Response Mechanism Of Melatonin To Copper Stress During Ripening Of Wine Grapes

Copper(Cu)is a micronutrient element necessary for the growth and development of most organisms.When the copper accumulation exceeds a certain limit,it will produce toxicity,affecting a series of biochemical reactions and physiological processes of plants including grapes.As a broad-spectrum and effective antioxidant,melatonin can significantly improve the tolerance of plants under biotic and abiotic stress.The previous study of our group found that melatonin can significantly alleviate the copper stress of grape,and the mechanism of melatonin involved in the stress response of the grape during the development of grape fruit and wine fermentation process is still unclear.Therefore,to elucidate the molecular biological mechanism of melatonin response to copper stress during the development of wine grape,it is of great significance for grape cultivation under copper stress.In this experiment,Cabernet Sauvignon was used as the material,and the grape fruit after the color change period was used as a test material.The fruit was sampled regularly until the fruit was ripe.First,the copper solution was used to stress,and the blank was used as the control.Then,the melatonin solution was used in different concentrations.deal with.Set 4 copper pesticide concentrations,4 melatonin solution concentrations,determine grape physiology and oxidative stress,antioxidant enzymes and enzyme activity gene expression and non-enzymatic antioxidant protection system response to copper stress,while passing HPLC-MS/MS was used to monitor the dynamic changes of endogenous melatonin in grape.The key enzymes of TDC,T5H,AANAT and ASMT in grape synthesis pathway were determined by Real-Time PCR at different gene stress levels.Change the law,find/confirm the key response enzymes of melatonin synthesis under copper stress conditions,and reveal the molecular mechanism of melatonin response to copper stress during the ripening process of wine grape fruit and fruit fermentation process.The results obtained from the study are as follows:(1)Moderate low copper stress promotes the formation of total phenols,total flavonoids and total flavanols;high concentrations of copper stress inhibit their formation.The total anthocyanin showed a rising content during the growth and development after the color change period,and it was sensitive to copper stress treatment.The lower concentration of copper stress had a certain promoting effect on the synthesis,and the high copper stress would be Its synthesis has an inhibitory effect,and the higher the copper stress concentration,the more obvious the inhibition effect.In general,almost all polyphenols are elevated in low concentrations of copper pesticides.Under the condition of high copper concentration,the content of polyphenols will be significantly reduced.(2)Exogenous spraying of copper pesticides for stress treatment will cause a surge in copper content in grape fruit.Low concentration of copper stress increased the antioxidant capacity of grape fruit to some extent.In the high copper concentration range,the antioxidant capacity of the wine is significantly reduced.The low concentration of copper pesticide stress treatment had a slight inhibitory effect on the synthesis of melatonin.On the contrary,the higher concentration of copper stress treatment had a significant effect on the synthesis of melatonin.The expression of key genes controlling melatonin synthesis is consistent with the performance of melatonin content.(3)Moderate low-concentration copper stress slightly inhibited the formation of MDA,and high concentrations of copper pesticides significantly stimulated MDA production.The low concentration of copper pesticide treatment group slightly inhibited the activity of SOD,CAT and POD,but the effect was not significant,while the high concentration of copper pesticide treatment group significantly increased the activity of three antioxidant enzymes.During the developmental period,moderately low concentrations of copper pesticides had little effect on the ratio of AsA/DHA and GSH/GSSG,and were consistent with changes in antioxidant enzyme activity.High concentrations of copper stress significantly reduced this.In contrast,high concentrations of copper stress can cause severe oxidative stress on grape fruit.(4)Copper stress-induced oxidative stress leads to a decrease in the ratio of AsA/DHA and GSH/GSSG,while exogenous spraying of melatonin can increase the content of key substances in the ascorbate-glutathione cycle and AsA/DHA and GSH/GSSG.ratio.Melatonin,as an oxidant,directly scavenges free radicals,not only increases the activity of antioxidant enzymes,but also increases the content and ratio of key substances in the ascorbate-glutathione cycle in non-enzymatic antioxidant systems,thereby helping plants resist Adversity stress.