Revisiting caffeine biosynthesis--speculations about the proximate source of its purine ring.

The prevailing hypothesis of caffeine biosynthesis starting from xanthosine was combined with Kremers' speculation on NAD as a biochemical precursor of caffeine and trigonelline in coffee. This bold sketch together with a few free-spirited ideas may channel future caffeine biosynthesis studies into novel directions.

Purine alkaloids in Paullinia.

Among the few purine alkaloid-containing genera consumed as stimulants, Paullinia is the least investigated with respect to both chemotaxonomy and within-the-plant allocation of caffeine and its allies. Since purine alkaloids (PuA) have been proved to be valuable marker compounds in chemotaxonomy, 34 species of Paullinia and related genera were screened for them, but only one, P. pachycarpa, was positive in addition to the already known P. cupana and P. yoco. The PuA allocation in P. pachycarpa was examined and found to be restricted to theobromine in the stem, leaves and flowers. Moreover, the theobromine concentration in the stem cortex increased significantly towards the base of the plant. Since the stem cortex of P. yoco is traditionally used by the natives of Colombia and Ecuador to prepare a caffeine-rich beverage, we suspected that within the genus Paullinia the PuA are preferentially allocated to the older parts of the stem and not to young shoots like e.g., in the coffee plant (Coffea spp.). Indeed, the axis (greenhouse) of P. cupana (guaraná), known for its caffeine-rich seeds, exhibited a basipetal PuA gradient (0.005-0.145%). Moreover, the analysis of young cortex samples (herbarium) and of one piece of old stem (museum collection) revealed the same for P. yoco, even though we found much less (0.5 vs 2.5%) caffeine in the old cortex as compared to the only two analyses in 1926 of similar material. However, this discrepancy may be explained by the high variability of the PuA pattern we detected among yoco, the diversity of which the Indians take advantage.

Non-destructive analysis of natural variability in bean caffeine content of Laurina coffee.

In order to conserve the germination power of coffee beans, only a tiny portion of endosperm was excised for caffeine determination. These non-destructive, single-bean analyses of the low-caffeine coffee mutant Laurina (C. arabica cv. laurina) revealed (a) a variation of the caffeine content from 0.36 to 1.08%, with lowest values found in the tissue culture-derived genotypes (somaclones), (b) a markedly lower mean caffeine content (0.56-0.61% dry wt) in the somaclones than in the parent line (0.68-0.71%), and (c) a high correlation between the caffeine contents of the bean and cotyledons emerged from it. Hence, breeding for low-caffeine coffee will be facilitated in future by simply analysing the cotyledons for this character. The combination of "somaclones" and single-seed analysis may be a useful and general strategy to tailor the phytochemical pattern of plants.Additionally, the young leaflets of Laurina, equally to the seeds furnished with a low caffeine content, were studied with respect to the N-methyltransferase (NMT) activities catalysing the last two steps in caffeine biosynthesis. The related activities were much lower than of the cultivar Catuai indicating that the lr lr alleles of Laurina, responsible for the reduction in half the "normal" caffeine content, interfere with the synthesis and not with the catabolism of caffeine.