Molecular Mechanism Of Ascorbic Acid Biosynthesis And Metabolism In Tea Plant[Camellia Sinensis(L.)O.Kuntze]

Tea plant[Camellia sinensis(L.)O.Kuntze]is an economic crop in China.The leaves of tea plants contain a variety of secondary metabolites which are beneficial to humans.Ascorbic acid(AsA)is one of the secondary metabolites in leaves of tea plants.The distribution of AsA is affected by developmental stages and external environment in leaves of tea plants.Fresh-cut leaves of tea plant are the production source of teas.AsA contents of fresh-cut leaves of tea plant are affected by different postharvest storage conditions.In addition,AsA content is also affected by nitrogen deficiency stress in leaves of tea plants.In higher plants,AsA content is regulated by AsA metabolism and synthesis.However,the metabolic pathways of AsA in tea leaves are unclear.Meanwhile,the roles of AsA-related genes and proteins in AsA metabolism urgently need to be analyzed.In this study,the mechanism of AsA metabolism was analyzed,and the roles of AsA-related genes and proteins in AsA metabolism were also analyzed in tea plant based on the physiological,biochemical,and molecular experimental evidences.The main results were as follows:1.The leaves at three developmental stages were harvested from‘Huangjingya',‘Yingshuang',and‘Baiyeyihao',prepared for the experimental material,respectively.The AsA levels of leaves at three developmental stages from three tea plant cultivars were detected using high-performance liquid chromatography(HPLC).A total of 18 genes involved in AsA biosynthesis and metabolism pathways were identified based on transcriptome and genome data of tea plant,and expression levels of these genes were analyzed to assess the AsA biosynthesis and metabolism pathways.Our results indicated that AsA biosynthesis and metabolism pathways might be regulated by expression levels of AsA-related genes during leaf development.In addition,AsA accumulations were closely related to AsA-related gene expressions.L-galactose pathway might be the primary pathway of AsA biosynthesis in tea plant leaves.2.Fresh-cut leaves of tea plant were harvested from‘Longjing43'exposed to different postharvest storage conditions(4? 25?,and 38?)after 1 and 4 h,prepared for the experimental material to explore the effects of different temperature treatments on AsA metabolism.The distribution of AsA in fresh-cut leaves of tea plant was analyzed using acidic-alcoholic AgNO3 method.The analyses of RT-qPCR and iTRAQ were performed to analyze the expression of genes and proteins involved AsA biosynthesis and metabolism pathways.Our results showed that AsA was mainly distributed in the mesophyll cells in fresh-cut leaves of tea plant.CsAPXl and CsDHAR2 proteins involved in AsA metabolism pathway were identified by iTRAQ analysis,and the CsAPXl and CsDHAR2 proteins were upregulated and differently expressed.The expressions of CsAPX1 and CsDHAR2 genes were induced by high or low temperature treatments during postharvest storage by RT-qPCR analysis.Our results suggested that CsAPX1 and CsDHAR2 proteins and genes played vital roles in AsA metabolism in fresh-cut leaves of tea plant during postharvest storage.3.Fresh-cut leaves of 'Longjing43' under light/dark treatments for 4 h during postharvest storage were prepared for experiments to analyze the potential effects of light condition on AsA metabolism.CsAPX1,CsGME,CsGalDH,and CsDHAR2 proteins were identified by iTRAQ analysis,and the four were upregulated and differently expressed.The fold change of CsAPX1 was the highest in the four identified proteins.The gene of CsAPX1 was cloned from tea plant cultivar‘Longjing43',which was 753 bp in length,and encoded 250 amino acids including active and heme-binding sites.CsAPX1 protein is located in cytoplasm onion epidermal cell of by subcellular localization experiment.Protein purification suggested that the molecular mass of recombinant CsAPX1 protein was approximately 34.45 kDa.Although AsA contents and APX activities of transgenic Arabidopsis plants hosting CsAPX1 and wild type were decreased,AsA contents and APX activity of transgenic Arabidopsis plants hosting CsAPX1 were higher than that of wild type under light/dark conditions.4.Nitrogen(N)is one of macronutrient for the growth of tea plant,and is related to the mechanism of secondary metabolites.The leaves of‘Longjing43' under N deficiency and sufficiency with hydroponic culture were harvested for experiments to analyze the N deficiency stress on AsA metabolism.Under N deficiency stress,CsAPXl and CsGLBl proteins were upregulated and differently expressed by iTRAQ analysis.CsAPXl and CsGLB1 genes expression levels were induced in tea plant.The recombinant vectors,pET-30a-CsAPX1 and pET-30a-CsGLB1,were expressed into Escherichia coli BL21(DE3)using prokaryotic expression system.The cell growth rates in E.coli hosting pET-30a-CsAPX1 and pET-30a-CsGLB1 were higher than that of empty vector pET-30a under N sufficiency condition,respectively.Under N deficiency condition,AsA content of transgenic Arabidopsis plants hosting CsAPX1 was higher than that of wild type.The expression levels of AtAPX1 and AtGLBl in transgenic Arabidopsis plants hosting CsAPXl were higher than that of wild type under N deficiency condition.Our results suggested that CsAPX1 regulated AsA metabolism through cooperating with nitrogen regulatory protein P-? in tea plant under nitrogen deficiency stress.In conclusion,L-galactose pathway might be the primary pathway of AsA biosynthesis in tea plant leaves.AsA was mainly distributed in the mesophyll cells in tea plant leaves.AsA metabolism of tea plant leaves was affected by high temperature,light,and N deficiency stresses.CsAPX1 gene and encoding protein played potential vital regulatory roles in AsA metabolism in tea plant leaves.AsA mechanism in tea plant leaves was systematically analyzed in this study.Our results contributed to analyze the roles of AsA-related genes and proteins in AsA mechanism in tea plant leaves under abiotic stress.