| Plants have complex metabolic networks, in which the balance of carbon(C) and nitrogen(N)metabolism is requisite for plant growth. Due to the imbalance of several metabolic processes caused by genic mutation and the environmental condition, the biosynthesis of quality-related components is significantly affected, which resulted in the variation of tea quality. Nitrogen(N) is necessary elements as the growth of tea, the change of N supply have an effect on metabolism of C、N metabolic balance of tea tree. This paper elaborated change of C、N metabolism in of young shoots and mature leaves during different periods by metabolomics, explained regulation of C、N metabolic balance under the N transfer from mature to young shoot. Furthermore, our research also revealed C 、 N metabolic regulation mechanism of ‘longjing’ tea tree under impact of nitrogen supply; our research also revealed C、N metabolic regulation mechanism of ‘zijuan’ tea tree which is the specific varieties under different nitrogen supply. This project expands metabolome and omics analytical methods to display the physiological metabolism in tea plant, and acquires a lot of information about the metabolites and transcriptional regulation. Hence, a new insight for understanding the regulation of C and N balance and biosynthesis of quality related compounds is obtained. The main results are as followed:1. To elucidate the mechanism underpinning the latter variation, the dynamic changes of primary and secondary metabolites and metabolic pathway in young shoots of different spring periods were evaluated by metabolomics approach. Samples of young shoots were collected on April 1, April 15 and April 28 representing the early, middle and late spring tea season. Primary and secondary metabolites were analyzed by means of ultra performance liquid chromatography coupled to quadrupole orthogonal time of flight mass spectrometer(UPLC-Q-TOF/MS) and two-dimensional gas chromatography coupled to time of flight mass spectrometry(GC×GC-TOF/MS). There were clear differences of metabolite profiling of amino acids, organic acids, carbohydrates, flavonoids and their glycosides in young shoots among the three periods. The contents of main free amino acid theanine, glutamine and glutamate decreased while levels of flavan-3-ols, quercetin and their glycosides increased, suggesting reinforced biosynthesis of carbohydrate and flavanoids, tricarboxylic acid cycle iand photorespiration in the late spring season. Blind sensory evaluation of made teas showed green tea processed from the early spring shoots was the best quality. The largely variation of metabolites and metabolic pathway of young shoots could not be explained simply by changes in climatic factors, which were recorded in the same time. Metabolomic analysis further showed distinctly reduced levels of amino acids,carbohydrates and flavonoids in mature leaves, suggesting remarkable remobilization of N and C reserves to sustain vigorous growth of the young shoot in spring. The balance of carbon and nitrogen metabolism was altered by a depletion of N supply in the young shoots, leading to the great fluctuation of metabolites and consequently green tea quality in the late spring season.2. In order to reveal the influence of C and N metabolism by supply of nitrogen, we select ‘longjing’tea tree as experimental material, and we using metabolomics combining quantitative PCR anddetermination of enzyme activity, analyzed C, N metabolic balance and the influence of the biosynthesis of quality components of young shoot, mature leaves and root in the different nitrogen levels(0.3, 1.5,4.5 mmol//L) for ‘longjing’ tea tree. Results show that flavonoid synthesis significantly were inhibited in the young shoot and mature leaves under high nitrogen, at the same time, genes involved in flavonoids synthesis are also significantly reduced in high nitrogen condition; in carbohydrate metabolism, we also found that the main sugar synthesis were limited in high nitrogen; in amino acid metabolism, all kinds of free amino acid contents significantly increased as total nitrogen content increases. Change of primary and secondary metabolites in tea plant root system are same as young shoot and mature leaves.3. To analyze C and N metabolism of ‘zijuan’ tea tree under supply of different nitrogen from aspect of gene expression, the activity of enzymes and metabolite, we selected ‘zijuan’ tea tree which have higher anthocyanin content as experimental material, studied C, N metabolic balance and the biosynthesis of quality components under the impact of different nitrogen levels(0.3, 1.5, 4.5 mmol/L)in young shoots, mature leaves, root based on metabolomics and transcriptome. Results showed that biosynthesis of anthocyanin were significantly inhibited in the ‘zijuan’ young shoots and mature under high nitrogen level. We also found genes in biosynthesis of anthocyanin were significantly reduced as the increase of nitrogen supply by analysis transcriptome and gene expression, the above result illustrates that there is positive correlation beween the metabolites and molecular regulation under impact of nitrogen level. In addition, we also found that flavonoid biosynthesis were significantly inhibited in young shoot and mature of ‘zijuan’, increase of PEPC enzyme activity suggested that C skeletons was more consumed. Biosynthesis of carbohydrate were significantly inhibited in mature of‘zijuan’ as the increase of nitrogen, but the content of much carbohydrate were increased in the young shoot of ‘zijuan’, the transfer of C, N from mature to young shoot, synthesis of the anthocyanin in‘zijuan’ young shoots inhibited in high nitrogen, increase of the photosynthesis, could be response for above result. In biosynthesis of amino acids, young shoot and mature of zijuan, the content of amino acids significantly increased as the increase of nitrogen level, gene expression and the transcriptome also suggests that the expression of genes involved nitrogen assimilation significantly increased as the increase of nitrogen supply. In ‘zijuan’ root, change of the primary and secondary metabolites and gene expression were identified with that in the shoots and mature leaves under supply of different nitrogen4. In order to explore different mechanism under influence of nitrogen supply between ‘zijuan’which is a specific varieties of purple buds tea tree and ordinary tea tree, we compared change of primary and secondary metabolism beteween ‘zijuan’ and ‘longjing’ young shoot. The results showed that compared with the ordinary varieties, anthocyanins metabolic pathway had a specific response mechanism in ‘zijuan’ as high anthocyanin content, synthesis of anthocyanins metabolites were inhibited as the increase of nitrogen supply; the synthesis of the flavonoids significantly were inhibited in ‘zijuan’ as same as the ordinary varieties as the increase of nitrogen; in carbohydrate metabolism,synthesis of carbohydrate in ‘zijuan’ were enhanced opposite to change of carbohydrate in the ordinaryvarieties as increase of nitrogen supply; in amino acid metabolism, free amino acid content significantly increased in ‘zijuan’ the same as change of amino acid in the ordinary varieties as incease of nitrogen supply. |
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