The Physiological Mechanism Of Topping Induced Chlorogenic Acid Biosynthesis And Accumulation In Tobacco Leaves

Chlorogenic acid is one of the important secondary metabolites in phenylpropane metabolism pathway.Its content is closely related to the formation of tobacco quality,and it can improve the resistance and antioxidant capacity of tobacco plants to cope with stress.In practice,topping can significantly improve the chlorogenic acid content of tobacco leaves,but its mechanism is not clear.Therefore,it is of great significance for improving the quality of tobacco to make clear the regulation mechanism of its synthesis.In this study,Honghuadajinyuan as experimental materials,First,the key metabolites of chlorogenic acid biosynthetic pathway were detected by targeted metabonomics in tobacco plants.The metabolic pathway analysis based on metabonomics combined with MetaboAnalyst 4.0 visual online software to depict the response of tobacco plants to metabolic level and analyze its potential mechanism.On this basis,we further analyzed the activities of key enzymes and the expression of synthetic key enzymes in key pathways of chlorogenic acid synthesis,and studied the regulatory mechanisms of chlorogenic acid biosynthesis after topping.Finally,by adding NAA and NPA,to clear effect of auxin on the content of chlorogenic acid after topping in tobacco leaves.It can provide a reference for clarify the physiological mechanism of chlorogenic acid synthesis and accumulation after topping and directional regulation of chlorogenic acid biosynthesis in tobacco to improve the quality of flue-cured tobacco.The main results are as follows:1.By targeting metabolism 13 kinds of phenylpropanoid metabolic components were detected,compared with no topping tobacco plants,topping tobacco cinnamic acid,1-O-p-Coumaroyl quinic acid,caffeic acid and chlorogenic acid content increased significantly,while L-phenylalanine,caffeic acid O-glucoside and coumarin content is decreased significantly,MetaboAnalyst analysis showed that these substances mainly involved in 16 metabolic pathways,The main pathways is phenylpropanoid biosynthesis.The activity of PAL,4CL,C4 H,HQT and HCT in the chlorogenic acid synthesis pathway increased significantly at third day after topping.It is further indicated that topping may enhance the transformation efficiency of phenylpropane pathway to chlorogenic acid synthesis branch,while the response weakens the flow rate of coumarin branching,which is competitive with chlorogenic acid pathway.At the same time,MetaboAnalyst analysis showed that topping induced tobacco chlorogenic acid synthesis key pathway is the catalytic reaction of HCT with coumarylquinic acid and then hydroxylated by C3 H.2.In order to further show that the reaction induced by topping chlorogenic acid accumulation and its regulation mechanism,this study respectively topping in the 30 th day after transplanting and in the 85 th day after transplanting,Comparing with the un-topping tobacco plants,topping in the 30 th day after transplanting could lead to the significant decrease of polyphenols such as chlorogenic acid in leaves,and the activity of PAL,PPO and POD increased,4CL activity decreased.The expression of PAL1,PAL4 and 4CL2 were up-regulated2.6,1.8 and 1.5 times,respectively.Topping in the 85 th day after transplanting could significantly improve the content of polyphenols in leaves of tobacco plants,PAL,C4 H,4CL and POD activity increased,the activity of PPO decreased,the expression of PAL1 was down-regulated 0.5 times.The expression levels of PAL2,PAL3,C4 H and 4CL1 were up-regulated in two periods.The above results show that the polyphenols biosynthesis in tobacco leaves induced by topping was related to the growth period.And it is regulated by the activity of PPO.3.In order to clarify the effect of changes in auxin content after topping on the accumulation of chlorogenic acid in tobacco leaves,in this experiment,the variation of chlorogenic acid and auxin content in tobacco leaf were studied by adding NAA and NPA treatment,and the activities of key enzymes of chlorogenic acid synthesis and degradation were analyzed.The results showed that the at the seedling stage endogenous IAA content decreased and chlorogenic acid content decreased after topping,while topping after the flower-bud appeared the IAA content of the tobacco leaves decreased after the third day,and the chlorogenic acid has the same changing rules.According to the NAA and NPA replenishment tests,the content of endogenous IAA in topping plants and NPA-treated tobacco leaves was decreased,the POD and PPO activities were increased,and the chlorogenic acid content was decreased;topping+NAA treatment elevated the content of chlorogenic acid and endogenous IAA in tobacco leaves,decreased POD and PPO the activity increased.The activity of PAL,4CL,C4 H,C3H and HQT increased in comparison with the untopped treatment.In conclusion,the content of chlorogenic acid was closely related to the content of endogenous auxin in tobacco leaves after topping,and regulated by the activity of POD and PPO.