Regulation And Mechanism Of Hydrogen Sulfide Signaling Molecule In Soybean-rhizobia Symbiotic Nitrogen Fixation System

Legume plants are not only diverse but also widely distributed all around the world.Most legume plants can form symbiotic mutualsm relationship with rhizobia in soil and fulfill the majority of their demand for nitrogen nutrition through symbiotic nitrogen fixation.Sinorhizobium fredii Q8 strain was isolated from the soybean nodule in farmland of Yangling,Shaanxi province,and has excellent nodulation and nitrogen fixation performance with Zhonghuang 13 variety of soybean?Glycine max?which is widely cultivated in northwest China.Using S.fredii Q8 strain and Zhonghuang 13 variety of soybean as the main research materials,this paper studied the role of H₂S signaling molecule and its related enzymes in the nodulation and nitrogen fixation processeses in soybean plants through transcriptome,gene knockout,complementary,fluorescence probe observation and other methods.Firstly,NaHS solution as an exogenous H₂S donor was used to treat soybean plants.The content of H₂S in plant roots and root nodules increased significantly after treated with NaHS.Meanwhile,the growth of soybean plants inoculated with S.fredii Q8 strain showed a significant improvement after treated with NaHS,with an increase in root length,plant height,dry weight of shoots and roots.The measurement of photosynthetic rate and chlorophyll fluorescence parameters showed that chlorophyll content and net photosynthetic rate in NaHS treated plant leaves were higher than those in the control group.In terms of symbiosis,the number of nodules in soybean plants treated with exogenous H₂S was also significantly higher than that in control plants.Moreover,exogenous H₂S treatment also promoted the nitrogen fixation ability of soybean root nodules.The results of nitrogenase activity determination showed that the nitrogenase activity in the root nodules of NaHS treated soybean plants was significantly higher than that of nontreated soybean plants.qRT-PCR analysis showed that exogenous H₂S treatment could promote the expression of symbiotic related genes such as rhizobial nodA,nodC,nodD,and GmENOD40,GmERN,GmNSP2b,GmNIN1a/2a/2b in soybean roots.In addition,the expressions of genes related to nitrogen metabolism,such as GmGOGAT,GmAS,GmNiR,GmSAT1 and GmLb,were also up-regulated in the roots of plants treated with NaHS.These results indicate that exogenous H₂S treatment can promote the symbiosis and nodulation between S.fredii Q8 and soybean plants,and promote the nitrogen fixation ability of soybean root nodules.Due to the improvement of nitrogen fixation,the nitrogen supply of soybean plants is relatively sufficient,which in return promotes the synthesis of chlorophyll and the progress of photosynthesis,thus improving the carbon assimilation capacity of soybean plants,and eventually promotes the growth of soybean plants.Secondly,the H₂S specific fluorescent probe showed that a large amount of endogenous H₂S was generated in the nitrogen fixation area of soybean root nodules.After the CSE gene of S.fredii Q8 encoding cystathionine?-lyase?CSE?was knocked out,the production of endogenous H₂S of the?CSE mutant strain was significantly reduced,and the production of H₂S in the root nodules formed by?CSE strain was also significantly inhibited.In the soybean root nodules inoculated with the?CSE strain,not only the content of endogenous H₂S was decreased,but also the nitrogenase activity was significantly lower than the root nodules formed by the wild-type WT strain and the complementary strain Cp?CSE.Transmission electron microscopy showed that although the external shape and size of the?CSE root nodules were not significantly different from that of the WT strain nodules,however the bacteroids and symbiosome membranes inside the root nodules in the nitrogen-fixation zone had similar changes to those under oxidative stress.Moreover,the deficiency of endogenous H₂S also led to the accumulation of hydrogen peroxide in root nodules and the occurrence of oxidative damage.Meanwhile,the decrease of H₂S content also stimulated the expression of genes encoding antioxidant enzymes and increased the activity of these enzymes.These results indicated that the endogenous H₂S generated in soybean root nodules could maintain the activity of nitrogenase through its antioxidant effect and promote the symbiotic nitrogen fixation of soybean.Lastly,the 3-mercaptopyruvate sulfertransferase?3MST?of rhizobia can affect the infection ability of S.fredii Q8 to 13 soybean variety.After knocking out the 3MST coding gene sseA,although the number of root nodules formed by the symbiosis between the?3MST strain and the soybean Zhonghuang 13 plant was significantly increased,they were all ineffective root nodules with extremely low leghemoglobin content and almost no nitrogen fixation ability.The microscopic structure and ultrastructure of root nodules were observed by paraffin section and transmission electron microscope.It was found that the infection and colonization ability of the?3MST strain in soybean nodules were destroyed,the number of bacteroid was very small and the morphology was obviously deformed.Meanwhile,the absence of 3MST also inhibited the secretion of NopX,a type III secretion system effector protein associated with nodulation process in S.fredii Q8.Transcriptome analysis showed that there were more differentially expressed genes?DEG?within 1 h after inoculation of the?3MST strain?KEGG enrichment analysis showed that the most significant effect of?3MST inoculation was in the plant-pathogen interaction pathway,with120 DEGs of total 325 genes.According to the qRT-PCR experiment,1h after inoculation of WT strain,the expression of pathogen defence-related genes in the root of soybean plant was inhibited to a significantly lower level,while in the soybean inoculated with?3MST strain,the expression of these genes was not inhibited,and a few defense genes were up-regulated on the contrary.