Impairment of Photorespiratory Carbon Flow into Rubber by the Inhibition of the Glycolate Pathway in Guayule (Parthenium argentatum Gray)

Cut shoots of guayule (Parthenium argentatum Gray) were treated with four inhibitors of the glycolate pathway (α-hydroxypyridinemethanesulfonic acid; isonicotinic acid hydrazide, glycine hydroxamate, and amino-oxyacetate, AOA) in order to evaluate the role of photorespiratory intermediates in providing precursors for the biosynthesis of rubber. Photorespiratory CO₂ evolution in guayule leaves was severely inhibited by AOA. Application of each of the four inhibitors has resulted in a significantly decreased incorporation of ¹⁴C into rubber fractions suggesting that the glycolate pathway is involved in the biosynthesis of rubber in guayule. However, the application of each of the glycolate pathway inhibitors showed no significant effect on photosynthetic CO₂ fixation in the leaves. The inhibitors individually also reduced the incorporation of labeled glycolate, glyoxylate, and glycine into rubber, while the incorporation of serine and pyruvate was not affected. The effective inhibition of incorporation of glycolate pathway intermediates in the presence of AOA was due to an inhibition of glycine decarboxylase and serine hydroxymethyltransferase. It is concluded that serine is a putative photorespiratory intermediate in the biosynthesis of rubber via pyruvate and acetyl coenzyme A.     

Markedly low requirement of added CO2 for photosynthesis by mesophyll protoplasts of pea (Pisum sativum): possible roles of photorespiratory CO2 and carbonic anhydrase

Mesophyll protoplasts of pea required only 74.1 μM CO₂ for maximal photosynthesis, unlike chloroplasts, which required up to 588 μM CO₂. Such a markedly low requirement for CO₂ could be because of an internal carbon source and/or a CO₂ concentrating mechanism in mesophyll protoplasts. Ethoxyzolamide (EZA), an inhibitor of internal carbonic anhydrase (CA) suppressed photosynthesis by mesophyll protoplasts at low CO₂ (7.41 μM) but had no significant effect at high CO₂ (741 μM). However, acetazolamide, another inhibitor of CA, did not exert as much dramatic effect as EZA. Three photorespiratory inhibitors, aminoacetonitrile or glycine hydroxamate (GHA) or aminooxyacetate inhibited markedly photosynthesis at low CO₂ but not at high CO₂. Inhibitors of glycolysis or tricarboxylic acid cycle (NaF, sodium malonate) or phosphoenolpyruvate carboxylase (3,3‐dichloro‐2‐dihydroxy phosphinoyl‐methyl‐2‐propenoate) had no significant effect on photosynthesis. The CO₂ requirement of protoplast photosynthesis and the sensitivity of photosynthesis to EZA were much higher at low oxygen (65 nmol ml^?1) than that at normal oxygen (212 nmol ml^?1). In contrast, the inhibitory effect of photorespiratory inhibitors on protoplast photosynthesis was similar in both normal and low oxygen medium. The marked elevation of glycine/serine ratio at low O₂ or in presence of GHA confirmed the suppression of photorespiratory decarboxylation by GHA. While demonstrating interesting difference between the response of protoplasts and chloroplasts to CO₂, we suggest that photorespiration could be a significant source of CO₂ for photosynthesis in mesophyll protoplasts at limiting CO₂ and at atmospheric levels of oxygen. Obviously, carbonic anhydrase is essential to concentrate or retain CO₂ in mesophyll cells.     

Light-enhanced dark respiration in leaves and mesophyll protoplasts of pea in relation to photorespiration,respiration and some metabolites content

The respiration rate of leaves and mesophyll protoplasts of pea (Pisum sativum L.), from plants which were previously kept in darkness for 24 h was doubled following a period of photosynthesis at ambient level of O₂ (21 %), whereas the low level of O₂ (1 % and 4 % for leaves and protoplasts, respectively) reduced this light-enhanced dark respiration (LEDR) to the rate as noted before the illumination. Similarly to respiration rate, the oxygen at used concentrations had no effect on the ATP/ADP ratio in the dark-treated leaves. However, the ATP/ADP ratio in leaves photosynthesizing at 21 % O₂ was higher (up to 40 %, dependence on CO₂ concentration in the range 40–1600 1 dm⁻³) than in those photosynthesizing at 1 % O₂ or darkened at air (21 % O₂). Also, at 1 % O₂ the accumulation of malate was suppressed (by about 40 %), to a value noted for leaves darkened at 21 % O₂. The dark-treatment of leaves reduced the ability of isolated mitochondria to oxidize glycine (by about twofold) and succinate, but not malate. Mitochondria from both the light- and dark-treated leaves did not differ in qualitative composition of free amino acids, however, there were significant quantitative differences especially with respect to aspartate, alanine, glutamate and major intermediates of the photorespiratory pathway (glycine, serine). Our results suggest that accumulation of photorespiratory and respiratory metabolites in pea leaves during photosynthesis at 1 % O₂ is reduced, hence the suppression of postillumination respiration rate.     

High-to-Low CO(2) Acclimation Reveals Plasticity of the Photorespiratory Pathway and Indicates Regulatory Links to Cellular Metabolism of Arabidopsis

Background

Photorespiratory carbon metabolism was long considered as an essentially closed and nonregulated pathway with little interaction to other metabolic routes except nitrogen metabolism and respiration. Most mutants of this pathway cannot survive in ambient air and require CO₂-enriched air for normal growth. Several studies indicate that this CO₂ requirement is very different for individual mutants, suggesting a higher plasticity and more interaction of photorespiratory metabolism as generally thought. To understand this better, we examined a variety of high- and low-level parameters at 1% CO₂ and their alteration during acclimation of wild-type plants and selected photorespiratory mutants to ambient air.

Methodology and Principal Findings

The wild type and four photorespiratory mutants of Arabidopsis thaliana (Arabidopsis) were grown to a defined stadium at 1% CO₂ and then transferred to normal air (0.038% CO₂). All other conditions remained unchanged. This approach allowed unbiased side-by-side monitoring of acclimation processes on several levels. For all lines, diel (24 h) leaf growth, photosynthetic gas exchange, and PSII fluorescence were monitored. Metabolite profiling was performed for the wild type and two mutants. During acclimation, considerable variation between the individual genotypes was detected in many of the examined parameters, which correlated with the position of the impaired reaction in the photorespiratory pathway.

Conclusions

Photorespiratory carbon metabolism does not operate as a fully closed pathway. Acclimation from high to low CO₂ was typically steady and consistent for a number of features over several days, but we also found unexpected short-term events, such as an intermittent very massive rise of glycine levels after transition of one particular mutant to ambient air. We conclude that photorespiration is possibly exposed to redox regulation beyond known substrate-level effects. Additionally, our data support the view that 2-phosphoglycolate could be a key regulator of photosynthetic-photorespiratory metabolism as a whole.     

Response of photosynthetic carbon assimilation in mesophyll protoplasts to restriction on mitochondrial oxidative metabolism: Metabolites related to the redox status and sucrose biosynthesis

The patterns of cellular metabolites related to redox status and sucrose biosynthesis in mesophyll protoplasts of pea (Pisum sativum L.) were examined in the absence or presence of oligomycin (inhibitor of oxidative phosphorylation) or antimycin A (inhibitor of cytochrome pathway) or salicylhydroxamic acid (SHAM) (inhibitor of alternative pathway). The increase on illumination in the rate of photosynthesis or cellular metabolites was more at optimal CO₂ (1.0 mM NaHCO₃) compared to that at limiting CO₂ (0.1 mM NaHCO₃). Furthermore, the inhibition of photosynthesis in presence of mitochondrial inhibitors was more pronounced at optimal CO₂ than that at limiting CO₂. There was a marked increase in steady-state levels of triose-P/PGA (phosphoglyceric acid) and glucose-6-phosphate (Glc-6-P) in the presence of oligomycin and antimycin A. In contrast, SHAM caused a marked increase in malate/OAA (oxaloacetate). We suggest that dissipation of excess redox equivalents generated in photosynthesis occurs through both cytochrome and alternative pathways, while sucrose biosynthesis is backed up by cytochrome pathway alone. Thus, mitochondrial respiration (through both cytochrome and alternative pathways of mitochondrial electron transport) optimizes chloroplast photosynthesis by modulating cellular metabolites related to both intracellular redox state and sucrose biosynthesis.     

光照下菜豆叶片抗氰呼吸与光合作用关系的分析北大核心CSCD

为了进一步了解光照下植物呼吸作用的内在机理以及呼吸作用和光合作用的关系,该文研究了在光照下菜豆(Phaseolus vulgaris)叶片抗氰呼吸与光合作用的关系。研究发现,将黑暗下生长的菜豆幼苗叶片转到光照下10 h,总呼吸、抗氰呼吸以及抗氰呼吸在总呼吸中的比例均逐步上升;光照也导致了叶片叶绿体光合放氧和CO2固定的出现及其速率的增加,但光合放氧和CO2固定速率的增加均滞后于抗氰呼吸的增加。将黑暗下生长的叶片转到光照下之前用抗氰呼吸的抑制剂水杨基氧肟酸(SHAM)处理叶片,发现用SHAM处理并没有导致叶片在光照下光合放氧和CO2固定速率的明显变化,这也提示了黑暗下生长的叶片转至光照的过程中,抗氰呼吸和光合作用没有产生偶联。进一步研究发现,在黑暗中对叶片施加短时间的光照能够增加抗氰呼吸在总呼吸中的比例,但短时间的光照对叶片光合CO2固定速率没有影响。这些结果表明了光照对抗氰呼吸的诱导可以不依赖于光合作用,光照可能是作为一种直接的信号去诱导抗氰呼吸。     

Reactivity of glyoxylate with hydrogen perioxide and simulation of the glycolate pathway of C3 plants and Euglena

The nonenzymatic reaction of glyoxylate and H₂O₂ was measured under physiological conditions of the pH and concentrations of reactants. The reaction of glyoxylate and H₂O₂ was secondorder, with a rate constant of 2.27 l mol^-1 s^-1 at pH 8.0 and 25° C. The rate constant increased by 4.4 times in the presence of Zn²⁺ and doubled at 35°C. We propose a mechanism for the reaction between glyoxylate and H₂O₂. From a comparison of the rates of H₂O₂ decomposition by catalase and the reaction with glyoxylate, we conclude that H₂O₂ produced during glycolate oxidation in peroxisomes is decomposed by catalase but not by the reaction with glyoxylate, and that photorespiratory CO₂ originates from glycine, but not from glyoxylate, in C₃ plants. Simulation using the above rate constant and reported kinetic parameters leads to the same conclusion, and also makes it clear that alanine is a satisfactory amino donor in the conversion of glyoxylate to glycine. Some serine might be decomposed to give glycine and methylene-tetrahydrofolate; the latter is ultimately oxidized to CO₂. In the simulation of the glycolate pathway of Euglena, the rate constant was high enough to ensure the decarboxylation of glyoxylate by H₂O₂ to produce photorespiratory CO₂ during the glycolate metabolism of this organism.Abbreviations Chl chlorophyll - GGT glutamate: glyoxylate aminotransferase (EC 2.6.1.4) - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - SGT serine: glyoxylate aminotransferase (EC 2.6.1.45) This is the ninth in a series on the metabolism of glycolate in Euglena gracilis. The eighth is Yokota et al. (1982)     

Ethoxyzolamide repression of the low photorespiration state in two submersed angiosperms

Net photosynthesis in the submersed angiosperms Myriophyllum spicatum L. and Hydrilla verticillata (L.f.) Royal was inhibited by 21% O₂, but the degree of inhibition was greater for plants in the high than in the low photorespiratory state. Increasing the CO₂ concentration from 50 through 2,500 l l^-1 decreased the O₂ inhibition of the high-photorespiration plants in a competitive manner, but it had no effect on the O₂ inhibition of plants in the low photorespiratory state. Carbonic-anhydrase activity increased by almost threefold with the induction of the low photorespiratory state. Ethoxyzolamide, an inhibitor of carbonic anhydrase, reduced the net photosynthesis of low-photorespiration Myriophyllum and Hydrilla plants by 40%, but their dark respiration was unaffected. This ethoxyzolamide inhibition of net photosynthesis exhibited a competitive response to CO₂ concentration, resulting in a decrease in the apparent affinity of photosynthesis for CO₂. The net photosynthesis of plants in the high photorespiratory state was inhibited only slightly by ethoxyzolamide, and this inhibition was independent of the CO₂ level. Ethoxyzolamide treatment caused an increase in the O₂ inhibition of net photosynthesis of plants in the low photorespiratory state. Ethoxyzolamide increased the low CO₂ compensation points of low-photorespiration Myriophyllum and Hydrilla, but the values for the high-photorespiration plants were unchanged. In comparison, the CO₂ compensation points of the terrestrial plants Sorghum bicolor (C₄), Moricandia arvensis (C₃-C₄ intermediate) and Nicotiana tabacum (C₃) were unaltered by ethoxyzolamide treatment. These data indicate that the low photorespiratory state in Myriophyllum and Hydrilla is repressed by ethoxyzolamide treatment, thus implicating carbonic anhydrase as a component of the photorespiration-reducing mechanism in these plants. The competitive interaction of CO₂ with ethoxyzolamide provides evidence that the low photorespiratory state in submersed angiosperms is the result of some type or types of CO₂ concentrating mechanism. In Myriophyllum it may be via bicarbonate utilization, but in Hydrilla it probably takes the form of an inducible C₄-type system.Abbreviations PEP phosphoenolpyruvate - RuBP ribulose bisphosphate     

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.     

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.     

Photorespiratory rates in wheat and maize as determined by o-labeling

A method was devised to quantify short-term photorespiratory rates in terrestrial plants using ¹⁸O-intermediates of the glycolate pathway, specifically glycolate, glycine, and serine. The pathway intermediates were isolated and analyzed on a GC/MS to determine molecular percent ¹⁸O-enrichment. Rates of glycolate synthesis were determined from ¹⁸O-labeling kinetics of the intermediates, derived rate equations, and nonlinear regression techniques. Glycolate synthesis in wheat (Triticum aestivum L.), a C₃ plant, and maize (Zea mays L.), a C₄ plant, was stimulated by high O₂ concentrations and inhibited by high CO₂ concentrations. The synthesis rates were 7.3, 2.1, and 0.7 micromoles per square decimeter per minute under a 21% O₂ and 0.035% CO₂ atmosphere for leaf tissue of wheat, maize seedlings, and 3-month-old maize, respectively. Photorespiratory CO₂ evolution rates were estimated to be 27, 6, and 2%, respectively, of net photosynthesis for the three groups of plants under the above atmosphere. The results from maize tissue support the hypothesis that C₄ plants photorespire, albeit at a reduced rate in comparison to C₃ plants, and that the CO₂/O₂ ratio in the bundle sheath of maize is higher in mature tissue than in seedling tissue. The pool size of the three photorespiratory intermediates remained constant and were unaffected by changes in either CO₂ or O₂ concentrations throughout the 10-minute labeling period. This suggests that photorespiratory metabolism is regulated by other mechanism besides phosphoglycolate synthesis by ribulose-1,5-bisphosphate carboxylase/oxygenase, at least under short-term conditions. Other mechanisms could be alternate modes of synthesis of the intermediates, regulation of some of the enzymes of the photorespiratory pathway, or regulation of carbon flow between organelles involved in photorespiration. The glycolate pool became nearly 100% ¹⁸O-labeled under an atmosphere of 40% O₂. This pool failed to become 100% ¹⁸O-enriched under lower O₂ concentrations.     

Evidence for 18O labeling of photorespiratory CO2 in photoautotrophic cell cultures of higher plants illuminated in the presence of 18O2

The ¹⁸O-enrichment of CO₂ produced in the light or during the post-illumination burst was measured by mass spectrometry when a photoautotrophic cell suspension of Euphorbia characias L. was placed in photorespiratory conditions in the presence of molecular ¹⁸O₂. The only ¹⁸O-labeled species produced was C¹⁸O¹⁶O; no C¹⁸O¹⁸O could be detected. Production of C¹⁸O¹⁶O ceased after addition of two inhibitors of the photosynthetic carbon-oxidation cycle, aminooxyacetate or aminoacetonitrile, and was inhibited by high levels of CO₂. The average enrichment during the post-illumination burst was estimated to be 46 ± 15% of the enrichment of the O₂ present during the preceding light period. Addition of exogenous carbonic anhydrase, by catalyzing the exchange between CO₂ and H₂O, drastically diminished the ¹⁸O-enrichment of the produced CO₂. The very low carbonio-anhydrase level of the photoautotrophic cell suspension probably explains why the ¹⁸O labeling of photorespiratory CO₂ could be observed for the first time. These data allow the establishment of a direct link between O₂ consumption and CO₂ production in the light, and the conclusion that CO₂ produced in the light results, at least partially, from the mitochondrial decarboxylation of the glycine pool synthesized through the photosynthetic carbon-oxidation cycle. Analysis of the C¹⁸O¹⁶O and CO₂ kinetics provides a direct and reliable way to assess in vivo the real contribution of photorespiratory metabolism to CO₂ production in the light.     

Evaluation of the light/dark C assay of photorespiration: tobacco leaf disk studies with glycidate and glyoxylate

Preincubation of illuminated tobacco (Nicotiana tabacum L.) leaf disks in glycidate (2,3-epoxypropionate) or glyoxylate inhibited photorespiration by about 40% as determined by the ratio of ¹⁴CO₂ evolved into CO₂-free air in light and in darkness. However, under identical preincubation conditions used for the light/dark ¹⁴C assays, the compounds failed to reduce photorespiration or stimulate net photosynthesis in tobacco leaf disks based on other CO₂ exchange parameters, including the CO₂ compensation concentration in 21% O₂, the inhibitory effect of 21% O₂ on net photosynthesis in 360 microliters per liter of CO₂ and the rate of net photosynthetic ¹⁴CO₂ uptake in air.

The effects of both glycidate and glyoxylate on the ¹⁴C assay are inconsistent with other measures of photorespiratory CO₂ exchange in tobacco leaf disks, and thus these data question the validity of the light to dark ratio of ¹⁴CO₂ efflux as an assay for relative rates of photorespiration (Zelitch 1968, Plant Physiol 43: 1829-1837). The results of this study specifically indicate that neither glycidate nor glyoxylate reduces photorespiration or stimulates net photosynthesis by tobacco leaf disks under physiological conditions of pO₂ and pCO₂, contrary to previous reports.

     

Sulfide-Resistant Respiration in Leaves of Elodea canadensis Michx: Comparison with Cyanide-Resistant Respiration

The rate of dark O₂ uptake of Elodea canadensis leaves was titrated with either cyanide or sulfide in the presence and in the absence of 5 millimolar salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase. The inhibition of O₂ uptake by SHAM alone was very small (3-6%), suggesting that actual respiration mainly occurred through the cytochrome pathway. O₂ uptake was slightly stimulated by cyanide at concentrations of 50 micromolar or higher, but in the presence of SHAM respiration was strongly suppressed. The effects of sulfide on O₂ uptake were similar to those of cyanide, except that the percent stimulation of O₂ uptake by sulfide alone was somewhat higher than that of cyanide. However, the estimates of the capacity of the alternative pathway were similar with both inhibitors. Another difference is that maximal inhibition of respiration in the presence of SHAM was observed with lower concentrations of sulfide (50 micromolar) than cyanide (250 micromolar). The results suggest that sulfide can be used as a suitable inhibitor of cytochrome c oxidase in studies with intact plant tissues, and that sulfide does not apparently inhibit the alternative oxidase.     

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.     

Carbon nutrition of seaweeds: Photosynthesis,photorespiration and respiration

Techniques are described for measuring gas exchange in seaweeds held in moist air (air suspension). In the species we have examined, oxygen has little or no effect on photosynthesis except at very low (50 μ1·1^?1) CO₂ concentration. Photorespiration could not be detected unless the seaweeds were treated simultaneously with high O₂ and low CO₂ or with the carbonic anhydrase inhibitor, diamox. However, sporulating and meristematic tissues exhibit oxygen-insensitive light respiration (CO₂production in light not associated with photorespiratory metabolism). Elevated pH in the surface water of seaweeds also caused light respiration. Oxygen-sensitive wound respiration was observed that could easily be mistaken for photorespiration. C₄ photosynthesis could not be detected. On the basis of several experimental approaches it was concluded that these seaweeds normally absorb bicarbonate rather than CO₂ from sea water. High CO₂ concentrations are required in gas streams aerating seaweed cultures in air or water suspension to maintain the bicarbonate concentration at levels normally found in sea water and to support normal levels of photosynthesis.     

Evidence for a significant contribution by peroxidase-mediated O2 uptake to root respiration of Brachypodium pinnatum

This study describes the O₂ uptake characteristics of intact roots of Brachypodium pinnatum. In the presence of 25 mM salicylhydroxamic acid (SHAM), concentrations of KCN below 3.5 νM had no effect on the rate of root respiration, whereas in the absence of 25 mM SHAM a significant inhibition of approx. 18% was observed. This indicates that an O₂-consuming reaction, not associated with the cytochrome pathway, the alternative pathway or the “residual component”, operates in the absence of any inhibitors in roots of B. pinnatum. We demonstrate here that this fourth O₂-consuming reaction is mediated by a peroxidase. A peroxidase which catalyzed O₂ reduction in the presence of NADH was readily washed from the roots of B. pinnatum. This peroxidase was stimulated by 5 mM SHAM, whereas ascorbic acid, catalase, catechol, gentisic acid, low concentrations potassium cyanide (3.5 μM), sodium azide, sodium sulfide, superoxide dismutase and high concentrations SHAM (25 mM) inhibited this reaction. Except for high concentrations of SHAM and concentrations of KCN higher than approx. 3.5 μM, these effectors could not be used to inhibit the peroxidase-mediated O₂ uptake in intact roots of B. pinnatum. Concentrations of SHAM below 10 mM stimulated O₂ uptake up to 15% of the control rate, depending on concentration, whereas 25 mM SHAM inhibited O₂ uptake by 35%. The stimulation at low concentrations resulted from a SHAM-stimulated peroxidase activity, whereas 25 mM SHAM completely inhibited both the peroxidase-mediated O₂ uptake and the activity of the alternative pathway. A method is presented for determining the relative contributions of each of the four O₂-consuming reactions, i.e. the cytochrome pathway, the alternative pathway, the “residual component” and the peroxidase-mediated O₂ uptake. The peroxidase-mediated O₂ uptake contributed 21% to the total rate of oxygen uptake in roots of B. pinnatum, the cytochrome pathway contributed 41%, the alternative pathway 14% and the “residual component” 24%.     

The effects of oligomycin on content of adenylates in mesophyll protoplasts,chloroplasts and mitochondria from Pb<Superscript>2+</Superscript> treated pea and barley leaves

The effects of oligomycin on photosynthesis and respiration in relation to ATP production in chloroplasts and mitochondria were investigated in protoplasts isolated from the detached pea (Pisum sativum L cv. Iłowiecki.) and barley (Hordeum vulgare L. cv. Gunilla) leaves treated 5 mM Pb(NO₃)₂. The oligomycin (OM), an inhibitor of oxidative phosphorylation at 0.1 μM concentration caused the inhibition of photosynthesis rate in the protoplasts from both the control and the Pb-treated pea leaves. The respiration rate and ATP/ADP ratio in the protoplasts and the activity of ATPase in mitochondria, were also diminished in the control protoplasts. These effects were not observed in the protoplasts and mitochondria isolated from the Pb-treated leaves. Oligomycin, an inhibitor of photophosphorylation at 10 μM concentration decreased ATPase activity in chloroplasts from both the control and the Pb- treated leaves. Using the method of rapid fractionation of barley protoplasts it was shown that the ATP/ADP ratio in the mitochondria from Pb-treated leaves was largely suppressed (from 1.8 to 0.4) by OM under nonphotorespiratory conditions (high CO₂), whereas under photorespiratory conditions (low CO₂) this ratio was high (5.3) and under OM decreased less (to 3.1). Our results indicate that oligomycin, in organelle isolated from Pb-treated leaves, had no inhibitory effect on the mitochondrial ATPase, whereas it inhibited chloroplasts ATPase. We suggest that Pb ions affected the catalytic cycle and/or conformational changes of ATPase in pea chloroplasts differently than in mitochondria. The differences in Pb responses may reflect fine mechanisms for the regulation of ATP production in the plant cells under stress conditions.     

Ribulosebisphosphate carboxylase activity and photosynthetic o(2) evolution rate in vicia guard-cell protoplasts

Activities of ribulose-1,5-bisphosphate carboxylase and rates of photosynthetic O₂ evolution were measured in guard-cell and mesophyll protoplasts from Vicia faba. The ribulose-1,5-bisphosphate carboxylase activity of guard-cell protoplasts was 30% of that of mesophyll protoplasts; however, the O₂ evolution rate was 3 times higher in guard-cell protoplasts than in mesophyll protoplasts on a chlorophyll basis. When the dark-adapted, guard-cell protoplasts were illuminated by red light, O₂ was evolved with an induction period, which became shorter when the protoplasts were reilluminated. High activity of irreversible NADP-glyceraldehyde-3-phosphate dehyrogenase was found in guard-cell protoplasts. Several lines of evidence revealed that there was virtually no contamination by mesophyll cells in guard-cell preparations. These results indicate that guard cells fix CO₂ photosynthetically and imply that the cells utilize a considerable proportion of reducing equivalents from water for reactions other than CO₂ fixation.     

Photorespiratory Properties of Mesophyll Protoplasts of Nicotiana plumbaginifolia

The photorespiratory activity of mesophyll protoplasts of Nicotiana plumbaginifolia has been clearly demonstrated by the presence of a Warburg-effect, the occurrence of an important CO₂-sensitive O₂ uptake and the effect of some photorespiratory inhibitors on photosynthetic activity. At a nonsaturating dissolved inorganic carbon (DIC) concentration (0.1 millimolar), we observed that the rate of CO₂ fixation was 60% lower at 50% O₂ compared to that measured at 2% O₂. Using ¹⁸O₂ and mass spectrometry, we measured O₂ exchange as a function of light intensity and of DIC concentration. Oxygen uptake measured at the CO₂ compensation point (47.4 micromoles O₂ per hour per milligram chlorophyll) was three-fold higher than that measured at a saturating CO₂ concentration. Cyanide or iodoacetamide, inhibitors of the Calvin cycle, were found to reduce the O₂ uptake to the same extent as CO₂ saturation. We conclude from these results that the major part of the CO₂-sensitive O₂ uptake is due to photorespiration. Further, we investigated the effect on net photosynthesis of some inhibitors of the glycolate pathway. At CO₂ saturation (10 millimolar DIC), 5 millimolar aminoacetonitrile (AAN), and 1 millimolar aminooxyacetate (AOA) did not cause any significant decrease in net photosynthesis. However, when these two inhibitors were added under a period of active photorespiration (10 minutes at the CO₂ compensation point at 20% O₂), we observed a decrease in the rate of net photosynthesis at 10 millimolar DIC measured afterward (respectively, 18 and 29%). This inhibition did not appear at 2% O₂, but was stronger at 50% O₂ (40% for AAN and 47% for AOA). With 0.05 millimolar butyl 2-hydroxy-3-butynoate (BHB) or 0.5 millimolar l-methionine-dl-sulfoximine (l-MSO), rates of net photosynthesis at 10 millimolar DIC were decreased by 10 to 15%. Additional decreases were observed after a period at the CO₂ compensation point at 20% O₂ (30% for BHB and 20% for l-MSO). From the sites of action of the four inhibitors tested, we suggest the inhibition of photosynthesis occurring after a period of active photorespiration to be due to the toxic accumulation of nonmetabolized phosphoglycolate.