Accumulation of trehalose within transgenic chloroplasts confers drought tolerance

Yeast trehalose phosphate synthase (TPS1) gene was introduced into the tobacco chloroplast or nuclear genomes to study resultant phenotypes. PCR and Southern blots confirmed stable integration of TPS1 into the chloroplast genomes of T-1, T-2 and T-3 transgenic plants. Northern blot analysis of transgenic plants showed that the chloroplast transformant expressed 169-fold more TPS1 transcript than the best surviving nuclear transgenic plant. Although both the chloroplast and nuclear transgenic plants showed significant TPS1 enzyme activity, no significant trehalose accumulation was observed in T-0/T-1 nuclear transgenic plants whereas chloroplast transgenic plants showed 15-25 fold higher accumulation of trehalose than the best surviving nuclear transgenic plants. Nuclear transgenic plants (T-0) that showed even small amounts of trehalose accumulation showed stunted phenotype, sterility and other pleiotropic effects whereas chloroplast transgenic plants (T-1, T-2, T-3) showed normal growth and no pleiotropic effects. Transgenic chloroplast thylakoid membranes showed high integrity under osmotic stress as evidenced by retention of chlorophyll even when grown in 6% PEG whereas chloroplasts in untransformed plants were bleached. After 7 hr drying, chloroplast transgenic seedlings (T-1, T-3) successfully rehydrated while control plants died. There was no difference between control and transgenic plants in water loss during dehydration but dehydrated leaves from transgenic plants (not watered for 24 days) recovered upon rehydration turning green while control leaves dried out. These observations suggest that trehalose functions by protecting biological membranes rather than regulating water potential. In order to prevent escape of drought tolerance trait to weeds and associated pleiotropic traits to related crops, it may be desirable to engineer crop plants for drought tolerance via the chloroplast genome instead of the nuclear genome.