|Vol. 18 >C. Research Notes>V. Gene and genome structure|
|39.||Accumulation of trehalose in transgenic indica rice using bifunctional fusion enzyme of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase of Escherichia coli|
|A. K. GARG1, J.-K. KIM2,
A. RANWALA3 and R. WU1
1)Department of Molecular Biology and Genetics
2)Department of Biological Science, Myongji University, Korea
3)Department of Horticulture, Cornell University, Ithaca, NY 14853
The explosive increase in world population, continuous deterioration
of arable land, shortage of water and environmental stress pose serious
threats to global agricultural production and food supplies. To improve
food production efficiency, we need to deploy favorable strategies to
counteract environmental stresses, such as salinity and drought. One such
protective mechanism could be exploitation and engineering of the genes
for trehalose biosynthesis in plants. In nature, trehalose is known to
act as a protectant against a variety of stresses in different organisms
such as bacteria, fungi, yeast and some invertebrates and vascular plants.
It is involved in osmoregulation, removal of free radicals and stabilization
of the hydrated structure of proteins to maintain membrane integrity and
protein stability under various stress conditions.
response imposed by limited water supply, most of the lines showed higher total water content than the wild-type control plant under drought stress. Some of the promising lines showed 2-to 3-fold higher plant dry biomass production under stress conditions. This was mainly due to better shoot and root growth ratio and maintenance of ion homeostasis, with minimal plant tissue injury. Under normal growth conditions, almost all of the transgenic plants were phenotypically similar to wild-type plants. The data on detailed molecular analysis for transgene copy number, integration site(s), gene expression pattern, inheritance and stability of transgene , will be reported elsewhere.
Trehalose accumulation and carbohydrate profile in several T3 transgenic plants (Pusa Basmati 1) were quantitatively determined by High-performance anion-exchange chromatography, coupled to pulsed amperometric detection (HPAEC-PAD). Leaf sample extracts were prepared from transgenic and non-transformed control plants. Trehalose, glucose, fructose and sucrose were quantified by Dionex HPAEC-PAD. Our results showed low but significant amounts of endogenous trehalose in rice (<30 microg/g FW). In transgenic lines, the non-stressed lines for chloroplast target of transgene accumulated more trehalose than stress-inducible lines, as expected. However, transgenic lines after drought stress showed 200-550 microg/g FW accumulation in highly tolerant lines. From our HPAEC-PAD results, it is quite clear that indeed transgenic rice plants can accumulate 4-to 13-fold higher levels of trehalose accumulation in leaf tissue under salinity or drought stress (Figure 1). Most of the tested transgenic rice plants which were highly tolerant to salinity and drought showed normal phenotype and seed setting. This may be because the fusion genes for trehalose biosynthesis are driven by a stress-inducible or a light-regulated promoter. It is essential to produce transgenic lines with normal growth pattern, and with desirable attributes for genetic improvement, unlike reports in dicots where multiple phenotypic alterations/pleiotropic effects (including drought tolerance) were observed when trehalose biosynthesis gene(s) were expressed constitutively (Holmstrom et al. 1996; Goddijn et al. 1997; Romero et al. 1997; Pilon-Smits et al. 1998; Yeo et al. 2000).
We thank Dr. Ju-Kon Kim for providing the plasmid constructs and TPSP monoclonal antibody. This work was partly supported by the Rockefeller Foundation, U.S.A.
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Seo H. S., Y.J. Koo, J.Y. Lim, J.T. Song, C.H. Kim, J.K. Kim, J.S. Lee, Y.D. Choi, 2000. Characterization of a bifunctional enzyme fusion of trehalose-6-phosphate synthetase and trehalose-6-phosphate phosphatase of Escherichia coli. Appl Environ Microbiol. 66: 2484-2490.
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|Vol. 18>C. Research Notes>V. Gene and genome structure|