Ureide metabolism in leaves of nitrogen-fixing soybean plants

In leaf pieces from nodulated soybean (Glycine max [L] Merr cv Maple Arrow) plants, [¹⁴C]urea-dependent NH₃ and ¹⁴CO₂ production in the dark showed an approximately 2:1 stoichiometry and was decreased to less than 11% of the control (12-19 micromoles NH₃ per gram fresh weight per hour) in the presence of 50 millimolar acetohydroxamate, a urease inhibitor. NH₃ and CO₂ production from the utilization of [2-¹⁴C] allantoin also exhibited a 2:1 stoichiometry and was reduced to a similar extent by the presence of acetohydroxamate with a concomitant accumulation of urea which entirely accounted for the loss in NH₃ production. The almost complete sensitivity of NH₃ and CO₂ production from allantoin and urea metabolism to acetohydroxamate, together with the observed stoichiometry, indicated a path of ureide assimilation (2.0 micromoles per gram leaf fresh weight per hour) via allantoate, ureidoglycolate, and glyoxylate with the production of two urea molecules yielding, in turn, four molecules of NH₃ and two molecules of CO₂.     

Ureide Catabolism in Soybeans : III. Ureidoglycolate Amidohydrolase and Allantoate Amidohydrolase Are Activities of an Allantoate Degrading Enzyme Complex

We demonstrate that allantoate is catabolized in soybean seedcoat extracts by an enzyme complex that has allantoate amidohydrolase and ureidoglycolate amidohydrolase activities. Soybean seedcoat extracts released ¹⁴CO₂ from [ureido-¹⁴C]ureidoglycolate under conditions in which urease is not detectable. CO₂ and glyoxylate are enzymically released in a one to one ratio indicating that ureidoglycolate amidohydrolase is the responsible activity. Ureidoglycolate amidohydrolase has a K_m of 85 micromolar for ureidoglycolate. Glyoxylate and CO₂ are enzymically released from allantoate at linear rates in a one to 2.3 ratio from 5 to 30 min. This ratio is consistent with the degradation of allantoate to two CO₂ and one glyoxylate with approximately 23% of the allantoate degraded reacting with 2-mercaptoethanol to yield 2-hydroxyethylthio, 2′-ureido, acetate (RG Winkler, JC Polacco, DG Blevins, DD Randall 1985 Plant Physiol 79: 787-793). That [¹⁴C]urea production from [2,7-¹⁴C]allantoate is not detectable indicates that allantoate-dependent glyoxylate production is enzymic and not a result of nonenzymic hydrolysis of a ureido intermediate (nonenzymic hydrolysis releases urea). These results and those from intact tissue studies (RG Winkler DG Blevins, JC Polacco, DD Randall 1987 Plant Physiol 83: 585-591) suggest that soybeans have a second amidohydrolase reaction (ureidoglycolate amidohydrolase) that follows allantoate amidohydrolase in allantoate catabolism. The rate of ¹⁴CO₂ release from [2,7-¹⁴C]allantoate is not reduced when the volume of the reaction mixture is increased, suggesting that the release of ¹⁴CO₂ is not dependent on the accumulation of free intermediates. That [2,7-¹⁴C]allantoate dependent ¹⁴CO₂ release is not proportionally diluted by unlabeled ureidoglycolate indicates that the reaction is carried out by an enzyme complex. This is the first report of ureidoglycolate amidohydrolase activity in any organism and the first in vitro demonstration in plants that the ureido-carbons of allantoate can be completely degraded to CO₂ without a urea intermediate.     

Ureide Catabolism of Soybeans : II. Pathway of Catabolism in Intact Leaf Tissue

Allantoin catabolism studies have been extended to intact leaf tissue of soybean (Glycine max L. Merr.). Phenyl phosphordiamidate, one of the most potent urease inhibitors known, does not inhibit ¹⁴CO₂ release from [2,7-¹⁴C]allantoin (urea labeled), but inhibits urea dependent CO₂ release ≥99.9% under similar conditions. Furthermore, ¹⁴CO₂ and [¹⁴C] allantoate are the only detectable products of [2,7-¹⁴C]allantoin catabolism. Neither urea nor any other product were detected by analysis on HPLC organic acid or organic base columns although urea and all commercially available metabolites that have been implicated in allantoin and glyoxylate metabolism can be resolved by a combination of these two columns. In contrast, when allantoin was labeled in the two central, nonureido carbons ([4,5-¹⁴C]allantoin), its catabolism to [¹⁴C]allantoate, ¹⁴CO₂, [¹⁴C]glyoxylate, [¹⁴C]glycine, and [¹⁴C]serine in leaf discs could be detected. These data are fully consistent with the metabolism of allantoate by two amidohydrolase reactions (neither of which is urease) that occur at similar rates to release glyoxylate, which in turn is metabolized via the photorespiratory pathway. This is the first evidence that allantoate is metabolized without urease action to NH₄⁺ and CO₂ and that carbons 4 and 5 enter the photorespiratory pathway.     

Glyoxylate Induced Changes in the Carbon and Nitrogen Metabolism of the Cyanobacterium Anabaena cylindrica

Addition of millimolar concentrations of glyoxylate to nitrogen-fixing cultures of Anabaena cylindrica, grown aerobically in the light, caused the following effects: an increase in the number of glycogen granules and in the excretion of carbohydrates; a decreased phycocyanin concentration, but an increase in the chlorophyll a to phycocyanin ratio. Also, an enhancement in the carbon to nitrogen ratio was noted, but this was restored if NH₄⁺ was added simultaneously. The most pronounced effect of glyoxylate addition was a 20-fold increase in the glycine pool. The effect of glyoxylate on N₂ fixation (acetylene reduction) was enhanced at high light intensities, but it did not affect the in vitro ribulose-1,5-bisphosphate carboxylase activity. However, addition of millimolar concentrations of glycolate did not cause changes in nitrogenase activity, CO₂ fixation, and NH₃ release comparable to those caused by glyoxylate. The primary mechanism of action of glyoxylate appears to be within the glycolate pathway of the vegetative cells and metabolically downstream from glycolate.     

Effects of glyoxylate on photosynthesis by intact chloroplasts

Because glyoxylate inhibits CO₂ fixation by intact chloroplasts and purified ribulose bisphosphate carboxylase/oxygenase, glyoxylate might be expected to exert some regulatory effect on photosynthesis. However, ribulose bisphosphate carboxylase activity and activation in intact chloroplasts from Spinacia oleracea L. leaves were not substantially inhibited by 10 millimolar glyoxylate. In the light, the ribulose bisphosphate pool decreased to half when 10 millimolar glyoxylate was present, whereas this pool doubled in the control. When 10 millimolar glyoxylate or formate was present during photosynthesis, the fructose bisphosphate pool in the chloroplasts doubled. Thus, glyoxylate appeared to inhibit the regeneration of ribulose bisphosphate, but not its utilization.

The fixation of CO₂ by intact chloroplasts was inhibited by salts of several weak acids, and the inhibition was more severe at pH 6.0 than at pH 8.0. At pH 6.0, glyoxylate inhibited CO₂ fixation by 50% at 50 micromolar, and glycolate caused 50% inhibition at 150 micromolar. This inhibition of CO₂ fixation seems to be a general effect of salts of weak acids.

Radioactive glyoxylate was reduced to glycolate by chloroplasts more rapidly in the light than in the dark. Glyoxylate reductase (NADP⁺) from intact chloroplast preparations had an apparent K_m (glyoxylate) of 140 micromolar and a V_max of 3 micromoles per minute per milligram chlorophyll.

     

Enzymology of the reduction of hydroxypyruvate and glyoxylate in a mutant of barley lacking peroxisomal hydroxypyruvate reductase

The use of LaPr 88/29 mutant of barley (Hordeum vulgare), which lacks NADH-preferring hydroxypyruvate reductase (HPR-1), allowed for an unequivocal demonstration of at least two related NADPH-preferring reductases in this species: HPR-2, reactive with both hydroxypyruvate and glyoxylate, and the glyoxylate specific reductase (GR-1). Antibodies against spinach HPR-1 recognized barley HPR-1 and partially reacted with barley HPR-2, but not GR-1, as demonstrated by Western immunoblotting and immunoprecipitation of proteins from crude leaf extracts. The mutant was deficient in HPR-1 protein. In partially purified preparations, the activities of HPR-1, HPR-2, and GR-1 could be differentiated by substrate kinetics and/or inhibition studies. Apparent K_m values of HPR-2 for hydroxypyruvate and glyoxylate were 0.7 and 1.1 millimolar, respectively, while the K_m of GR-1 for glyoxylate was 0.07 millimolar. The K_m values of HPR-1, measured in wild type, for hydroxypyruvate and glyoxylate were 0.12 and 20 millimolar, respectively. Tartronate and P-hydroxypyruvate acted as selective uncompetitive inhibitors of HPR-2 (K_i values of 0.3 and 0.4 millimolar, respectively), while acetohydroxamate selectively inhibited GR-1 activity. Nonspecific contributions of HPR-1 reactions in assays of HPR-2 and GR-1 activities were quantified by a direct comparison of rates in preparations from wild-type and LaPr 88/29 plants. The data are evaluated with respect to previous reports on plant HPR and GR activities and with respect to optimal assay procedures for individual HPR-1, HPR-2, and GR-1 rates in leaf preparations.     

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.     

Evidence for the presence in tobacco leaves of multiple enzymes for the oxidation of glycolate and glyoxylate

The enzymic oxidation of glycolate to glyoxylate and glyoxylate to oxalate by preparations purified from tobacco (Nicotiana tabacum var Havana Seed) leaves was studied. The K_m values for glycolate and glyoxylate were 0.26 and 1.0 millimolar, respectively. The ratio of glycolate to glyoxylate oxidation was 3 to 4 in crude extracts but decreased to 1.2 to 1.5 on purification by (NH₄)₂SO₄ fractionation and chromatography on agarose A-15 and hydroxylapatite. This level of glyoxylate oxidation activity was higher than that previously found for glycolate oxidase (EC 1.1.3.1). The ratio of the two activities was changed by reaction with the substrate analog 2-hydroxy-3-butynoate (HBA) which at all concentrations inhibited glyoxylate oxidation to a greater extent than glycolate oxidation. The ratio of the two activities could also be altered by changing the O₂ concentration. Glycolate oxidation increased 3.6-fold when the O₂ atmosphere was increased from 21 to 100%, whereas glyoxylate oxidation increased only 1.6-fold under the same conditions. These changes in ratio during purification, on inhibition by HBA, and under varying O₂ concentrations imply that tobacco leaves contain at least two enzymes capable of oxidizing glycolate and glyoxylate.     

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.     

Carbon and nitrogen limitations on soybean seedling development

Carbon and nitrogen limitations on symbiotically grown soybean seedlings (Glycine max [L.] Merr.) were assessed by providing 0.0, 1.0, or 8.0 millimolar NH₄NO₃ and 320 or 1,000 microliters CO₂/liter for 22 days after planting. Maximum development of the Rhizobium-soybean symbiosis, as determined by acetylene reduction, was measured in the presence of 1.0 millimolar NH₄NO₃ under both levels of CO₂. Raising NH₄NO₃ from 0.0 to 8.0 millimolar under 320 microliters CO₂/liter increased plant dry weight by 251% and Kjeldahl N content by 287% at 22 days after planting. Increasing NH₄NO₃ from 1.0 to 8.0 millimolar under 320 microliters CO₂/liter increased total dry weight and Kjeldahl N by 100 and 168%, respectively, on day 22. Raising CO₂ from 320 to 1,000 microliters CO₂/liter during the same period had no significant effect on Kjeldahl N content of plants grown with 0.0 or 1.0 millimolar NH₄NO₃. The maximum CO₂ treatment effects were observed in plants supplied with 8.0 millimolar NH₄NO₃, where dry weight and Kjeldahl N content were increased 64% and 20%, respectively. An increase in shoot CO₂-exchange rate associated with the CO₂-enrichment treatment was reflected in a significant increase in leaf dry weight and starch content for plants grown with 1,000 microliters CO₂/liter under all combined N treatments. These data show directly that seedling growth in symbiotically grown soybeans was limited primarily by N availability. The failure of the CO₂-enrichment treatment to increase total plant N significantly in Rhizobium-dependent plants indicates that root nodule development and functioning in such plants was not limited by photosynthate production.     

Extraction and partial characterization of the glycine decarboxylase multienzyme complex from pea leaf mitochondria

Glycine decarboxylase has been successfully solubilized from pea (Pisum sativum) leaf mitochondria as an acetone powder. The enzyme was dependent on added dithiothreitol and pyridoxal phosphate for maximal activity. The enzyme preparation could catalyze the exchange of CO₂ into the carboxyl carbon of glycine, the reverse of the glycine decarboxylase reaction by converting serine, NH₄⁺, and CO₂ into glycine, and ¹⁴CO₂ release from [1-¹⁴C]glycine. The half-maximal concentrations for the glycine-bicarbonate exchange reaction were 1.7 millimolar glycine, 16 millimolar NaH¹⁴CO₂, and 0.006 millimolar pyridoxal phosphate. The enzyme (glycine-bicarbonate exchange reaction) was active in the assay conditions for 1 hour and could be stored for over 1 month. The enzymic mechanism appeared similar to that reported for the enzyme from animals and bacteria but some quantitative differences were noted. These included the tenacity of binding to the mitochondrial membrane, the concentration of pyridoxal phosphate needed for maximum activity, the requirement for dithiothreitol for maximum activity, and the total amount of activity present. Now that this enzyme has been solubilized, a more detailed understanding of this important step in photorespiration should be possible.     

Photorespiratory ammonia does not inhibit photosynthesis in glutamate synthase mutants of Arabidopsis

Exposure of ferredoxin-dependent glutamate synthase (EC 1.4.7.1) mutants of Arabidopsis thaliana to photorespiratory conditions resulted in the accumulation of NH₄⁺ and the inhibition of photosynthesis. However, upon transfer from 2% O₂, 350 microliters per liter CO₂, to 21% O₂, 350 microliters per liter CO₂, net photosynthesis declined at a slower rate in methionine sulfoximine treated leaf discs relative to controls. The recovery of photosynthesis was also more rapid in MSO-treated leaf discs although ammonia levels were more than threefold higher. Photosynthesis in leaf discs treated with azaserine was inhibited more than controls when transferred to 21% O₂ and recovered less than controls when returned to 2% O₂ although NH₄⁺ levels were not significantly different. The results obtained are consistent with the view that the rapid inhibition of photosynthesis in the glutamate synthase mutants in photorespiratory conditions is not due to the accumulation of NH₄⁺ but rather to the depletion of amino donors for glyoxylate and the consequent effects of glyoxylate on the lack of return of carbon to the chloroplast.     

Effect of Glyoxylate on the Sensitivity of Net Photosynthesis to Oxygen (the Warburg Effect) in Tobacco

The addition of glyoxylate to tobacco (Nicotiana tabacum) leaf discs inhibited glycolate synthesis and photorespiration and increased net photosynthetic ¹⁴CO₂ fixation. This inhibition of photorespiration was investigated further by studying the effect of glyoxylate on the stimulation of photosynthesis that occurs when the atmospheric O₂ level was decreased from 21 to 3% (the Warburg effect). The Warburg effect is usually ascribed to the increased glycolate synthesis and metabolism that occurs at higher O₂ concentrations. Photosynthesis in control discs increased from 59.1 to 94.7 micromoles of CO₂ per gram fresh weight per hour (a 60% increase) when the O₂ level was lowered from 21 to 3%, while the rate for discs floated on 15 millimolar glyoxylate increased only from 82.0 to 99.7 micromoles of CO₂ per gram fresh weight per hour (a 22% increase). The decrease in the O₂ sensitivity of photosynthesis in the presence of glyoxylate was explained by changes in the rate of glycolate synthesis under the same conditions.

The rate of metabolism of the added glyoxylate by tobacco leaf discs was about 1.35 micromoles per gram fresh weight per hour and was not dependent on the O₂ concentration in the atmosphere. This rate of metabolism is about 10% the amount of stimulation in the rate of CO₂ fixation caused by the glyoxylate treatment on a molar carbon basis. Glyoxylate (10 millimolar) had no effect on the carboxylase/oxygenase activity of isolated ribulose diphosphate carboxylase. Although the biochemical mechanism by which glyoxylate inhibits glycolate synthesis and photorespiration and thereby decreases the Warburg effect is still uncertain, these results show that cellular metabolites can regulate the extent of the Warburg effect.

     

Photorespiration-deficient Mutants of Arabidopsis thaliana Lacking Mitochondrial Serine Transhydroxymethylase Activity

Three allelic mutants of Arabidopsis thaliana which lack mitochondrial serine transhydroxymethylase activity due to a recessive nuclear mutation have been characterized. The mutants were shown to be deficient both in glycine decarboxylation and in the conversion of glycine to serine. Glycine accumulated as an end product of photosynthesis in the mutants, largely at the expense of serine, starch, and sucrose formation. The mutants photorespired CO₂ at low rates in the light, but this evolution of photorespiratory CO₂ was abolished by provision of exogenous NH₃. Exogenous NH₃ was required by the mutants for continued synthesis of glycine under photorespiratory conditions. These and related results with wild-type Arabidopsis suggested that glycine decarboxylation is the sole site of photorespiratory CO₂ release in wild-type plants but that depletion of the amino donors required for glyoxylate amination may lead to CO₂ release from direct decarboxylation of glyoxylate. Photosynthetic CO₂ fixation was inhibited in the mutants under atmospheric conditions which promote photorespiration but could be partially restored by exogenous NH₃. The magnitude of the NH₃ stimulation of photosynthesis indicated that the increase was due to the suppression of glyoxylate decarboxylation. The normal growth of the mutants under nonphotorespiratory atmospheric conditions indicates that mitochondrial serine transhydroxymethylase is not required in C₃ plants for any function unrelated to photorespiration.     

Effect of Methionine Sulfoximine on the Accumulation of Ammonia in C(3) and C(4) Leaves : The Relationship between NH(3) Accumulation and Photorespiratory Activity

Additions of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase (GS), result in an increase in NH₃ in seedling leaves of C₃ (wheat [Triticum aestivum cv. Kolibri] and barley [Hordeum vulgare var Perth]) and C₄ (corn [Zea mays W6A × W182E] and sorghum [Sorghum Vulgare var MK300]) plants. NH₃ accumulation is higher in C₃ (about 17.8 micromoles per gram fresh weight per hour) than in C₄ (about 4.7 micromoles) leaves. Under ideal conditions, when photosynthesis is not yet inhibited by the accumulation of NH₃, the rate of NH₃ accumulation is about 16% of the apparent rate of photosynthesis. A maximum accumulation of NH₃ was elicited by 2.5 millimolar MSX and was essentially independent of the addition of NO₃⁻ during either the growth or experimental period. When O₂ levels in the air were reduced to 2%, MSX resulted in some accumulation of NH₃ (6.0 micromoles per gram fresh weight per hour). At these levels of NH₃, there was no significant inhibition of rates of CO₂ fixation. There was also a minor, but significant, accumulation of NH₃ in corn roots treated with MSX. Inhibitors of photorespiration (isonicotinic hydrazide, 70 millimolar; 2-pyridylhydroxymethanesulfonic acid, 20 millimolar) or transaminase reactions (aminooxyacetate, 1 millimolar) inhibited the accumulation of NH₃ in both C₃ and C₄ leaves. These results support the hypothesis that GS is important in the assimilation of NH₃ in leaves and that the glycine-serine conversion is a major source of that NH₃.     

Identification and Characterization of Glycolate Oxidase and Related Enzymes from the Endocyanotic Alga Cyanophora paradoxa and from Pea Leaves

Glycolate oxidase (GO) has been identified in the endocyanom Cyanophora paradoxa which has peroxisome-like organelles and cyanelles instead of chloroplasts. The enzyme used or formed equimolar amounts of O₂ or H₂O₂ and glyoxylate, respectively. Aerobically, the enzyme did not reduce the artificial electron acceptor dichlorophenol indophenol. However, after an inhibitor of glycolate dehydrogenase, KCN (2 millimolar), was added to the assay medium, considerable aerobic glycolate:dichlorophenol indophenol reductase activity was detectable. The leaf GO inhibitor 2-hydroxybutynoate (30 micromolar), which binds irreversibly to the flavin moiety of the active site of leaf GO, inhibited Cyanophora GO and pea (Pisum sativum L.) GO to the same extent. This suggests that the active sites of both enzymes are similar. Cyanophora GO and pea GO cannot oxidize d-lactate. In contrast to GO from pea or other organisms, the affinity of Cyanophora GO for l-lactate is very low (K_m 25 millimolar). Another important difference is that Cyanophora GO produced sigmoidal kinetics with O₂ as varied substrate, whereas pea GO produced normal Michaelis-Menten kinetics. It is concluded that there is considerable inhomogeneity among the glycolate-oxidizing enzymes from Cyanophora, pea, and other organisms. The specific catalase activity in Cyanophora was only one-tenth of that in leaves. NADH-and NADPH-dependent hydroxypyruvate reductase (HPR) and glyoxylate reductase activities were detected in Cyanophora. NADH-HPR was markedly inhibited by hydroxypyruvate above 0.5 millimolar. Variable substrate inhibition was observed with glyoxylate in homogenates from different algal cultures. It is proposed that Cyanophora has multiple forms of HPR and glyoxylate reductase, but no enzyme clearly resembling leaf peroxisomal HPR was identified in these homogenates. Moreover, no serine:glyoxylate aminotransferase activity was detected. These results collectively indicate the possibility that the glycolate metabolism in Cyanophora deviates from that in leaves.     

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.     

Soybean cultivars 'Williams 82' and 'Maple Arrow' produce both urea and ammonia during ureide degradation

The ability of two soybean (Glycine max L. [Merrill]) cultivars, 'Williams 82' and 'Maple Arrow', which were reported to use different ureide degradation pathways, to degrade the ureides allantoin and allantoate was investigated. Protein fractions and total leaf homogenates from the fourth trifoliate leaves of both cultivars were examined for the ability to evolve either (14)CO(2) or [(14)C]urea from (14)C-labelled ureides in the presence of various inhibitors. (14)CO(2) evolution from [2,7-(14)C]allantoate was catalysed by 25-50% saturated ammonium sulphate fractions of both cultivars. This activity was inhibited by acetohydroxamate (AHA), which has been used to inhibit plant ureases, but not by phenylphosphorodiamidate (PPD), a more specific urease inhibitor. Thus, in both cultivars, allantoate may be metabolized by allantoate amidohydrolase. This activity was sensitive to EDTA, consistent with previous reports demonstrating that allantoate amidohydrolase requires manganese for full activity. Total leaf homogenates of both cultivars evolved both (14)CO(2) and [(14)C]urea from [2,7-(14)C] (ureido carbon labelled) allantoin, not previously reported in either 'Williams 82' or in 'Maple Arrow'. In situ leaf degradation of (14)C-labelled allantoin confirmed that both urea and CO(2)/NH(3) are direct products of ureide degradation. Growth of plants in the presence of PPD under fixing and non-fixing conditions caused urea accumulation in both cultivars, but did not have a significant impact on total seed nitrogen. Urea levels were higher in N-fixing plants of both cultivars. Contrary to previous reports, no significant biochemical difference was found in the ability of these two cultivars to degrade ureides under the conditions used.     

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.     

Oxalate metabolism by tobacco leaf discs

The turnover rate of oxalate in leaf discs of Nicotiana tabacum, var Havana Seed, during photosynthesis was estimated to be 1 to 2 micromoles per gram fresh weight per hour. Radioactivity from the enzymic oxidation of [¹⁴C]oxalate rapidly appeared in neutral sugars (mainly sucrose), organic acids (mainly malate), and amino acids. Only 5% of the radioactivity was released to the atmosphere as ¹⁴CO₂, and no formate or formaldehyde could be detected. The metabolism of oxalate was not increased by raising the O₂ concentration from 1% to 21% to 60%, nor was the formation of [¹⁴C]oxalate from [2-¹⁴C]glyoxylate changed under the same conditions as was previously observed in vitro (Havir 1983 Plant Physiol 71: 874-878). While oxalate is not an inert end product of the glycolate pathway, it contributes little to the formation of photorespiratory CO₂.