Alteration in cell permeability as a mechanism of action of certain quinone pesticides

The permeability of the Chlorella pyrenoidosa membrane was studied by following the efflux of ¹⁴C-intracellular material from cells which had been allowed to incorporate ¹⁴CO₂ photosynthetically. It was observed that the efflux increased upon treatment with low concentrations (3-30 μM) of 2, 3-dichloro-1, 4-naphthoquinone (dichlone), 2-amino-3-chloro-1, 4-naphthoquinone (06K-quinone), and 2, 3, 5, 6-tetrachloro-1, 4-benzoquinone (chloranil). Dichlone caused a greater loss of intracellular material than chloranil or 06K-quinone. The rate of loss as well as the total loss of ¹⁴C increased with an increase in the concentration of the quinones. In the dichlone-treated cells, the leakage was observed within 1 minute of the addition of the chemical and the effect on cell permeability was irreversible. Cells exposed to dichlone in the light or under anaerobic conditions released significantly greater amounts of ¹⁴C-material than cells treated in the dark or under aerobic conditions. The aqueous ethanol-soluble fraction of the cell was found to be the source of the released material. The proportion of the ethanol-soluble ¹⁴C that leaked out of the cell varied with the time of ¹⁴C-assimilation prior to treatment with dichlone. In the dichlone-treated cells, practically all the ¹⁴C-sucrose, alanine, glutamine, serine, and glycine leaked out, whereas glutamic, aspartic, succinic, and fumaric acids were lost only partially. Essentially no ¹⁴C-lipids were lost from the cells during dichlone treatment.     

Studies on Effect of Certain Quinones: I. Electron Transport, Photophosphorylation, and CO(2) Fixation in Isolated Chloroplasts

The effect of quinone herbicides and fungicides on photosynthetic reactions in isolated spinach (Spinacia oleracea) chloroplasts was investigated. 2,3-Dichloro-1,4-naphthoquinone (dichlone), 2-amino-3-chloro-1,4-naphthoquinone (06K-quinone), and 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil) inhibited ferricyanide reduction as well as ATP formation. Benzoquinone had little or no effect on these reactions. The two reactions showed a differential sensitivity to these inhibitors. Dichlone was a strong inhibitor of both photosystems I and II; photosystem I was more sensitive to 06K-quinone than was photosystem II, whereas the reverse was true of chloranil. Chloranil and 06K-quinone inhibited ferricyanide reduction and the coupled photophosphorylation to the same extent, whereas dichlone affected photophosphorylation to a greater extent than the ferricyanide reduction.     

Metabolism of Separated Leaf Cells: III. Effects of Calcium and Ammonium on Product Distribution During Photosynthesis with Cotton Cells

Separated mesophyll cells from cotton (Gossypium hirsutum var. Stoneville 1613 Glandless) were isolated with pectinase and mechanical agitation. The separated cells had rates of light-dependent CO₂ fixation between 50 to 100 μmoles CO₂ per mg chlorophyll per hour. The presence of Ca²⁺ in the incubation medium did not significantly affect the type of photosynthetic products formed, but 2 mm Ca²⁺ did cause a 50% decrease in the appearance of photosynthetic products in the incubation medium. The movement of all types of products (sugars, organic, and amino acids) out of the cells was reduced similarly by the Ca²⁺. Light had no affect on the movement of products out of the cells, whereas 1 mm ethylenediaminetetra-acetate greatly increased the movement. The addition of 1.6 mm NH₄Cl to the cell suspensions caused a large increase in the amount of fixed ¹⁴C appearing in the amino acid fraction and a decrease in the sugar fraction. These metabolic changes in the cells were reflected in the movement of products out of the cells so that the incubation medium also contained a larger amount of label in amino acids and a smaller amount in sucrose. Although the cell plasma membrane restricted the movement of soluble products, it did not discriminate significantly between the types of products moved.     

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.     

Oxygen inhibition of photosynthesis and stimulation of photorespiration in soybean leaf cells

The occurrence of photorespiration in soybean (Glycine max [L.] Merr.) leaf cells was demonstrated by the presence of an O₂-dependent CO₂ compensation concentration, a nonlinear time course for photosynthetic ¹⁴CO₂ uptake at low CO₂ and high O₂ concentrations, and an O₂ stimulation of glycine and serine synthesis which was reversed by high CO₂ concentration. The compensation concentration was a linear function of O₂ concentration and increased as temperature increased. At atmospheric CO₂ concentration, 21% O₂ inhibited photosynthesis at 25 C by 27%. Oxygen inhibition of photosynthesis was competitive with respect to CO₂ and increased with increasing temperature. The Km (CO₂) of photosynthesis was also temperature-dependent, increasing from 12 μm CO₂ at 15 C to 38 μm at 35 C. In contrast, the Ki (O₂) was similar at all temperatures. Oxygen inhibition of photosynthesis was independent of irradiance except at 10 mm bicarbonate and 100% O₂, where inhibition decreased with increasing irradiance up to the point of light saturation of photosynthesis. Concomitant with increasing O₂ inhibition of photosynthesis was an increased incorporation of carbon into glycine and serine, intermediates of the photorespiratory pathway, and a decreased incorporation into starch. The effects of CO₂ and O₂ concentration and temperature on soybean cell photosynthesis and photorespiration provide further evidence that these processes are regulated by the kinetic properties of ribulose-1,5-diphosphate carboxylase with respect to CO₂ and O₂.     

Characterization of an Electron Transport Pathway Associated with Glucose and Fructose Respiration in the Intact Chloroplasts of Chlamydomonas reinhardtii and Spinach

The role of an electron transport pathway associated with aerobic carbohydrate degradation in isolated, intact chloroplasts was evaluated. This was accomplished by monitoring the evolution of ¹⁴CO₂ from darkened spinach (Spinacia oleracea) and Chlamydomonas reinhardtii chloroplasts externally supplied with [¹⁴C]fructose and [¹⁴C]glucose, respectively, in the presence of nitrite, oxaloacetate, and conventional electron transport inhibitors. Addition of nitrite or oxaloacetate increased the release of ¹⁴CO₂, but it was shown that O₂ continued to function as a terminal electron acceptor. ¹⁴CO₂ evolution was inhibited up to 30 and 15% in Chlamydomonas and spinach, respectively, by 50 μm rotenone and by amytal, but at 500- to 1000-fold higher concentrations, indicating the involvement of a reduced nicotinamide adenine dinucleotide phosphate-plastoquinone oxidoreductase. ¹⁴CO₂ release from the spinach chloroplast was inhibited 80% by 25 μm 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. ¹⁴CO₂ release was sensitive to propylgallate, exhibiting approximately 50% inhibition in Chlamydomonas and in spinach chloroplasts of 100 and 250 μm concentrations, respectively. These concentrations were 20- to 50-fold lower than the concentrations of salicylhydroxamic acid (SHAM) required to produce an equivalent sensitivity. Antimycin A (100 μm) inhibited approximately 80 to 90% of ¹⁴CO₂ release from both types of chloroplast. At 75 μm, sodium azide inhibited ¹⁴CO₂ evolution about 50% in Chlamydomonas and 30% in spinach. Sodium azide (100 mm) combined with antimycin A (100 μm) inhibited ¹⁴CO₂ evolution more than 90%. ¹⁴CO₂ release was unaffected by uncouplers. These results are interpreted as evidence for a respiratory electron transport pathway functioning in the darkened, isolated chloroplast. Chloroplast respiration defined as ¹⁴CO₂ release from externally supplied [1-¹⁴C]glucose can account for at least 10% of the total respiratory capacity (endogenous release of CO₂) of the Chlamydomonas reinhardtii cell.     

Compartmental modelling of photorespiration and carbon metabolism of water stressed leaves

Abstract Carbon fluxes in photosynthesis and photorespiration of water stressed leaves have been analysed in a steady state model based on the ribulose diphosphate carboxylase (RuDP carboxylase) and RuDP oxygenase enzyme activities and the CO₂ and O₂ concentrations in the leaf. Agreement between predicted and observed photorespiration (Lawlor & Fock, 1975) and C flux in the glycollate pathway is good over much of the range of water stress, but not at severe stress. An alternative source of respiratory CO₂ is suggested to explain the discrepancy. The model suggests that resistance to CO₂ fixation is mainly in the carboxylation reactions, not in CO₂ transport. Using the steady state model, the kinetics of ¹⁴C incorporation into photosynthetic and photorespiratory intermediates are simulated. The predicted rate of ¹⁴C incorporation is faster than observed and delay terms in the model are used to simulate the slow rates of mixing and metabolic reactions. Inactive pools of glycine and serine are suggested to explain the observed specific activities of glycine and serine. Three models of carbon flux between the glycollate pathway, the photosynthetic carbon reduction cycle and sucrose synthesis are considered. The most satisfactory simulation is for glycollate pathway carbon feeding into the PCR cycle pool of 3-phosphoglyceric acid which provides the carbon for sucrose synthesis. Simulation of the specific activity of CO₂ released in photorespiration suggests that a source of unlabelled carbon may contribute to photorespiration.     

Photosynthetic characteristics of photoautotrophically grown tobacco callus cells

Haploid callus cells of tobacco (Nicotiana tabacum) were grown photoautotrophically on a solid agar medium in the absence of sucrose in Petri plates in an atmosphere of 1% or 3% CO₂ in air. The averages of dry weight increases for four to five consecutive passages were 2.3- to 3.6-fold per 3-week passage for different subclones. Photosynthetic ¹⁴CO₂ assimilation was maximum at about 1% CO₂ with half-maximal rates obtained at 0.2% CO₂. At saturating CO₂ concentration the average rate of CO₂ fixation was about 5 μmole per gram fresh weight per hour or about 125 μmole per mg of chlorophyll per hour.     

Glycolate pathway in green algae

By three criteria, the glycolate pathway of metabolism is present in unicellular green algae. Exogenous glycolate-1-¹⁴C was assimilated and metabolized to glycine-1-¹⁴C and serine-1-¹⁴C. During photosynthetic ¹⁴CO₂ fixation the distributions of ¹⁴C in glycolate and glycine were similar enough to suggest a product-precursor relationship. Five enzymes associated with the glycolate pathway were present in algae grown on air. These were P-glycolate phosphatase, glycolate dehydrogenase (glycolate:dichloroindophenol oxidoreductase), l-glutamate:glyoxylate aminotransferase, serine hydroxymethylase, and glycerate dehydrogenase. Properties of glycerate dehydrogenase and the aminotransferase were similar to those from leaf peroxisomes. The specific activity of glycolate dehydrogenase and serine hydroxymethylase in algae was 1/5 to 1/10 that of the other enzymes, and both these enzymes appear ratelimiting for the glycolate pathway.     

Quinone-enhanced Ascorbate Reduction of Nitric Oxide: Role of Quinone Redox Potential

The quinones 1,4-naphthoquinone (NQ), methyl-1,4-naphthoquinone (MNQ), trimethyl-1,4-benzoquinone (TMQ) and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ-0) enhance the rate of nitric oxide (NO) reduction by ascorbate in nitrogen-saturated phosphate buffer (pH 7.4). The observed rate constants for this reaction were determined to be 16±2,215±6,290±14 and 462±18?M^-1?s^-1, for MNQ, TMQ, NQ and UQ-0, respectively. These rate constants increase with an increase in quinone one-electron redox potential at neutral pH, E₇₁. Since NO production is enhanced under hypoxia and under certain pathological conditions, the observations obtained in this work are very relevant to such conditions.     

Inhibition of photorespiration and increase of net photosynthesis in isolated maize bundle sheath cells treated with glutamate or aspartate

Net photosynthetic ¹⁴CO₂ fixation by isolated maize (Zea mays) bundle sheath strands was stimulated 20 to 35% by the inclusion of l-glutamate or l-aspartate in the reaction mixture. Maximal stimulation occurred at a 7.5 mm concentration of either amino acid. Since the photosynthetic rate and the glutamate-dependent stimulation in the rate were equally sensitive to a photosynthetic electron transport inhibitor, 3-(p-chlorophenyl)-1,1-dimethylurea, it was concluded that glutamate did not stimulate CO₂ fixation by supplying needed NADPH (NADH) through glutamate dehydrogenase. Treatment of the bundle sheath strands with glutamate inhibited glycolate synthesis by 59%. Photorespiration in this tissue, measured as the O₂ inhibition of CO₂ fixation (the Warburg effect), was inhibited by treatment with glutamate. The stimulation in net photosynthetic CO₂ fixation probably results from the decrease in photorespiratory CO₂ loss. This metabolic regulation of the rate of glycolate synthesis and photorespiration observed with isolated bundle sheath strands could account for the inability to detect rapid photorespiration in the mature intact maize leaf.     

Effects of carbon dioxide and oxygen on the regulation of photosynthetic carbon metabolism by ammonia in spinach mesophyll cells

Photosynthetic carbon metabolism of isolated spinach mesophyll cells was characterized under conditions favoring photorespiratory (PR; 0.04% CO₂ and 20% O₂) and nonphotorespiratory (NPR; 0.2% CO₂ and 2% O₂) metabolism, as well as intermediate conditions. Comparisons were made between the metabolic effects of extracellularly supplied NH₄⁺ and intracellular NH₄⁺, produced primarily via PR metabolism. The metabolic effects of ¹⁴CO₂ fixation under PR conditions were similar to perturbations of photosynthetic metabolism brought about by externally supplied NH₄⁺; both increased labeling and intracellular concentrations of glutamine at the expense of glutamate and increased anaplerotic synthesis through α-ketoglutarate. The metabolic effects of added NH₄⁺ during NPR fixation were greater than those during PR fixation, presumably due to lower initial NH₄⁺ levels during NPR fixation. During PR fixation, addition of ammonia caused decreased pools and labeling of glutamate and serine and increased glycolate, glyoxylate, and glycine labeling. The glycolate pathway was thus affected by increased rates of carbon flow and decreased glutamate availability for glyoxylate transamination, resulting in increased usage of serine for transamination. Sucrose labeling decreased with NH₄⁺ addition only during PR fixation, suggesting that higher photosynthetic rates under NPR conditions can accommodate the increased drain of carbon toward amino acid synthesis while maintaining sucrose synthesis.     

Anaerobic Formation of d-Lactate and Partial Purification and Characterization of a Pyruvate Reductase from Chlamydomonas reinhardtii

d-Lactate accumulation in Chlamydomonas reinhardtii was dependent on anaerobic conditions. As much as 50% of the ¹⁴C after 2 minutes of photosynthetic ¹⁴CO₂ fixation moved into d-lactate from sugar phosphates if the cells became anaerobic for short time periods. No lactate accumulated in the dark until the O₂ concentration decreased to less than 0.1%. Lactate was determined to be of the d-configuration using stereospecific lactate dehydrogenases. d-Lactate produced anaerobically by algae grown on 5% CO₂ was only slowly metabolized aerobically in the light or dark, and in the dark, only a trace of the lactate was excreted.     

Photosynthesis in Tall Fescue : IV. Carbon Assimilation Pattern in two Genotypes of Tall Fescue Differing in Net Photosynthesis Rates

We previously reported that the net photosynthetic rate of a decaploid genotype (I-16-2) of tall fescue (Festuca arundinacea Schreb.) was 32 to 41 versus 22 milligrams CO₂ per square decimeter per hour in a hexaploid genotype (V6-802) (Randall, Nelson, Asay Plant Physiol 59: 38-41). The high rate was later correlated with increases in total ribulose 1,5-bisphosphate carboxylase protein (17%) and activity (27%) (Joseph, Randall, Nelson Plant Physiol 68: 894-898). This report characterizes photosynthesis with respect to light saturation and early products of photosynthesis in an attempt to identify regulatory metabolic site(s) in these two genotypes. Analysis of the early products of photosynthesis indicated that both genotypes fixed CO₂ via the Calvin-Benson cycle with phosphoglyceric acid as the initial primary product. Both genotypes had similar ¹⁴C-labeled intermediates. Sucrose was the primary sink of ¹⁴CO₂ assimilation. After 10 min of ¹⁴CO₂ assimilation with attached leaves, sucrose accounted for 89% (decaploid) and 81% (hexaploid) of the total ¹⁴C incorporated. In 10 min, this amounted to 1.3 (decaploid) and 0.8 (hexaploid) μmol [¹⁴C]sucrose formed g fresh weight⁻¹ and reflected the observed differences in photosynthetic rates. There was limited labeling of starch (1%) and fructan (1%). Results of total nonstructural carbohydrates and P_i analysis also demonstrated sucrose was the predominant carbohydrate in fescue leaves. Quantitative differences in sucrose and P_i between the two genotypes may reflect changes in partitioning and this possibility is discussed.     

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.     

Studies on brain-cortex slices: Differences in the oxidation of 14C-labelled glucose and pyruvate revealed by the action of triethyltin and other toxic agents

1. The rate of appearance of ¹⁴CO₂ from [6-¹⁴C]glucose and [3-¹⁴C]pyruvate was measured. Pyruvate is oxidized to carbon dioxide twice as fast as glucose, although the oxygen uptake is almost the same with each substrate. 2. The presence of 30μm-2,4-dinitrophenol increases the output of ¹⁴CO₂ from [6-¹⁴C]glucose sixfold whereas the oxygen uptake is not quite doubled. Similar results are obtained with 0·1m-potassium chloride. The stimulating action of these two agents on the output of ¹⁴CO₂ from [3-¹⁴C]pyruvate is much less than on that from [6-¹⁴C]glucose. 3. The effects of oligomycin, ouabain and triethyltin on the respiration of control and stimulated brain-cortex slices were studied. Triethyltin (1·3μm) inhibited the oxidation of [6-¹⁴C]glucose more than 70%, but did not inhibit the oxidation of[3-¹⁴C]pyruvate. [3-¹⁴C]pyruvate. 4. The production of lactic acid by brain-cortex slices incubated with glucose is twice as great as that with pyruvate. Lactic acid increases two and a half times in the presence of either triethyltin or oligomycin when the substrate is glucose, but is no different from the control when the substrate is pyruvate. 5. With kidney slices the production of lactic acid from glucose is very low. It is increased by oligomycin but not by triethyltin. 6. The results are discussed in terms of the oxidation of the extramitochondrial NADH₂ produced during glycolysis.     

Photosynthetic Capacities and Growth Characteristics of Nicotiana tabacum (cv. Xanthi) Cell Suspension Cultures

Photoheterotrophic growth of cell suspensions of Nicotiana tabacum L. (cv. Xanthi) in organic culture medium enriched in sucrose (30 g per liter) showed a classical sigmoid growth curve. The cells developed functional chloroplast structures during the exponential growth phase, when their chlorophyll content increased steadily. A limited drop (30%) in the chlorophyll amount and structural changes of the plastids (starch accumulation) were observed during the lag phase. The measurements of photosynthetic capacities (O₂ evolution and CO₂ fixation) during the growth cycle revealed changes in the photosynthetic ratio (O₂/CO₂), which was near 1 during the lag and stationary phases and near 2 during exponential growth. During exponential growth there was also a rapid NO₃^? uptake. Analysis of label distribution among the products of ¹⁴CO₂ fixation showed that both CO₂ assimilation pathways, linked to the ribulose-biphosphate carboxylase (the autotrophic pathway) and to phosphoenolpyruvate carboxylase (the non-autotrophic pathway) were operative with an important increase of the capacity of the latter during the exponential growth phase. Maximum rate of oxygen evolution, either endogenous or with p-benzoquinone as Hill reagent, as well as the increased CO₂ Fixation capacity via the non-autotrophic pathway during the exponential phase were concomitant with a high cyanide inhibited O₂ uptake.     

Glyoxylate decarboxylation during photorespiration

At 25° C under aerobic conditions with or without gluamate 10% of the [1-¹⁴C]glycollate oxidised in spinach leaf peroxisomes was released as ¹⁴CO₂. Without glutamate only 5% of the glycollate was converted to glycine, but with it over 80% of the glycollate was metabolised to glycine. CO₂ release was probably not due to glycine breakdown in these preparations since glycine decarboxylase activity was not detected. Addition of either unlabelled glycine or isonicotinyl hydrazide (INH) did not reduce ¹⁴CO₂ release from either [1-¹⁴C]glycollate or [1-¹⁴C]glyoxylate. Furthermore, the amount of available H₂O₂ (Grodzinski and Butt, 1976) was sufficient to account for all of the CO₂ release by breakdown of glyoxylate. Peroxisomal glycollate metabolism was unaffected by light and isolated leaf chloroplasts alone did not metabolise glycollate. However, in a mixture of peroxisomes and illuminated chloroplasts the rate of glycollate decarboxylation increased three fold while glycine synthesis was reduced by 40%. Although it was not possible to measure available H₂O₂ directly, the data are best explained by glyoxylate decarboxylation. Catalase reduced CO₂ release and enhanced glycine synthesis. In addition, when a model system in which an active preparation of purified glucose oxidase generating H₂O₂ at a known rate was used to replace the chloroplasts, similar rates of ¹⁴CO₂ release and [¹⁴C]glycine synthesis from [1-¹⁴C]glycollate were measured. It is argued that in vivo glyoxylate metabolism in leaf peroxisomes is a key branch point of the glycollate pathway and that a portion of the photorespired CO₂ arises during glyoxylate decarboxylation under the action of H₂O₂. The possibility that peroxisomal catalase exerts a peroxidative function during this process is discussed.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - INH isonicotinylhydrazide - PHMS pyridyl-2-yl--hydroxymethane sulphonic acid     

The influence of sucrose and an elevated CO2 concentration on photosynthesis of photoautotrophic peanut (Arachis hypogaea L.) cell cultures

Using photoautotrophic cells ofArachis hypogaea (L.) growing at ambient CO₂, it was shown that exogenous sucrose supplied to the liquid medium reduced¹⁴CO₂ fixation (supplied as NaH¹⁴CO₃). This was mostly due to a reduced labelling in P-esters, and to a lesser extent, in the serine/glycine moiety. However, radioactivity in the neutral sugar fraction was increased upon supplement of exogenous sucrose. The reduced labelling of P-esters and serine/glycine agrees with a lower concentration and specific activity of Rubisco in the sucrose supplied treatments as compared to the control. Following a transfer into a sugar free nutrient medium the concentration and activity of Rubisco is increased. The concentration of PEPCase was not influenced by sucrose application, although its specific activity was increased.At elevated CO₂ concentration (2.34% v/v) the Rubisco concentration and specific activity was at the same level as in the control (0.03% v/v CO₂). However, the concentration and the specific activity of PEPCase was increased and dry weight increase was about 8–9-fold higher than at ambient CO₂.     

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.