Expression of a Bifunctional Fusion of the Escherichia coli Genes for Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in Transgenic Rice Plants Increases Trehalose Accumulation and Abiotic Stress Tolerance without Stunting Growth

Abstract

Trehalose plays an important role in stress tolerance in plants. Trehalose-producing, transgenic rice (Oryza sativa) plants were generated by the introduction of a gene encoding a bifunctional fusion (TPSP) of the trehalose-6-phosphate (T-6-P) synthase (TPS) and T-6-P phosphatase (TPP) of Escherichia coli, under the control of the maize (Zea mays) ubiquitin promoter (Ubi1). The high catalytic efficiency (Seo et al., 2000) of the fusion enzyme and the single-gene engineering strategy make this an attractive candidate for high-level production of trehalose; it has the added advantage of reducing the accumulation of potentially deleterious T-6-P. The trehalose levels in leaf and seed extracts from Ubi1::TPSP plants were increased up to 1.076 mg g fresh weight Ϫ1 . This level was 200-fold higher than that of transgenic tobacco (Nicotiana tabacum) plants transformed independently with either TPS or TPP expression cassettes. The carbohydrate profiles were significantly altered in the seeds, but not in the leaves, of Ubi1::TPSP plants. It has been reported that transgenic plants with E. coli TPS and/or TPP were severely stunted and root morphology was altered. Interestingly, our Ubi1::TPSP plants showed no growth inhibition or visible phenotypic alterations despite the high-level production of trehalose. Moreover, trehalose accumulation in Ubi1::TPSP plants resulted in increased tolerance to drought, salt, and cold, as shown by chlorophyll fluorescence and growth inhibition analyses. Thus, our results suggest that trehalose acts as a global protectant against abiotic stress, and that rice is more tolerant to trehalose synthesis than dicots.is a nonreducing diglucoside that is found in various organisms, including bacteria, algae, fungi, yeast (Saccharomyces cerevisiae), insects, and some plants (Elbein, 1974). Trehalose serves not only as a carbohydrate reserve, but also as a protective agent against a variety of physical and chemical stresses in various organisms (van Laere, 1989;Wiemken, 1990;Eleutherio et al., 1993;Strøm and Kassen, 1993). Trehalose is known to have high water retention activity, which maintains the fluidity of membranes under dry conditions (Leslie et al., 1995). Thus, this sugar allows desert plants to tolerate naturally occurring stresses during cycles of dehydration and rehydration (Drennan et al., 1993;Mü ller et al., 1995).A role for trehalose in stress tolerance has been demonstrated for cryptobiotic plant species, such as the desiccation-tolerant Selaginella lepidophylla. In this case, trehalose accumulation represented 12% of the plant dry weight during dehydration, which probably protected the proteins and membrane structures. Upon rehydration, S. lepidophylla regained complete viability and the trehalose levels declined (Goddijn and van Dun, 1999). Plants accumulate a number of osmoprotective agents, such as Pro, in response to NaCl stress. During osmotic stress in rice (Oryza sativa), trehalose or similar carbohydrates appear to be more important than Pro. It has been shown that treatmen...

A bifunctional TPS–TPP enzyme from yeast confers tolerance to multiple and extreme abiotic-stress conditions in transgenic Arabidopsis

“…For example, the overexpression of the TPS1 gene in tobacco, potato and tomato improved drought tolerance (Holmström et al 1996;Romero et al 1997;Yeo et al 2000;Cortina and Culiáñez-Macià 2005). Transgenic rice expressing the E. coli TPS-TPP were tolerant to drought, chilling and salt stress (Garg et al 2002;Jang et al 2003). Similarly, in the present study we showed that Arabidopsis plants overexpresing the TPS1-TPS2 gene displayed improved tolerance to several abiotic stresses.…”

“…Saccharomyces cerevisiae can grow in the presence of 0.9 M NaCl when a plant or yeast TPS1 gene is overexpressed (Zentella et al 1999). Rice plants can tolerate 100 mM NaCl after overexpressing an E. coli TPS-TPP gene fusion (Garg et al 2002;Jang et al 2003). We showed that plants expressing the yeast TPS-TPP bifunctional enzyme could tolerate signiWcantly higher -350 mM).…”

“…On the other hand, the expression of yeast TPS1-TPS2 in Arabidopsis did not provoke growth or morphological alterations, even if the strong and constitutive 35S promoter was used. Similarly, when the bifunctional E. coli TPS-TPP enzyme was overexpressed in plants it resulted in rice plants with a normal morphology and growth (Garg et al 2002;Jang et al 2003).…”

“…Previous work have shown that accumulation of trehalose in transgenic rice allowed recovery of plants after 6 h of 4°C treatment (Jang et al 2003) or 3 days at 10°C (Garg et al 2002). Recently, it has been shown that a TPP coding gene from rice is induced by chilling-stress (Pramanik and Imai 2005).…”

“…Expression in rice of a gene fusion of otsA (TPS1 homologue) and otsB (TPS2 homologue) driven by a stress-regulated promoter conferred resistance to several abiotic stresses without causing morphological changes (Garg et al 2002;Jang et al 2003).…”