Exploring The Role Of MiR160a In Modulating Soybean Salt Tolerance Via The Target Gene GmARF16

Soil salinization is a global issue that hinders agricultural production.In China,there is avast area of saline-alkali land,with approximately 9 million mu in the Yellow River Delta alone.Moreover,there is an annual increase of around 100,000 mu of saline-alkali land.With the growing population and the increasing concerns over arable land resources and food security,saline-alkali land has become an important reserve resource for"expanding capacity,improving quality,and increasing efficiency"in agricultural cultivation.In October2021,general secretary Xi Jinping pointed out during his inspection of the Yellow River Delta that"we should strengthen fundamental research on genetic resources,farmland protection and utilization,and transform breeding concepts.We need to shift from managing saline-alkali land to adapting crops,towards the selection and cultivation of salt-tolerant plants for saline-alkali land".Therefore,exploring the genetic and molecular mechanisms of crop responses to salt stress,cloning highly efficient salt-tolerant genes,and facilitating the cultivation of salt-tolerant crops are not only the core challenges in crop science research but also crucial strategic demands for the country.As the most economical grain and oil crop,soybean is an important basic and strategic material related to the national economy and people’s livelihood.China is the main consumer and importer of soybeans,and the dependence on foreign countries exceeds 85%,which makes China’s soybeans face a serious crisis.The contradiction between supply and demand of soybean has a great impact on China’s food security.Expanding planting area and increasing soybean yield is the main way to increase soybean total output.By exploring salt-tolerant genes,breeding new varieties of salt-tolerant soybeans is helpful to promote the full utilization of saline-alkali land resources,expand soybean planting area,and alleviate the domestic soybean industry crisis.However,few salt-tolerant genes have been identified and the mechanism of salt-tolerant genes is still unclear.In this paper,we used genetics,molecular biology and genomics techniques to reveal miR160a-GmARF16-GmMYC2,a regulatory module of soybean salt tolerance,and analyzed the natural variation of GmARF16gene in 559 germplasm resources to find an excellent haplotype of salt tolerance.It provides important candidate gene resources for breeding new soybean salt-tolerant varieties.The specific research results are as follows:1.The expression of miR160a is induced by salt stress and can positively regulate the salt tolerance of soybeanThe transcriptome data of miRNA precursors under salt stress showed that members of mi160 family were induced by salt stress.By measuring the expression levels of 6 members of miR160 family at different time after salt stress,it was found that miR160a had the most significant response to salt stress.Stable miR160a overexpression（miR160a-OE）and silenced（STTM160a）transformation plants and Mosaic plants were obtained using soybean cotyledon node stable genetic transformation and hairy root transformation systems,respectively.It was found that miR160a overexpression could improve soybean salt tolerance,while miR160a silenced soybean salt tolerance decreased.miR160a can positively regulate the salt tolerance of soybean.2.miR160a affects the salt tolerance of soybean by regulating the target gene GmARF16Using bioinformatics methods,13 potential target genes of miR160a were identified.Among them,the expression level of GmARF16 under salt stress was significantly negatively correlated with that of miR160a（r=-0.80452）,and miR160a negatively regulated the expression of GmARF16 in stable transformation plants of miR160a.RLM-5’RACE results showed that miR160a could specifically recognize GmARF16.These results indicate that GmARF16 is the target gene of miR160a.The function of GmARF16 was verified by the stable genetic transformation of soybean cotyledon node and hairy root transformation system,and it was found that GmARF16 negatively regulated the salt tolerance of soybean.The genetic complementarity experiment between miR160a stable genetic plant and GmARF16 hairy root transformation plant showed that miR160a and GmARF16 were in the same genetic pathway.When the binding site of GmARF16 and miR160a was mutated（the coding amino acid remained unchanged）,the salt tolerance of m GmARF16 transgenic plants was further reduced.These results indicated that miR160a regulated the salt tolerance of soybean by inhibiting the expression of GmARF16.3.GmMYC2,as a downstream target gene of GmARF16,negatively regulates salt tolerance by influencing proline contentCombined analysis using transcriptome sequencing（RNA-seq）and chromatin immunoprecipitation sequencing（Ch IP-seq）identified 51 downstream genes regulated by GmARF16.Among them,GmMYC2 is a b HLH transcription factor whose homologous gene negatively regulates salt tolerance in Arabidopsis Thaliana.It was found by Ch IP-q PCR,EMSA and LUC that GmARF16 can activate the expression of GmMYC2 by directly binding to the GACA element of GmMYC2 promoter,suggesting that GmMYC2 may be involved in the regulation of soybean salt tolerance as a target gene of GmARF16.The salt tolerance function of GmMYC2 transgenic hairy root was analyzed,and it was found that GmMYC2negatively regulated the salt tolerance of soybean.Genetic complementation experiments were carried out between GmMYC2 hairy root transformation plants and stable transformation plants of miR160a and GmARF16 respectively,indicating that GmMYC2,as a downstream gene of GmARF16,plays an important role in the salt tolerance regulation pathway of soybean.Meanwhile,overexpression of GmMYC2 in hairy roots of soybean resulted in a significant decrease in proline content,and inhibition of GmMYC2 expression significantly increased the accumulation of proline.Under salt stress,the proline accumulation in STTM160a and GmARF16-OE transgenic plants decreased,while the proline content in GmARF16-RNAi transgenic plants increased,indicating that miR160a-GmARF16-GmMYC2module regulates the adaptability of soybeans to salt stress by regulating the synthesis of proline.4.The natural variation of GmARF16 gene has different salt tolerance and geographical adaptabilityFour haplotypes of GmARF16 gene were found by genetic population analysis of 559 soybean varieties.Among them,GmARF16^Hap1,GmARF16^Hap2 and GmARF16^Hap4differ in the5’UTR,while GmARF16^Hap3 has a non-synonymous base substitution in the coding area,causing it to change from encoding"arginine"to"lysine".There was no significant difference in transcription activity in the 5’UTR region between different haplotypes by double luciferase detection system.Compared with GmARF16^Hap2,the activation ability of GmARF16^Hap3 to GmMYC2 and its binding ability to promoter were significantly reduced,and the tolerance of GmARF16^Hap3 transgenic plants to salt stress was better than that of GmARF16^Hap2.Further analysis showed that GmARF16^Hap3 was absent in wild species,but the proportion of domestic species and cultivated species increased significantly,and GmARF16^Hap3 was mainly distributed in Huang-Huai-Hai and its surrounding areas where soil salinization was more serious,suggesting that GmARF16^Hap3 had been artificially selected to adapt to salt stress in the evolutionary process.In summary,we proposed a new miR160a-GmARF16-GmMYC2 salt tolerance regulatory module,which regulates soybean salt tolerance by influencing proline content,and GmARF16^Hap3 is an excellent salt-tolerant haplotype,which can be used as a potential target for soybean improvement in high salt areas.