Metabolism of Caffeine and Related Purine Alkaloids in Leaves of Tea {Camellia sinensis L.)

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1 Plant Cell Physiol. 38(4): (1997) JSPP 1997 Metabolism of Caffeine and Related Purine Alkaloids in Leaves of Tea {Camellia sinensis L.) Hiroshi Ashihara 1, Fiona M. Gillies 2 and Alan Crozier 2 ' 3 1 Department of Biology, Faculty of Science, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo, 112 Japan 2 Bower Building, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, U.K. Purine alkaloid catabolism pathways in young, mature and aged leaves of tea (Camellia sinensis L.) were investigated by incubating leaf sections with l4 C-labelled theobromine, caffeine, theophylline and xanthine. Incorporation of label into CO 2 was determined and methanolsoluble metabolites were analysed by high-performance liquid chromatography-radiocounting and thin layer chromatography. The data obtained demonstrate that theobromine is the immediate precursor of caffeine, which accumulates in tea leaves because its conversion to theophylline is the rate limiting step in the purine alkaloid catabolism pathway. The main fate of [8-14 C]theophylline incubated with mature and aged leaves, and to a lesser extent young leaves, is conversion to 3-methylxanthine and onto xanthine which is degraded to 14 CO 2 via the purine catabolism pathway. However, with young leaves, sizable amounts of [8-14 C]- theophylline were salvaged for the synthesis of caffeine via a 3-methylxanthine» theobromine * caffeine pathway. Trace amounts of [2-14 C]xanthine were also salvaged for caffeine biosynthesis in young leaves, by conversion to 3- methylxanthine, and this was enhanced in the presence of 5 mm allopurinol which inhibits purine catabolism. Feeds of [2-14 C]xanthine to young leaves also indicated that 3- methylxanthine, as well as being salvaged for theobromine and caffeine production, is also converted, via TV-l-methylation, to theophylline. Key words: Caffeine Camellia sinensis Purine alkaloids Theobromine Theophylline Xanthine. The mono-, di- and trimethylxanthines that are found in a limited number of higher plant species, including coffee (Coffea arabica L.), tea (Camellia sinensis L.) and mate (Ilex paraguariensis), are referred to as purine alkaloids. The biosynthesis of the purine alkaloids caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) has been investigated in tracer studies that have utilised radiolabelled precursors such as [8-14 C]adenine and [8-14 C]guanine (see Suzuki et al. 1992). In tea, the leaves of which typically contain ca. 2-5% caffeine (g dry weight)" 1 (Takeda 1994, Ashihara et al. 1995), caffeine biosynthesis 3 To whom correspondence should be addressed. involves an AMP * IMP XMP * (or GMP * guanosine) -» xanthosine -+ 7-methylxanthosine 7-methylxanthine -> theobromine -> caffeine pathway (Fig. 1, see Suzuki et al. 1992). In addition to this major route, there is evidence from in vitro studies that several minor pathways may also operate (Kato et al. 1996). Furthermore, Schulthess et al. (1996) have recently proposed that caffeine biosynthesis in C. arabica starts with the metabolically channelled conversion of XMP -* 7-methyl-XMP 7- methylxanthosine. In tea, the highest levels of caffeine biosynthesis occur in young leaves (Ashihara and Kubota 1986, Fujimori et al. 1991),flowers (Fujimori and Ashihara 1990) and fruits (Terrasaki et al. 1994) and these rates decline markedly as the tissues age. In contrast to caffeine biosynthesis, relatively little is known about caffeine degradation and the catabolism of purine alkaloids in tea. In Coffea arabica, it has been shown that caffeine is metabolised very slowly, via theophylline and 3-methylxanthine, to xanthine which is degraded by the purine catabolism pathway to CO 2 and NH 3 (Kalberer 1964, 1965, Ashihara et al. 1996b). This paper reports on a study of caffeine catabolism in which the in vivo metabolism of 14 C-labelled xanthine, theobromine, theophylline and caffeine were investigated in young, mature and aged leaves of Darjeeling tea. Incorporation of label in I4 CO 2 was determined and methanol-soluble metabolites were analysed by reversed phase HPLC-radiocounting (RC) and TLC. In some experiments allopurinol was used to inhibit xanthine dehydrogenase/oxidase activity (Nguyen 1986), thereby preventing the degradation of xanthine by the purine catabolism pathway (see Ashihara et al. 1996b). Materials and Methods Plant material Leaves of tea (Camellia sinensis L. cv. Darjeeling) were obtained, in July to October 1995, from two-yearold plants growing under a natural photoperiod in a greenhouse at the University of Glasgow. The developmental stages of the leaves was categorized as (i) young leaves, (ii) mature leaves and (iii) aged leaves. Young leaves were the most recently emerged, weighed ca. 50 mg (FW) and were ca. 28 mm long and 13 mm in width. Mature leaves comprised the fully expanded second and third leaf below the apex (ca. 80 mm long and 30 mm in width, weight ca. 400 mg), while similarly sized aged leaves, from near the base of the shoot, were dark green and weighed ca. 500 mg. Chemicals The following radiochemicals were purchased 413

2 414 Metabolism of caffeine in tea leaves from the commercial sources indicated: [8- I4 C]caffeine (specific activity 1.91 MBq/jmor 1, Moravek Biochemicals Inc., Brea, CA, U.S.A.), [2-14 C]theobromine (2.07 MBq^mol" 1, Moravek), [2-14 C]xanthine (1.94MBq//mol"', Moravek) and [8- l4 C]theophylline (2.04MBq^mol~', American Radiolabelled Chemicals, Inc. St. Louis, MO, U.S.A.). Radiolabelled uric acid, allantoin and allantoic acid were prepared in vitro from [2- l4 C]xanthine using xanthine oxidase (buttermilk), uricase (Candida utilis) and allantoinase (peanut). These enzymes and all other chemicals were purchased from Sigma (St. Louis, MO, U.S.A.). Metabolism ofradiolabelled purine alkaloids and xanthine Segments of tea leaves (ca. 4 mm x 4 mm, 100 mg fresh weight, unless noted otherwise in Table legends) were incubated in 2 ml medium, comprising 30 mm potassium phosphate, ph5.6, 10 mm sucrose and a radiolabelled substrate, in a 30 ml Erlenmeyer flask, in a shaking water bath at 27 C. The 30-ml Erlenmeyer flask had a centre well containing a small glass tube into which was inserted a piece of filter paper wetted with 0.1 ml of 20% potassium hydroxide (w/v). At the end of the incubation period, the glass tube and filter paper from the centre well were transferred to a 50 ml-flask containing 10 ml of distilled water and, after thorough shaking, radioactivity in a 0.5 ml-aliquot was determined by liquid scintillation counting in order to estimate the amount of I4 CO2 released. The tea leaf segments were separated from the incubation medium by filtering through a tea strainer, washed with 50 ml distilled water then transferred to 1 ml of extraction medium, comprising 20 mm sodium diethyldithiocarbamate in 80% methanol and stored at 20 C. The leaf segments were subsequently ground in a pestle and mortar with the same extraction medium (ca. 5 ml) after which the resultant tissue homogenate was centrifuged at 12,000 xg for 5 min and the supernatant and pellet separated. The pellet was resuspended in extraction medium (ca. 5 ml) and recentrifuged. The supernatant fractions, containing the methanol-soluble metabolites, were combined, reduced to dryness in vacuo and aliquots were analysed by HPLC-RC and TLC. HPLC-RC analysis of radiolabelled metabolites An Altex 332 liquid chromatograph (Berkeley, CA, U.S.A.) was used to deliver a 25 min, 0-40% gradient of methanol in 50 mm sodium acetate, ph5.0, at a flow rate of 1 ml min" 1. Samples were introduced off-column via an Altex 210 valve with a 1 ml loop. Reversed phase HPLC utilised a 250- x 4.6-mm i.d. universal ferruleless column (Capital HPLC Specialists, Broxburn, Lothian, U.K.), packed in-house with a 5-j/m ODS Hypersil support (Shandon, Runcorn, Cheshire, U.K.). Column eluate was directed first to a Spectra Physics 8450 absorbance monitor operating at 270 nm and then to a Reeve Analytical 9701 radioactivity monitor (Glasgow, U.K.) fitted with a 200-//1 heterogeneous glass flow cell containing a cerium-activated lithium glass scintillator. Signals from both detectors were processed by a dual channel 2700 data handling system (Reeve Analytical). The gradient elution, reversed phase HPLC system separates 11 different purine derivatives and is also able to resolve 14 C-labelled uric acid, allantoin and allantoic acid produced enzymically from [2- l4 C]xanthine (Ashihara et al. 1996b). Samples containing mono-methylxanthines were also analysed isocratically using a mobile phase of 10% methanol in 50 mm sodium acetate, ph5.0, which provides an enhanced separation of 1-, 3- and 7-methylxanthine (Ashihara et al. 1996b). TLC analysis of radiolabelled metabolites The methanolsoluble metabolites were also analysed by TLC using 200x200 mm sheets of micro crystalline cellulose (Spotfilm, Tokyo Kasei Kogyo Co., Tokyo, Japan). The solvent system used was n- butanol/acetic acid/water (4:1:2, v/v) which separates the radioactive metabolites which had been identified with the HPLC-RC. The Rf values of allantoin, xanthine, 3-methylxanthine, theobromine, theophylline and caffeine were 0.23, 0.35, 0.47, 0.59, 0.68 and 0.78, respectively. Radiolabelled spots, detected after 2-3 weeks exposure using Kodak X-OMAT AR film (Eastman Kodak Co., Rochester, NY, U.S.A.), were scraped off the TLC plates, eluted from the cellulose support with distilled water and the radioactivity was measured by liquid scintillation counting (Ashihara et al. 1996a). Results Metabolism of [8-"]caffeine and [2-' 4 C]theobromine Incubation of tea leaf segments with [2-' 4 C]theobromine resulted in the conversion of the radiolabelled substrate to [ l4 C]caffeine, with no detectable incorporation of radioactivity into other metabolites, including CO 2 (Table 1, Fig. 2A). Theobromine, thus, appears to act as the immediate precursor of caffeine. The highest rate of [ 14 C]caffeine production from [2-14 C]theobromine was observed in mature leaves. Only a small amount of 14 CO 2 was released from [8- l4 C]caffeine incubated with young, mature and aged tea leaves over a 48 h period and no methanol-soluble radiolabelled metabolites were detected (Table 1, Fig.2B). These observations indicate that the rate of caffeine catabolism is extremely low and as a consequence caffeine accumulates as the major purine alkaloid in tea leaves. Incubation of tea leaf segments with 5 mm allopurinol resulted in reduced conversions of [2-14 C]theobromine to [ 14 C]caffeine (Table 1). This suggests that allopurinol, an inhibitor of xanthine dehydrogenase/oxidase (Nguyen 1986), also inhibits S-adenosylmethionine:theobromine-7V-l-methyltransferase activity (see Fig. 1). Metabolism of [8-' 4 C]theophylline-Unlike [2-14 C]- caffeine and [2- l4 C]theobromine, [8-14 C]theophylline was metabolized to a variety of compounds (Table 2, Fig. 3). Metabolism of [8- l4 C]theophylline was especially rapid in mature and aged leaves where more than 75% of the total radioactivity taken up by the tea leaf segments was catabolised and recovered as 14 C-labelled CO 2, 3-methylxanthine, xanthine and allantoin, with trace amounts of label also being incorporated into theobromine and caffeine. The addition of 5 mm allopurinol to the incubation medium resulted in a marked decline in 14 CO 2 production and a concomitant increase in [ l4 C]xanthine, with little effect on the level of radioactivity associated with theobromine and caffeine in the mature and aged leaf incubations. These data indicate that theophylline is catabolised, via 3- methylxanthine, to xanthine which is degraded to CO 2 by the conventional purine catabolic pathway. Young tea leaves catabolised [8-14 C]theophylline more slowly than mature and aged leaves, with a marked reduction in 14 CO 2 output, indicating that this is a consequence of the purine catabolism pathway being less active than in the older leaves. Approximately 20% of total radioactivity

3 Metabolism of caffeine in tea leaves 415 Table 1 Summary of the metabolism of [2-14 C]theobromine and [8-14 C]caffeine by young, mature and aged leaves of Camellia sinensis Substrate Leaves Treatment [2- l4 C]Theobromine Young Mature Aged Distribution of recovered radioactivity (% of total ±SE) Tb Cf CO ± ± ± ± ± ± ± ± ± ± ±0.1 Total radioactivity recovered (kbq±s.e.) 0.64± ± ± ±0.08 O.58±O.O8 O.58±O.O3 [8-14 C]Caffeine Young 99.9 ± ± ±0.04 Aged 99.9 ± ± ±0.51 Mature 99.6± ± ±1.0O Leaf segments incubated with [2-14 C]theobromine (3.7 IcBq) in the presence and absence of 5 mm allopurinol for 24 h and with [8- H C]- caffeine (36.7 kbq) in the absence of allopurinol for 48 h. Total radioactivity recovered presented as kbq±s.d. (n=3). Levels of theobromine (Tb) and caffeine (Cf) and CO 2 are expressed as a percentage of the total radioactivity recovered at the end of the incubation period, = not detected. SAM --. / Xanthosine N Ribose O Ribose 7-Methylxanthosine taken up by the young leaf segments was associated with [ l4 C]caffeine and, to a lesser extent, [ l4 C]theobromine. The conversion of [8- l4 C]theophylline to caffeine, but not theobromine, was inhibited, and the size of the [ 14 C]3-methylxanthine pool enhanced, by the addition of 5 mm allopurinol to the incubation medium (Table 2). This is in keeping with the observation made in the previous section, that allopurinol lowers the rate of conversion of theobro- Ribose- II N r SAM 7-Methylxanthine SAH 3,7-Dimethylxanthine (Theobromine) rv SAM r SAH CH 3 1,3,7-Trimethy Ixan thine (Caffeine) Fig. 1 Pathway for the biosynthesis of caffeine from xanthosine in leaves of C. sinensis L. The numbers relate to the enzymes involved in the individual steps. (I) S-adenosylmethionine: xanthosine-n-7-methyltransferase; (II) Af-methyl nucleosidase; (III) S-adenosylmethionine:7-methylxanthine-/V-3-methyltransferase; (IV) S-adenosylmethionine:theobromine-7V-l-methyltransferase. EC numbers have not yet been assigned to these enzymes.

4 416 Metabolism of caffeine in tea leaves Retention time (mins) 30 Fig. 2 Reversed-phase HPLC-RC analysis of aliquots of methanol extracts from young leaves of C. sinensis following incubation with 3.67 kbq [2-14 C]theobromine for 24 h (A) and 36.7 kbq [8- l4 C]caffeine for 48 h (B). Tb, theobromine; Cf, caffeine. Sample size: trace A-230 Bq, trace B-300 Bq. mine to caffeine. The increased incorporation of label into 3-methylxanthine is probably a consequence of the reduced rate of turnover of theobromine. Metabolism of [2-' 4 C]xanthine The incubation of young, mature and aged tea leaves with [2-14 C]xanthine resulted in the release of very large amounts of I4 CO 2 with most of the remaining radioactivity being associated with residual [2-14 C] xanthine and allantoin (Table 3, Fig. 4). Catabolism of [2-14 C]xanthine to I4 CO 2 was slightly lower in young leaves, which unlike their mature and aged counterparts, incorporated small amounts of label into caffeine, theophylline and theobromine. Inclusion of 5 mm allopurinol in the incubation medium resulted in a significant reduction in the production of 14 CO 2 and [ 14 C]allan Retention time (mins) Fig. 3 Reversed-phase HPLC-RC analysis of aliquots of methanol extracts from young (A), mature (B) and aged (C) leaves of C. sinensis following incubation with 36.7 kbq [8- l4 C]theophyIline for 24 h. AH, allantoin; X, xanthine; 3-mX, 3-methylxanthine; Tb, theobromine; Tp, theophylline; Cf, caffeine. Sample size: A-400 Bq, B-280 Bq, C-290 Bq. toin and a much larger pool of unmetabolised [2- l4 C]xanthine. In young leaves treated with allopurinol, the reduced activity of the purine catabolism pathway was also associat- Table 2 Summary of the metabolism of [2-14 C]theobromine (36.7 kbq) by young, mature and aged leaves of Camellia sinensis in the presence and absence of 5 mm allopurinol for 24 h Leaves Young Mature Aged Treatment Tb 47.3± ± ± ± ± ±0.1 Distributiorl of recovered radioactivity (% of total ±SE) 3-mX X All CO 2 Tb 11.6± ± ± ± ± ± ± ± ± ± ±1.4 5O.7± ± ± ± ± ± ±0.1 1O.8± ± ± ± ± ± ± ± ± ± ± ±0.1 Cf 18.2± ± ± ± ± Total radioactivity recovered (kbq±s.e.) 4.00± ± ± ± ± ±0.06 Total radioactivity recovered presented as kbq per 50 mg fresh weight (young leaves) and per 80 mg fresh weight (mature and aged leaves)±s.e. (n = 3)). Levels of residual [8-14 C]theophylline (Tp) and radiolabelled metabolites, 3-methylxanthine (3-mX), xanthine (X), allantoin (All), CO2, theobromine (Tb) and caffeine (Cf) are expressed as a percentage of the total radioactivity recovered at the end of the 24 h incubation period. = not detected.

5 Metabolism of caffeine in tea leaves 417 g 5 a (A) / n \ \D ) All i I Al! X X l 3-mX Tp Tp I Cf I Retention time (mins) Fig. 4 Reversed-phase HPLC-RC analysis of aliquots of methanol extracts from young leaves of C. sinensis following incubation with 36.7 kbq [2- l4 C]xanthine (A) and 36.7 kbq [2 14 C]xanthine in the presence of 5 mm allopurinol (B) for 24 h. AH, allantoin; X, xanthine; 3-mX, 3-methylxanthine; Tb, theobromine; Tp, theophylline; Cf, caffeine. Sample size: A-370 Bq, B-450 Bq. ed with a very large increase in the radioactivity incorporated into 3-methylxanthine, theophylline, theobromine and caffeine, with these compounds representing more than 40% of the recovered radioactivity compared to 1.4% in control leaf segments (Table 3, Fig. 4). From the amounts of total radioactivity recovered, it can be seen that the uptake of [2-14 C]xanthine by young leaves was much higher than in mature and aged leaves and that this uptake was inhibited, to varying degrees, by the presence of 5 mm allopurinol in the incubation medium (Table 3). This contrasts with the uptake of [2-14 C]theobromine, [8- l4 C]- caffeine (Table 1), and [8- l4 C]theophylline (Table 2), which were not affected to any great extent by either leaf age or allopurinol. 30 Discussion The data obtained in this investigation, demonstrate that, as in C. arabica leaves (Ashihara et al. 1996a, b), theobromine is the immediate precursor of caffeine in C. sinensis. A previous investigation, in which [ l4 C]adenine was used as a substrate, indicated that caffeine biosynthesis is most active in young tea leaves (Ashihara and Kubota 1986). It was unexpected, therefore, that in the present study, the highest rate of [ l4 C]caffeine production from [2- l4 C]theobromine was observed in mature leaves (Table 1). This may genuinely reflect that the conversion of theobromine to caffeine is more rapid in mature leaves, but because of a rate limiting step earlier in the pathway the overall capacity for caffeine biosynthesis is highest in young leaves. Alternatively, the seemingly greater rates of conversion of [2-14 C]theobromine to [ 14 C]caffeine could be due to dilution of the exogenously supplied radiolabelled theobromine by endogenous theobromine, which in tea is present at higher concentrations in young leaves than in mature leaves (Ashihara and Kubota 1986). Caffeine accumulates in tea leaves in concentrations of ca. 2-5% of the dry weight of the leaf (Takeda 1994, Ashihara et al. 1995) and the data presented in this paper demonstrate that this is because caffeine is degraded very slowly, as a consequence of its conversion to theophylline being very effectively blocked (Table 1, Fig. 2). In aged and mature leaves and, to a lesser extent in young leaves, [8-14 C]theophylline is demethylated rapidly via 3-methylxanthine to xanthine, which enters the purine catabolism pathway and is broken down to CO 2 and NH 3. However, most notably in young leaves, [8-14 C]theophylline is also incorporated, in sizable amounts, into theobromine and caffeine (Table 2, Fig 3). In mature and aged C. sinensis leaves, [2- l4 C]xanthine acts almost exclusively as a substrate for the purine ca- Table 3 Summary of the metabolism of [2-14 C]xanthine (36.7 kbq by young, mature and aged leaves of Camellia sinensis in the presence and absence of 5 mm allopurinol for 24 h Leaves Young Mature Aged Treatment X 2.9± ± ± ± ± ±0.7 Distribution of recovered radioactivity (%of total ±SE) All 27.3±13.7 CO2 68.3± mX 0.6±0.4 Tp 0. 3±0.2 Tb 0.1± ± ± ± ± ± ± 7.7± ± 25.4± ± ± ± 17.0± Cf 0.4± ±1.6 Total radioactivity recovered (kbq±s.e.) 7.96± ± ± ± ± ±0.34 Total radioactivity recovered presented as kbq per 50 mg fresh weight (young leaves) and per 80 mg fresh weight (mature and aged leaves)±s.e. (n = 3)). Levels of residual [2-'"C]xanthine (X) and radiolabelled metabolites, allantoin (All), CO 2, 3-methylxanthine (3- mx), theophylline (Tp) theobromine (Tb) and caffeine (Cf) are expressed as a percentage of the total radioactivity recovered at the end of the 24 h incubation period. = not detected.

6 418 Metabolism of caffeine in tea leaves tabolism pathway and, when this route is partially blocked by allopurinol treatment, it is not further metabolised, to any extent, to alternative products. [14C]Allantoin levels and I4CC>2 output indicate that the purine catabolism pathway is also active in young leaves incubated with [2-14C]xanthine. However, young leaves also convert [2-uC]xanthine into small, but readily detectable, quantities of 3-methylxanthine, theophylline, theobromine and caffeine and, in the presence of allopurinol, there is a substantial increase in the incorporation of label into these purine alkaloids (Table 3, Fig. 4). The data obtained in this study indicate that the catabolism and salvage pathways illustrated in Figure 5 operate in leaves of C. sinensis. Theobromine is converted to caffeine which accumulates as its further catabolism to theophylline is blocked in young, mature and aged leaves. The main fate of [8-I4C]theophylline incubated with mature and old leaves, and to a lesser extent young leaves, is conversion to xanthine, via 3-methylxanthine, and entry into the purine catabolism pathway. However in [8-14C]theophylline feeds to young leaves, a significant amount of label is salvaged for the synthesis of caffeine via a 3methylxanthine» theobromine» caffeine pathway. Trace amounts of [2-14C]xanthine are also salvaged for caffeine biosynthesis, and this is increased when purine catabolism is blocked by allopurinol. Salvage of xanthine occurs as a consequence of its conversion to 3-methylxanthine which is metabolised to caffeine via theobromine. Interestingly, the [2-l4C]xanthine feeds to young leaves show that 3-methylO II xanthine, as well as yielding theobromine, is also converted via 1-methylation to theophylline. Supporting evidence for the operation of the pathways illustrated in Figure 5 comes from in vitro studies with N-methyltransferase activity from young leaves of C. sinensis which have shown that (i) xanthine is metabolized to 3-methylxanthine (Negishi et al. 1985), (ii) 3-methylxanthine is converted to theophylline, theobromine and caffeine (Kato et al. 1996) and (iii) theophylline does not act as a methyl acceptor and therefore does not undergo direct conversion to caffeine (Kato et al. 1996). Caffeine homeostasis is similar in leaves of C. sinensis and C. arabica in that caffeine accumulates because its conversion to theophylline is the rate limiting step in the purine alkaloid catabolism pathway (Ashihara et al. 1996b). In both species conversion of [8-14C]theophylline * 3-methylxanthine -* xanthine links theophylline to the purine alkaloid catabolism pathway which results in breakdown to CO2 and NH 3. In young tea leaves there is detectable salvage of 3-methylxanthine and xanthine and resynthesis of caffeine via theobromine. However, there is no evidence for the operation of similar pathways in coffee leaves. Instead, young, aged and mature C. arabica leaves, treated with 5 mm allopurinol, convert xanthine to 7-methylxanthine which does not appear to be further metabolised to any extent (Ashihara et al. 1996b). In general, the caffeine content of C. sinensis leaves at 2-5% dry weight, (Takeda 1994) is higher than the \-2% of caffeine found in C. arabica leaves (Mazzafera et al. 1991). CH3 f CH3 Caffeine CH 3 Theobromine CH 3 Theophylline CH3 3-Methylxanthine HN H N Purine Catabolism Pathway Xandiine Fig. 5 Purine alkaloid catabolism and salvage pathways operating in leaves of C. sinensis. Arrow with two vertical bars represents a blocked conversion. Double headed arrows indicate reversible conversions.

7 Metabolism of caffeine in tea leaves 419 It is possible that this may be a consequence of the salvage of purine alkaloid catabolites for caflfeine synthesis which occurs in tea but not in coffee leaves. The physiological significance of the accumulation of caffeine in young tea leaves remains to be established, although it has been proposed that caffeine may act as a chemical defence that protects young soft tissues from predators, such as insect larvae (see Harborne 1993). This work was supported in part by a Grant-in-Aid for Scientific Research (No ) from the Ministry of Education, Science, Sports and Culture of Japan to H.A., UK-Japan travel grants from the British Council to H.A. and A.C. and a Japanese Society for the Promotion of Science Fellowship for Priority Area Research in Japan (Grant RC10506) to F.M.G. References Ashihara, H. and Kubota, H. (1986) Patterns of adenine metabolism and caffeine biosynthesis in different parts of tea seedlings. Physiol. Plant. 68: Ashihara, H. and Kubota, H. (1987) Biosynthesis of purine alkaloids in Camellia plants. Plant Cell Physiol. 28: Ashihara, H., Monteiro, A.M., Gillies, F.M. and Crozier, A. (1996a) Caffeine biosynthesis in leaves of coffee (Coffea arabica L.) Plant Physiol. Ill: Ashihara, H., Monteiro, A.M., Moritz, T., Gillies, F.M. and Crozier, A. (1996b) Catabolism of caffeine and related purine alkaloids in leaves of Coffea arabica L. Planta 198: Ashihara, H., Shimizu, H., Takeda, Y., Suzuki, T., Gillies, F.M. and Crozier, A. (1995) Caffeine metabolism in high and low caffeine containing cultivars of Camellia sinensis. Z. Naturforsch. 50c: Fujimori, N. and Ashihara, H. (1990) Adenine metabolism and the synthesis of purine alkaloids in flowers of Camellia plants. Phytochemistry 29: Fujimori, N. and Ashihara, H. (1993) Biosynthesis of caffeine in flower buds of Camellia sinensis. Ann. Bot. 71: Fujimori, N., Suzuki, T. and Ashihara, H. (1991) Seasonal variations in biosynthetic capacity for the synthesis of caffeine in tea leaves. Phytochemistry 30: Harborne, J.B. (1993) Introduction to Ecological Biochemistry, 4th Ed., pp Academic Press, London. Kalberer, P. (1964) Unersuchungen zum Abbau des Kaffeins in den Blattern von Coffea arabica. Ber. Schweiz Bot. Ges. 74: Kalberer, P. (1965) Breakdown of caffeine in leaves of Coffea arabica L. Nature 205: Kato, M., Kanehara, T., Shimizu, H., Suzuki, T., Gillies, F.M., Crozier, A. and Ashihara, H. (1996) Caffeine biosynthesis in young leaves of Camellia sinensis: in vitro studies on N-methyltransferase activity involved in the conversion of xanthosine to caffeine. Physiol. Plant. 98: Mazzafera, P., Crozier, A. and Magalhaes, A.C. (1991) Caffeine metabolism in Coffea arabica and other species of coffee. Phytochemistry 30: Negishi, O., Ozawa, T. and Imagawa, H. (1985) Methylation of xanthosine by tea-leaf extracts and biosynthesis of caffeine. Agric. Biol. Chem. 49: Nguyen, J. (1986) Plant xanthine dehydrogenase: distribution, properties and function. Physiol. Veg. 24: Schulthess, B.H., Morath, P. and Baumann, T.W. (1996) Caffeine biosynthesis starts with the metabolically channelled formation of 7-methyl- XMP A new hypothesis. Phytochemistry 41: Suzuki, T., Ashihara, H. and Waller, G.R. (1992) Purine and purine alkaloid metabolism in Camellia and Coffea plants. Phytochemistry 31: Suzuki, T. and Waller, G.R. (1984) Biosynthesis and degradation of caffeine, theobromine and theophylline in Coffea arabica L. fruits. J. Agric. Food Chem. 32: Takeda, Y. (1994) Differences in caffeine and tannin contents between tea cultivars, and application to tea breeding. Jap. Agric. Res. Quart. 28: Terrasaki, Y., Suzuki, T. and Ashihara, H. (1994) Purine metabolism and the biosynthesis of purine alkaloids in tea fruits during development. Life Sa. Adv. (Plant Physiol.) 13: (Received November 13, 1996; Accepted January 21, 1997)