Trehalose-6-phosphate Synthase Genes Research Articles

Cloning and Characterization of Functional Trehalose-6-Phosphate Synthase Gene in Maize

Trehalose is a non-reducing disaccharide of glucose that functions as a compatible solute in the stabilization of biological structures under heat and desiccation stress in bacteria, fungi, and some “resurrection plants”. In the plant kingdom, trehalose is biosynthesized by trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). Over-expression of exogenous and endogenous genes encoding TPS and TPP is reported to be effective for improving abiotic stress tolerance in tobacco, potato, tomato, rice, and Arabidopsis. On the basis of bioinformatics prediction, we cloned a fragment containing an open reading frame of 2,820 bp from maize, which encodes a protein of 939 amino acids. Phylogenetic analysis showed that this gene belongs to the class I subfamily of the TPS gene family. Analysis of conserved domains revealed the presence of a TPS domain and a TPP domain. Yeast complementation with TPS and TPP mutants demonstrated that this protein has the activity of trehalose-6-phosphate synthase. Semi-quantitative RT-PCR and real-time quantitative PCR indicated that the expression of this gene is upregulated in response to both salt and cold stress.

Isolation and characterization of the trehalose‐6‐phosphate synthase gene from Locusta migratoria manilensis

Abstract Trehalose plays an important role in protecting organisms from various stresses. Trehalose‐6‐phosphate synthase (TPS) is the key enzyme in trehalose synthesis, but in insects only a few TPS genes have been identified and their function has not been well characterized. To better understand the function of TPS in insects, a complete TPS complementary DNA (cDNA) clone was obtained from the fat body of the locust Locusta migratoria manilensis (GenBank accession number: EU131894). The full‐length cDNA is 2 806 bp, including an open reading frame of 2 442 bp, which encodes an 813 amino acids protein with a calculated molecular weight of 91 976 Daltons and an isoelectric point of 6.14. The deduced amino acid sequence is highly similar to other published insect TPS and its C‐terminal also has a region homologous to trehalose phosphate phsophatase (TPP). Semi‐quantitative analysis indicated that the TPS transcript was expressed not only in fat body, but also in gut, hemolymph and leg muscle. These data may facilitate studies of TPS function in insects and improve our understanding of trehalose metabolism.

Metabolic and genetic perturbations accompany the modification of galactomannan in seeds of Medicago truncatula expressing mannan synthase from guar (Cyamopsis tetragonoloba L.)

Galactomannan gums are widely used in the food and oil industries, and there is considerable interest in applying biotechnological approaches to improve their physical properties. A mannan synthase from guar (Cyamopsis tetragonoloba) was expressed under the control of a bean beta-phaseolin promoter in transgenic Medicago truncatula. Although the expression of exogenous mannan synthase caused a slight decrease in galactomannan levels in Medicago, the molecular weight and viscosity of the polymer were significantly increased, although the mannose to galactose ratio and degree of polydispersity remained unchanged. At the same time, expression of about 2.8% of the genes was altered significantly in the seeds of transgenic Medicago lines analysed by Affymetrix genome chip, with a particularly striking induction of putative trehalose phosphate synthase genes. Mannan synthase expression also caused large alterations in the levels of a number of sugars and sugar alcohols, suggesting that over-expression of a processive glycosyltransferase perturbs the mechanisms of sugar sensing and/or homeostasis, possibly involving signalling via trehalose-6-phosphate.

A trehalose 6-phosphate synthase gene of the hemocytes of the blue crab, Callinectes sapidus: cloning, the expression, its enzyme activity and relationship to hemolymph trehalose levels

Trehalose in ectoderms functions in energy metabolism and protection in extreme environmental conditions. We structurally characterized trehalose 6-phosphate synthase (TPS) from hemocytes of the blue crab, Callinectes sapidus. C. sapidus Hemo TPS (CasHemoTPS), like insect TPS, encodes both TPS and trehalose phosphate phosphatase domains. Trehalose seems to be a major sugar, as it shows higher levels than does glucose in hemocytes and hemolymph. Increases in HemoTPS expression, TPS enzyme activity in hemocytes, and hemolymph trehalose levels were determined 24 h after lipopolysaccharide challenge, suggesting that both TPS and TPP domains of CasHemoTPS are active and functional. The TPS gene has a wide tissue distribution in C. sapidus, suggesting multiple biosynthetic sites. A correlation between TPS activity in hemocytes and hemolymph trehalose levels was found during the molt cycle. The current study provides the first evidence of presence of trehalose in hemocytes and TPS in tissues of C. sapidus and implicates its functional role in energy metabolism and physiological adaptation.

Electroporation of maize embryogenic calli with the trehalose-6-phosphate synthase gene from Arabidopsis thaliana

Trehalose is a non-reducing disaccharide of glucose that occurs in a large number of organisms, playing an important role in desiccation and heat stress protection. Trehalose accumulation has proven to be an effective way of increasing drought tolerance in both model plants such as tobacco and important crops such as potato or rice. In this work we aim to genetically engineer maize with the Arabidopsis thaliana trehalose phosphate synthase gene (AtTPS1), involved in trehalose biosynthesis via electroporation. A cassette harboring the AtTPS1 gene under the control of the CaMV35S promoter and the Bialaphos resistance gene Bar as a selective agent was inserted in the plasmid vector pGreen0229 and used to transform maize inbred line Pa91 via electroporation. Fifteen putative transgenic plants (T0 generation) were obtained. Transgene integration in T0 plants was analyzed by Southern-blot analysis. T0 plants had normal phenotypes, although smaller than wild type plants. Contrary to wild type plants, when sexual organs emerged, tassels appeared at least 15 days earlier than ears in the same plant, rendering impossible the self-pollination of the T0 plant. These plants were then crossed with wild type plants and in some cases T1 seeds were obtained. T1 seeds presented deformities, especially the lack of endosperm, but it was still possible to germinate some of these seeds. The so obtained plants were tested by Northern blot but no AtTPS1 gene expression was detected, a fact possibly due to the incomplete insertion of the AtTPS1 gene or an extremely low gene expression level.

Gene families and evolution of trehalose metabolism in plants.

The genomes of Arabidopsis thaliana L., rice (Oryza sativa L.) and poplar (Populus trichocarpa Torr. & A.Gray) contain large families of genes encoding trehalose-phosphate synthase (TPS) and trehalose-phosphatase (TPP). The class I subfamily of TPS genes encodes catalytically active TPS enzymes, and is represented by only one or two genes in most species. A. thaliana is atypical in having four class I TPS genes, three of which (AtTPS2-4) encode unusual short isoforms of TPS that appear to be found only in members of the Brassicaceae family. The class II TPS genes encode TPS-like proteins with a C-terminal TPP-like domain, but there is no experimental evidence that they have any enzymatic activity and their function is unknown. Both classes of TPS gene are represented in the genomes of chlorophyte algae (Ostreococcus species) and non-flowering plants [Physcomitrella patens (Hedw.) Bruch & Schimp.(B.S.G.) and Selaginella moellendorffii (Hieron. in Engl. & Prantl.)]. This survey shows that the gene families encoding the enzymes of trehalose metabolism are very ancient, pre-dating the divergence of the streptophyte and chlorophyte lineages. It also provides a frame of reference for future studies to elucidate the function of trehalose metabolism in plants.

Insights on the evolution of trehalose biosynthesis

BackgroundThe compatible solute trehalose is a non-reducing disaccharide, which accumulates upon heat, cold or osmotic stress. It was commonly accepted that trehalose is only present in extremophiles or cryptobiotic organisms. However, in recent years it has been shown that although higher plants do not accumulate trehalose at significant levels they have actively transcribed genes encoding the corresponding biosynthetic enzymes.ResultsIn this study we show that trehalose biosynthesis ability is present in eubacteria, archaea, plants, fungi and animals. In bacteria there are five different biosynthetic routes, whereas in fungi, plants and animals there is only one. We present phylogenetic analyses of the trehalose-6-phosphate synthase (TPS) and trehalose-phosphatase (TPP) domains and show that there is a close evolutionary relationship between these domains in proteins from diverse organisms. In bacteria TPS and TPP genes are clustered, whereas in eukaryotes these domains are fused in a single protein.ConclusionWe have demonstrated that trehalose biosynthesis pathways are widely distributed in nature. Interestingly, several eubacterial species have multiple pathways, while eukaryotes have only the TPS/TPP pathway. Vertebrates lack trehalose biosynthetic capacity but can catabolise it. TPS and TPP domains have evolved mainly in parallel and it is likely that they have experienced several instances of gene duplication and lateral gene transfer.

Differential Multisite Phosphorylation of the Trehalose-6-phosphate Synthase Gene Family in Arabidopsis thaliana

Multisite protein phosphorylation plays a fundamental role in metabolic regulation. To detect and quantify in vitro kinase phosphorylation activities, we developed a highly selective LC-MS/MS-based method using high resolution multiple reaction monitoring on a triple quadrupole mass spectrometer. This method eliminates the need for stable isotope labeling and enables multiparallel kinase target assays. Using these assays, we made the first observation of in vitro phosphorylation of different trehalose-6-phosphate synthase (TPS) isozymes. TPSs possess putative Ca2+-independent, sucrose non-fermenting 1-related protein kinase 1 (SnRK1) phosphorylation sites. Sixteen synthetic peptides from six different Arabidopsis thaliana TPS isozymes containing the SnRK1 consensus recognition motif were phosphorylated simultaneously in vitro, and their phosphorylation dynamics were determined. We achieved absolute quantification of TPS peptide phosphorylation by tuning the mass spectrometer to the corresponding synthetic standard phosphopeptides. The selectivity of the mass spectrometer in the multiple reaction monitoring mode compensates for the low ionization efficiency of phosphopeptides in the presence of a complex matrix. Results are in close agreement with recent in vivo studies of TPS phosphorylation and regulation and reveal significant differences in the phosphorylation levels of different TPS members within the TPS gene family ranging over 3 orders of magnitude. Substituting EGTA for CaCl2 in the reaction mixture reduced the formation of some of the phospho-TPS peptides drastically, indicating that Ca2+-dependent kinases are active in the presence of Ca2+-independent SnRKs. This agrees with the proposed overlap of the consensus motifs of these kinases and enables delineation between Ca2+-independent and Ca2+-dependent phosphorylation. Results demonstrate that multiparallel kinase target assays are sensitive enough to provide evidence for differential multisite phosphorylation of homologous TPS proteins and their highly conserved putative phosphorylation sites.

Genetic engineering of maize towards desiccation tolerance: Electroporation with the trehalose gene

Trehalose is a non-reducing disaccharide of glucose that occurs in a large number of organisms such as bacteria, fungi, nematodes or crustaceans. Trehalose plays an important role in desiccation and heat stress protection since it has been shown to stabilize proteins and cell membranes under these stress conditions. Trehalose accumulation has proven to be an effective way of increasing drought tolerance in both model plants such as tobacco and important crops such as potato or rice. In this work we aim to improve desiccation tolerance in maize, one of the, most agronomical important crops, by increasing trehalose accumulation through transformation with the Arabidopsis thaliana trehalose phosphate synthase gene (AtTPSl) is involved in trehaIose-6-phosphate synthase and hence on trehalose biosynthesis. A cassette harboring the AtTPSl gene under the control of the CaMV35S promoter and the Bialaphos resistance gene Bar as a selective agent (conferring resistance to the PPT) was inserted in the plasmid vector pGreenO229 and used to transform maize inbred line Pa91. Immature zygotic embryos were collected 14-20 days after pollination and embryogenic calli culture were initiated. Embryog?nie calli were electroporated with 20?g of plasmid DNA using a Biorad Gene Puiser II at 374 V, for 1 second. Embryogenic calli were electroporated and selected PPT. Eighty putative transgenic plants were obtained and analyzed by PCR for the presence of the AtTPS1 gene.

Photosynthetic response to dehydration and high temperature in trehalose-producing transgenic tobacco

Transgenic tobacco plants harboring E. coli TPS (trehalose-6-phosphate synthase) gene (ots A) were generated by leaf disc transformation. Transgenic plants were identified by PCR, Northern hybridization, and trehalose synthesis. All lines of transgenic plants manifested stunted growth and extended generation time in varying degree. Transgenic plants typically exhibited some morphological changes in leaf shape and branching pattern. The stunted growth was not accompanied with early senescence or necrosis. Homozygous plants obtained in F2 generation were used to examine their photosynthetic responses to dehydration and high temperature. All transgenic plants showed enhanced tolerance against dehydration as shown by improved retention in fresh weight after PEG-treatment. However, decreases in water potential by dehydration through PEG-treatment were in parallel phase in both nontransformants and transgenic plants although the initial water potential was observed higher in all transgenic plants. Upon dehydration, decrease in Pmax occurred in similar phase in both nontransformants and transgenic plants, but no significant changes in Chl fluorescence parameters were observed in both nontransformants and transgenic plants. It appears that trehalose confers transgenic plants improved water retaining ability against dehydration, but without keeping their photosynthetic capacity. On the other hand, trehalose-producing plants showed improved ability of holding photosynthetic activity after heating at 40° ~ 45°C. Both Pmax and Chl fluorescence parameters (Fo and Fv/Fm) remained more favorable in transgenic plants after heat treatment than nontransformants. The results suggest that engineering trehalose-producing plants would be more effective in conferring enhanced tolerance against high temperature than dehydration in term of maintaining photosynthetic productivity.