Metabolism of purine bases, nucleosides and alkaloids in theobromine-forming Theobroma cacao leaves

Abstract

We examined the purine alkaloid content and purine metabolism in cacao ( Theobroma cacao L.) plant leaves at various ages: young small leaves (stage I), developing intermediate size leaves (stage II), fully developed leaves (stage III) from flush shoots, and aged leaves (stage IV) from 1-year-old shoots. The major purine alkaloid in stage I leaves was theobromine (4.5 μmol g –1 fresh weight), followed by caffeine (0.75 μmol g –1 fresh weight). More than 75% of purine alkaloids disappeared with subsequent leaf development (stages II–IV). In stage I leaves, 14C-labelled adenine, adenosine, guanine, guanosine, hypoxanthine and inosine were converted to salvage products (nucleotides and nucleic acids), to degradation products (ureides and CO 2) and to purine alkaloids (3- and 7-methylxanthine, 7-methylxanthosine and theobromine). In contrast, 14C-labelled xanthine and xanthosine were not used for nucleotide synthesis. They were completely degraded, but nearly 20% of [8- 14C]Xanthosine was converted in stage I leaves to purine alkaloids. These observations are consistent with the following biosynthetic pathways for theobromine: (a) AMP → IMP → 5′-xanthosine monophosphate → xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine; (b) GMP → guanosine → xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine; (c) xanthine → 3-methylxanthine → theobromine. Although no caffeine biosynthesis from 14C-labelled purine bases and nucleosides was observed during 18 h incubations, exogenously supplied [8- 14C]Theobromine was converted to caffeine in young leaves. Conversion of theobromine to caffeine may, therefore, be slow in cacao leaves. No purine alkaloid synthesis was observed in the subsequent growth stages (stages II–IV). Significant degradation of purine alkaloids was found in leaves of stages II and III, in which [8- 14C]Theobromine was degraded to CO 2 via 3-methylxanthine, xanthine and allantoic acid. [8- 14C]Caffeine was catabolised to CO 2 via theophylline (1,3-dimethylxanthine) or theobromine.