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Invertase
Kinds of Invertase Terms modified by Invertase Selected AbstractsCOVALENT IMMOBILIZATION OF INVERTASE ON CHEMICALLY ACTIVATED POLY (STYRENE-2-HYDROXYETHYL METHACRYLATE) MICROBEADSJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2008HAYDAR ALTINOK ABSTRACT A carrier for invertase enzyme was synthesized from styrene (S) and 2- hydroxyethyl methacrylate (HEMA) in the form of microbeads. These poly (styrene-2-hydroxyethyl methacrylate), P(S-HEMA) microbeads were activated by epichlorohydrin (ECH) treatment for covalent immobilization. The free and immobilized invertase were assayed in the hydrolysis of sucrose to glucose, and the obtained results were compared. The optimum pH was 4.5 for free and 5.5 for immobilized invertase. The optimum temperature of invertase shifted from 45C to 55C upon immobilization. For free and immobilized enzymes, kinetic parameters were calculated as 4.1 × 10,3 mol L,1and 9.2 × 10,3 mol L,1for Km, and 6.6 × 10,2 mol L,1 min,1and 4.1 × 10,1 mol L,1 min,1for Vmax, respectively. After 1 month of storage at 4C, free enzyme retained 36% of its initial activity, while for the ECH-activated P(S-HEMA) immobilized enzyme, P(S-HEMA)-E, this value was observed as 67%. In repeated batch use, i.e., 20 times in 3 days, 78% retention of the initial activity was observed for P(S-HEMA)-E system. PRACTICAL APPLICATIONS Immobilization of enzymes are very important for many industrial applications, e.g., food, medicine, pharmacology, etc. Invertase converts sucrose to glucose and fructose, which have wide applications in food industry especially as sweeteners. Glucose,fructose mixture has much lower crystallinity compared to sucrose and therefore used in the production of noncrystallizing jams and creams. They are also used as liquid sweeteners. Immobilization enables repeated use, provides significant reduction in the operation costs, facilitates easy separation and speeds up recovery of enzyme and extends the stability of enzyme by protecting the active material from deactivation. Industrial application of immobilized invertase may decrease the production cost of glucose,fructose mixture because it could be used repeatedly for long periods. Although invertase is not a very expensive enzyme, the technique can also be applied to expensive ones for biotechnological productions. [source] Calystegines in Calystegia sepium do not Inhibit Fungal Growth and Invertase Activity but Interact with Plant InvertasePLANT BIOLOGY, Issue 2 2004D. Höke Abstract: Calystegines are alkaloidal glycosidase inhibitors. They accumulate predominantly in young and meristemic parts of Calystegia sepium (Convolvulaceae). C. sepium, bindweed, infests meadows and cereal fields and is difficult to control chemically. Fungal pathogens against C. sepium are established as mycoherbicides. Stagonospora convolvuli LA39 attacks C. sepium and does not affect crop plants, but young plants of C. sepium are less susceptible to the fungus. The interaction of Stagonospora convolvuli with calystegines was investigated. Further, endophytic fungi of several classes were isolated from wild-grown Calystegia sepium leaves, and selected strains were tested for interaction with calystegines. Fungal growth on agar containing calystegines was not affected considerably. Plants in climate chambers were infected with an endophyte, Phomopsis, and with the fungal pathogen, Stagonospora convolvuli. Calystegine levels were measured in infected and non-infected plant tissues. Accumulation depended on developmental stage of the plant tissue and was not influenced by infection. Acid invertase was measured from fungal mycelia and from infected and non-infected plant tissues. Fungal acid invertase activity was not inhibited by 10 mM calystegine B2, while invertase from C. sepium leaves was inhibited. It is concluded that calystegines do not inhibit fungal development and sucrose consumption under the conditions of the present investigation, but may act by redirection of plant carbohydrate metabolism. [source] A proteomic analysis of 14-3-3 binding proteins from developing barley grainsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2006Ross D. Alexander Abstract 14-3-3 proteins are important eukaryotic regulatory proteins. Barley (Hordeum vulgare,L.) 14-3-3A was over-expressed, immobilised and used to affinity purify 14-3-3 binding proteins from developing barley grains. Binding was shown to be phosphorylation-dependent. These proteins were fractionated by PAGE and identified by MALDI-TOF MS. In total, 54,14-3-3 binding proteins were identified, 49,of these interactions are novel to plants. These proteins fell into a number of functional categories. The largest category was for carbohydrate metabolism, including plastidic enzymes for starch synthesis and modification. 14-3-3 was shown to be present in isolated plastids. Four of five enzymes involved in sucrose biosynthesis from triose phosphates were identified, suggesting co-ordinated regulation of this pathway. Invertase and sucrose synthase, which break down sucrose to hexoses, were found. Sucrose synthase activity was shown to be inhibited by exogenous 14-3-3 in a dosage-dependent manner. The second-largest functional group was for proteins involved in stress and defence responses; for example, RGH2A, closely related to the MLA powdery mildew resistance protein, was found. This work illustrates the broad range of processes in which 14-3-3 may be involved, and augments previous data demonstrating key roles in carbohydrate metabolism and plant defence. [source] Reversible Immobilization of Invertase on Sepabeads Coated with Polyethyleneimine: Optimization of the Biocatalyst's StabilityBIOTECHNOLOGY PROGRESS, Issue 6 2002Rodrigo Torres Invertase from S. cerevisiae has been immobilized by ionic adsorption on Sepabeads fully coated with PEI. The enzyme was strongly adsorbed on the support (no desorption of the invertase was found under conditions in which all of the enzyme was released from conventional anionic exchanger supports (e.g., DEAE-agarose)). Nevertheless, the enzyme could still be desorbed after its inactivation, and new fresh enzyme could be adsorbed on the supports without detrimental effects on enzyme loading. This is a multimeric enzyme, its minimal oligomerization active state being the dimer, but under certain conditions of pH and concentration it may give larger multimers. Very interestingly, results suggested that the adsorption of the enzyme on this large and flexible polymeric bed was able to freeze some of the different oligomeric structures of the enzyme. Thus, we have found that the enzyme immobilized at certain pH values (pH 8.5) and high enzyme concentration, in which the main enzyme structure is the tetramer, was more stable than immobilized preparations produced in conditions under which oligomerization was not favorable (dimers at low enzyme concentration) or it was too high (e.g., hexamers-octamers at low pH value). The optimal enzyme preparation remained fully active after a 15-day incubation at 50 °C and pH 4.5 (conditions of standard industrial use) and presented an optimal temperature approximately 5 °C higher than that of soluble enzyme. [source] Effect of Cu stress on the invertase activity and root growth in two populations of Rumex dentatus L. with different Cu toleranceENVIRONMENTAL TOXICOLOGY, Issue 4 2008Yu Huang Abstract There has been no study on key enzymes in sucrose cleavage in metallophyte plants so far, which may be crucial for the plants' root growth and heavy metal tolerance maintenance. Acid invertases are rate-limiting enzymes in sucrose metabolism. Here, we tested the hypothesis that the roots of copper-tolerant plants should manifest a higher activity of acid invertases than nontolerant plants both for supporting growth and for their maintaining tolerance under Cu stress. Two populations of Rumex dentatus L., one from an ancient waste heap at a Cu mine (Cu-tolerant population), and the other from a noncontaminated site (Cu nontolerant population), were used in the experiments. The seedlings of Rumex dentatus L. were exposed to 0, 10, and 40 ,M CuCl2 for 14 days. Cu exposure had a stronger inhibition on root growth and thus resulted in a lower root/shoot ratio in the plants of nontolerant population compared with the Cu-tolerant population. Cu exposure showed a stronger inhibition of acid invertase activity of Cu nontolerant plants than Cu tolerant plants, whereas neutral/alkaline invertase was insensitive to Cu. A positive correlation between the activity of acid invertases and the root growth and root/shoot ratio was observed. The results suggested that the higher activities in acid invertases of Cu-tolerant population might at least partly associate with the plants' Cu tolerance, and their higher activities in acid invertases in turn played an role in maintenance of the Cu tolerance by supplying carbon and energy for tolerance mechanisms. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2008. [source] Donor and acceptor substrate selectivity among plant glycoside hydrolase family 32 enzymesFEBS JOURNAL, Issue 20 2009Wim Van den Ende Plant family 32 glycoside hydrolase enzymes include hydrolases (cell wall invertases, fructan exohydrolases, vacuolar invertases) and fructosyltransferases. These enzymes are very similar at the molecular and structural levels but are functionally different. Understanding the basis of the functional diversity in this family is a challenging task. By combining structural and site-directed mutagenesis data, Asp239 in AtcwINV1 was identified as an amino acid critical for binding and stabilizing sucrose. Plant fructan exohydrolases lack such an Asp239 equivalent. Substitution of Asp239 led to the loss of invertase activity, while its introduction in fructan exohydrolases increased invertase activity. Some fructan exohydrolases are inhibited by sucrose. The difference between the inhibitor (fructan exohydrolase) and the substrate (invertase) binding configurations of sucrose can be explained by the different orientation of Trp82. Furthermore, the evolutionary hydrolase/transferase transition could be mimicked and the difference between S-type fructosyltransferases (sucrose as donor) and F-type fructosyltransferases (fructan as donor) could be unravelled. [source] Identification and classification of genes required for tolerance to high-sucrose stress revealed by genome-wide screening of Saccharomyces cerevisiaeFEMS YEAST RESEARCH, Issue 2 2006Akira Ando Abstract Yeasts used in bread making are exposed to high concentrations of sucrose during sweet dough fermentation. Despite its importance, tolerance to high-sucrose stress is poorly understood at the gene level. To clarify the genes required for tolerance to high-sucrose stress, genome-wide screening was undertaken using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 273 deletions that yielded high sucrose sensitivity, approximately 20 of which were previously uncharacterized. These 273 deleted genes were classified based on their cellular function and localization of their gene products. Cross-sensitivity of the high-sucrose-sensitive mutants to high concentrations of NaCl and sorbitol was studied. Among the 273 sucrose-sensitive deletion mutants, 269 showed cross-sensitivities to sorbitol or NaCl, and four (i.e. ade5,7, ade6, ade8, and pde2) were specifically sensitive to high sucrose. The general stress response pathways via high-osmolarity glycerol and stress response element pathways and the function of the invertase in the ade mutants were similar to those in the wild-type strain. In the presence of high-sucrose stress, intracellular contents of ATP in ade mutants were at least twofold lower than that of the wild-type cells, suggesting that depletion of ATP is a factor in sensitivity to high-sucrose stress. The genes identified in this study might be important for tolerance to high-sucrose stress, and therefore should be target genes in future research into molecular modification for breeding of yeast tolerant to high-sucrose stress. [source] Production, purification and thermal characterisation of invertase from a newly isolated Fusarium sp. under solid-state fermentationINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 7 2008Iram Shaheen Summary Production of invertase employing a newly isolated Fusarium sp. under solid-state fermentation was optimised. Different process parameters were optimised. The maximum enzyme activity under optimum conditions was 47.23 ± 2.12 U gds,1 with nitrogen additives. The enzyme was purified by ammonium sulphate precipitation, diethylaminoethyl cellulose ion-exchange chromatography and Sephadex gel filtration. This protocol gave 20.25-fold purification and 5.53% recovery. The optimum pH and temperature for activity were 5.0 and 50 °C. The Km and Vmax values for the enzyme were 8.33 mm and 21.48 ,mol min,1, respectively. A detailed kinetic study of thermal inactivation has been carried out. Enthalpy of activation (,H*) decreased when entropy (,S*) of activation increased at higher temperatures. Moreover, free energy of denaturation (,G*) increased at higher temperature making the enzyme thermally stable. A possible explanation for the thermal inactivation of invertase at higher temperatures is also discussed. [source] Diastase and invertase activities in Andalusian honeysINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 1 2007Salud Serrano Summary Forty-nine samples of unheated, commercially purchased Andalusian (southern Spain) honeys were examined for diastase (, - and , -amylase) and invertase (, -glucosidase) activities. The honeys were from Citrus sp. (5), Sunflower (2), Rosmarinus (3), Eucalyptus sp. (4) or multifloral in origin (35). Mean value for diastase was 20.48 ± 10.14 (range 3.99,49.42) expressed as diastase number in Gothe's scale, for invertase it was 12.34 ± 8.09 (range 1.2,36.8) expressed as invertase number. Correlation was found between invertase and diastase activities (r = 0.853). These results were compared with literature data and differences are discussed. [source] DROUGHT STRESS: Role of Carbohydrate Metabolism in Drought-Induced Male Sterility in Rice Anthers,JOURNAL OF AGRONOMY AND CROP SCIENCE, Issue 5 2010G. N. Nguyen Abstract Rice plants exposed to three consecutive days of water stress (,0.5 MPa) show a reduction in male fertility and grain set, which is attributed to increased levels of reactive oxygen species (ROS) and activation of a programmed cell death. This current research was conducted to further investigate the association of sugar metabolism with microspore abortion in rice anthers. Biochemical assays showed that sucrose, glucose and fructose contents were found to be significantly increased in anthers from water stressed plants compared with the control. qRT-PCR analyses and in situ hybridization of metabolic genes (sugar transporters, invertase and phosphotransferase/kinases) demonstrated that the supply of sugars for developing microspores and the initial steps of sugar utilization e.g. glycolysis, were not repressed. However, it appears that the accumulation of sugars in stressed anthers might involve a reduction of mitochondrial activity during the tricarboxylic acid cycle, which could result in excessive production of ROS and a depletion of the ATP pool. These results also suggest that higher levels of sugars at all stages of anther development seemed to be associated with some measure of protection to the anthers against oxidative stress. Induced expression of sugar transporter genes might have maintained the high levels of sugar in the tapetum and the locules, which alleviated oxidant damage caused by excessive ROS generation. Thus, the increased level of sugars might potentially be a natural response in providing protection against oxidant damage by strengthening the antioxidant system in anthers. [source] COVALENT IMMOBILIZATION OF INVERTASE ON CHEMICALLY ACTIVATED POLY (STYRENE-2-HYDROXYETHYL METHACRYLATE) MICROBEADSJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2008HAYDAR ALTINOK ABSTRACT A carrier for invertase enzyme was synthesized from styrene (S) and 2- hydroxyethyl methacrylate (HEMA) in the form of microbeads. These poly (styrene-2-hydroxyethyl methacrylate), P(S-HEMA) microbeads were activated by epichlorohydrin (ECH) treatment for covalent immobilization. The free and immobilized invertase were assayed in the hydrolysis of sucrose to glucose, and the obtained results were compared. The optimum pH was 4.5 for free and 5.5 for immobilized invertase. The optimum temperature of invertase shifted from 45C to 55C upon immobilization. For free and immobilized enzymes, kinetic parameters were calculated as 4.1 × 10,3 mol L,1and 9.2 × 10,3 mol L,1for Km, and 6.6 × 10,2 mol L,1 min,1and 4.1 × 10,1 mol L,1 min,1for Vmax, respectively. After 1 month of storage at 4C, free enzyme retained 36% of its initial activity, while for the ECH-activated P(S-HEMA) immobilized enzyme, P(S-HEMA)-E, this value was observed as 67%. In repeated batch use, i.e., 20 times in 3 days, 78% retention of the initial activity was observed for P(S-HEMA)-E system. PRACTICAL APPLICATIONS Immobilization of enzymes are very important for many industrial applications, e.g., food, medicine, pharmacology, etc. Invertase converts sucrose to glucose and fructose, which have wide applications in food industry especially as sweeteners. Glucose,fructose mixture has much lower crystallinity compared to sucrose and therefore used in the production of noncrystallizing jams and creams. They are also used as liquid sweeteners. Immobilization enables repeated use, provides significant reduction in the operation costs, facilitates easy separation and speeds up recovery of enzyme and extends the stability of enzyme by protecting the active material from deactivation. Industrial application of immobilized invertase may decrease the production cost of glucose,fructose mixture because it could be used repeatedly for long periods. Although invertase is not a very expensive enzyme, the technique can also be applied to expensive ones for biotechnological productions. [source] Dependence of Fructooligosaccharide Content on Activity of Fructooligosaccharide-Metabolizing Enzymes in Yacon (Smallanthus sonchifolius) Tuberous Roots during StorageJOURNAL OF FOOD SCIENCE, Issue 6 2007A. Narai-Kanayama ABSTRACT:, Tuberous roots of yacon (Smallanthus sonchifolius) accumulate about 10%, on a fresh weight basis, of inulin-type fructooligosacharides (FOSs), known as a food ingredient with various healthy benefits. However, we have a great difficulty to ensure these benefits because FOSs with a lower degree of polymerization (DP) decreased remarkably, and fructose increased when the tuberous roots were stored after harvesting even under previously recommended storage conditions of low temperature with high humidity. In the present study, to elucidate the involvement of FOS-metabolizing enzymes in FOS reduction during storage at 90% relative humidity and 8°C, we extracted a crude protein from yacon tuberous roots and measured the activities of invertase (,-fructofuranosidase, EC 3.2.1.26), sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT, EC 2.4.1.100), and fructan 1-exohydrolase (1-FEH, EC 3.2.1.80). The enzyme activities acting on sucrose, both invertase and 1-SST, were weakened after storage for a month. In addition, the activity of 1-FEH acting on short FOSs such as 1-kestose (GF2) and 1-nystose (GF3) was higher than that of 1-FFT. These results suggest that the continuous decline in FOSs of low DP during storage was dependent mainly on the 1-FEH activity. On the other hand, FOSs with a DP of , 9 only slightly decreased in stored yacon tuberous roots during storage, though distinct 1-FEH activity was observed in vitro toward a high-DP inulin-type substrate, indicating that highly polymerized FOSs content was unlikely to be closely connected with the 1-FEH activity. [source] Cell-wall Invertases from Rice are Differentially Expressed in Caryopsis during the Grain Filling StageJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 4 2008Yong-Qin Wang Abstract Cell-wall invertase plays an important role in sucrose partitioning between source and sink organs in higher plants. To investigate the role of cell-wall invertases for seed development in rice (Oryza sativa L.), cDNAs of three putative cell-wall invertase genes OsCIN1, OsCIN2 and OsCIN3 were isolated. Semi-quantitative reverse transcription-polymerase chain reaction analysis revealed different expression patterns of the three genes in various rice tissues/organs. In developing caryopses, they exhibited similar temporal expression patterns, expressed highly at the early and middle grain filling stages and gradually declined to low levels afterward. However, the spatial expression patterns of them were very different, with OsCIN1 primarily expressed in the caryopsis coat, OsCIN2 in embryo and endosperm, and OsCIN3 in embryo. Further RNA in situ hybridization analysis revealed that a strong signal of OsCIN2 mRNA was detected in the vascular parenchyma surrounding the xylem of the chalazal vein and the aleurone layer, whereas OsCIN3 transcript was strongly detected in the vascular parenchyma surrounding the phloem of the chalazal vein, cross-cells, the aleurone layer and the nucellar tissue. These data indicate that the three cell-wall invertase genes play complementary/synergetic roles in assimilate unloading during the grain filling stage. In addition, the cell type-specific expression patterns of OsCIN3 in source leaf blades and anthers were also investigated, and its corresponding physiological roles were discussed. [source] Carbon Metabolism Alterations in Sunflower Plants Infected with the Sunflower Chlorotic Mottle VirusJOURNAL OF PHYTOPATHOLOGY, Issue 5 2003M. C. Arias Abstract Sunflower chlorotic mottle virus (SuCMoV) causes chlorotic mottling symptoms and important growth reductions and yield losses in sunflower (Helianthus annuus L., cv. Contiflor 7). This paper describes the effects of SuCMoV on some aspects of carbon metabolism of sunflower plants. After symptoms became evident, CO2 fixation rates decreased, nevertheless, soluble sugars and starch increased in infected leaves. High H2O2 accumulation, lipid peroxidation and chlorophyll degradation were, like the other changes, observed only after symptom expression. Increased soluble carbohydrate accumulation was not related to changes in , -amylase (EC 3.2.1.1) activity, nor in the activities of enzymes associated with sugar import and hydrolysis such as invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13), suggesting it did not derive from starch hydrolysis nor increased sugar import. Rather, it may derive from recycling of cell components associated with the development of oxidative damage. The physiological alterations caused by this virus share many common features with the development of senescence. [source] Proteinaceous inhibitors of carbohydrate-active enzymes in cereals: implication in agriculture, cereal processing and nutrition,JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 11 2006Nathalie Juge Abstract Enzymes that degrade, modify, or create glycosidic bonds are involved in carbohydrate biosynthesis and remodelling. Microbial carbohydrate-active enzymes form the basis of current green technology in the food, feed, starch, paper and pulp industries and the revolution in genomics may offer long-term gains on the quality and quantity of the raw materials. Proteinaceous inhibitors of carbohydrate-active enzymes (,-amylase, limit-dextrinase, polygalacturonase, pectin lyase, pectin methylesterase, invertase and xyloglucan endoglucanase) naturally occur in plants where they are involved in various roles from plant defence to metabolism. Xylanase inhibitors represent the latest addition to this growing family. In this review, we will focus on the inhibitors of carbohydrate-active enzymes present in cereals, mostly represented by ,-amylase and xylanase inhibitors, and summarise the existing knowledge on their structure, function, and implication in cereal processing, agriculture and nutrition. Copyright © 2006 Society of Chemical Industry [source] Population structure and phylogeography of Solanum pimpinellifolium inferred from a nuclear geneMOLECULAR ECOLOGY, Issue 7 2004Ana Lucía Caicedo Abstract Phylogeographical studies are emerging as a powerful tool for understanding the population structure and evolution of wild relatives of crop species. Because of their value as genetic resources, there is great interest in exploring the distribution of variation in wild relatives of cultivated plants. In this study, we use sequence variation from the nuclear gene, fruit vacuolar invertase (Vac), to investigate the population history of Solanum pimpinellifolium. Solanum pimpinellifolium is a close relative of the cultivated tomato and has repeatedly served as a source of valuable traits for crop improvement. We sequenced the second intron of the Vac gene in 129 individuals, representing 16 populations from the northern half of Peru. Patterns of haplotype sharing among populations indicate that there is isolation by distance. However, there is no congruence between the geographical distribution of haplotypes and their genealogical relationships. Levels of outcrossing decrease towards the southernmost populations, as previously observed in an allozyme study. The geographical pattern of Vac variation supports a centre of origin in northern Peru for S. pimpinellifolium and a gradual colonization along the Pacific coast. This implies that inbreeding populations are derived from outcrossing ones and that variation present at the Vac locus predates the spread of S. pimpinellifolium. The expansion of cities and human agricultural activity in the habitat of S. pimpinellifolium currently pose a threat to the species. [source] Inhibitors of plant invertases do not affect the structurally related enzymes of fructan metabolismNEW PHYTOLOGIST, Issue 3 2009Ute Kusch Summary ,,Plant fructan active enzymes (FAZYs), including the enzymes involved in inulin metabolism, namely sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT; EC 2.4.1.100) and fructan 1-exohydrolase (1-FEH; EC 3.2.1.153), are evolutionarily related to acid invertases (AIs), that is, plant cell wall invertase (CWI) and vacuolar invertase (VI). Acid invertases are post-translationally controlled by proteinaceous inhibitors. Whether FAZYs are subject to similar controls is not known. ,,To probe their possible interactions with invertase inhibitors, we transiently expressed chicory (Cichorium intybus) FAZYs, as well as several previously characterized invertase inhibitors from nonfructan species, and the C. intybus cell wall/vacuolar inhibitor of fructosidase (CiC/VIF), a putative invertase inhibitor of a fructan-accumulating plant, in leaves of Nicotiana benthamiana. ,,Leaf extracts containing recombinant, enzymatically active FAZYs were used to explore the interaction with invertase inhibitors. Neither heterologous inhibitors nor CiC/VIF affected FAZY activities. CiC/VIF was confirmed as an AI inhibitor with a stronger effect on CWI than on VI. Its expression in planta was developmentally regulated (high in taproots, and undetectable in leaves and flowers). In agreement with its target specificities, CiC/VIF was associated with the cell wall. ,,It is concluded that subtle structural differences between AIs and FAZYs result in pronounced selectivity of inhibitor action. [source] Molecular physiology of adventitious root formation in Petunia hybrida cuttings: involvement of wound response and primary metabolismNEW PHYTOLOGIST, Issue 3 2009Amir H. Ahkami Summary ,,Adventitious root formation (ARF) in the model plant Petunia hybrida cv. Mitchell has been analysed in terms of anatomy, gene expression, enzymatic activities and levels of metabolites. This study focuses on the involvement of wound response and primary metabolism. ,,Microscopic techniques were complemented with targeted transcript, enzyme and metabolite profiling using real time polymerase chain reaction (PCR), Northern blot, enzymatic assays, chromatography and mass spectrometry. ,,Three days after severance from the stock plants, first meristematic cells appeared which further developed into root primordia and finally adventitious roots. Excision of cuttings led to a fast and transient increase in the wound-hormone jasmonic acid, followed by the expression of jasmonate-regulated genes such as cell wall invertase. Analysis of soluble and insoluble carbohydrates showed a continuous accumulation during ARF. A broad metabolite profiling revealed a strong increase in organic acids and resynthesis of essential amino acids. ,,Substantial changes in enzyme activities and metabolite levels indicate that specific enzymes and metabolites might play a crucial role during ARF. Three metabolic phases could be defined: (i) sink establishment phase characterized by apoplastic unloading of sucrose and being probably mediated by jasmonates; (ii) recovery phase; and (iii) maintenance phase, in which a symplastic unloading occurs. [source] Trehalose metabolism in root nodules of the model legume Lotus japonicus in response to salt stressPHYSIOLOGIA PLANTARUM, Issue 4 2006Miguel López The effect of NaCl stress (50 mM) and validamycin A treatment (30 ,M) on growth and nitrogen fixation of Lotus japonicus was investigated in plants cultured under symbiotic and hydroponics conditions for teen weeks (flowering stage). Validamycin A was used as a potent trehalase inhibitor, and was able to produce a five-fold increase in the level of trehalose during salt treatment, concomitant with an enhance in biomass (20%) in salinized plants. Alterations of nodule metabolism related to some carbohydrates and some enzyme activities were also examined. The shoot and total plant dry weight were severely affected by saline conditions decreasing by 40% and only 15,20% in plant treated without or with validamycin A, respectively. Nitrogenase activity (E.C. 1.7.9.92) was inhibited almost 40% by salt stress and no effect of validamycin was observed. Based on these results, L. japonicus might be considered as a salt-sensitive legume. In addition, the saline conditions also inhibited the enzyme activities of sucrose synthase (E.C. 2.4.1.13), alkaline invertase (E.C. 3.2.1.26) and trehalose-phosphate synthetase (E.C. 2.4.1.15). The validamycin A treatment mainly decreased enzyme activities: sucrose synthase, trehalose-phosphate phosphatase (E.C. 3.1.3.12) and trehalase (E.C. 3.2.1.28). On the other hand, a high concentration of the carbohydrates, starch, sucrose and glucose, seems not to be the mechanism induced in L. japonicus to protect nodules exposed to NaCl because all these sugars decreased in such conditions. Results of the present study support the possible role of trehalose as an osmoprotectant under salt stress. [source] Abscisic acid activates acid invertases in developing grape berryPHYSIOLOGIA PLANTARUM, Issue 2 2005Qiu-Hong Pan Acid invertases play a key role in sugar metabolism, and the plant hormone abscisic acid (ABA) enhances sugar accumulation in crop sink organs, but information about the relationship between ABA and acid invertases has been limited. The present experiments were done with both in vivo pre-incubation of the grape (Vitis vinifera × V. labrusca L.) berry tissues in ABA-containing medium and in vivo infiltration of ABA into the intact berries. The results show that ABA activates both the soluble and cell wall-bound acid invertases during fruit development by enhancing their activities and amounts as assessed by immunoblotting or enzyme-linked immunosorbent assay. This activation was pH, time course and ABA dose dependent. The serine/threonine protein kinase inhibitors K252a, staurosporine and H7 and acid phosphatase increased the activation of ABA-induced acid invertase, but the tyrosine protein kinase inhibitor quercetin strongly suppressed the ABA-induced effects, suggesting that a complex reversible protein phosphorylation is involved in the ABA-induced activation of acid invertases. The effects of the protein kinase inhibitors were dependent on the in vivo state of the tissues but independent of the expression of acid invertases. Two ABA analogues, (,)-ABA and trans-ABA, had no effect on acid invertases, showing that the ABA-induced activation of acid invertases is specific to the physiologically active form of ABA. These data suggest that ABA may be involved in fruit development by activating acid invertases. [source] In situ analysis of enzymes involved in sucrose to hexose-phosphate conversion during stolon-to-tuber transition of potatoPHYSIOLOGIA PLANTARUM, Issue 2 2002Niek J.G. Appeldoorn An in situ study of enzymes involved in sucrose to hexose-phosphate conversion during in vitro stolon-to-tuber transition of potato (Solanum tuberosum L. cv. Bintje) was employed to follow developmental changes in spatial patterns. In situ activity of the respective enzymes was visualized by specific activity-staining techniques and they revealed distinct spatially and developmentally regulated patterns. Two of the enzymes studied were also subject to in situ investigations at the transcriptional level. During the stages of stolon formation high hexokinase (EC 2.7.1.1) and acid (cell wall-bound) invertase (EC 3.2.1.26) activities were restricted to the mitotically active (sub)apical region, suggesting a possible importance of these enzymes for cell division. At the onset of tuberization sucrose synthase (EC 2.4.1.13) and fructokinase (EC 2.7.1.4) were strongly induced (visualized at transcriptional and translational level) and the acid invertase activities disappeared from the swelling subapical region as expected. The high degree of similarity in the spatial pattern and the temporal induction of sucrose synthase and fructokinase suggests a tightly co-ordinated coarse (up)regulation, which may be subject to a sugar-modulated mechanism(s) by which genes involved in the metabolic sucrose-starch converting potential are co-ordinately regulated during tuber growth. The overall activity of uridine-5-diphosphoglucose pyrophosphorylase (EC 2.7.7.9) was present in all tissues during stolon and tuber development, implying that its coarse control is not subject to (in)direct developmental regulation. [source] Calystegines in Calystegia sepium do not Inhibit Fungal Growth and Invertase Activity but Interact with Plant InvertasePLANT BIOLOGY, Issue 2 2004D. Höke Abstract: Calystegines are alkaloidal glycosidase inhibitors. They accumulate predominantly in young and meristemic parts of Calystegia sepium (Convolvulaceae). C. sepium, bindweed, infests meadows and cereal fields and is difficult to control chemically. Fungal pathogens against C. sepium are established as mycoherbicides. Stagonospora convolvuli LA39 attacks C. sepium and does not affect crop plants, but young plants of C. sepium are less susceptible to the fungus. The interaction of Stagonospora convolvuli with calystegines was investigated. Further, endophytic fungi of several classes were isolated from wild-grown Calystegia sepium leaves, and selected strains were tested for interaction with calystegines. Fungal growth on agar containing calystegines was not affected considerably. Plants in climate chambers were infected with an endophyte, Phomopsis, and with the fungal pathogen, Stagonospora convolvuli. Calystegine levels were measured in infected and non-infected plant tissues. Accumulation depended on developmental stage of the plant tissue and was not influenced by infection. Acid invertase was measured from fungal mycelia and from infected and non-infected plant tissues. Fungal acid invertase activity was not inhibited by 10 mM calystegine B2, while invertase from C. sepium leaves was inhibited. It is concluded that calystegines do not inhibit fungal development and sucrose consumption under the conditions of the present investigation, but may act by redirection of plant carbohydrate metabolism. [source] Transgenic salt-tolerant sugar beet (Beta vulgaris L.) constitutively expressing an Arabidopsis thaliana vacuolar Na+/H+ antiporter gene, AtNHX3, accumulates more soluble sugar but less salt in storage rootsPLANT CELL & ENVIRONMENT, Issue 9 2008HUA LIU ABSTRACT In Arabidopsis thaliana, six vacuolar Na+/H+ antiporters (AtNHX1-6) were identified. Among them, AtNHX1, 2 and 5 are functional Na+/H+ antiporters with the most abundant expression levels in seedling shoots and roots. However, the expression of AtNHX3 in Arabidopsis can only be detected by RT-PCR, and its physiological function still remains unclear. In this work, we demonstrate that constitutive expression of AtNHX3 in sugar beet (Beta vulgaris L.) conferred augmented resistance to high salinity on transgenic plants. In the presence of 300 or 500 mm NaCl, transgenic plants showed very high potassium accumulation in the roots and storage roots. Furthermore, the transcripts of sucrose phosphate synthase (SPS), sucrose synthase (SS) and cell wall sucrose invertase (SI) genes were maintained in transgenic plants. The accumulation of soluble sugar in the storage roots of transgenic plants grown under high salt stress condition was also higher. Our results implicate that AtNHX3 is also a functional antiporter responsible for salt tolerance by mediating K+/H+ exchange in higher plants. The salt accumulation in leaves but not in the storage roots, and the increased yield of storage roots with enhanced constituent soluble sugar contents under salt stress condition demonstrate a great potential use of this gene in improving the quality and yield of crop plants. [source] Arabidopsis AtcwINV3 and 6 are not invertases but are fructan exohydrolases (FEHs) with different substrate specificitiesPLANT CELL & ENVIRONMENT, Issue 4 2005BARBARA DE CONINCK ABSTRACT The genome of Arabidopsis thaliana contains six putative cell-wall type invertase genes (AtcwINV1-6). Heterologous expression of AtcwINV1, 3 and 6 cDNAs in Pichia pastoris revealed that the enzymes encoded by AtcwINV3 and 6 did not show invertase activity. Instead, AtcwINV3 is a 6-FEH and AtcwINV6 is a fructan exohydrolase (FEH) that can degrade both inulin and levan-type fructans. For AtcwINV6 it is proposed to use the term (6&1) FEH. In contrast, AtcwINV1 is a typical invertase. FEH activity was also detected in crude extracts of different parts of Arabidopsis. To verify that the FEH activity of AtcwINV3 and 6 were not artefacts of the heterologous expression system, the protein corresponding to AtcwINV3 was isolated from whole Arabidopsis plants and indeed showed only 6-FEH activity and no invertase activity. Although no fructans can be detected in Arabidopsis plants, it is shown that kestoses (trimers) can be synthesized in crude leaf extracts. The putative physiological significance of FEH in so-called non-fructan plants is discussed. [source] Inulin metabolism in dicots: chicory as a model systemPLANT CELL & ENVIRONMENT, Issue 6 2002A. Van Laere Abstract Fructans are fructose-based oligo- and polymers that serve as reserve carbohydrates in many plant species. The original Edelman and Jefford model for GFn inulin type fructan biosynthesis was confirmed by the de novo synthesis of fructans in vitro as well as by heterologous expression of the respective cDNAs in non-fructan plants. Now the model can be extended for the biosynthesis of Fn inulin type fructans. Recent progress has now succeeded in elucidating the biochemistry and molecular biology of fructan biodegradation in chicory, an economically important species used for commercial inulin extraction. Unlike fructan biosynthetic genes that originated from vacuolar type invertase, plant fructan exohydrolases seem to have evolved from a cell wall invertase ancestor gene that later obtained a low iso-electric point and a vacuolar targeting signal. Using chicory as a model system, expression analysis revealed that fructan enzymes are mainly controlled at the transcriptional level. [source] X-ray diffraction structure of a cell-wall invertase from Arabidopsis thalianaACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2006Maureen Verhaest Cell-wall invertases play crucial roles during plant development. They hydrolyse sucrose into its fructose and glucose subunits by cleavage of the ,1,,2 glycosidic bond. Here, the structure of the Arabidopsis thaliana cell-wall invertase 1 (AtcwINV1; gene accession code At3g13790) is described at a resolution of 2.15,Ĺ. The structure comprises an N-terminal fivefold ,-propeller domain followed by a C-terminal domain formed by two ,-sheets. The active site is positioned in the fivefold ,-propeller domain, containing the nucleophile Asp23 and the acid/base catalyst Glu203 of the double-displacement enzymatic reaction. The function of the C-terminal domain remains unknown. Unlike in other GH 32 family enzyme structures known to date, in AtcwINV1 the cleft formed between both domains is blocked by Asn299-linked carbohydrates. A preliminary site-directed mutagenesis experiment (Asn299Asp) removed the glycosyl chain but did not alter the activity profile of the enzyme. [source] Comparison of enzymes involved in sugar metabolism from Shang-24 (Vinifera quinguangularis) and Cabernet Sauvignon (Vinifera vinifera) at veraisonAUSTRALIAN JOURNAL OF GRAPE AND WINE RESEARCH, Issue 1 2009Q.-H. PAN Abstract Background and Aims:, Sugar is a main contributor to the quality of grape berries, but little is known about the characteristics of sugar metabolism in Chinese wild grapes. Here, enzymes related to sugar metabolism were investigated in berries of both Shang-24 (Vitis quinguangularis Rehd), a wild grape native to China, and Cabernet Sauvignon (V. vinifera L.). Methods and Results:, Analyses using high performance liquid chromatography and spectrophotometer showed that Shang-24 contained lower levels of glucose and fructose, compared with Cabernet Sauvignon, but had higher activities of enzymes related to sugar hydrolysis, particularly soluble acid invertase (SAI) and ,-amylase. Analyses of enzyme kinetics, enzyme-linked immunosorbent assay and Western blot revealed that SAI and ,-amylase in Shang-24 had low Km values and that high levels of both enzymes were present. Furthermore, a novel peptide of SAI of 105 kDa was detected in Shang-24 along with a peptide of 60 kDa that also was present in Cabernet Sauvignon. Conclusions:, It is thus suggested that biochemical characteristics of SAI and ,-amylase in Shang-24 differ from those in Cabernet Sauvignon, and the novel peptide may be related to high activity of SAI in Shang-24. Significance of the Study:, These data provide an essential basis for further study of the genetic regulation of sugar and its metabolism in grape berries. [source] Assimilate transport in grapevines -effect of phloem disruptionAUSTRALIAN JOURNAL OF GRAPE AND WINE RESEARCH, Issue 3 2001J.J.(KOBUS) HUNTER Abstract Assimilate translocation in mature grapevines (cv. Gewürztraminer and cv. Harslevelü) under field conditions was investigated during the growth season by quantifying individual sugars and organic acids in mature leaves, shoot bark and berries, as affected by girdling the shoot just above the bunches. Tissue was sampled at berry set, pea size, veraison and ripeness stages of the vine. Invertase activity was determined in the shoot bark at ripeness. In the leaves, malic acid concentrations reached lowest levels at pea size, but increased thereafter. Tartaric acid decreased after peaking at pea size stage. Tartaric acid concentrations increased with girdling. Despite the increase in leaf age, sucrose concentrations remained virtually stable during the season, emphasising the importance of mature leaves for nourishing bunches. Girdling resulted in a build-up of sucrose in the leaves. In the bark, malic and tartaric acid stayed more or less the same during the growth period, but increased above the girdle. As a result of phloem disruption, sucrose also increased. The increase in glucose and tartaric acid is believed to result from catabolic cleavage of sucrose by invertase. Invertase activity was evident in the bark (of mature Harslevelü vines) at ripeness, which may indicate involvement in osmotic adjustments and gradients in the bark/phloem structure. In the berries, malic and tartaric acids reached peak concentrations at pea size. The volume increase during the ripening period, and in the case of malic acid also respiratory loss, resulted in a decrease in organic acid concentration. Malic acid continued to decrease after the initial decline, whereas tartaric acid stayed virtually stable. Girdling had no marked effect on organic acid accumulation in the berries. Sucrose concentrations were low during the first part of the season, but increased thereafter. Sucrose concentrations during ripening increased with girdling, which may represent a concentration effect and/or import from the rest of the vine. Sucrose concentrations (in mature Harslevelü vines) were indeed lower below than above the girdle. Comparison of sucrose concentrations in the leaves, bark and berries showed the existence of a decreasing concentration gradient, in line with the source:sink transport concept. An equally prominent decrease in sucrose:glucose ratio in the berries from the start of the ripening period indicates that vacuolar integrity (compartmentation) was affected in the ripening berry, most probably allowing hydrolysis of sucrose by invertase and decreasing osmotic potential within the berry. The results provide further evidence for the hypothesis of an osmotic gradient driven transport to the berry. [source] Molecular biology of grape berry ripeningAUSTRALIAN JOURNAL OF GRAPE AND WINE RESEARCH, Issue 2 2000SIMON P. ROBINSON Abstract Grapevines produce non-climacteric fruit that exhibit a double sigmoidal pattern of growth. Ripening occurs during the second growth phase when grapes change colour, start to soften, accumulate reducing sugars, metabolise organic acids and synthesise flavour compounds. Unlike many other fruit, grapes ripen while the berries are still expanding, and as with most non-climacteric fruit, ripening does not appear to be controlled by ethylene. Sugars and amino acids that accumulate in grapes during ripening are imported via the phloem, while many secondary metabolites are synthesised within the berry itself. Grapes import sucrose but accumulate hexoses. Conversion of sucrose to hexoses is most likely catalysed by invertase. cDNAs encoding vacuolar invertases have been isolated from grape berries. Expression of these genes and an increase in invertase activity occur before veraison, so it seems unlikely that synthesis of this enzyme is a controlling factor for sugar accumulation during ripening. Proteins that transport sugars into the berry vacuole may regulate sugar accumulation, and cDNAs encoding both sucrose and hexose transporters have been isolated from ripening grape berries. Determination of the role of these transporters may reveal the pathway of sugar accumulation in grapes. Anthocyanins are only synthesised in the skin of red grapes after veraison. Analysis of the patterns of expression of genes in the flavonoid pathway has shown that there is a dramatic increase in expression of many of these genes in skin cells at veraison. Expression of the gene encoding a glycosyl transferase involved in the lasts steps of anthocyanin synthesis was absolutely correlated with anthocyanin synthesis and may explain the lack of anthocyanin synthesis in white grapes and in the flesh of most red grapes. We infer that the synthesis of anthocyanins is regulated at the transcription level and is likely to be controlled by regulatory genes. Softening of fruit generally results from changes in the properties of cell walls. Analysis of the cell walls of grapes during ripening suggests that there are no dramatic changes in polysaccharide composition but modification of specific components may contribute to softening. A number of proteins are newly synthesised in grapes during ripening and several of these proteins have now been identified. The most abundant are pathogenesis-related (PR) proteins, including chitinases and thaumatin-like proteins. Expression of genes encoding a number of PR proteins increased dramatically in grapes during ripening. It is not clear what role the PR proteins play during ripening but they may provide resistance to pathogens. Differential screening of a post-veraison grape berry cDNA library has also identified ripening-related genes, some of which encode proline-rich cell wall proteins. Other grape ripening-related genes have homologues that are induced by stress in other plants. These studies indicate that a dramatic change in gene expression occurs in grape berries at veraison and suggest that ripening involves a coordinated increase in transcription of a number of different genes. [source] Crystallization and preliminary X-ray diffraction analysis of the fructofuranosidase from Schwanniomyces occidentalisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2009Aitana Polo Schwanniomyces occidentalis invertase is an extracellular enzyme that releases ,-fructose from the nonreducing termini of various ,- d -fructofuranoside substrates. Its ability to produce 6-kestose by transglycosylation makes this enzyme an interesting research target for applications in industrial biotechnology. The enzyme has been expressed in Saccharomyces cerevisiae. Recombinant and wild-type forms, which showed different glycosylation patterns, were crystallized by vapour-diffusion methods. Although crystallization trials were conducted on both forms of the protein, crystals suitable for X-ray crystallographic analyses were only obtained from the wild-type enzyme. The crystals belonged to space group P212121, with unit-cell parameters a = 105.78, b = 119.49, c = 137.68,Ĺ. A diffraction data set was collected using a synchrotron source. Self-rotation function and sedimentation-velocity experiments suggested that the enzyme was dimeric with twofold symmetry. [source] | |||||||||||||||||||||||||||||||