The Mechanism Of GSK3 Kinase Regulating Nodule Development In Soybean In Response To Salt Stress

Legumes establish symbiotic associations with rhizobia to form nodules.The rhizobia in the nodules can convert nitrogen gas into ammonia by using the carbon source provided by the plant,and transport it from the nodules to the whole plant,so that the legumes can obtain nitrogen.Legumes need to undergo a series of complex biological processes to form symbiotic nitrogen-fixing nodules,including mutual recognition between legumes and rhizobia,rhizobial infection,nodule initiation,nodule growth,nitrogen fixation and nodule senescence.However,these nodule development processes are highly regulated by a variety of abiotic stresses,that salt stress inhibits the rhizobial infection,nodule development and the efficiency of nitrogen fixation.The genetic and molecular mechanisms of this phenomenon have not been elucidated.It is reported that Glycogen Synthase Kinase 3（GSK3）kinases play key roles in coordinating plant growth and stress responses,but it is still unclear whether GSK3 kinase is related to salt stress inhibiting nodule development.In this study,we explore the mechanism of salt stress inhibiting symbiosis and nodulation in soybean（Glycine max）and found the following results:1.GmSK2-8 is strongly induced by salt stress,and the expression of GmSK2-8 is more specific than other GSK3 members during nodule development.Functional analyses were performed in hairy roots and stable transgenic plants after inoculation with USDA110.GmSK2-8 inhibits rhizobial infection,the formation of nodule primordia,the expression of symbiotic responsive genes,and the number of nodules formed finally.2.GmNSP1a,a homolog of GRAS transcription factor Nodulation Signaling Pathway1（NSP1）,was identified as an interactor of GmSK2-8.Soybean contains two NSP1proteins,and the other is GmNSP1b.A variety of in vitro and in vivo experiments verified that GmSK2-8 can interact with both GmNSP1a and GmNSP1b.Histochemical analyses showed that the spatial expression patterns of GmSK2-8 and GmNSP1a/1b overlapped in the root hairs and nodule primordia in the early stages of nodule development.3.Analyses of transgenic plants that overexpression,knock-down or genome-editing of two GmNSP1 genes showed that the two GmNSP1 genes are required for rhizobial infection,the formation of nodule primordium,and the expression of symbiotic responsive genes.These results indicate that two GmNSP1 genes play key roles in the process of establishing symbiotic associations between soybean and rhizobium.4.Biochemical analyses showed that GmSK2-8 strongly phosphorylates GmNSP1,and inhibits the DNA binding activity of GmNSP1a by phosphorylation.Further analyses revealed that five amino acid residues in the LHRI domain of GmNSP1a are the key phosphorylation sites.GmSK2-8 weakens the phosphorylation of the GmNSP1a^L5A,and cannot inhibit its DNA binding activity.These results indicate that GmSK2-8phosphorylates the LHRI domain of GmNSP1a,thereby inhibiting its DNA binding activity.5.Through the responses of GmSK2-8 overexpression and RNAi transgenic plants to salt stress,it is concluded that GmSK2-8 mediates the inhibitory effect of salt stress on nodule development.Furthermore,Chromatin Immunoprecipitation（ChIP）assays demonstrated that salt stress inhibited the DNA binding activity of GmNSP1a on the promoters of symbiotic responsive genes（such as Gm ERN1a）in vivo.Salt stress also induced the phosphorylation of GmNSP1a,and the mutant form GmNSP1a^L5A showed a weakened response to salt stress.These results indicate that GmSK2-8-GmNSP1 module plays an important role in salt stress inhibiting nodule development.In summary,this study found GmSK2-8 regulates the DNA binding activity of transcription factor GmNSP1 by phosphorylation,therefore mediates salt-inhibited soybean–rhizobium interactions and nodulation.This study provides important targets for improving symbiotic nitrogen fixation under environmental stress conditions in soybean and possibly other legumes,and is of great significance for developing green sustainable agriculture.