Screening And Functional Analysis Of Differentially Expressed Proteins Of Alfalfa Under Salt Stress

Medicago sativa is an important perennial legume forage grass with rich nutritional value.Salt stress is the main environmental factor limiting alfalfa growth and development.It is of great importance to illuminate the molecular mechanisms underlying salt stress response at different development stages of alfalfa.Based on this,some key proteins involved in salt stress response are expected to be screened for genetic engineering breeding.This will provide theoretical basises and feasible methods for breeding salt-tolerant alfalfa.In present study,the regulation mechanisms underlying salt tolerance of roots and leaves in alfalfa cultivar zhongmu NO.1 were disclosed by means of a proteomic approach and key salt-responsive proteins were selected for further study by transgenic experiment of Arabidopsis thaliana,Medicago truncatula,and Medicago sativa.The main results are as follows:We performed physiological and proteomic analysis of the root tissue of Medicago sativa cv.Zhongmu No.1 treated with 100 and 200 mM NaCl for 3 weeks.Compaired with control group,decreased root length,seedling growth and chlorophyll contents,in addition to increased Na⁺concentration were observed in salt-stressed alfalfa,showing a concentration-dependent progress.Additional,H₂O₂ content,SOD and POD activity were increased due to high salt stress.51 differentially expressed proteins?DEPs?were identified using gel-based proteomic approach.These DEPs were involved in antioxidant and defense,energy metabolism,protein processing,folding and degradation,secondary metabolism,signal transduction and amino acid metabolism.Of them,MsANN2,a key salt-response protein,was selected and their expressions in mRNA and protein level were increased under 100 and 200 mM NaCl stress.The comparative proteome of leaves from Medicago sativa cv.Zhongmu No.1?ZM1,salt-tolerant?and Medicago truncatula cv.Jemalong A17?A17,salt-sensitive?was performed using the iTRAQ approach.A total of 438 differentially expressed proteins?DEPs?were identified,among which 282 and120 DEPs were specific to A17 and ZM1,respectively.In salt tolerant ZM1,key DEPs were primarily enriched in antioxidant system,starch and sucrose metabolism,and secondary metabolism.ZM1possessed a greater ability to remove reactive oxygen species and methylglyoxal under salt stress,as demonstrated by enhancement of the antioxidant system and secondary metabolism.Moreover,ZM1orchestrated starch and sucrose metabolism to accumulate various soluble sugars?sucrose,maltose,glucose,and trehalose?,which in turn facilitate osmotic homeostasis.Salt stress dramatically inhibited photosynthesis of A17 due to the downregulation of the light-harvesting complex and photosystem II related protein.Quantitative analyses of photochemical efficiency,antioxidant enzyme activities,hydrogen peroxide,malondialdehyde,and soluble sugar contents were consistent with the alterations predicted on the basis of DEPs functions.The salt tolerance evaluation of MsANN2?NCBI ID:MK689219?transgenic plants were performed.The wild type?WT?and over-expressed MsANN2 plants?OE?were subjected to 75 mM NaCl salt stress at germination stages for 3 weeks.Fresh weight and root legth were higher in OE plants than in WT.Under salt stress?200 mM NaCl for 2 weeks?,relative water content and chlorophyll content in leaves were higher in OE than in WT at seedling stage.The results suggested that the performance of MsANN2transgenic lines of Arabidopsis thaliana were better than that of wild type at different stages of seedling?germination-seedling stage;the mature seedling stage?under salt stress.Moreover,MsANN2 transgenic alfalfa plants showed a higher survival rate and more stable membrane than that of the wild type.Furthermore,MsANN2 transgenic Medicago truncatula showed relatively strong salt-tolerant characteristics,as manifested by the increased activities of SOD,POD and CAT and decreased contents of O₂^-,H₂O₂ and MDA at germination stage,compared with wild type.