Caffeine Production in Tobacco Plants by Simultaneous Expression of Three Coffee N-methyltrasferases and Its Potential as a Pest Repellant

Abstract

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.

Simultaneous activation of salicylate production and fungal resistance in transgenic Chrysanthemum producing caffeine

“…8 The resulting transformed plantlets were finally selected on MS medium containing 250 mg l -1 cefotaxime and 10 mg l -1 kanamycin. The wild-type and transgenic plants (lines C#1, C#2 and C#3) were grown in a closed greenhouse at 25°C under natural day conditions.…”

“…Caffeine content was determined as described using HPLC. 8 Infection test of gray mold (Botrytis cinerea) was performed with healthy detached leaves as followings: spores of B. cinerea (1 x 10 5 spores ml -1 ) suspended in 5 mm diameter x 3 mm thick agar disks (1% agar, 2.5% glucose) were deposited on the adaxial side of each chrysanthemum leaflets and incubated in a wet chamber at 100% relative humidity for 15 days at 20°C (light period 12 h day -1 ). Susceptibility was determined by measuring the diameter of necrotic lesions.…”

“…In three transgenic lines, caffeine was accumulated at 3 μg g -1 fresh weight of the tissue, being comparable with reported values in transgenic tobacco plants. 8,9 Transgenic and wild type chrysanthemum plants were tested for resistance against Botrytis cinerea, a necrotrophic fungus that affects many plant species. In the wild type plant, lesions appeared 72 h after inoculation, and rapidly developed from the infected site to outward leaves (Fig.…”

“…Transgenic tobacco plants producing caffeine were previously shown to acquire resistance against pathogen and herbivore attacks. 8,9 Molecular analyses suggested that the effect of caffeine was not direct by the toxicity to the attackers, but indirect by the stimulation of a self-defense system. 9 It was conceivable that since endogenous caffeine is mildly toxic the host plant counteracted the situation by activating the self-defense.…”

“…8 The resulting plants exhibited a high resistance against herbivores 8 and viral and bacterial pathogens. 9 A novel feature of this observation was that the caffeine effect appeared not to be direct toxicity, but indirect stimulation of the defense network, or systemic-acquired resistance of the host plant.…”