Accepted 17 Jun. 2008Received 16 Mar. 2008

Trehalose is a non-reducing disaccharide that is present in diverse organisms ranging from bacteria and fungi to invertebrates, in which it serves as an energy source, osmolyte or protein/membrane protectant. The occurrence of trehalose and trehalose biosynthesis pathway in plants has been discovered recently. Multiple studies have revealed regulatory roles of trehalose-6-phosphate, a precursor of trehalose, in sugar metabolism, growth and development in plants. Trehalose levels are generally quite low in plants but may alter in response to environmental stresses. Transgenic plants overexpressing microbial trehalose biosynthesis genes have been shown to contain increased levels of trehalose and display drought, salt and cold tolerance. In.silico expression profiling of all Arabidopsis trehalose-6-phosphate synthases (TPSs) and trehalose-6-phosphate phosphatases (TPPs) revealed that certain classes of TPS and TPP genes are differentially regulated in response to a variety of abiotic stresses. These studies point to the importance of trehalose biosynthesis in stress responses.     

Accepted 17 Jun. 2008Received 16 Mar. 2008

Trehalose is a non-reducing disaccharide that is present in diverse organisms ranging from bacteria and fungi to invertebrates, in which it serves as an energy source, osmolyte or protein/membrane protectant. The occurrence of trehalose and trehalose biosynthesis pathway in plants has been discovered recently. Multiple studies have revealed regulatory roles of trehalose-6-phosphate, a precursor of trehalose, in sugar metabolism, growth and development in plants. Trehalose levels are generally quite low in plants but may alter in response to environmental stresses. Transgenic plants overexpressing microbial trehalose biosynthesis genes have been shown to contain increased levels of trehalose and display drought, salt and cold tolerance. In.silico expression profiling of all Arabidopsis trehalose-6-phosphate synthases (TPSs) and trehalose-6-phosphate phosphatases (TPPs) revealed that certain classes of TPS and TPP genes are differentially regulated in response to a variety of abiotic stresses. These studies point to the importance of trehalose biosynthesis in stress responses.     

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.     

The C. elegans lethal gut-obstructed gob-1 gene is trehalose-6-phosphate phosphatase

We identified the gob-1 (gut-obstructed) gene in a forward genetic screen for intestinal defects in the nematode Caenorhabditis elegans. gob-1 loss of function results in early larval lethality, at least in part because of a blocked intestinal lumen and consequent starvation. The gob-1 gene is first expressed in the 8E cell stage of the embryonic intestine, and the GATA factor ELT-2 is sufficient but not necessary for this early phase of gob-1 expression; gob-1 expression later becomes widespread in embryos, larvae, and adults. GOB-1 is a member of the HAD-like hydrolase superfamily and shows a robust and specific phosphatase activity for the substrate trehalose-6-phosphate. Trehalose is a glucose disaccharide found in bacteria, fungi, plants, insects, and nematodes but not in mammals. Trehalose plays a number of critical roles such as providing flexible energy reserves and contributing to thermal and osmotic stress resistance. In budding yeast and in plants, the intermediate in trehalose synthesis, trehalose-6-phosphate, has additional critical but less well-defined roles in controlling glycolysis and carbohydrate metabolism. Strong loss-of-function mutants in the C. elegans tps-1 and tps-2 genes (which encode the two trehalose phosphate synthases responsible for trehalose-6-phosphate synthesis) completely suppress the lethality associated with gob-1 loss of function. The suppression of gob-1 lethality by ablation of TPS-1 and TPS-2, the upstream enzymes in the trehalose synthesis pathway, suggests that gob-1 lethality results from a toxic build-up of the intermediate trehalose-6-phosphate, not from an absence of trehalose. GOB-1 is the first trehalose-6-phosphate phosphatase to be identified in nematodes and, because of its associated lethality and distinctive sequence properties, provides a new and attractive target for anti-parasitic drugs.     

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.     

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.     

Analysis of trehalose-6-phosphate synthase (TPS) gene family suggests the formation of TPS complexes in rice

Trehalose-6-phosphate (T6P), an intermediate in the trehalose biosynthesis pathway, is emerging as an important regulator of plant metabolism and development. T6P levels are potentially modulated by a group of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) homologues. In this study, we have isolated 11 TPS genes encoding proteins with both TPS and TPP domains, from rice. Functional complement assays performed in yeast tps1 and tps2 mutants, revealed that only OsTPS1 encodes an active TPS enzyme and no OsTPS protein possesses TPP activity. By using a yeast two-hybrid analysis, a complicated interaction network occurred among OsTPS proteins, and the TPS domain might be essential for this interaction to occur. The interaction between OsTPS1 and OsTPS8 in vivo was confirmed by bimolecular fluorescence complementation and coimmunoprecipitation assays. Furthermore, our gel filtration assay showed that there may exist two forms of OsTPS1 (OsTPS1a and OsTPS1b) with different elution profiles in rice. OsTPS1b was particularly cofractionated with OsTPS5 and OsTPS8 in the 360 kDa complex, while OsTPS1a was predominantly incorporated into the complexes larger than 360 kDa. Collectively, these results suggest that OsTPS family members may form trehalose-6-phosphate synthase complexes and therefore potentially modify T6P levels to regulate plant development.     

Extensive expression regulation and lack of heterologous enzymatic activity of the Class II trehalose metabolism proteins from Arabidopsis thaliana

Trehalose metabolism has profound effects on plant growth and metabolism, but the mechanisms involved are unclear. In Arabidopsis , 21 putative trehalose biosynthesis genes are classified in three subfamilies based on their similarity with yeast TPS1 (encoding a trehalose-6-phosphate synthase, TPS) or TPS2 (encoding a trehalose-6-phosphate phosphatase, TPP). Although TPS1 (Class I) and TPPA and TPPB (Class III) proteins have established TPS and TPP activity, respectively, the function of the Class II proteins (AtTPS5-AtTPS11) remains elusive. A complete set of promoter- β -glucurinidase/green fluorescent protein reporters demonstrates their remarkably differential tissue-specific expression and responsiveness to carbon availability and hormones. Heterologous expression in yeast furthermore suggests that none of the encoded enzymes displays significant TPS or TPP activity, consistent with a regulatory rather than metabolic function for this remarkable class of proteins.     

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.     

Enzymes of alpha,alpha-Trehalose Metabolism in Soybean Nodules

Metabolism of trehalose, α,d-glucopyranosyl-α,d-glucopyranoside, was studied in nodules of Bradyrhizobium japonicum-Glycine max [L.] Merr. cv Beeson 80 symbiosis. The nodule extract was divided into three fractions: bacteroid soluble protein, bacteroid fragments, and cytosol. The bacteroid soluble protein and cytosol fractions were gel-filtered. The key biosynthetic enzyme, trehalose-6-phosphate synthetase, was consistently found only in the bacteroids. Trehalose-6-phosphate phosphatase activity was present both in the bacteroid soluble protein and cytosol fractions. Trehalase, the most abundant catabolic enzyme was present in all three fractions and showed two pH optima: pH 3.8 and 6.6. Two other degradative enzymes, phosphotrehalase, acting on trehalose-6-phosphate forming glucose and glucose-6-phosphate, and trehalose phosphorylase, forming glucose and β-glucose-1-phosphate, were also detected in the bacteroid soluble protein and cytosol fractions. Trehalase was present in large excess over trehalose-6-phosphate synthetase. Trehalose accumulation in the nodules would appear to be predicated on spatial separation of trehalose and trehalase.     

The orlA gene from Aspergillus nidulans encodes a trehalose-6-phosphate phosphatase necessary for normal growth and chitin synthesis at elevated temperatures

A cosmid carrying the orIA gene from Aspergillus nidulans was identified by complementation of an orlA1 mutant strain with DNA from the pKBY2 cosmid library. An orlA1 complementing fragment from the cosmid was sequenced. orlA encodes a predicted polypeptide of 227 amino acids (26 360 Da) that is homologous to a 211-amino-acid domain from the polypeptide encoded by the Saccharomyces cerevisiae TPS2 gene and to almost the entire Escherichia coli of otsB-encoded polypeptide. TPS2 and otsB each specify a trehalose-6-phosphate phosphatase, an enzyme that is necessary for trehalose synthesis. orlA disruptants accumulate trehalose-6-phosphate and have reduced trehalose-6-phosphatate phosphatase levels, indicating that the gene encodes a tre-halose-6-phosphatate phosphatase. Disruptants have a nearly-wild-type morphology at 32°C. When germinated at 42°C, the conidia and hyphae from disruptants are chitin deficient, swell excessively, and lyse. The lysis is almost completely remedied by osmotic stabilizers and is partially remedied by N-acetylglucosamine (GlcNAc). The activity of glutamine:fructose-6-phosphate amido-transferase (GFAT), the first enzyme unique to aminosugar synthesis, is reduced and is labile in orIA disruption strains. The findings are consistent with the hypothesis that trehalose-6-phosphate reduces the temperature stability of GFAT and other enzymes of chitin metabolism at elevated temperatures. The results extend to filamentous organisms the observation that mutations in fungal trehalose synthesis are highly pleiotropic and affect aspects of carbohydrate metabolism that are not directly related to trehalose synthesis.     

Genetic engineering of drought resistant potato plants by introduction of the trehalose-6-phosphate synthase (TPS1) gene from Saccharomyces cerevisiae

In yeast, trehalose-6-phosphate synthase is a key enzyme for trehalose biosynthesis, encoded by the structural gene TPS1. Trehalose affects sugar metabolism as well as osmoprotection against several environmental stresses, such as heat and desiccation. The TPS1 gene of Saccharomyces cerevisiae was engineered under the control of the CaMV 35S promoter for constitutive expression in transgenic potato plants by Ti-plasmid of Agrobacterium-mediated transformation. The resulting TPS1 transgenic potato plants exhibited various morphological phenotypes in culture tubes, ranging from normal to severely retarded growth, including dwarfish growth, yellowish lancet-shaped leaves, and aberrant root development. However, the plants recovered from these negative growth effects when grown in a soil mixture. The TPS1 transgenic potato plants showed significantly increased drought resistance. These results suggest that the production of trehalose not only affects plant development but also improves drought tolerance.     

A temperature-sensitive mutant of Sacchromyces cerevisiae defective in the specific phosphatase of trehalose biosynthesis

Abstract A temperature-sensitive mutant of Saccharomyces cerevisiae has been isolated which accumulates a large pool of trehalose-6-phosphate when shifted to temperatures above 34°C nonpermissive for growth. This indicates that its defect is in the second enzyme of trehalose biosynthesis, the hydrolase that converts trehalose-6-phosphate to trehalose. Trehalose is made continouosly when yeast is growing on high glucose or when it is starved for a nitrogen source, and accumulates as cells enter the stationary phase. Revertants of the mutant able to grow at 37°C arise spontaneously and no longer accumulate trehalose-6-phosphate at this temperature. Also the kinetics of trehalose-6-phosphate accumulation in the mutant following a 25–37°C shift resemble the kinetics of inhibition of RNA and protein synthesis. It is probable therefore that accumulation of high levels of this metabolic intermediate is inhibitory to growth.     

Trehalose transport and metabolism in Escherichia coli.

Trehalose metabolism in Escherichia coli is complicated by the fact that cells grown at high osmolarity synthesize internal trehalose as an osmoprotectant, independent of the carbon source, although trehalose can serve as a carbon source at both high and low osmolarity. The elucidation of the pathway of trehalose metabolism was facilitated by the isolation of mutants defective in the genes encoding transport proteins and degradative enzymes. The analysis of the phenotypes of these mutants and of the reactions catalyzed by the enzymes in vitro allowed the formulation of the degradative pathway at low osmolarity. Thus, trehalose utilization begins with phosphotransferase (IITre/IIIGlc)-mediated uptake delivering trehalose-6-phosphate to the cytoplasm. It continues with hydrolysis to trehalose and proceeds by splitting trehalose, releasing one glucose residue with the simultaneous transfer of the other to a polysaccharide acceptor. The enzyme catalyzing this reaction was named amylotrehalase. Amylotrehalase and EIITre were induced by trehalose in the medium but not at high osmolarity. treC and treB encoding these two enzymes mapped at 96.5 min on the E. coli linkage map but were not located in the same operon. Use of a mutation in trehalose-6-phosphate phosphatase allowed demonstration of the phosphoenolpyruvate- and IITre-dependent in vitro phosphorylation of trehalose. The phenotype of this mutant indicated that trehalose-6-phosphate is the effective in vivo inducer of the system.     

Overproduction of trehalose: heterologous expression of Escherichia coli trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in Corynebacterium glutamicum

Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum.     

Trehalose Metabolism-Related Genes in Maize

Maize is a cereal crop that is grown widely throughout the world in a range of agro-ecological environments. Trehalose is a nonreducing disaccharide of glucose that has been associated with tolerance to different stress conditions, including salt and drought. Bioinformatic analysis of genes involved in trehalose biosynthesis and degradation in maize has not been reported to date. Through systematic analysis, 1 degradation-related and 36 trehalose biosynthesis-related genes were identified. The conserved domains and phylogenetic relationships among the deduced maize proteins and their homologs, isolated from other plant species such as Arabidopsis and rice, were revealed. Using a comprehensive approach, the intron/exon structures and expression patterns of all identified genes and their responses to salt stress, jasmonic acid, and abscisic acid treatment were analyzed. Microarray data demonstrated that some of the genes show differential, organ-specific expression patterns in the 60 different developmental stages of maize. It was discovered that some of the key enzymes such as hexokinase, trehalose-6-phosphate synthase, and trehalose-6-phosphate phosphatase are encoded by multiple gene members with different expression patterns. The results highlight the complexity of trehalose metabolism and provide useful information for improving maize stress tolerance through genetic engineering.