A comparative study on the protective role of trehalose and LEA proteins against abiotic stresses in transgenic Chinese cabbage (<Emphasis Type="Italic">Brassica campestris</Emphasis>) overexpressing<Emphasis Type="Italic">CaLEA</Emphasis> or<Emphasis Type="Italic">otsA</Emphasis>

Trehalose and LEA proteins, representative low MW chemicals that are synthesized under dehydration, are known to protect plants from drought stress. To compare their effectiveness on enhancing tolerance against various abiotic stresses, we generated transgenic Chinese cabbage plants overexpressingE. ctdi trehalose-6-phosphate synthase gene (otsA) or hot pepper (Capsicum annuum) LEA protein gene(CaLEA). Both transgenic plants exhibited altered phenotype including stunted growth and aberrant root development When subjected to drought, salt or heat stress, these plants showed remarkably improved tolerance against those stresses compared with nontransformants. After dehydration treatment, leaf turgidity and fresh weight was better maintained in both transgenic plants. GaUEA-plants performed somewhat better under dehydrated condition. When treated with 250 mM NaCI, both otsA-plants and CaLEA-plants remained equally healthier than nontransformants in maintaining leaf turgidity and delaying necrosis. Furthermore, leaf Chi content and Fv/Fm was maintained considerably higher in both transgenic plants than nontransformants. After heat-treatment at 45°C, both transgenic plants appeared much less damaged in external shape and PSII function, but LEA proteins were more protective. Our results indicate that although both trehalose and LEA proteins are effective in protecting plants against various abiotic stresses, LEA proteins seem to be more promising in generating stress-tolerant transgenic plants.     

Trehalose‐6‐phosphate phosphatases from Arabidopsis thaliana: identification by functional complementation of the yeast tps2 mutant

It is currently thought that most flowering plants lack the capacity to synthesize trehalose, a common disaccharide of bacteria, fungi and invertebrates that appears to play a major role in desiccation tolerance. Attempts have therefore been made to render plants more drought-resistant by the expression of microbial genes for trehalose synthesis. It is demonstrated here that Arabidopsis thaliana itself possesses genes for at least one of the enzymes required for trehalose synthesis, trehalose-6-phosphate phosphatase. The yeast tps2 mutant, which lacks this enzyme, is heat-sensitive, and Arabidopsis cDNA able to complement this effect has been screened for. Half of the yeast transformants that grew at 38.6°C were also able to produce trehalose. All of these expressed one of two Arabidopsis cDNA, either AtTPPA or AtTPPB, which are both homologous to the C-terminal part of the yeast TPS2 gene and other microbial trehalose-6-phosphate phosphatases. Yeast tps2 mutants expressing AtTPPA or AtTPPB contained trehalose-6-phosphate phosphatase activity that could be measured both in vivo and in vitro. The enzyme dephosphorylated trehalose-6-phosphate but not glucose-6-phosphate or sucrose-6-phosphate. Both genes are expressed in flowers and young developing tissue of Arabidopsis. The finding of these novel Arabidopsis genes for trehalose-6-phosphate phosphatase strongly indicates that a pathway for trehalose biosynthesis exists in plants.     

Improved drought tolerance without undesired side effects in transgenic plants producing trehalose

Most organisms naturally accumulating trehalose upon stress produce the sugar in a two-step process by the action of the enzymes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). Transgenic plants overexpressing TPS have shown enhanced drought tolerance in spite of minute accumulation of trehalose, amounts believed to be too small to provide a protective function. However, overproduction of TPS in plants has also been found combined with pleiotropic growth aberrations. This paper describes three successful strategies to circumvent such growth defects without loosing the improved stress tolerance. First, we introduced into tobacco a double construct carrying the genes TPS1 and TPS2 (encoding TPP) from Saccharomyces cerevisiae. Both genes are regulated by an Arabidopsis RuBisCO promoter from gene AtRbcS1A giving constitutive production of both enzymes. The second strategy involved stress-induced expression by fusing the coding region of ScTPS1 downstream of the drought-inducible Arabidopsis AtRAB18 promoter. In transgenic tobacco plants harbouring genetic constructs with either ScTPS1 alone, or with ScTPS1 and ScTPS2 combined, trehalose biosynthesis was turned on only when the plants experienced stress. The third strategy involved the use of AtRbcS1A promoter together with a transit peptide in front of the coding sequence of ScTPS1, which directed the enzyme to the chloroplasts. This paper confirms that the enhanced drought tolerance depends on unknown ameliorated water retention as the initial water status is the same in control and transgenic plants and demonstrates the influence of expression of heterologous trehalose biosynthesis genes on Arabidopsis root development.     

Overexpression of a trehalose-6-phosphate synthase/phosphatase fusion gene enhances tolerance and photosynthesis during drought and salt stress without growth aberrations in tomato

Trehalose is a non-reducing disaccharide of glucose that confers tolerance against abiotic stresses in many diverse organisms, including higher plants. It was previously reported that overexpression of the yeast trehalose-6-phosphate synthase gene in tomato results in improved tolerance against abiotic stresses. However, these transgenic tomato plants had stunted growth and pleiotropic changes in appearance. In this study, transgenic tomato plants were generated by the introduction of a gene encoding a bifunctional fusion of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase genes from Escherichia coli under the control of the CaMV35S promoter. Transgenic plants accumulated higher levels of trehalose in their leaves and exhibited enhanced drought and salt tolerance and photosynthetic rates under salt stress conditions than wild-type plants. All of the transgenic plants had normal growth patterns and appearances. Therefore, the system described in this study can be used for practical application of the gene in crop improvement.     

Overexpression of the trehalose-6-phosphate synthase gene <Emphasis Type="Italic">OsTPS1</Emphasis> enhances abiotic stress tolerance in rice

Trehalose plays an important role in metabolic regulation and abiotic stress tolerance in a variety of organisms. In plants, its biosynthesis is catalyzed by two key enzymes: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). The genome of rice (Oryza sativa) contains 11 OsTPS genes, and only OsTPS1 shows TPS activity. To demonstrate the physiological function of OsTPS1, we introduced it into rice and found that OsTPS1 overexpression improved the tolerance of rice seedling to cold, high salinity and drought treatments without other significant phenotypic changes. In transgenic lines overexpressing OsTPS1, trehalose and proline concentrations were higher than in the wild type and some stress-related genes were up-regulated, including WSI18, RAB16C, HSP70, and ELIP. These results demonstrate that OsTPS1 may enhance the abiotic stress tolerance of plants by increasing the amount of trehalose and proline, and regulating the expression of stress-related genes. Furthermore, we found that overexpression of some Class II TPSs also enhanced plant tolerance of abiotic stress. This work will help to clarify the role of trehalose metabolism in abiotic stress response in higher plants.     

Expression of the yeast trehalose-6-phosphate synthase gene in transgenic tobacco plants: pleiotropic phenotypes include drought tolerance

The yeast trehalose-6-phosphate synthase gene (TPS1) was engineered under the control of the cauliflower mosaic virus regulatory sequences (CaMV35S) for expression in plants. Using Agrobacterium-mediated transfer, the gene was incorporated into the genomic DNA and constitutively expressed in Nicotiana tabacum␣L. plants. Trehalose was determined in the transformants, by anion-exchange chromatography coupled to pulsed amperometric detection. The non-reducing disaccharide accumulated up to 0.17 mg per g fresh weight in leaf extracts of transgenic plants. Trehalose-accumulating plants exhibited multiple phenotypic alterations, including stunted growth, lancet-shaped leaves, reduced sucrose content and improved drought tolerance. These pleiotropic effects, and the fact that water loss from detached leaves was not significantly affected by trehalose accumulation, suggest that synthesis of this sugar, rather than leading to an osmoprotectant effect, had altered sugar metabolism and regulatory pathways affecting plant development and stress tolerance. Received: 8 July 1996 / Accepted: 10 October 1996     

Overexpression of a <Emphasis Type="Italic">Panax ginseng</Emphasis> tonoplast aquaporin alters salt tolerance,drought tolerance and cold acclimation ability in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

Water movement across cellular membranes is regulated largely by a family of water channel proteins called aquaporins (AQPs). Since several abiotic stresses such as, drought, salinity and freezing, manifest themselves via altering water status of plant cells and are linked by the fact that they all result in cellular dehydration, we overexpressed an AQP (tonoplast intrinsic protein) from Panax ginseng, PgTIP1, in transgenic Arabidopsis thaliana plants to test its role in plant’s response to drought, salinity and cold acclimation (induced freezing tolerance). Under favorable conditions, PgTIP1 overexpression significantly increased plant growth as determined by the biomass production, and leaf and root morphology. PgTIP1 overexpression had beneficial effect on salt-stress tolerance as indicated by superior growth status and seed germination of transgenic plants under salt stress; shoots of salt-stressed transgenic plants also accumulated greater amounts of Na⁺ compared to wild-type plants. Whereas PgTIP1 overexpression diminished the water-deficit tolerance of plants grown in shallow (10 cm deep) pots, the transgenic plants were significantly more tolerant to water stress when grown in 45 cm deep pots. The rationale for this contrasting response, apparently, comes from the differences in the root morphology and leaf water channel activity (speed of dehydration/rehydration) between the transgenic and wild-type plants. Plants overexpressed with PgTIP1 exhibited lower (relative to wild-type control) cold acclimation ability; however, this response was independent of cold-regulated gene expression. Our results demonstrate a significant function of PgTIP1 in growth and development of plant cells, and suggest that the water movement across tonoplast (via AQP) represents a rate-limiting factor for plant vigor under favorable growth conditions and also significantly affect responses of plant to drought, salt and cold stresses.     

Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression

Endogenously synthesized trehalose is a stress protectant in Escherichia coli. Externally supplied trehalose does not serve as a stress protectant, but it can be utilized as the sole source of carbon and energy. Mutants defective in trehalose synthesis display an impaired osmotic tolerance in minimal growth media without glycine betaine, and an impaired stationary-phaseinduced heat tolerance. Mechanisms for stress protection by trehalose are discussed. The genes for trehalose-6-phosphate synthase (otsA) and anabolic trehalose-6-phosphate phosphatase (otsB) constitute an operon. Their expression is induced both by osmotic stress and by growth into the stationary phase and depend on the sigma factor encoded by rpoS (katF). rpoS is amber-mutated in E. coli K-12 and its DNA sequence varies among K-12 strains. For trehalose catabolism under osmotic stress E. coli depends on the osmoticcally inducible periplasmic trehalase (TreA). In the absence of osmotic stress, trehalose induces the formation of an enzyme II^Tre (TreB) of the group translocation system, a catabolic trehalose-6-phosphate phosphatase (TreE), and an amylotrehalase (TreC) which converts trehalose to free glucose and a glucose polymer.     

Overexpression of NHX1s in transgenic Arabidopsis enhances photoprotection capacity in high salinity and drought conditions

In this study, we observed that transgenic plants overexpressing NHX1 from different organisms grew well in the presence of 200 mM NaCl and also under water deprivation, while the wild-type plants exhibited chlorosis and growth inhibition. The photosynthesis activity of five kinds of transgenic plants was higher than the wild-type plants. The leaf water potential was less negative for wild-type than for transgenic plants. Moreover, these transgenic plants accumulated more Na⁺ and K⁺ in their leaf tissue than the wild-type plants. The toxic effects of Na⁺ accumulation in the cytosol are reduced by its sequestration into the vacuole. In addition, the thermal dissipation and ROS scavenging components increased in all transgenic Arabidopsis plants compared with that in non-transgenics. The salt tolerance of transgenic plants was passed on to the offsprings to the T₅ generation. Furthermore, it should be noted that in transgenic Arabidopsis plants, overexpression of NHX1s from dicots showed higher salt and drought tolerance than that from wheat.     

Trehalose-6-phosphate-mediated phenotypic change in Acinetobacter baumannii

The stress protectant trehalose is synthesized in Acinetobacter baumannii from UPD-glucose and glucose-6-phosphase via the OtsA/OtsB pathway. Previous studies proved that deletion of otsB led to a decreased virulence, the inability to grow at 45°C and a slight reduction of growth at high salinities indicating that trehalose is the cause of these phenotypes. We have questioned this conclusion by producing ∆otsA and ∆otsBA mutants and studying their phenotypes. Only deletion of otsB, but not deletion of otsA or otsBA, led to growth impairments at high salt and high temperature. The intracellular concentrations of trehalose and trehalose-6-phosphate were measured by NMR or enzymatic assay. Interestingly, none of the mutants accumulated trehalose any more but the ∆otsB mutant with its defect in trehalose-6-phosphate phosphatase activity accumulated trehalose-6-phosphate. Moreover, expression of otsA in a ∆otsB background under conditions where trehalose synthesis is not induced led to growth inhibition and the accumulation of trehalose-6-phosphate. Our results demonstrate that trehalose-6-phosphate affects multiple physiological activities in A. baumannii ATCC 19606.     

Effects of drought on water content and photosynthetic parameters in potato plants expressing the trehalose-6-phosphate synthase gene of Saccharomyces cerevisiae

Two transgenic potato lines, T1 and T2, expressing the trehalose-6-phosphate synthase (TPS1) gene of yeast were isolated. In our experimental approach, we applied two novelties, namely the fusion of the drought-inducible promoter StDS2 to TPS1 and a marker-free transformation method. In contrast to the expected drought-induced expression, only a very low constitutive TPS1 expression was detected in the transgenic lines, probably due to chromosomal position effects. The observed expression pattern, however, was sufficient to alter the drought response of plants. Detached leaves of T1 and T2 showed an 8 h delay in wilting compared to the non-transformed control. Potted plants of T1 and T2 kept water 6 days longer than control plants and maintained high stomatal conductance and a satisfactory rate of net photosynthesis. During drought treatment, CO₂ assimilation rate measured at saturating CO₂ level was maintained at maximum level for 6–9 days in transgenic plants while it decreased rapidly after 3 days in the wild type plants. Under optimal growth conditions, lower CO₂ fixation was detected in the transgenic than in the control plants. Stomatal densities of T1 and T2 leaves were reduced by 30–40%. This may have contributed to the lower CO₂ fixation rate and altered drought response. Ibolya Stiller, and Sándor Dulai contributed equally to this work.     

Ectopic overexpression of vacuolar and apoplastic <Emphasis Type="Italic">Catharanthus roseus</Emphasis> peroxidases confers differential tolerance to salt and dehydration stress in transgenic tobacco

CrPrx and CrPrx1 are class III peroxidases previously cloned and characterized from Catharanthus roseus. CrPrx is known to be apoplastic in nature, while CrPrx1 is targeted to vacuoles. In order to study their role in planta, these two peroxidases were expressed in Nicotiana tabacum. The transformed plants exhibited increased peroxidase activity. Increased oxidative stress tolerance was also observed in transgenics when treated with H₂O₂ under strong light conditions. However, differential tolerance to salt and dehydration stress was observed during germination of T1 transgenic seeds. Under these stresses, the seed germination of CrPrx-transformed plants and wild-type plants was clearly suppressed, whereas CrPrx1 transgenic lines showed improved germination. CrPrx-transformed lines exhibited better cold tolerance than CrPrx1-transformed lines. These results indicate that vacuolar peroxidase plays an important role in salt and dehydration stress over cell wall-targeted peroxidase, while cell wall-targeted peroxidase renders cold stress tolerance.     

The Arabidopsis TCH4 xyloglucan endotransglycosylase. Substrate specificity, pH optimum, and cold tolerance.

As a first step toward the exploitation of the disaccharide trehalose as a stress-protective and preservative agent in plants, we engineered trehalose biosynthesis in tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) by introducing the otsA and otsB genes from Escherichia coli, which encode trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively. In leaves of transgenic tobacco plants, very low levels of trehalose accumulation were obtained (0.11 mg g-1 fresh weight), whereas in transgenic potato tubers, no trehalose accumulated at all. Plant trehalase activity was shown to affect the accumulation of trehalose in these plants. An increase in trehalose accumulation, up to 0.41 and 4.04 mg g-1 fresh weight in tobacco leaves and potato micro-tubers, respectively, was noted when the potent trehalase inhibitor validamycin A was added to in vitro plants and to hydroponically grown greenhouse plants. Stunted growth and the formation of lancet-shaped leaves by trehalose-accumulating tobacco plants suggest a negative effect of trehalose biosynthesis on N. tabacum development. It is surprising that experiments with wild-type plants cultured in the presence of validamycin A indicate that, despite current belief, the capacity to synthesize trehalose may not be restricted to primitive phyla of vascular plants and certain "resurrection plants," but may exist throughout the angiosperms.     

Increased Chilling Tolerance Following Transfer of a betA Gene Enhancing Glycinebetaine Synthesis in Cotton (Gossypium hirsutum L.)

A betA gene encoding choline dehydrogenase from Escherichia coli was transformed into cotton (Gossypium hirsutum L.) via Agrobacterium-mediated transformation. Transgenic cotton plants exhibited improved tolerance to chilling due to accumulation of glycinebetaine (GB). The results of our experiment showed that GB contents of leaves of transgenic lines 1, 3, 4, and 5, both before and after chilling stress, were significantly higher than those of wild-type (WT) plants. At 15°C, transgenic lines 1, 3, 4, and 5 exhibited higher germination capacity as determined by the germination speed and final germination percentage and, displayed less inhibition in seedling shoot growth rate than WT plants. Under chilling stress, transgenic lines 4 and 5 maintained higher relative water content, upper carbon dioxide (CO₂) fixation capacity and PSII electron transfer rate, better osmotic adjustment (OA), a lower percentage of ion leakage, and less lipid membrane peroxidation when compared with WT plants. Chilling resistance of the transgenic lines was demonstrated to be positively correlated with GB content under chilling stress. The high levels of GB in transgenic cotton plants might not only protect the integrity of cell membrane from chilling damage, but also be involved in OA which alleviated chilling induced water stress. Moreover, under chilling-stressed conditions, transgenic cotton plants enhanced stomatal conductance, PSII electron transport rate, and further leaf photosynthesis through accumulating high levels of GB.     

Accumulation of Trehalose by Overexpression of tps1, Coding for Trehalose-6-Phosphate Synthase, Causes Increased Resistance to Multiple Stresses in the Fission Yeast Schizosaccharomyces pombe

Recent studies have shown that heat shock proteins and trehalose synthesis are important factors in the thermotolerance of the fission yeast Schizosaccharomyces pombe. We examined the effects of trehalose-6-phosphate (trehalose-6P) synthase overexpression on resistance to several stresses in cells of S. pombe transformed with a plasmid bearing the tps1 gene, which codes for trehalose-6P synthase, under the control of the strong thiamine-repressible promoter. Upon induction of trehalose-6P synthase, the elevated levels of intracellular trehalose correlated not only with increased tolerance to heat shock but also with resistance to freezing and thawing, dehydration, osmostress, and toxic levels of ethanol, indicating that trehalose may be the stress metabolite underlying the overlap in induced tolerance to these stresses. Among the isogenic strains transformed with this construct, one in which the gene coding for the trehalose-hydrolyzing enzyme, neutral trehalase, was disrupted accumulated trehalose to a greater extent and was more resistant to the above stresses. Increased trehalose concentration is thus a major determinant of the general stress protection response in S. pombe.     

Cleavage of trehalose-phosphate in Bacillus subtilis is catalysed by a Phospho-α-(1–1)-glucosidase encoded by the treA gene

A 2.5 kb DNA fragment contain a gene encoding a phospho-α-(1–1)-glucosidase (phosphotrehalase), designated treA, was isolated from a Bacillus subtilis chromosomal library by complementation of the tre-12 mutation. The major TreA activity was found in the cytoplasm. TreA exhibits high sequence similarity to thermostable oligo 1,6 β-glucosidases of several species and the trehalose-6-phosphate hydrolase TreC of Escherichia coli. TreA activity is induced by trehalose and repressed by glucose, fructose or mannitol. Induction by trehalose and repression by glucose are concentration dependent. The highest activity of TreA occurs 90min before the end of the exponential growth phase in crude cell extracts. The enzyme is able to cleave para-nitrophenyl-glucopyranoside and trehalose-6-phosphate but not trehalose. These results indicate that treA encodes a specific phospho-α-(1–1)-giucosidase which cleaves trehalose-6-phosphate in the cytoplasm after transport and phosphorylation of trehalose. The 5′ flanking region of treA contains an open reading frame which was partially sequenced, whose product shows about 40% identity to sucrose Enzyme II of the phospho-transferase transport system from several organisms.     

Over-expression of tobacco NtHSP70-1 contributes to drought-stress tolerance in plants

HSP70, a heat shock protein, is a molecular chaperone responsive to various environmental stresses. Here, NtHSP70-1 (AY372069) was a drought-/ABA-inducible gene. We monitored the expression of CaERD15 (early responsive to dehydration, DQ267932) with exposing plants to progressive drought stress. Its activity was used as an indicator of water-deficit conditions. To analyze the protective role of HSP70, we obtained transgenic tobacco plants that constitutively expressed elevated levels of the tobacco HSP70, NtHSP70-1, as well as transgenic plants containing either the vector alone or else having NtHSP70-1 in the antisense orientation. Plants with enhanced levels of NtHSP70-1 in their transgenic sense lines exhibited tolerance to water stress. Under progressive drought, the amount of leaf NtHSP70-1 was correlated with maintenance of optimum water content, with contents being higher in the leaves of dehydrated transgenic sense plants than in those of either the control (vector-only) or the transgenic antisense plants. Moreover, the expression of CaERD15 was considerably reduced in tobacco plants that over-expressed NtHSP70-1. These results suggest that elevated levels of NtHSP70-1 can confer drought-stress tolerance.     

Overexpression of wheat dehydrin DHN-5 enhances tolerance to salt and osmotic stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Late Embryogenesis Abundant (LEA) proteins are associated with tolerance to water-related stress. A wheat (Triticum durum) group 2 LEA proteins, known also as dehydrin (DHN-5), has been previously shown to be induced by salt and abscisic acid (ABA). In this report, we analyze the effect of ectopic expression of Dhn-5 cDNA in Arabidopsis thaliana plants and their response to salt and osmotic stress. When compared to wild type plants, the Dhn-5 transgenic plants exhibited stronger growth under high concentrations of NaCl or under water deprivation, and showed a faster recovery from mannitol treatment. Leaf area and seed germination rate decreased much more in wild type than in transgenic plants subjected to salt stress. Moreover, the water potential was more negative in transgenic than in wild type plants. In addition, the transgenic plants have higher proline contents and lower water loss rate under water stress. Also, Na⁺ and K⁺ accumulate to higher contents in the leaves of the transgenic plants. Our data strongly support the hypothesis that Dhn-5, by its protective role, contributes to an improved tolerance to salt and drought stress through osmotic adjustment.