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PubMed2002

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 mgg 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.

Carbohydrate Metabolism↗
PubMed2001

Role of trehalose phosphate synthase in anoxia tolerance and development in Drosophila melanogaster.

Recent studies have shown that trehalose plays a protective role in yeast in a variety of stresses, including heat, freezing and thawing, dehydration, hyperosmotic shock, and oxidant injury. Because (a) heat shock and anoxia share mechanisms that allow organisms to survive, (b) Drosophila melanogaster is tolerant to anoxia, and (c) trehalose is present in flies and is metabolically active, we asked whether trehalose can protect against anoxic stress. Here we report on a new role of trehalose in anoxia resistance in Drosophila. We first cloned the gene trehalose-6-phosphate synthase (tps1), which synthesizes trehalose, and examined the effect of tps1 overexpression as well as mutation on the resistance of Drosophila to anoxia. Upon induction of tps1, trehalose increased, and this was associated with increased tolerance to anoxia. Furthermore, in vitro experiments showed that trehalose reduced protein aggregation caused by anoxia. Homozygous tps1 mutant (P-element insertion into the third intron of the gene) leads to lethality at an early larval stage, and excision of the P-element rescues totally the phenotype. We conclude that trehalose contributes to anoxia tolerance in flies; this protection is likely to be due to a reduction of protein aggregation.

Adaptation, Physiological↗
PubMed2005

The OtsAB pathway is essential for trehalose biosynthesis in Mycobacterium tuberculosis.

The disaccharide trehalose is the major free sugar in the cytoplasm of mycobacteria; it is a constituent of cell wall glycolipids, and it plays a role in mycolic acid transport during cell wall biogenesis. The pleiotropic role of trehalose in the biology of Mycobacterium tuberculosis and its absence from mammalian cells suggests that its biosynthesis may provide a useful target for novel drugs. However, there are three potential pathways for trehalose biosynthesis in M. tuberculosis, and the aim of the present study was to introduce mutations into each of the pathways to determine whether or not they are functionally redundant. The results show that the OtsAB pathway, which generates trehalose from glucose and glucose-6-phosphate, is the dominant pathway required for M. tuberculosis growth in laboratory culture and for virulence in a mouse model. Of the two otsB homologues annotated in the genome sequence of M. tuberculosis, only OtsB2 (Rv3372) has a functional role in the pathway. OtsB2, trehalose-6-phosphate phosphatase, is strictly essential for growth and provides a tractable target for high throughput screening. Inactivation of the TreYZ pathway, which can generate trehalose from alpha-1,4-linked glucose polymers, had no effect on the growth of M. tuberculosis in vitro or in mice. Deletion of the treS gene altered the late stages of pathogenesis of M. tuberculosis in mice, significantly increasing the time to death in a chronic infection model. Because the TreS enzyme catalyzes the interconversion of trehalose and maltose, the mouse phenotype could reflect either a requirement for synthesis of additional trehalose or, conversely, a requirement for breakdown of stored trehalose to liberate free glucose.

Animals↗
PubMed2004

Cryopreservation of stem cells using trehalose: evaluation of the method using a human hematopoietic cell line.

While stem cell cryopreservation methods have been optimized using dimethylsulfoxide (DMSO), the established techniques are not optimal when applied to unfertilized human embryonic cells. In addition, important questions remain regarding the toxicity and characteristics of DMSO for treatment of stem cells for clinical use. The objective of this study was to establish an optimal method for cryopreservation of stem cells using low concentrations (0.2 M) of trehalose, a nontoxic disaccharide of glucose, which possesses excellent protective characteristics, in place of current methods utilizing high concentrations (1-2 M) of DMSO. A human hematopoietic cell line was used in this investigation as a surrogate for human stem cells. Trehalose was loaded into cells using a genetically engineered mutant of the pore-forming protein alpha-hemolysin from Staphylococcus aureus. This method results in a nonselective pore equipped with a metal-actuated switch that is sensitive to extracellular zinc concentrations, thus permitting controlled loading of trehalose. Preliminary experiments characterized the effects of poration on TF-1 cells and established optimal conditions for trehalose loading and cell survival. TF-1 cells were frozen at 1 degrees C/min to -80 degrees C with and without intra- and extracellular trehalose. Following storage at -80 degrees C for 1 week, cells were thawed and evaluated for viability, differentiation capacity, and clonogenic activity in comparison to cells frozen with DMSO. Predictably, cells frozen without any protective agent did not survive freezing. Colony-forming units (CFU) generated from cells frozen with intra- and extracellular trehalose, however, were comparable in size, morphology, and number to those generated by cells frozen in DMSO. There was no observable alteration in phenotypic markers of differentiation in either trehalose- or DMSO-treated cells. These data demonstrate that low concentrations of trehalose can protect hematopoietic progenitors from freezing injury and support the concept that trehalose may be useful for freezing embryonic stem cells and other primitive stem cells for therapeutic and investigational use.

Animals↗
PubMed2003

Trehalose synthesis and metabolism are required at different stages of plant infection by Magnaporthe grisea.

The relationship of trehalose metabolism to fungal virulence was explored in the rice blast fungus Magnaporthe grisea. To determine the role of trehalose synthesis in pathogenesis, we identified and deleted TPS1, encoding trehalose-6-phosphate synthase. A Deltatps1 mutant failed to synthesize trehalose, sporulated poorly and was greatly attenuated in pathogenicity. Appressoria produced by Deltatps1 did not develop full turgor or elaborate penetration hyphae efficiently. To determine the role of subsequent trehalose breakdown, we deleted NTH1, which encodes a neutral trehalase. Nth1 mutants infected plants normally, but showed attenuated pathogenicity due to a decreased ability to colonize plant tissue. A second trehalase was also identified, required both for growth on trehalose and mobilization of intracellular trehalose during infection-related development. TRE1 encodes a cell wall-localized enzyme with characteristics of both neutral and acidic trehalases, but is dispensable for pathogenicity. Our results indicate that trehalose synthesis, but not its subsequent breakdown, is required for primary plant infection by M.grisea, while trehalose degradation is important for efficient development of the fungus in plant tissue following initial infection.

Amino Acid Sequence↗
PubMed2005

Mouse sperm desiccated and stored in trehalose medium without freezing.

Mouse sperm with and without trehalose were desiccated under nitrogen gas and stored at 4 degrees C and 22 degrees C. After rehydration, sperm were injected into oocytes using intracytoplasmic sperm injection and embryonic development was followed. Sperm were dried for 5.0, 6.25, or 7.5 min, stored at 22 degrees C for 1 wk with and without trehalose. The percentages of blastocysts that developed from sperm with trehalose were 51%, 31%, and 20%, respectively, which was significantly higher than sperm without trehalose (10%, 3%, and 5%, respectively). Desiccation and storage in medium with trehalose significantly increased sperm developmental potential compared to medium without trehalose. Sperm dried for 5 min produced more blastocysts than sperm dried for 6.25 or 7.5 min. When sperm were dried in trehalose for 5 min and stored for 1 wk, 2 wk, 1 mo, or 3 mo at 4 degrees C, the percentages of blastocysts were 73%, 84%, 63%, and 39%; whereas those stored at 22 degrees C for 1 wk, 2 wk, or 1 mo were significantly lower (53%, 17%, and 6%, respectively). Embryos from sperm partially desiccated in trehalose for 5 min and stored at 4 degrees C for 1 or 3 mo were transferred to 10 pseudopregnant recipients. Implantation rates were 81% and 48%; live fetuses were 26% and 5%, respectively. One of the recipients delivered three live fetuses. The results show that trehalose has a significant beneficial effect in preserving the developmental potential of mouse sperm following partial desiccation and storage at temperatures above freezing.

Animals↗
PubMed1992

The 70-kilodalton heat-shock proteins of the SSA subfamily negatively modulate heat-shock-induced accumulation of trehalose and promote recovery from heat stress in the yeast, Saccharomyces cerevisiae.

In the yeast, Saccharomyces cerevisiae, the disaccharide trehalose is a stress-related metabolite that accumulates upon exposure of cells to heat shock or a variety of non-heat inducers of the stress response. Here, we describe the influence of mutations in individual heat-shock-protein genes on trehalose metabolism. A strain mutated in three proteins of the SSA subfamily of 70-kDa heat-shock proteins (hsp70) overproduced trehalose during heat shock at 37 degrees C or 40 degrees C and showed abnormally slow degradation of trehalose upon temperature decrease from 40 degrees C to 27 degrees C. The mutant cells were unimpaired in the induction of thermotolerance; however, the decay of thermotolerance during recovery at 27 degrees C was abnormally slow. Since both a high content of trehalose and induced thermotolerance are associated with the heat-stressed state of cells, the abnormally slow decline of trehalose levels and thermotolerance in the mutant cells indicated a defect in recovery from the heat-stressed state. A similar albeit minor defect, as judged from measurements of trehalose degradation during recovery, was detected in a delta hsp104 mutant, but not in a strain deleted in the polyubiquitin gene, UB14. In all our experiments, trehalose levels were closely correlated with thermotolerance, suggesting a thermoprotective function of trehalose. In contrast, heat-shock proteins, in particular hsp70, appear to be involved in recovery from the heat-stressed state rather than in the acquisition of thermotolerance. Cells partially depleted of hsp70 displayed an abnormally low activity of neutral trehalase when shifted to 27 degrees C after heat shock at 40 degrees C. Trehalase activity is known to be under positive control by cAMP-dependent protein kinases, suggesting that hsp70 directly or indirectly stimulate these protein-kinase activities. Alternatively, hsp70 may physically interact with neutral trehalase, thereby protecting the enzyme from thermal denaturation.

Glucose↗
PubMed1987

Heat-induced accumulation and futile cycling of trehalose in Saccharomyces cerevisiae.

Heat shock resulted in rapid accumulation of large amounts of trehalose in Saccharomyces cerevisiae. In cultures growing exponentially on glucose, the trehalose content of the cells increased from 0.01 to 1 g/g of protein within 1 h after the incubation temperature was shifted from 27 to 40 degrees C. When the temperature was readjusted to 27 degrees C, the accumulated trehalose was rapidly degraded. In parallel, the activity of the trehalose-phosphate synthase, the key enzyme of trehalose biosynthesis, increased about sixfold during the heat shock and declined to the normal level after readjustment of the temperature. Surprisingly, the activity of neutral trehalase, the key enzyme of trehalose degradation, also increased about threefold during the heat shock and remained almost constant during recovery of the cells at 27 degrees C. In pulse-labeling experiments with [14C]glucose, trehalose was found to be turned over rapidly in heat-shocked cells, indicating that both anabolic and catabolic enzymes of trehalose metabolism were active in vivo. Possible functions of the heat-induced accumulation of trehalose and its rapid turnover in an apparently futile cycle during heat shock are discussed.

Chromatography, High Pressure Liquid↗
PubMed1990

Regulation of trehalose metabolism by Streptomyces griseus spores.

Spores of Streptomyces griseus contain trehalose and trehalase, but trehalose is not readily hydrolyzed until spore germination is initiated. Trehalase in crude extracts of spores, germinated spores, and mycelia of S. griseus had a pH optimum of approximately 6.2, had a Km value for trehalose of approximately 11 mM, and was most active in buffers having ionic strengths of 50 to 200 mM. Inhibitors or activators or trehalase activity were not detected in extracts of spores or mycelia. Several lines of evidence indicated that trehalose and trehalase are both located in the spore cytoplasm. Spores retained their trehalose and most of their trehalase activity following brief exposure to dilute acid. Protoplasts formed by enzymatic removal of the spore walls in buffer containing high concentrations of solutes also retained their trehalose and trehalase activity. Protoplasts formed in buffer containing lower levels of solutes contained low levels of trehalose. The mechanism by which trehalose metabolism is regulated in S. griseus spores is unresolved. A low level of hydration of the cytoplasm of the dormant spores and an increased level of hydration during germination may account for the apparent inactivity of trehalase in dormant spores and the rapid hydrolysis of trehalose upon initiation of germination.

Disaccharides↗
PubMed2006

The natural osmolyte trehalose is a positive regulator of the heat-induced activity of yeast heat shock transcription factor.

In Saccharomyces cerevisiae, the intracellular concentration of trehalose increases rapidly in response to many environmental stresses, including heat shock. These high trehalose levels have been correlated with tolerance to adverse conditions and led to the model that trehalose functions as a chemical cochaperone. Here, we show that the transcriptional activity of Hsf1 during the heat shock response depends on trehalose. Strains with low levels of trehalose have a diminished transcriptional response to heat shock, while strains with high levels of trehalose have an enhanced transcriptional response to heat shock. The enhanced transcriptional response does not require the other heat-responsive transcription factors Msn2/4 but is dependent upon heat and Hsf1. In addition, the phosphorylation levels of Hsf1 correlate with both transcriptional activity and the presence of trehalose. These in vivo results support a new role for trehalose, where trehalose directly modifies the dynamic range of Hsf1 activity and therefore influences heat shock protein mRNA levels in response to stress.

DNA-Binding Proteins↗
PubMed2005

Interaction between trehalose and alkaline-earth metal ions.

We investigated the interaction between trehalose and alkaline-earth metal ions. The nuclear relaxation times of carbon atoms of trehalose were shortened by addition of the alkaline-earth chloride salts, MgCl2, CaCl2, and SrCl2, indicating that trehalose formed metal-complexes with the alkaline-earth metal chlorides. From the data of the 1H-1H coupling constants of trehalose in the presence of the alkaline-earth chlorides, it appeared that trehalose formed complexes with MgCl2, and CaCl2 at the various complexing sites: Mg2+ was coordinated to O-4 and O-4' of trehalose, and Ca2+ to O-2 and O-3. We succeeded in the preparation of two types of crystals of the trehalose/CaCl2. One was a crystal consisting of trehalose, CaCl2, and water in a ratio of 1:1:1. The other was an anhydrous crystal containing trehalose and CaCl2 in a ratio of 1:2. Several applications of the complexing between trehalose and the metal ions for food processing are proposed.

Animals↗
PubMed2005

[Optimization on trehalose loading technique as protective conditioning for lyophilization of human platelets].

This study was aimed to further optimize trehalose loading technique including loading temperature, loading time, loading solution and loading concentration of trehalose, based on the established parameters. Loading efficiency in plasma was compared with that in buffer at 37 degrees C; the curves of intracellular trehalose concentration versus loading time at 37 degrees C and 16 degrees C were measured; curves of mean platelet volume (MPV) versus loading time and loading concentration were investigated and compared. According to results obtained, the loaing time, loading temperature, loading solution and trehalose concentration were ascertained for high loading efficiency of trehalose into human platelet. The results showed that the loading efficiency in plasma was markedly higher than that in buffer at 37 degrees C, the loading efficiency in plasma at 37 degrees C was significantly higher than that at 16 degrees C and reached 19.51% after loading for 4 hours, but 6.16% at 16 degrees C. MPV at 16 degrees C was increased by 43.2% than that at 37 degrees C, but had no distinct changes with loading time and loading concentration. In loading at 37 degrees C, MPV increased with loading time and loading concentration positively. Loading time and loading concentration displayed synergetic effect on MPV. MPV increased with loading time and concentration while trehalose loading concentration was above 50 mmol/L. It is concluded that the optimization parameters of trehalose loading technique are 37 degrees C (temperature), 4 hours (leading time), plasma (loading solution), 50 mmol/L (feasible trehalose concentration). The trehalose concentration can be adjusted to meet the requirement of lyophilization.

Blood Platelets↗
PubMed2001

Trehalose improves survival of electrotransfected mammalian cells.

BACKGROUND: Electropermeabilization is widely used for introduction of DNA and other foreign molecules into eukaryotic cells. However, conditions yielding the greatest molecule uptake and gene expression can result in low cell survival. In this study, we assessed the efficiency of trehalose for enhancing cell viability after excessive electropermeabilization. This disaccharide was chosen because of its capability of stabilizing cell membranes under various stressful conditions, such as dehydration and freezing. MATERIALS AND METHODS: Various mammalian cell lines were electropermeabilized by single exponentially decaying electric pulses of few kV/cm strength and of several-microsecond duration. Propidium iodide (PI) and a plasmid encoding green fluorescent protein (GFP), respectively, served as reporter molecules. The effects of trehalose on PI-uptake, GFP gene expression, transfection yield, and short- and long-term viability were analyzed by flow cytometry and electronic cell counting. RESULTS: The substitution of inositol by trehalose in pulse media protected cells against field-induced cell lysis. The protection effect saturated at about 40-50 mM trehalose. Transfection yield and gene expression were not significantly affected by trehalose. But the transfection efficiency was generally higher in the presence of trehalose, mainly because of the increased cell survival. CONCLUSIONS: We demonstrated that trehalose-substituted media are superior to standard trehalose-free pulse media for improving cell survival and achieving higher electrotransfection efficiency.

Animals↗
PubMed2000

The Kluyver effect for trehalose in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells are able to grow using trehalose as a sole source of carbon and energy. However, the biomass yield obtained with trehalose was higher, and the specific growth rate lower, than that obtained with glucose or maltose. The respiratory inhibitor antimycin A prevented cell growth on trehalose, and no ethanol or glycerol was formed during batch growth on this carbon source. Thus, S. cerevisiae exhibits the KLUYVER effect for trehalose: this disaccharide is assimilated and respired, but, in contrast to glucose or maltose, it cannot be fermented. The high-affinity trehalose-H+ symporter encoded by the AGT1 gene is required for growth on trehalose. Analysis of the differences in the metabolism of maltose and trehalose (both disaccharides of glucose transported by active transport systems) indicated that the absence of trehalose fermentation is a consequence of low sugar influx into the cells during growth on this carbon source.

Antifungal Agents↗
PubMed2002

Stimulation of trehalose efflux from cockroach (Periplaneta americana) fat body by hypertrehalosemic hormone is dependent on protein kinase C and calmodulin.

Protein kinase C and calmodulin play key roles in cockroach fat body during activation of phosphorylase and trehalose efflux by HTH-II. The data support the view that an increase in cytosolic Ca2+ is prerequisite for enhanced activity of protein kinase C and calmodulin. Chelation of Ca2+ (i) with BAPTA blocks HTH-II-induced trehalose efflux from the fat body whereas thapsigargin, which raises [Ca2+]i to the same level as HTH-II, produces only a small, yet significant increase in trehalose efflux. Sphingosine, an inhibitor of protein kinase C, inhibits HTH-II-induced trehalose efflux in a concentration-dependent manner. Trehalose efflux is not activated by the protein kinase C activators OAG or PMA alone but in the presence of thapsigargin both agents increase trehalose efflux to a level comparable to that obtained with HTH-II. Thapsigargin has only a moderate activating effect on phosphorylase but in combination with OAG produces an activation indistinguishable from that provoked by HTH-II. Each of the structurally different calmodulin inhibitors, trifluoperazine, W-7, and calmidazolium, blocks completely the action of HTH-II on trehalose efflux, thus confirming the importance of calmodulin in HTH-II initiated trehalose efflux.

Animals↗
PubMed2006

Cryoprotection with L- and meso-trehalose: stereochemical implications.

Two unnatural stereoisomers of alpha,alpha-trehalose (L- and meso-trehalose) were synthesized and evaluated as cryoprotectants in order to determine the functional consequences of relative or absolute stereochemistry on their physicochemical properties. Adherent yeast cell cultures were frozen in 10% solutions of D-, L-, and meso-trehalose for periods of 7-28 days, then evaluated by a MTT viability assay. D- and L-trehalose were equally effective in maintaining high rates of cell survival, thus demonstrating the absence of chiral discrimination at the carbohydrate-lipid interface, whereas meso-trehalose was inferior in cryoprotection efficacy. Differential scanning calorimetry revealed a difference in the glass transition temperatures (Tg) of D- and meso-trehalose of nearly 75 degrees C. This can be attributed to differences in conformational behavior, as portrayed by torsional energy maps for rotation about the glycosidic bonds of D- and meso-trehalose. We conclude that the biostabilizing properties of alpha,alpha-trehalose depend on relative stereochemical factors, but are independent of absolute stereochemical configuration.

Calorimetry, Differential Scanning↗
PubMed2000

Essential role of trehalose in the synthesis and subsequent metabolism of corynomycolic acid in Corynebacterium matruchotii.

A previous paper indicated that corynomycolates synthesized by the fluffy layer fraction prepared from Corynebacterium matruchotii cells appeared exclusively as alpha-trehalose 6-monocorynomycolate (TMM) (T. Shimakata, K. Tsubokura, T. Kusaka, and K. Shizukuishi, 1985, Arch. Biochem. Biophys. 238, 497-508). In the present communication, the role of trehalose in the synthesis and subsequent metabolism of corynomycolic acids was reexamined. Consequently the following facts were clarified: (i) trehalose 6-phosphate (T-6-P), but not trehalose, stimulated corynomycolate synthesis from palmitate in the presence of ATP; the immediate product was TMM, which showed a rapid turnover. Since the turnover was blocked by addition of alpha-trehalose, only TMM accumulated among corynomycolate-containing substances. These results strongly suggested that T-6-P is an essential component as the acceptor in corynomycolate-synthetic system; (ii) TMM was the precursor not only to alpha-trehalose 6,6'-dicorynomycolate (TDM) and free corynomycolic acids but also to cell wall corynomycolate; (iii) addition of alpha-trehalose blocked the transfer of the corynomycolate moiety from TMM to cell wall corynomycolate, TDM, and free corynomycolic acids to a similar extent. These results clearly indicate that trehalose plays an essential role in the metabolism of corynomycolate after Claisen condensation and subsequent reduction in C. matruchotii.

Adenosine Triphosphate↗
PubMed2001

Directed evolution of operon of trehalose-6-phosphate synthase/phosphatase from Escherichia coli.

Trehalose is a nonspecific protective agent for biomacromolecules. Trehalose-6-phosphate synthase (OtsA)/phosphatase (OtsB), which is encoded by the gene operon otsBA located at -42 of the Escherichia coli genome, is the main enzyme system that catalyzes the synthesis of trehalose in E. coli. We cloned the operon and modified it by directed evolution. Unlike in the previously reported work, we modified the whole operon and screened the positive mutant simultaneously. Thus we believe that the gene complex solves the negative effects between two enzymes if one of them diversifies its structure or functions and finds the form most suitable for trehalose synthesis. It thus mimics the natural process, in which the functional improvement of organisms is related to alterations in coordinated enzymes. The evolution procedure was carried out in a sequence of error-prone PCR, shuffling PCR, and then strict screening of the mutants. After screening of a library of more than 4000 colonies, about 15 positive colonies were analyzed, resulting in a higher concentration of trehalose than control. One of them, E. coli TS7, shows 12.3-fold higher trehalose synthesis ability than E. coli DH5alpha. In contrast, we introduced the cDNA sequence of the tps1 gene from Saccharomyces cerevisiae, which has 54% identity with the gene otsA, as one of the templates in shuffling PCR. By hybrid evolution and screening, we obtained 10 positive colonies with higher concentrations of trehalose than control. E. coli TS22 appears to have 5.3-fold higher trehalose synthesis ability than E. coli DH5alpha and 1.6-fold more than E. coli DEF3(pOTS11). This result demonstrated that coevolution and hybrid evolution, as powerful protocols in protein engineering, are effective in modifying enzyme. It indicates that repeating the process of genomic evolution in nature is feasible.

Directed Molecular Evolution↗