Mechanism Of Acetylation Regulation And Symbiotic Salt Tolerance In Rhizobium Of Coastal Salt-tolerant Plant Sesbania Rostrata

Soil salinization has become the main factor restricting the security of food production and the improvement of land use efficiency.The advantageous water-heat conditions of coastal saline land make it a key area in the development and use of saline land,and are of great significance for sustainable agriculture development.The cultivation of salt-tolerant crops has become a relatively effective measure to combat soil salinity,and Sesbania rostrata has become a pioneering plant for improving coastal saline soils due to its salt tolerance,drought tolerance,waterlogging tolerance,high yield and adaptation to nutrient-poor environments.Azorhizobium caulinodans ORS571 is able to form nodules on the stems or roots of S.rostrata,forming a rhizobiaS.rostrata symbiosis that can be used as a classical remediation measure for saline soils.Protein acetylation can regulate protein function,thus rapidly altering physiological metabolism of bacteria in response to environmental changes.The physiological regulation of bacteria by protein acetylation involves central and secondary metabolism,transcription,chemotaxis,stress,and virulence,which are closely related to the establishment of A.caulinodans-S.rostrata symbiosis for salt tolerance.The mechanism of A.caulinodans-S.rostrata symbiosis for salt tolerance need to be further explored.In this study,the physiological regulation mechanism of A.caulinodans in symbiosis with S.rostrata was analyzed by protein acetylation omics.The methods of enhancing the salt stress tolerance of S.rostrata were investigated by using potting experiments of vermiculite,and the related salt-tolerance mechanisms were revealed.The main results of the study are as follows:(1)A systematic analysis of the acetylation-regulating enzymes in A.caulinodans ORS571 was carried out by using bioinformatics methods.There were four deacetylases in A.caulinodans ORS571.AZC＿0414,AZC＿2049 and AZC＿3413,AZC＿1935 belonged to two types of protein superfamily,class II and class III(SIR2family),all of which have conserved domain and functional sites.Among them,AZC＿1935 was the homologous protein of Cob B in E.coli.The expression levels of the four deacetylases in the stable phase were slightly higher than that in the logarithmic growth phase,with little overall difference.The expression level of AZC＿0414 was the highest,being seven times higher than the other three deacetylases.In addition,ORS571 encoded more than 20 acetyltransferases.The structural domain of the protein encoded by AZC＿4657 was the most conserved and was homologous to Yfi Q in E.coli.The gene expression level of AZC＿4657 was much higher than the other selected acetyltransferases.(2)Acetylation-regulated enzymes such as acetyltransferases and deacetylases are involved in a series of physiological processes such as morphology,chemotaxis,stress,and kin recognition.Deacetylase deletion mutants Δ1935,Δ3413,Δ0414,Δ2049,4Δand acetyltransferase mutant Δ4657 were obtained by gene knockout technology.The results showed that AZC＿1935,AZC＿0414 and AZC＿2049 in ORS571 exerted deacetylase activity by qualitative and quantitative enzyme activity assays.The growth of each mutant was not significantly different compared to the wild type,but overexpression of AZC＿1935 resulted in an 18.6% increase in the percentage of elongated cells.The survival rates of mutant strain Δ1935 under heat and oxidative stress were 17.4% and 22.8%,respectively,which were significantly lower than those of wild type 79.5% and 73.7%.The chemotaxis ability of the mutant Δ0414 was significantly increased,and produced clear colony boundaries with the wild type and other mutants during swimming.The cell growth under salt stress of Δ0414、Δ3413 、Δ2049 and 4Δ were weaker than that of the wild type.The survival rate of acetyltransferase mutant Δ4657 under heat and oxidative stress was 22.4% and 15.2%,respectively,significantly lower than wild type.(3)The global acetylated protein dataset of A.caulinodans ORS571 was obtained by using antibody enrichment and chromatography-tandem mass spectrometry.There are 2302 acetylation sites from 982 proteins,accounting for 20.8% of the total proteins.Acetylated proteins identified in ORS571 were widely distributed in the biosynthesis of cofactors and proteins,translation,carbohydrate metabolism,signal transduction,membrane transport,quorum sensing,etc.Some of the identified acetylated proteins were also involved in cellular processes characteristic of rhizobia,such as resistance,symbiosis,nitrogen fixation,salt tolerance and chemotaxis.Analysis of the acetylated motifs showed the preferences for the amino acid residues around acetylated lysines.The core chemotaxis protein Che Y1 was identified as an acetylated protein,and a mutation of the acetylated site of Che Y1 significantly impaired the strain’s motility.(4)Inoculation with A.caulinodans ORS571 could improve salt tolerance of S.rostrata.Effects of inoculation with A.caulinodans ORS571 and exogenous addition of γ-aminobutyric acid on the growth and development of S.rostrata under salt stress were investigated using potting experiments of vermiculite.Inoculation with ORS571 under 100 m M salt stress significantly increased the height,dry weight aboveground,dry weight underground and chlorophyll content of S.rostrata by 1.91,3.87,3.91,and2.68 times,respectively.Under 200 m M salt stress,inoculation with ORS571 significantly increased the height,dry weight aboveground,dry weight underground and chlorophyll content of S.rostrata by 1.57,3.31,2.25,and 3.15 times,respectively.Inoculation with rhizobia increased the antioxidant enzyme activity and proline content,and reduced malondialdehyde levels in the leaves.Under 100 m M salt stress,γ-aminobutyric acid treatment increased the height,dry weight aboveground,dry weight underground and chlorophyll content by 1.44,1.81,1.46 and 2.35 times,respectively.Under 200 m M salt stress,γ-aminobutyric acid treatment increased the height,dry weight aboveground,dry weight underground and chlorophyll content by 1.76,2.17,1.74 and 3.47 times,respectively.γ-aminobutyric acid treatment also increased the antioxidant enzyme activity and reduced the malondialdehyde level in the leaves of S.rostrata.In addition,exogenous addition of γ-aminobutyric acid could increase the catalase activity and chlorophyll content of the ORS571-S.rostrata symbiotic system.In summary,the mechanism of acetylation regulation in ORS571 was systematically investigated in this study,and the different physiological processes involved in acetylation were confirmed.A large number of acetylated protein substrates were identified and analyzed by acetylation omics,and methods to improve salt tolerance in ORS571 were investigated,providing a theoretical basis for exploring the physiological mechanisms of salt tolerance in rhizobia-S.rostrata symbiosis.