Study On The Role Of Hydrogen Sulfide Signaling Molecules In The Symbiotic Nitrogen Fixation System Of Robinia Pseudoacacia

Robinia pseudoacacia is a woody legume that is a pioneer species of ecological greening,but nitrogen deficiency is the main limiting factor in vegetation restoration.Therefore,how to mobilize the nitrogen fixation potential of legumes and rhizobium symbiotic nitrogen fixation system is of great significance.As the new gas signal molecule,Hydrogen Sulfide?H₂S?plays an important role in regulating plant physiological functions,and can regulate plants to adapt to high salt stress,ion toxicity,drought stress and temperature stress,and promote plant growth and development.As a gas signal molecule,Nitric Oxide?NO?can affect the formation and senescence of leguminous nodules,and can regulate the expression of key genes in the nodulation process and affect the nitrogen fixation ability of legumes.Does H₂S also involve the regulation of nodule formation and symbiotic nitrogen fixation in legumes?If so,what role does H₂S play in symbiosis?This study intends to investigate the role of H₂S in the symbiosis system of Rhizobium from plant physiology,nodule structure,symbiotic gene expression and transcriptome analysis after inoculated Rhizobium in Robinia pseudoacacia by plant experiments.The main findings are as follows:?1?Treatment with H₂S donor NaHS promoted the phenotypic growth of Robinia pseudoacacia plants and improved physiological indexes such as root length,plant height,dry weight and chlorophyll content of leaves increased significantly.At the same time,H₂S can also affect the infection event,promote the formation and development of the root nodule of the Robinia pseudoacacia,and thus increase the nitrogenase activity of the nodule.?2?The microstructure of the nodule is observe by paraffin section and transmission electron microscopy.It was suggest that the H₂S donor NaHS could promote the development of nodule during the formation of nodule.In the mature stage,the nitrogen-fixation area in the nodules increased,and the infected cells arranged more closely and in quantity.Moreover,the cell shape is more round and full,more energetic;ultrastructural observation of the bacteria also have the same result.?3?Quantitative real-time PCR data of symbiotic related genes indicated that H₂S donor NaHS treatment resulted in calcium ion and calmodulin-dependent protein kinase gene?CCaMK?,calcium channel protein?Castor?,intranuclear coiled-coil protein gene?CYCLOP?.The expression levels of E3 ubiquitin ligase gene?Cerberus?were up-regulated,symbiotic receptor kinase gene?SYMRK?and histidine protein kinase?LHK?were up-regulation during the maturity of the nodule.NaHS treatment resulted in the symbiotic related gene nodC,nifH and other expression levels are up-regulate in the nodule of Robinia pseudoacacia.?4?Through transcriptome analysis,it was suggest that H₂S treatment significantly encodedtheexpressionofmembraneprotein,peroxidase,oxidoreductase,phosphotransferase and histidine protein kinase,and the metabolism of saccharides such as sucrose and starch.Synthetic,nitrogen metabolism,ATP binding,synthesis of secondary metabolites such as alkaloids,plant and microbial interactions,transmembrane signal transduction,metabolism of various amino acids,and glutathione metabolism are significantly up-regulation.Indicated that H₂S It can provide more energy through sugar metabolism to meet the consumption of symbiotic nitrogen fixation process,improve nitrogen fixation capacity,participate in antioxidant defense reaction,and promote growth of Robinia pseudoacacia.In addition,there are significant up-regulations of transcription factors,transporters,spliceosome,and RNA transport.The regulation of symbiotic nitrogen fixation by H₂S is a complex and elaborate process.The results showed that H₂S could promote the development of nodule by up-regulation the expression of symbiotic genes and many metabolic pathway genes in plants and rhizobium.Moreover,the chlorophyll content increases,promotes photosynthesis,increases biomass accumulation,and ultimately promotes the growth of Robinia pseudoacacia.