Molecular Evolution And Functional Divergence Of Homocysteine S-Methyltransferases(HMT) Gene Family In Soybean

Methionine is an essential amino acid in humans,which is mainly obtained from plants.In plants,methionine is mainly present in seeds in the form of stored proteins.Homocysteine??methyltransferase（HMT）is a key enzyme in the synthesis of methionine in plant seeds.HMT catalyze homocysteine and S-methylmethionine or S-adenosylmethionine??into methionine.Studies in Arabidopsis have found that the function of the HMT gene family has diverged.In order to explore the evolutionary history and functional differentiation in soybean of the HMT gene family,we performed detailed molecular evolution and functional differentiation analysis of the gene family.The research results are as follows:First,the phylogenetic relationship of the plant HMT gene family revealed that the HMT genes diverged into two branches,Class 1 and Class 2,in the ancestors of terrestrial plants.Class 1 contained only seed plants,while Class 2 contained all terrestrial plants.Further,the analysis of gene structure also showed that HMTs were relatively conservative in protein structure,and the number and structure of exons and introns.In addition,the family was under strongly purified selection during the evolution.Nevertheless,the results of functional divergence showed that type I functional differentiation between Class1 and Class2 was detected with six diverged sites,while the type II was not detected.The results suggest that the evolving process of HMT gene family is relatively conservative with some non-polar sub-functional divergence.Second,expression differentiation reflected the divergence of gene functions.In this study,tissue-specific expression and stress-response expression was performed on GmaHMT 1,GmaHMT 2,GmaHMT 3 and GmaHMT 4,respectively.The results of tissue-specific expression showed that the expression of GmaHMT 1,GmaHMT 2 and GmaHMT3 in the productive organs was higher than that in the vegetative tissues.There is some fluctuations in different develoipmental stages;however,the expression of GmaHMT 4 is significantly higher in leaves than the flowers and pods.In addition,the expression patterns of GmaHMTs to response different stresses also have divergence.First,as for the drought stress,GmaHMT 1,GmaHMT 2 and GmaHMT3 had similar response patterns,in which their expression levels was significantly down-regulated at low concentrations and were up-regulated at 25%PEG6000,while GmaHMT4 was significantly down-regulated at all of the concentrations.Under salt stress,the expression trends of the four genes were similar to them in the drought stress.Differently,the expression of the four genes was significantly down-regulated under all the salt concentrations.However,under the acid-base stress,the expression of GmaHMT genes was different,in which most of them were down-regulated except that GmaHMT 1 and GmaHMT 3 were induced at pH10.Therefore,GmaHMTs have experienced different divergence in tissue-specific and stress-responsive expression.The promoters of GmaHMT gene was also analyzed and the results showed that the promoters of GmaHMT 1,GmaHMT 2 and GmaHMT 3,and GmaHMT 4 diverged,which might be the reasons that why the expression of GmaHMT4 is different to GmaHMT 1,GmaHMT 2 and GmaHMT 3.Finally,comparative study of the fermentation of different GmaHMTs in Escherichia coli BL₂₁（DE3）was also performed.The results showed that the yield of methionine produced by GmaHMT 3 and GmaHMT 4 was significantly higher than that of GmaHMT 1 and GmaHMT 2,and the GmaHMT2 was the lowest.In addition,catalysis of GmaHMTs was also performed.The results showed that GmaHMT1 could not use SMM as the methyl donor to synthesize methionine,while GmaHMT2,GmaHMT3 and GmaHMT4 could.This result suggests that the catalytic activities and methyl donors of GmaHMTs have diverged.In summary,our results showed that the HMT gene family in plants has diverged in their molecular evolution,gene expression,enzyme activity and methyl donor.The divergence of HMT genes are mainly related to the synthesis of methionine in plants,especially in seeds.Therefore our studies will provide a theoretical basis for the improvement of methionine in plants,especially in soybeans.