Molecular Mechanisms Of Drought Stress Response In Malus Rootstocks Based On Quantitative Phosphoproteomics Technology

The response of plants to drought stress is a very complex phenomenon,and the individual characteristics of different species are also different.The drought resistance mechanism of herbaceous plants has been studied,and the physiological and molecular mechanisms of perennial woody plants in drought resistance are still unclear.Apple is one of the world's important gardening crops.In order to study the physiological and biochemical characteristics of apple rootstock under drought stress and the mechanism of phosphorylation signal transduction,the phosphorylated proteins were identified.In this paper,two genotype rootstocks of M26 and Malus baccata as materials.Through the determination of physiological and biochemical indexes,and the identification of phosphorylated proteins by nano LC-ESI-MS/MS tandem mass spectrometry combined with Ti O2 enrichment,the response mechanism of apple stock was expounded.The main results are as follows:1.With the increase of drought degree,the two apple genotypes responded to the continuous drought stress by increasing the content of soluble sugar(SS),soluble protein(SP)and free proline(FP).At the same time,the accumulation of osmolytes in the leaves of the M.baccata was higher than that of M26 during the continuous drought stress,indicating that the drought resistance of the M.baccata was better than that of M26.2.With the persistence of drought stress,the content of malondialdehyde(MDA)in leaves was increased.The antioxidant enzyme system and As A-GSH circulatory system of apple rootstock can eliminate the oxidative damage of reactive oxygen species in the cell membrane to a certain extent.Superoxide dismutase(SOD),peroxidase(POD),ascorbate peroxidase(APX),glutathione reductase(GR),ascorbic acid(As A)and reduced glutathione(GSH)and other indicators in the pre-stress period continued to rise,while in the late decline.The peak appeared on day 6,ie,moderate drought stress,and the ability of eliminating active oxygen is higher than that of M26.3.The activities of amylase(AMY)and invertase(INV),which were involved in the process of carbon metabolism,increased rapidly to the peak(moderate drought stress)and then decreased slowly in the early stage of drought stress,while the activities of nitrate reductase(NR)and glutamine synthetase(GS)related to nitrogen metabolism were continuously decreased.Furthermore,the activities of the four enzymes were higher in M.baccata than in M26.4.The leaf proteome of the two genotype rootstocks was identified by phosphorylation proteome under moderate drought stress.The results showed that 595 unique phosphorylation peptides,682 phosphorylation sites(593 p S,80 p T and 9 p Y)and 446 phosphorylated proteins were identified in the two genotypes.Six significant enrichment phosphorylated motifs were obtained by the Motif-X program,which was recognized as the basic motif for the mitogen activated protein kinase(MAPK),cyclin dependent kinase(CDK),class of cyclin dependent kinase(CDK-like),tyrosine kinase II(CK-II),calmodulin kinase II(Ca MK-II),protein kinase A(PKA)and protein kinase C(PKC).5.According to the difference analysis of two genotype phosphorylated proteins,12 PLSC phosphorylated proteins were identified to have significant changes in phosphorylation level(PLSC)in M26,and 46 PLSC phosphorylated proteins were identified in M.baccata.There were 9 PLS phosphorylated proteins with the same genotype.6.The results of differential analysis of phosphorylated proteins between the two genotypes showed that the 5 differential phosphorylated proteins,one microtubuleassociated protein 70-1-like and the three unknown phosphorylated proteins identified in M26 were shared with M.baccata.The acyl-Co A binding domain protein was different from the phosphorylated peptides and the phosphorylated sites in both genotypes.These results showed that the drought resistance mechanism of M26 was the same as M.baccata under moderate drought stress,and mainly related to lipid metabolism and cytoskeleton.Most of the remaining 30 differential phosphorylated proteins identified in the M.baccata were involved in the carbon and nitrogen metabolism process.7.According to the physiology and phosphorylation proteomics analysis,it was found that under the drought stress,the phosphorylated protein mainly clears the reactive oxygen species and the synthetic osmotic substances to regulate the damage caused by the drought stress by regulating the metabolism of lipid and the process of carbon and nitrogen metabolism.