The Molecular Mechanism Of GmSNE3 Ubiquitin Ligase Regulating Soybean Nodulation In Response To Halosulfuron-Methyl Stress

As a leguminous plant,soybean can establish symbiotic relationships with rhizobia in the soil to form symbiotic nodules,and convert nitrogen in the air into ammonia through nitrogen fixation,providing sufficient nitrogen for soybean growth.The symbiotic nodulation of soybean and rhizobium is an extremely complex process,involving a large number of gene regulation.However,this process is easily affected by various abiotic stresses,leading to root nodule failure.Halosulfuron-methyl(HSM)is a sulfonylurea herbicide,which is widely used to control broad-leaved weeds and sedge weeds in corn,rice,wheat,sugarcane and other grass crops.Previous studies have found that HSM has a long residual period,and its residues in the soil can significantly inhibit symbiotic nodulation between soybean and rhizobium.However,the molecular mechanism underlying this phenomenon is still unclear.It has been found that E3 ubiquitin ligase plays a key role in abiotic stress response and symbiotic nodulation of leguminous plants,but it is unclear whether E3 ubiquitin ligase participates in the inhibition of soybean nodulation by HSM stress.This study revealed the molecular mechanism of E3 ubiquitin ligase regulating soybean nodulation under HSM stress through omics,physiological,biochemical,and molecular biological methods.The main findings are as follows:1.HSM(0.01,0.05 and 0.5 mg/L)stress significantly reduced the content of chlorophyll and carotenoid in soybean leaves,seriously affected the chlorophyll a fluorescence,significantly increased the content of glutathione,hydrogen peroxide and malondialdehyde and the activity of antioxidant enzymes.In addition,HSM significantly inhibited the activities of major enzymes involved in the TCA cycle in soybean leaves,includingα-Ketoglutarate dehydrogenase,isocitrate dehydrogenase,aconitase,malate dehydrogenase and succinate dehydrogenase.The proteomics results showed that the contents of various differentially expressed proteins involved in chlorophyll synthesis,photosynthesis system and TCA cycle in soybean leaves were significantly reduced under HSM stress.In addition,metabolomics results showed that the metabolites involved in the TCA cycle were also significantly reduced under HSM stress.These results showed that HSM stress had toxic effects on soybean mainly through destroying photosynthesis system,inhibiting chlorophyll synthesis,interrupting TCA cycle and causing oxidative damage.2.Under 0.01 mg/L HSM stress,the number of soybean nodules significantly decreased by 38%.Further,transcriptomic analysis was conducted on the symbiotic nodulation process between soybean and rhizobium under 0.01 mg/L HSM stress.The results showed that the differentially expressed genes involved in flavonoid synthesis,phytohormone biosynthesis and phytohormone signal transduction were all significantly down-regulated.This is consistent with the reduction of flavonoid content and plant hormone content during symbiosis under HSM stress in this study.After 7 days of HSM stress at 0.01 mg/L,the contents of IAA,ABA,ZR,JA,and GA3 in the soybean root inoculated with rhizobium significantly decreased by 56%,59%,74%,57%,and 55%,respectively,while the content of flavonoids decreased by 42%.In addition,under HSM stress,the expression of multiple genes involved in the symbiotic nodulation process is inhibited.In particular,the expression levels of tumor factor receptor(NFR5A)and symbiotic signaling pathway gene(NSP1)were significantly reduced by 9.0 and 7.9 times,respectively.These results suggest that HSM may inhibit soybean nodulation by inhibiting the synthesis of root flavonoids during symbiosis,destroying the balance of plant endogenous hormones during symbiosis,destroying symbiotic signal transduction and blocking the transmission of hormone signals during symbiosis.3.In order to further reveal the molecular mechanism of HSM inhibiting soybean nodulation.The interaction proteins of the key nodulation protein GmNSP1 were screened by yeast two-hybrid experiment,and 17 interaction proteins were screened,including E3 ubiquitin ligase GmSNE3 protein(KAH1261024.1).Yeast retransformation validation test,bimolecular fluorescence complementation test(BIFC)and pull-down test showed that GmSNE3 could interact with GmNSP1 in vitro and in vivo.In addition,the expression level of GmSNE3 in nodules is significantly lower than that in stems,leaves and roots,and it remains at a low expression level during the symbiosis of soybean and rhizobium.These results suggest that GmSNE3 may play a negative role in the formation of soybean nodules.4.After 4 and 7 days of HSM stress,the expression of GmSNE3 in soybean roots was significantly increased by 3.3 and 2.2 times,and overexpression of GmSNE3 significantly reduced the number of nodules in soybean plants by 56.1%.In addition,qRT-PCR analysis showed that the important nodulation genes(NIN,ERN1,ENOD40)downstream of GmNSP1 in the roots of GmSNE3 overexpression plants were significantly down-regulated.Similarly,under HSM stress,GmNSP1 downstream important nodulation genes(NIN,ERN1,ENOD40)expression level also showed a significant decrease.These results showed that the inhibition of soybean nodulation by HSM stress was caused by the interaction between GmSEN3 and GmNSP1 to inhibit the expression of GmNSP1 downstream genes.5.Physiological and biochemical analysis showed that GmSNE3 had E3 ubiquitin ligase activity.Further research found that GmSNE3 can mediate the ubiquitination degradation of GmNSP1 protein,and HSM stress can promote the ubiquitin degradation of GmNSP1 protein.These results reveal the mechanism of HSM inhibiting soybean nodulation: HSM stress can enhance the degradation of GmNSP1 by inducing GmSNE3,thus making GmNSP1 unable to regulate its downstream important nodulation genes,and ultimately leading to the reduction of nodulation.To sum up,this study identified an E3 ubiquitin ligase(GmSNE3)that interacts with the key protein of symbiotic nodulation,GmNSP1,and revealed its regulatory role in the inhibition of soybean nodulation under HSM stress.The study founed that HSM promotes the ubiquitination degradation of GmNSP1 by inducing GmSNE3,thereby inhibiting the regulation of GmNSP1 on downstream target genes,and ultimately leading to the reduction of nodulation.These results provide a new understanding of the molecular mechanism of halosulfuron-methyl stress inhibiting symbiotic nodulation,provide a theoretical basis for the environmental risk assessment and use of halosulfuron-methyl herbicide,provide genetic resources for the development of halosulfuron-methyl tolerant soybeans,and provide important target genes and theoretical basis for cultivating efficient symbiotic nodulation soybeans under residual pollution of halosulfuron-methyl,It is of great significance for improving soybean yield and quality,as well as developing green and sustainable agriculture.