Caffeine is synthesized from xanthosine through N-methylation and ribose removal steps. In the present study, three types of cDNAs encoding N-methyltransferases were isolated from immature fruits of coffee (Coffea arabica) plants, and designated as CaXMT1, CaMXMT2, and CaDXMT1, respectively. The bacterially expressed encoded proteins were characterized for their catalytic properties. CaXMT1 catalyzed formation of 7-methylxanthosine from xanthosine with a K m value of 78 m, CaMXMT2 catalyzed formation of 3,7-dimethylxanthine (theobromine) from 7-methylxanthine with a K m of 251 m, and CaDXMT1 catalyzed formation of 1,3,7-trimethylxanthine (caffeine) from 3,7-dimethylxanthine with a K m of 1,222 m. The crude extract of Escherichia coli was found to catalyze removal of the ribose moiety from 7-methylxanthosine, leading to the production of 7-methylxanthine. As a consequence, when all three recombinant proteins and E. coli extract were combined, xanthosine was successfully converted into caffeine in vitro. Transcripts for CaDXMT1 were predominantly found to accumulate in immature fruits, whereas those for CaXMT1 and CaMXMT2 were more broadly detected in sites encompassing the leaves, floral buds, and immature fruits. These results suggest that the presently identified three N-methyltransferases participate in caffeine biosynthesis in coffee plants and substantiate the proposed caffeine biosynthetic pathway: xanthosine 3 7-methylxanthosine 3 7-methylxanthine 3 theobromine 3 caffeine.

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The caffeine biosynthetic pathway in coffee plants has been proposed to involve three distinct N -methyltransferases, xanthosine methyltransferase (XMT), 7- N -methylxanthine methyltransferase (MXMT; theobromine synthase), and 3,7-dimethylxanthine methyltransferase (DXMT; caffeine synthase). We previously isolated all corresponding cDNAs designated as CaXMT1 , CaMXMT1 , CaMXMT2 and CaDXMT1 , respectively, and showed that caffeine was indeed synthesized in vitro by the combination of their gene products. In order to regulate caffeine biosynthesis in planta , we suppressed expression of CaMXMT1 by the double stranded RNA interference (RNAi) method. For this purpose, we first established a protocol for efficient somatic embryogenesis of Coffea arabica and C. canephora , and then Agrobacterium -mediated transformation techniques. The RNAi transgenic lines of embryogenic tissues derived from C. arabica and transgenic plantlets of C. canephora demonstrated a clear reduction in transcripts for CaMXMT1 in comparison with the control plants. Transcripts for CaXMT1 and CaDXMT1 were also reduced in the most cases. Both embryonic tissues and plantlets exhibited a concomitant reduction of theobromine and caffeine contents to a range between 30% and 50% of that of the control. These results suggest that the CaMXMT1 -RNAi sequence affected expression of not only CaMXMT1 itself, but also CaXMT1 and CaDXMT1 , and that, since the reduction in theobromine content was proportional to that for caffeine, it is involved in the major synthetic pathway in coffee plants. The results also indicate that the method can be practically applied to produce decaffeinated coffee plants.

Caffeine (1,3,7-trimethylxanthine) is derived from xanthosine through three successive transfers of methyl groups and a single ribose removal in coffee plants. The methyl group transfer is catalyzed by N-zmethyltransferases, xanthosine methyltransferase (XMT), 7-methylxanthine methyltransferase (MXMT) and 3,7-dimethylxanthine methyltransferase (DXMT). We previously cloned three genes encoding each of these N-methyltransferases from coffee plants, and reconstituted the final sequence of the caffeine synthetic pathway in vitro. In the present study, we simultaneously expressed these coffee genes in tobacco plants (Nicotiana tabacum), using a multiple-gene transfer method, and confirmed successful caffeine production up to 5 microg g(-1) fresh weight in leaves of the resulting transgenic plants. Their effects on feeding behavior of tobacco cutworms (Spodoptera litura), which damage a wide range of crops, were then examined. Leaf disc choice test showed that caterpillars selectively fed on the wild-type control materials, or positively avoided the transgenic materials. The results suggest a novel approach to confer self-defense by producing caffeine in planta. A second generation of transgenic crops containing caffeine may save labor and agricultural costs and also mitigate the environmental load of pesticides in future.