Ray Wu

Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses

Trehalose is a nonreducing disaccharide of glucose that functions as a compatible solute in the stabilization of biological structures under abiotic stress in bacteria, fungi, and invertebrates. With the notable exception of the desiccation-tolerant “resurrection plants,” trehalose is not thought to accumulate to detectable levels in most plants. We report here the regulated overexpression of Escherichia coli trehalose biosynthetic genes (otsA and otsB) as a fusion gene for manipulating abiotic stress tolerance in rice. The fusion gene has the advantages of necessitating only a single transformation event and a higher net catalytic efficiency for trehalose formation. The expression of the transgene was under the control of either tissue-specific or stress-dependent promoters. Compared with nontransgenic rice, several independent transgenic lines exhibited sustained plant growth, less photo-oxidative damage, and more favorable mineral balance under salt, drought, and low-temperature stress conditions. Depending on growth conditions, the transgenic rice plants accumulate trehalose at levels 3–10 times that of the nontransgenic controls. The observation that peak trehalose levels remain well below 1 mg/g fresh weight indicates that the primary effect of trehalose is not as a compatible solute. Rather, increased trehalose accumulation correlates with higher soluble carbohydrate levels and an elevated capacity for photosynthesis under both stress and nonstress 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 tissue-specific or stress-dependent overproduction of trehalose.

Expression of a Late Embryogenesis Abundant Protein Gene, HVA1, from Barley Confers Tolerance to Water Deficit and Salt Stress in Transgenic Rice

A late embryogenesis abundant (LEA) protein gene, HVA1, from barley (Hordeum vulgare L.) was introduced into rice suspension cells using the Biolistic-mediated transformation method, and a large number of independent transgenic rice (Oryza sativa L.) plants were generated. Expression of the barley HVA1 gene regulated by the rice actin 1 gene promoter led to high-level, constitutive accumulation of the HVA1 protein in both leaves and roots of transgenic rice plants. Second-generation transgenic rice plants showed significantly increased tolerance to water deficit and salinity. Transgenic rice plants maintained higher growth rates than nontransformed control plants under stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by stress and by improved recovery upon the removal of stress conditions. We also found that the extent of increased stress tolerance correlated with the level of the HVA1 protein accumulated in the transgenic rice plants. Using a transgenic approach, this study provides direct evidence supporting the hypothesis that LEA proteins play an important role in the protection of plants under water-or salt-stress conditions. Thus, LEA genes hold considerable potential for use as molecular tools for genetic crop improvement toward stress tolerance.

Construction of expression vectors based on the rice actin 1 (Act1) 5' region for use in monocot transformation.

SummaryIt has been previously reported that the 5′ region of the rice actin 1 gene (Act1) promoted high-level expression of a β-glucuronidase reporter gene (Gus) in transformed rice cells. In this paper we describe the construction of Act1-based expression vectors for use in monocot transformation. As part of the development of these vectors, we have evaluated the influence of the Act1 first intron, the Act1-Gus junction-encoded N-terminal amino acids, and the sequence context surrounding the Act1 and Gus translation initiation site on Act1-Gus gene expression in rice and maize cells. We have found that addition of Act1 intron 1 to the transcription unit of a Gus reporter gene under control of the cauli-flower mosaic virus (CaMV) 35S promoter stimulated GUS activity more than 10-fold in transformed rice cells. Optimization of the sequence context around the Gus translation initiation site resulted in a 4-fold stimulation of Gus expression in transformed rice cells. By utilizing both the Act1 intron 1 and optimized Gus translation initiation site, a 40-fold stimulation in Gus expression from the CaMV 35S promoter has been achieved in transformed rice cells; very similar results were obtained in transformed maize cells. Taken together these results suggest that the Act1-based expression vectors described here should promote the expression of foreign genes in most, if not all, transformed monocot cells to levels that have not previoulsy been attainable with alternative expression vectors.

Overexpression of a Δ1-pyrroline-5-carboxylate synthetase gene and analysis of tolerance to water- and salt-stress in transgenic rice

Abstract A Δ 1 -pyrroline-5-carboxylate synthetase (P5CS) cDNA from mothbean ( Vigna aconitifolia L.) was introduced into rice ( Oryza sativa L.) genome by the biolistic method. Expression of this P5CS transgene under the control of a stress-inducible promoter led to stress-induced overproduction of the P5CS enzyme and proline accumulation in transgenic rice plants. Second-generation (R 1 ) transgenic rice plants showed an increase in biomass under salt-stress and water-stress conditions as compared to the non-transformed control plants.

Regeneration of herbicide resistant transgenic rice plants following microprojectile-mediated transformation of suspension culture cells

SummarySuspension cells of Oryza sativa L. (rice) were transformed, by microprojectile bombardment, with plasmids carrying the coding region of the Streptomyces hygroscopicus phosphinothricin acetyl transferase (PAT) gene (bar) under the control of either the 5′ region of the rice actin 1 gene (Act1) or the cauliflower mosaic virus (CaMV) 35S promoter. Subsequently regenerated plants display detectable PAT activity and are resistant to BASTATM, a phosphinothricin (PPT)-based herbicide. DNA gel blot analyses showed that PPT resistant rice plants contain a bar-hybridizing restriction fragment of the expected size. This report shows that expression of the bar gene in transgenic rice plants confers resistance to PPT-based herbicide by suppressing an increase of ammonia in plants after spraying with the herbicide.

Efficient regeneration of transgenic plants from rice protoplasts and correctly regulated expression of the foreign gene in the plants

SummaryRice is one of the most important crops in the world with 35% of the total population (over two billion people) depending on it as their source of food. It is therefore essential to develop efficient methods for the transformation and regeneration of rice plants in order to delineate the exact regulatory sequences responsible for gene expression and to transfer beneficial genes into this plant. Here, for the first time, we present definitive evidence for the regeneration of a large number of transgenic rice plants after introduction of the bacterial β-glucuronidase gene into rice protoplasts. The presence of integrated copies of this gene was detected in the genome of transgenic plants by DNA hybridization analysis. Furthermore, under the control of regulatory regions from a maize alcohol dehydrogenase sequence, β-glucuronidase gene expression was detected in the roots of transgenic plants. This expression was stimulated up to six fold under anaerobic conditions.

Arginine decarboxylase transgene expression and analysis of environmental stress tolerance in transgenic rice.

Arginine decarboxylase (ADC) cDNA from oat (Avena sativa L.) was introduced into rice (Oryza sativa L.) genome by an Agrobacterium-mediated transformation method. Expression of the ADC transgene under the control of an ABA-inducible promoter led to stress-induced upregulation of ADC activity and polyamine accumulation in transgenic rice plants. Second-generation (Rl) transgenic rice plants showed an increase in biomass under salinity-stress conditions, as compared to the non-transformed control plants.

Transient expression of foreign genes in rice, wheat and soybean cells following particle bombardment

The development of an efficient transformation system is a prerequisite for the molecular analysis of gene expression in plants. In crop plants, this development has been hindered by difficulties encountered both in whole plant regeneration from protoplasts and in the general insusceptibility of monocots to Agrobacterium-mediated transformation. We have circumvented these difficulties by transferring foreign genes directly into the intact cells (with cell walls) of three important crop plants including rice, wheat and soybean by a particle bombardment device. Oryza sativa and Triticum monococcum cells were bombarded with accelerated tungsten particles coated with plasmids containing a β-glucuronidase gene as the reporter. Blue transformed cells were detected in an in situ enzyme assay. The number of blue cells was next used as a convenient criterion to study several factors affecting gene transfer efficiency. After optimal conditions were defined, gene transfer into intact cells of O. sativa, T. monococcum and Glycine max was successfully carried out with chloramphenicol acetyltransferase (CAT) gene as the reporter.

Nucleotide sequence analysis of DNA: II. Complete nucleotide sequence of the cohesive ends of bacteriophage λ DNA☆☆☆

Abstract The base sequence of the cohesive ends of bacteriophage φ80 DNA has been shown to be identical to the base sequence of the cohesive ends of bacteriophage lambda DNA.

Constitutive expression of a cowpea trypsin inhibitor gene, CpTi, in transgenic rice plants confers resistance to two major rice insect pests.

The gene encoding a cowpea trypsin inhibitor (CpTI), which confers insect resistance in trangenic tobacco, was introduced into rice. Expression of the CpTi gene driven by the constitutively active promoter of the rice actin 1 gene (Act1) leads to high-level accumulation of the CpTI protein in transgenic rice plants. Protein extracts from transgenic rice plants exhibit a strong inhibitory activity against bovine trypsin, suggesting that the proteinase inhibitor produced in transgenic rice is functionally active. Small-scale field tests showed that the transgenic rice plants expressing the CpTi gene had significantly increased resistance to two species of rice stem borers, which are major rice insect pests. Our results suggest that the cowpea trypsin inhibitor may be useful for the control of rice insect pests.