Fine control of caffeine biosynthesis in tissue cultures of Camellia sinensis

To determine whether caffeine biosynthesis is controlled by the availability of purine precursors and/or methyl-donors, we examined the effect of some purine compounds on purine alkaloid accumulation, using tea callus cultures. No stimulation of caffeine biosynthesis was observed when the calli were cultured with 0.5 mM adenosine, guanosine or hypoxanthine for 3 weeks. However, 0.5 mM paraxanthine doubled the caffeine level relative to controls. Adenosine stimulated the growth of callus and reduced the caffeine concentration 3 months after inoculation. These results indicate that methylation of xanthosine by 7-methylxanthosine synthase is the most plausible rate-limiting step of caffeine biosynthesis; the supply of non-methylated purine precursors or availability of S-adenosyl-l-methionine are not the principal controlling factors of caffeine biosynthesis. Adenosine salvage to adenine nucleotide synthesis may contribute to the growth of tea calli, but not to caffeine biosynthesis.