Identification Of Proteins Related To Wheat Grain Different Developmental Stages And Respnse To Stress

Wheat grain development affects grain size and weight,and further affects wheat production.Abiotic stresses is another major factor that cause grain reductions in the world.In field,crops generally suffer from a combination of various abiotic stresses.Abiotic stresses,especially cold stress and cold-drought stress are important factors to cause wheat yield reductions in China.The middle and late stages of bread wheat grain development,as well cold and cold-drought responsive proteins were identified based on 2-DE and i TRAQ methods.The main results obtained in this study are summarized as following:1.Proteomic approaches were applied in four grain developmental stages of the Chinese bread wheat Yunong 201 and its ethyl methanesulfonate(EMS)mutant line Yunong 3114.2-DE and tandem MALDI-TOF/TOF-MS were used to analyze proteome characteristics during middle and late grain development of the Chinese bread wheat Yunong 201 and its EMS mutant line Yunong 3114 with larger grain sizes.We identified 130 differentially accumulated protein spots representing 88 unique proteins,and four main expression patterns displayed a dynamic description of middle and late grain formation.Those identified protein species participated in eight biochemical processes: stress/defense,carbohydrate metabolism,protein synthesis/assembly/degradation,storage proteins,energy production and transportation,photosynthesis,transcription/translation,signal transduction.Comparative proteomic characterization demonstrated 12 protein spots that co-accumulated in the two wheat cultivars with different expression patterns,and 6 cultivar-specific protein spots including serpin,small heat shock protein,beta-amylase,alpha-amylase inhibitor,dimeric alpha-amylase inhibitor precursor,and cold regulated protein.These cultivar-specific protein spots possibly resulted in differential yield-related traits of the two wheat cultivars.2.By comparing differentially accumulated proteins of two cultivars(UC1110 and PI 610750)and their derivative,the F10 recombinant inbred line(RIL)population differing in cold-tolerance,a total of 20 common protein spots representing 16 unique proteins were successfully identified based on the 2-DE and MALDI-TOF/TOF-MS methods.Of the 20 protein spots,14 spots showed significantly enhanced abundance in cold-sensitive parental cultivar UC1110 and its 20 descendant lines when compared with cold-tolerant parental cultivar PI 610750 and its 20 descendant lines.Six reduced abundance protein spots were also detected.The identified protein spots are involved in stress/defense,carbohydrate metabolism,protein metabolism,nitrogen metabolism,energy metabolism,and photosynthesis.The 20 differently expressed protein spots were chosen for quantitative real-time polymerase chain reaction(q RT-PCR)to investigate the expression changes at the RNA level.The results indicated that the transcriptional expression patterns of 11 genes were consistent with their protein expression models.Among the three unknown proteins,Spot 20(PAP6-like)showed high sequence similarities with PAP6.q RT-PCR results implied that cold and salt stresses increased the expression of PAP6-like in wheat leaves.Furthermore,VIGS(virus-induced gene silencing)-treated plants generated for PAP6-like were subjected to cold limitation,and they showed more serious droop and wilt but increased rate of relative electrolyte leakage and reduced relative water content(RWC)when compared to viral control plants.These results indicate that PAP6-like possibly plays an important role in conferring cold tolerance in wheat.3.Wheat seedlings exposed to drought-cold stress for 24 h showed inhibited growth,increased lipid peroxidation,relative electrolyte leakage,and soluble sugar contents.To determine the wheat protein response to drought-cold stress,i TRAQ-based quantitative proteomic and liquid chromatography tandem mass spectrometry(LC-MS/MS)methods were employed to determine the proteomic profiles of the roots and leaves of wheat seedlings exposed to drought-cold stress conditions.We identified 250 and 258 proteins with significantly altered abundance in the roots and leaves,respectively.These proteins were classified into several main groups,as follows: protein metabolism,stress/defense,carbohydrate metabolism,lipid metabolism,transcription-related processes,energy production,cell-wall and cytoskeleton metabolism,membrane and transportation,signal transduction,other metabolic processes,and unknown biological processes.Nine proteins were simultaneously presented in both roots and leaves exposed to drought-cold stress,and the majority of proteins identified differed from one another and displayed differently altered abundance.These findings uncovered organ-specific differences in adaptation to drought-cold stress.Exogenous abscisic acid(ABA)application conferred the plant with protection against drought-cold stress and significantly increased catalase and peroxidase enzyme activities,as well as the transcription of glutathione S-transferase and other 11 sample genes in the roots or leaves,respectively.These results suggested that ABA is a potentially vital factor that contributes to the drought-cold signaling pathway and a promising target for growth recovery.This study provided valuable information for dissection of molecular and genetics basis of yield-related traits as well as cold and cold-drought response in bread wheat.Some vital candidate proteins of grain developmental and cold and cold-drought responsive pathways could provide important genetic resources for wheat resistant breeding.