Molecular Mechanism Of Enhancing Drought Resistance Of White Clover Through Melatonin Biosynthesis Regulated By TrMAT1

After the 19 th National Congress of the Communist Party of China,the state put forward the concept of"mountains,rivers,forests,fields,lakes and grasses.",and researchers’attention to"grass and animal husbandry"related to"grass"has also reached a new height.White clover is a perennial herb.It is widely used in grassland animal husbandry,landscaping,ecological restoration and other fields because of its good palatability,rich nutrition,high digestibility,long green period and strong expansion ability.It is a’dominant’legume cultivated in Southwest China and is also widely planted in some counties and cities in the three northeastern provinces.However,its root distribution is shallow and vulnerable to drought.Melatonin promotes plant growth,directly or indirectly regulates the production and removal of reactive oxygen species,thereby affecting the metabolic process of plants in response to stress and improving their stress resistance.However,there is a lack of research on the effect of melatonin on the growth of white clover under drought stress.Therefore,in this study,drought-sensitive white clover’Latino’was selected as plant material to study the effect of exogenous melatonin on the growth of white clover under drought stress,and the drought regulation mechanism of its upstream gene through melatonin was analyzed by transgenic technology.The main results and conclusions are as follows:（1）The content of melatonin in white clover without exogenous melatonin treatment increased by 30.35%at 7 d of drought compared with that at 0 d.At 14 d of drought,the melatonin content in the plant was 31.05%lower than that at 0 d.The results showed that in the middle of drought,melatonin responded to drought stress and the content increased,which was a regulator of drought stress response.（2）After treatment with 40μM,80μM,120μM,160μM and 200μM melatonin,the endogenous melatonin content of white clover increased significantly with the increase of melatonin concentration,and reached a maximum of 55.3 ng/g FW at160μM.However,there was no significant change in the endogenous melatonin content of white clover from 120μM to 200μM.（3）Melatonin treatment alleviated the damage of drought to white clover.The results were as follows:exogenous MT enhanced the synthesis of proline and the accumulation of soluble sugar in white clover,reduced osmotic potential,and regulated the osmotic adjustment ability of white clover in drought environment,thereby increasing the relative water content of plants.In the protection of photosynthetic pigments,exogenous MT delayed the decrease of chlorophyll a,chlorophyll b and total chlorophyll content.In terms of antioxidant,after exogenous MT treatment,the activities of SOD,POD and CAT in leaves increased,and the contents of O₂^-and MDA decreased significantly,indicating that the antioxidant effect of white clover leaves was significantly enhanced,and the senescence of leaves was delayed.At the same time,the expression of senescence-related genes SAG101 and SAG102 decreased significantly,and the relative growth rate of plants increased significantly.During the increase of melatonin concentration from 40μM to 120μM,the mitigation effect of melatonin treatment increased significantly,while the mitigation effect on drought damage did not change significantly from 120μM to 200μM.（4）Compared with wild type,the expression of MAT1 gene in TrMAT1overexpression white clover increased significantly,the content of SAM increased significantly,and the content of MT increased significantly.TrMAT1 mutant white clover,TrMAT1 gene expression decreased significantly,SAM content decreased significantly,MT content decreased significantly.It indicated the authenticity of TrMAT1→SAM→MT metabolic pathway.（5）Compared with wild type,the drought resistance of TrMAT1 overexpressing white clover was significantly enhanced,which showed that the relative water content of leaves increased,the relative conductivity decreased,the SOD activity of leaves increased,the MDA content of leaves decreased,the Pro content of leaves increased,and the relative expression of senescence-related gene SAG101 decreased.The previous physiological changes in mutant plants were just the opposite.It shows that TrMAT1 has a significant regulatory function in drought resistance of white clover.