Chapter Four - Biosynthesis and Catabolism of Purine Alkaloids
Introduction
Purine alkaloids, methylxanthines and methyluric acids, are secondary plant metabolites derived from purine nucleotides. These structures are based on xanthine or uric acid skeletons (Fig. 4.1). Methyl groups originating from S-adenosyl-l-methionine (SAM) are attached to nitrogen atoms at positions 1, 3, 7, 9 or an oxygen atom at position 2. The most widely distributed methylxanthine in the plant kingdom is caffeine (1,3,7-trimethylxanthine) which accumulates in leaves and seeds of tea (Camellia sinensis), coffee (Coffea arabica) and a limited number of other species. Sizable amounts of theobromine (3,7-dimethylxanthine) are stored in the seeds of cacao (Theobroma cacao; Zheng, Koyama, Nagai, & Ashihara, 2004), while theacrine (1,3,7,9-tetramethyl uric acid) accumulates in the leaves of a novel wild Chinese tea, kucha (Camellia assamica var. kucha; Lu et al., 2009, Zheng et al., 2002).
Although caffeine was isolated from tea and coffee in the early 1820s, the biosynthetic pathway of caffeine from purine nucleotides was not fully established until a highly purified N-methyltransferase (caffeine synthase) was obtained from tea leaves and a gene encoding this enzyme was cloned ~ 180 years later (Kato et al., 2000, Kato et al., 1999). The biosynthetic pathway of caffeine from xanthosine is now well understood in coffee and tea plants. Caffeine catabolism usually starts with the formation of theophylline, although little is known about the demethylases involved in the conversion. The recent advances in caffeine research involving the use of caffeine synthase genes to produce transgenic plants have opened up the possibilities of making decaffeinated coffee and tea plants as well as the use of caffeine as a natural pesticide in species of agricultural importance.
In this chapter, we summarize current information on the occurrence, biosynthesis and catabolism of purine alkaloids. The ecological role of caffeine, as well as the potential value of genetically modified caffeine-reduced and caffeine-induced plants, is also discussed. Purine alkaloid biosynthesis in plants has been reviewed recently (Ashihara, Kato and Crozier, 2011, Ashihara, Ogita and Crozier, 2011, Ashihara et al., 2008). Consumption of purine alkaloids can have a diversity of impacts on human health (Lean, Ashihara, Clifford, & Crozier, 2012). The metabolism and pharmacological function of purine alkaloids in animals, humans and plants and their impact on human nutrition as dietary constituents of tea, cacao, coffee and many soft drinks are discussed in detail in books edited by Fredholm (2011) and Crozier, Ashihara, and Tomas-Barberan (2012).
Section snippets
Occurrence of Purine Alkaloids in Plant Kingdom
Accumulation of purine alkaloids occurs in several plant species used for beverages and foods. In an earlier review, we noted that purine alkaloids, including caffeine, had been detected in at least 80 species in 13 orders of plant kingdom (Ashihara & Crozier, 1999). This was based on a review by Kihlman (1977) that quoted Willaman and Schubert (1961) and O'Connell (1969), who reported that caffeine occurs in more than 63 species which were distributed among 17 families and 28 genera. More
Biosynthesis of Purine Alkaloids
The xanthine and uric acid skeletons of purine alkaloids are derived from purine nucleotides. Results from studies on in situ metabolism of radioactive precursors and from the identification of enzymes and genes have established that the main caffeine biosynthetic pathway is a four-step sequence consisting of three methylation reactions and a nucleosidase reaction starting with xanthosine acting as the initial purine substrate (Fig. 4.2).
Catabolism of Caffeine
Caffeine appears to be the end product in most purine alkaloid-forming plants. However, limited amounts of caffeine are very slowly degraded with the removal of the three methyl groups, resulting in the formation of xanthine (Fig. 4.4). Catabolism of caffeine has been studied using 14C-labelled caffeine (Ashihara, Gillies and Crozier, 1997, Ashihara, Takasawa and Suzuki, 1997, Mazzafera, 2004, Suzuki and Waller, 1984a, Suzuki and Waller, 1984b). Caffeine catabolism begins with its conversion to
Distribution in tissues
In young tea leaves, immune-histochemical localization with primary anti-caffeine antibodies and conjugated secondary antibodies on leaf sections proved at the tissue level caffeine is localized within vascular bundles, mainly the precursor phloem (van Breda, Merwe, Robbertse, & Apostolides, 2013). Photosynthetic cells, that is, palisade and spongy parenchyma, also contain caffeine but in much lower concentrations, that is, amounts undetectable by immune-labelling and confocal scanning
Biotechnology of Purine Alkaloids
Using the gene sequences of N-methyltransferases involved in caffeine biosynthesis, two types of transgenic plants have been established. One is the construction of genetically modified decaffeinated coffee and tea plants, in which caffeine production is suppressed. The other is the introduction of caffeine biosynthesis into non-caffeine-producing plants (Table 4.3). The first approach involves introducing antisense or the double-stranded RNA interference (RNAi) constructs for the caffeine
In Planta Function of Purine Alkaloids
It has long been thought that purine alkaloids are the waste end products of purine nucleotides. Degradation of caffeine in most species is relatively slow even in aged leaves, and it appears not to act as a nitrogen reserve since considerable amounts remain in detached leaves following abscission (Suzuki et al., 1992). It has, however, also been proposed that purine alkaloids have an ecological role providing a chemical defence in planta against insect and vertebrate herbivores as well as
Conclusions and Perspectives
It is over a decade since our first review article on purine alkaloids was published in the Advances in Botanical Research (Ashihara & Crozier, 1999). Subsequently, there has been significant progress in the cloning of genes encoding N-methyltransferases involved in caffeine biosynthesis from tea and coffee plants which has provided to support the presence of the biosynthetic pathway initiated from xanthosine; xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine. Detailed
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