ORGANIC COMPOUNDS RELEASED FROM A NATURAL POPULATION OF EPIBENTHIC BLUEGREEN ALGAE1

Extracellular release of dissolved organic compounds by the bluegreen algal community of a brackish marsh was studied using ¹⁴C techniques. Mannitol and trehalose were identified as the most commonly released compounds. The proportions of these two extracellular compounds varied in response to light intensity and the water potential of the environment. The presence of mannitol, in particular, suggests that excretion of organic compounds in natural situations is a function of osmotic adjustment.     

C Nuclear Magnetic Resonance Study of Mannitol Cycle and Trehalose Synthesis during Glucose Utilization by the Ectomycorrhizal Ascomycete Cenococcum graniforme

¹³C nuclear magnetic resonance spectroscopy has been used to follow the utilization of glucose for the synthesis of carbohydrates in the ectomycorrhizal ascomycete Cenococcum graniforme. The fate of ¹³C label was analyzed in vivo and in mycelial extracts. The major carbohydrates produced from [1-¹³C]glucose and [6-¹³C]glucose were mannitol and trehalose. Mannitol was mainly synthesized via a direct route from glucose. Scrambling of the ¹³C label was observed to occur in trehalose during glycolysis. From the analysis of the scrambling patterns, it is concluded that the mannitol cycle was operative and that a large part of the carbon of glucose was used to form trehalose after cycling through the mannitol pool. The activities of NAD-mannitol-l-P dehydrogenase (EC 1.1.1.17) and NADP-mannitol dehydrogenase (EC 1.1.1.138), which participate in the mannitol cycle relative to the activity of glycolytic enzymes, provide evidence that the cycle is important for NADPH production.     

The soluble carbohydrates of Euglena gracilis—their identity and response to increased external solute concentration

Abstract Mannitol and trehalose were the predominant soluble carbohydrates in Euglena gracilis strain z growing heterotrophically in complete darkness or in light. The ratio of trehalose to mannitol was correlated with the water activity of the medium. That is, extracts of Euglena gracilis adapted to grow in media supplemented with either sodium chloride or glucose, thereby reducing the water activity, yielded molar ratios of trehalose to mannitol 10 times greater than extracts of cultures grown under conditions in common usage.     

树舌灵芝中分离得到非还原性二糖—海藻糖

Trehalose, a non-reducing disaccharide, was separated for the first time from Ganoderma applanatum fruit body. The structure of trehalose was determined by electrospray ionization mass spectrometric and NMR data. The content of trehalose was determined by improved anthrone-sulphuric acid colorimetric method and it was 0.48% of dried weight of G. applanatum fruit body. Mannitol was simultaneously obtained during separation of trehalose from G. applanatum.     

TRANSPIRATION FROM THE SPOROPHORE OF AGARICUS BISPORUS ‘WHITE’

The transpiration from normal, intact, growing sporophores of the cultivated mushroom, Agaricus bisporus cv ‘White’ was determined by a gravimetric method. A simple method was devised to estimate the surface area of a sporophore. Under different conditions of temperature and relative humidity, the quotient of transpiration/cm² sporophore surface area and evaporation/cm² free-water surface area did not significantly differ from 1. Transpiration from the underside of an open-veil mushroom was related to the planar area rather than to the total exposed gill area. Normally growing sporophores transpired up to 3 mg/cm²/hr. It was estimated that during development to the open-veil stage, a sporophore transpired a quantity of water equal to ca. one-half of its fresh weight. There was no evidence of factors other than environmental affecting the evaporation of water from the surface of the normally growing sporophore. Our data were not extensive enough, however, to provide evidence for or against Schütte's hypothesis that transpiration in a mature agaric fructification may be intimately linked with a physiological process.     

An analysis of trehalose, glycerol, and mannitol accumulation during heat and salt stress in a salt marsh isolate of Aureobasidium pullulans

Polyhydroxy compounds from Aureobasidium pullulans exposed to stress treatments of heat, salt, and simultaneous heat and salt were isolated, identified, and quantified. Results from both thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) showed that concentrations of trehalose, mannitol, and glycerol increased under stress conditions that induce osmotic- and thermotolerance in A. pullulans. The cellular concentration of trehalose increased in heat-stressed and in simultaneously heat- and salt-stressed cells but not in cells subjected to salt stress alone. Mannitol increased under all stress conditions examined, while an increase in intracellular glycerol was apparent only in salt-stressed cells. The significance of these findings in relation to stress tolerance in salt marsh environments is discussed.     

Purification and Characterization of Trehalose Phosphorylase from Micrococcus varians

Trehalose phosphorylase (EC 2.4.1.64), which catalyzes the reversible reaction of phosphorolysis and synthesis of trehalose, was purified to homogeneity from a cell-free extract of Micrococcus varians strain No. 39. The enzyme was shown to have a molecular weight of 570,000 to 580,000 by gel filtration, and to have a subunit of molecular weight of 105,000 by SDS–polyacrylamide gel electrophoresis. The stoichiometry of the reaction between trehalose, Pi, glucose, and β-glucose 1-phosphate was 1: 1: 1: 1 (molar ratio). The enzyme had high specificity for trehalose, glucose, and β-glucose 1-phosphate. The K_ms for trehalose, Pi, glucose, and β-glucose 1-phosphate were 10, 3.1, 23, and 38mM, respectively. The k_cats were 200s^?1 for trehalose phosphorolysis and 660s^?1 for trehalose synthesis. The enzyme was inhibited by validamycin A, validoxylamine A, 1-deoxynojirimycin, and Cu^{2 +} during trehalose phosphorolysis, and by Cu^{2 +}, Zn^{2 +}, and Ni^{2 +} during trehalose synthesis. Inhibition competitive against trehalose was noted with validamycin A, validoxylamide A, and 1-deoxynojirimycin. Initial velocity, product inhibition, and dead-end inhibition studies suggested that both trehalose phosphorolysis and trehalose synthesis proceeded through an ordered Bi Bi mechanism.     

The composition of fresh and stored oyster mushrooms (Pleurotus ostreatus)

Soluble carbohydrate, protein, polysaccharide and cell wall composition were assayed in freshly harvested Pleurotus ostreatus sporophores and those stored for 4 days at 2° or 18°. Mannitol and trehalose were present at 1.8 and 6.5% dry wt respectively in fresh sporophores, and at reduced levels in those stored at 18°. In sporophores stored at 2°, trehalose levels increased by up to 122%. Soluble polysaccharide appeared to be composed of glycogen-like material, which was susceptible to post-harvest breakdown, and components containing mannose and other sugars. The total protein content was 42% dry wt; no protein degradation was seen in sporophores stored at 2°, but about 25% of the protein disappeared during storage at 18°. Cell wall polysaccharide was utilised during storage. Respiration rate was about 8–10 ml CO₂/g dry wt/hr at harvest and declined to about 5 ml/g dry wt/hr after 40 hr storage at 18°.     

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.     

Recrystallization of Ice Crystals in Trehalose Solution at Isothermal Condition

An isothermal ice recrystallization behavior in trehalose solution was investigated. The isothermal recrystallization rate constants of ice crystals in trehalose solution were obtained at ?5 °C, ?7 °C, and ?10 °C. Then the results were compared to those of a sucrose solution used as a control sample. Simultaneous estimation of water mobility in the freeze-concentrated matrix was conducted by ¹H spin–spin relaxation time T₂ to investigate mechanisms causing the different ice crystal recrystallization behaviors of sucrose and trehalose. At lower temperatures, lower recrystallization rates were obtained for both trehalose and sucrose solutions. The ice crystallization rate constants in trahalose solution tended to be smaller than those in sucrose solution at the same temperature. Although different ice contents (less than 3.6%) were observed between trehalose and sucrose solutions at the same temperature, the recrystallization behaviors of ice crystals were not markedly different. The ¹H spin–spin relaxation time T₂ of water components in a freeze-concentrated matrix for trehalose solution was shorter than in a sucrose solution at the same temperature. Results show that the water mobility of trehalose solutions in freeze-concentrated matrix was less than that of sucrose solutions, which was suggested as the reason for retarded ice crystal growth in a trehalose solution. Results of this study suggest that the replacement of sucrose with trehalose will not negatively affect deterioration caused by ice crystal recrystallization in frozen foods and cryobiological materials.     

Lipid profile of Pleurotus sajor caju

The lipid profile of Pleurotus sajor caju was studied in relation to mycelial and sporophore growth and different cultural factors. The growth was characterised by lipid synthesis during mycelial growth and utilisation during sporophore growth. The degree of instauration increased during mycelial growth and decreased during sporophore formation. The fatty acid composition of mycelium and sporophore was similar, linoleic acid (C_18:2) being the most dominant acid in both. C:N ratio had a significant (P<0.05) positive effect on mycelial dry weight; however, per cent total lipids was similar. Non-polar lipids became more unsaturated as the temperature was raised from 10° to 25°C and pH from 3.0 to 6.0, but declined when the cultures were aerated. Mycelial dry weight increased significantly (P<0.05) when the liquid medium was supplemented with lipids. In general, fatty acids with carbon chain length C₁₆ and C₁₈ stimulated the growth of mycelium. Supplementation of solid substrate (cotton seed hulls) with safflower oil, soybean oil or rice bran significantly (P<0.05) increased the yield of sporophores. Total lipids and ratio of non-polar to polar lipids were not affected by lipid supplementation.     

Self-aggregation of trehalose in the mixed solvents of 1,3-dimethylimidazolium ionic liquid and water

Abstract

Conformational variations of solvated trehaloses in binary mixtures of 1,3-dialkylimidazolium ([dmim]Cl) ionic liquids and trehalose as well as ternary mixtures of trehalose, [dmim]Cl and water have been studied by molecular dynamics (MD) simulations with and without polarisable force fields. The interaction energy between anion Cl^? and water is stronger than that between water itself in the [dmim]Cl-water mixtures. Isolated water clusters were found in the binary [dmim]Cl-water mixtures with 60.0 and 75.0% mole fraction of water, but a continuous water network appears when the concentration of the mixture increases to 99.9%. In the case of binary mixtures of trehalose and [dmim]Cl, both non-polarisable and polarisable models demonstrated that the pyranose rings of trehalose displayed chair conformations. MD simulations with polarisation model could sample larger conformation space than that with non-polarisable model. A self-aggregation behaviour of trehalose was found in the ternary trehalose-[dmim]Cl-water mixtures, which can be rationalised by the stronger non-bonded interaction energy between trehalose molecules and anion Cl^? than that between trehalose molecules and water.     

Exogenously-supplied trehalose protects thylakoid membranes of winter wheat from heat-induced damage

The effects of trehalose pretreatment on thylakoid membranes of winter wheat were investigated under heat stress. Under normal growth conditions, the winter wheat synthesized 502 μg g⁻¹(f.m.) trehalose, which increased to 1250 μg g⁻¹(f.m.) under heat stress and to 1658 μg g⁻¹(f.m.) in trehalose-pretreated seedlings. Under heat stress, proteins in the thylakoid membranes and the photosynthetic capacity were protected by trehalose pretreatment. Moreover, the electrolyte leakage, contents of malondialdehyde, superoxide anion and hydrogen peroxide, and lipoxygenase activity in trehalose-pretreated seedlings were lower than in the non-pretreated plants.     

Protease activity inAgaricus bisporus during periodic fruiting (flushing) and sporophore development

Protease activity from sporophores and mycelium of the mushroomAgaricus bisporus was assayed during periodic cropping (flushing) and from sporophores during maturation. When the sporophores were harvested at the same developmental stages (pins or buttons) during cropping, proteolytic activity of the sporophores was found to oscillate with the same periodicity as the flushing cycle. For pin mushrooms (an early stage of development), peaks of activity occurred during the interflush periods, whereas for button mushrooms (a later stage of development) peak proteolytic activity coincided with the periods of maximum production. The proteolytic activity in the mycelium remained low and varied little with time. Of the tissues within the sporophore, gill tissue had a higher activity than the stipe or pileus. The changes in activity during sporophore development or maturation depended on the period in the flushing cycle when the sporophore was initiated. The results are discussed in relation to the possible role and regulation of flush co-ordinated proteases.     

An experimental study of the use of ultrasound to facilitate the loading of trehalose into platelets

Trehalose is widely used as a freeze-drying protectant in biomaterial preservation. For this purpose, trehalose has to be loaded into the cells but this is difficult and many methods have been tried. The application of ultrasound can temporarily permeabilize cell membranes, which offers a non-chemical, non-viral, and non-invasive method of cellular drug delivery. Ultrasound is employed here to enhance the loading of trehalose into human platelets. Two frequencies were used, 25 kHz and 800 kHz. The estimated intensity of ultrasound in the sample was varied from 0 to 1.5 W/cm². The trehalose concentration in the platelets was 11.27 ± 2.53 mmol/L when Wolkers et al.’s method was used without ultrasound. The application of 0.8 W/cm², 800 kHz ultrasound for 1 h increased the concentration of trehalose loaded by 54%. The application of 0.8 W/cm², 25 kHz ultrasound for 30 min increased the trehalose concentration that was loaded by 172%. The number and mean volume of the platelets following ultrasonic radiation in these two cases remained normal as compared with fresh untreated platelets. Morphological examination of the radiated platelets showed slight changes. Although further work is needed, ultrasound has been shown to be efficient for the loading of trehalose into platelets.     

Induction of an Increase in Nerve Growth Factor Content in the Cell-conditioned Medium of Mouse L-M Cells by Basic Peptides

Thermostable trehalose synthase, which catalyzes the conversion of maltose into trehalose by intramolecular transglucosylation, was purified from a cell-free extract of the thermophilic bacterium Thermus aquaticus ATCC 33923 to an electrophoretically homogeneity by successive column chromatographies. The purified enzyme had a molecular weight of 105,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.6 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was methionine. The optimum pH and temperature were pH 6.5 and 65°C, respectively. The enzyme was stable from pH 5.5 to 9.5 and up to 80°C for 60min. The trehalose synthase from Thermus aquaticus is more thermoactive and thermostable than that from Pimelobacter sp. R48. The yield of trehalose from maltose by the enzyme was independent of the substrate concentration, and tended to increase at lower temperatures. The maximum yield of trehalose from maltose by the enzyme reached 80–82% at 30–40°C. The activity was inhibited by Cu²⁺ , Hg²⁺, Zn²⁺, and Tris.     

Comparative analysis of trehalose production by Debaryomyces hansenii and Saccharomyces cerevisiae under saline stress

The comparative analysis of growth, intracellular content of Na⁺ and K⁺, and the production of trehalose in the halophilic Debaryomyces hansenii and Saccharomyces cerevisiae were determined under saline stress. The yeast species were studied based on their ability to grow in the absence or presence of 0.6 or 1.0 M NaCl and KCl. D. hansenii strains grew better and accumulated more Na⁺ than S. cerevisiae under saline stress (0.6 and 1.0 M of NaCl), compared to S. cerevisiae strains under similar conditions. By two methods, we found that D. hansenii showed a higher production of trehalose, compared to S. cerevisiae; S. cerevisiae active dry yeast contained more trehalose than a regular commercial strain (S. cerevisiae La Azteca) under all conditions, except when the cells were grown in the presence of 1.0 M NaCl. In our experiments, it was found that D. hansenii accumulates more glycerol than trehalose under saline stress (2.0 and 3.0 M salts). However, under moderate NaCl stress, the cells accumulated more trehalose than glycerol. We suggest that the elevated production of trehalose in D. hansenii plays a role as reserve carbohydrate, as reported for other microorganisms.     

Mannitol biosynthesis is required for plant pathogenicity by Alternaria alternata

Mannitol has been hypothesized to play a role in antioxidant defense. In previous work, we confirmed the presence of the two mannitol biosynthetic enzymes, mannitol dehydrogenase (MtDH) and mannitol 1-phosphate 5-dehydrogenase (MPDH), in the fungus Alternaria alternata and created disruption mutants for both enzymes. These mutants were used to investigate the role of mannitol in pathogenicity of A. alternata on its host, tobacco. Conidia of all mutants were viable and germinated normally. GC-MS analysis demonstrated elevated levels of trehalose in the mutants, suggesting that trehalose may substitute for mannitol as a storage compound for germination. Tobacco inoculation showed no reduction in lesion severity caused by the MtDH mutant as compared with wild type; however, the MPDH mutant and a mutant in both enzymes caused significantly less disease. Microscopy analysis indicated that the double mutant was unaffected in the ability to germinate and produce appressoria on tobacco leaves and elicited a defense response from the host, indicating that it was able to penetrate and infect the host. We conclude that mannitol biosynthesis is required for pathogenesis of A. alternata on tobacco, but is not required for spore germination either in vitro or in planta or for initial infection.     

Fertile sporophore production of Typhula phacorrhiza in the field is related to temperatures near freezing

Two field tests and one lab test were conducted to examine the environmental factors affecting sporophore production in Typhula phacorrhiza and to compare these results with those documented for T. ishikariensis and T. incarnata. In the 2001 lab test where lighting, soil moisture, and soil-sand media were tested in 50 mL screw-cap tubes incubated at 4 degrees C, the limiting factor for Typhula sporophore production was found to be moisture. In the fall 2001 field test, 100 sclerotia of six isolates from three Typhula spp. were placed into pots filled with a sand and soil mixture. The pots were monitored weekly, and maximum sporophore production for all six isolates and for watered and unwatered pots was observed at 11 weeks, which was soon after mean daily temperatures fell below 0 degrees C. In the second field test in fall 2003, five isolates of the three species were tested with similar procedures, but peak sporophore production was observed after 6 weeks, and again only after mean daily temperatures fell below 0 degrees C. In the field, sporophore production of T. phacorrhiza seems to require the same environmental cues as those of T. ishikariensis or T. incarnata, namely high moisture and temperatures near freezing.     

Temporal accumulation of mannitol and arabitol in Geotrichum candidum

The temporal depletion and accumulation of polyols were investigated in the fungus Geotrichum candidum. The major intracellular polyols were tentatively identified by paper chromatography as mannitol and arabitol. Inositol was also present in small quantities, and trehalose was also detected in appreciable concentrations.Germination and vegetative growth depended on the type and concentration of the sole exogenous carbon source. Mannitol occurred in arthrospores at 9.4% of the dry weight after several days growth in 2% (w/v) glucose solid medium, and became depleted during germination and vegetative growth in liquid medium containing 2% (w/v) glucose, 2% (w/v) sodium acetate or 25% (w/v) glucose as sole carbon source. This hexitol latter accumulated during arthrosporulation. The depletion and accumulation of ethanol-soluble carbohydrate believed to be primarily trehalose was temporally similar to that of mannitol. Arabitol accumulated intracellularly during germination and vegetative growth in sodium acetate medium and 25% glucose medium. This pentitol was not detected intracellularly at any culture age during growth in 2% glucose medium.Prolonged incubation of the culture in 25% glucose medium after stationary phase was reached resulted in the gradual disappearance of arabitol from the arthrospores simultaneously with an increase in intracellular mannitol. In comparison, ethanol-soluble carbohydrate did not change with prolonged incubation in this medium.