Glycolate metabolism is under nitrogen control in chlorella

The utilization of nitrate and ammonia as nitrogen sources had different effects on the metabolism of glycolate in Cholorella sorokiniana. During photolithotrophic growth with nitrate as nitrogen source, glycolate was metabolized via the glycine-serine pathway. Ammonia, produced as a result of glycolate metabolism, was reassimilated by glutamine synthetase. Two isoforms of this enzyme were present at different relative abundance in C. sorokiniana wild type and in a mutant with an increased capacity for the metabolism of glycolate (strain OR).

During photolithotrophic growth in the presence of ammonia as sole nitrogen source, several lines of evidence indicated that glycolate was metabolized to malate, pyruvate, tricarboxylic acid cycle intermediates and related amino acids in C. sorokiniana wild-type cells. Malate synthase was induced and glycine decarboxylase and serine-glyoxylate aminotransferase were repressed in cells grown with ammonia. An inverse correlation was observed between aminating NADPH-glutamate dehydrogenase and the in vivo glycine decarboxylation rate.

     

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.     

A study of the control of glycolate excretion in chlorella

Chlorella pyrenoidosa cells grown on 5% CO(2) excreted glycolate when incubated in light with 10 mm bicarbonate, but excreted no glycolate under the same conditions when they were maintained on air for 7 hours prior to the assay. Incubation of 5% CO(2)-grown and air-grown cells with 10 mm isonicotinyl hydrazide or 10 mm alpha-hydroxypyridinemethane sulfonate during the assay stimulated the excretion of glycolate by CO(2)-grown cells severalfold that of air-grown cells.Adaptation of CO(2)-grown Chlorella to growth on air did not affect the levels of glycolate dehydrogenase in the cells and did not affect the rate of dark oxidation and metabolism of exogeneous (14)C-glycolate by the cells. These results indicate that the lack of glycolate excretion by air-grown or air-adapted cells of Chlorella cannot be explained by a concomitant change in the level of glycolate dehydrogenase.     

The plant-like C2 glycolate cycle and the bacterial-like glycerate pathway cooperate in phosphoglycolate metabolism in cyanobacteria

The occurrence of a photorespiratory 2-phosphoglycolate metabolism in cyanobacteria is not clear. In the genome of the cyanobacterium Synechocystis sp. strain PCC 6803, we have identified open reading frames encoding enzymes homologous to those forming the plant-like C2 cycle and the bacterial-type glycerate pathway. To study the route and importance of 2-phosphoglycolate metabolism, the identified genes were systematically inactivated by mutagenesis. With a few exceptions, most of these genes could be inactivated without leading to a high-CO(2)-requiring phenotype. Biochemical characterization of recombinant proteins verified that Synechocystis harbors an active serine hydroxymethyltransferase, and, contrary to higher plants, expresses a glycolate dehydrogenase instead of an oxidase to convert glycolate to glyoxylate. The mutation of this enzymatic step, located prior to the branching of phosphoglycolate metabolism into the plant-like C2 cycle and the bacterial-like glycerate pathway, resulted in glycolate accumulation and a growth depression already at high CO(2). Similar growth inhibitions were found for a single mutant in the plant-type C2 cycle and more pronounced for a double mutant affected in both the C2 cycle and the glycerate pathway after cultivation at low CO(2). These results suggested that cyanobacteria metabolize phosphoglycolate by the cooperative action of the C2 cycle and the glycerate pathway. When exposed to low CO(2), glycine decarboxylase knockout mutants accumulated far more glycine and lysine than wild-type cells or mutants with inactivated glycerate pathway. This finding and the growth data imply a dominant, although not exclusive, role of the C2 route in cyanobacterial phosphoglycolate metabolism.     

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.     

Metabolic Influences on Tyrosine Excretion in Bacillus subtilis

The biosynthetic pathway for tyrosine synthesis is regulated both by repression of enzyme synthesis and by feedback inhibition of enzyme activity in Bacillus subtilis. Nevertheless, wild-type cells produce significantly more tyrosine than is required for protein synthesis, and part of this is excreted into the medium. Alteration of nutritional and other environmental conditions of cultivation strongly influenced the amount of tyrosine excretion. The excretion of tyrosine by wild-type cells was compared with that of a regulatory mutant having a feedback-insensitive prephenate dehydrogenase. Tyrosine excretion varied directly with the in vitro activity of prephenate dehydrogenase and inversely with temperature in the two strains. The regulatory mutant excreted five times as much tyrosine as the wild type at all growth temperatures examined. The carbon source used for growth significantly influenced the level of tyrosine excretion. The specific activity of prephenate dehydrogenase was also affected by the carbon source. Incorporation studies with isotopic tyrosine and fluorometric determinations of tyrosine concentrations extractable in hot water were used to measure operationally the tyrosine pools in the mutant and wild-type strains. The effects of various environmental conditions on the synthesis and excretion of tyrosine led to the conclusion that metabolic controls governing end product contrations exist which are completely independent of regulation by feedback inhibition and repression.     

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.     

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.     

Formation and metabolism of glycolate in the cyanobacterium Coccochloris peniocystis

The formation and metabolism of glycolate in the cyanobacterium Coccochloris peniocystis was investigated and the activities of enzymes of glycolate metabolism assayed. Photosynthetic ¹⁴CO₂ incorporation was O₂ insensitive and no labelled glycolate could be detected in cells incubated at 2 and 21% O₂. Under conditions of 100% O₂ glycolate comprised less than 1% of the acid-stable products indicating ribulose 1,5 bisphosphate (RuBP) oxidation only occurs under conditions of extreme O₂ stress. Metabolism of [1-¹⁴C] glycolate indicated that as much as 62% of ¹⁴C metabolized was released as ¹⁴CO₂ in the dark. Metabolism of labelled glycolate, particularly incorporation of ¹⁴C into glycine, was inhibited by the amino-transferase inhibitor amino-oxyacetate. Metabolism of [2-¹⁴C] glycine was not inhibited by the serine hydroxymethyltransferase inhibitor isonicotinic acid hydrazide and little or no labelled serine was detected as a result of ¹⁴C-glycolate metabolism. These findings indicate that a significant amount of metabolized glycolate is totally oxidized to CO₂ via formate. The remainder is converted to glycine or metabolized via a glyoxylate cycle. The conversion of glycine to serine contributes little to glycolate metabolism and the absence of hydroxypyruvate reductase confirms that the glycolate pathway is incomplete in this cyanobacterium.Abbreviations AAN aminoacetonitrile - AOA aminooxyacetate - DIC dissolved inorganic carbon - INH isonicotinic acid hydrazide - PEP phosphoenolpyruvate - PEPcase phosphoenolpyruvate carboxylase - PG phosphoglycolate - PGA phosphoglyceric acid - PGPase phosphoglycolate phosphatase - PR photorespiration - Rubisco ribulose-1,5-bisphosphate carboxylase oxygenase - TCA trichloroacetic acid - RuBP ribulose-1,5-bisphosphate     

Limitations on the Utilization of Glycolate by Chlamydomonas reinhardtii

Growth and shorter term incorporation measurements with both wild type Chlamydomonas reinhardtii and a mutant (F-60, lacking phosphori-bulokinase activity) indicate that the rate of glycolate utilization is always relatively low. Growth support with external glycolate is restricted to cells with full photosynthetic capacity. A high concentration of glycolate is required for optimal growth support and incorporation of [¹⁴C]glycolate. Glycolate incorporation is low at pH 3.8 even with the relatively free permeability. The F-60 mutant can take up only small quantities of glycolate in spite of photosynthetic electron transport and photophosphorylation competencies. This requirement for photosynthetic carbon metabolism indicates a significant difference in the glycolate pathway of this alga. No growth condition significantly increases glycolate incorporation rates. There is no evidence that one of the primary enzymes, glycolate dehydrogenase, is limiting utilization; measurements of glycolate uptake and excretion do not always correlate with its activity. Since the maximal utilization rate of glycolate is low, control of glycolate formation is important in preventing the loss of this fixed carbon from the algal cell.     

Different metabolic fate of two carbons of glycolate in its conversion to serine in Euglena gracilis z

In our preceding work (A. Yokota, Y. Nakano, and S. Kitaoka, 1978, Agric. Biol. Chem. 42, 121-129), extensive decarboxylation of glycolate carboxyl carbon during its metabolism in Euglena gracilis suggested occurrence of a metabolic pathway of glycolate different from that of higher C3 plants. In the present report, we establish the Euglena glycolate pathway from characteristics of the decarboxylation of the carboxyl carbon and from the metabolic fate of hydroxymethyl carbon of glycolate. The ratio of the decarboxylation of the carboxyl carbon of glycolate to the total metabolized carbon increased with increasing metabolic rate in an asymptotic fashion. Thus, the ratio was 20% at the metabolic rate of 0.05 nmol of glycolate/10(6) cells/min, but it was over 60% at the rate of more than 0.35 nmol/10(6) cells/min after 2 min of incubation. Metabolic products were also changed depending on the rate of metabolism of glycolate; glycine was the main product at the low rate of glycolate metabolism and the contribution of glycine was reversed by the increased contribution of evolved CO2 at the high rates. At the metabolic rate of 1.5 nmol of glycolate/10(6) cells/min, the rate of the decarboxylation was 1.0 nmol of CO2/10(6) cells/min, which could not be explained by the extremely low activity of glycine synthase in Euglena. Experiments with [2-14C]glycolate showed that exogenously added formate and methionine caused accumulation of radioactive formate. Based on these results, we have proposed that the glycolate metabolism of E. gracilis consists of glycine and formate pathways and that the relative contribution of both pathways to the glycolate metabolism depends on the metabolic rate of glycolate.     

Uptake, intracellular binding, and excretion of polyamines during growth of Neurospora crassa

In Neurospora crassa mycelia, the amounts of the main polyamines, putrescine and spermidine, are approximately 0.8 and 18 nmol/mg, dry weight. We wished to know what determines these pool sizes. In the growth medium, externally added polyamines enter cells largely by a nonsaturable, diffusional system. In a mutant unable to polyamines, internal and external spermidine appear to equilibrate across the cell membrane during growth. However, this was true only after an intracellular "sink," with a capacity equal to the amount of spermidine found in wild-type cells, had been saturated. We speculate that internal anionic binding sites, detectable in permeabilized cells, sequester virtually all of the spermidine normally found in exponentially growing N. crassa. Further evidence for this view was that in mature, stationary cultures, excess spermidine is excreted. Putrescine is also excreted if its concentration in the cell is abnormally high. The control of pool size by intracellular binding and excretion may be an advantage in this pathway, because feedback inhibition does not prevail, enzyme regulation is by comparison slow, and excessive polyamines are toxic.     

PHOTORESPIRATION IN CONTINUOUS CULTURE OF DUNALIELLA TERTIOLECTA (CHLOROPHYTA): RELATIONSHIPS BETWEEN SERINE, GLYCINE, AND EXTRACELLULAR GLYCOLATE

The concentrations of extracellular glycolate and intracellular free pools of serine and glycine were monitored in nitrogen-limited continuous cultures of Dunaliella tertiolecta (Butcher) UTEX LB999, grown at two different irradiances on a light:dark cycle. Under steady-state conditions, this microalga excreted into the medium a large amount of glycolate during the light phase, up to 100 nmol·(10⁶ cells)⁻¹ for a cell concentration of around 1.5 10⁸ cells·L⁻¹, but glycolate disappeared from the dissolved phase in the dark. Cells grown at 70 and those grown at 430 μmol photons·m⁻²·s⁻¹ differed in maximal glycolate concentration, intracellular serine and glycine concentrations, and serine:glycine ratio. Reversal of these photon flux densities to which the cultures were exposed caused rapid modification of the extracellular glycolate and intracellular serine and glycine pools. These results suggest that photorespiratory metabolism in D. tertiolecta could be approximately quantified by measuring the changes in dissolved glycolate and intracellular serine and glycine concentrations, extending previous results from cultured phytoplankton and suggesting methods for field studies.     

Effects of Aminooxyacetate and Aminoacetonitrile on Glycolate and Ammonia Release by the Cyanobacterium Anabaena cylindrica

Aminooxyacetate and aminoacetonitrile cause increased excretion of glycolate by the cyanobacterium Anabaena cylindrica. Both compounds also reduce NH₄-N release induced by methionine sulfoximine in non-nitrogen-fixing cultures. Changes in amino acid pool sizes together with changes in activities of some enzymes related to glycolate metabolism show that glyoxylate to glycine conversion and glycine to serine conversion are inhibited by aminooxyacetate and aminoacetonitrile, respectively. The results also verify that photorespiratory glycolate metabolism via amination of glyoxylate is operative in A. cylindrica.     

Involvement of photorespiration and glycolate pathway in carbonic anhydrase induction and inorganic carbon concentration in Chlamydomonas reinhardtii

Interaction between induction of carbonic anhydrase (CA) activity, induction of inorganic carbon (C_i) concentrating mechanisms and the photorespiratory glycolate pathway has been studied in wild type 6145c and photorespiratory mutant 18–7F (low in phosphoglycolate phosphatase activity) cells of C. reinhardtii . Cell transfer from high CO₂ (5%, v/v) to low CO₂ (0.03%) provoked an increase of extracellular and total (extracellular plus intracellular) CA in both wild type and mutant cells. During adaptation to low CO₂ conditions, both strains excreted ammonium to the medium at a similar rate in the presence of l -methionine- d-l -sulfoximine (MSX), an inhibitor of glutamine synthetase (GS). MSX also provoked ammonium excretion by air adapted wild type and mutant cells, even though both strains had high levels of CA activity and of C_i concentrating activities.
GS increased in both strains after transfer from high to low CO₂ conditions. However, this increase was abolished by aminooxyacetate, an inhibitor of the glyoxylate-serine aminotransferase, and by glycolaldehyde, an inhibitor of triose phosphate to ribulose 1,5-bisphosphate conversion. CA synthesis did not occur in the presence of either aminooxyacetate or glycolaldehyde. Algae grown in high CO₂ in the presence of aminooxyacetate did not induce C_i concentrating mechanisms. Integration of these three processes, i.e., CA synthesis, C_i-concentration, and photorespiratory glycolate pathway is proposed in the framework of carbon metabolism of the alga.     

EXCRETION OF GLYCOLATE BY A SPECIES OF CHLORELLA (CHLOROPHYCEAE)1

The claim that Chlorella sp. (CCAP 211/8p), sometimes referred to as C. fusca, Shihira and Krauss, does not excrete glycolate has been reexamined. Chlorella sp. grown on 5% CO₂in air, excreted glycolate when incubated in light in 10 mM bicarbonate. Excretion ceased 30–60 min after transfer of the cells to air and no excretion could be detected with air-grown cells or with cells grown on 5% CO₂in media buffered at pH 8.0. Incubation with 10 mM isonicotinyl hydrazide, a glycolate pathway inhibitor, caused excretion in air-grown cells and stimulated excretion in CO₂-grown cells indicating that both the rate of glycolate synthesis and metabolism is higher in CO₂grown cells than in air-grown cells. Enhanced glycolate synthesis and excretion in CO₂-grown cells is correlated with law photosynthetic rate in 10 mM bicarbonate, and the photosynthetic rate of these cells doubles over a period of 2–2.5 h after initial transfer from high CO₂to bicarbonate. This correlation of photosynthetic induction with cessation of glycolate excretion is similar to that reported in a bluegreen alga and thought to occur in other green algae. These results indicate that glycolate excretion and its regulation in this species of Chlorella is not different from that in other algae.     

The Effect of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) on Photosynthesis, Glycolate Excretion ('Photorespiration') and Amino Acid Metabolism in the Blue-Green Alga Anacystis

When photosynthesis of the blue-green alga Anacystis nidulans was measured as ¹⁴CO₂-fixation, the inhibitory effect of DCMU at low concentrations was greatest when mainly Photosystem 1 (PS 1) (excitation at 446 or 687 nm) was operative. At concentrations above 10-⁶M the inhibition on ¹⁴CO₂-fixation was greatest when mainly Photosystem 2 (PS 2) was operative (excitation at 619). During excitation of PS 1, the excretion of glycolate was stimulated at low concentrations of DCMU (5 × 10^-8M and lower), while higher concentrations inhibited excretion. All concentrations of DCMU inhibited glycolate excretion when mainly PS 2 was excited. The curves showing the relative effect of DCMU on the two photosystems, measured as PS 1/PS 2, had opposite shapes for ¹⁴CO₂-fixation and glycolate excretion. An increase in ¹⁴CO₂-fixation coincided with a decrease in glycolate excretion and vice versa. It appears that the increased rate of photosynthesis when mainly PS 1 was operative relative to that when mainly PS 2 was excited, increases the consumption of glycolate in an oxidation process associated with the excitation of PS 1, resulting in less excretion of glycolate to the medium. The influence of DCMU inhibition on labelled amino acid pools connected to the glycolate pathway (glycine-serine) is quite similar to that for ¹⁴CO₂-fixation. At concentrations below 10^-6M DCMU, inhibition of ¹⁴CO₂- incorporation into the amino acids was greatest when PS 1 was excited, while at the higher concentrations tested, inhibition was greater when PS 2 was excited. We conclude that the metabolism of glycine and serine is closely connected to the rate of photosynthesis.     

Glycolate metabolism in cyanobacteria

A comparative analysis of glycolate excretion in 11 cyanobacteria showed that 8 strains, although grown and assayed in air, excreted glycolate. The largest quantities were excreted by the filamentous strains Plectonema boryanum 73110 and Anabaena cylindrica (Lemm). The carbon lost by excretion was at most 9% of the net fixed carbon in air for heterocystous cyanobacteria but increased (up to 60%) in some strains under a high pO₂ (0.03 kPa CO₂ in pure O₂). A. cylindrica excreted glycolate at a maximum level of 2 and 10 μmol (mg chl a )⁻¹ h⁻¹ in air and at high pO₂, respectively. The excretion continued for several hours. Increases in light intensity and pO₂ and a shift in pH from 7 to 9 increased the amount of glycolate excreted. A. cylindrica also showed the most O₂-sensitive fixation of CO₂. In vitro activity of phosphoglycolate phosphatase (EC 3.1.3.18) was found in all strains tested, with the highest activities noted for Gloeobacter violaceus 7.82 and Gloeothece 6909 and for young cultures of A. cylindrica . The lowest activities were found in Anabaena 7120 and Anacystis nidulans 625, strains excreting no or only minor quantities of glycolate.     

Experimental evolution of a metabolic pathway for ethylene glycol utilization by Escherichia coli.

Spontaneous mutants of Escherichia coli able to grow on ethylene glycol as a sole source of carbon and energy were obtained from mutants that could grow on propylene glycol. Attempts to obtain ethylene glycol-utilizing mutants from wild-type E. coli were unsuccessful. The two major characteristics of the ethylene glycol-utilizing mutants were (i) increased activities of propanediol oxidoreductase, an enzyme present in the parental strain (a propylene glycol-positive strain), which also converted ethylene glycol into glycolaldehyde; and (ii) constitutive synthesis of high activities of glycolaldehyde dehydrogenase, which converted glycolaldehyde to glycolate. Glycolate was metabolized via the glycolate pathway, which was present in the wild-type cells; this was indicated by the induction in ethylene glycol-grown cells of glycolate oxidase, the first enzyme in the pathway. Glycolaldehyde dehydrogenase was partially characterized as an enzyme of this new metabolic pathway in E. coli, and glycolate was identified as the product of the reaction. This enzyme used NAD and NADP as coenzymes, although the NADP-dependent activity was about 10 times lower than the NAD-dependent activity. Uptake of [14C]ethylene glycol was dependent on the presence of the enzymes capable of metabolism of ethylene glycol. Glycolaldehyde and glycolate were identified as intermediate metabolites in the pathway.     

High glycolate oxidase activity is required for survival of maize in normal air

A mutant in the maize (Zea mays) Glycolate Oxidase1 (GO1) gene was characterized to investigate the role of photorespiration in C4 photosynthesis. An Activator-induced allele of GO1 conditioned a seedling lethal phenotype when homozygous and had 5% to 10% of wild-type GO activity. Growth of seedlings in high CO2 (1%-5%) was sufficient to rescue the mutant phenotype. Upon transfer to normal air, the go1 mutant became necrotic within 7 d and plants died within 15 d. Providing [1-14C]glycolate to leaf tissue of go1 mutants in darkness confirmed that the substrate is inefficiently converted to 14CO2, but both wild-type and GO-deficient mutant seedlings metabolized [1-14C]glycine similarly to produce [14C]serine and 14CO2 in a 1:1 ratio, suggesting that the photorespiratory pathway is otherwise normal in the mutant. The net CO2 assimilation rate in wild-type leaves was only slightly inhibited in 50% O2 in high light but decreased rapidly and linearly with time in leaves with low GO. When go1 mutants were shifted from high CO2 to air in light, they accumulated glycolate linearly for 6 h to levels 7-fold higher than wild type and 11-fold higher after 25 h. These studies show that C4 photosynthesis in maize is dependent on photorespiration throughout seedling development and support the view that the carbon oxidation pathway evolved to prevent accumulation of toxic glycolate.