-1EDTA,30%sucrose)containing 1 mmol L-1Phenylmethanesulfonyl fluoride(PMSF).After centrifugation for 15 min at 13,000 r min-1,the supernatants were transferred to new tubes,to which 40 mL of cold Trichloroacetic acid(TCA)/acetone solution was added.The samples were stored at-20°C overnight,followed by centrifugation at 7830 r min-1for 30 min,after which the supernatantswereremoved.Next,40 mLofpre-cooled acetone was added,after which the samples were centrifuged at 7830 r min-1at 4°C for 30 min.This process was repeated until the acetone was completely colorless.The precipitate was dried at room temperature.The protein samples were incorporated into 500 μL STD buffer(4%SDS,1 mmol L-1DTT,150 mmol L-1Tris-HCl,pH 8.0),incubated at room temperature,shocked for 1 h,and centrifuged at 13,400 r min-1for 30 min.Protein concentrations were determined using the bicinchoninic acid(BCA)method.

2.6.Protein digestion and iTRAQ labeling

The FASP protocol[24]was applied for the digestion of protein.The resulting mixture of peptides was labeled with 4-plex/8-plex iTRAQ reagent following the manufacturer's instructions(Applied Biosystems,Massachusetts,USA).Each protein sample of about 200 μg was mixed with 30 μL of STD buffer(4%SDS,100 mmol L-1DTT,150 mmol L-1Tris-HCl,pH 8.0).The detergent,DTT and other low-molecular-weight components were subjected to repeated ultrafiltration(Microcon units,30 kD)usingUA buffer(8 mol L-1urea,150 mmol L-1Tris-HCl,pH 8.0).Cysteine residues were blocked by addition of 100 μL of 0.05 mol L-1iodoacetamide in UA buffer and the samples were incubated in darkness for 20 min.The filterswerewashedthreetimeswith100 μLofUAbufferandthen twice with 100 μL of DS buffer(50 mmol L-1triethylammonium bicarbonate at pH 8.5).In the final step,2 μg trypsin(Promega(Beijing)Biotech Co.,Ltd.,Beijing,China)in40 μLofDSbufferwas used to digest the protein suspension at 37°C overnight and peptides were collected as filtrate.The concentration of the peptideswasmeasuredbyUVlightat280 nmusinganextinction coefficient of 1.1 per 0.1%(g L-1),based on tryptophan and tyrosine vertebrate protein frequencies.

For labeling,70 μL of ethanol was used to dissolve each iTRAQ reagent and the solution was mixed with peptide mixtures.Samples were labeled as(Sample 1)-114,(Sample 2)-115,(Sample 3)-116,and(Sample 4)-117,after which they were multiplexed and vacuum-dried.

2.7.Peptide fractionation with strong cation exchange(SCX)chromatography

SCX chromatography was used for fractionation of iTRAQ-labeled peptidesusingan AKTA PurifierSystem (GE Healthcare).About 2 mL of buffer A(10 mmol L-1KH2PO4in 25%ACN,pH 2.7)was used to acidify and reconstitute the dried peptide mixture.It was then loaded onto a polysulfoethyl column(4.6 mm × 100.0 mm,5 μm,200 ?,PolyLCInc.,Columbia,MD,USA).Buffer B(500 mmol L-1KCl,10 mmol L-1KH2PO4in 25%ACN,pH 2.7)was used for elution of the peptides at a flow rate of 1 mL with a gradient of 0–10%for 2 min,10%–20%for 25 min,20%–45%for 5 min and 50%–100%for 5 min.Elution was monitored by measurement of eluate absorbance at 214 nm,collecting fractions at 1-min intervals.The collected fractions(about 30 fractions)were finally combined into 10 pools and desalted on C18 Cartridges(Empor SPE Cartridges C18(standard density),bed I.D.7 mm,volume 3 mL,Sigma).Each fraction was concentrated by vacuum centrifugation and reconstituted in 40 μL of 0.1%(v/v)trifluoroacetic acid.After vacuum centrifugation,all samples were stored at-80°C until Liquid chromatography(LC)-MS/MS analysis.

2.8.Liquid chromatography(LC)-electrospray ionization(ESI)tandem MS(MS/MS)analysis

Experiments were performed with a Q Exactive mass spectrometer coupled to Easy nLC(Proxeon Biosystems,now Thermo FisherScientific).Eachfractionof10 μLwassubjectedtonanoLCMS/MS.A 5-μg sample of peptide mixture was loaded onto a C18-reversed phase column(Thermo Scientific Easy Column,10 cm length,75 μminnerdiameter,3 μmresin)inbufferA(0.1%formic acid)and was isolated with a linear gradient of buffer B(80%acetonitrile and 0.1%formic acid)at a flow rate of 250 nL min(controlled by IntelliFlow technology)for 140 min.MS data was obtained using a data-dependent top10 method,which picked the most rich precursor ions from the overview examine(m/z 300–1800)for HCD fragmentation.On the basis of predictive automatic gain control the target value was determined.The dynamic exclusion duration was 60 s.Survey scans were obtained at a resolution of 70,000 at m/z 200.The normalized collision energy was 30 eV.The underfill ratio(the minimum percentage of the target value likely to be reached at maximum fill time)was defined as 0.1%.The instrument was run with peptide recognition mode enabled.

2.9.Sequence database searching and data analysis

MS/MS spectra were searched using MASCOT engine(Matrix Science,London,UK;version 2.2)embedded into Proteome Discoverer 1.3(Thermo Electron,San Jose,CA.)against Uniprot Human database(133,549 sequences,download at October 24,2014)and the decoy database.The following options were used for protein identification:peptide mass tolerance,20 ppm;MS/MS tolerance,0.1 Da;enzyme,trypsin;missed cleavage,2;fixed modification,carbamidomethyl(C),iTRAQ4/8plex(K),iTRAQ4/8plex(N-term);variable modification,oxidation(M);false discovery rate(FDR)≤0.01.

2.10.Biological analysis of selected differentially expressed proteins

The UniProtKB database(Release 2014_12)was used for sequence data retrieval of all differentially expressed proteins in FASTA format.The NCBI BLAST+client software(ncbiblast-2.2.28+-win32.exe)was used to search for homologous sequencesin theSwissProtdatabase(mouse).Allthe retrieved sequences were locally searched to acquire functional annotations to be transferred to the query sequences in Shanghai Applied Protein Technology Co.Ltd.For each query sequence,the top 10 BLAST hits having E-values＜1e-3 were retrieved and entered into Blast2GO(version 2.8.0)for GO mapping and annotation[25,26].An annotation configuration with a 1e-6 E-value filter predetermined EC weights,a GO weight of 5,and an annotation threshold of 75 were chosen.Re-annotation was performed for some of the unannotated sequences with more lenient parameters.To identify the functional annotations of protein motifs,all sequences without blast hits or annotation were searched against European Bioinformatics Institute(EBI)databases using InterProScan4 and the InterProScan GO terms were merged with the annotation set.The proteins after the annotation and annotation augmentation steps were blasted against KEGG GENES to retrieve their KOs and proteins were mapped to pathways in KEGG[27,28].Using gene symbols,data for query proteins were retrieved from the IntAct molecular interaction database[29].For further analysis the results were obtained in XGMML format and transferred into Cytoscape.

3.Results

3.1.Changes in agronomic and quality traits following high temperature stress

Heat treatment was applied to Gaocheng 8901 wheat from anthesis to harvest.Wheat kernels showed significant difference in response to heat stress(Fig.1-A).Heat stress caused the kernel to become narrowed and reduced its weight,while the length of the kernel remained unchanged(Fig.1-B,C,D).These results suggest that heat stress may affect grain filling and reduce the weight of wheat kernels,ultimately reducing yield.

Wheat kernel proteins are classified as albumin,globulin,gliadin,and gluten according to their solubility.Gluten and prolamin are the main components of wheat storage protein[30].High-temperature stress reduced the SDS-sedimentation value of wheat flour,indicating reduced flour quality(Fig.2-A).The protein content of wheat grain subjected to heat stress was increased,while its starch content was reduced(Fig.2-B,C).The reduced starch content of wheat grain subjected to heat stress may have been associated with the simultaneous decrease in thousand-kernelweight.Thecontentsofgluten,gliadin,globulin,and albumin were significantly increased by high-temperature stress(Fig.2-D).However,the reduction in kernel weight was consistent with the reduction in starch content after hightemperature treatment.

3.2.Grain protein identification by iTRAQ and functional annotation

Proteomic analysis(iTRAQ)performed to characterize the effect of heat stress on wheat cultivar Gaocheng 8901 revealed 2493 proteins that had quantitative information in the tags of each channel(Table S1-a).The sequences of the quantified proteins were extracted in batches from the UniProt Knowledgebase(UniProtKB).Acorrelationanalysisofthe proteomesfromthreedifferentbiologicalsamplesyieldedr＞0.7,which indicated good reliability between repetitions(Fig.S1).

Fig.1–Changes in kernel traits following high temperature stress(A).Kernel shapes of wheat cultivar Gaocheng 8901 following high-temperature stress and normal growth.(B,C,D).Differences in kernel traits of wheat cultivar Gaocheng 8901 following high-temperature stress and normal growth.Thousand-kernel weight was determined using three biological replicates,each of which included three measurements of 1000 randomly selected kernels.Kernel width and length were determined using three biological replicates,each of which included three measurements of 100 randomly selected kernels.**Significantly different at P＜0.01;CK,normal growth conditions;T,heat stress.

Fig.2–Changes in starch content,protein content and the contents of four storage proteins.(A).SDS-sedimentation of wheat cultivars following heat stress and growth under normal conditions.(B).Changes in starch content following high-temperature stress.(C).Total protein content following high-temperature stress.(D).Changes in the contents of four storage proteins following high-temperature stress.Note:*denotes a significant difference(P＜0.05)and**denotes an extremely significant difference(P＜0.01).CK,normal growth conditions;T,heat stress.

Identified proteins were tested for differential expression based on standard criteria:expression ratio＞1.2 and P＜0.05.This analysis revealed that 207 proteins were differentially expressed in wheat cultivar Gaocheng 8901 following heat stress,including 116 upregulated and 91 downregulated proteins(Table S1-b).The iTRAQ results were validated by qRT-PCR to measure the relative transcript abundance of three differentially expressed proteins:heat shock protein 90(HSP90;upregulated under heat stress),LMW-GS(downregulated under heat stress),and starch branching enzyme IIb(SBE IIb;downregulated under heat stress)(Fig.S2,Table S2).The qRT-PCR results were consistent with the results of the grain proteome analysis.

3.3.Sequence alignment and functional annotation of differentially expressed proteins

Comparison of the identified protein sequences with the sequences in the NCBI nr database revealed a similarity range of 47%–100%,with the alignment similarity of most of the target protein sequences exceeding 93%(Fig.S3).Comparison of the identified proteins using Blast2GO(Version 2.8.0)revealed that 106 protein sequences were annotated with 279 GO function entries,with an average GO level of 5.168(Fig.S4).After the final annotation,184 protein sequences were annotated with 579 GO function entries(Table S3,protein2GO page).

3.4.GO annotationandKEGG pathwayannotationof differentially expressed proteins

Functionalannotation ofthedifferentiallyexpressed proteins revealed cellular component information for 125 proteins,mainly membrane proteins and organelle-forming proteins,and molecular function information for 202 proteins;including ATP-binding,DNA-binding,and ribosome-binding proteins as well as proteins involved in kinase activity;and biological process information for 252 proteins involved mainly in starch metabolism,sucrose metabolism,stress response,transcription,and redox(Table S3-BP,MF,CC).Differentially expressed proteinsin kernelsofcultivar Gaocheng 8901 were classified at GO level 2 as follows(Fig.3).1)Biological process:response to stimuli(45 proteins),cellular processes(60),and metabolic processes(93).2)Molecular function:catalyzing activities(69),binding function(71),structural molecule activity(12),and regulating enzyme activity(7).3)Cellular component:cell(71),membrane(16),macromolecular complex(13),and organelle(43)(Table S3-Level2_BP,Level2_CC Level2_MF).Some of the differentially expressed proteins were found to perform multiple cellular functions.Comparison of the target protein sequences with the plant protein sequences in the KEGG GENES database using KAAS revealed 83 KEGG signaling/metabolic pathways associated with 78 differentially expressed protein sequences(Table S4-map2query).

3.5.GO enrichment analysis and KEGG pathway enrichment analysis of differentially expressed proteins

The GO enrichment analysis showed that the set of differentially expressed proteins was enriched in the following terms:response to abiotic stimuli(21 proteins),response to stress(40),response to stimulus(45)and plasma membrane(7)(Fig.4-A;Table S3-GO enrich).The differentially expressed proteins participated mainly in stimulus and stress response.The KEGG pathway enrichment analysis of the 83 KEGG signaling/metabolic pathways associated with 78 differentially expressed protein sequences(Table S4-TopMapStat)revealed that the set of proteins was enriched in 51 cellular pathways(Fig.4-B;Table S4-Enrichment).

3.6.Analysis of protein–protein interaction networks

Fig.3–GO annotation terms for the set of differentially expressed proteins from wheat cultivar Gaocheng 8901 at GO level.

Fig.4–GO enrichment analysis and KEGG pathway enrichment analysis of the set of differentially expressed proteins from wheat cultivar Gaocheng 8901.(A)Significantly enriched GO terms for the set.(B)KEGG pathway enrichment analysis for the set.Reference,the total number of proteins associated with this GO term for all identified proteins,Test,the number of proteins associated with this GO term in differentially expressed proteins.

CytoScape software and the IntAct database were used to generateprotein–protein interaction networksfrom the proteins found to be differentially expressed following heat stress,to revealdirectinteractionsbetween thetarget proteins and other proteins.Elicitor responsive gene3(ERG3),brassinosteroid-insensitive 1(BRI1),chaperone protein(CLPB1),histone cell cycle regulator(HIR1)and pre-mRNA processing factor(RSZ22)were found to be involved in known interaction networks.These results suggest that ERG3,BRI1,CLPB1,HIR1,and RSZ22 may perform critical regulatory functions during or following heat stress(Fig.5).

4.Discussion

4.1.Analysis of differentially expressed proteins in Gaocheng 8901 following heat stress

Proteins associated with energy metabolism,growth and development,and stress response were found to be regulated in response to high temperature stress during wheat kernel filling.These results provide an empirical foundation for future studies assessing the heat resistance of wheat and other plants in the future.

4.1.1.Energy-related and metabolism-related proteins

Differentially expressed proteins associated with energy and metabolism were found to be regulated in Gaocheng 8901 under heat stress. Chaperone protein ClpB2 and peptidylprolyl isomerase were upregulated,whereas DNA methyltransferase and sucrose synthetase (SS) were downregulated.

ClpB2 is a molecular chaperone protein that facilitates protein folding and prevents protein aggregation.However,once protein accumulation has occurred,the molecular chaperones do not promote protein depolymerization.The bacterial ClpB protein and its eukaryotic homolog Hsp104 are essential proteins in the heat shock reaction that have the ability to depolymerize aggregated proteins [31].Peptidylprolyl isomerase regulates transcription factor activity,and the activity of transcription factor c-Myb binding DNA is negatively regulated by Cyp40,a member of the peptide prolylisomerase family[32].Peptidylprolyl isomerase catalyzes the cis-trans isomerization of peptidyl proline and plays an important role in protein folding and unfolding;it may also be involved in assembly and disassembly of protein complexes,protein transport and regulation of protein activity,but many questions about its various cellular roles remain[33].

Fig.5–Interaction networks of differentially expressed proteins in wheat cultivar Gaocheng 8901.Yellow nodes denote differentially expressed proteins and green nodes denote proteins interacting directly with the differentially expressed proteins.The nodes are elicitor responsive gene 3(ERG3),brassinosteroid-insensitive 1(BRI1),chaperone protein(CLPB1),histone cell cycle regulator(HIR1)and splicing arginine serine-rich 7(RSZ22).

DNA methyltransferase uses S-adenosylmethionine(SAM)as a methyl(CH3-)donor to transfer methyl groups to DNA bases,and is an important catalyst of DNA methylation.DNA methylation does not change the primary structure of DNA,but it can produce stable genetic changes in offspring.DNA methylation is involved in a very large number of biological processes,including gene expression regulation,embryonic development,cell differentiation,genomic imprinting,and X chromosome inactivation[34].Sucrose is the main photosynthetic product of the leaves,and as a key enzyme necessary for sucrose to enter various metabolic pathways,SS plays an important role in plant growth and development[35].SS expression was reduced by heat stress,indirectly inhibiting carbohydrate synthesis and thus reducing wheat yield.

4.1.2.Proteins associated with growth and development

Heat treatment altered the expression levels of several proteins related to growth and development.Glucose 6-phosphate dehydrogenase,serine carboxypeptidases(SCP),phosphoglycerate kinase(PGK),vacuolar processing enzyme(VPE),trypsin,and glutamine synthetase(GS)were upregulated under high-temperature stressundeafter,whereas 40S and 60S ribosomal protein were downregulated.

Glucose-6-phosphate dehydrogenase is a key regulatory enzyme in the plant pentose phosphate pathway,an important pathway for carbohydrate metabolism.The pentose phosphate pathway generates NADPH[36,37],nucleic acid pentose phosphates,and pentoses and plays an important role in maintaining redox balance in plant cells[38].SCP is a protease of the α/β hydrolase family that is involved in the processing,modification and degradation of peptides and proteins during plant growth and development.Biosynthesis of secondary metabolites,catalytic acyl transfer,and seed germination-related protein degradation play important roles in many biochemical pathways[39].PGK is a key enzyme in the glycolytic pathway that is necessary to the survival of all living organisms.In the present study,PGK was downregulated after high temperature stress.VPE is an aspartic acidspecific cysteine protease in plant vacuoles that is responsible for the maturation and activation of precursor proteins[40].In rice,VPE plays an important role in regulating the maturation of rice gluten.VPE is involved in senescence,terminal differentiation,and biological stress-induced programmed cell death[41].Trypsin,also known as protein hydrolase,can selectively hydrolyze the peptide chain formed by lysine or arginine.Trypsin can digest denatured proteins,but has no effect on undenatured proteins.In plant growth and development,nitrogen assimilation is an important physiological process in which inorganic nitrogen must be assimilated into organic forms such as glutamine and glutamic acid to be absorbed and utilized by plants.GS is a key enzyme in ammonia assimilation[42].Trypsin and GS were downregulated under high-temperature stress.Ribosomal proteins influence the efficiency and stability of ribosomes and are involved in DNA repair,apoptosis,and regulation of gene expression[43].Hurkman et al.[14]have found changes in 40S and 60S ribosomal protein in winter wheat flowering 10 days to 20 days after high-temperature treatment.In the present study,40S and 60S ribosomal protein were upregulated1 after high-temperature stress.

4.1.3.Stress-associated proteins

Glycine-rich proteins(GRPs),heat shock proteins,and other stress-associated proteins were upregulated in wheat cultivar Gaocheng 8901 following heat treatment,whereas 14-3-3 protein and leucine-rich repeat(LRR)were downregulated.GRPs are proteins with repetitive glycines in their structure.GRP expression is generally strictly controlled and is associated with development and various environmental factors.GRP expression is also regulated by various abiotic stresses including ABA,high-salt,drought,low temperature,injury,and waterlogging[14,44–47].Heat shock proteins can participate in protein folding and assembly,but the functions of many heat shock proteins are unknown.In the present study,heat shock proteins showed large variation after heat stress.Laino et al.also identified several of the same proteins by 2-DE,and the expression pattern was consistent with that observed in this study[15].

14-3-3 protein is a primary regulator of primary metabolism and cellular signal transduction.In plants,Jahn et al.[48]showed that 14-3-3 protein is involved in the regulation of H+ATPase activity on the plasma membrane and affects proton transport.Chen et al.[49]reported that 14-3-3 protein is regulated by biological or abiotic stress.Yang et al.[8]obtained similar results by 2-DE.

LRR proteins contain repeats consisting of 20 to 29 amino acids that form a regular spiral structure consisting of alpha helices and beta folds.Since the first report of a LRR protein in humans in 1985,researchers have discovered numerous proteins with LRR structures in animals,fungi,and plants.The conserved domain in LRR proteins is a LxxLxLxx(N/C)xL structure containing 11 amino acid residues(where “x”can be any amino acid)[50,51].This structure plays an important regulatory role in the process of protein interaction and plant stress transduction[52,53].

4.1.4.Protein storage and processing

High-temperaturestressoftenleadstochangesinthe expression levels of proteins involved in grain storage and processing in cereals.In this study,two storage proteins,12S globulin and 19 kDa globulin,were upregulated following high temperature stress.This finding suggests that these proteins may be directly or indirectly involved in the plant stress response,in agreement with previous studies[14,15].The mechanism underlying differential globulin expression in response to heat stress is unknown.Hurkman et al.[14]suggested that globulin can respond to high-temperature stress through unknown mechanisms or act indirectly as a target protein for other proteins under high-temperature stress.

Gliadin and gluten are major storage proteins in wheat kernels.Seven gliadin proteins were differentially expressed under high temperature stress,of which two were upregulated(one α-gliadin and one γ-gliadin)and five downregulated(three α-alcohols and two γ-gliadins).These results are consistent with previous findings[12,13,19].Yang et al.[19]supposed that increases in the abundance of certain gliadins occur simultaneously with corresponding decreases in the abundances of other gliadins.They also found that seven LMW-GS were differentially expressed(six up-and one downregulated).Thus,changes in the abundances of gliadins and LMW-GS altered glutenin and gliadin accumulation under heat stress,indirectly affecting wheat quality and field.

4.1.5.Other types of protein

Primarygenescontrollingkernelhardness(HDT2,UDP glucose transferase,and cytoplasmic malate dehydrogenase)weredownregulatedinGaocheng8901followinghightemperature stress,whereas histone H2A.1,histone H2B.2,xylanase inhibitors,defensins,and lipid transfer protein precursors were upregulated.Plasma membrane intrinsic protein, α-N-acetylglucosaminidase subtype X1, αgalactosidase and pyruvate carboxylase were downregulated.

4.2.KEGG pathway enrichment analysis for the set of differentially expressed proteins

The set of differentially expressed proteins was enriched in primarily three pathways:protein processing in the endoplasmic reticulum(22 proteins),starch and sucrose metabolism(five proteins)and reaction on the ribosome(13 proteins).These results suggest that heat stress affects protein synthesis in the endoplasmic reticulum,the metabolism of starch and sucrose,and reactions on the ribosome(Figs.S5,S6,and S7;Table S4-Enrichment).In particular,elucidation of the effect of heat shock on the proteome of the wheat kernel will illuminate the molecular basis of heat stress response in plants.

4.3.Analysis of protein–protein interaction networks

Because most of the proteins in an organism act in functional networks,protein–protein interaction networks can reveal the rolesand relativeimportanceofproteinsin biological processes.In the present study,five proteins differentially expressed under high-temperature stress were involved in known protein–protein interaction networks(Fig.5).These proteins may perform critical regulatory functions under heat stress.ERG3,BRI1,and RSZ22 interacted with multiple proteins simultaneously and may participate in several biological processes during high-temperature stress.In contrast,HIR1 and CLPB1 each interacted with a single protein.

EGR3isanenzymethatbindstotheendoplasmic reticulum membrane and catalyzes the formation of double bonds between C5 and C6 on the γ-7-sterol B ring in yeast,plants,and vertebrates[44].RSZ22 is pre-mRNA processing factor[54].CLPB1,known as AtHsp101 in Arabidopsis,is the product of the HOT1 gene and is the CLP/Hsp100 protein that has been studied most intensively in plants.CLPB1 is an HSP that accumulates rapidly under high temperature stress and hasavitalregulatoryeffecton plantgrowth [55].In Arabidopsis,BRI1 is a transmembrane receptor kinase that responds to brassinolide.BRI1 protein abundance determines the degree of response to brassinolide and the number of brassinolide binding sites;BRI1 can mediate transmembrane signaling by steroids[56].HIR,a transcription inhibitor,participates in the ASF1-mediated silencing pathway[57].Mazzoni et al.found that histone genes were down-regulated in Saccharomyces cerevisiae mutants overexpressing HIR1,suggesting a connection between HIR1 and apoptosis[58].

5.Conclusions

UsingiTRAQ,weidentified207differentiallyexpressed proteins in wheat cultivar Gaocheng 8901 cultivated at normal and high temperature.Differentially expressed proteins were enriched in 51 signaling pathways and participated mainly in protein synthesis in the endoplasmic reticulum,starch and sucrose metabolism,and reactions on the ribosome.Five differentially expressed proteins were involved in protein-protein interaction networks that may operate under heat stress and could influence grain yield and quality.In this study,we identified differentially expressed proteins in wheat cultivar Gaocheng 8901 using iTRAQ.These results illuminate the molecular mechanisms through which wheat plants respond to high-temperature stress.

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2018.04.003.

Acknowledgments

This work was supported by the National Key Research and Development Program of China(2016YFD0100502).We thank the Shanghai Applied Protein Technology Co.Ltd.and the Chinese Academy of Sciences Shanghai Institutes for Proteomic Study Center for sample analysis.