March, 2003

by Ray Wu and Ajay Garg
Department of Molecular Biology and Genetics
Cornell University, USA
ray.wu@cornell.edu 

Rice is a major source of food for more than 2.7 billion people on a daily basis. Rice is planted on about one-tenth of the earth's arable land and is the single largest source of food energy to half of humanity. Of the 130 million hectares of land where rice is grown, about 30 percent contain levels of salt too high to allow normal rice yield. Another 20 percent of this land is periodically subject to drought conditions that routinely affect food production. About 10 percent of the locations where rice is grown occasionally experience temperatures that are too low for healthy plant development. It is difficult to improve rice tolerance against these abiotic stresses because they involve not a single gene but a network of genes. Fortunately, recent developments in transgenic approaches offer new opportunities to elucidate the functions of many useful candidate genes from different organisms and to improve the resilience and yield of rice plants. Moreover, developing salt-tolerant transgenic rice plants can introduce new areas of land that currently contain salt too high to grow rice. It is expected that genetically engineered, improved rice varieties will help combat world hunger and poverty.

In general, plants respond to environmental stresses (drought, excessive salinity, and low temperature) through a wide variety of biochemical and physiological adaptive changes, such as the accumulation of compatible solutes (glycine betaine, proline, polyamines, and trehalose) and synthesis of many regulatory proteins. One such compound is trehalose, a non-reducing disaccharide of glucose, which plays an important role in stress protection in a large variety of organisms ranging from bacteria and fungi to invertebrate animals. For example, brine shrimp eggs, commercially marketed as fish food or as pet "sea monkeys," can remain dehydrated for years in a state of suspended animation due to their trehalose content. Trehalose also acts as a storage carbohydrate, and it possesses the unique feature of reversible water absorption capacity to protect biological structures from damage during desiccation. When water dissipates from the shell of macromolecules (such as protein) during severe dehydration, trehalose can act as a water substitute on the surface of the dried protein.1 Thus, the native folding and biological activity of proteins are maintained, and denaturation and aggregation are prevented. These protective properties of trehalose are clearly superior to those of other sugars, such as sucrose, making trehalose an ideal stress protectant.

Despite the wide distribution of trehalose in microorganisms and invertebrates, trehalose had until recently only been found in a few plant species, notably highly desiccation-tolerant, resurrection plants [club mosses Selaginella lepidophylla and the angiosperm Myrothamnus flabellifolius], so named because of their unique ability to fully recover from a state of almost complete loss of water. These resurrection plants can accumulate trehalose at levels approaching 1% of dry weight under non-stress
conditions, whereas the majority of plants do not appear to accumulate easily-detectable amounts of trehalose. However, genes that encode enzymes of trehalose synthesis, i.e., trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) (Figure 1), have been recently identified in a number of plants. This suggests that the ability to synthesize low amounts of endogenous trehalose may be widely distributed in the plant kingdom.

Recently, several research groups have attempted to study, via genetic engineering, the role of trehalose in abiotic stress protection and carbohydrate metabolism in plants. In all previous studies, engineering constitutive overexpression of TPS- and/or TPP-encoding genes from yeast or Escherichia coli in tobacco or potato resulted in enhanced trehalose levels and drought tolerance. However, constitutive overexpression of these genes also leads to unfavorable developments, such as stunted plant growth, lancelet leaves, and altered roots, as well as changes in carbohydrate metabolism under normal growth conditions.2,3,4,5 Recently, we reported an alternate strategy to engineer increased trehalose accumulation in rice in such a way that trehalose synthesis occurs only when there is abiotic stress. We used a stress-inducible promoter to drive the overexpression of Escherichia coli trehalose biosynthetic genes (otsA and otsB) as a fusion gene (TPSP) for providing abiotic stress tolerance in rice.6 The TPSP fusion gene7 has the dual advantages of necessitating only a single transformation event to introduce both genes simultaneously into the rice genome, while at the same time increasing the catalytic efficiency for trehalose formation by the bifunctional enzyme.

Figure 1. Trehalose synthesis and degradation pathway in bacteria and plants

We introduced these two genes, which are responsible for the synthesis of trehalose, into an important variety of rice plant (Pusa Basmati 1) by Agrobacterium-mediated gene transfer and created a large number of transgenic rice plants that are completely fertile and grow well under normal growth conditions.(6)

The genetically-engineered rice plants produced higher amounts of trehalose. Importantly, since our custom-designed inducible promoter that drives the fusion gene is expressed only under stress, the plants grow normally without any undesirable effects. This is in contrast to previous experiments in which researchers have constitutively expressed an individual TPS or TPP gene so that it is turned on all the time, which stunts the growth of plants.

Furthermore, the transgenic rice plants exhibited sustained plant growth, less photo-oxidative damage, and more favorable mineral balance under salt, drought, and low-temperature stress conditions as compared to non-transgenic plants, many of which died due to salt stress. Depending on growth conditions, the transgenic rice plants accumulate trehalose at levels 3 to 10 times that of the non-transgenic controls. The observation that peak trehalose levels remain well below 1 mg/g fresh leaf or root weight indicates that the primary effect of trehalose is not just serving as a compatible solute. Rather, increased trehalose accumulation correlates with higher soluble carbohydrate levels and an elevated capacity for photosynthesis under both stress and non-stress conditions, consistent with a suggested role in modulating sugar sensing and carbohydrate metabolism. These findings demonstrate the feasibility of engineering rice for increased tolerance of abiotic stress and enhanced productivity through stress-dependent or tissue-specific overproduction of trehalose.6

Our laboratory has experimented with six other genes, each of which provides some degree of stress tolerance. What is special about the two genes responsible for the overproduction of trehalose is that the degree of protection from stresses has been much higher than with the other genes reported previously.

In conclusion, we have demonstrated that engineering trehalose overproduction in rice can be achieved by stress-inducible or tissue-specific expression of a bifunctional TPSP fusion gene without any detrimental effect on plant growth or grain yield. During abiotic stress, transgenic plants accumulated increased amounts of trehalose and showed high levels of tolerance to salt, drought, and low-temperature stresses, as compared to non-transgenic plants. These results demonstrate the potential use of our transgenic approach in developing new rice cultivars with increased abiotic stress tolerance and enhanced rice productivity. In principle, this same technique can be used to confer stress tolerance on other high-value, sensitive crops such as wheat and corn.

References
1. Crowe JH, Hoekstra FA, Crowe LM. 1992. Anhydrobiosis. Annu Rev Physiol 54:579-599.

2. Goddijn OJ et al. 1997. Inhibition of trehalase activity enhances trehalose accumulation in transgenic plants. Plant Physiol 113:181-190.

3. Holmstrom KO et al. 1996. Drought tolerance in tobacco. Nature 379:683-684.

4. Pilon-Smits EAH et al. 1998. Trehalose-producing transgenic tobacco plants show improved growth performance under drought stress. J Plant Physiol 152: 525-532.

5. Romero C et al. 1997. Expression of the yeast trehalose-6-phosphate synthase gene in transgenic tobacco plants: pleiotropic phenotypes include drought tolerance. Planta 201: 293-297.

6. Garg AK et al. 2002. Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses. Proc Natl Acad Sci USA 99: 15898-15903.

7. Seo HS et al. 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.