Function And Expression Regulation Of Aluminum-responsive Genes MsMATE And MsSTOP1 In Medicago Sativa

Aluminum?Al?toxicity is one of the important limiting factors for crop growth and yield in acidic soils,with the inhibition of root elongation as a typical symptom.The main mechanism of Al resistance in many plants is the chelation of activated Al³⁺by organic acids.Genes encoding organic acid transporters enhance Al resistance,which depends on their mediation of exudation of organic acids in plants.Alfalfa?Medicago sativa L.?is one of the most important legume forage,but is hypersensitive to the Al stress.Thus,alfalfa is hard to adapt to the acidic soil in south China,which is an important restriction factor for the modem animal husbandry development.Exploring of potential mechanisms of Al resistance for breeding new varieties to adapt to the acidic soil would be significant for the alfalfa industry development in China.In this study,microarray hybridization,quantitative PCR,yeast one-hybrid,EMSA and complementation of mutant assay in Arabidopsis were employed to investigate two Al stress responsive genes,MsMATE and MsSTOP1,in alfalfa.The main research conclusions are as followings:1.The root elongation of alfalfa was dramatically inhibited under Al stress,and meristematic zones were the major target of Al stress identified by hematoxylin staining.Microarray hybridization and bioinformatics analysis showed that Al induced drastic oxidative stress reaction in both roots and shoots of alfalfa,which would result in oxidative damage.At the same time,the genes related to DNA replication,transcription,translation,ribosomal biogenesis,and protein modification were down regulated,which would affect the regular physiological activities in both roots and shoots.Moreover,Al stress inhibited the genes associated with cytokinin response and cell cycle in roots,which could explain the inhibition of root elongation,while the genes related to auxin response,photosynthesis,and epigenetic modification were down regulated which could be the reason for etiolated leaves and short plants.To withstand the oxidative stress reaction induced by Al stress,expression of many antioxidant genes were observed at a higher level in both roots and shoots.Besides,numbers of genes related to cell wall remediation were showed up-regulated expression in roots,while this up-regulated expression performance of genes in shoots focused on those associated with different biological processes,such as phenylpropanoid biosynthesis,alpha-linolenic acid metabolism,flavonoid biosynthesis and metal transport in shoot.It suggests that Al stress induces different damage mechanism in different tissues,and respective detoxification mechanism also exists in different tissues of alfalfa.2.Moreover,under Al stress a predicted MATE gene was significantly up-regulated in roots of alfalfa.The MATE gene in alfalfa,defined as Ms MATE,exhibited highly homology with the genes in other plants,which had been recognized as the coding genes of citrate transporters related to Al tolerance.In this research,MsMATE gene in alfalfa was cloned,which contained 13 exons and 12 introns,with a 1524 bp open reading frame coding 507 amino acids.MsMATE was expressed in roots,stems and leaves,but at a low basal level in root tips.Al stress dramatically induced the expression of MsMATE expression in the manner of time dependence and concentration dependence.Besides,MsMATE was also response to H⁺stress,but not to other metal ions.The results of MsMATE transgene in STOP1-KO mutant Arabidopsis showed that MsMATE gene didn't mediate citrate exudation and failed to enhance Al tolerance in transgenic lines.The results from electrophysiological characterization in X.laevis oocytes suggested that MsMATE of alfalfa as a citrate transporter showed significantly lower activity than Arabidopsis AtMATE transporter,which could be the main reason of MsMATE transgene in STOP1-KO mutant Arabidopsis failed to enhance Al tolerance.3.Transcription factor STOP1 of Arabidopsis plays an essential role in Al resistance mechanism,which could regulate the expression of AtMATE gene.MsSTOP1,STOP1 in alfalfa,was also investigated in this study to explore the relevant Al resistance mechanism.MsSTOP1 showed highly homology with the proteins in other plants,which had been proved to be transcription factors with four C₂H₂ zinc finger domains related to Al tolerance.MsSTOP1 was constitutively expressed in roots,stems,and leaves of alfalfa.MsSTOP1 could respond to Al and H⁺stress,but not to other metal ions.The results of overexpression of MsSTOP1 gene in Arabidopsis STOP1-KO mutant showed that MsSTOP1 could facilitate the citrate exudation and recover partial Al tolerance by regulating the expression of AtMATE in Arabidopsis,but the other two Al tolerance genes,AtALS3 and AtALMT,were not regulated by MsSTOP1.Moreover,the results of yeast one-hybrid and EMSA showed that MsSTOP1 could also regulate the expression of MsMATE gene.All the results suggested that MsSTOP1 of alfalfa had some effects on Al tolerance.The specific binding of MsMATE promoter sequence GGTGC with MsSTOP1 was confirmed by yeast one-hybrid and electrophoretic mobility shift assay.In summary,this research investigated the expression profiles under Al stress,and explored the expression,function and regulation of MsMATE and MsSTOP1 genes in alfalfa.The results suggest that the transcription factor MsSTOP1 of alfalfa takes part in the regulation of Al tolerance mechanisms like Arabidopsis AtSTOP1,which also regulates the expression of MsMATE gene in alfalfa.MsMATE gene responds mightily to Al stress,but shows significantly lower activity in citrate exudation than Arabidopsis,which could be one reason for Al sensibility of alfalfa.