Proteomic And Metabolomics Analysis Of Flowers And Seeds Of Medicago Sativa L.Cv."Aohan" Response To Boron Stress

Boron(B)has been recognized as an essential element for increasing seed yield and yield components in alfalfa(Medicago sativa L.).However,the specific functions and molecular mechanisms for B in alfalfa seeds are not clear.To investigate this question,proteomic,metabonomic and physiological studies were carried out on flowers and mature seeds of Medicago sativa L.cv."Aohan" grown under B deficiency(0%),B normal(0.08%)and B toxicity(0.16%)condition.Main results are as follows:1.To investigate the boron(B)effect of alfalfa for seed production,a field experiment with five B treatments(0,0.04,0.08,0.12 and 0.16%)was carried out with Medicago sativa L.cv."Aohan",and compared the pollen number and pollen viability of flower,seed yield,yield components,seed quality and dynamic changes of B accumulation amount of alfalfa.The results showed that foliar boron application improve the pollen number and pollen viability of flower,seed yield,yield components and seed quality.The results suggested that the optimum B application rate was 0.08%.When the spraying concentration reached 0.16%,toxic effects have been produced.Therefore select 0%(boron deficiency treatment),0.08%(normal boron treatment)and 0.16%(high boron treatment)to conduct the next test.2.Determined the changes of the antioxidant enzymatic activities and lipid peroxidation of flowers and mature seeds of Medicago sativa L.cv."Aohan" grown under B deficiency(0%),B normal(0.08%)and B toxicity(0.16%)condition.For alfalfa flowers,the activities of SOD,POD and CAT,the contents of MDA and proline decreased significantly after boron stress.For alfalfa seeds,the activities of SOD,POD and CAT,and the contents of MDA decreased significantly after boron deficiency stress,but increased significantly after high boron stress.The contents of proline and soluble sugar increased significantly after boron deficiency stress,but decreased significantly after high boron stress.3.A combination of two-dimensional electrophoresis(2DE)and mass spectrometry(MS)approaches was used to investigate expressed proteins in Medicago sativa L.cv."Aohan" flowers and seeds response to B stress.Twelve differentially expressed proteins that mainly involved in carbohydrate metabolism and lipid metabolism were successfully identified in B deficient flowers,and eleven differentially expressed proteins that mainly involved in carbohydrate metabolism and amino acid metabolism were successfully identified in B deficient seeds.Fourteen differentially expressed proteins that mainly involved in carbohydrate metabolism,amino acid metabolism,lipid metabolism and secondary metabolites were successfully identified in B toxic flowers,and eleven differentially expressed proteins that mainly involved in carbohydrate metabolism,amino acid metabolism and lipid metabolism were successfully identified in B toxic seeds.Acyl-protein thioesterase and NAD(P)-binding rossmann-fold protein played a key roles in Merdicago sativa L.cv."Aohan" flowers response to B stress,and NAD-dependent aldehyde dehydrogenase family protein and NAD(P)-binding rossmann-fold protein played a key roles in alfalfa Medicago sativa L.cv."Aohan" seeds response to B stress.4.Metabolomics analysis based on GC-MS was applied to investigate expressed metabolites in alfalfa Medicago sativa L.cv."Aohan" flowers and seeds.Fifty differentially expressed metabolites were successfully identified in alfalfa flowers,when thirteen differentially expressed metabolites were successfully identified in alfalfa seeds by T-test analysis and OPLS-DA model under boron deficiency stresss.Boron deficiency stress increased the contents of metabolites that involed in carbohydrateted metabolism and amino acid metabolism.Ninety-seven differentially expressed metabolites were successfully identified in alfalfa flowers,and sixteen differentially expressed metabolites were successfully identified in alfalfa seeds under high boron stress.Boron toxity decreased the contents of metabolites that involed in carbohydrateted metabolism,amino acid metabolism,lipid metabolism,purine metabolism and pyrimidline metabolism in alfalfa flowers but increased the contents of metabolites that involed in amino acid metabolism in alfalfa seeds.5.The comprehensive analysis reveals that alfalfa flowers and seeds through the regulation of amino acid metabolism,carbohydrate metabolism,lipid metabolism and TCA cycle to adapt to the boron stress.The result of this work might be helpful to understand the basic metabolic regulation mechanism of alfalfa reproductive organs in response to B stress,and give insight into potential strategies for improving seed yield,quality and stress tolerance in alfalfa grown for seed production.