Identification Of Transcription Factors Regulating Soybean CLCs Genes And Their Physiological Functions In Salt Stress Adaptation

Plants are usually affected by a variety of unfavorable environmental factors during their growth,such as salt,drought,high and low temperature stresses,among them salt stress is one of the main factors limiting agricultural production.Currently,20%of the arable land is affected by salt stress.Soybean（Glycine max）,the most widely planted cereal legume in the world,is considered to be a moderately salt-tolerant crop,but its growth was also inhibited under salt stress,germination,and seed setting rates are reduced,resulting in reduced yields.The ion toxicity formed under salt stress will have serious adverse effects on plant growth,development,and yield.Among them,there are many studies on Na⁺toxicity,but only a few reports on anion toxicity represented by Cl^-.In terms of cultivated soybean,under salt stress,both Na⁺and Cl^-could produce ion toxicity to growing soybean plants,but the toxicity of Cl^-was greater than that of Na⁺,and the degree of soybean injury was positively correlated with the content of Cl^-in leaves and stems.Therefore,the molecular mechanism of Cl^-uptake,transport,and regulation in soybean plants under salt stress has become an important aspect of plant salt tolerance research.Transcription factors play an important regulatory role in the response and adaptation of plants to salt stress.In terms of transcriptional regulation of genes alleviating salt ion toxicity in plants,transcription factors have been reported to enhance the adaptability of plants to salt stress by regulating the expression of cation（Na⁺and K⁺）transporter or channel genes,and then regulating the absorption,distribution,and homeostasis of these cations in plants.However,the transcriptional regulation mechanism of chloride channel protein family genes（CLCs）mediating anion（Cl^-）transport has not been reported yet.In this study,8 CLCs of soybean subfamily I have been reported to be involved in the adaptation process of plants to salt stress.Gene co-expression network and transcriptional regulatory database was used to identify upstream transcriptional regulators;and Y1H,transient expression in tobacco leaves,EMSA,q RT-PCR,transcriptional activation activity,subcellular localization,salt tolerance identification of gene-overexpressed soybean hairy root-composite plants and transgenic Arabidopsis were performed,focused on the exploration of the physiological functions and mechanisms of two types important transcription factors involved in the regulation of soybean salt tolerance.The main results are as follows:The plant gene co-expression database Genevestigator was used to construct the co-expression network of 8 CLCs of cultivated soybean subfamily I,and the transcription factor regulation database Plant Reg Map predicted all possible transcriptional regulators upstream of soybean CLCs.107 transcription factor encoding genes were preliminarily screened,among which,10 transcription factors predicted upstream of GmCLC1,24 upstream of GmCLC-b1,8 upstream of GmCLC-b2,21 upstream of GmCLC-c1,and 7 upstream of GmCLC-c2,7 upstream of GmCLC-d1,22 upstream of GmCLC-d2 and 8 upstream of GmCLC-g.Yeast one-hybrid experiments and transient expression analysis in tobacco leaves were performed and 8 transcription factors were identified upstream of GmCLC1,GmCLC-b1,GmCLC-c1,GmCLC-c2,and GmCLC-d2:Glyma.04G054200（WRKY）,Glyma.08G152500（b HLH）,Glyma.13G187500（MYB）,Glyma.10G257900（C2H2）,Glyma.04G044900（C2H2）,Glyma.05G234600（MYB）,Glyma.20G203700（ERF）,and Glyma.04G170100（MYB）.Based on the salt tolerance function of soybean GmCLC1 and GsCLC-c2,this study firstly explored the function of GmbHLH3,a candidate transcription factor of GmCLC1.Through point mutation of promoter sequence,Y1H,transient expression in tobacco,GUS activity assay,and EMSA,it was found that GmbHLH3 transcription factor could bind to the G-box element at the promoter of GmCLC1 gene to activate the expression of the downstream gene.In addition,the expression level of GmCLC1 in the roots of GmbHLH3-overexpressing soybean hairy root-composite plants was significantly higher than that of Vector plants.The salt tolerance of OEGmbHLH3 composite plants was significantly enhanced in terms of growth phenotype,plant height,number of dead cells in leaves,MDA content in leaves,and REL values in roots and leaves of plants.The accumulation of Cl^-and NO₃^-in roots of OEGmbHLH3 plants significantly increased under salt treatment,and the transport and accumulation of Cl^-to shoots（stems and leaves）were significantly decreased,resulting in a significant decrease of Cl^-/NO₃^-values in roots,stems and leaves.The Na⁺content in roots,stems and leaves and Na⁺/K⁺values in stems and leaves of OEGmbHLH3 plants also showed similar changes.GmbHLH3-transgenic Arabidopsis also showed significantly enhanced salt tolerance,and the changes of Cl^-and Na⁺contents in roots,NO₃^-and K⁺contents in roots and shoots were consistent with those of soybean composite plants.In addition,At CLCc,At CLCg,At NCED3 and At P5CS1 were activated and up-regulated in salt-treated GmbHLH3-transgenic Arabidopsis.The phenotype of the GmbHLH3-RNAi soybean hairy root-composite plants was consistent with that of the Vector plants under salt stress,and did not show reduced salt tolerance.GmERF160 activates the reporter gene by binding to the promoter of GmCLC-c2 as demonstrated by Y1H,tobacco transient expression and GUS activity assay.Further prediction of the upstream transcription factor of wild soybean GsCLC-c2,a gene with high homology to GmERF160 was identified and named Gs ERF160.Gs ERF160 and GmERF160genes were up-regulated under salt stress,especially in roots.The functions of Gs/GmERF160 genes were analyzed by soybean hair-root composite plants and transgenic Arabidopsis.It was found that soybean composite plants and transgenic Arabidopsis showed less salt damage under salt stress,indicating that Gs/GmERF160 can positively regulate the salt tolerance of plants and activate the expression of GmCLC-c2 in the roots of soybean composite plants.Under salt treatment,the content of Cl^-in roots of Gs/GmERF160-overexpressing soybean composite plants was significantly increased,while the content of Cl^-in stems and leaves was significantly decreased,the content of NO₃^-in roots,stems and leaves of overexpressed plants was significantly increased,resulting in a significant decrease in the value of Cl^-/NO₃^-in stems and leaves.The salt tolerance of Gs/GmERF160-transgenic Arabidopsis plants was also significantly enhanced,which was related to the significantly lower Cl^-/NO₃^-and Na⁺/K⁺values in shoots under salt treatment than those in wild type（WT）.On this basis,a Gm EIN3 protein that interacted with GmERF160 was identified.In summary,the transcription factor GmbHLH3 can confer enhanced salt tolerance in GmbHLH3-overexpressing soybean hairy root-composite plants by upregulating the GmCLC1 expression,mediating the Cl^-and NO₃^-absorption and their transportation to shoots to maintain relatively low Cl^-/NO₃^-and Na⁺/K⁺values,and ABA-mediated signaling pathway or proline metabolism pathway may also be involved in the process of above-mentioned salt damage alleviation.The transcription factor Gs/GmERF160,identified upstream of Gs/GmCLC-c2 gene in wild and cultivated soybeans,also participates in the regulation of anion homeostasis in plants under salt stress.It mainly regulates the distribution of Cl^-,NO₃^-,etc.in Gs/GmERF160-overexpressing hairy root-composite plants or transgenic Arabidopsis by activating and enhancing the expression of GmCLC-c2,and maintaining significantly lower Cl^-/NO₃^-value in shoots,thereby enhancing salt tolerance.