The regulation of caffeine production in Camellia sinensis L. cv. Iranian and Darjeeling tea

In the first part of this project, the distribution of caffeine and associated purine alkaloids in tea seedlings (C. sinensis cv. Iranian tea No. 100, and C. sinensis cv. Darjeeling tea) were investigated. Analysis was carried out by high performance liquid chromatography. The major purine alkaloid in tea seedling is caffeine. More than 90% of the caffeine was detected in the leaves. The highest amounts of caffeine were found in apical bud of Iranian and Darjeeling tea both of which contained ca. 38 mg caffeine per gram fresh weight. The lowest levels of caffeine were found in roots and cotyledons. Theobromine, a precursor of caffeine biosynthesis, was found in leaves, but in amounts far lower than those of caffeine. Trace amounts of paraxanthine were detected occasionally in leaves of Iranian tea. The second part of this project investigated the biosynthesis of caffeine in tea leaves using 14C-labelled adenine and guanine. Both substrates were converted to caffeine but adenine was a more effective precursor than guanine. Time-course and pulse-chase studies showed a transient incorporation of label into theobromine, the immediate precursor of caffeine, prior to a more substantial rise in [14C]caffeine. Biosynthesis of caffeine occurred more extensively in young leaves, declining substantially in mature and aged leaves. Although the exact pathway was not elucidated in this study, the available data supports the operation of an AMP → IMP → xanthosine → theobromine → caffeine pathway. [8-14C]Caffeine was degraded only slowly to 14CO2, via the purine catabolism pathway, which explains the accumulation of endogenous caffeine in tea leaves. Caffeine catabolism was, however, more extensive in leaves incubated in light than in darkness. In the third section of this project, the effects of the phytohormones, abscisic acid, indole-3-acetic acid, gibberellic acid and zeatin, on the metabolism of [8- 14C]adenine and caffeine by the first leaves of Iranian and Darjeeling tea in light and darkness, were investigated. Overall the phytohormones had little effect on the conversion of adenine to caffeine. However, incubations with [8-14C] caffeine showed that in darkness the phytohormones, most notably abscisic acid and indole-3-acetic acid, increased the rate of catabolism of caffeine after 12 h. In light the effect was less marked with caffeine degradation being enhanced by zeatin, and to a lesser degree abscisic acid and gibberellic acid, but only after in incubation period of 24 h