Molecular Mechanism Of Melatonin Synthetase OsASMT1 Regulating Salt Stress Tolerance In Rice

Salt stress is an important abiotic stress that seriously threatens plant growth and agricultural production.Melatonin is a plant hormone and antioxidant that plays an important role in plant growth,development,and stress response.However,the role and molecular mechanism of endogenous melatonin in salt tolerance in rice are still elusive.This study constructed transgenic plants of OsASMT1,a key gene in the melatonin synthesis pathway in rice,and analyzed the function and molecular mechanism of endogenous melatonin in salt tolerance in rice seedlings.The main results are as follows:1.By measuring the melatonin content and related gene expression levels in wild-type rice seedlings under salt stress,it was found that the endogenous melatonin content in rice seedlings significantly increased compared to normal growth conditions after salt stress,and the expression of OsASMT1 was significantly induced by salt stress,indicating that OsASMT1 may be involved in the response of rice seedlings to salt stress.2.By analyzing the expression pattern of OsASMT1,it was found that OsASMT1 was expressed in various tissues of rice and may be involved in the regulation of rice growth,development,stress,and hormone response.Subcellular localization analysis showed that OsASMTl protein was localized in the nucleus and cytoplasm.3.OsASMT1 CRISPR-Cas9 mutant lines and overexpression lines were created using Agrobacterium-mediated genetic transformation.The two independent OsASMTl-Cas9 mutant lines caused protein frameshift and premature termination mutations,and the expression level of OsASMT1 in the two independent overexpression lines was significantly higher than that of wild type.4.Through prokaryotic protein expression and in vitro enzyme activity detection,as well as measuring the melatonin content in transgenic plants,it was found that OsASMT1 had melatonin synthesis enzyme activity both in vitro and in vivo.5.Salt stress treatment and phenotype analysis of OsASMT1 transgenic lines showed that the survival rate of OsASMT1 overexpression lines was significantly higher than that of the wild type,while the survival rate of OsASMT1-Cas9 mutant lines was significantly lower than that of the wild type.Exogenous melatonin treatment could rescue the salt-sensitive phenotype of OsASMT1-Cas9 mutant lines,indicating that OsASMT1 positively regulates rice tolerance to salt stress through melatonin synthesis.6.Physiological index analysis of OsASMT1 transgenic lines showed that melatonin may enhance rice tolerance to salt stress through promoting the expression of proline synthesis genes and the accumulation of proline,promoting the expression of Na+ transport genes and Na+efflux,increasing the expression level of antioxidant enzyme genes,and enhancing the activity of antioxidant enzymes.7.Detection of agronomic trait of OsASMT1 transgenic lines showed that the 1000-grain weight and yield per plant of OsASMT1 overexpression lines were significantly higher than those of wild type,while the tiller number,1000-grain weight,and yield per plant of OsASMT1-Cas9 mutant lines were significantly lower than those of wild type.These results indicate that OsASMT1 may promote melatonin synthesis and increase the 1000-grain weight of rice,thus promoting rice grain yield.In summary,the results of this study revealed that salt stress can induce the expression of the melatonin synthesis enzyme OsASMT1 and promote endogenous melatonin synthesis in rice.Melatonin enhances rice tolerance to salt stress through increasing proline synthesis,activating the antioxidant system,and enhancing Na+efflux.In addition,OsASMT1 positively regulates rice grain yield.Therefore,OsASMT1 can be used as a target gene to simultaneously improve rice yield and salt tolerance,providing a reference for breeding new high-yield and salt-tolerant rice varieties.