Mechanism of glycolate transport in spinach leaf chloroplasts

The incorporation of ¹⁴CO₂ into glycolate by intact spinach leaf (Spinacia oleracea L. var. Kyoho) chloroplasts exposed to ¹⁴CO₂ (NaH¹⁴CO₃, 1 millimolar) in the light was determined as a function of O₂ concentrations in the reaction media. A hyperbolic saturation curve was obtained, apparent K_m (O₂) of 0.28 millimolar, indicating that glycolate is produced predominantly by ribulose-1,5-bisphosphate carboxylase/oxygenase. A concentration gradient of glycolate was invariably observed between chloroplast stroma and the outside media surrounding chloroplasts during photosynthetic ¹⁴CO₂ fixation under an O₂ atmosphere.     

Aminooxyacetate stimulation of glycolate formation and excretion by chlamydomonas

Aminooxyacetate (1 millimolar) did not inhibit photosynthetic ¹⁴CO₂ fixation by Chlamydomonas reinhardtii Dangeard, (−) strain (N.90) but greatly stimulated the biosynthesis and excretion of glycolate. Similar results were obtained from cells grown with 5% CO₂ or low CO₂ (air). After 2 minutes with air-grown cells, [¹⁴C]glycolate increased from 0.3% of the total ¹⁴C fixed by the control to 11.7% in the presence of aminooxyacetate and after 10 minutes from 3.8% to 41.1%. Ammonium nitrate (0.2 millimolar) in the media blocked the aminooxyacetate stimulation of glycolate excretion. Chromatographic analyses of the labeled products in the cells and supernatant media indicated that aminooxyacetate also completely inhibited the labeling of alanine while some pyruvate accumulated and was excreted. A high percentage (35%) of initial ¹⁴CO₂ fixation was into C₄ acids. Initial products of ¹⁴CO₂ fixation included phosphate esters as well as malate, aspartate, and glutamate in treated or untreated cells. Lactate was also a major early product of photosynthesis, and its labeling was reduced by aminooxyacetate. Inasmuch as lactate was not excreted, glycolate excretion seemed to be specific. When photosynthesis was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, labeled organic and amino acids but not phosphate esters were lost from the cells. Aminooxyacetate did not inhibit the enzymes associated with glycolate synthesis from ribulose bisphosphate.     

Excretion of Glycolate, Mesotartrate and Isocitrate Lactone by Synchronized Cultures of Ankistrodesmus braunii

Fixation of ¹⁴CO₂ by synchronized cultures of Ankistrodesmus braunii was highest for young growing cells, low for mature cells, and lowest for dividing cells. The amount of ¹⁴C excreted during photosynthesis followed the same trend. Cells at the end of the growing phase, after 10 hours of a 16-hour light phase, excreted nearly 35% of the total ¹⁴C fixed as one product, glycolate. Dividing cells from the dark phase, when tested in the light, excreted only 4% as much glycolate-¹⁴C as the young growing cells. Dividing cells also excreted as much mesotartrate as glycolate and also some isocitrate lactone and an unidentified acid. None of these excreted acids were found inside the cells in significant amounts. Methods for isolation and identification of the excreted acids are present. With ¹⁴C-labeled algae, it was shown that the excretion of glycolate was light-dependent and inhibited by 1,1-dimethyl-3-(p-chlorophenyl) urea. The excretion of labeled mesotartrate, isocitrate lactone, and an unknown acid, but not glycolate, also occurred in the dark. The excreted mesotartrate was predominantly carboxyl-labeled even after long periods of ¹⁴CO₂ fixation. Since glycolate is known to be uniformly labeled, glycolate could not be the precursor of the carboxyl-labeled mesotartrate. The reason for the specific excretion of glycolate, mesotartrate, and isocitrate lactone is not known, but the metabolism of all three acids by the algae may be limited and each can form dilactides or lactones by dehydration. In this context isocitrate lactone was excreted rather than the free acid.     

Glycolate Metabolism in Low and High CO(2)-Grown Chlorella pyrenoidosa and Pavlova lutheri as Determined by O-Labeling

Photorespiration in Chlorella pyrenoidosa Chick. was assayed by measuring ¹⁸O-labeled intermediates of the glycolate pathway. Glycolate, glycine, serine, and excreted glycolate were isolated and analyzed on a gas chromatograph/mass spectrometer to determine isotopic enrichment. Rates of glycolate synthesis were determined from ¹⁸O-labeling kinetics of the intermediates, pool sizes, derived rate equations, and nonlinear regression techniques. Glycolate synthesis was higher in high CO₂-grown cells than in air-grown cells when both were assayed under the same O₂ and CO₂ concentrations. Synthesis of glycolate, for both types of cells, was stimulated by high O₂ levels and inhibited by high CO₂ levels. Glycolate synthesis in 1.5% CO₂-grown Chlorella, when exposed to a 0.035% CO₂ atmosphere, increased from about 41 to 86 nanomoles per milligram chlorophyll per minute when the O₂ concentration was increased from 21% to 40%. Glycolate synthesis in air-grown cells increased from 2 to 6 nanomoles per milligram chlorophyll per minute under the same gas levels. Synthesis was undetectable when either the O₂ concentration was lowered to 2% or the CO₂ concentration was raised to 1.5%. Glycolate excretion was also sensitive to O₂ and CO₂ concentrations in 1.5% CO₂-grown cells and the glycolate that was excreted was ¹⁸O-labeled. Air-grown cells did not excrete glycolate under any experimental condition. Indirect evidence indicated that glycolate may be excreted as a lactone in Chlorella. Photorespiratory ¹⁸O-labeling kinetics were determined for Pavlova lutheri, which unlike Chlorella and higher plants did not directly synthesize glycine and serine from glycolate. This alga did excrete a significant proportion of newly synthesized glycolate into the media.     

ALGAL EXCRETION AND BACTERIAL ASSIMILATION IN HOT SPRING ALGAL MATS1

Benthic algal-bacterial mats are present in the effluents of alkaline hot springs at temperatures between 50 and 73 C. The thin surface layer is composed of the unicellular blue-green alga Synechococcus lividus. Also present in the surface layer and forming thick, orange mats beneath it, are filamentous, phototrophic, gliding bacteria of the genus Chloroflexis, also capable of heterotrophic growth. The very low species diversity and the constancy of the hot spring environment, make these mats a good ecosystem for studying the transfer of nutrients from the algae to the bacteria. To determine whether the alga might supply organic materials to the bacterium, excretion by natural populations of S. lividus was studied in the field by means of short-term radioisotope experiments. Under optimal conditions for photosynthesis, between 3 and 12% of the total ¹⁴C fixed was excreted as ¹⁴C-labeled organic compounds. Variations in cell density at concentrations of S. lividus approximating those found in the mat had no effect on the percentage excretion. However, at cell densities below a threshold, level, the percentage excretion increased with diminishing cell density. Except at very low light intensities the percentage of fixed carbon excreted, was very similar for all light intensities tested. Excretion at temperatures approaching the upper limit for growth was not significantly different from the percentage excretion values observed at lower temperatures. ¹⁴C-labeled organic compounds excreted during algal photosynthesis could be subsequently assimilated by natural populations of the bacteria present in the mat.     

Fate of Immediate Methane Precursors in Low-Sulfate, Hot-Spring Algal-Bacterial Mats

The fates of acetate and carbon dioxide were examined in several experiments designed to indicate their relative contributions to methane production at various temperatures in two low-sulfate, hot-spring algal-bacterial mats. [2-¹⁴C]acetate was predominantly incorporated into cell material, although some ¹⁴CH₄ and ¹⁴CO₂ was produced. Acetate incorporation was reduced by dark incubation in short-term experiments and severely depressed by a 2-day preincubation in darkness. Autoradiograms showed that acetate was incorporated by long filaments resembling phototrophic microorganisms of the mat communities. [³H]acetate was not converted to C³H₄ in samples from Octopus Spring collected at the optimum temperature for methanogenesis. NaH¹⁴CO₃ was readily converted to ¹⁴CH₄ at temperatures at which methanogenesis was active in both mats. Comparisons of the specific activities of methane and carbon dioxide suggested that of the methane produced, 80 ± 6% in Octopus Spring and 71 ± 21% in Wiegert Channel were derived from carbon dioxide. Addition of acetate to 1 mM did not reduce the relative importance of carbon dioxide as a methane precursor in samples from Octopus Spring. Experiments with pure cultures of Methanobacterium thermoautotrophicum suggested that the measured ratio of specific activities might underestimate the true contribution of carbon dioxide in methanogenesis.     

ALGAL EXCRETION OF 14C-LABELED COMPOUNDS AND MICROBIAL INTERACTIONS IN CYANIDIUM CALDARIUM MATS1

Acid spring effluents are often covered with mats of the eucaryotic phycocyanin-containing alga. Cyanidium caldarium. The primary bacterial component of such mats is an acidophilic strain of Bacillus coagulans, and the primary fungal component is Dactylaria gallopava. Because of the limited species diversity, C. caldarium mats appeared to be an excellent system for studying algal excretion and various microbial interactions in nature. From 2 to 6% of the NaH¹⁴CO₃ taken up by natural or laboratory populations of the alga was excreted as ¹⁴C-labeled materials. The maximum excretion occurred at temperature, light, and pH values optimum for NaH¹⁴CO₃ uptake. However, when excretion was expressed as a percentage of NaH14 CO₃ uptake, a higher percentage of the radioactivity was excreted at nonoptimal conditions for NaH¹⁴CO₃ uptake. Fungal biomass was directly proportional to algal density, but bacterial numbers varied widely and did not correlate with algal numbers. The bacterial and fungal components could be grown in mixed culture with either growing C. caldarium cultures or in an extract prepared, by healing algal cells.     

Glyoxylate and glutamate effects on photosynthetic carbon metabolism in isolated chloroplasts and mesophyll cells of spinach

Addition of millimolar sodium glyoxylate to spinach (Spinacia oleracea) chloroplasts was inhibitory to photosynthetic incorporation of ¹⁴CO₂ under conditions of both low (0.2 millimolar or air levels) and high (9 millimolar) CO₂ concentrations. Incorporation of ¹⁴C into most metabolites decreased. Labeling of 6-P-gluconate and fructose-1,6-bis-P increased. This suggested that glyoxylate inhibited photosynthetic carbon metabolism indirectly by decreasing the reducing potential of chloroplasts through reduction of glyoxylate to glycolate. This hypothesis was supported by measuring the reduction of [¹⁴C]glyoxylate by chloroplasts. Incubation of isolated mesophyll cells with glyoxylate had no effect on net photosynthetic CO₂ uptake, but increased labeling was observed in 6-P-gluconate, a key indicator of decreased reducing potential. The possibility that glyoxylate was affecting photosynthetic metabolism by decreasing chloroplast pH cannot be excluded. Increased ¹⁴C-labeling of ribulose-1,5-bis-P and decreased 3-P-glyceric acid and glycolate labeling upon addition of glyoxylate to chloroplasts suggested that ribulose-bis-P carboxylase and oxygenase might be inhibited either indirectly or directly by glyoxylate. Glyoxylate addition decreased ¹⁴CO₂ labeling into glycolate and glycine by isolated mesophyll cells but had no effect on net ¹⁴CO₂ fixation. Glutamate had little effect on net photosynthetic metabolism in chloroplast preparations but did increase ¹⁴CO₂ incorporation by 15% in isolated mesophyll cells under air levels of CO₂.     

Metabolism of Glycolate in Isolated Spinach Leaf Peroxisomes : KINETICS OF GLYOXYLATE, OXALATE, CARBON DIOXIDE, AND GLYCINE FORMATION

The flow of glyoxylate derived from glycolate into various metabolic routes in the peroxisomes during photorespiration was assessed. Isolated spinach leaf peroxisomes were fed [¹⁴C] glycolate in the absence or presence of exogenous glutamate, and the formation of radioactive glyoxylate, CO₂, glycine, oxalate, and formate was monitored at time intervals. In the absence of glutamate, 80% of the glycolate was consumed within 2 hours and concomitantly glyoxylate accumulated; CO₂, oxalate, and formate each accounted for less than 5% of the consumed glycolate. In the presence of equal concentration of glutamate, glycolate was metabolized at a similar rate, and glycine together with some glyoxylate accumulated; CO₂, oxalate, and formate each accounted for an even lesser percentage of the consumed glycolate. CO₂ and oxalate were not produced in significant amounts even in the absence of glutamate, unless glycolate had been consumed completely and glyoxylate had accumulated for a prolonged period. These in vitro findings are discussed in relation to the extent of CO₂ and oxalate generated in leaf peroxisomes during photorespiration.     

An investigation of glycolate excretion in two species of blue-green algae

Summary The amount of ¹⁴C-glycolate excreted by Oscillatoria sp. and Anabaena flos-aquae is less than 1% of the ¹⁴C fixed by the algae during photosynthesis. Transfer of cells grown on 5% CO₂ in air to a medium of low bicarbonate concentration or treatment of the cells with isonicotinyl hydrazide (INH) during photosynthesis, caused little increase in glycolate excretion. -Hydroxysulfonates failed to stimulate massive excretion of glycolate. Although these blue-green algae excreted little glycolate, a significant proportion of the photosynthetically fixed carbon was excreted in the form of basic, neutral and acidic compounds, and such excretion was greater in 5% CO₂-grown cells than in air-grown cells.     

Effects of glycine hydroxamate, carbon dioxide, and oxygen on photorespiratory carbon and nitrogen metabolism in spinach mesophyll cells

The effects of added glycine hydroxamate on the photosynthetic incorporation of ¹⁴CO₂ into metabolites by isolated mesophyll cells of spinach (Spinacia oleracea L.) was investigated under conditions favorable to photorespiratory (PR) metabolism (0.04% CO₂ and 20% O₂) and under conditions leading to nonphotorespiratory (NPR) metabolism (0.2% CO₂ and 2.7% O₂). Glycine hydroxamate (GH) is a competitive inhibitor of the photorespiratory conversion of glycine to serine, CO₂ and NH₄⁺. During PR fixation, addition of the inhibitor increased glycine and decreased glutamine labeling. In contrast, labeling of glycine decreased under NPR conditions. This suggests that when the rate of glycolate synthesis is slow, the primary route of glycine synthesis is through serine rather than from glycolate. GH addition increased serine labeling under PR conditions but not under NPR conditions. This increase in serine labeling at a time when glycine to serine conversion is partially blocked by the inhibitor may be due to serine accumulation via the “reverse” flow of photorespiration from 3-P-glycerate to hydroxypyruvate when glycine levels are high. GH increased glyoxylate and decreased glycolate labeling. These observations are discussed with respect to possible glyoxylate feedback inhibition of photorespiration.     

Rate of Glycolate Formation During Photosynthesis at High pH

The products of C¹⁴O₂ fixation by Chlamydomonas and Chlorella were studied under conditions most favorable for glycolate synthesis. The highest percentage of the C¹⁴ was incorporated into glycolate in the pH range of 8 to 9. After 1 to 2 minutes as much as 40% of the C¹⁴ was found in glycolate products and only a trace of C¹⁴ was present as phosphoglycerate. Below pH 8 the rate of photosynthesis was much faster, but only a small percent of the C¹⁴ was incorporated into glycolate in 1 or 2 minutes, while a high percent of the C¹⁴ accumulated in phosphoglycerate. C¹⁴ labeling of glycolate even at pH 8 or above did not occur at times shorter than 10 seconds. During the first seconds of photosynthesis, nearly all of the C¹⁴ was found in phosphoglycerate and sugar phosphates. Thus glycolate appears to be formed after the phosphate esters of the photosynthetic carbon cycle.

Washing Chlamydomonas with water 2 or 3 times resulted in the loss of most of their free phosphate. When a small aliquot of NaHC¹⁴O₃ was added to washed algae in the absence of this buffering capacity, the pH of the algal medium became 8 or above and much of the fixed C¹⁴ accumulated in glycolate.

     

Glycolate Formation and Excretion by Chlorella pyrenoidosa and Netrium digitus

Conditions are described whereby suspensions of Chlorella pyrenoidosa and Netrium digitus photosynthetically biosynthesize and excrete glycolate continuously in high yields. Aminooxyacetic acid, an inhibitor of pyridoxal phosphate-linked enzymes, increased the excretion of glycolate approximately 4-fold in 1 hour (8 millimolar) and 20-fold in 4 hours (40 millimolar) in the presence of 0.2% CO₂ in air. The amount of glycolate excreted in the presence of aminooxyacetate and an atmosphere of 0.2% CO₂ in air equaled or exceeded the amount excreted in 0.2% CO₂ in O₂ minus aminooxyacetate. CO₂ and light were required for glycolate excretion. Aminooxyacetate also stimulated photosynthetic glycolate excretion in an atmosphere of 0.2% CO₂ in nitrogen or helium, although the stimulation was not as great as when air or O₂ was present.

The excreted glycolate was converted to H₂ and CO₂ by the combined action of glycolic oxidase and the formic hydrogenlyase complex found in Escherichia coli in total conversion yields of 80%.

     

Glycolate Metabolism and Excretion by Chlamydomonas reinhardtii

The flux of glycolate through the C₂ pathway in Chlamydomonas reinhardtii was estimated after inhibition of the pathway with aminooxyacetate (AOA) or aminoacetonitrile (AAN) by measurement of the accumulation of glycolate and glycine. Cells grown photoautotrophically in air excreted little glycolate except in the presence of 2 mm AOA when they excreted 5 micromoles glycolate per hour per milligram clorophyll. Cells grown on high CO₂ (1-5%) when transferred to air produced three times as much glycolate, with half of the glycolate metabolized and half excreted. The lower amount of glycolate produced by the air-grown cells reflects the presence of a CO₂ concentrating mechanism which raises the internal CO₂ level and decreases the ribulose-1,5-bisP oxygenase reaction for glycolate production. Despite the presence of the CO₂ concentrating mechanism, there was still a significant amount of glycolate produced and metabolized by air-grown Chlamydomonas. The capacity of these cells to metabolize between 5 and 10 micromoles of glycolate per hour per milligram chlorophyll was confirmed by measuring the biphasic uptake of added labeled glycolate. The initial rapid (<10 seconds) phase represented uptake of glycolate; the slow phase represented the metabolism of glycolate. The rates of glycolate metabolism were in agreement with those determined using the C₂-cycle inhibitors during CO₂ fixation.     

Chemical inhibition of the glycolate pathway in soybean leaf cells

Isolated soybean (Glycine max [L.] Merr.) leaf cells were treated with three inhibitors of the glycolate pathway in order to evaluate the potential of such inhibitors for increasing photosynthetic efficiency. Preincubation of cells under acid conditions in α-hydroxypyridinemethanesulfonic acid increased ¹⁴CO₂ incorporation into glycolate, but severely inhibited photosynthesis. Isonicotinic acid hydrazide (INH) increased the incorporation of ¹⁴CO₂ into glycine and reduced label in serine, glycerate, and starch. Butyl 2-hydroxy-3-butynoate (BHB) completely and irreversibly inhibited glycolate oxidase and increased the accumulation of ¹⁴C into glycolate. Concomitant with glycolate accumulation was the reduction of label in serine, glycerate, and starch, and the elimination of label in glycine. The inhibitors INH and BHB did not eliminate serine synthesis, suggesting that some serine is synthesized by an alternate pathway. The per cent incorporation of ¹⁴CO₂ into glycolate by BHB-treated cells or glycine by INH-treated cells was determined by the O₂/CO₂ ratio present during assay. Photosynthesis rate was not affected by INH or BHB in the absence of O₂, but these compounds increased the O₂ inhibition of photosynthesis. This finding suggests that the function of the photorespiratory pathway is to recycle glycolate carbon back into the Calvin cycle, so if glycolate metabolism is inhibited, Calvin cycle intermediates become depleted and photosynthesis is decreased. Thus, chemicals which inhibit glycolate metabolism do not reduce photorespiration and increase photosynthetic efficiency, but rather exacerbate the problem of photorespiration.     

The synthesis of phosvitin in vitro by slices of liver from the laying hen

1. Slices of liver from laying hens incorporated Na₂¹⁴CO₃ and NaH₂³²PO₄ into phosvitin. Slices of liver from immature birds did not do so to any appreciable extent. The ³²P was incorporated into O-phosphorylserine in the phosvitin molecule. 2. Kidney, spleen, muscle, large and small intestine, ovary and oviduct from laying birds did not incorporate Na₂¹⁴CO₃ into phosvitin. 3. Slices of liver from laying hens carried out a net synthesis of phosphoprotein under the standard conditions of incubation. Slices from the livers of immature pullets did not do so. 4. Liver from the laying hen incorporated [2-¹⁴C]glycine, [3-¹⁴C]serine and [2-¹⁴C]glutamic acid into phosvitin. Part of the glycine was shown to be present as serine in the final product. 5. Slices of liver from immature birds treated with oestradiol synthesized phosvitin from [2-¹⁴C]glycine, but the addition of oestrogens in vitro to slices from untreated immature birds did not promote synthesis during a 3 hr. incubation period.     

Inhibition of glycine decarboxylation and serine formation in tobacco by glycine hydroxamate and its effect on photorespiratory carbon flow

Glycine hydroxamate is a competitive inhibitor of glycine decarboxylation and serine formation (referred to as glycine decarboxylase activity) in particulate preparations obtained from both callus and leaf tissue of tobacco. In preparations from tobacco callus tissues, the K_i for glycine hydroxamate was 0.24 ± 0.03 millimolar and the K_m for glycine was 5.0 ± 0.5 millimolar. The inhibitor was chemically stable during assays of glycine decarboxylase activity, but reacted strongly when incubated with glyoxylate. Glycine hydroxamate blocked the conversion of glycine to serine and CO₂in vivo when callus tissue incorporated and metabolized [1-¹⁴C]glycine, [1-¹⁴C]glycolate, or [1-¹⁴C]glyoxylate. The hydroxamate had no effect on glyoxylate aminotransferase activities in vivo, and the nonenzymic reaction between glycine hydroxamate and glyoxylate did not affect the flow of carbon in the glycolate pathway in vivo. Glycine hydroxamate is the first known reversible inhibitor of the photorespiratory conversion of glycine to serine and CO₂.     

Aspartate Carbamyltransferase : Site of End-Product Inhibition of the Orotate Pathway in Intact Cells of Cucurbita pepo

Lovatt et al. (1979 Plant Physiol 64: 562-569) have previously demonstrated that end-product inhibition functions as a mechanism regulating the activity of the orotic acid pathway in intact cells of roots excised from 2-day-old squash plants (Cucurbita pepo L. cv Early Prolific Straightneck). Uridine (0.5 millimolar final concentration) or one of its metabolites inhibited the incorporation of NaH¹⁴CO₃, but not [¹⁴C]carbamylaspartate or [¹⁴C]orotic acid, into uridine nucleotides (ΣUMP). Thus, regulation of de novo pyrimidine biosynthesis was demonstrated to occur at one or both of the first two reactions of the orotic acid pathway, those catalyzed by carbamylphosphate synthetase (CPSase) and aspartate carbamyltransferase (ACTase). The results of the present study provide evidence that ACTase alone is the site of feedback control by added uridine or one of its metabolites. Evidence demonstrating regulation of the orotic acid pathway by end-product inhibition at ACTase, but not at CPSase, includes the following observations: (a) addition of uridine (0.5 millimolar final concentration) inhibited the incorporation of NaH¹⁴CO₃ into ΣUMP by 80% but did not inhibit the incorporation of NaH¹⁴CO₃ into arginine; (b) inhibition of the orotate pathway by added uridine was not reversed by supplying exogenous ornithine (5 millimolar final concentration), while the incorporation of NaH¹⁴CO₃ into arginine was stimulated more than 15-fold when both uridine and ornithine were added; (c) incorporation of NaH¹⁴CO₃ into arginine increased, with or without added ornithine when the de novo pyrimidine pathway was inhibited by added uridine; and (d) in assays employing cell-free extracts prepared from 2-day-old squash roots, the activity of ACTase, but not CPSase, was inhibited by added pyrimidine nucleotides.     

Two photosynthetic mechanisms mediating the low photorespiratory state in submersed aquatic angiosperms

The submersed angiosperms Myriophyllum spicatum L. and Hydrilla verticillata (L.f.) Royal exhibited different photosynthetic pulse-chase labeling patterns. In Hydrilla, over 50% of the ¹⁴C was initially in malate and aspartate, but the fate of the malate depended upon the photorespiratory state of the plant. In low photorespiration Hydrilla, malate label decreased rapidly during an unlabeled chase, whereas labeling of sucrose and starch increased. In contrast, for high photorespiration Hydrilla, malate labeling continued to increase during a 2-hour chase. Thus, malate formation occurs in both photorespiratory states, but reduced photorespiration results when this malate is utilized in the light. Unlike Hydrilla, in low photorespiration Myriophyllum, ¹⁴C incorporation was via the Calvin cycle, and less than 10% was in C₄ acids.

Ethoxyzolamide, a carbonic anhydrase inhibitor and a repressor of the low photorespiratory state, increased the label in glycolate, glycine, and serine of Myriophyllum. Isonicotinic acid hydrazide increased glycine labeling of low photorespiration Myriophyllum from 14 to 25%, and from 12 to 48% with high photorespiration plants. Similar trends were observed with Hydrilla. Increasing O₂ increased the per cent [¹⁴C]glycine and the O₂ inhibition of photosynthesis in Myriophyllum. In low photorespiration Myriophyllum, glycine labeling and O₂ inhibition of photosynthesis were independent of the CO₂ level, but in high photorespiration plants the O₂ inhibition was competitively decreased by CO₂. Thus, in low but not high photorespiration plants, glycine labeling and O₂ inhibition appeared to be uncoupled from the external [O₂]/[CO₂] ratio.

These data indicate that the low photorespiratory states of Hydrilla and Myriophyllum are mediated by different mechanisms, the former being C₄-like, while the latter resembles that of low CO₂-grown algae. Both may require carbonic anhydrase to enhance the use of inorganic carbon for reducing photorespiration.

     

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

Glycolate was excreted from the 5% CO₂-grown cells of Euglena gracilis Z when placed in an atmosphere of 100% O₂ 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 CO₂ 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% O₂ and 20,000 lux; this led the cells to excrete glycolate outside the cells. Exogenous glycolate was metabolized only to CO₂ but not to glycine and serine. The physiologic role of the glycolate metabolism and excretion under such conditions is discussed.