Metabolism of Hydroxypyruvate in a Mutant of Barley Lacking NADH-Dependent Hydroxypyruvate Reductase, an Important Photorespiratory Enzyme Activity

A mutant of barley (Hordeum vulgare L.), LaPr 88/29, deficient in NADH-dependent hydroxypyruvate reductase (HPR) activity has been isolated. The activities of both NADH (5%) and NADPH-dependent (19%) HPR were severely reduced in this mutant compared to the wild type. Although lacking an enzyme in the main carbon pathway of photorespiration, this mutant was capable of CO₂ fixation rates equivalent to 75% of that of the wild type, in normal atmospheres and 50% O₂. There also appeared to be little disruption to the photorespiratory metabolism as ammonia release, CO₂ efflux and ¹⁴CO₂ release from l-[U-¹⁴C]serine feeding were similar in both mutant and wild-type leaves. When leaves of LaPr 88/29 were fed either [¹⁴C]serine or ¹⁴CO₂, the accumulation of radioactivity was in serine and not in hydroxypyruvate, although the mutant was still able to metabolize over 25% of the supplied [¹⁴C]serine into sucrose. After 3 hours in air the soluble amino acid pool was almost totally dominated by serine and glycine. LaPr 88/29 has also been used to show that NADH-glyoxylate reductase and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-dependent HPR activity is due to the NADH-dependent enzyme. We also suggest that the alternative NADPH activity can metabolise a proportion, but not all, of the hydroxypyruvate produced during photorespiration and may thus form a useful backup to the NADH-dependent enzyme under conditions of maximal photorespiration.     

Effect of CO(2) Concentration on Glycine and Serine Formation during Photorespiration

Amount and products of photosynthesis during 10 minutes were measured at different ¹⁴CO₂ concentrations in air. With tobacco (Nicotiana tabacum L. cv. Maryland Mammoth) leaves the percentage of ¹⁴C in glycine plus serine was highest (42%) at 0.005% CO₂, and decreased with increasing CO₂ concentration to 7% of the total at 1% CO₂ in air. However, above 0.03% CO₂ the total amount of ¹⁴C incorporated into the glycine and serine pool was about constant. At 0.005% or 0.03% CO₂ the percentage and amount of ¹⁴C in sucrose was small but increased greatly at higher CO₂ levels as sucrose accumulated as an end product. Relatively similar data were obtained with sugar beet (Beta vulgaris L. cv. US H20) leaves. The results suggest that photorespiration at high CO₂ concentration is not inhibited but that CO₂ loss from it becomes less significant.     

Dark Respiration during Photosynthesis in Wheat Leaf Slices

The metabolism of [¹⁴C]succinate and acetate was examined in leaf slices of winter wheat (Triticum aestivum L. cv Frederick) in the dark and in the light (1000 micromoles per second per square meter photosynthetically active radiation). In the dark [1,4-¹⁴C]succinate was rapidly taken up and metabolized into other organic acids, amino acids, and CO₂. An accumulation of radioactivity in the tricarboxylic acid cycle intermediates after ¹⁴CO₂ production became constant indicates that organic acid pools outside of the mitochondria were involved in the buildup of radioactivity. The continuous production of ¹⁴CO₂ over 2 hours indicates that, in the dark, the tricarboxylic acid cycle was the major route for succinate metabolism with CO₂ as the chief end product. In the light, under conditions that supported photorespiration, succinate uptake was 80% of the dark rate and large amounts of the label entered the organic and amino acids. While carbon dioxide contained much less radioactivity than in the dark, other products such as sugars, starch, glycerate, glycine, and serine were much more heavily labeled than in darkness. The fact that the same tricarboxylic acid cycle intermediates became labeled in the light in addition to other products which can acquire label by carboxylation reactions indicates that the tricarboxylic acid cycle operated in the light and that CO₂ was being released from the mitochondria and efficiently refixed. The amount of radioactivity accumulating in carboxylation products in the light was about 80% of the ¹⁴CO₂ release in the dark. This indicates that under these conditions, the tricarboxylic acid cycle in wheat leaf slices operates in the light at 80% of the rate occurring in the dark.     

Biochemical components of the photosynthetic CO2 compensation point of higher plants.

Barley, Panicum milioides and Panicum maximum were exposed to ¹⁴CO₂ near their photosynthetic CO₂ compensation points and their respective ¹⁴C-products were determined. In short exposure times Panicum maximum had 100% of its ¹⁴C in malate and aspartate whereas Panicum milioides and barley had 16 and 3% of their respective ¹⁴C in C₄ organic acids. Near the respective CO₂ compensation points a linear relationship occurs in plotting the ratio of glycine, serine, and glycerate to C₄ organic acids. The ratio of ribulose 1,5-bisphosphate oxygenase to phosphoenolpyruvate carboxylase is linear with their CO₂ compensation points. The photosynthetic CO₂ compensation point apparently is controlled by the activity of enzymes producing photorespiration metabolites and the activity of phospheonolpyruvate carboxylase.     

Metabolism of Oat Leaves during Senescence : VII. The Interaction of Carbon Dioxide and Other Atmospheric Gases with Light in Controlling Chlorophyll Loss and Senescence

In air largely freed from CO₂, senescence of isolated oat (Avena sativa cv Victory) seedling leaves is no longer prevented by white light; instead, the leaves lose both chlorophyll and protein as rapidly as in the dark. Senescence in light is also accelerated in pure O₂, but it is greatly delayed in N₂; 100% N₂ preserves both protein and chlorophyll in light and in darkness. In light in air, most of the compounds tested that had previously been found to delay or inhibit senescence in darkness actually promote the loss of chlorophyll, but they do not promote proteolysis. Under these conditions, proteolysis can therefore be separated from chlorophyll loss. But in light minus CO₂, where chlorophyll loss is rapid in controls, two of these same reagents prevent the chlorophyll loss. Unlike the many reagents whose action in light is thus the opposite of that in darkness, abscisic acid, which promotes chlorophyll loss in the dark, also promotes it in light with or without CO₂. Kinetin, which prevents chlorophyll loss in the dark, also prevents it in light minus CO₂. In general, therefore, the responses to light minus CO₂ are similar to the responses to darkness, and (with the exception of abscisic acid and kinetin) opposite to the response to light in air.     

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.     

Oxygen effect on photosynthetic and glycolate pathways in young maize leaves

To study the effect of O₂ on the photosynthetic and glycolate pathways, maize leaves were exposed to ¹⁴CO₂ during steady-state photosynthesis in 21 or 1% O₂. At the two O₂ concentrations after a ¹⁴CO₂ pulse (4 seconds) followed by a ¹²CO₂ chase, there was a slight difference in CO₂ uptake and in the total amount of ¹⁴C fixed, but there were marked changes in ¹⁴C distribution especially in phosphoglycerate, ribulose bisphosphate, glycine, and serine. The kinetics of ¹⁴C incorporation into glycine and serine indicated that the glycolate pathway is inhibited at low O₂ concentrations. In 1% O₂, labeling of glycine was reduced by 90% and that of serine was reduced by 70%, relative to the control in 21% O₂. A similar effect has been observed in C₃ plants, except that, in maize leaves, only 5 to 6% of the total ¹⁴C fixed under 21% O₂ was found in glycolate pathway intermediates after 60 seconds chase. This figure is 20% in C₃ plants. Isonicotinyl hydrazide did not completely block the conversion of glycine to serine in 21% O₂, and the first carbon atom of serine was preferentially labeled during the first seconds of the chase. These results supported the hypothesis that the labeled serine not only derives from glycine but also could be formed from phosphoglycerate, labeled in the first carbon atom during the first seconds of photosynthesis.     

Effects of Arsenite, Sulfite, and Sulfate on Photosynthetic Carbon Metabolism in Isolated Pea (Pisum sativum L., cv Little Marvel) Chloroplasts

Photosynthetic CO₂-fixation in isolated pea (Pisum sativum L., cv Little Marvel) chloroplasts during induction is markedly inhibited by 0.4 millimolar sulfite. Sulfate at the same concentration has almost no effect. The ¹⁴CO₂-fixation pattern indicates that the primary effect of sulfite is inhibition of the reaction catalyzed by ribulose bisphosphate carboxylase and a stimulation of export of intermediates out of the chloroplasts. Inhibition of light modulation of stromal enzyme activity does not appear to account for the toxicity of SO₂ in this Pisum variety. Arsenite at 0.2 millimolar concentrations inhibits light activation and inhibits photosynthetic CO₂ fixation. The ¹⁴CO₂-fixation pattern indicates that the primary effect of arsenite is inhibition of light activation of reductive pentose phosphate pathway enzyme activity.     

Biochemistry of photosynthesis in species of triticum of differing ploidy

Illuminated flag leaves of Triticum monococcum(2X), T. urartu(2X), T. dicoccum(4X), T. dicoccoides(4X), and T. aestivum(6X) were exposed to ¹⁴CO₂ for 10 seconds and subsequently allowed to continue photosynthesis in the ambient air for periods of up to 2 minutes. The relative distribution of ¹⁴C among water-soluble products in the leaves was similar for each species at each sampling time. After the 10-second pulse of ¹⁴CO₂, radioactivity was mainly in phosphate esters with less than 5% in C₄ acids. Subsequently, radioactivity increased in sucrose, glycine, and serine at the expense of that in phosphate esters. By 2 minutes, between 18% and 29% of the ¹⁴C was in glycine plus serine. The results suggest rapid photorespiration in all species and an absence of C₄ photosynthesis.     

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.     

The effect of methionine on the metabolism of serine and glycine in vitamin B-12-deficient rats

The effect of methionine supplementation on glycine and serine metabolism was studied in vitamin B-12-deficient rats which received only 0.2% methionine in the diet. In the perfused liver, incorporation of the C-2 of glycine to the C-3 of serine was increased by addition of methionine to the perfusate. The oxidation of [1-¹⁴C]glycine to ¹⁴CO₂ was however depressed. Unlike methionine, glycine did not have any significant effect on the liver folate coenzyme distribution. Oxidation of [3-¹⁴C]serine to ¹⁴CO₂ both in vivo and in perfused liver was increased by methionine. A major portion of the C-3 radioactivity however was recovered in glucose. Data presented indicate that the rate of oxidation of [2-¹⁴C]histidine to ¹⁴CO₂ is more sensitive indicator of folate deficiency than the rate of oxidation of [3-¹⁴C] serine to ¹⁴CO₂ although both are presumably tetrahydrofolate dependent.     

Changes in specific radioactivity of sunflower leaf metabolites during photosynthesis in 14CO2 and 12CO2 at three concentrations of CO2

Summary Sunflower (Helianthus annuus L.) leaf discs were exposed to ¹⁴CO₂ or ¹⁴CO₂ followed by ¹²CO₂ at 21% O₂ and three different CO₂ concentrations. After intervals of up to 15 min, the specific activity of some photosynthetic intermediates was determined. At all CO₂ concentrations, the specific activity of 3-phosphoglyceric acid (3-PGA) increased most rapidly and after 15 min of ¹⁴CO₂ feeding was 92% (967 ppm CO₂), 87% (400 ppm CO₂) and 53% (115 ppm CO₂) of CO₂ supplied to the assimilation chamber. The specific activity of glycine, serine and the photorespiratory CO₂ was similar at all CO₂ concentrations, in aggreement with their proposed close metabolic relationship in the glycolate pathway. However, the kinetics of serine and glycine labelling suggested that serine was not totally derived from glycine. Because the specific activity of these glycolate-pathway intermediates was very differnet from that of 3-PGA at all CO₂ concentrations, not all of the carbon traversing this pathway came directly from the Calvin cycle. The non-equilibration of the 3-PGA with the feeding gas reflects the recycling of C from the glycolate pathway into the photosynthetic reduction cycle. Measurements of the rates of CO₂ evolution in the light and estimates of the C flux through the glycolate pathway suggest that the photorespiratory activity was high and similar at 115 ppm CO₂ and 400 ppm CO₂ but inhibited at 967 ppm CO₂.     

Photosynthetic products of division synchronized cultures of euglena

Rates and products of photosynthetic ¹⁴CO₂ fixation by division synchronized cultures of Euglena gracilis strain Z were determined over the cycle. Rate of ¹⁴CO₂ fixation doubled in a continuous manner throughout the light phase followed by a slight reduction of photosynthetic capacity in the dark phase. Greater ¹⁴C incorporation into the nucleic acid-polysaccharide fraction occurred with mature cells. Products of ¹⁴CO₂ fixation varied markedly over the cycle: although with mature cells ¹⁴C-labeled sucrose was not detected, with dividing cells this was the main sugar labeled; in young cells ¹⁴C maltose was formed. Cells removed at end of dark phase accumulated ¹⁴C in glycolate, whereas at other stages over the cycle less ¹⁴C was present in glycolate, and this was accompanied by a rapid incorporation of ¹⁴C into glycine and serine. Glycerate was an early and major product of photosynthesis with cells at the mature stage of the cycle.     

Metabolism of amino acids in Ascaridia galli: decarboxylation reactions

Production of ¹⁴CO₂ from 12 carbon-labelled amino acids by Ascaridia galli was studied. Appreciable amounts of CO₂ were evolved from alanine, aspartate, glutamate, serine, leucine and valine by intestines, ovaries, cuticle and intact worms, in that order, but not from lysine, proline and tyrosine. Maximum CO₂ produced by whole worms was from serine, while with isolated organs it was from alanine. For cuticle, the decarboxylations of alanine, aspartate and glutamate were found to be associated with the mitochondrial fraction.     

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.     

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.     

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.     

Carbon dioxide fixation by detached cereal caryopses

Immature detached cereal caryopses from barley (Hordeum vulgare L. var distichum cv Midas) and wheat (Triticum aestivum L. cv Sicco) were shown to be capable of fixing externally supplied ¹⁴CO₂ in the light or dark. Green cross cells and the testa contained the majority of the ¹⁴C-labeled material. Some ¹⁴C-labeled material was also found in the outer, or transparent, layer and in the endosperm/embryo fraction. More ¹⁴C was recovered from caryopses when they were incubated in ¹⁴CO₂ without the transparent layer, thus suggesting that this layer is a barrier to the uptake of CO₂. In all cases, significant amounts of ¹⁴C-labeled material were found in caryopses after dark incubation with ¹⁴CO₂. Interestingly, CO₂ fixation in the chlorophyll-less mutant Albino lemma was significantly greater in the light than in the dark. The results indicate that intact caryopses have the ability to translocate ¹⁴C-labeled assimilate derived from external CO₂ to the endosperm/embryo. Carboxylating activity in the transparent layer appears to be confined to phosphoenolpyruvate carboxylase activity but that in the chloroplast-containing cross-cells may be accounted for by both ribulose-1,5-bisphosphate carboxylase-oxygenase and phosphoenolpyruvate carboxylase activity. Depending on a number of assumptions, the amount of CO₂ fixed is sufficient to account for about 2% of the weight of starch found in the mature caryopsis.     

Carbon Dioxide Fixation in the Carbon Economy of Developing Seeds of Lupinus albus (L.)

The effects of CO₂ concentration and illumination on net gas exchange and the pathway of ¹⁴CO₂ fixation in detached seeds from developing fruits of Lupinus albus (L.) have been studied.

Increasing the CO₂ concentration in the surrounding atmosphere (from 0.03 to 3.0% [v/v] in air) decreased CO₂ efflux by detached seeds either exposed to the light flux equivalent to that transmitted by the pod wall (500 to 600 micro-Einsteins per square meter per second) in full sunlight or held in darkness. Above 1% CO₂ detached seeds made a net gain of CO₂ in the light (up to 0.4 milligrams of CO₂ fixed per gram fresh weight per hour) but ¹⁴CO₂ injected into the gas space of intact fruits (containing around 1.5% CO₂ naturally) was fixed mainly by the pod and little by the seeds.

Throughout development seeds contained ribulose-1,5-bisphosphate carboxylase activity (EC 4.1.1.39), especially in the embryo (up to 99 micromoles of CO₂ fixed per gram fresh weight per hour) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) in both testa (up to 280 micromoles of CO₂ fixed per gram fresh weight per hour) and embryo (up to 355 micromoles of CO₂ fixed per gram fresh weight per hour).

In kinetic experiments the most significant early formed product of ¹⁴CO₂ fixation in both light and dark was malate but in the light phosphoglyceric acid and sugar phosphates were also rapidly labeled. ¹⁴CO₂ fixation in the light was linked to the synthesis of sugars and amino acids but in the dark labeled sugars were not formed.

     

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.