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.

In E. coli, biosynthesis of trehalose is a two-step process, consisting of the conversion of UDP-glucose and glucose-6-phosphate into trehalose-6-phosphate by trehalose-6-phosphate synthase (TPS), and subsequently, dephosphorylation by trehalose-6-phosphate phosphatase (TPP). An operon (OtsBA) that harbors the OtsA and OtsB genes encode for TPS and TPP, respectively. Since the substrates for TPS and TPP are readily available in the cytosol of plant cells, introduction of TPS/TPP enzyme activity in this cell compartment is expected to lead to the formation of trehalose-6-phosphate and conversion into trehalose by utilizing a wide array of phosphatases. The role of trehalose (and even its actual presence) in plants remains an open question although evidence for its regulatory role is emerging. However, the level of trehalose in agronomically important crop species has not been reported. Therefore, we are interested to introduce trehalose biosynthetic fusion genes (OtsA and OtsB) from E. coli (Seo et al. 2000) into rice to test whether an important crop plant can accumulate trehalose, and to evaluate the capacity of trehalose to protect plant tissues against various abiotic stresses.

For genetic engineering of indica rice (Pusa Basmati 1), two plasmids were constructed using the OtsA and OtsB fusion genes in frame that encodes for TPS and TPP, respectively. The fused genes (TPSP) were either driven by a stress-inducible promoter, ABRC complex for cytosolic expression, or linked to a rice rbcS promoter with transit peptide sequences for chloroplast targeting of the transgene. Using the Agrobacterium-mediated transformation method, the gene was incorporated into rice genomic DNA and we obtained a total of 104 putative transgenic lines. Twenty-eight independent lines were obtained with an ABA-inducible promoter (ABA lines) while 76 lines with rbcS promoter plus a transit peptide (rbcS lines). Basta herbicide resistance test on 104 putative lines showed marker (bar) transgene in approximately 90% of these plants. Southern blot analysis from primary transformants showed 91% in ABA lines and 75% in rbcS lines to contain the TPSP transgene. About 40% of the transgenic plants harbor a single copy, and 70-80% of plants harbor 1-3 copies of the transgene. Genetic segregation analysis from 78 independent T1 lines for the marker gene showed Mendelian segregation in approximately 60% of the lines. Immunoblot analysis from T1 non-stressed seedlings showed more TPSP recombinant fusion protein in rbcS lines than in ABA lines as detected by TPSP monoclonal antibodies. Regulated expression of the fusion transgene had no deleterious effect on plant morphology or growth in several independent transgenic rice lines in T1, T2, T3 and subsequent generations.

A total of 56 homozygous, T3 generation, independent transgenic lines were evaluated for salt and/or drought tolerance, along with several non-transformed control plants. Many single-copy transgenic lines showed markedly enhanced tolerance to 100 mM NaCl treatment after four weeks of continued salt stress. Growth, water content, plant stress injury index, photo-synthesis (PS II) and ion uptake and translocation were measured under salt-stress conditions. The transgenic lines had vigorous root and shoot growth and robust plant type under salt-stress conditions, whereas the control non-transformed plants showed major growth inhibition.

In another experiment, when the same set of transgenic lines was also evaluated for drought-stress

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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