Opinion TRENDS in Plant Science Vol.6 No.9 September 2001 407 the widespread medical interest in caffeine as a dietary Caffeine: a well known component, these developments have received little attention in the plant literature, with the topic being all but neglected in recent biochemistry text books9–12. but little mentioned Caffeine is synthesized from xanthosine via a xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine pathway; compound in plant the first, third and fourth steps are catalysed by N-methyltransferases that use S-adenosyl-L- methionine (SAM) as the methyl donor13. A recent science important development has been the cloning and expression in E. coli of a gene from tea leaves that encodes caffeine synthase, an extremely labile Hiroshi Ashihara and Alan Crozier N-methyltransferase that catalyses the last two steps in this pathway14. In addition, coffee leaf cDNAs of theobromine synthase, which catalyses the penultimate Caffeine, a purine alkaloid, is a key component of many popular drinks, most methylation step, have been similarly cloned and notably tea and coffee, yet most plant scientists know little about its expressed in E. coli15,16. There are also preliminary biochemistry and molecular biology. A gene from tea leaves encoding caffeine reports on the cloning of an N-methyltransferase from synthase, an N-methyltransferase that catalyses the last two steps of coffee that catalyses the initial methylation step in the caffeine biosynthesis, has been cloned and the recombinant enzyme pathway17,18. These advances in our knowledge of the produced in E. coli. Similar genes have been isolated from coffee leaves but metabolism of caffeine and related compounds in plants the recombinant protein has a different substrate specificity to the tea and the potential biotechnological applications of enzyme. The cloning of caffeine biosynthesis genes opens up the possibility of purine alkaloid research are highlighted in this article. using genetic engineering to produce naturally decaffeinated tea and coffee. Distribution of purine alkaloids Caffeine (1,3,7-trimethylxanthine) is one of the few Purine alkaloids have a limited distribution within plant products with which the general public is readily the plant kingdom. In some species, the main purine familiar, because of its occurrence in beverages such as alkaloid is theobromine or methyluric acids rather coffee and tea, as well as various soft drinks. A growing than caffeine13. Among the purine-alkaloid- belief that the ingestion of caffeine can have adverse containing plants, most studies have been carried out effects on health has resulted in an increased demand with species belonging to the genera Camellia and for decaffeinated beverages1. Unpleasant short-term Coffea. In C. sinensis (Fig. 2), caffeine is found in the side effects from caffeine include palpitations, highest concentrations in young leaves of first-flush gastrointestinal disturbances, anxiety, tremor, shoots of var. sinensis (2.8% of the dry weight). increased blood pressure and insomnia2,3. In spite of Theobromine is the predominant purine alkaloid in numerous publications on the long-term consequences young leaves of cocoa tea (Camellia ptilophylla) of caffeine consumption on human health, no clear (5.0–6.8%) and Camellia irrawadiensis (<0.8%). picture has emerged, with reports of both protective The beans of most cultivars of Arabica coffee and deleterious effects4. (C. arabica) (Fig. 3) contain ~1.0% caffeine, whereas Caffeine was discovered in tea (Camellia sinensis) Coffea canephora cv. Robusta (1.7%) and cv. Guarini and coffee (Coffea arabica) in the 1820s (Ref. 5). (2.4%), Coffea dewevrei (1.2%) and Coffea liberica (1.4%) Along with other methylxanthines, including contain higher concentrations. By contrast, the caffeine theobromine (3,7-dimethylxanthine), paraxanthine contents of the seeds of other species, such as Coffea Hiroshi Ashihara (1,7-dimethylxanthine) and methyluric acids (Fig. 1), eugenioides (0.4%), Coffea salvatrix (0.7%) and Coffea Metabolic Biology Group, Dept Biology, Faculty of caffeine is a member of a group of compounds known racemosa (0.8%), are lower than that of C. arabica. Science, Ochanomizu collectively as purine alkaloids. There are two Young expanding leaves of C. arabica plants also University, Otsuka, hypotheses about the role of the high concentrations of contain caffeine, with traces of theobromine. In model Bunkyo-ku, Tokyo 112-8610, Japan. caffeine that accumulate in tea, coffee and a few other systems, weak intermolecular complexes form 19 e-mail: plant species. The ‘chemical defence theory’proposes between caffeine and polyphenols , and it has been [email protected] that caffeine in young leaves, fruits and flower buds proposed that caffeine is sequestered in the vacuoles 20 Alan Crozier acts to protect soft tissues from predators such as insect of coffee leaves as a chlorogenic acid complex . Plant Products and larvae6 and beetles7. The ‘allelopathic theory’proposes Mature leaves of C. liberica, C. dewevrei and Coffea Human Nutrition Group, that caffeine in seed coats is released into the soil and abeokutae convert caffeine to the methyluric acids, Division of Biochemistry 8 and Molecular Biology, inhibits the germination of other seeds . The potential theacrine (1,3,7,9-tetramethyluric acid), liberine Faculty of Biomedical and ecological role of caffeine is described in Ref. 6. [O(2),1,9-trimethyluric acid] and methylliberine Life Sciences, University It is only within the past five years that the [O(2),1,7,9-tetramethyluric acid] (Fig. 1). of Glasgow, Glasgow, biosynthetic and catabolic pathways that regulate the Purine alkaloids are also present in the leaves of UK G12 8QQ. e-mail: build-up of caffeine in the vacuoles of cells of tea and maté (Ilex paraguariensis), which is used in rural areas [email protected] coffee plants have been elucidated fully. In contrast with of South America, such as the Brazilian Panthanal and http://plants.trends.com 1360-1385/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)02055-6 408 Opinion TRENDS in Plant Science Vol.6 No.9 September 2001 O O O O O O CH CH CH H CH 1 7 3 3 H 3 3 H3C N N H3C H3C N H3C N H3C N N HN N N N N N O O O 3 O N N O N N O N N O N N O N N O N N H C CH 3 CH H3C CH CH3 CH3 CH3 CH3 3 3 3 Caffeine Theobromine Paraxanthine Theacrine Liberine Methylliberine TRENDS in Plant Science Fig. 1. Structures of the the Pampas in Argentina, to produce a herbal tea purine ring of caffeine can be produced exclusively methylxanthines caffeine, (http://www.vtek.chalmers.se/~v92tilma/tea/mate.html). by this route in young tea leaves25. The formation of theobromine and Young maté leaves contain 0.8–0.9% caffeine and caffeine by this pathway is closely associated with paraxanthine, and the methyluric acids 0.08–0.16% theobromine. Theobromine is the the SAM cycle (also known as the activated-methyl theacrine, liberine and dominant purine alkaloid in seeds of cocoa (Theobroma cycle) because the three methylation steps in the methylliberine. cacao), with cotyledons of mature beans containing caffeine biosynthesis pathway use SAM as the 2.2–2.7% theobromine and 0.6–0.8% caffeine. Caffeine methyl donor (Fig. 4). During this process, SAM is (4.3%) is the major methylxanthine in cotyledons of converted to SAH, which in turn is hydrolysed to guaraná (Paulliania cupana), extracts of which are L-homocysteine and adenosine. The adenosine is used as a refreshing pick-me-up (http://www.rain- used to synthesize the purine ring of caffeine and tree.com/guarana.htm) and which is, in a dilute form, the L-homocysteine is recycled to replenish SAM sold extensively in Brazil as a carbonated drink. Seeds levels. Because 3 moles of SAH are produced via the of cola (Cola nitida) also contain caffeine (2.2%)21. SAM cycle for each mole of caffeine that is Caffeine has recently been detected in flowers of synthesized, this pathway has the capacity to be the several citrus species, with the highest concentrations sole source of both the purine skeleton and the (0.2%) in pollen22, and is also a fungal metabolite, being methyl groups required for caffeine biosynthesis in the principal alkaloid in sclerotia of Claviceps young tea leaves25. sorhicola, a Japanese ergot pathogen of Sorghum23. Purine ring methylation Biosynthesis of purine alkaloids Xanthosine is the initial purine compound in the Origin of the purine ring of caffeine caffeine biosynthesis pathway, acting as a substrate Caffeine is a trimethylxanthine whose xanthine for the methyl group donated by SAM. Tracer skeleton is derived from purine nucleotides that are experiments with labelled precursors and leaf discs converted to xanthosine, the first committed from tea and coffee plants have shown that the major intermediate in the caffeine biosynthesis pathway. route to caffeine is xanthosine → 7-methylxanthosine → There are at least four routes from purine 7-methylxanthine → theobromine → caffeine, nucleotides to xanthosine (Fig. 4). The available although alternative minor routes might also evidence indicates that the most important routes operate26. However, as well as entering the caffeine are the production of xanthosine from inosine biosynthesis pathway, xanthosine is also converted to ′ 5 -monophosphate, derived from de novo purine xanthine, which is degraded to CO2 and NH3 via the nucleotide biosynthesis, and the pathway in purine catabolism pathway27,28 (Fig. 5). which adenosine, released from S-adenosyl-L- The first methylation step in the caffeine homocysteine (SAH), is converted to xanthosine biosynthesis pathway, the conversion of xanthosine via adenine, adenosine 5′-monophosphate, inosine 5′-monophosphate and xanthosine 5′-monophosphate13,24,25. Recently published data indicate that the conversion of SAH to xanthosine is such that the Fig.