Plasma Membrane Proteomic Analysis Provides Insight Into Mechanisms Of Aluminum Resistance And Functional Characterization And Utilization Of Related Differentially Expressed Proteins In Tamba Black Soybean

Alfalfa（Medicago sativa L.）,recognized as“the king of forage”is the most widely distributed,protein-rich perennial high-quality legumes in the world.In recent years,herbivorous animal husbandry has developed rapidly in southern C hina,and the demand for alfalfa has increased sharply.However,most of the soils in the south are acidic,in which aluminum ions(mainly Al³⁺)are vital factors affecting plant growth in acidic soils.Alfalfa is especially sensitive to Al toxicity,thereby greatly limiting the development of herbivorous animal husbandry in the south.Therefore,the cultivation of new Al-resistant varieties is the key to solving the problem of alfalfa cultivation in south of China.Alfalfa plants are lack of the Al-resistant germplasm adapting to acid soil,and it is difficult to obtain Al-resistant varieties through conventional breeding methods.Therefore,introducing Al-resistant genes by means of molecular breeding is an essential way to cultivate Al-resistant alfalfa varieties.O ur previous studies found that Tamba black soybean（TBS）,which is a leguminous plant like alfalfa,is suitable to grow in acidic soil.The important mechanism of TBS resistant to Al to xicity involves secreting citrate from the root tips and chelating Al³⁺in the rhizosphere under Al stress.Furthermore,studies indicated that the proteins related to Al-resistance,including organic acid channel proteins,H⁺-ATPase,cell wall modificatio n-related proteins and Al signal transduction,are mostly located in the plasma membrane.Therefore,TBS was used to reveal Al-resistance mechanism via plasma membrane proteomics in this study.The genes,including GmALS3,GmPMT13,GmPMT75 and GmPMT16,were cloned,and their function were identified through genetic transformation into tobacco or arabidopsis.In addition,the Al-resistance genes were also transformed into alfalfa,and alfalfa plants resistant to Al were screened.The main results are as follows:（Medicago sativa L.）is a perennial leguminous forage that is widely distributed world.It is well-known as“the queen of forage”for it is rich in protein.In recent years,herbivorous animal husbandry in southern C hina has developed rapidly,and the demand for alfalfa has increased sharply.However,most of the soils in the south are acidic,in which aluminum ions(mainly Al³⁺)are important factors affecting plant growth in acidic soils.Alfalfa is very sensitive to Al toxicity,thereby greatly limiting the development of herbivorous animal husbandry in the south.Therefore,the cultivation of new Al-resistant varieties is the key to solve the problem.Alfalfa plants lack Al-resistant germplasm adapting to acid soil,it is difficult to obtain Al-resistant varieties through conventional breeding methods.Therefore,introducing Al-resistant genes by means of molecular breeding is an important way to cultivate Al-resistant alfalfa varieties.O ur previous studies found that Tamba black soybean（TBS）,which is a leguminous plant like alfalfa,is suitable to grow in acidic soil.The important mechanism of TBS resistant to Al toxicity involves secretion of citrate from the root tip and chelates Al³⁺in the rhizosphere under Al stress.Furthermore,some studies indicated that proteins related to plant Al-resistance,including organic acid channel proteins,H⁺-ATPase,cell wall modification-related proteins and Al signal transduction,are most located in the plasma membrane.Therefore,TBS was used to reveal Al-resistance mechanism via plasma membrane proteomics in this study,and the genes,including GmALS3,GmPMT13,GmPMT75 and GmPMT16 of TBS were cloned,and their function were identified through genetic transformation into tobacco or arabidopsis.In addition,the Al-resistant genes were also transformed into alfalfa,and alfalfa plants resistant to Al were screened.The main results are as follows:1.Plasma membrane proteomics reveals the Al tolerance mechanism of TBSIn this study,tandem mass tag（TMT）-based quantitative proteomic methods were used to identify the differentially expressed plasma membrane（PM）proteins in Tamba black soybean（TBS）root tips under Al stress.In addition,parallel reaction monitoring（PRM）was used to verify the protein quantitative data.The results showed that 907 PM proteins were identified in Al-treated plants.Among them,90 proteins were differentially expressed（DEPs）with 46 upregulated and 44 downregulated compared to untreated plants（fold change>1.3 or<0.77,p<0.05）.Functional enrichment based on GO,KEGG and protein domain revealed that the DEPs were associated with membrane trafficking and transporters,modifying cell wall composition,defense response and signal transduction.Functiona l enrichment based on GO,KEGG and prote in doma in revealed that the DEPs were associated with membrane trafficking and transporters,modifying cell wall composition,defense response and signal transduction.In conclusion,our results highlight the involvement of GmMATE13,GmMATE75,GmMATE87 and H⁺-ATPase in Al-induced citrate secretion in PM of TBS roots,and ABC transporters and Ca²⁺have been implicated in internal detoxification and signaling of Al,respectively.GmMATE13,GmPMT16,GmALS3 and GmMATE75 are upregulated significant ly in plasma membrane proteomics under Al stress,followed by which may play a crucial role in coping with Al stress.It is necessary to further study the function of genes corresponding to these proteins in Al stress in order to lay a foundation for germp lasm improvement of alfalfa.2.The cloning and functional identification of GmALS3 from TBSAn ABC transporter gene GmALS3 from plasma membrane proteomics was significantly upregulated under Al stress.The function of GmALS3 was verified by RNAi silencing and heterologous overexpression.The results of expression pattern analysis showed that the expression of GmALS3 was significantly higher in the roots（especially in the root tips）than that of stems,leaves and cotyledons（p<0.05）.The expression of GmALS3 in the stems was also significantly upregulated under Al stress.In addition,GmALS3 was located on the plasma membrane.After silencing GmALS3with RNAi,the Al-resistance of TBS reduced significantly,and the Al content in the roots increased significantly（p<0.05）.On the contrary,the heterologous overexpression of GmALS3 significantly improved the Al resistance of tobacco,and the accumulation of Al in roots decreased significantly.These results suggest that GmALS3is a vital Al-resistant gene,which is involved in the Al-resistance process of TBS.The overexpression of GmALS3 can improve the Al-resistance of plants and provide important genetic resources for cultivating alfalfa varieties suitable for growing in acidic soil.3.The cloning and functional identification of GmPMT16 Gene from TBSWe functionally characterized an Al-inducible methyltransferase gene,GmPMT16,which was expressed in different tissues of TBS and upregulated in roots and stems under Al stress.The results of subcellular localization showed that GmPMT16 was located in the plasma membrane and nucleus.Silent GmPMT16 significantly inhibited citrate secretion and Al-resistance in TBS hairy roots.Moreover,the activity of methyltransferase,melatonin and citrate secretion in the root tip of the tobacco overexpressing GmPMT16 were 1.18-1.30 times,1.21-1.36 times and 1.52-2.04 times than those of the wild types（WT）,respectively.These results suggest that GmFMT16 is a methyltransferase gene,which is involved in regulating the secretion of citrate in plant roots and affecting the content of melatonin in root tip.O verexpression of GmPMT16can improve the Al tolerance of plants.4.Cloning and functional identification of GmMATE13 and GmMATE75 genesCitrate secretion is a valuable Al-resistance mechanism of TBS under Al stress.In this study,the MATE family genes GmMATE13 and GmMATE75 screened by plasma membrane proteomics were cloned.The analysis of gene expression showed that GmMATE13 and GmMATE75 were mainly expressed in roots.Subcellular localization showed that GmMATE13 and GmMATE75 were localized on the plasma membrane.GmMATE13 and GmMATE75 were identified through the overexpression recovery experiment of Arabidopsis mutants.The results showed that overexpression of GmMATE13 and GmMATE75 alleviated the inhibition of root growth of atmate mutant under Al stress,and the citrate secretion was significantly higher than that of atmate mutants.These results suggested that GmMATE13 and GmMATE75 are citrate channel proteins localized in the plasma membrane.5.New breeding materials obtained by genetic transformation of Al-resistant genes into alfalfaPrevious studies have shown that GmALS3,GmMATE75,GmMATE13 and GmMATE75 are Al-resistant genes in TBS.Hence,these genes were transformed into alfalfa and their Al-resistance was further verified.The results showed the contents of MDA,H₂O₂ and Al in root tips of alfalfa overexpressing GmALS3 were significantly lower than those of WT,while the content of soluble protein was significantly higher than those of WT,and Al tolerance was significantly enhanced.Evans blue and chrome azurol S staining in root tips of alfalfa overexpressing GmPMT16 became lighter than that of WT,MDA content decreased significantly,soluble protein content increased significantly,Al content decreased significantly,citrate secretion increased significantly,and Al tolerance increased significantly.The citriate secretion of alfalfa overexpressing GmMATE13 or GmMATE75 was significantly higher than that of WT,and the content of MDA and Al in root tip of transgenic alfalfa was lower than that of WT.However,the Al toxicity of transgenic alfalfa root tip was similar to WT,which indicating that citrate secretion of alfalfa overexpressing GmMATE13 or GmMATE75 was not enough to alleviate Al toxicity.In order to improve the effectiveness of MATE channel proteins and achieve the purpose of detoxification of citrate in alfalfa,H⁺-ATPase and citrate synthesis-related genes should be introduced into alfalfa plants based on overexpressing GmMATE13 or GmMATE75.These results show that the transgenic alfalfa plants obtained can be used as precious breeding materials for Al-resistant alfalfa varieties by overexpressing GmALS3,GmPMT16,GmMATE13 or GmMATE75.In summary,this study reveals the molecular mechanism of Al tolerance using plasma membrane proteomics of TBS.The functions of upregulated proteins GmALS3,GmMATE13,GmMATE75 and GmPMT16 were identified,and were genetically transformed into alfalfa.The obtained alfalfa plants provide new materials for breeding the cultivation of Al-resistant alfalfa varieties.