Phd Info

Molecular Breeding 11: 1–13, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.

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Accumulation of trehalose within transgenic chloroplasts confers drought tolerance
Seung-Bum Lee 1, Hawk-Bin Kwon 2,3, Soo-Jin Kwon 2, Soo-Chul Park 2, Mi-Jeong Jeong 2, Sang-Eun Han 2, Myung-Ok Byun 2 and Henry Daniell 1,*
1 Molecular Biology and Microbiology Department, University ofCentral Florida, 336 Biomolecular Science (Bldg #20), Orlando, FL 32816-2360, USA; 2Molecular Genetics Division, National Institute of Agricultural Science and Technology, Suwon, 441-707, Korea; 3Current address: Division of Applied Biological Sciences, Sunmoon University, Asan, 336-840, Korea; *Author for correspondence (e-mail: daniell@mail.ucf.edu; phone: 407-823-0952; fax: 407-823-0956)Received 18 July 2001; accepted in revised form 13 November 2001

Key words: Abiotic stress tolerance, Chloroplast genetic engineering, Clean-gene technology, Drought tolerance, Genetically Modified Crops Abstract 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 integrationof 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 observedin 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 normalgrowth 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 betweencontrol 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 andassociated 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. Introduction Water stress due to drought, salinity or freezing is a major limiting factor in plant growth and development. Trehalose is a non-reducing disaccharide of glucose and its synthesis is mediated by the trehalose6-phosphate(T6P) synthase and trehalose-6-phosphate phosphatase complex in Saccharomyces cerevisiae. In S. cerevisiae, this complex consists of at least three subunits performing either T6P synthase (TPS1), T6P phosphatase (TPS2) or regulatory activities (TPS3 or TSL1, Thevelein and Hohmann (1995) and Singer and Lindquist (1998)). Trehalose is found in diverse organisms including algae, bacteria, insects,yeast, fungi, animal and plants (Elbein 1974). Because of its accumulation under various stress conditions such as freezing, heat, salt or drought, there is general consensus that trehalose protects against dam-

2 ages imposed by these stresses (Mackenzie et al. 1988; De Vigilio et al. 1994; Sharma 1997). Trehalose is also known to accumulate in anhydrobiotic organisms that survive complete...