Changes In The Expression Of Genes Related To The Biosynthesis Of Catechins In Tea Plants(Camellia Sinensis L.)

Tea trees named "Camellia sinensis (L.) O. Kuntze" originated from China. Tea is one of the most well known beverages in the world. Biological studies have demonstrated that tea flavonoids especially epigallocatechin gallate (EGCG) are beneficial for human health. The better quality of tea, the more economic benefit. Catechins are the main astringent substance in tea and synthesised mainly via the phenylpropanoid pathway. And genes involved in this pathway included PAL, C4H,4CL, CHS, CHI, F3H, F3'H, F3'5'H, DFR, LAR, ANS and ANR. These results provide insight into the possible mechanisms regulating catechin biosynthesis in tea plants and may lead to a better understanding of differences in catechin content and the underlying genetic mechanisms.In this study, in order to investigate the correlation between the changes in the concentration of total catechins (TC) and expression levels of those genes related to metabolism pathway, cultivar Longjingchangye "LJCY" grown both in greenhouse, sunshade net and open field, cultivar Wuniuzao "WNZ" grown both in anti frost fan and open field, eight cultivars of tea plants harvested both in spring and autumn and the dynamic changes of a special kind of tea plants Anjibaicha "AJBC" were used to investigate differences in TC concentrations, by using high-performance liquid chromatography (HPLC). The expression levels of genes associated with catechin biosynthesis were investigated using reverse transcription-quantitative polymerase chain reaction (qRT-PCR). The main results are as follows:1. "LJCY" tea plants were grown in a greenhouse, sunshade net and in an open field, to study changes in TC concentrations using HPLC and changes in the levels of expression of genes related to the biosynthesis of catechins using qRT-PCR. The results indicated that tea plants grown in a greenhouse and sunshade net had different levels of expression of those genes that participate in the biosynthesis of catechins. The levels of expression of the "early" genes for phenylalanine ammonialyase(PAL), flavonoid 3'-hydroxylase (F3'H), flavanone 3-hydroxylase (F3H), and flavonoid 3',5'-hydroxylase (F3'5'H) were down-regulated by 72.12%,83.68%, 82.95% and 88.94%, respectively, when plants were grown in a greenhouse. But it is noteworthy that the levels of expression of the genes for dihydroflavonol 4-reductase (DFR), leucocyanidin reductase (LAR) and anthocyanidin reductase (ANR) increased by 161.74%,96.34% and 36.89%, respectively, compared to plants grown in the open field. When plants were grown in a sunshade net, genes for PAL, F3'H and LAR were up-regulated by 50.00%,171.00% and 42.00%. But the down-regulated expression in DFR, ANR and ANS genes were found. HPLC analysis of catechins showed that plants grown in a greenhouse and sunshade net contained higher levels of TC examined compared to plants grown in the open field.2. Wuniuzao"WNZ" tea plants were grown in an anti frost fan and in an open field, to study changes in catechin concentrations using HPLC and changes in the levels of expression of genes related to the biosynthesis of catechins using qRT-PCR. HPLC analysis of catechins showed that plants grown in an anti frost fan contained higher levels of TC examined compared to plants grown in the open field. The results of qRT-PCR indicated that WNZ grown in an anti frost fan had different levels of expression of those genes that participate in the biosynthesis of catechins. The levels of expression of the genes related to biosynthesis of catechins were stable expressed but PAL, cinnamate 4-hydroxylase(C4H), F3'5'H, DFR and LAR, when plants were grown in an anti frost fan. But it is noteworthy that the levels of expression of the genes for PAL and LAR increased by 97.33% and 211.57%, respectively, compared to plants grown in the open field.3. In this study, eight cultivars of tea plants harvested both in spring and autumn were used to investigate differences in catechin concentrations, by using HPLC. The expression levels of genes associated with catechin biosynthesis were investigated using qRT-PCR. The results indicated that TC concentrations were significantly higher in tea plants harvested in autumn than in those harvested in spring, based on higher concentrations of epigallocatechin (EGC) in autumn tea (P<0.01). The expression of the genes PAL, F3H, F3'5'H, DFR and ANS is closely related to the TC content of tea plants in both spring and autumn. Positive correlations between PAL, C4H, F3H and DFR expression and EGC accumulation in autumn tea were identified, with correlation coefficients of 0.710,0.763,0.884 and 0.707, respectively. A negative correlation between ANS expression level and EGC concentrations in tea plants harvested in spring was noted (r=-0.732). Additionally, negative correlations between F3H and ANS expression levels and the catechin content were identified in spring tea, whereas the correlations were positive in autumn tea. Significant differences in F3H and ANS expression levels between spring and autumn tea indicate that F3H and ANS are potentially key genes affecting catechin accumulation in tea plants.4. In this study, the dynamic changes of catechin levels and the expression levels of catechin-related genes in a special kind of tea plants Anjibaicha "AJBC" which expressed albino of tea growing leaves were intensively examined during tea leaves colour changing in tea. Accumulation of catechins were measured with using HPLC. It was found that the concentrations of TC, EGC, epiccatechin gallate (ECG) and EGCG decreased to their lowest levels in the all albino stage but a rapid increase in the re-greening stage. EGCG was the highest of the catechins compared to all catechins, and catechin (C) the lowest. The expression of genes involved in catechin biosynthesis in bud leaves of AJBC were measured by qRT-PCR. Gene expression profiling by qRT-PCR showed that the transcript abundance of flavonoid biosynthetic genes followed a tightly regulated biphasic pattern, and was affected by albinism. The expression levels of CHI, F3'5'H, ANS and ANR were negative regulated with the concentration of TC and the correlation coefficients were-0.845,-0.873,-0.681 and-0.879, respectively. The expression of PAL (r=0.957) and DFR (r=0.954) were positive significantly correlated with the content of TC. The results suggested that PAL and DFR might be key regulators in the control of catechin biosynthesis in albino tea plants.