Mining And Functional Analysis Of Spring Freezing Response Proteins In Wheat

In recent years,the frequent occurrence of spring freeze in wheat caused by low temperature poses a serious threat to wheat production and has become one of the main disasters of wheat yield loss.This study revealed the alleviating mechanism of exogenous abscisic acid on wheat low temperature stress in spring from phenotypic,physiological and molecular levels,and used Label-free technology to mine the key proteins in response to spring freeze.The main results are as follows:Exogenous ABA pretreatment significantly improved the frost resistance of wheat Zhengmai 9023 at the anther connective tissue formation phase(ACFP)and tetrad developmental phase.After 3-day freeze stress,ABA pre-treatment significantly increases the content of soluble sugar,soluble protein,and proline,activities of superoxide dismutase(SOD)and peroxidase(POD)and decreases hydrogen peroxide(H2O2)and malondialdehyde(MDA)levels.Under freeze stress conditions,ABA treatment at the ACFP and tetrad developmental phase resulted in a marked increase in leaf ascorbic acid(ASA)and glutathione(GSH)levels.Temporal and spatial expression patterns of eight genes encoding ASA–GSH synthesis-related enzymes and eight stress-responsive genes were measured using quantitativereal-time PCR(q RT-PCR).The results showed that ABA temporally regulated the transcript levels of these genes.Moreover,these genes were differentially expressed in ABA-treated wheat seedlings between the ACFP and tetrad developmental phase during freeze stress.These results implied that exogenous ABA increases the levels of GSH and ASA in freeze stressed wheat seedlings in time and developmental phase specific manners.In this study,we found that the ABA-independent and ABA-dependent pathways were not completely independent in this study and that ABA-independent stress-responsive genes were also induced by exogenous ABA in at least one stress exposure time point.Compared with the research results showed that plant hormones induce an increase in GSH and ASA levels,possibly by differentially regulating the expression levels of genes encoding ASA–GSH synthesis enzymes.The results of this study provide the first insights on the molecular mechanism of ABA-induced spring freeze tolerance in wheat.The differential proteomics was used to analyze the low temperature treatment and normal growth of wheat varieties Zhengmai 7698,and a new response protein(delta-1-pyrroline-5-carboxylate synthase,P5CS)was identified.The gene of this protein was cloned in wheat and named Ta P5 CS.The subcellular localization vector of the gene was constructed and transformed into wheat protoplast,and the results showed that the gene was localized in cell membrane.The q RT-PCR results showed that wheat Ta P5 CS gene was induced by four abiotic stresses: drought,high salinity,low temperature and high temperature.Moreover,the Ta P5 CS gene was expressed in eight tissues of wheat,including blade ring,leaf,leaf sheath,root,glumes,spikelet,stalk and stem node,and was expressed in high abundance in spikelet,stalk and stem node,indicating that Ta P5 CS gene was ubiquitous in wheat tissues and had tissue expression specificity.Functional studies showed that transgenic wheat plants overexpressing Ta P5 CS showed enhanced resistance to spring freeze,and homozygous mutant Ta P5 CS were more sensitive to spring freeze than wild-type plants in comparison.Plants overexpressing Ta P5 CS accumulated more proline and MDA,as well as higher activities of SOD,POD and CAT than the control plants.In addition,the overexpression of Ta P5 CS could significantly reduce the production of H2O2,and the expression level of antioxidant genes was also higher than that of the control plants.The results indicated that Ta P5 CS may positively regulate wheat resistance to spring freeze through ROS-mediated pathway.Co-immunoprecipitation(CO-IP)was used to identify the interaction proteins of Ta P5 CS,and the results showed that these proteins were involved in multiple metabolic pathways such as antioxidant pathways,which laid a foundation for further understanding the molecular mechanism of Ta P5 CS regulating spring freeze in wheat.