Mechanism Of S-nitrosylation Of ACO Homolog 4 Improves Salt Tolerance In Tomato

According to FAO statistics,there are more than 1 billion hectares of salinized land in the world,accounting for about 24%of the world’s total arable land.The total area of saline-alkali land in China is about 99 million hectares,ranking third in the world.These saline-alkali lands have not been effectively utilized,and they are huge potential resources for agricultural development.In addition to natural saline-alkali soil,there are 36 million hectares of secondary salinized land in my country.How to release the production potential of saline-alkali land is an international frontier and key scientific issue.Tomato（Solanum lycopersicum L.）is one of the important vegetable crops widely cultivated in the world.It originated in the Andes region of South America.Wild tomato can grow in the soil environment with high salinity.Although the cultivated tomato lost part of its salt tolerance genes during the domestication process,it still has good potential for genetic improvement of salt tolerance.The identification of salt-tolerant genes and signaling pathways,and the analysis of biochemical functions and molecular mechanisms are important biological foundations for the creation of new tomato salt-tolerant germplasm.NO and ethylene are important signaling molecules in plant stress response,regulating multiple abiotic stress resistance including salt stress.NO and ethylene signals are mutually induced and antagonistic,which is determined by plant tissues and organs,developmental stage and living environment.At present,the production,interaction and function of NO and ethylene signals in plants under salt stress remain unclear,and direct molecular evidence for their interaction is lacking.In this study,the production of NO and ethylene signals in tomato under salt stress was taken as the starting point,and the following conclusions were reached:1.Using 100m M Na Cl as salt stress,NO fluorescence detection and ethylene content determination were carried out on the root tips of tomato plants.It was found that salt stress could induce tomato roots to produce NO and ethylene signals,and ethylene production was partly dependent on NO.2.Wild-type WT tomato,GSNOR transgenic tomato,ethylene synthesis mutant rin and ethylene signal mutant nor were used as test materials to study the change of ethylene content under salt stress by adding exogenous reagents such as NO donor,ethylene donor,ethylene synthesis inhibitor and ethylene antagonist.It was found that GSNOR could negatively regulate ethylene synthesis.At the same time,through the determination of three indexes of salt tolerance,it was found that NO and ethylene signals were beneficial to improve the salt resistance of tomato,and NO as the upstream signal of ethylene function.3.Through the analysis of the activity of ethylene synthesis-related enzymes（ACS and ACO）,it was found that salt stress could induce the increase of enzyme activity,but the exogenous addition of NO scavenger only weakened the ACO activity.We took GSNOR transgenic tomato as the test material,combined with exogenous drug（exogenous NO donor）scientific test method to further verify the ACO activity changes,and confirmed that the key target of salt-induced NO signal regulation of ethylene synthesis is ACC oxidase（ACO）.4.By referring to the tomato S-nitrosodination proteomics previously obtained by our research group,ACOh4 was selected as the target gene,His-ACOh4 recombinant protein was constructed,and mass spectroscopic analysis was conducted.Cys172 was found to be a potential S-nitrosodination modification site in ACOh4.By constructing His-ACOh4,His-ACOh4^C172S and His-ACOh4^C172W recombinant proteins,ACO activity and ethylene content were determined,which further verified that Cys172 could be modified by S-nitroso.At the same time,using a combination of protoplast transfection and in vitro and in vivo biotin switching method,it was found that salt-induced NO can improve the enzyme activity of ACOh4 by S-nitrososylation of Cys172 and promote ethylene synthesis.5.Through the combination of q RT-PCR analysis and western blot analysis,it was found that GSNOR negatively regulated the expression of ACOh4 in tomato roots under salt stress,and the addition of exogenously added NO scavengers could inhibit the expression of ACOh4induced by salt stress,indicating that salt-induced NO could also activate the expression of ACOh4 gene.6.VIGs technology was used to construct ACOh4 inhibitory strain,and the gene expression and ethylene content of Ac OH4 were significantly reduced.By comparing with the salt tolerance analysis of WT,it was found that inhibiting the expression of ACOh4 could significantly reduce the salt tolerance,increase the accumulation of Na⁺,and reduce the ratio of K⁺-Na⁺,while the addition of NO only had a weak resistance induction effect on the transgenic lines.Further analysis showed that the efflux capacity of Na⁺and H⁺of the inhibitory strains was reduced,and the expression of key genes of SOS signaling pathway was weakened.In conclusion,inhibition of ACOh4 expression can significantly reduce ethylene synthesis and salt resistance in tomato roots,which is related to Na⁺and H⁺effluence in roots and K⁺-Na⁺balance in plants regulated by ACOh4.In conclusion,this study for the first time elucidates the key enzyme（ACOh4）that induces ethylene synthesis under salt stress and its regulatory mechanism,and analyzes the interaction relationship and molecular physiological functions of NO and ethylene in regulating tomato salt resistance.The research results can provide theoretical basis for genetic improvement of salt-tolerant tomato varieties and research and development of salt-tolerant cultivation technology.