Cloning and characterization of genes encoding trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2) from Zygosaccharomyces rouxii

In many organisms, trehalose protects against several environmental stresses, such as heat, desiccation, and salt, probably by stabilizing protein structures and lipid membranes. Trehalose synthesis in yeast is mediated by a complex of trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2). In this study, genes encoding TPS1 and TPS2 were isolated from Zygosaccharomyces rouxii (designated ZrTPS1 and ZrTPS2, respectively). They were functionally identified by their complementation of the tps1 and tps2 yeast deletion mutants, which are unable to grow on glucose medium and with heat, respectively. Full-length ZrTPS1 cDNA is composed of 1476 nucleotides encoding a protein of 492 amino acids with a molecular mass of 56 kDa. ZrTPS2 cDNA consists of 2843 nucleotides with an open reading frame of 2700 bp, which encodes a polypeptide of 900 amino acids with a molecular mass of 104 kDa. The amino acid sequence encoded by ZrTPS1 has relatively high homology with TPS1 of Saccharomyces cerevisiae and Schizosaccharomyces pombe, compared with TPS2. Western blot analysis showed that the antibody against S. cerevisiae TPS1 recognizes ZrTPS1. Under normal growth conditions, ZrTPS1 and ZrTPS2 were highly and constitutively expressed, unlike S. cerevisiae TPS1 and TPS2. Salt stress and heat stress reduced the expression of the ZrTPS1 and ZrTPS2 genes, respectively.     

Trehalose metabolism genes in Caenorhabditis elegans and filarial nematodes

The sugar trehalose is claimed to be important in the physiology of nematodes where it may function in sugar transport, energy storage and protection against environmental stresses. In this study we investigated the role of trehalose metabolism in nematodes, using Caenorhabditis elegans as a model, and also identified complementary DNA clones putatively encoding genes involved in trehalose pathways in filarial nematodes. In C. elegans two putative trehalose-6-phosphate synthase (tps) genes encode the enzymes that catalyse trehalose synthesis and five putative trehalase (tre) genes encode enzymes catalysing hydrolysis of the sugar. We showed by RT-PCR or Northern analysis that each of these genes is expressed as mRNA at all stages of the C. elegans life cycle. Database searches and sequencing of expressed sequence tag clones revealed that at least one tps gene and two tre genes are expressed in the filarial nematode Brugia malayi, while one tps gene and at least one tre gene were identified for Onchocerca volvulus. We used the feeding method of RNA interference in C. elegans to knock down temporarily the expression of each of the tps and tre genes. Semiquantitative RT-PCR analysis confirmed that expression of each gene was silenced by RNA interference. We did not observe an obvious phenotype for any of the genes silenced individually but gas-chromatographic analysis showed >90% decline in trehalose levels when both tps genes were targeted simultaneously. This decline in trehalose content did not affect viability or development of the nematodes.     

Physiological roles of trehalose in Leptinotarsa larvae revealed by RNA interference of trehalose-6-phosphate synthase and trehalase genes

Trehalose is proposed to serve multiple physiological roles in insects. However, its importance remains largely unconfirmed. In the present paper, we knocked down either a trehalose biosynthesis gene (trehalose-6-phosphate synthase, LdTPS) or each of three degradation genes (soluble trehalases LdTRE1a, LdTRE1b or membrane-bound LdTRE2) in Leptinotarsa decemlineata by RNA interference (RNAi). Knockdown of LdTPS decreased trehalose content and caused larval and pupal lethality. The LdTPS RNAi survivors consumed a greater amount of foliage, obtained a heavier body mass, accumulated more glycogen, lipid and proline, and had a smaller amount of chitin compared with the controls. Ingestion of trehalose but not glucose rescued the food consumption increase and larval mass rise, increased survivorship, and recovered glycogen, lipid and chitin to the normal levels. In contrast, silencing of LdTRE1a increased trehalose content and resulted in larval and pupal lethality. The surviving LdTRE1a RNAi hypomorphs fed a smaller quantity of food, had a lighter body weight, depleted lipid and several glucogenic amino acids, and contained a smaller amount of chitin. Neither trehalose nor glucose ingestion rescued these LdTRE1a RNAi defects. Silencing of LdTRE1b caused little effects. Knockdown of LdTRE2 caused larval death, increased trehalose contents in several tissues and diminished glycogen in the brain-corpora cardiaca-corpora allata complex (BCC). Feeding glucose but not trehalose partially rescued the high mortality rate and recovered glycogen content in the BCC. It seems that trehalose is involved in feeding regulation, sugar absorption, brain energy supply and chitin biosynthesis in L. decemlineata larvae.     

The function of trehalose biosynthesis in plants

Trehalose (alpha-D-glucopyranosyl-1,1-alpha-D-glucopyranoside) occurs in a large variety of organisms, ranging from bacteria to invertebrate animals, where it serves as an energy source or stress protectant. Until recently, only few plant species, mainly desiccation-tolerant 'resurrection' plants, were considered to synthesise trehalose. Instead of trehalose, most other plants species accumulate sucrose as major transport sugar and during stress. The ability to synthesize sucrose has probably evolved from the cyanobacterial ancestors of plastids and may be linked to photosynthetic function. Although most plant species do not appear to accumulate easily detectable amounts of trehalose, the discovery of genes for trehalose biosynthesis in Arabidopsis and in a range of crop plants suggests that the ability to synthesise trehalose is widely distributed in the plant kingdom. The apparent lack of trehalose accumulation in these plants is probably due to the presence of trehalase activity. After inhibition of trehalase, trehalose synthesis can be detected in Arabidopsis. Since trehalose induces metabolic changes, such as an accumulation of storage carbohydrates, rapid degradation of trehalose may be required to prevent detrimental effects of trehalose on the regulation of plant metabolism. In addition, the precursor of trehalose, trehalose-6-phosphate, is probably involved in the regulation of developmental and metabolic processes in plants.     

植物海藻糖-6-磷酸合成酶基因研究进展

海藻糖-6-磷酸代谢通路是植物响应非生物胁迫生理调控网络中的重要组成部分,海藻糖-6-磷酸合成酶基因TPS（Trehalose-6-phosphate synthase）是植物合成海藻糖的关键基因。为了更加充分地理解TPS基因在植物应答非生物胁迫、调节开花时间等方面的作用,本文首先对植物TPS基因家族成员的系统进化关系及序列结构信息进行了研究,重点论述了TPS基因在植物响应干旱、盐害、高温、低温胁迫中的调控功能及分子作用机制,最后对TPS基因参与植物开花调控的研究进展进行了综述。本文深入总结了目前植物TPS基因响应非生物胁迫及开花调控的研究进展,并对今后TPS同源基因的研究方向和应用价值进行了展望。     

Mode of action of the gcr9 and cat3 mutations in restoring the ability of Saccharomyces cerevisiae tps1 mutants to grow on glucose

Mutations in the TPS1 gene, which encodes trehalose-6-P synthase, cause a glucose-negative phenotype in Saccharomyces cerevisiae. Antimycin A or disruption of the QCR9 gene, which encodes one subunit of the cytochrome bc ₁ complex, restore the ability to grow in glucose-containing media. Under these conditions the cell excreted a large amount of glycerol, corresponding to about 20% of the glucose taken up. Suppression appears to be achieved by diversion of accumulated glycolytic intermediates to the production of glycerol, thereby providing NAD⁺ and phosphate for the glyceraldehyde-3-P dehydrogenase reaction. Analysis of the mutation scil-1, which also suppresses the glucose-negative phenotype of tps1 mutants, showed that glucose transport was decreased in scil-1 mutants. The gene SCI1 was cloned and its nucleotide sequence revealed it to be identical to CAT3/SNF4. The suppression mediated by scil-1 is attributable to a decrease in glycolytic flux.This paper is dedicated to Professor Friedrich K. Zimmermann on the occasion of his sixtieth birthday     

Proteolytic activation of α,α-trehalose 6-phosphate synthase in Candida utilis

Total trehalose 6-phosphate synthase activity increased in cell-free extracts from Candida utilis following short-term preincubation of the enzyme samples at 37 degrees C. This endogenous activation was prevented by the inhibitors of serine-type proteases, phenylmethylsulfonyl fluoride, antipain or chymostatin, but not by other protease inhibitors such as pepstatin. Fractionation of the cell extracts by Sephadex G-200 gel filtration revealed that the activity of one of the two synthase enzymes present in these cells was enhanced after the activation treatment. These observations indicate the existence of a proteolytically activatable enzyme form in the trehalose 6-phosphate synthase complex of this yeast in addition to the previously characterized enzyme, whose activity appears to be inactivated by reversible phosphorylation.     

The fdp1 and cif1 mutations are caused by different single nucleotide changes in the yeast CIF1 gene

Abstract The allelism between the mutations cif1 and fdp1 from Saccharomyces cerevisiae has been demonstrated using PCR techniques and complementation of function. The cif1 mutation results in a shortened version of the protein while the fdp1 mutation introduces a charged residue in a highly hydrophobic stretch.     

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.     

Arginine mediated purification of trehalose-6-phosphate synthase (TPS) from Candida utilis: Its characterization and regulation

Background

Trehalose is the most important multifunctional, non-reducing disaccharide found in nature. It is synthesized in yeast by an enzyme complex: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP).

Methods

In the present study TPS is purified using a new methodology from Candida utilis cells by inclusion of 100 mM l-arginine during cell lysis and in the mobile phase of high performance gel filtration liquid chromatography (HPGFLC).

Results

An electrophoretically homogenous TPS that was purified was a 60 kDa protein with 22.1 fold purification having a specific activity of 2.03 U/mg. Alignment of the N-terminal sequence with TPS from Saccharomyces cerevisiae confirmed the 60 kDa protein to be TPS. Optimum activity of TPS was observed at a protein concentration of 1 μg, at a temperature of 37 °C and pH 8.5. Aggregation mediated enzyme regulation was indicated. Metal cofactors, especially MnCl₂, MgCl₂ and ZnSO₄, acted as stimulators. Metal chelators like CDTA and EGTA stimulated enzyme activity. Among the four glucosyl donors, the highest V_max and lowest K_m values were calculated as 2.96 U/mg and 1.36 mM when adenosine di phosphate synthase (ADPG) was used as substrate. Among the glucosyl acceptors, glucose-6-phosphate (G-6-P) showed maximum activity followed by fructose-6-phosphate (F-6-P). Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity with different glucosyl donors.

General significance

Substrate specificity, V_max and K_m values provided an insight into an altered trehalose metabolic pathway in the C. utilis strain where ADPG is the preferred substrate rather than the usual substrate uridine diphosphaphate glucose (UDPG). The present work employs a new purification strategy as well as highlights an altered pathway in C. utilis.     

Trehalose 6-phosphate synthase from Selaginella lepidophylla: purification and properties

A protein of 440 kDa with trehalose 6-phosphate synthase activity was purified with only one purification step by immobilized metal affinity chromatography, from fully hydrated Selaginella lepidophylla plants. The enzyme was purified 50-fold with a yield of 89% and a specific activity of 7.05 U/mg protein. This complex showed two additional aggregation states of 660 and 230 kDa. The three complexes contained 50, 67, and 115 kDa polypeptides with pI of 4.83, 4.69, and 4.55. The reaction was highly specific for glucose 6-phosphate and UDP-glucose. The optimum pH was 7.0 and the enzyme was stable from pH 5.0 to 10. The enzyme was activated by low concentrations of Ca2+, Mg2+, K+, and Na+ and by fructose 6-phosphate, fructose, and glucose. Proline had an inhibitory effect, while sucrose and trehalose up to 0.4M did not have any effect on the activity. Neither the substrates nor final product had an inhibitory effect.     

A yeast gene for trehalose-6-phosphate synthase and its complementation of an Escherichia coli otsA mutant

Abstract A Saccharomyces cerevisiae gene for trehalose-6-phosphate synthase (TPS1) was sequenced. The gene appeared to code for a protein of 495 amino acid residues, giving the protein a molecular mass of 56 kDa. The TPS1 gene was able to restore both osmotolerance and trehalose accumulation during salt stress in an Escherichia coli strain mutated in the otsA gene encoding trehalose-6-phosphate synthase. Complementation studies with E. coli galU mutants showed that the TPS1-encoded trehalose-6-phosphate synthase is UDP-glucose-dependent. Sequence analysis and data base searches showed that TPS1 is allelic to GGS1, byp1, cif1 and fdp1 . A possible gene for trehalose-6-phosphate synthase in Methanobacterium thermoautotrophicum was identified.     

A genetically encoded Förster resonance energy transfer sensor for monitoring in vivo trehalose-6-phosphate dynamics

Trehalose-6-phosphate is a pivotal regulator of sugar metabolism, growth, and osmotic equilibrium in bacteria, yeasts, and plants. To directly visualize the intracellular levels of intracellular trehalose-6-phosphate, we developed a series of specific Förster resonance energy transfer (FRET) sensors for in vivo microscopy. We demonstrated real-time monitoring of regulation in the trehalose pathway of Escherichia coli. In Saccharomyces cerevisiae, we could show that the concentration of free trehalose-6-phosphate during growth on glucose is in a range sufficient for inhibition of hexokinase. These findings support the hypothesis of trehalose-6-phosphate as the effector of a negative feedback system, similar to the inhibition of hexokinase by glucose-6-phosphate in mammalian cells and controlling glycolytic flux.