Effect of Boron on the Incorporation of Glucose from UDP-Glucose into Cotton Fibers Grown in Vitro

Boron is required for fiber growth and development in cotton ovules cultured in vitro. Incorporation of [¹⁴C]glucose by such fiber from supplied UDP-[¹⁴C]glucose into the hot alkali-insoluble fraction is rapid and linear for about 30 minutes. Incorporation of [¹⁴C]glucose from such substrate by fibers grown in boron-deficient ovule cultures is much less than in the case with fibers from ovules cultured with boron in the medium. Total products (alkali-soluble plus alkali-insoluble fractions) were also greater in fibers from ovules cultured with boron. The fraction insoluble in acetic-nitric reagent was a small part of the total glucans; however, in the boron-sufficient fibers, there was significantly more of this fraction than in fibers from boron-deficient ovule cultures. The hot water-soluble glucose polymers from the labeled fibers had a significant fraction of the total [¹⁴C]glucose incorporated from UDP-[¹⁴C]glucose. Both β-1,4- and β-1,3- water-soluble polymers were formed in the boron-sufficient fibers, whereas the same water-soluble fraction from the boron-deficient fibers was predominantly β-1,3-polymers. The incorporation of [¹⁴C]glucose from GDP-[¹⁴C]glucose by the fibers attached to the ovules was insignificant.     

Effects of low water potentials on some aspects of carbohydrate metabolism in Chlorella pyrenoidosa

Summary Chlorella pyrenoidosa was subjected to low water potentials and the resulting changes in carbohydrate metabolism were measured.Water deficit reduced the incorporation of ¹⁴C-glucose into methanol insoluble compounds, principally starch and increased that into sucrose. Even moderate water deficit, for example potentials of -2.5 and -5 atm, greatly reduced the incorporation of ¹⁴C-glucose into uridine diphosphate glucose, while ¹⁴C levels of the hexose monophosphates changed little, indicating a direct stimulus of sucrose synthesis. This increased sucrose synthesis was one of the earliest effect of water deficit, because potentials of -2.5 and -5 atm did not reduce respiration and glucose uptake.At lower water potentials (-10 atm or less) there was reduced ¹⁴C incorporation into all sugar phosphates. This resulted from a combination of reduced ¹⁴C-glucose uptake and increased sucrose synthesis.Water potentials as low as -20 atm had little effect on acetate uptake, or on the ¹⁴C levels in the intermediates of the TCA cycle. This confirmed that low water potentials do not directly inhibit respiratory pathways in Chlorella.The results are discussed in relation to the effect of water deficit on levels of various metabolites in vascular plants, which have been reported by other workers.     

Glycogen synthesis by cell-free extracts from the dimorphic yeast Candida albicans

A particulate glucosyltransferase prepared from budding and filamentous cultures of Candida albicans used uridine diphosphate glucose as sole glucosyl donor in a reaction (measured by following the incorporation of [¹⁴C]-glucose from UDP [¹⁴C]-glucose into polymer) stimulated by glucose-6-phosphate and inhibited by adenosine triphosphate and guanosine triphosphate. The radiolabelled reaction product was solubilized by -amylase, and, on oxidation with periodate followed by reduction with borohydride and acid hydrolysis, yielded erythritol and glycerol in the ratio of 4 to 1. The radiolabelled glucosyl residues were attached to an endogenous acceptor of high molecular weight.     

Synthesis of beta-(1-->3)-Glucan from Extracellular Uridine Diphosphate Glucose as a Wound Response in Suspension-cultured Soybean Cells

Soybean (Glycine max) suspension-cultured cells were incubated with 600 micromolar uridine diphosphate [¹⁴C]glucose, and the incorporation into alkali-insoluble material was studied. When the cells were kept in suspension by shaking on a linear shaker, the incorporation was very low. The incorporation was stimulated 30-fold when the cells were continually resuspended by stirring with a narrow glass rod. The stirring procedure was shown to damage some of the cells, and the incorporation appeared to be a wound response. The alkali-insoluble material formed was a β-(1→3)-glucan, and it was synthesized from uridine diphosphate glucose which did not penetrate through the plasma membrane of intact cells. The synthetase activity was probably induced by the stirring procedure. No evidence for cellulose synthesis from extracellular uridine diphosphate glucose was obtained.     

Regulation of cell wall synthesis in Avena stem segments by gibberellic Acid

Gibberellic acid induces (a) increased elongation of Avena sativa stem segments, (b) increased formation of cell wall material, measured on the basis of dry weight, and (c) increased incorporation of ¹⁴C-glucose into all fractions of the cell wall material. This increased incorporation of radioactivity correlates well with increased formation of cell wall material and shows a time-course pattern similar to the time course of the elongation response. Approximately one hour after the application of gibberellic acid, the rates both of growth and of incorporation of radioactivity accelerate to about 2-fold over the control rate. Gibberellic acid does not stimulate the incorporation of labeled glucose into the cell wall material simply by increasing the rate of uptake of glucose by internodal cells. The stimulation of the incorporation of ¹⁴C-glucose into cell wall material, which reflects the stimulation of cell wall synthesis, seems to be an important and relatively early effect of gibberellic acid in this system and probably contributes significantly to the elongation response elicited by the hormone.     

Biosynthesis of Insoluble Glucans From Uridine-Diphosphate-d-Glucose With Enzyme Preparations From Phaseolus aureus and Lupinus albus

Particulate, and digitonin-solubilized, enzyme systems from Phaseolus aureus and Lupinus albus catalyze the biosynthesis of aqueous-insoluble glucans from UDP-d-glucose. The digitonin treatment greatly increases the enzymic activity of (per unit protein) both the 34,000g pellet and the supernatant liquid as compared with that of the original particles. Most of the polymer produced (90-95%) is soluble in hot, dilute alkali; the interglucosidic linkages of the alkali-soluble and alkali-insoluble polymers are identical. The optimum concentration for the incorporation of radioactivity from UDP-d-glucose-¹⁴C into soluble glucan is high; at 10⁻³ m at least 50% of the added radioactive glucosyl donor is incorporated.     

beta-1, 3-Glucan Synthase from Lilium longiflorum Pollen

A particulate fraction from pollen tubes and ungerminated pollen of Lilium longiflorum incorporated ¹⁴C-glucose from UDP-glucose-¹⁴C into a lipid fraction and into β-1, 3-glucan. Partial hydrolysis of the glucan yielded laminaribiose as the only radioactive disaccharide. The preferred substrate was UDP-glucose, and enzyme activity was stimulated by glucose and by β-linked di- and trisaccharides. Enzyme from growing pollen tubes synthesized β-1, 3-glucan more rapidly and produced a higher proportion of alkali-insoluble glucan than did enzyme from ungerminated pollen. The onset of pollen tube growth may be dependent on altered activity of β-1, 3-glucan synthase.     

Studies on the Biosynthesis of Kanamycins

Streptomyces kanamyceticus produces ¹⁴C-kanamycin and other labeled kanamycin-related compounds in the presence of ¹⁴C-glucose or ¹⁴C-glucosamine. One of the latter is paromamine. In order to find a condition to obtain high incorporation rate of radioactive precursors, the culture phase for the addition of labeled compounds was studied. The maximal incorporation rate was obtained by addition of ¹⁴C-glucosamine in the later phase of the culture and the specific activity of resultant kanamycin A was found to be much higher than in the case of the addition of ¹⁴C-glucose. In some experiments one third of radioactivity of the added ¹⁴C-glucosamine was taken into kanamycin, and the distribution was limited only in 6-amino-6-deoxy-D-glucose moiety of kanamycin A when ¹⁴C-glucosamine was added. In contrast, when ^1JC-glucose was added, approximately equal distribution of radioactivity was observed in each of three moieties of kanamycin A molecule.     

Incorporation of UDPGlucose into Cell Wall Glucans and Lipids by Intact Cotton Fibers

The [¹⁴C] moiety from [³H]UDP[¹⁴C]glucose was incorporated by intact cotton fibers into hot water soluble, acetic-nitric reagent soluble and insoluble components, and chloroform-methanol soluble lipids; the [³H] UDP moiety was not incorporated. The ³H-label can be exchanged rapidly with unlabeled substrate in a chase experiment. The cell wall apparent free space of cotton fibers was in the order of 30 picomoles per milligram of dry fibers; 25 picomoles per milligram easily exchanged and about 5 picomoles per milligram more tightly adsorbed. At 50 micromolar UDPglucose, 70% of the [¹⁴C]glucose was found in the lipid fraction after both a short labeling period and chase. The percent of [¹⁴C]glucose incorporated into total glucan increased slightly with chase, but the fraction of total glucans incorporated into insoluble acetic-nitric reagent (cellulose) did increase within a 30-minute chase period. The data supports the concept that glucan synthesis, including cellulose, as well as the synthesis of steryl glucosides, acetylated steryl glucosides, and glucosyl-phosphoryl-polyprenol from externally supplied UDPglucose occurs at the plasma membrane-cell wall interface. The synthase enzymes for such synthesis must be part of this interfacial membrane system.     

Compartmentation and Fluxes of Sugars in Roots of Phaseolus Vulgaris Under Phosphate Deficiency

The influence of phosphate deficiency on the sugar accumulation and sugar partitioning in the root cells of bean (Phaseolus vulgaris L.) was studied. Bean plants were cultured 17 - 19 d on a phosphate-sufficient and phosphate-deficient nutrient medium. Phosphate deficit in the growth medium resulted in increased sugar concentration for about 30 % in the apoplastic and cytoplasmic compartments as well as in the vacuoles of root cells. However, the distribution of sugars between apoplast and cytoplasm compartment and vacuole was not affected by decreased phosphate concentration. About 20 % of sugars were found in the apoplast and cytoplasm, about 80 % in the vacuole. Low phosphate concentration enhanced influx of exogenous ¹⁴C-sucrose into meristematic and elongation zones of root. The ¹⁴C-labelled sugar content in the root tips increased for about 60 % as compared to control plants. Phosphate deficiency increased also ¹⁴C-glucose uptake and content in the root tips. However, the amount of ¹⁴CO₂ liberated during respiration of P-deficient roots (after feeding with uniformly labelled ¹⁴C-glucose) was lower than ¹⁴CO₂ respired by control plants, thus a large part of accumulated sugars seems to be metabolically inactive. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of anoxia and post-anoxia recovery on carbohydrate metabolism in the jaw muscle of the crab Chasmagnathus granulatus maintained on carbohydrate-rich or high-protein diets

The present study assesses the effects of 1-h anoxia and 3-h post-anoxia recovery period on the activity of pyruvate kinase (PK), ¹⁴CO₂ produced from ¹⁴C-glucose and ¹⁴C-lactate, ATP, and glycogen levels in the jaw muscle of Chasmagnathus granulatus fed either a carbohydrate-rich (HC) or high-protein (HP) diet.In the HC control group the jaw muscle PK activity was higher than in HP-fed crabs. In jaw muscle from control HP-fed crabs the lactate oxidation was higher than in HC-fed animals. We observed increased PK activity and ATP concentration and a reduction in the glycogen concentration, ¹⁴CO₂ production from ¹⁴C-lactate in HP-fed crab jaw muscle during anoxia. In crabs fed an HC diet the PK activity decreased in anoxia, the ¹⁴CO₂ production from ¹⁴C-glucose increased, and the ¹⁴CO₂ production from ¹⁴C-lactate did not change.During recovery, a low oxidation capacity for lactate was found in jaw muscle of both dietary groups. PK activity and ¹⁴CO₂ production from ¹⁴C-glucose were high during the recovery period only in the jaw muscle from HP-fed crabs.Recovery decreased the ATP concentration of both dietary groups as compared to anoxia and normoxia, and did not restore the glycogen concentration in the jaw muscle.     

Substrates for anaerobic CO2-production by the goldfish,Carassius auratus (L.): Decarboxylation of14C-labeled metabolites

Summary Goldfish acclimated to normal oxygen levels and to 20°C were made anoxic and injected i.p. with U-¹⁴C-glucose, 6-¹⁴C-glucose, U-¹⁴C-lactate, 3-¹⁴C-lactate, 1-¹⁴C-acetate or 3,4-¹⁴C-glutamate. Radioactivity released into the water (total¹⁴C and¹⁴CO₂) was monitored over a period of about 12 h. With the exception of 3,4-¹⁴C-glutamate from which only 4% was released, the release of¹⁴C from the other compounds was found to be over 30%. The fraction of the radioactivity released as CO₂ varied with the compound injected but was high during the first 4 h after injection. It is argued that the acid-stable¹⁴C component is ethanol, which arises by the combined action of a modified pyruvate dehydrogenase and of alcohol dehydrogenase in muscle (Shoubridge and Hochachka 1980; Mourik et al. 1982).¹⁴CO₂ release from 3-¹⁴C-lactate, 6-¹⁴C-glucose, 3,4-¹⁴C-glutamate and 1-¹⁴C-acetate cannot be explained by ethanol fermentation. Neither was there a stoichiometric relation between¹⁴CO₂ and¹⁴C-ethanol release after U-¹⁴C-glucose and U-¹⁴C-lactate injection. It is concluded that at least 20% of the CO₂ released is produced by Krebs cycle activity.     

Studies on the Mode of Action of Polyoxin D

In the previous papers we reported that the antibiotic Polyoxin D induced the characteristic swelling of the mycelia of fungi,^1,2) and strongly inhibited the incorporation of ¹⁴C-glucosamine into the fungal cell wall chitin.³⁾ The present work has been conducted to further investigate the influence of this antibiotic on the fungal cell wall biosynthesis.

Polyoxin D did not inhibit the incorporation of ¹⁴C-glucose, ¹⁴C-amino acids and ¹⁴C-sodium acetate into the cell wall. In addition, UDP-N-acetylglucosamine, a precurcor of chitin biosynthesis of cell wall, was accumulated in the Polyoxin D-treated mycelia of Cochliobolus miyabeanus more than 150 to 160% of that accumulated in the untreated one.

Chitin synthetase prepared from Piricularia oryzae which is not treated with Polyoxin D was completely inhibited by the addition of 0.1 ppm of Polyoxin D. The fungitoxicity of Polyoxins A to G was positively related to their inhibition of ¹⁴C-glucosamine incorporation into the cell wall chitin of C. miyabeanus. From above results, it became evident that the antibiotic Polyoxin complex inhibited the biosynthesis of fungal cell wall chitin.     

A metabolic flux analysis to study the role of sucrose synthase in the regulation of the carbon partitioning in central metabolism in maize root tips

In order to understand the role of sucrose synthase (SuSy) in carbon partitioning, metabolic fluxes were analyzed in maize root tips of a double mutant of SuSy genes, sh1 sus1 and the corresponding wild type, W22. [U-¹⁴C]-glucose pulse labeling experiments permitted the quantification of unidirectional fluxes into sucrose, starch and cell wall polysaccharides. Isotopic steady-state labeling with [1-¹³C]-, [2-¹³C]- or [U-¹³C]-glucose followed by the quantification by ¹H-NMR and ¹³C-NMR of enrichments in carbohydrates and amino acids was also performed to determine 29 fluxes through central metabolism using computer-aided modeling. As a consequence of the suppression of SUS1 and SH1 isozymes, maize root tips diameter was significantly decreased and respiratory metabolism reduced by 30%. Our result clearly established that, in maize root tips, starch is produced from ADP-Glc synthesized in the plastid and not in the cytosol by sucrose synthase. Unexpectedly, the flux of cell wall synthesis was increased in the double mutant. This observation indicates that, in maize root tips, SH1 and SUS1 are not specific providers for cellulose biosynthesis.     

Sugar Transport in Immature Internodal Tissue of Sugarcane: II. Mechanism of Sucrose Transport

The mechanism by which sucrose is transported into the inner spaces of immature internodal parenchyma tissue of sugarcane (Saccharum officinarum L. var. H 49-5) was studied in short term experiments (15 to 300 seconds). Transport of sucrose, glucose, and fructose was each characterized by a V_max of 1.3 μmoles/gram fresh weight·2 hours, and each of these three sugars mutually and competitively inhibited transport of the other two. When ¹⁴C-glucose was supplied exogenously, ¹⁴C-glucose 6-phosphate and ¹⁴C-glucose were the first labeled compounds to appear in the tissue; no ¹⁴C-sucrose was detected until after 60-second incubation. After 15-second incubation in ¹⁴C-sucrose, all intracellular radioactivity was in glucose, fructose, glucose 6-phosphate, and fructose 6-phosphate; trace amounts of ¹⁴C-sucrose were found after 30 seconds and after 5 minutes, 71% of the intracellular radioactivity was in sucrose. Although it was possible that sucrose was transported intact into the inner space and then immediately hydrolyzed, it was shown that the rate of hydrolysis under these conditions was too low to account for the rate of hexose accumulation. Pretreatment of the tissue with rabbit anti-invertase antiserum eliminated sucrose transport, but had no effect on glucose transport. Since the antibodies did not penetrate the plasmalemma, it was concluded that sucrose was hydrolyzed by an invertase in the free space prior to transport. The glucose and fructose moieties, or their phosphorylated derivatives, were then transported into the inner space and sucrose was resynthesized. No evidence for the involvement of sucrose phosphate in transport was found in these experiments.     

Phospholipase D1 Mediates AMP-Activated Protein Kinase Signaling for Glucose Uptake

Background

Glucose homeostasis is maintained by a balance between hepatic glucose production and peripheral glucose utilization. In skeletal muscle cells, glucose utilization is primarily regulated by glucose uptake. Deprivation of cellular energy induces the activation of regulatory proteins and thus glucose uptake. AMP-activated protein kinase (AMPK) is known to play a significant role in the regulation of energy balances. However, the mechanisms related to the AMPK-mediated control of glucose uptake have yet to be elucidated.

Methodology/Principal Findings

Here, we found that AMPK-induced phospholipase D1 (PLD1) activation is required for ¹⁴C-glucose uptake in muscle cells under glucose deprivation conditions. PLD1 activity rather than PLD2 activity is significantly enhanced by glucose deprivation. AMPK-wild type (WT) stimulates PLD activity, while AMPK-dominant negative (DN) inhibits it. AMPK regulates PLD1 activity through phosphorylation of the Ser-505 and this phosphorylation is increased by the presence of AMP. Furthermore, PLD1-S505Q, a phosphorylation-deficient mutant, shows no changes in activity in response to glucose deprivation and does not show a significant increase in ¹⁴C-glucose uptake when compared to PLD1-WT. Taken together, these results suggest that phosphorylation of PLD1 is important for the regulation of ¹⁴C-glucose uptake. In addition, extracellular signal-regulated kinase (ERK) is stimulated by AMPK-induced PLD1 activation through the formation of phosphatidic acid (PA), which is a product of PLD. An ERK pharmacological inhibitor, PD98059, and the PLD inhibitor, 1-BtOH, both attenuate ¹⁴C-glucose uptake in muscle cells. Finally, the extracellular stresses caused by glucose deprivation or aminoimidazole carboxamide ribonucleotide (AICAR; AMPK activator) regulate ¹⁴C-glucose uptake and cell surface glucose transport (GLUT) 4 through ERK stimulation by AMPK-mediated PLD1 activation.

Conclusions/Significance

These results suggest that AMPK-mediated PLD1 activation is required for ¹⁴C-glucose uptake through ERK stimulation. We propose that the AMPK-mediated PLD1 pathway may provide crucial clues to understanding the mechanisms involved in glucose uptake.     

Formation of bound gibberellins inPharbitis nil

Summary Developing seeds ofPharbitis nil accumulate free as well as bound gibberellins until a maximum level is reached at approximately 25 days after anthesis. Seeds from CCC-treated parent plants have a strongly reduced level of free as well as bound gibberellins. When different spray reagents were used it was found that trichloroacetic acid in particular was suitable to locate non-hydrolysed bound GA fractions on thin-layer plates. Chromatography showed two major bound GA fractions, determined with spray reagents as well as by means of hydrolysis.³H-GA₁ applied to youngPharbitis plants was converted to two water-soluble compounds present in the aqueous phase. The rate of conversion was significantly enhanced when³H-GA₁ and¹⁴C-glucose were applied to the same plants. Chromatography indicated that one of the conversion products of³H-GA₁ became at least partly associated with the applied¹⁴C-glucose (or its products). This suggestion was also supported by the fact that mild acid hydrolysis of the aqueous fraction resulted in the reappearance of³H-GA₁ and a conversion product of³H-GA₁, including a¹⁴C-radioactivity peak cochromatographing with¹⁴C-glucose. However, the conversion products obtained with³H-GA₁ applied to plants appeared to be chromatographycally different from any of the bound-GA fraction established by means of hydrolysis or spray reagents in developing seeds.Abbreviation GA(s) gibberellin(s).     

Effect of 2,4-Dichlorophenoxyacetic Acid on Growth and on β-Glucan Synthetases of Peroxyacetyl Nitrate Pretreated Avena Coleoptile Sections

Coleoptile sections from Avena sativa L. were exposed to non-lethal concentrations of peroxyacetyl nitrate (PAN). The sections were then incubated in solutions of 50 mM glucose plus 2.5 mM potassium phosphate with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). Growth after 4 hours was measured. A corresponding series of experiments was carried out and the effect of the 2,4-D treatments on enzymes utilizing uridine diphosphate glucose (¹⁴C-glucose) to form glucolipid and β-glucans including cellulose was determined. Growth in the PAN-treated sections was inhibited less at optimal and superoptimal auxin levels than at low auxin levels. Glucolipid synthetase activity was only slightly inhibited by PAN pretreatment and was reduced by increasing levels of auxin. Responses of alkali-soluble glucan and cellulose synthetases were similar to growth in both control and PAN treated tissues. It was concluded that the earlier reported response of cell wall metabolism in vivo probably is due to effects on these enzyme levels.