Response Mechanisms Of Amino Acid Metabolism In Roots Of Malus Prunifolia Under Alkali Stress

The soil p H value in excellent apple production area,the northwest plateau in China,is higher than 8.0.Soil alkalization has a negative impact on plant production,resulting in dwarfing plants,yellowing leaves,hindering the absorption of nutrient elements,which hinders food security and economic benefits of farmers,and has greatly affected the development of agriculture.Under stress,in order to activate the defense mechanism and reduce the damage caused by alkali stress,the synthetic pathway of metabolites and the expression of genes will change.As a smotic regulation substances and the precursor of other amino acid,amino acids play a positive role in improving adaptability to alkali stress.Malus prunifolia has been verified to be a resource with disease resistance,drought resistance and alkali tolerance.Studying on the changes of metabolic pathway,DEDs and amino acid synthesis of this resource under alkali stress,and exploring the molecular mechanism of alkali tolerance could provide a theoretical basis for improving the alkali tolerance of apple seedlings and discovery alkali-tolerant germplasm resources.In this study,tissue culture seedlings of M.prunifolia were used as materials,and the changes in seedlings after alkali stress,such as the growth,photosynthetic characteristics,root membrane damage,and so on,were detectived.Based on the transcriptome sequencing data of M.prunifolia roots under alkali stress for different time,the response of amino acid metabolism pathway of M.prunifolia to alkali stress was analyzed.Further,the GS genes in Malus were analyzed in order to explore the alkali tolerance mechanism of M.prunifolia from the aspect of plant physiology and molecular science.The results showed that:1.Compared with the control,the fresh weight and dry weight of M.prunifolia seedlings decreased by 14.2% and 16.1% respectively,the contents of chlorophyll,carotenoid and the chlorophyll fluorescence parameters decreased significantly,and the photosynthetic parameters,such as net photosynthetic rate and stomatal conductance root conductivity,decreased significantly.The relative electrolyte leakage of root of seedlings increased significantly due to alkali stress.Meanwhile,the contents of phenylalanine,glycine,tyrosine,leucine,proline,isoleucine,valine and proline in seedlings were changed differently,and the contents of most amino acids showed an increasing trend with the time of alkali stress.2.Transcriptome sequencing was used to analyze the gene expression in roots of M.prunifolia under alkali stress at 6 h,12 h,24 h and 48 h.Theresults showed that DEGs were mainly annotated to several pathways,including response to stimulus,carbohydrate metabolism,signal transduction mechanisms,secondary metabolites biosynthesis and biosynthesis of amino acids.Futher,the pathway of amino metabolism was analyzed.The results showed that at 48 h of alkali stress,the number of DEGs upregulated in these pathways was obviously increased,phenylalanine metabolism(9 up-regulated DEGs),cyano-amino acid metabolism(6 up-regulated DEGs),arginine biosynthesis(4 up-regulated DEGs),cysteine and methionine metabolism(7 up-regulated DEGs),tyrosine metabolism(7up-regulated DEGs),alanine,aspartic acid and glutamate metabolism(6 up-regulated DEGs),glycine,serine and threonine metabolism(6 up-regulated DEGs).3.Based on the statistics and analysis of transcription factors at different time,it was found that the first five transcription factors with the largest number of responses to alkali stress included WRKY,NAC,b HLH,AP2-ERF and MYB,and the number of transcription factors was higher at 48 h,which were 5,9,13,14 and 17,respectively.4.The identification and bioinformatics analysis of GS gnen members showed that there were 7 GS genes members in Malus,in which MD09G1270600,MD17G1268700,MD16G1001400,MD13G1006600 and MD14G1238700 belonged to GS1 subfamily,and located in cytoplasm.While MD13G1180400 and MD16G1181300 belonged to GS2 subfamily,and located in chloroplast.GS genes in Malus were distributed on 5chromosomes,containing 29-46 phosphorylation sites each gene,and most members with relatively conservative gene structure could response to alkali stress.