Role of periplasmic trehalase in uptake of trehalose by the thermophilic bacterium Rhodothermus marinus

Trehalose uptake at 65°C in Rhodothermus marinus was characterized. The profile of trehalose uptake as a function of concentration showed two distinct types of saturation kinetics, and the analysis of the data was complicated by the activity of a periplasmic trehalase. The kinetic parameters of this enzyme determined in whole cells were as follows: K_m = 156 ± 11 μM and V_max = 21.2 ± 0.4 nmol/min/mg of total protein. Therefore, trehalose could be acted upon by this periplasmic activity, yielding glucose that subsequently entered the cell via the glucose uptake system, which was also characterized. To distinguish the several contributions in this intricate system, a mathematical model was developed that took into account the experimental kinetic parameters for trehalase, trehalose transport, glucose transport, competition data with trehalose, glucose, and palatinose, and measurements of glucose diffusion out of the periplasm. It was concluded that R. marinus has distinct transport systems for trehalose and glucose; moreover, the experimental data fit perfectly with a model considering a high-affinity, low-capacity transport system for trehalose (K_m = 0.11 ± 0.03 μM and V_max = 0.39 ± 0.02 nmol/min/mg of protein) and a glucose transporter with moderate affinity and capacity (K_m = 46 ± 3 μM and V_max = 48 ± 1 nmol/min/mg of protein). The contribution of the trehalose transporter is important only in trehalose-poor environments (trehalose concentrations up to 6 μM); at higher concentrations trehalose is assimilated primarily via trehalase and the glucose transport system. Trehalose uptake was constitutive, but the activity decreased 60% in response to osmotic stress. The nature of the trehalose transporter and the physiological relevance of these findings are discussed.     

Trehalose Toxicity in Cuscuta reflexa: Cell Wall Synthesis Is Inhibited upon Trehalose Feeding

α,α-Trehalose induced a rapid blackening of the terminal 2.5-centimeter region of excised Cuscuta reflexa Roxb. vine. The incorporation of radioactivity from [¹⁴C]glucose into alkali-insoluble fraction of shoot tip was markedly inhibited by 12 hours of trehalose feeding to an excised vine. This inhibition was confined to the apical segment of the vine in which cell elongation occurred. The rate of blackening of shoot tip explants was hastened by the addition of gibberellic acid A₃, which promoted elongation growth of isolated Cuscuta shoot tips. The symptom of trehalose toxicity was duplicated by 2-deoxyglucose, which has been shown to be a potent inhibitor of cell wall synthesis in yeast. The observations suggest that trehalose interferes with the synthesis of cell wall polysaccharides, the chief component of which was presumed to be cellulose.     

Three Enzymes for Trehalose Synthesis in Bradyrhizobium Cultured Bacteria and in Bacteroids from Soybean Nodules

α,α-Trehalose is a disaccharide accumulated by many microorganisms, including rhizobia, and a common role for trehalose is protection of membrane and protein structure during periods of stress, such as desiccation. Cultured Bradyrhizobium japonicum and B. elkanii were found to have three enzymes for trehalose synthesis: trehalose synthase (TS), maltooligosyltrehalose synthase (MOTS), and trehalose-6-phosphate synthetase. The activity level of the latter enzyme was much higher than those of the other two in cultured bacteria, but the reverse was true in bacteroids from nodules. Although TS was the dominant enzyme in bacteroids, the source of maltose, the substrate for TS, is not clear; i.e., the maltose concentration in nodules was very low and no maltose was formed by bacteroid protein preparations from maltooligosaccharides. Because bacteroid protein preparations contained high trehalase activity, it was imperative to inhibit this enzyme in studies of TS and MOTS in bacteroids. Validamycin A, a commonly used trehalase inhibitor, was found to also inhibit TS and MOTS, and other trehalase inhibitors, such as trehazolin, must be used in studies of these enzymes in nodules. The results of a survey of five other species of rhizobia indicated that most species sampled had only one major mechanism for trehalose synthesis. The presence of three totally independent mechanisms for the synthesis of trehalose by Bradyrhizobium species suggests that this disaccharide is important in the function of this organism both in the free-living state and in symbiosis.     

Cloning and truncation modification of trehalose-6-phosphate synthase gene from Selaginella pulvinata

A homologous sequence was amplified from resurrection plant Selaginella pulvinta by RACE technique, proved to be the full-length cDNA of trehalose-6-phosphate synthase gene by homologous alignment and yeast complementation assay, and nominated as SpTPS1 gene. The open reading frame of this gene was truncated 225 bp at the 5′-end, resulting the N-terminal truncation modification of 75 amino acids for its encoding protein. The TPS1 deletion mutant strain YSH290 of the brewer's yeast transformed by the truncated gene SpTPS1Δ and its original full-length version restored growth on the medium with glucose as a sole carbon source and displayed growth curves with no significant difference, indicating their encoding proteins functioning as TPS enzyme. The TPS activity of the mutant strain transformed by the truncated gene SpTPS1Δ was about six fold higher than that transformed by its original version, reasoning that the extra N-terminal extension of the full-length amino acid sequence acts as an inhibitory domain to trehalose synthesis. However, the trehalose accumulation of the mutant strain transformed by the truncated gene SpTPS1Δ was only 8% higher than that transformed by its original version. This result is explained by the feedback balance of trehalose content coordinated by the comparative activities between trehalose synthase and trehalase. The truncated gene SpTPS1Δ is suggested to be used in transgenic operation, together with the inhibition of trehalase activity by the application of validamycin A or genetic deficiency of the endogenous trehalase gene, for the enhancement of trehalose accumulation and improvement of abiotic tolerance in transgenic plants.     

Effects of validamycin A,a potent trehalase inhibitor,and phytohormones on trehalose metabolism in roots and root nodules of soybean and cowpea

Trehalose, a common microbial disaccharide, has been reported to be toxic to plants, and plant trehalase has therefore been hypothesized to function as a detoxifying enzyme. To test this, aseptically grown soybean (Glycine max L. Merr.) plantlets were supplied with trehalose. The plants accumulated trehalose only when validamycin A, a potent trehalase inhibitor, was added as well. Under these conditions, they accumulated trehalose to up to 8% of the dry weight in their primary leaves without any detectable impairment of growth or health. We have previously shown that in soybean nodules, trehalose is generated by the symbiotic bacteria, and trehalase is strongly induced. However, direct exposure of plants to trehalose did not affect their trehalase activity, whereas a treatment with auxin strongly increased it, indicating that the enzyme level is regulated by hormones rather than by its substrate. Addition of validamycin A to nodules caused an increase in the amount of trehalose and a decrease in the sucrose and starch pools, but nitrogen fixation was not affected. Similar results were obtained with cowpea (Vigna unguiculata L.) plantlets and nodules. These results indicate that plant trehalase is functional in metabolizing trehalose from exogenous and endogenous sources, even though the disaccharide has no obvious toxic effects.Abbreviations ABA abscisic acid - ARA acetylene-reduction activity (assay for nitrogenase) - 2,4-D 2,4-dichlorophenoxyacetic acid - DW dry weight - FW fresh weight - GA₃ gibberellic acid - -NAA, -NAA -,-naphthaleneacetic acid We are indebted to Prof. Dr. W. Broughton (University of Geneva, Switzerland) for kindly providing us cowpea seeds and the symbiont strain Rhizobium sp. NGR 234. Validamycin A was a gift of Dr. J.-P. Métraux, Ciba, Basel. This work was supported by the Swiss National Foundation.     

Trehalose Toxicity in Cuscuta reflexa: SUCROSE CONTENT DECREASES IN SHOOT TIPS UPON TREHALOSE FEEDING

Trehalose, an α,α-diglucoside, induced a rapid blackening and death of shoot tips of Cuscuta reflexa (dodder) cultured in vitro. The onset of toxic symptom was delayed if any of the several sugars which support the in vitro growth of Cuscuta was supplied with trehalose. The rate of trehalose uptake or its accumulation in the tissue was not affected by sugar cofeeding. The levels of total and reducing sugars declined appreciably in the trehalose-fed shoot tip explants compared to control tissue cultured in absence of a carbon source. This was not due to an increased rate of respiration of the trehalose-treated tissue. In shoot tips cultured in presence of both trehalose and sucrose, the decline in total and reducing sugars was curtailed. There was a marked fall in the level of sucrose; and invertase activity was higher in trehalose-fed shoot tips. The incorporation of label from [¹⁴C]glucose into sucrose in the shoot tip explant was reduced as early as 12 h of trehalose feeding. The results suggest that increased utilization of sucrose as well as an inhibition of its synthesis contribute to the drastic fall in the sucrose content upon trehalose feeding.     

The ram1 Mutant of Arabidopsis Exhibits Severely Decreased β-Amylase Activity

Despite extensive biochemical analyses, the biological function(s) of plant β-amylases remains unclear. The fact that β-amylases degrade starch in vitro suggests that they may play a role in starch metabolism in vivo. β-Amylases have also been suggested to prevent the accumulation of highly polymerized polysaccharides that might otherwise impede flux through phloem sieve pores. The identification and characterization of a mutant of Arabidopsis var. Columbia with greatly reduced levels of β-amylase activity is reported here. The reduced β-amylase 1 (ram1) mutation lies in the gene encoding the major form of β-amylase in Arabidopsis. Although the Arabidopsis genome contains nine known or putative β-amylase genes, the fact that the ram1 mutation results in almost complete loss of β-amylase activity in rosette leaves and inflorescences (stems) indicates that the gene affected by the ram1 mutation is responsible for most of the β-amylase activity present in these tissues. The leaves of ram1 plants accumulate wild-type levels of starch, soluble sugars, anthocyanin, and chlorophyll. Plants carrying the ram1 mutation also exhibit wild-type rates of phloem exudation and of overall growth. These results suggest that little to no β-amylase activity is required to maintain normal starch levels, rates of phloem exudation, and overall plant growth.     

Physiological Role of β-Phosphoglucomutase in Lactococcus lactis

A β-phosphoglucomutase (β-PGM) mutant of Lactococcus lactis subsp. lactis ATCC 19435 was constructed using a minimal integration vector and double-crossover recombination. The mutant and the wild-type strain were grown under controlled conditions with different sugars to elucidate the role of β-PGM in carbohydrate catabolism and anabolism. The mutation did not significantly affect growth, product formation, or cell composition when glucose or lactose was used as the carbon source. With maltose or trehalose as the carbon source the wild-type strain had a maximum specific growth rate of 0.5 h⁻¹, while the deletion of β-PGM resulted in a maximum specific growth rate of 0.05 h⁻¹ on maltose and no growth at all on trehalose. Growth of the mutant strain on maltose resulted in smaller amounts of lactate but more formate, acetate, and ethanol, and approximately 1/10 of the maltose was found as β-glucose 1-phosphate in the medium. Furthermore, the β-PGM mutant cells grown on maltose were considerably larger and accumulated polysaccharides which consisted of α-1,4-bound glucose units. When the cells were grown at a low dilution rate in a glucose and maltose mixture, the wild-type strain exhibited a higher carbohydrate content than when grown at higher growth rates, but still this content was lower than that in the β-PGM mutant. In addition, significant differences in the initial metabolism of maltose and trehalose were found, and cell extracts did not digest free trehalose but only trehalose 6-phosphate, which yielded β-glucose 1-phosphate and glucose 6-phosphate. This demonstrates the presence of a novel enzymatic pathway for trehalose different from that of maltose metabolism in L. lactis.     

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 Arabidopsis pi4kIIIβ1β2 double mutant is salicylic acid-overaccumulating: a new example of salicylic acid influence on plant stature

Growth is the best visible sign of plant comfort. If plants are under stress, a difference in growth with control conditions can indicate that something is going wrong (or better). Phytohormones such as auxin, cytokinins, brassinosteroids or giberellins, are important growth regulators and their role in plant growth was extensively studied. On the other hand the role of salicylic acid (SA), a phytohormone commonly connected with plant defense responses, in plant growth is under-rated. However, studies with SA-overaccumulating mutants directly showed an influence of SA on plant growth. Recently we characterized an Arabidopsis SA-overaccumulating mutant impaired in phosphatidylinositol-4-kinases (pi4kIIIβ1β2). This mutant is dwarf. The crossing with mutants impaired in SA signaling revealed that pi4kIIIβ1β2 stunted rosette is due to high SA, while the short root length is not. This brings into evidence that upper and lower parts of the plants, even though they may share common phenotypes, are differently regulated.     

Induction of trehalase in Arabidopsis plants infected with the trehalose-producing pathogen Plasmodiophora brassicae

Various microorganisms produce the disaccharide trehalose during their symbiotic and pathogenic interactions with plants. Trehalose has strong effects on plant metabolism and growth; therefore, we became interested to study its possible role in the interaction of Arabidopsis thaliana with Plasmodiophora brassicae, the causal agent of clubroot disease. We found that trehalose accumulated strongly in the infected organs (i.e., the roots and hypocotyls) and, to a lesser extent, in the leaves and stems of infected plants. This accumulation pattern of trehalose correlated with the expression of a putative trehalose-6-phosphate synthase (EC 2.4.1.15) gene from P. brassicae, PbTPS1. Clubroot formation also resulted in an induction of the Arabidopsis trehalase gene, ATTRE1, and in a concomitant increase in trehalase (EC 3.2.1.28) activity in the roots and hypocotyls, but not in the leaves and stems of infected plants. Thus, induction of ATTRE1 expression was probably responsible for the increased trehalase activity. Trehalase activity increased before trehalose accumulated; therefore, it is unlikely that trehalase was induced by its substrate. The induction of trehalase may be part of the plant's defense response and may prevent excess accumulation of trehalose in the plant cells, where it could interfere with the regulation of carbon metabolism.     

Contribution of trehalose biosynthetic pathway to drought stress tolerance of Capparis ovata Desf

Trehalose and the trehalose biosynthetic pathway are important contributors and regulators of stress responses in plants. Among recent findings for trehalose and its metabolism, the role of signalling in the regulation of growth and development and its potential for use as a storage energy source can be listed. The xerophytic plant Capparis ovata (caper) is well adapted to drought and high temperature stress in arid and semi‐arid regions of the Mediterranean. The contribution of trehalose and the trehalose biosynthetic pathway to drought stress responses and tolerance in C. ovata are not known. We investigated the effects of PEG‐mediated drought stress in caper plants and analysed physiological parameters and trehalose biosynthetic pathway components, trehalose‐6‐phosphate synthase (TPS), trehalose‐6‐phosphate phosphatase (TPP), trehalase activity, trehalose and proline content in drought stress‐treated and untreated plants. Our results indicated that trehalose and the trehalose biosynthetic pathway contributed to drought stress tolerance of C. ovata. Overall growth and leaf water status were not dramatically affected by drought, as both high relative growth rate and relative water content were recorded even after 14 days of drought stress. Trehalose accumulation increased in parallel to induced TPS and TPP activities and decreased trehalase activity in caper plants on day 14. Constitutive trehalose levels were 28.75 to 74.75 μg·g·FW^?1, and drought stress significantly induced trehalose accumulation (385.25 μg·g·FW^?1 on day 14) in leaves of caper. On day 14 of drought, proline levels were lower than on day 7. Under drought stress the discrepancy between trehalose and proline accumulation trends might result from the mode of action of these osmoprotectant molecules in C. ovata.     

Phospholipase D δ knock-out mutants are tolerant to severe drought stress

Phospholipase D (PLD) is involved in different plant processes, ranging from responses to abiotic and biotic stress to plant development. Phospholipase Dδ (PLDδ) is activated in dehydration and salt stress, producing the lipid second messenger phosphatidic acid. In this work we show that pldδ Arabidopsis mutants were more tolerant to severe drought than wild-type plants. PLDδ has been shown to be required for ABA regulation of stomatal closure of isolated epidermal peels. However, there was no significant difference in stomatal conductance at the whole plant level between wild-type and pldδ mutants. Since PLD hydrolyses structural phospholipids, then we looked at membrane integrity. Ion leakage measurements showed that during dehydration of leaf discs pldδ mutant has less membrane degradation compared to the wild-type. We further analyzed the mutants and showed that pldδ have higher mRNA levels of RAB18 and RD29A compared to wild-type plants under normal growth conditions. Transient expression of AtPLDδ in Nicotiana benthamiana plants induced a wilting phenotype. These findings suggest that, in wt plants PLDδ disrupt membranes in severe drought stress and, in the absence of the protein (PLDδ knock-out) might drought-prime the plants, making them more tolerant to severe drought stress. The results are discussed in relation to PLDδ role in guard cell signaling and drought tolerance.     

Oats Tolerant of Pseudomonas syringae pv. tabaci Contain Tabtoxinine-beta-Lactam-Insensitive Leaf Glutamine Synthetases

Pseudomonas syringae pv. tabaci, a commonly recognized leaf pathogen of tobacco, can infest the rhizosphere of many plants, including oats. Normal oat plants do not survive this infestation as a consequence of the complete and irreversible inactivation of all of their glutamine synthetases by tabtoxinine-β-lactam (TβL), a toxin released by pv. tabaci. We have identified a population of oat (Avena sativa L. var Lodi) plants that are tolerant of pv. tabaci. The tolerant plants had no detectable TβL-detoxification mechanisms. Pathogen growth on these plant roots was not inhibited. These plants contain leaf glutamine synthetases (GS₁ and GS₂) that were less sensitive to inactivation by TβL in vitro; these GSs have normal K_m values for glutamate and ATP when compared with those of GS in control plants. Root glutamine synthetase of the tolerant plants was inactivated in vivo during infestation by the pathogen or by TβL in vitro. When growing without pv. tabaci, the tolerant plants contained normal levels of glutamine synthetase in their roots and leaves and normal levels of protein, ammonia, glutamate, and glutamine in their leaves. However, when the tolerant plants' rhizosphere was infested with pv. tabaci, the plant leaves contained elevated levels of glutamine synthetase activity, protein, ammonia, glutamate, and glutamine. No changes in glutamate dehydrogenase activity were detected in leaves and roots of pathogen-infested tolerant plants.     

Extracellular trehalose utilization by Saccharomyces cerevisiae

Two haploid strains of Saccharomyces cerevisiae viz. MATα and MATa were grown in glucose and trehalose medium and growth patterns were compared. Both strains show similar growth, except for an extended lag phase in trehalose grown cells. In both trehalose grown strains increase in activities of both extracellular trehalase activities and simultaneous decrease in extracellular trehalose level was seen. This coincided with a sharp increase in extracellular glucose level and beginning of log phase of growth. Alcohol production was also observed. Secreted trehalase activity was detected, in addition to periplasmic activity. It appeared that extracellular trehalose was hydrolyzed into glucose by extracellular trehalase activity. This glucose was utilized by the cells for growth. The alcohol formation was due to the fermentation of glucose. Addition of extracellular trehalase caused reduction in the lag phase when grown in trehalose medium, supporting our hypothesis of extracellular utilization of trehalose.     

Analysis of Hydroxycinnamic Acid Degradation in Agrobacterium fabrum Reveals a Coenzyme A-Dependent,Beta-Oxidative Deacetylation Pathway

The soil- and rhizosphere-inhabiting bacterium Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to have species-specific genes involved in ferulic acid degradation. Here, we characterized, by genetic and analytical means, intermediates of degradation as feruloyl coenzyme A (feruloyl-CoA), 4-hydroxy-3-methoxyphenyl-β-hydroxypropionyl–CoA, 4-hydroxy-3-methoxyphenyl-β-ketopropionyl–CoA, vanillic acid, and protocatechuic acid. The genes atu1416, atu1417, and atu1420 have been experimentally shown to be necessary for the degradation of ferulic acid. Moreover, the genes atu1415 and atu1421 have been experimentally demonstrated to be essential for this degradation and are proposed to encode a phenylhydroxypropionyl-CoA dehydrogenase and a 4-hydroxy-3-methoxyphenyl-β-ketopropionic acid (HMPKP)–CoA β-keto-thiolase, respectively. We thus demonstrated that the A. fabrum hydroxycinnamic degradation pathway is an original coenzyme A-dependent β-oxidative deacetylation that could also transform p-coumaric and caffeic acids. Finally, we showed that this pathway enables the metabolism of toxic compounds from plants and their use for growth, likely providing the species an ecological advantage in hydroxycinnamic-rich environments, such as plant roots or decaying plant materials.     

Function and regulation of the acid and neutral trehalases of Mucor rouxii

Two different trehalose-hydrolysing activities, known as acid or non-regulatory trehalases, and neutral or regulatory trehalases, have been recognised in a number of fungal species. The true role of these apparently redundant hydrolases remained obscure for many years. However, recent evidence suggests that neutral trehalases would be specialised in the mobilisation of cytosolic trehalose, while acid trehalases would only hydrolyse extracellular trehalose. Results obtained with Mucor rouxii, a Zygomycete initially thought to posses only neutral trehalase activity, reinforced this hypothesis. M. rouxii grows efficiently in trehalose as the sole carbon source. Trehalose-grown or carbon-starved cells exhibit a high trehalase activity of optimum pH 4.5, bound to the external surface of the cell wall, in contrast with the neutral (pH 6.5) trehalase, which occurs in the cytosol. Other differences between the neutral and the acid trehalases are the temperature optimum (35°C and 45°C, respectively) and thermal stability (half-life of 2.5 min and 12 min at 45°C, respectively). The neutral trehalase, but not the acid trehalase, is activated in vitro by cAMP-dependent phosphorylation, stimulated by Ca²⁺, and inhibited by EDTA. It shows maximal activity at germination and decreases as growth proceeds. In contrast the activity of the acid trehalase is totally repressed in glucose-grown cultures and increases upon exhaustion of the carbon source, and is strongly induced by extracellular trehalose.     

Enhanced freeze tolerance of baker’s yeast by overexpressed trehalose-6-phosphate synthase gene (TPS1) and deleted trehalase genes in frozen dough

Several recombinant strains with overexpressed trehalose-6-phosphate synthase gene (TPS1) and/or deleted trehalase genes were obtained to elucidate the relationships between TPS1, trehalase genes, content of intracellular trehalose and freeze tolerance of baker’s yeast, as well as improve the fermentation properties of lean dough after freezing. In this study, strain TL301_TPS1 overexpressing TPS1 showed 62.92 % higher trehalose-6-phosphate synthase (Tps1) activity and enhanced the content of intracellular trehalose than the parental strain. Deleting ATH1 exerted a significant effect on trehalase activities and the degradation amount of intracellular trehalose during the first 30 min of prefermentation. This finding indicates that acid trehalase (Ath1) plays a role in intracellular trehalose degradation. NTH2 encodes a functional neutral trehalase (Nth2) that was significantly involved in intracellular trehalose degradation in the absence of the NTH1 and/or ATH1 gene. The survival ratio, freeze-tolerance ratio and relative fermentation ability of strain TL301_TPS1 were approximately twice as high as those of the parental strain (BY6-9α). The increase in freeze tolerance of strain TL301_TPS1 was accompanied by relatively low trehalase activity, high Tps1 activity and high residual content of intracellular trehalose. Our results suggest that overexpressing TPS1 and deleting trehalase genes are sufficient to improve the freeze tolerance of baker’s yeast in frozen dough. The present study provides guidance for the commercial baking industry as well as the research on the intracellular trehalose mobilization and freeze tolerance of baker’s yeast.