CAFFEINE BIOSYNTHESIS AND PURINE METABOLISM IN LEAVES OF MASCAROCOFFEA SPECIES

Caffeine, a purine alkaloid, was not detected in leaves of two Mascarocoffea species, Coffea millotii and Coffea perrieri. Trigonelline, a pyridine alkaloid, occurred in these species, but the levels (3−4 μmol g fresh weight) were much lower than that of Robusta coffee (Coffea canephora) (36 μmol g fresh weight). Feeding experiments with [8-C]adenine indicated that purine alkaloid biosynthesis was terminated at 7-methylxanthine formation and as a consequence theobromine and caffeine were not produced in Coffea millotii and Coffea perrieri. The adenine salvage activity was lower, but its degradation activity was higher in leaves of these Mascarocoffea species than those in Coffea canephora. The metabolic fate of the purine nucleosides, [8-C]inosine, [8-C]guanosine and [8-C]xanthosine was investigated in leaves of Coffea millotii. The biosynthesis of 7-methylxanthine, but not theobromine or caffeine, from these precursors was detected. Large amounts of these purine nucleosides were catabolized via allantoin. Limited amounts of [8-C]inosine and [8-C]guanosine were salvaged and utilized for RNA synthesis, however, no [8-C]xanthosine salvage was observed. Little or no C-metabolites were observed when [8C]theobromine and [8-C]caffeine were applied to leaf disks of Coffea millotii. From the results obtained in this study, possible metabolic pathways of purines in Mascarocoffea species are discussed.


Introduction
[7][8] In Arabica coffee (Coffea arabica) and Robusta coffee (C.canephora) plants which are used for coffee beverages, rapid biosynthesis of caffeine have been demonstrated in young leaf and fruit tissues, but the degradation of caffeine is very slow, as a result, caffeine is accumulated in mature leaves and seeds. 9The activity of biosynthesis and degradation of caffeine in leaves of low-caffeine-containing Coffea species, C. salvatrix, C. eugenioides, and C. bengalensis has been investigated by Ashihara and  Crozier. 10The biosynthesis of caffeine is less pronounced in leaves of these Coffea species than in C. arabica.Degradation of caffeine was slow in both of high-and low caffeine coffee species, except in C. eugenioides which rapidly catabolises caffeine. 10,11 the present studies, we used two Mascarocoffea spices, C. millotii (Fig. 1) and C. perrieri and the level of caffeine and trigonelline and the activity of caffeine biosynthesis are compared with those in caffeine-accumulating C. canephora.As expected, caffeine was found only in C. canephora.In contrast, trigonelline occurred in all three Coffea samples examined.Since purine and purine alkaloid metabolism have not yet been investigated in Mascarocoffea species, we examined by the feeding experiments with 14 C-labelled adenine, inosine, guanosine, xanthosine, theobromine and caffeine.Possible pathways of purine metabolism in Mascarocoffea species is discussed.

Determination of purine alkaloids and trigonelline
Endogenous levels of purine alkaloids and trigonelline were determined according to Zheng and Ashihara 12 .Samples were ground with 80% methanol in a mortar and pestle.After incubation at 60°C for 30 min, the homogenates were centrifuged and the supernatant was collected.After complete evaporation of the methanol, the extracts were dissolved in distilled water.The aliquots (10-50 μL) of water-soluble fraction were used for determination by HPLC after centrifugation.

C-Feeding experiments
All radiochemicals used were obtained from Moravek Biochemicals Inc, Brea, CA, USA.The labeled compounds were administered essentially as described in previous reports. 11,13,14 I brief, leaves were sterilized with 1 % sodium hypochlorite solution and then washed with sterilized distilled water, and leaf disks (~4 mm x 4 mm) were prepared aseptically.Samples (~100 mg f.w.), and 2.0 mL of 30 mM potassium phosphate buffer (pH 5.6) containing 10 mM sucrose and 1 % sodium ascorbate, were placed in the main compartment of a 30 mL Erlenmeyer flask fitted with a glass tube containing a piece of filter paper impregnated with 0.1 mL of 20 % KOH in the centre well.Each reaction was started by addition of 14 C-labelled purine compounds.
The flasks were incubated in an oscillating water bath at 27°C.After incubation, the glass tube was removed from the center well and placed in a 50 mL Erlenmeyer flask containing 10 mL of distilled water.At the same time, the leaf disks were separated from the incubation medium by filtering through a tea strainer.The samples were washed with distilled water and then frozen with liquid N2 and stored at -80° C. The KH 14 CO3 that had been absorbed by the filter paper was allowed to diffuse overnight into the distilled water, and 0.5 mL aliquots of the resulting solution were used for the determination of radioactivity incorporated into CO2.
Metabolites from 14 C-labelled purine bases and nucleosides were extracted successively with 4 mL of cold 6 % perchloric acid solution.Lipids in the insoluble materials were removed with a mixture of ethanol and diethyl ether (1:1, v/v) at 50°C for 15 min.The precipitate was washed with the same mixture, then with distilled water.RNA in the insoluble fraction was hydrolysed with 0.3 M KOH at 37°C for 18 h, and RNA hydrolysates was obtained. 14e perchloric acid-soluble metabolites and the RNA hydrolysates were neutralized with KOH and radioactive metabolites were separated by TLC using cellulose plates and the solvent systems shown in a previous paper. 14adioactivity of liquid samples and on the TLC plates was determined using a multipurpose scintillation counter (Beckman, LS 6500) and a bio-imaging analyzer (Type FLA-2000, Fuji Photo Film Co. Ltd.), respectively.

Endogenous levels of caffeine and trigonelline
Table 1 shows the leaf sizes and fresh weights of two species of Mascarocoffea and Coffea canephora.We used well-developed young leaves in this study.Accumulation of caffeine was found only in C. canephora while caffeine could not be detected in C. millotii and C. perrieri.In contrast, trigonelline was found in the leaves of all samples.The level of trigonelline in C. perrieri and C. millotii leaves were respectively 8-and 12-fold lower than the level in C. canephora leaves.

Comparison of purine alkaloid biosynthesis
Among exogenously administered purine precursors, it has been known that adenine is the most effective precursor for the biosynthesis of caffeine. 15Therefore, we first examined the metabolic fate of [8-14 C]adenine in two Mascarocoffea species, C. millotii and C. perrieri and compared with the metabolism in C. canephora.As shown in Table 2, a limited amount of radioactivity from [8-14 C]adenine accumulated as methylxanthines (purine alkaloids).Although radioactivity was found in 7methylxanthine in all three species, incorporation into theobromine and caffeine was found only in C. canephora.In C. canephora, more radioactivity was found in 7methylxanthine and theobromine than in caffeine.This is probably due to the fact that the precursors could not be transformed into caffeine during the incubation period, although caffeine is the end-product.

Comparison of adenine salvage and degradation
In addition to purine alkaloids, the radioactivity from [8-14 C]adenine was distributed in various metabolites (Table 2).Since only small amounts of [8-14 C]adenine (2−8 % of total radioactivity) were detected in the leaf disks, exogenously supplied [8-14 C]adenine was not retained as adenine, but rapidly metabolized to other compounds.Substantial amounts of radioactivity from [8-14 C]adenine was incorporated into the salvage products, namely, nucleotides and RNA.However, compared with C. canephora (73 % of total radioactivity), the rate of adenine salvage (38−51 %) was low in Mascarocoffea species.
As shown in other plants, adenine was converted to AMP by adenine phosphoribosyltransferase (EC 2.4.2.7, step 1 in Fig. 2) and then entered the adenine nucleotide pool comprising AMP, ADP and ATP.Some ATP was utilized for RNA synthesis. 10In addition to the methylxanthines shown above, radioactivity was also found in xanthosine, hypoxanthine and xanthine.In C. millotii and C. perrieri, significant amounts of radioactivity (25−37 %) were incorporated in the purine degradation products, ureides (allantoin and allantoic acid) and CO2.

Comparison of purine metabolism in caffeine reduced Coffea plants
Biosynthesis and degradation of caffeine occur in low caffeine Coffea plants including native and transgenic plants. 9In most cases, the activity of N-methyltransferase is reduced.There are at least two distinct N-methyltransferases, 7-methylxanthosine synthase and dual functional caffeine synthase which catalyzes the last two steps, namely, 7methylxanthine → theobromine → caffeine (steps 13 and 14 in Fig. 2). 9The reduction of activity of N-methyltransferase activity was suggested in all cases.29]  In the present study, we found that the purine alkaloid synthesis was stopped at 7-methylxanthine, thus dual functional caffeine synthase activity appeared to be missing in Mascarocoffea species.]Inosine (10 μM, specific activity 1.9 GBq mmol -1 ), [8- 14 C]guanosine (10 μM, specific activity 1.9 GBq mmol -1 ) and [8- 14 C]xanthosine (9 μM, specific activity 2.1 GBq mmol -1 ) were administered for 18 h.The mean incorporation of radioactivity into individual metabolites is expressed as the percentage of total radioactivity taken up by the segments ± SD.Total uptake is expressed as kBq 100 mg f.w. - , nd, not detected.
In contrast, Silvarolla et al. 30 reported that theobromine was accumulated in a naturally decaffeinated C. arabica plant from Ethiopia designated "AC".They found that the leaves accumulated radioactivity in theobromine when [ 14 C]adenine was fed to the "AC" plants, with none being incorporated into caffeine.In contrast, no difference was found in degradation of [ 14 C]caffeine between in the "AC" plants and caffeine accumulating normal C. arabica plants.These results indicate that the low caffeine content in the "AC" plants is due to the lack of the activity of Nmethyltransferase which catalyzes the last step in the caffeine biosynthesis pathway (step 14 in Fig. 2).Although no data was shown, they mentioned that no caffeine synthase activity was detected in the leaves, and concluded that the caffeine synthase gene had mutated in the AC plants.In this case, theobromine synthase which is distinct from a dual-functional caffeine synthase may be functional.
The reduction of caffeine synthase has also been suggested in low-caffeine hybrid coffee by 14  ) and [8-14 C] caffeine (10 μM, specific activity 2.0 GBq mmol -1 ) were administered for 18 h.The mean incorporation of radioactivity into individual metabolites is expressed as the percentage of total radioactivity taken up by the segments ± SD.Total uptake is expressed as kBq 100 mg f.w. - , nd, not detected.
Similar results were also demonstrated in anti-sense and RNA interference transgenic plants of C. canephora in which the expression of an N-methyltransferase gene was suppressed.Compared with wild-type control plants, total purine alkaloid biosynthesis from adenine and conversion of theobromine to caffeine were both reduced in the transgenic plants.In the transgenic plants, metabolism of [8-14 C]adenine shifted from purine alkaloid synthesis to purine catabolism or salvage for nucleotides.
From these results, we can speculate that in most caffeinefree Coffea plants including Mascarocoffea species, caffeine synthase-related genes are varied and, as a result, there is reduced the enzyme activity in planta.In these plants, the metabolic flow of purine metabolites to the purine alkaloids synthesis appeared to be shifted to the purine catabolism.
theobromine and [8-14 C]caffeine in the disks of C. millotii is presented in Table

Table 1 .
Leaf sizes (length and width) and purine alkaloid contents in three different Coffea species used in this research.nd, not detected.

Table 2 .
Comparison of adenine metabolism in leaf segments of millotii, C. perrieri, and C. canephora.

Table 4 .
C-feeding experiments, the hybrid named GCAs which are new tetraploid interspecific hybrids developed in Madagascar from C. eugenioides, C. canephora and C. arabica.Selected GCA contained low caffeine (<0.4 % dry wt.) and no detectable theobromine in seeds.Low caffeine accumulation in GCA plants is due mainly to the low caffeine biosynthesis activity, possibly due to extremely weak Nmethyltransferase reactions.No significant catabolic activity of caffeine was found in the GCA, in common with C. arabica.Metabolism of theobromine and caffeine in Coffea mellotii leaf segments.