Physiological And Molecular Mechanisms Of Exogenous Melatonin Promoting Seed Germination Of Cotton Under Salt Stress

Cotton is an important cash crop in China,and seed germination is an important stage in its growth process,which is very vulnerable to biotic and abiotic stress.Salt damage is one of the important factors affecting cotton yield and quality.Melatonin(N-acetyl-5-methoxytryptamine)is a small-molecule indole substances widely existed in animals and plants.It has two kinds of characteristics,namely,hydrophilic and libidophile.It is easy to enter the inside of seeds and has remarkable antioxidant function and can improve seed vigor and germination rate under stress.At present,there are many studies on the regulation effect of melatonin on seed germination of vegetables and fruit trees under stress,but few studies on the regulation effect of melatonin on seed germination of cotton.Therefore,the physiological regulating mechanism of melatonin on cotton seed germination was studied by measuring seed germination index,antioxidant enzyme system,osmotic regulatory substance,cell membrane permeable substance,melatonin and hormone content,metabolic enzyme activity with 'GXM9' as material under salt stress(150 mM NaCl).Combined with metabolomics and transcriptome analysis,the molecular regulating mechanism of melatonin on cotton seed germination under salt stress was explored.It provides a basis for the development and utilization of melatonin and the regulation of cotton cultivation.The main research results are as follows:1.Exogenous melatonin effectively alleviated the inhibitory effect of salt stress on cotton seed germination.Salt stress significantly reduced the germination rate,germination potential and radical length of cotton seeds,and fresh weight,dry weight after seed germination also decreased significantly.The seed germination rate,radicle length and biomass varied after 24 h immersion with different melatonin concentration.Low concentration of melatonin(10,20 ?M)promoted cotton seed germination,while high concentration of melatonin(50,100 ?M)did not significantly promote the germination of cotton seeds,or even inhibited the germination.Among them,20 ?M melatonin had the most significant effect on cotton seed germination.2.Exogenous melatonin increased antioxidant enzyme activity and osmotic regulatory substance of cotton seed germination under salt stress.Salt stress reduced the activities of SOD,POD and CAT,and decreased the content of proline,soluble sugar and protein,and increased the contents of Na+ and Cl-,the contents of electrolyte permeability,H2O2 and MDA increased during seed germination.After applying different concentrations of melatonin,the SOD,POD,CAT activity of cotton seeds increased first and then decreased,and the contents of proline,soluble sugar and soluble protein increased;the electrolyte permeability and H2O2,MDA content also decreased continuously.Among them,the regulation effect of 20 ?M melatonin was the most obvious.3.Exogenous melatonin increased amylase activity and the content of melatonin and ABA in cotton seed germination under salt stress.The content of?-amylase and ?-galactosidase decreased significantly under salt stress,starch content increased significantly,ABA content increased,GA and melatonin content decreased.After the application of melatonin,the contents of ?-amylase and ?-galactosidase increased significantly,and the starch content decreased significantly.ABA content decreased,GA content increased,and melatonin content also increased significantly.At the same time,the expression levels of ABA and GA signaling genes GhABF2,GhGID1B and GhGID1C increased significantly,while the expression levels of GhDPBF2 decreased.4.Exogenous melatonin regulates the metabolic pathway of cotton seed germination under salt stress.Through metabolomic analysis,it was found that S had 163 different metabolites compared with CK.Compared with S,SM has 46 different metabolites,which are mainly enriched in tryptophan metabolic pathway,and some of them are also enriched in lysine biosynthesis,phosphate lipid metabolism,glycerol phospholipid metabolism and purine metabolism.KEGG enrichment analysis showed that exogenous melatonin increased the content of amino acids and lipids and provided energy for seed germination.5.Exogenous melatonin regulated the expression of transcriptional genes in cotton seed germination under salt stress.Based on transcriptome analysis,there were 6817 differentially expressed genes in S and CK at 6 h after seed germination,and 3379 differentially expressed genes in SM and S.At 12 h after seed germination,there were 15998 differentially expressed genes in S and CK,and 2815 differentially expressed genes in SM and S.After 24 h of seed germination,there were 19820 differentially expressed genes in S and CK.and 5076 differentially expressed genes in SM and S.Different genes of seed germination(S and CK;SM and S)are mainly expressed in above tones.The enrichment analysis of the differentially expressed genes by KEGG showed that the differentially expressed genes treated by S and CK were mainly enriched in the MAPK signaling pathway and the tricarboxylic acid cycle,while the differentially expressed genes treated by SM and S were mainly enriched in the pathways of plant hormone signal transduction,the tricarboxylic acid cycle and fatty acid degradation.In summary,exogenous melatonin increased the antioxidant enzyme activity of cotton seeds,increased the accumulation of osmotic regulatory substances,and enhanced the permeability of cell membrane.Exogenous melatonin decreased ABA content and increased GA content under salt stress.Exogenous melatonin promotes seed germination by regulating hormone signal transduction,energy cycle and hormone-related gene expression.