Exogenous Glycolate Research Articles

Introduction of Exogenous Glycolate Catabolic Pathway Can Strongly Enhances Photosynthesis and Biomass Yield of Cucumber Grown in a Low-CO2 Environment.

Carbon dioxide (CO2) is very important for photosynthesis of green plants. CO2 concentration in the atmosphere is relatively stable, but it drops sharply after sunrise due to the tightness of the greenhouse and the absorption of CO2 by vegetable crops. Vegetables in greenhouses are chronically CO2 starved. To investigate the feasibility of using genetic engineering to improve the photosynthesis and yield of greenhouse cucumber in a low CO2 environment, five genes encoding glyoxylate carboligase (GCL), tartronic semialdehyde reductase (TSR), and glycolate dehydrogenase (GlcDH) in the glycolate catabolic pathway of Escherichia coli were partially or completely introduced into cucumber chloroplast. Both partial pathway by introducing GlcDH and full pathway expressing lines exhibited higher photosynthetic efficiency and biomass yield than wild-type (WT) controls in low CO2 environments. Expression of partial pathway by introducing GlcDH increased net photosynthesis by 14.9% and biomass yield by 44.9%, whereas the expression of the full pathway increased seed yield by 33.4% and biomass yield by 59.0%. Photosynthesis, fluorescence parameters, and enzymatic measurements confirmed that the introduction of glycolate catabolic pathway increased the activity of photosynthetic carbon assimilation-related enzymes and reduced the activity of photorespiration-related enzymes in cucumber, thereby promoting the operation of Calvin cycle and resulting in higher net photosynthetic rate even in low CO2 environments. This increase shows an improvement in the efficiency of the operation of the photosynthetic loop. However, the utilization of cucumber of low concentration CO2 was not alleviated. This study demonstrated the feasibility of introducing the pathway of exogenous glycolate catabolic pathway to improve the photosynthetic and bio-yield of cucumber in a low CO2 environment. These findings are of great significance for high photosynthetic efficiency breeding of greenhouse cucumber.

Oxalate accumulation and regulation is independent of glycolate oxidase in rice leaves

Cellular oxalate, widely distributed in many plants, is implicated to play important roles in various functions and is also known to affect food qualities adversely in fruits and vegetables. How oxalate is regulated in plants is currently not well understood. Glycolate oxidase (GLO) has long been considered as an important player in oxalate accumulation in plants. To gain further insight into the biochemical and molecular mechanisms, the possible roles of GLO in the process were studied. Drastically different levels of oxalate could be achieved by treating rice with various nitrogen forms (nitrate versus ammonium). While nitrate stimulated oxalate accumulation, ammonium reduced its level. Such treatments resulted in similar pattern changes for some other related organic acids, such as glycolate, oxaloacetate, and malate. By feeding plants with exogenous glycolate it was possible almost completely to restore the ammonium-decreased oxalate level. Under the two treatments few differences were observed for GLO mRNA levels, protein levels, and in vitro activities. Both K(m) for glycolate/glyoxylate and K(i) for oxalate remained almost the same for GLO purified from either nitrate- or ammonium-fed leaves. A further in vivo study, with transgenic plants carrying an estradiol-inducible GLO antisense gene, showed that, while the estradiol-induced antisense expression remarkably reduced both GLO protein levels and activities, oxalate levels were not significantly altered in the estradiol-treated transgenic plants. Taken together, it is suggested that oxalate accumulation and regulation is independent of GLO in rice leaves.

Identification of Genes Required for Recycling Reducing Power during Photosynthetic Growth

Photosynthetic organisms have the unique ability to transform light energy into reducing power. We study the requirements for photosynthesis in the alpha-proteobacterium Rhodobacter sphaeroides. Global gene expression analysis found that approximately 50 uncharacterized genes were regulated by changes in light intensity and O\2 tension, similar to the expression of genes known to be required for photosynthetic growth of this bacterium. These uncharacterized genes included RSP4157 to -4159, which appeared to be cotranscribed and map to plasmid P004. A mutant containing a polar insertion in RSP4157, CT01, was able to grow via photosynthesis under autotrophic conditions using H2 as an electron donor and CO2 as a carbon source. However, CT01 was unable to grow photoheterotrophically in a succinate-based medium unless compounds that could be used to recycle reducing power (the external electron acceptor dimethyl sulfoxide (DMSO) or CO2 were provided. This suggests that the insertion in RSP4157 caused a defect in recycling reducing power during photosynthetic growth when a fixed carbon source was present. CT01 had decreased levels of RNA for genes encoding putative glycolate degradation functions. We found that exogenous glycolate also rescued photoheterotrophic growth of CT01, leading us to propose that CO2 produced from glycolate metabolism can be used by the Calvin cycle to recycle reducing power generated in the photosynthetic apparatus. The ability of glycolate, CO2, or DMSO to support photoheterotrophic growth of CT01 suggests that one or more products of RSP4157 to -4159 serve a previously unknown role in recycling reducing power under photosynthetic conditions.

Enhancement of Ethylene Release from Leaf Tissue during Glycolate Decarboxylation

When leaf discs of Xanthium strumarium L. and Salvia splendens L. are incubated in sealed flasks in the light, more C(2)H(4) gas is released in the presence of added CO(2) (30-200 millimolar NaHCO(3)) than without CO(2). In Salvia, the maximum rate of C(2)H(4) release occurs when sufficient CO(2) (above 125 millimolar NaHCO(3)) is added to saturate photosynthesis confirming previous studies. The maximum rate of C(2)H(4) release from illuminated discs is similar to the rate in the dark with or without CO(2) in both species. Glycolate enhances a CO(2)-dependent C(2)H(4) evolution from illuminated leaf discs. However, the maximum rate of C(2)H(4) release with glycolate is the same as that observed with saturating CO(2). When photosynthesis is inhibited by darkness or by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, glycolate has no effect.Studies with [2,3-(14)C]-1-aminocyclopropane-1-carboxylic acid (ACC) show that the pattern of C(2)H(4) release and the specific activity of the (14)C(2)H(4) in the presence and absence of glycolate is similar to that described above, indicating that glycolate does not alter uptake of the exogenously supplied precursor (ACC) or stimulate C(2)H(4) release from an endogenous source at appreciable rates. Glycolate oxidase in vitro generates H(2)O(2) which stimulates a slow breakdown of ACC to C(2)H(4), but since exogenous glycolate is oxidized to CO(2) in both the light and the dark it is argued that the glycolate-dependent increase in C(2)H(4) release from illuminated leaf discs is not mediated directly by the action of enzymes of glycolate catabolism. The effects of glycolate and CO(2) are not easily explained by changes in stomatal resistance. The data support the view that glycolate decarboxylation at subsaturating levels of CO(2) in the light stimulates C(2)H(4) release by raising the CO(2) level in the tissue.

Synthesis, Excretion, and Metabolism of Glycolate under Highly Photorespiratory Conditions in Euglena gracilis Z

Glycolate was excreted from the 5% CO(2)-grown cells of Euglena gracilis Z when placed in an atmosphere of 100% O(2) under illumination at 20,000 lux. The amount of excreted glycolate reached 30% of the dry weight of the cells during incubation for 12 hours. The content of paramylon, the reserve polysaccharide of E. gracilis, was decreased during the glycolate excretion, and of the depleted paramylon carbon, two-thirds was excreted to the outside of cells and the remaining metabolized to other compounds, both as glycolate. The paramylon carbon entered Calvin cycle probably as triose phosphate or 3-phosphoglycerate, but not as CO(2) after the complete oxidation through the tricarboxylic acid cycle. The glycolate pathway was partially operative and the activity of the pathway was much less than the rate of the synthesis of glycolate in the cells under 100% O(2) and 20,000 lux; this led the cells to excrete glycolate outside the cells. Exogenous glycolate was metabolized only to CO(2) but not to glycine and serine. The physiologic role of the glycolate metabolism and excretion under such conditions is discussed.

Glycollate Metabolism in the Obligate Chemolithotroph Thiobacillus neapolitanus Grown in Continuous Culture

CO2-limited chemostat cultures of Thiobacillus neapolitanusexcreted 13·5; nmol glycollate min−1 (mg protein)−1. This confirmed the earlier finding that glycollate excretion by T. neapolitanusis dependent on the CO2/O2 ratio applied in the chemostat and thus is probably due to the oxygenase activity of d-ribulose-1,5-bisphosphate (RuBP) carboxylase. RuBP-dependent oxygen uptake and activity of phosphoglycollate phosphatase were demonstrated in cell-free extracts of T. neapolitanus. Thiobacillus neapolitanuswas able to metabolize exogenous glycollate only to a limited extent. The proportion of dry weight that was derived from [1-14C]glycollate by both CO2- and thiosulphate-limited chemostat cultures was only 0·7 % of the total. Short-term labelling experiments with [1-14C]glycollate and enzyme studies indicated that T. neapolitanuscan metabolize glycollate via two pathways. The enzymes of the glycine-serine pathway were demonstrated in cell-free extracts, but on the basis of kinetic experiments it was concluded that this pathway does not play a major role in glycollate metabolism. Malate was the major primary labelling product. Glyoxylate, formed from glycollate, may be incorporated into malate via malate synthase, which was detected in cell-free extracts of T. neapolitanus. The activities of the enzymes needed for the metabolism of glycollate were independent of growth conditions applied in the chemostat. The results are discussed in relation to the possible causes of obligate chemolithotrophy.

Stimulation of Malate Synthesis by Glycolate in Tomato Leaves in Relation to CO2 Concentration

The mechanism by which malate synthesis from CO2 is increased under low concentrations of CO2 was investigated in C3 plants. A number of metabolites were administered to illuminated tomato leaves, and their effects on the incorporation of 14CO2 into malate were determined. Compared with water as a control, glycolate, glyoxylate, D,L-glycerate, glycine, phosphoglycolate and L-serine increased malate synthesis by factors of 6.8, 3.8, 3.3, 2.5, 2.3 and 2.2, respectively. The effect of exogenous glycolate on malate synthesis from CO2 was dependent on its concentration up to 100 mu, but was independent of ambient CO2 concentration. The feeding of l-14C-glycolate in the light indicated that glycolate stimulated the carbon flow from CO2 to malate. The analysis of the products of 14CO2 fixation in illuminated leaves supplied with glycolate showed increases in malate and sugar and decreases in serine and phosphate esters. However, this stimulated malate synthesis ceased when malonate was supplied simultaneously with glycolate. Treatment with glycolate did not affect the dark 14CO2-fixation, but increased the 14C-malate synthesis, with a corresponding decrease in 14C-aspartate and 14C-glutamate. These results suggest that exogenous glycolate activates malate dehydrogenase in leaves, and that the increased glycolate formation at low CO2 concentrations is associated with the increased malate synthesis from CO2.

The metabolism of photosynthetically fixed carbon by isolated chloroplasts from Codium fragile (Chlorophyta: Siphonales) and by Elysia viridis (Mollusca: Sacoglossa)

The metabolism of photosynthetically fixed 14C by isolated chloroplasts from Codium fragile is compared with that by Elysia viridis (which contains C. fragile chloroplasts). There are marked differences between the two in the formation and subsequent metabolism of both soluble and insoluble products. Less than 6% of the carbon fixed by the isolated chloroplasts during a 15 min pulse of 14C-bicarbonate in the light is released into the medium over the succeeding 24 h. During photosynthesis glycollate and glucose monophosphate are the only labelled compounds released; after the pulse very little glycollate is released and over 24 h only glucose monophosphate and an unidentified compound are found in the medium. In E. viridis photosynthetically fixed carbon can be recovered from compounds of all major classes found in animals. Soon after the pulse, hexoses are the most heavily labelled compounds, but two hours later amino acids are more heavily labelled than hexoses. The unidentified compound is not found in the animals. E. viridis can absorb and metabolize exogenous glycollate and glucose. Earlier authors' suggestions that glucose is the compound which moves from the chloroplast to the animal cell are discussed, and it is proposed that both glucose monophosphate and glucose are formed outside the chloroplast from triose phosphate exported from it.

THE CONVERSION OF GLYCERATE INTO PYRUVATE BY CHLORELLA EXTRACTS

SUMMARYCell‐free extracts from autotrophically grown Chlorella convert glycerate to pyruvate by the reactions of glycolysis. It has already been demonstrated that exogenous glycollate is metabolized to glycerate by the reactions of the glycollate pathway in Chlorella. The activities of the constituent enzymes of glycolysis are great enough to account for the further metabolism of glycerate formed during the metabolism of exogenous glycollate by Chlorella.

Pathways of glycollate metabolism in the blue-green alga Anabaena cylindrica.

1. Exogenous glycollate was assimilated by the blue-green alga Anabaena cylindrica. 2. About 50% of the C-1 carbon of 14C-1-glycollate (i.e.25% of the total carbon) was released as 14CO2 in the dark and also in the light in the presence of DCMU. Most of the 14CO2 released in the light in the absence of DCMU was refixed. 3. Assimilation was almost completely inhibited by α-hydroxy-2-pyridinemethane sulphonic acid, an inhibitor of enzymic glycollate oxidation. Cell extracts catalyzed the oxidation of glycollate to glyoxylate at rates sufficient to account for the in vivo assimilation. 4. Isonicotinylhydrazide, an inhibitor of the conversion of glycine to serine in higher plant/green algae glycollate metabolism, did not significantly affect glycollate metabolism in A. cylindrica. Short-term labelling experiments with 14C-1-glycollate in the light and dark did not show a significant metabolism of 14C via glycine and serine. However, the enzymes for the metabolism of glyoxylate via glycine, serine and hydroxypyruvate to glycerate were demonstrated in cell extracts, although the activity of the enzyme catalyzing the metabolism of serine to hydroxypyruvate was not sufficient to account for the in vivo rate of glycollate assimilation. 5. Cell extracts catalyzed the enzymic condensative decarboxylation of glyoxylate to tartronic semialdehyde and also the enzymic reduction of tartronic semialdehyde to glycerate. The activities in extracts were sufficient to account for the total in vivo photoassimilation of glycollate. The specific activity of malate synthase was insufficient to account for the total photometabolism of glycollate but exceeded the in vivo rate in the dark. 6. On the basis of the inhibitor and kinetic experiments and in terms of the enzymes detected, it appears that in the light glycollate is metabolized mainly via glyoxylate → tatronic semialdehyde → glycerate → 3-phosphoglycerate → (glycolytic pathway) → pyruvate → alanine plus tricarboxylic acid cycle and related compounds. The bulk of the CO2 released in the light is probably refixed via the Calvin cycle. In the dark, the glyoxylate, produced from exogenous glycollate, appears to be metabolized mainly by malate synthase directly to malate.

THE UPTAKE AND OXIDATION OF GLYCOLIC ACID BY BLUE‐GREEN ALGAE1

SUMMARYTwo species of blue‐green algae Anabaena flosaquae and Oscillatoria sp. were shown to assimilate glycolic acid. In the presence of DCMU in light, approximately 50% of it wax oxidized to carbon dioxide; 90% was oxidized in the dark. Glycolate assimilation was increased fivefold by lowering the pH of the medium from 9.0 to 5.0, and the rate of uptake increased with increasing concentration of exogenous glycolate up to a saturation concentration of 12–14 mM. α‐Hydroxysulfonates markedly inhibited glycolate uptake and oxidation but iso‐nicotinyl hydrazide had little effect. These results indicate that glycolate oxidation occurs in vivo, but that the glycolate pathway in these algae differs some‐what from that of higher plants.

Metabolism of glycolic acid-1-14C in barley leaves with or without added CO2

Detached primary leaves of barley (Hordeum vulgare L. var. Dayton) were supplied with glycolate-1 -14C in light or darkness with or without CO2, and the radioactivity of soluble metabolites and evolved CO2 was measured at intervals. The results of these experiments suggest that exogenously supplied glycolate is metabolized by two distinct pathways in illuminated leaves. It may be converted to sucrose outside the chloroplasts, presumably via the glycolate pathway. Alternatively glycolate carbon, through some product of its metabolism, enters the chloroplasts and becomes incorporated into intermediates of the Calvin cycle, from which photorespired CO2 is derived. Endogenous glycolate, originating from Calvin cycle intermediates, may leave the chloroplasts and become a substrate for sucrose synthesis. Exchange of carbon therefore appears to take place between the Calvin cycle inside and the glycolate pathway outside the chloroplasts. This exchange gives a net flow of carbon out of the chloroplasts during photosynthetic CO2 fixation. However, at low external CO2 concentration the flow of carbon from the glycolate pathway into the Calvin cycle in chloroplasts is greatly enhanced, providing substrate for CO2 production. Exogenous glycolate apparently does not directly enter the site of CO2 production in light. However, it is converted to CO2 in darkness, without the participation of the Calvin cycle.

The Growth ofChlorella pyrenoidosaon Glycollate

The utilization of glycollate as a substrate for photoheterotrophic growth by a strain of Chlorella pyrenoidosa has been demonstrated. Glycollate stimulated growth of this alga in basal medium over the pH range 4-0 to 8-0 in the light, but did not support growth in the dark. Stimulation of growth in the light occurred over a wide range of glycollate concentrations and was optimal at 30 ml. Enzyme and inhibitor experiments suggested that the synthesis of cell constituents during growth on glycollate is achieved by the same pathway which operates during the metabolism of exogenous glycollate by autotrophically-grown cells. For algae to metabolize and grow on exogenous glycollate the cells must be readily permeable to this compound. When the cells readily take up exogenous glycollate, the level of activity of enzymes in the cell, in particular glycollate :DCPIP oxidoreductase, may regulate the over-all rate of glycollate metabolism.

Zur Glykolatoxydation einzelliger Gr�nalgen

O2-uptake and CO2-release by a chlorophyll-free, carotenoid-containing mutant of Chlorella vulgaris increase on addition of Na-glycolate by factors of 4–5 and 5–6, respectively (Fig. 1). In an enzyme preparation of that alga (sonification, centrifugation, precipitation with 0–30% (NH4)2SO4, dialysis) activity of glycolate oxidase can be demonstrated by O2-uptake (Fig. 2a) as well as by reduction of the artificial electron acceptor DCPIP (Fig. 2b). The same holds true for whole cells as well as equally prepared enzyme preparations of heterotrophically or autotrophically grown wildtype Chlorella vulgaris, provided the cells are cracked by a “French press” instead of a sonicator (Figs. 3a-c and 4a-c). Glyoxylate is the main reaction product (Table). Oxidation of exogenous glycolate is rapidly performed by whole cells of Scenedesmus quadricauda and of Ankistrodesmus convolutus, too, but hardly or not at all by Chlorella pyrenoidosa and Ankistrodesmus braunii. No definite influence of the level of CO2 applied during growth is found: Chlorella vulgaris and Ankistrodesmus convolutus show a rapid oxidation of glycolate after growth under 0,03 and 1,5% CO2 in air, whereas Chlorella pyrenoidosa and Ankistrodesmus braunii do not show an enhanced O2-uptake on addition of glycolate after either condition (Fig. 5). Various developmental stages of Chlorella pyrenoidosa respond differently to addition of glycolate, the extra O2-consumption varying between about 25% (mature cells) and 50–60% (young cells) of the endogenous rate (Fig. 6). It thus appears that species of unicellular green algae within the same genus have strong or weak glycolate oxidase activity and that several external factors have only a modifying effect on that enzyme.

The pathway of glycollate utilization in Chlorella pyrenoidosa.

1. Exogenous glycollate was rapidly metabolized in both the light and the dark by photoautotrophically grown Chlorella pyrenoidosa. 2. The incorporation of (14)C from [1-(14)C]glycollate by these cells was inhibited by the tricarboxylic acid-cycle inhibitors monofluoroacetate, diethylmalonate and arsenite, and also by alpha-hydroxypyrid-2-ylmethanesulphonate and isonicotinylhydrazine. 3. Short-term kinetic experiments showed over 80% of the total (14)C present in the soluble fraction from the cells to be in glycine and serine after 10s. This percentage decreased with time whereas the percentage radioactivity in glycerate increased for up to 30s then remained steady. The percentage of the total radioactivity present in citrate increased over the experimental period. Malate was the only other tricarboxylic acid-cycle intermediate to become labelled. 4. The kinetic and inhibitor experiments supported the following pathway of glycollate incorporation: glycollate --> glyoxylate --> glycine --> serine --> hydroxypyruvate --> glycerate --> 3-phosphoglycerate --> 2-phosphoglycerate --> phosphoenolpyruvate --> pyruvate --> acetyl-CoA. 5. The specific activities of the enzymes catalysing this metabolic sequence in cell-free extracts were great enough to account for the observed rate of glycollate metabolism of 0.25mumol/h per mg dry wt. of cells in the light.

Enzymes of the glycollate pathway in relation to greening in Euglena gracilis

The kinetics of chlorophyll formation during the greening of dark-grown Euglena gracilis was accompanied by marked increases in activity of the enzymes of glycollate metabolism; glycollate: DCPIP(1) oxidoreductase and phosphoglycollate phosphatase (E.C. 3.1.3.18.). Inhibitors of protein synthesis indicated a de novo synthesis of these enzymes during the development of the photosynthetic system. The inhibitory effects of chloramphenicol and cycloheximide, together with a non-aqueous localisation of glycollate: DCPIP oxidoreductase in photoautotrophically-grown cells, indicated that this enzyme is synthesized and located in the cytoplasm, while phosphoglycollate phosphatase was synthesized in the chloroplast. Glycollate: DCPIP oxidoreductase did not increase above the low level in heterotrophic cells when exogenous glycollate, in the presence or absence of glucose, was supplied in the dark.

The metabolism of glycine and glycollate by pea leaves in relation to photosynthesis.

Abstract 1. 1. The metabolism of 14 C-labelled glycollate and glycine both to sucrose and to a polyglucan via serine has been demonstrated in excised pea leaves. The effect of light, DCMU, isonicotinyl hydrazide and the partial pressure of CO 2 on the pathway has been studied. 2. 2. The metabolism of [ 14 C]glycine and serine formed during a preceding period of photosynthesis has been followed during a subsequent period of photosynthesis in 12 CO 2 . The radioactivity in the glycine and serine declined whilst that in sucrose rose, suggesting that in photosynthesis some sucrose is formed via glycine and serine. The effect of changes in partial pressure of carbon dioxide indicates that the photosynthetic pathway may be spatially separated from that which metabolises exogenous glycine and glycollate.