Seasonal variation in δ13C and δ18O of cellulose from growth rings of Pinus radiata

Seasonal variation in δ¹³C and δ¹⁸O of cellulose (δ¹³C_c and δ¹⁸O_c) was measured within two annual rings of Pinus radiata growing at three sites in New Zealand. In general, both δ¹³C_c and δ¹⁸O_c increased to a peak over summer. The three sites differed markedly in annual water balance, and these differences were reflected in δ¹³C_c and δ¹⁸O_c. Average δ¹³C_c and δ¹⁸O_c from each site were positively related, so that the driest site had the most enriched cellulose. δ¹³C_c and δ¹⁸O_c were also related within each site, although both the slope and the closeness of fit of the relationship varied between sites. Supporting the theory, the site with the lowest average relative humidity also had the greatest change in δ¹⁸O_c‰ change in δ¹³C_c. Specific climatic events, such as drought or high rainfall, were recorded as a peak or a trough in enrichment, respectively. These results suggest that seasonal and between‐site variation in δ¹³C_c and δ¹⁸O_c are driven by the interaction between variation in climatic conditions and soil water availability, and plant response to this variation.     

Pyrophosphate as an Energy Donor in the Cytosol of Plant Cells: an Enigmatic Alternative to ATP

Pyrophosphate serves as an alternative energy donor to ATP for sucrose mobilisation via sucrose synthase, for glycolysis via pyrophosphate: fructose-6-phosphate phosphotransferase, and for tonoplast energisation via the tonoplast proton-pumping pyrophosphatase. This review considers the possible roles of these pyrophosphate-driven reactions. Correlative evidence based on expression patterns, the distribution of proteins and activities in various tissues, and comparisons of the in vitro properties of the enzymes with the in vivo metabolite levels indicates an important role in young growing tissues and in stress conditions including anaerobiosis, but interpretation is complicated by the reversibility of the pyrophosphate-driven reactions and by their duplication by ATP-dependent reactions. The review then considers the evidence emerging from experiments using reversed genetics to alter expression of sucrose synthase, the pyrophosphate: fructose-6-phosphate phosphotransferase, and the tonoplast proton-pumping pyrophosphatase. This approach has revealed that sucrose synthase plays an essential role in sucrose breakdown in potato tubers, and that pyrophosphate: fructose-6-phosphate phosphotransferase catalyses a near-equilibrium reaction with a net flux in the direction of glycolysis. However, it does not support a special role of the latter enzymes in stress responses. Interpretation is complicated by compensation, which can include expression of other members of a gene family, use of alternative pathways, and relaxation of the feed back regulation in response to decreased expression of the enzyme. In an alternative approach, ectopic overexpression of soluble pyrophosphatase from E. coli has been used as a tool to decrease the levels of pyrophosphate in the cytosol. Constitutive overexpression leads to dramatic changes in sucrose and starch synthesis, sink-source relations and plant growth, phloem-specific overexpression of soluble pyrophosphatase leads to an inhibition of phloem transport, leaf mesophyll-specific overexpression leads to a small stimulation of sucrose synthesis, and potato tuber-specific overexpression leads to an inhibition of starch accumulation.     

Phototransformation of protochlorophyll(ide) by a proplastid fraction from Euglena

The phototransformation of protochlorophyll(ide) (Pchl(ide)) to chlorophyll(ide) (Chl(ide)) can be demonstrated in a proplastid fraction from Euglena gracilis Klebs var. bacillaris Cori if appropriate conditions are employed. Pigments were measured fluorometrically in acetone extracts of cell or organelles. Pchl(ide) and the phototransformation to Chl(ide) are at their highest levels in cells grown in darkness on normal or low vitamin B₁₂-containing medium (pH 3.5) to the late exponential phase (1.2–1.4 × 10⁶ cells ml^?1). Late exponential cells on low B¹² medium yield a proplastid fraction that contains Pchl(ide) which is phototransformed to Chl(ide) when illuminated with red light (5.6 W m^?2 for 4 min) in the presence of 10 mM Hepes, 20 mM TES, 0.5 mM potassium phosphate (pH 7.4), 70 mM sorbitol, 5 mM DTT, 5 mM ATP, 5 mM fructose-1, 6-bisphosphate, 10 mM malate and 2 mM MgCl²; intact organelles appear to be involved since deletion of osmoticum gives a lower activity, and addition of NAD(P)H is without effect. Phototransformation of Pchl(ide) to Chl(ide) in red light shows Bunsen-Roscoe reciprocity between fluence rate and duration of illumination. Although mitochondria are present, they do not appear to be involved since inhibitors of respiration and uncouplers of oxidative phosphorylation fail to block the phototransformation. The percentage phototransformation of Pchl(ide) to Chl(ide) in late exponential normal B¹² cells is 61 ± 10, and is 52 ± 3 in low B¹² cells. About 67% of the activity in low B¹² cells is recovered in the proplastid fraction incubated with the complete incubation mixture in saturating light. In both types of cells and in the proplastid fraction, the stoichiometry of conversion of Pchl(ide) to Chl(ide) is about 1:1 (mol/mol).     

Herbivore-induced shifts in carbon and nitrogen allocation in red oak seedlings

* A dual-isotope, microcosm experiment was conducted with Quercus rubra (red oak) seedlings to test the hypothesis that foliar herbivory would increase belowground carbon allocation (BCA), carbon (C) rhizodeposition and nitrogen (N) uptake. Plant BCA links soil ecosystems to aboveground processes and can be affected by insect herbivores, though the extent of herbivore influences on BCA is not well understood in woody plants. * Microcosms containing 2-yr-old Q. rubra seedlings and soil collected from the Coweeta Hydrologic Laboratory (NC, USA) were subjected to herbivory or left as undamaged controls. All microcosms were then injected with 15N-glycine and pulsed with 13CO2. * Contrary to our hypothesis, herbivore damage reduced BCA to fine roots by 63% and correspondingly increased allocation of new C to foliage. However, 13C recoveries in soil pools were similar between treatments, suggesting that exudation of C from roots is an actively regulated component of BCA. Herbivore damage also reduced N allocation to fine roots by 39%, apparently in favor of storage in taproot and stem tissues. * Oak seedlings respond to moderate insect herbivore damage with a complex suite of allocation shifts that may simultaneously increase foliar C, maintain C rhizodeposition and N assimilation, and shift N resources to storage.     

Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells

The aim of this work was to establish the influence of fructose 2,6-bisphosphate (Fru-2,6-P₂) on non-photosynthetic carbohydrate metabolism in plants. Heterotrophic callus lines exhibiting elevated levels of Fru-2,6-P₂ were generated from transgenic tobacco (Nicotiana tabacum L.) plants expressing a modified rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Lines containing increased amounts of Fru-2,6-P₂ had lower levels of hexose phosphates and higher levels of 3-phosphoglycerate than the untransformed control cultures. There was also a greater redistribution of label into the C6 position of sucrose and fructose, following incubation with [1-¹³C]glucose, in the lines possessing the highest amounts of Fru-2,6-P₂, indicating a greater re-synthesis of hexose phosphates from triose phosphates in these lines. Despite these changes, there were no marked differences between lines in the metabolism of ¹⁴C-substrates, the rate of oxygen uptake, carbohydrate accumulation or nucleotide pool sizes. These data provide direct evidence that physiologically relevant changes in the level of Fru-2,6-P₂ can affect pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in vivo, and are consistent with PFP operating in a net glycolytic direction in the heterotrophic culture. However, the results also show that activating PFP has little direct effect on heterotrophic carbohydrate metabolism beyond increasing the rate of cycling between hexose phosphates and triose phosphates. Received: 29 March 2000 / Accepted: 13 June 2000     

Immobilization of Haloferax mediterranei aldolase by cross-linking in a proteinic matrix: stability and halophilic characteristics

Fructose-1,6-bisphosphate (FBP) aldolase (EC 4.1.2.13) of Haloferax mediterranei was immobilized by treating the cell extract in the presence of 10% BSA, with the cross-linking reagent, 0.5% glutaraldehyde for 15min, with the retention of 60% of its original activity. The immobilized preparation exhibited a shift in the temperature optimum from 55°C to 65°C. The enzyme showed enhanced stability towards inactivation by radiation and storage (0–5°C) on immobilization. Immobilization also made the enzyme less halophilic, reducing its denaturation on prolonged storage in a non-salt medium, as well as exhibiting optimal activity at a lower KCl concentration (0.5m) as compared to the soluble enzyme (1–2m).     

Flux regulation in glycogen-induced oscillatory glycolysis in cell-free extracts of Saccharomyces carlsbergensis

To localise the controlling point of the glycolytic system, the temporal changes of concentrations of glycolytic intermediates have been analysed after addition of glycogen to a substrate-depleted yeast extract. Three sequential metabolic states are clearly observable: a transition state at which there is continuous accumulation of the intermediates before the glyceraldehydephosphate dehydrogenase (GAPDH, EC 1.2.1.12) step; a stationary state with all glycolytic intermediates having concentrations oscillating at nearly stationary mean values; and a depletion state at which the intermediates before the GAPDH step are being depleted due to the exhaustion of glycogen. In all these states, the mean ethanol production rate and the concentration of ATP and the intermediates beyond the GAPDH-step are maintained fairly constant, while the glycogen consumption rate and intermediate concentrations of the upper part of the glycolytic system change considerably: the glycogen consumption rate varies 4-fold and fructose-bis-phosphate concentration more than 10-fold. Doubling of the initial glycogen concentration and the addition of a great excess of fructose-bis-phosphate do not affect the ethanol production rate and the mean glycerate-3-phosphate (3-PGA) and pyruvate levels. By contrast, ethanol production was accelerated by an increase of the net ATP consumption rate resulting from either the addition of apyrase or by substitution of trehalose for glycogen. Neither the mean absolute ATP level nor the adenylate energy charge were measurably affected, however all this data can be interpreted in terms of a very strong stoichiometric regulation and stabilization of the lower part of the glycolytic system.     

Initial characterization of sucrose-6-phosphate hydrolase from Streptococcus mutans and its apparent identity with intracellular invertase.

An intracellular enzyme catalyzing the hydrolysis of sucrose-6-phosphate to glucose-6-phosphate and fructose has been identified in extracts of $\text{Streptococcus}\text{mutans}$ 6715-10. The preparation was purified chromatographically and found to have an apparent molecular weight of 42,000. The enzyme has as a K_m for sucrose-6-phosphate of 0.21 mM, a pH optimum of 7.1, is quite stable and requires no added cofactors or metal ions. Sucrose is a competitive inhibitor of sucrose-6-phosphate hydrolysis (K_i = 8. 12 mM). A previously described intracellular invertase copurifies with the enzyme and could not be separated from it by disc gel electrophoresis. It is concluded that intracellular invertase is a sucrose-6-phosphate hydrolase with a low catalytic activity for hydrolysis of sucrose.     

Crystal structure of Bifidobacterium Longum phosphoketolase; key enzyme for glucose metabolism in Bifidobacterium

The crystal structure of Bifidobacterium longum phosphoketolase, a thiamine diphosphate (TPP) dependent enzyme, has been determined at 2.2 Å resolution. The enzyme is a dimer with the active sites located at the interface between the two identical subunits with molecular mass of 92.5 kDa. The bound TPP is almost completely shielded from solvent except for the catalytically important C2-carbon of the thiazolium ring, which can be accessed by a substrate sugar through a narrow funnel-shaped channel. In silico docking studies of B. longum phosphoketolase with its substrate enable us to propose a model for substrate binding.

Structured summary

MINT-7985878: PKT (uniprotkb:Q6R2Q7) and PKT (uniprotkb:Q6R2Q7) bind (MI:0407) by X-ray crystallography (MI:0114)     

Fermentative glycolysis with purified Escherichia coli enzymes for in vitro ATP production and evaluating an engineered enzyme

Each of the twelve enzymes for glycolytic fermentation, eleven from Escherichia coli and one from Saccharomyces cerevisiae, have been over-expressed in E. coli and purified with His-tags. Simple assays have been developed for each enzyme and they have been assembled for fermentation of glucose to ethanol. Phosphorus-31 NMR revealed that this in vitro reaction accumulates fructose 1,6-bisphosphate while recycling the cofactors NAD⁺ and ATP. This reaction represents a defined ATP-regeneration system that can be tailored to suit in vitro biochemical reactions such as cell-free protein synthesis. The enzyme from S. cerevisiae, pyruvate decarboxylase 1 (Pdc1; EC 4.1.1.1), was identified as one of the major ‘flux controlling’ enzymes for the reaction and was replaced with an evolved version of Pdc1 that has over 20-fold greater activity under glycolysis reaction conditions. This substitution was only beneficial when the ratio of glycolytic enzymes was adjusted to suit greater Pdc1 activity.     

Structure and synthesis of polyisoprenoids used in N-glycosylation across the three domains of life

N-linked protein glycosylation was originally thought to be specific to eukaryotes, but evidence of this post-translational modification has now been discovered across all domains of life: Eucarya, Bacteria, and Archaea. In all cases, the glycans are first assembled in a step-wise manner on a polyisoprenoid carrier lipid. At some stage of lipid-linked oligosaccharide synthesis, the glycan is flipped across a membrane. Subsequently, the completed glycan is transferred to specific asparagine residues on the protein of interest. Interestingly, though the N-glycosylation pathway seems to be conserved, the biosynthetic pathways of the polyisoprenoid carriers, the specific structures of the carriers, and the glycan residues added to the carriers vary widely. In this review we will elucidate how organisms in each basic domain of life synthesize the polyisoprenoids that they utilize for N-linked glycosylation and briefly discuss the subsequent modifications of the lipid to generate a lipid-linked oligosaccharide.     

Photodynamic activity of the boronated chlorin e6 amide in artificial and cellular membranes

Photodynamic tumor-destroying activity of the boronated chlorin e6 derivative BACE (chlorin e6 13(1)-N-{2-[N-(1-carba-closo-dodecaboran-1-yl)methyl]aminoethyl}amide-15(2), 17(3)-dimethyl ester), previously described in Moisenovich et al. (2010) PLoS ONE 5(9) e12717, was shown here to be enormously higher than that of unsubstituted chlorin e6, being supported by the data on much higher photocytotoxicity of BACE in M-1 sarcoma cell culture. To validate membrane damaging effect as the basis of the enhanced tumoricidal activity, BACE was compared with unsubstituted chlorin e6 in the potency to photosensitize dye leakage from liposomes, transbilayer lipid flip-flop, inactivation of gramicidin A ionic channels in planar lipid membranes and erythrocyte hemolysis. In all the models comprising artificial and cellular membranes, the photodynamic effect of BACE exceeded that of chlorin e6. BACE substantially differed from chlorin e6 in the affinity to liposomes and erythrocytes, as monitored by fluorescence spectroscopy, flow cytometry and centrifugation. The results support the key role of membrane binding in the photodynamic effect of the boronated chlorin e6 amide.     

The in vitro biosynthesis and metabolism of progesterone and estrogens by chimpanzee placental tissue

The biosynthesis and metabolism of progesterone and estrogens have been studied in chimpanzee placental tissue in vitro. The conversion of androstenedione-4-14C to estrone and estradiol-17β and of pregnenolone-7α-3H to progesterone has been demonstrated. In addition, the following metabolites were isolated following incubation of either pregnenolone-7α-3H or progesterone-4-¹⁴C: 20α-dihydroprogesterone, 20β-dihydroprogesterone, 6β-hydroxyprogesterone, 5α-pregnane-3,20 dione. The compound 5α-pregnan-3β o1-20-one was identified only after incubation with pregnenolone-7α-3H, while 5β-pregnane-3, 20 dione was identified only after incubation with progesterone-4-¹⁴C. No estrogens could be demonstrated following the incubation of placental preparations with either of the C₂₁ substrates.     

Growth and gas exchange responses of Brazilian pepper (Schinus terebinthifolius) and native South Florida species to salinity

Schinus terebinthifolius Raddi (Schinus) is an invasive exotic species widely found in disturbed and native communities of Florida. This species has been shown to displace native species as well as alter community structure and function. The purpose of this study was to determine if the growth and gas exchange patterns of Schinus, under differing salinity conditions, were different from native species. Two native upland glycophytic species (Rapanea punctata and Randia aculeata) and two native mangrove species (Rhizophora mangle and Laguncularia racemosa) were compared with the exotic. Overall, the exotics morphologic changes and gas exchange patterns were most similar to R. mangle. Across treatments, increasing salinity decreased relative growth rate (RGR), leaf area ratio (LAR) and specific leaf area (SLA) but did not affect root/shoot ratios (R:S). Allocation patterns were however significantly different among species. The largest proportion of Schinus biomass was allocated to stems (47%), resulting in plants that were generally taller than the other species. Schinus also had the highest SLA and largest total leaf area of all species. This meant that the exotic, which was taller and had thinner leaves, was potentially able to maintain photosynthetic area comparable to native species. Schinus response patterns show that this exotic exhibits some physiological tolerance for saline conditions. Coupled with its biomass allocation patterns (more stem biomass and large area of thin leaves), the growth traits of this exotic potentially provide this species an advantage over native plants in terms of light acquisition in a brackish forested ecosystem.