Trehalose Biosynthetic Pathway Research Articles

Fine tuning of trehalose biosynthesis and hydrolysis as novel tools for the generation of abiotic stress tolerant plants.

The impact of abiotic stress on plant growth and development has been and still is a major research topic. An important pathway that has been linked to abiotic stress tolerance is the trehalose biosynthetic pathway. Recent findings showed that trehalose metabolism is also important for normal plant growth and development. The intermediate compound – trehalose-6-phosphate (T6P) – is now confirmed to act as a sensor for available sucrose, hereby directly influencing the type of response to the changing environmental conditions. This is possible because T6P and/or trehalose or their biosynthetic enzymes are part of complex interaction networks with other crucial hormone and sugar-induced signaling pathways, which may function at different developmental stages. Because of its effect on plant growth and development, modification of trehalose biosynthesis, either at the level of T6P synthesis, T6P hydrolysis, or trehalose hydrolysis, has been utilized to try to improve crop yield and biomass. It was shown that alteration of the amounts of either T6P and/or trehalose did result in increased stress tolerance, but also resulted in many unexpected phenotypic alterations. A main challenge is to characterize the part of the signaling pathway resulting in improved stress tolerance, without affecting the pathways resulting in the unwanted phenotypes. One such specific pathway where modification of trehalose metabolism improved stress tolerance, without any side effects, was recently obtained by overexpression of trehalase, which results in a more sensitive reaction of the stomatal guard cells and closing of the stomata under drought stress conditions. We have used the data that have been obtained from different studies to generate the optimal plant that can be constructed based on modifications of trehalose metabolism.

Carbohydrate‐active enzymes revealed in Coptotermes formosanus (Isoptera: Rhinotermitidae) transcriptome

Coptotermes formosanus is one of the most destructive wood-feeding termites. To understand the molecular mechanisms that regulate the development of the termite, a normalized C. formosanus cDNA library was constructed using mixed RNA isolated from workers, soldiers, nymphs and alates of both sexes. The sequencing of this library generated 131 636 expressed sequence tags (ESTs) and 25 939 assembled unigenes. The carbohydrate-active enzymes (CAZymes) revealed in this library were analysed in the present report. A total of 509 putative CAZymes were identified. Diverse cellulolytic enzymes were uncovered from both the host termite and from symbionts harboured by the termite, which were possibly the result of the high efficiency of cellulose utilization. CAZymes associated with trehalose biosynthetic and metabolic pathways were also identified, which are potential regulators of the physiological activities of trehalose, an important insect blood sugar. Representative CAZyme coding genes in glycoside hydrolase family 1 (GH1) were quantitatively analysed. The results showed that the five GH1 β-glucosidase genes were expressed differentially among different castes and one of them was female alate-specific. Overall, the normalized EST library provides a comprehensive genetic resource of C. formosanus and will serve a diverse range of research areas. The CAZymes represent one of the repositories of enzymes useful for physiological studies and applications in sugar-based biofuel production.

How Do Sugars Regulate Plant Growth?

Autotrophic organisms synthesize sugars, principally sucrose in the reactions of photosynthesis. This ancient primary process drives the growth and development of all organisms as a source of energy and carbon source. But can sugars actually regulate growth processes themselves beyond just supplying them with the carbon and energy they need? Well, the answer to this question and articulated in the mini review by Ponnu et al. (2011) is an emphatic yes. Sugars perform diverse functions in living organisms. In plants the pathway that synthesizes the non-reducing disaccharide trehalose, however, has become highly specialized around the signaling function of the precursor of trehalose, trehalose 6-phosphate (T6P). The model that has developed quite rapidly over the last couple of years or so is that T6P signals the availability of sucrose and then through the SnRK1 protein kinase regulatory system orchestrates changes in gene expression that enable sucrose to be used in growth processes. Plants cannot grow, it would seem, without this regulatory system that connects sugars with growth and development. Not only is there regulation of the amount of growth and development by T6P seen in many diverse tissue types, but T6P appears also to control qualitative aspects of growth and development that affect organ form and function such as in inflorescence, leaf, tuber, and seed development. The authors present the context for trehalose metabolism in plants going back to studies from the early 1980s which reported the surprising toxicity of trehalose when fed in high concentrations to plants. Although only recently appreciated this was actually an indication of the strong regulation on growth processes imparted by T6P. The authors then bring readers up to date with latest knowledge about the numerous trehalose phosphate synthase (TPS) and trehalose phosphate phosphatase (TPP) genes found in plants. A large section in then devoted to the regulation of plant carbohydrate metabolism including impacts on photosynthesis, starch and cell wall metabolism, SnRK1, and the emerging role of the bZIP11 transcription factor in the T6P/SnRK1 regulatory system. Of all the genes involved in the trehalose biosynthetic pathway the specific roles of TPS1 and ra3 TPP are the best characterized and these are described in a final section that emphasizes the importance of the pathway in development. Whilst many questions still remain, for example, what is the function of trehalose itself in plants, what is the role of the many TPS-like genes without apparent catalytic activity and does T6P only function in a cell autonomous fashion or are there specific T6P transporters in the cell? Encouragingly some of the previous barriers to progress are coming down as we understand more about the targets of the pathway, its cell specificity and regulation, and as measurements of T6P become less labor intensive and technically demanding. There is promise too that knowledge of T6P biology in plants will soon deliver into tackling global concerns about food security. Indeed, links between T6P and plant abscisic acid stress hormone signaling as well as the increased photosynthetic capacity upon overexpression of TPS may allow engineering plants that can overcome periods of water shortage and/or result in higher yields under non-optimal conditions.

Sugars in crop plants

We review current knowledge of the most abundant sugars, sucrose, maltose, glucose and fructose, in the world's major crop plants. The sucrose-accumulating crops, sugar beet and sugar cane, are included, but the main focus of the review is potato and the major cereal crops. The production of sucrose in photosynthesis and the inter-relationships of sucrose, glucose, fructose and other metabolites in primary carbon metabolism are described, as well as the synthesis of starch, fructan and cell wall polysaccharides and the breakdown of starch to produce maltose. The importance of sugars as hormone-like signalling molecules is discussed, including the role of another sugar, trehalose, and the trehalose biosynthetic pathway. The Maillard reaction, which occurs between reducing sugars and amino acids during thermal processing, is described because of its importance for colour and flavour in cooked foods. This reaction also leads to the formation of potentially harmful compounds, such as acrylamide, and is attracting increasing attention as food producers and regulators seek to reduce the levels of acrylamide in cooked food. Genetic and environmental factors affecting sugar concentrations are described.

Structural Insights on the New Mechanism of Trehalose Synthesis by Trehalose Synthase TreT from Pyrococcus horikoshii

Many microorganisms produce trehalose for stability and survival against various environmental stresses. Unlike the widely distributed trehalose–biosynthetic pathway, which utilizes uridine diphosphate glucose and glucose-6-phosphate, the newly identified enzyme trehalose glycosyltransferring synthase (TreT) from hyperthermophilic bacteria and archaea synthesizes an α,α-trehalose from nucleoside diphosphate glucose and glucose. In the present study, we determined the crystal structure of TreT from Pyrococcus horikoshii at 2.3 Å resolution to understand the detailed mechanism of this novel trehalose synthase. The conservation of essential residues in TreT and the high overall structural similarity of the N-terminal domain to that of trehalose phosphate synthase (TPS) imply that the catalytic reaction of TreT for trehalose synthesis would follow a similar mechanism to that of TPS. The acceptor binding site of TreT shows a wide and commodious groove and lacks the long flexible loop that plays a gating role in ligand binding in TPS. The observation of a wide space at the fissure between two domains and the relative shift of the N-domain in one of the crystal forms suggest that an interactive conformational change between two domains would occur, allowing a more compact architecture for catalysis. The structural analysis and biochemical data in this study provide a molecular basis for understanding the synthetic mechanism of trehalose, or the nucleotide sugar in reverse reaction of the TreT, in extremophiles that may have important industrial implications.

Identification of the trehalose biosynthetic loci of Pseudomonas syringae and their contribution to fitness in the phyllosphere

Surprisingly little is known of the trehalose biosynthetic pathways in pseudomonads, despite the importance of trehalose to protecting cells from environmental stresses such as low water availability. The genome of the foliar pathogen Pseudomonas syringae pv. tomato strain DC3000 contains genes for two trehalose biosynthetic pathways, TreS and TreYZ, and lacks genes for the more common OtsAB pathway. Deletion of either the treS (PSPTO_2760-2762) or treY/treZ (PSPTO_3125-3134) locus eliminated trehalose accumulation and reduced bacterial growth under hyperosmotic conditions. In evaluating the role of trehalose in P. syringae fitness on leaves, we found that a double deletion mutant lacking these loci exhibited poorer survival than the wild type on tomato leaves over a 2-week period in a growth chamber. Similarly, this mutant exhibited reduced survival on leaves of susceptible and resistant cultivars of the host plant tomato and of the non-host plant soybean over a 10-day period in field plots. Thus, the trehalose biosynthetic loci in P. syringae, which are highly conserved among pseudomonads, contributed to DC3000 fitness on leaves, supporting a role for trehalose in P. syringae survival and population maintenance in the phyllosphere.

Role of trehalose-6P phosphatase ( TPS2) in stress tolerance and resistance to macrophage killing in Candida albicans

Disruption of the TPS2 gene encoding the only trehalose-6P phosphatase activity in Candida albicans caused a pleiotropic defective phenotype, maintaining the cell wall integrity and the ability to form chlamydospores. A homozygous tps2Δ/ tps2Δ showed reduced growth at high temperatures and a marked sensitivity to heat shock (42 °C) and severe oxidative exposure (50 mM H 2O 2). Reintroduction of the TPS2 gene reversed these alterations. A more detailed study of the antioxidant response showed that exponential tps2Δ null cells displayed an adaptive response to oxidative stress as well as cross-tolerance between temperature and oxidative stress. Differential measurement of trehalose and trehalose-6P, using reliable new HPLC methodology, revealed a significant accumulation of trehalose-6P in tps2Δ cells, which was enhanced after oxidative exposure. In contrast, the level of trehalose-6P in parental cells was virtually undetectable, and oxidative treatment only induced the synthesis of free trehalose. A transitory increase in the expression of TPS2 and TPS1 genes was promoted in wild-type cells in response to acute (50 mM) but not gentle (5 mM) oxidative exposure. TPS1 and TPS2 oxidative-induced transcriptions were completely absent from the tps2Δ mutant. Exponential blastoconidia from both parental and tps2Δ/ tps2Δ strains were completely phagocytosed by murine and human macrophages, triggering a subsequent proinflammatory response manifested by the release of TNF-α. Reflecting the lower resistance to oxidative stress displayed by the tps2Δ mutant, intracellular survival in resting and IFN- γ and LPS-stimulated macrophages was also diminished. Taken together, our results confirm the mainly protective role played by the trehalose biosynthetic pathway in the cellular response to oxidative stress and subsequently in the resistance to phagocytosis in C. albicans, a defensive mechanism in which TPS2 would be involved.

Trehalose synthase converts glycogen to trehalose

Trehalose (alpha,alpha-1,1-glucosyl-glucose) is essential for the growth of mycobacteria, and these organisms have three different pathways that can produce trehalose. One pathway involves the enzyme described in the present study, trehalose synthase (TreS), which interconverts trehalose and maltose. We show that TreS from Mycobacterium smegmatis, as well as recombinant TreS produced in Escherichia coli, has amylase activity in addition to the maltose <--> trehalose interconverting activity (referred to as MTase). Both activities were present in the enzyme purified to apparent homogeneity from extracts of Mycobacterium smegmatis, and also in the recombinant enzyme produced in E. coli from either the M. smegmatis or the Mycobacterium tuberculosis gene. Furthermore, when either purified or recombinant TreS was chromatographed on a Sephacryl S-200 column, both MTase and amylase activities were present in the same fractions across the peak, and the ratio of these two activities remained constant in these fractions. In addition, crystals of TreS also contained both amylase and MTase activities. TreS produced both radioactive maltose and radioactive trehalose when incubated with [(3)H]glycogen, and also converted maltooligosaccharides, such as maltoheptaose, to both maltose and trehalose. The amylase activity was stimulated by addition of Ca(2+), but this cation inhibited the MTase activity. In addition, MTase activity, but not amylase activity, was strongly inhibited, and in a competitive manner, by validoxylamine. On the other hand, amylase, but not MTase activity, was inhibited by the known transition-state amylase inhibitor, acarbose, suggesting the possibility of two different active sites. Our data suggest that TreS represents another pathway for the production of trehalose from glycogen, involving maltose as an intermediate. In addition, the wild-type organism or mutants blocked in other trehalose biosynthetic pathways, but still having active TreS, accumulate 10- to 20-fold more glycogen when grown in high concentrations (> or = 2% or more) of trehalose, but not in glucose or other sugars. Furthermore, trehalose mutants that are missing TreS do not accumulate glycogen in high concentrations of trehalose or other sugars. These data indicate that trehalose and TreS are both involved in the production of glycogen, and that the metabolism of trehalose and glycogen is interconnected.

Metabolic engineering of Corynebacterium glutamicum for trehalose overproduction: role of the TreYZ trehalose biosynthetic pathway.

Trehalose has many potential applications in biotechnology and the food industry due to its protective effect against environmental stress. Our work explores microbiological production methods based on the capacity of Corynebacterium glutamicum to excrete trehalose. We address here raising trehalose productivity through homologous overexpression of maltooligosyltrehalose synthase and the maltooligosyltrehalose trehalohydrolase genes. In addition, heterologous expression of the UDP-glucose pyrophosphorylase gene from Escherichia coli improved the supply of glycogen. Gene expression effects were tested on enzymatic activities and intracellular glycogen content, as well as on accumulated and excreted trehalose. Overexpression of the treY gene and the treY/treZ synthetic operon significantly increased maltooligosyltrehalose synthase activity, the rate-limiting step, and improved the specific productivity and the final titer of trehalose. Furthermore, a strong decrease was noted in glycogen accumulation. Expression of galU/treY and galU/treYZ synthetic operons showed a partial recovery in the intracellular glycogen levels and a significant improvement in both intra- and extracellular trehalose content.

Exogenous trehalose alters Arabidopsis transcripts involved in cell wall modification, abiotic stress, nitrogen metabolism, and plant defense

Trehalose exists in most living organisms and functions as a storage carbohydrate and as an osmoprotectant in yeast, fungi, and bacteria. Trace amount of endogenous trehalose was detected in flowering plants, and the trehalose biosynthetic pathway was essential for embryo maturation in Arabidopsis. Conversely, exogenous trehalose was toxic to higher plants and severely curtailed root and shoot growth. In the current study, 30 mM trehalose was added to 2‐week‐old liquid cultures containing Arabidopsis thaliana (Columbia ecotype) seedlings. Densely stained granular particles were detected in the extracellular space of cotyledons and roots of trehalose‐treated seedlings using transmission electron microscopy. Expression levels of 91 transcripts were altered by 1–6 h of trehalose treatment using DNA microarray analysis, and 65 of these encoded either known proteins or putative proteins with known functions. The exogenous trehalose treatment altered transcript levels of transcription factors, cell wall modification, nitrogen metabolism, and stress‐related, defense‐related, and fatty acid biosynthesis genes. Many of the transcripts altered by exogenous trehalose treatment were associated with the ethylene and methyl jasmonate‐signaling pathways. The above findings suggested that trehalose, or metabolites derived from trehalose, are important regulators of plant gene expression in higher plants.

Arabidopsis trehalose-6-phosphate synthase 1 is essential for normal vegetative growth and transition to flowering.

In resurrection plants and yeast, trehalose has a function in stress protection, but the absence of measurable amounts of trehalose in other plants precludes such a function. The identification of a trehalose biosynthetic pathway in angiosperms raises questions on the function of trehalose metabolism in nonresurrection plants. We previously identified a mutant in the Arabidopsis trehalose biosynthesis gene AtTPS1. Plants homozygous for the tps1 mutation do not develop mature seeds (Eastmond et al., 2002). AtTPS1 expression analysis and the spatial and temporal activity of its promoter suggest that this gene is active outside the seed-filling stage of development as well. A generally low expression is observed in all organs analyzed, peaking in metabolic sinks such as flower buds, ripening siliques, and young rosette leaves. The arrested tps1/tps1 embryonic state could be rescued using a dexamethasone-inducible AtTPS1 expression system enabling generation of homozygous mutant plants. When depleted in AtTPS1 expression, such mutant plants show reduced root growth, which is correlated with a reduced root meristematic region. Moreover, tps1/tps1 plants are retarded in growth and remain generative during their lifetime. Absence of Trehalose-6-Phosphate Synthase 1 in Arabidopsis plants precludes transition to flowering.

The importance of a functional trehalose biosynthetic pathway for the life of yeasts and fungi

The view of the role of trehalose in yeast has changed in the last few years. For a long time considered a reserve carbohydrate, it gained new importance when its function in the acquisition of thermotolerance was demonstrated. More recently the cellular processes in which the trehalose biosynthetic pathway has been implicated range from the control of glycolysis to sporulation and infectivity by certain fungal pathogens. There is now enough experimental evidence to conclude that trehalose 6-phosphate, an intermediate of trehalose biosynthesis, is an important metabolic regulator in such different organisms as yeasts or plants. Its inhibition of hexokinase plays a key role in the control of the glycolytic flux in Saccharomyces cerevisiae but other, likely important, sites of action are still unknown. We present examples of the phenotypes produced by mutations in the two steps of the trehalose biosynthetic pathway in different yeasts and fungi, and whenever possible examine the molecular explanations advanced to interpret them.

Role of trehalose in growth at high temperature of Salmonella enterica serovar Typhimurium.

Moderate osmolality can stimulate bacterial growth at temperatures near the upper limit for growth. We investigated the mechanism by which high osmolality enhances the thermotolerance of Salmonella enterica serovar Typhimurium, by isolating bacteriophage MudI1734-induced insertion mutations that blocked the growth-stimulatory effect of 0.2 M NaCl at 45 degrees C. One of these mutations proved to be in the seqA gene (a regulator of initiation of DNA synthesis). Because this gene is cotranscribed with pgm (which encodes phosphoglucomutase), it is likely to be polar on the expression of the pgm gene. Pgm catalyzes the conversion of glucose-6-phosphate to glucose-1-phosphate during growth on glucose, and therefore loss of Pgm results in a deficiency in a variety of cellular constituents derived from glucose-1-phosphate, including trehalose. To test the possibility that the growth defect of the seqA::MudI1734 mutant at high temperature in medium of high osmolality is due to the block in trehalose synthesis, we determined the effect of an otsA mutation, which inactivates the first step of the trehalose biosynthetic pathway. The otsA mutation caused a growth defect at 45 degrees C in minimal medium containing 0.2 M NaCl that was similar to that caused by the pgm mutation, but otsA did not affect growth rate in this medium at 37 degrees C. These results suggest that the growth defect of the seqA-pgm mutant at high temperature could be a consequence of the block in trehalose synthesis. We found that, in addition to the well-known osmotic control, there is a temperature-dependent control of trehalose synthesis such that, in medium containing 0.2 M NaCl, cells grown at 45 degrees C had a fivefold higher trehalose pool size than cells grown at 30 degrees C. Our observations that trehalose accumulation is thermoregulated and that mutations that block trehalose synthesis cause a growth defect at high temperature in media of high osmolality suggested that this disaccharide is crucial for growth at high temperature either for turgor maintenance or for protein stabilization.