SEASONALVARIATIONS ON FLAVONOID BIOSYNTHETIC PATHWAY PROCESSES IN Camellia sinensis (L) O Kuntze OF SELECTED KENYAN CULTIVARS

Abstract

Globally, production of black tea has been on an increasing trend. This has posed a challenge on the market prices due to glut in the market. Thus need for diversification of tea products. Flavonoids are good antioxidants hence health-promoting properties. They are also relevant in determining the quality of food products. The quality of beverage tea is determined by the accumulation of flavonoids which are profoundly influenced by changes in environmental parameters and germplasm. The objective of this study was to investigate the effect of seasonal variations on tea flavonoids. Five cultivars comprising green-(TRFK 31/8, TRFK 6/8, and NRIT St. 536) and purple-leafed (TRFK 306 and TRFK 91/1) grown at Tea Research Institute (TRI), Timbilil center, Kericho county and Kangaita center, Kirinyaga county were evaluated for flavonoid profiles over the hot-dry, cool-wet and warm-wet seasons in the year 2014. Stomatal density (SD) was determined to find the effect on the biosynthesis of flavonoids. It involved scoring of leaf imprints of the 3rd tea leaf under the light microscope (Mg ×25). Results showed significant (P< 0.05) variation of stomatal density among the cultivars and all the interactions. Higher stomatal density was recorded in the cool-wet season in assam varieties; TRFK 31/8, 306 and 91/1. For biochemical analysis, three leaves plus apical bud were analyzed for catechins using HPLC (high performance liquid chromatography). There was significant (P<0.05) variation in total catechins (TC) due to cultivars and seasons. The cultivar TRFK 6/8 showed signifigant high accumulation of total catechins during the warm-wet season. EGCG (epigallocatechin gallate) which is the most abundant catechin in tea leaves was significantly higher during the warm-wet season in Timbilil. Further, three leaves plus apical bud of the purple-leafed cultivar TRFK 306 and TRFK 91/1 were sampled for anthocyanins. Total monomeric anthocyanin was determined using pH differential method of two buffers systems. Results revealed significant (P<0.05) variation among the cultivars and all the interactions. Anthocyanins content was significantly accumulated during the warm-wet season compared with other seasons. The hybrid TRFK 306 showed significantly higher contents compared with the parent (wild tea) TRFK 91/1. For gene expression studies, three leaves plus apical bud were analysed for eight gene transcripts; CsPAL, CsCHS, CsCHI CF3H, CsF3’5’H, CsDFR, CsANR and CsANS. The raw threshold cycle (Ct) values were normalized against a housekeeping gene encoding CsActin and the values used to calculate the differences in expression using the Relative Expression Software Tool (REST). Results revealed that there was differential expression of gene transcripts CsPAL, CsCHI, CF3H and CsF3’5’H in purple-leafed and green-leafed cultivars. In all the seasons, purple leafed cultivar TRFK 306 showed; up regulation of CsCHI and down regulation of CsCHS, down regulation of CsPAL in cool-wet and warm-wet seasons, and down regulation of CsF3’5H only in hot-dry season. Overall, the results of the study demonstrated that, stomatal density was higher in cool-wet season in assam cultivars; TRFK 306 accumulated higher contents of total monomeric anthocyanis during the warm-wet season. While TRFK 6/8 commercially grown for quality tea showed significantly higher total catechins during the warm-wet season. For molecular analysis, results showed differentially expression of flavonoids gene transcripts in the two selected cultivars (TRFK 306 and 31/8). The observed season-specific differences in flavonoids content in different tea cultivars are an indication of the potential for product diversification.