The value of mutants unable to carry out photorespiration

Manipulation of the CO₂ concentration of the atmosphere allows the selection of photorespiratory mutants from populations of seeds treated with powerful mutagens such as sodium azide. So far, barley lines deficient in activity of phosphoglycolate phosphatase, catalase, the glycine to serine conversion, glutamine synthetase, glutamate synthase, 2-oxoglutarate uptake and serine: glyoxylate aminotransferase have been isolated. In addition one line of pea lacking glutamate synthase activity and one barley line containing reduced levels of Rubisco are available. The characteristics of these mutations are described and compared with similar mutants isolated from populations of Arabidopsis. As yet, no mutant lacking glutamine synthetase activity has been isolated from Arabidopsis and possible reasons for this difference between barley and Arabidopsis are discussed. The value of these mutant plants in the elucidation of the mechanism of photorespiration and its relationships with CO₂ fixation and amino acid metabolism are highlighted.Abbreviations GS cytoplasmic glutamine synthetase - GS₂ chloroplastic glutamine synthetase - PFR Photon fluence rate - Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP Ribulose-1,5-bisphosphate - SGAT serine:glyoxylate aminotransferase     

Metabolic regulation of carbon flux during C4 photosynthesis

The potential for glycolate and glycine metabolism and the mechanism of refixation of photorespiratory CO₂ in leaves of C₄ plants were studied by parallel inhibitor experiments with thin leaf slices, different leaf cell types and isolated mitochondria of C₃ and C₄ Panicum species. CO₂ evolution by leaf slices of P. bisulcatum, a C₃ species, fed glycolate or glycine was light-independent and O₂-sensitive. The C₄ P. maximum and P. miliaceum leaf slices fed glycolate or glycine evolved CO₂ in the dark but not in the light. In C₄ species, dark CO₂ evolution was abolished by the addition of phosphoenolpyruvate (PEP)⁴. The addition of maleate, a PEP carboxylase inhibitor, resulted in photorespiratory CO₂ efflux by C₄ leaf slices in the light also. However, PEP and maleate had no effect on either glycolate-dependent O₂ uptake by the C₄ leaf slices or on glycolate and glycine metabolism in C₃ leaf slices. The rate of photorespiratory CO₂ evolution in the C₃ Panicum species was 3 times higher than that observed with the C₄ species. The ratio of glycolate-dependent CO₂ evolution to O₂ uptake in both groups was 1:2. Isolated C₄ mesophyll protoplasts or their mitochondria did not metabolize glycolate or glycine. However, both C₃ mesophyll protoplasts and C₄ bundle sheath strands readily metabolized glycolate and glycine in a light-independent, O₂-sensitive manner, and the addition of PEP or maleate had no effect. C₄ bundle sheath- and C₃-mitochondria were capable of oxidizing glycine. This oxidation was linked to the mitochondrial electron transport chain, was coupled to three phosphorylation sites and was sensitive to electron transport inhibitors. C₄ bundle sheath- and C₃-mitochondrial glycine decarboxylation was stimulated by oxaloacetate and NAD had no effect. In marked contrast, mitochondria isolated from C₄ mesophyll cells were incapable of oxidizing or decarboxylating added glycine. The results suggest that in leaves of C₄ plants bundle sheath cells are the primary site of O₂-sensitive photorespiratory CO₂ evolution and the PEP carboxylase present in the mesophyll cells has the Potential for efficiently refixing CO₂ before it escapes out of the leaf. The relative role of the PEP carboxylase mediated CO₂ pump and reassimilation of photorespiratory CO₂ are discussed in relation to the apparent lack of photorespiration in leaves of C₄ species.Abbreviations BSA bovine serum albumin - Chl chlorophyll - PEP phosphoenolpyruvate - Rbu-P ₂ ribulose 1,5-bisphosphate - Rib-5-P ribose-5-phosphate - Ru-5-P ribuluse-5-phosphate - FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone Journal Series Paper, New Jersey Agricultural Experiment Station     

Barley mutants lacking chloroplast glutamine synthetase-biochemical and genetic analysis

Eight mutants of barley (Hordeum vulgare cv Maris Mink) lacking the chloroplast isozyme of glutamine synthetase (EC 6.3.1.2.) were isolated by their inability to grow under photorespiratory conditions. The cytoplasmic isozyme of glutamine synthetase was present in the leaves of all the mutants, with activities comparable to the wild-type (10-12 nanokatals per gram fresh weight). The mutant plants developed normally and were fully fertile under conditions that minimize photorespiration. In 1% O₂ the rate of CO₂ fixation in leaves of one of the mutants, RPr 83/32, was the same as the wild-type, but in air this rate declined to 60% of the wild-type after 30 minutes. During this time the ammonia concentration in leaves of the mutant rose from 1 to 50 micromoles per gram fresh weight. Such ammonia accumulation in air was found in all the mutant lines. In back-crosses with the parent line, F₁ plants were viable in air. In the F₂ generation, nonviability in air and the lack of chloroplast glutamine synthetase co-segregated, in both the lines tested. These two lines and four others proved to be allelic; we designate them gln 2a-f. The characteristics of these mutants conclusively demonstrate the major role of chloroplast glutamine synthetase in photorespiration and its associated nitrogen recycling.     

Utilization of O2 in the metabolic optimization of C4 photosynthesis

The combined effects of O₂ on net rates of photosynthesis, photosystem II activity, steady‐state pool size of key metabolites of photosynthetic metabolism in the C₄ pathway, C₃ pathway and C₂ photorespiratory cycle and on growth were evaluated in the C₄ species Amaranthus edulis and the C₃ species Flaveria pringlei. Increasing O₂ reduced net CO₂ assimilation in F. pringlei due to an increased flux of C through the photorespiratory pathway. However, in A. edulis increasing O₂ up to 5–10% stimulated photosynthesis. Analysis of the pool size of key metabolites in A. edulis suggests that while there is some O₂ dependent photorespiration, O₂ is required for maximizing C₄ cycle activity to concentrate CO₂ in bundle sheath cells. Therefore, the response of net photosynthesis to O₂ in C₄ plants may result from the balance of these two opposing effects. Under 21 versus 5% O₂, growth of A. edulis was stimulated about 30% whereas that of F. pringlei was inhibited about 40%.     

The re-assimilation of ammonia produced by photorespiration and the nitrogen economy of C3 higher plants

Photorespiration involves the conversion of glycine to serine with the release of ammonia and CO₂. In C₃ terrestrial higher plants the flux through glycine and serine is so large that it results in the production of ammonia at a rate far exceeding that from reduction of new nitrogen entering the plant. The photorespiratory nitrogen cycle re-assimilates this ammonia using the enzymes glutamine synthetase and glutamine:2-oxoglutarateaminotransferase.     

Affixing the O to Rubisco: discovering the source of photorespiratory glycolate and its regulation

The source of glycolate in photorespiration and its control, a particularly active and controversial research topic in the 1970s, was resolved in large part by several discoveries and observations described here. George Bowes discovered that the key carboxylation enzyme Rubisco (ribulosebisphosphate carboxylase/oxygenase) is competitively inhibited by O₂ and that O₂ substitutes for CO₂ in the initial `dark' reaction of photosynthesis to yield glycolate-P, the substrate for photorespiration. William Laing derived an equation from basic enzyme kinetics that describes the CO₂, O₂, and temperature dependence of photosynthesis, photorespiration, and the CO₂ compensation point in C₃ plants. Jerome Servaites established that photosynthesis cannot be increased by inhibiting the photorespiratory pathway prior to the release of photorespiratory CO₂, and Douglas Jordan discovered substantial natural variation in the Rubisco oxygenase/carboxylase ratio. A mutant Arabidopsis plant with defective glycolate-P phosphatase, isolated by Chris Somerville, definitively established the role of O₂ and Rubisco in providing photorespiratory glycolate. Selection techniques to isolate photorespiration-deficient plants were devised by Jack Widholm and by Somerville, but no plants with reduced photorespiration were found. Somerville's approach, directed mutagenesis of Arabidopsis plants, was subsequently successful in the isolation of numerous other classes of mutants and revolutionized the science of plant biology. This revised version was published online in August 2006 with corrections to the Cover Date.     

Photorespiration Rate in Spinach Leaves Under Moderate NaCl Stress

We analyzed the effect of NaCl stress on photorespiration of spinach leaves by calculating the rate of carboxylation/oxygenation of ribulose-1,5-bisphosphate carboxylase/oxygenase, and by measuring the content of amino acids produced through photorespiration. After 20 d of NaCl stress the carboxylation rate was reduced while the oxygenation rate was not affected. The contents of serine, glycine, and alanine increased relevantly. The amount of glutamine also increased after 20 d but the amount of glutamate did not. Hence photorespiration may be stimulated under moderate NaCl stress. A relevant electron transport rate was observed under CO₂-free air, which may indicate refixation of photorespiratory CO₂. When NaCl accumulation proceeded for more than 20 d, photosynthesis was reduced and the content of photo-respiratory amino acids started to decrease, but the oxygenation rate did not change. This revised version was published online in September 2006 with corrections to the Cover Date.     

On the significance of C3—C4 intermediate photosynthesis to the evolution of C4 photosynthesis

Abstract Evidence is drawn from previous studies to argue that C₃—C₄ intermediate plants are evolutionary intermediates, evolving from fully-expressed C₃ plants towards fully-expressed C₄ plants. On the basis of this conclusion, C₃—C₄ intermediates are examined to elucidate possible patterns that have been followed during the evolution of C₄ photosynthesis. An hypothesis is proposed that the initial step in C₄-evolution was the development of bundle-sheath metabolism that reduced apparent photorespiration by an efficient recycling of CO₂ using RuBP carboxylase. The CO₂-recycling mechanism appears to involve the differential compartmentation of glycine decarboxylase between mesophyll and bundle-sheath cells, such that most of the activity is in the bundlesheath cells. Subsequently, elevated phosphoenolpyruvate (PEP) carboxylase activities are proposed to have evolved as a means of enhancing the recycling of photorespired CO₂. As the activity of PEP carboxylase increased to higher values, other enzymes in the C₄-pathway are proposed to have increased in activity to facilitate the processing of the products of C₄-assimilation and provide PEP substrate to PEP carboxylase with greater efficiency. Initially, such a ‘C₄-cycle’ would not have been differentially compartmentalized between mesophyll and bundlesheath cells as is typical of fully-expressed C₄ plants. Such metabolism would have limited benefit in terms of concentrating CO₂ at RuBP carboxylase and, therefore, also be of little benefit for improving water- and nitrogen-use efficiencies. However, the development of such a limited C₄-cycle would have represented a preadaptation capable of evolving into the leaf biochemistry typical of fully-expressed C₄ plants. Thus, during the initial stages of C₄-evolution it is proposed that improvements in photorespiratory CO₂-loss and their influence on increasing the rate of net CO₂ assimilation per unit leaf area represented the evolutionary ‘driving-force’. Improved resourceuse efficiency resulting from an efficient CO₂-concentrating mechanism is proposed as the driving force during the later stages.     

Predominant localization of mitochondria enriched with glycine-decarboxylating enzymes in bundle sheath cells of Alternanthera tenella, a C3–C4 intermediate species

Mesophyll protoplasts and bundle sheath cells were prepared by enzymatic digestion of leaves of Alternanthera tenella, a C₃-C₄ intermediate species. The intercellular distribution of selected photosynthetic, photorespiratory and respiratory (mitochondrial) enzymes in these meso-phyll and bundle sheath cells was studied. The activity levels of photosynthetic enzymes such as PEP carboxylase (EC 4.1.1.31) or NAD-malic enzyme (EC 1.1.1.39) and photorespiratory enzymes such as glycolate oxidase (EC 1.1.3.1) or NADH-hydroxypyruvate reductase (EC 1.1.1.29) were similar in the two cell types. The activity levels of mitochondrial TCA cycle enzymes such as citrate synthase (EC 4.1.3.7) or fumarase (EC 4.2.1.2) were 2- to 3-fold higher in bundle sheath cells. On the other hand, the activity levels of mitochondrial photorespiratory enzymes, namely glycine decarboxylase (EC 2.1.2.10) and serine hydroxymethyltransferase (EC 2.1.2.1), were 6-9-fold higher in bundle sheath cells than in mesophyll protoplasts. Such preferential localization of mitochondria enriched with the glycine-decarboxylating system in the inner bundle sheath cells would result in efficient refixa-tion of CO₂ from not only photorespiration but also dark respiration before its exit from the leaf. We propose that predominant localization of mitochondria specialized in glycine decarboxylation in bundle sheath cells may form the basis of reduced photorespiration in this C₃-C₄ intermediate species.     

Photosynthetic/photorespiratory characteristics of C3−C4 intermediate species

The extent of photorespiration, the inhibition of apparent photosynthesis (APS) by 21% O₂, and the leaf anatomical and ultrastructural features of the naturally occurring C₃–C₄ intermediate species in the diverse Panicum, Moricandia, and Flaveria genera are between those features of representative C₃ and C₄ plants. The greatest differences between the photosynthetic/photorespiratory CO₂ exchange characteristics of the C₃–C₄ intermediates and C₃ plants occur for the parameters which are measured at low pCO₂ (i.e., the CO₂ compensation concentration and rates of CO₂ evolution into CO₂-free air in the light). The rates of APS by the intermediate species at atmospheric pCO₂ are similar to those of C₃ plants.The mechanisms which are responsible for reducing photorespiration in the C₃–C₄ intermediate species are poorly understood, but two proposals have been advanced. One emphasizes the importance of limited C₄ photosynthesis which reduces O₂ fixation by ribulose 1,5-bisphosphate carboxylase/oxygenase, and, thus, reduces photorespiration by a CO₂-concentrating mechanism, while the other emphasizes the importance of the internal recycling of photorespiratory CO₂ evolved from the chloroplast/mitochondrion-containing bundle-sheath cells. There is no evidence from recent studies that limited C₄ photosynthesis is responsible for reducing photorespiration in the intermediate Panicum and Moricandia species. However, preliminary results suggest that some, but not all, of the intermediate Flaveria species may possess a limited C₄ cycle. The importance of a chlorophyllous bundle-sheath layer in the leaves of intermediate Panicum and Moricandia species in a mechanism based on the recycling of photorespiratory CO₂ is uncertain.Therefore, although they have yet to be clearly delineated, different strategies appear to exist in the C₃–C₄ intermediate group to reduce photorespiration. Of major importance is the finding that some mechanism(s) other than Crassulacean acid metabolism or C₄ photosynthesis has (have) evolved in at least the majority of these terrestrial intermediate species to reduce the seemingly wasteful metabolic process of photorespiration.Abbreviations APS apparent (net) photosynthesis - CAM Crassulacean acid metabolism - CE carboxylation efficiency - T CO₂ compensation concentration - IRGA infrared gas analysis - P_i orthophosphate - PEP phosphoenolpyruvate - RuBP ribulose 1,5-bisphosphate Published as Paper No. 7383, Journal Series, Nebraska Agricultural Experiment Station.     

Photorespiratory metabolism and immunogold localization of photorespiratory enzymes in leaves of C3 and C3-C4 intermediate species of Moricandia

Photorespiratory metabolism of the C₃-C₄ intermediate species Moricandia arvensis (L.) DC has been compared with that of the C₃ species, Moricandia moricandioides (Boiss.) Heywood. Assays of glycollate oxidase (EC 1.1.3.1), glyoxylate aminotransferases (EC 2.6.1.4, EC 2.6.1.45) and hydroxypyruvate reductase (EC 1.1.1.29) indicate that the capacity for flux through the photorespiratory cycle is similar in both species. Immunogold labelling with monospecific antibodies was used to investigate the cellular locations of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), glycollate oxidase, and glycine decarboxylase (EC 2.1.2.10) in leaves of the two species. Ribulose 1,5-bisphosphate carboxylase/oxygenase was confined to the stroma of chloroplasts and glycollate oxidase to the peroxisomes of all photosynthetic cells in leaves of both species. However, whereas glycine decarboxylase was present in the mitochondria of all photosynthetic cells in M. moricandioides, it was only found in the mitochondria of bundle-sheath cells in M. arvensis. We suggest that localized decarboxylation of glycine in the leaves of M. arvensis will lead to improved recapture of photorespired CO₂ and hence a lower rate of photorespiration.Abbreviations kDa kilodalton - RuBP ribulose-1,5-bisphosphate     

Photorespiration in C3–C4 intermediate species of Alternanthera and Parthenium: Reduced ammonia production and increased capacity of CO2 refixation in the light

The pattern of photorespiratory ammonia (PR–NH₃) formation and its modulation by exogenous bicarbonate or glycine were investigated in C₃–C₄ intermediates of Alternanthera (A. ficoides and A. tenella) and Parthenium hysterophorus in comparison to those of C₃ or C₄ species. The average rates of PR–NH₃ accumulation in leaves of the intermediates were slightly less than (about 25% reduced) those in C₃ species, and were further low in C₄ plants (40% of that in C₃). The levels of PR–NH₃ in leaf discs decreased markedly when exogenous bicarbonate was present in the incubation medium. The inhibitory effect of bicarbonate on PR–NH₃ accumulation was pronounced in C₃ plants, very low in C₄ species and was moderate in the C₃–C₄ intermediates. Glycine, an intermediate of photorespiratory metabolism, raised the levels of PR–NH₃ in leaves of not only C₄ but also C₃–C₄ intermediates, bringing the rates close to those of C₃ species. The rate of mitochondrial glycine decarboxylation in darkness in C₃–C₄ intermediates was partially reduced (about 80% of that in C₃ species), corresponding to the activity-levels of glycine decarboxylase and serine hydroxymethyltransferase in leaves. The intermediates had a remarkable capacity of reassimilating photorespiratory CO₂ in vivo, as indicated by the apparent refixation of about 85% of the CO₂ released from exogenous glycine in the light. We suggest that the reduced photorespiration in the C₃–C₄ intermediate species of Alternanthera and Parthenium is due to both a limitation in the extent of glycine production/decarboxylation and an efficient refixation/recycling of internal CO₂.Abbreviations GDC glycine decarboxylase - GS glutamine synthetase - GOGAT glutamate-oxoglutarate aminotransferase - -HPMS -hydroxy-2-pyridinemethanesulfonic acid - INH isonicotinyl hydrazide - MSO L-methionine sulfoximine - PR–NH₃ photorespiratory-ammonia - SHMT serine hydroxymethyltransferase     

Relationship between leaf development,carboxylase enzyme activities and photorespiration in the C4-plant Portulaca oleracea L.

Summary Ribulose diphosphate (RuDP) and (PEP) phosphoenolpyruvate carboxylase enzyme activities were studied in young, mature, and senescent Portulaca oleracea leaves. While the absolute amount of both the C₃ (RuDP) and C₄ (PEP) carboxylase is less in senescent leaves than in mature leaves, RuDP carboxylase activity is reduced to a lesser degree. In senescent leaves, PEP carboxylase activity equals 10% of that in mature tissue, but RuDP carboxylase is 27% of that in mature leaves. The same ontogenetic series was also used to determine photorespiration rates and responses to several gas treatments. Young and mature leaves were unaffected by changes in the light regime or oxygen concentrations, and exhibited typical C₄-plant light/dark ¹⁴CO₂ evolution ratios. Senescent leaves, on the other hand, have photorespiration ratios similar to C₃-plants. In addition, senescent leaves were affected by minus CO₂, 100% O₂ and N₂ in a manner expected of C₃-plants, but not C₄-plants. These results are discussed in terms of a relative increase in activity of the C₃ cycle in later developmental stages in this plant.Abbreviation RuDP ribulose diphosphate - PEP phosphoenolpyruvate - PGA phosphoglyceric acid     

Variation with age in the photosynthetic carbon fixation pattern by leaves of Amaranthus paniculatus and Oryza sativa: Change in the primary carboxylation but no shift from C4 or C3 pathway

Abstract The pattern of photosynthetic carbon fixation by leaves of Amaranthus paniculatus L. (a C₄ plant) and Oryza sativa L. (a C₃ plant) varied with age. Younger leaves of A. paniculatus incorporated ¹⁴CO₂ into malate and aspartate while senescent leaves fixed predominantly into phosphoglycerate (PGA) and sugar phosphates. Only developing leaves of O. sativa formed malate/aspartate whereas mature and senescent leaves produced PGA/sugar phosphates as the initial labelled products. Correspondingly the ratio of phosphoenolpyruvate/ribulose bisphosphate (RuBP) carboxylase activities was higher in younger leaves of A. paniculatus and developing leaves of O. sativa than in older leaves. However, pulse chase experiments revealed that the main donors of carbon to end products, irrespective of leaf stage, were C₄ acids and PGA in A. paniculatus and O. sativa respectively. The results suggest that although an apparent change from initial β-carboxylation to RuBP carboxylation occurs during leaf ontogeny in both the plants, the overall leaf photosynthesis remains C₄ or C₃. The high rate of ¹⁴CO₂ incorporation into PGA/sugar phosphates by senescent leaves of A. paniculatus is suggested to be partly due to the increased intercellular spaces in their mesophyll, allowing greater access of CO₂ directly to RuBP carboxylase in the bundle sheath.     

The isolation and characterisation of a mutant of the C4 plant Amaranthus edulis deficient in NAD-malic enzyme activity

A mutant of Amaranthus edulis (Speg.) lacking activity of the C₄ leaf form of NAD-malic enzyme (ME; EC 1.1.1.39) has been isolated. Homozygous mutant (5% wild-type ME activity) and heterozygous (50% wild-type ME activity) F₂ plants were shown to contain both the α and β NAD-ME subunits in similar amounts to those detected in the wild-type leaves. The rate of photosynthetic CO₂ assimilation was reduced in the homozygous mutant to 5% of that observed for the wild-type leaves. Other C₄ enzymes were not down-regulated in the mutant plants. There was little difference in photosynthetic rate of the heterozygous plants compared to the wild-type, suggesting that NAD-ME exerts little control over the rate of C₄ photosynthesis, and that in the wild-type the enzyme has a very low control coefficient. The activity loss in the heterozygote may therefore be compensated by regulatory mechanisms that increase the activity of the enzyme in vivo. Data for bundle-sheath strands indicated that although the homozygous mutants were able to oxidise malate via the Krebs cycle, they were unable to convert malate to pyruvate and alanine via NAD-ME. Received: 2 April 1998 / Accepted: 7 May 1998     

Leaf development,gas exchange characteristics,and photorespiratory activity in maize seedlings

Five decades ago, a novel mode of CO₂ assimilation that was later described as C₄-photosynthesis was discovered on mature leaves of maize (Zea mays L.) plants. Here we show that 3- to 5-day-old developing maize leaves recapitulate the evolutionary advance from the ancient, inefficient C₃ mode of photosynthesis to the C₄ pathway, a mechanism for overcoming the wasteful process of photorespiration. Chlorophyll fluorescence measurements documented that photorespiration was high in 3-day-old juvenile primary leaves with non-specialized C₃-like leaf anatomy and low in 5-day-old organs with the typical “Kranz-anatomy” of C₄ leaves. Photosynthetic gas (CO₂)-exchange measurements on 5-day-old leaves revealed the characteristic features of C₄ photosynthesis, with a CO₂ compensation point close to zero and little inhibition of photosynthesis by the normal oxygen concentration in the air. This indicates a very low photorespiratory activity in contrast to control experiments conducted with mature C₃ sunflower (Helianthus annuus L.) leaves, which display a high rate of photorespiration.     

Decarboxylation of glycine contributes to carbon isotope fractionation in photosynthetic organisms

Carbon isotope effects were investigated for the reaction catalyzed by the glycine decarboxylase complex (GDC; EC 2.1.2.10). Mitochondria isolated from leaves of pea (Pisum sativum L.) and spinach (Spinacia oleracea L.) were incubated with glycine, and the CO₂ evolved was analyzed for the carbon isotope ratio (δ¹³C). Within the range of parameters tested (temperature, pH, combination of cofactors NAD⁺, ADP, pyridoxal 5-phosphate), carbon isotope shifts of CO₂ relative to the C₁-carboxyl carbon of glycine varied from +14‰ to −7‰. The maximum effect of cofactors was observed for NAD⁺, the removal of which resulted in a strong ¹²C enrichment of the CO₂ evolved. This indicates the possibility of isotope effects with both positive and negative signs in the GDC reaction. The measurement of δ¹³C in the leaves of the GDC-deficient barley (Hordeum vulgare L.) mutant (LaPr 87/30) plants indicated that photorespiratory carbon isotope fractionation, opposite in sign when compared to the carbon isotope effect during CO₂ photoassimilation, takes place in vivo. Thus the key reaction of photorespiration catalyzed by GDC, together with the key reaction of CO₂ fixation catalyzed by ribulose-1,5-bisphosphate carboxylase, both contribute to carbon isotope fractionation in photosynthesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of phosphinothricin (glufosinate) on photosynthesis and photorespiration of C3 and C4 plants

Phosphinothricin (glufosinate), an irreversible inhibitor of glutamine synthetase, causes an inhibition of photosynthesis in C₃ (Sinapis alba) and C₄ (Zea mays) plants under atmospheric conditions (400 ppm CO₂, 21% O₂). This photosynthesis inhibition is proceeding slower in C₄ leaves. Under non-photorespiratory conditions (1000 ppm CO₂, 2% O₂) there is no inhibition of photosynthesis. The inhibition of glutamine synthetase by phosphinothricin results in an accumulation of NH₄ ⁺. The NH₄ ⁺-accumulation is lower in C₄ plants than in C₃ plants. The inhibition of glutamine synthetase through phosphinothricin in mustard leaves results in a decrease in glutamine, glutamate, aspartate, asparagine, serine, and glycine. In contrast to this, a considerable increase in leucine and valine following phosphinothricin treatment is measured. With the addition of either glutamine, glutamate, aspartate, glycine or serine, photosynthesis inhibition by phosphinothricin can be reduced, although the NH₄ ⁺-accumulation is greatly increased. This indicates that NH₄ ⁺-accumulation cannot be the primary cause for photosynthesis inhibition by phosphinothricin. The investigations demonstrate the inhibition of transmination of glyoxylate to glycine in photorespiration through the total lack of amino donors. This could result in a glyoxylate accumulation inhibiting ribulose-1,5-bisphosphate-carboxylase and consequently CO₂-fixation.Abbreviations GOGAT glutamine-2-oxoglutarate-amidotransferase - GS glutamine synthetase - PPT phosphinothricin - MSO methionine sulfoximine - RuBP ribulose-1,5-bisphosphate     

Photorespiration-induced reduction of ribulose bisphosphate carboxylase activation level

Leaf photosynthesis and ribulose bisphosphate carboxylase activation level were inhibited in several mutants of the C₃ crucifer Arabidopsis thaliana which possess lesions in the photorespiratory pathway. This inhibition occurred when leaves were illuminated under a photorespiratory atmosphere (50% O₂, 350 microliters per liter CO₂, balance N₂), but not in nonphotorespiratory conditions (2% O₂, 350 microliters per liter CO₂, balance N₂). Inhibition of carboxylase activation level was observed in strains with deficient glycine decarboxylase, serine transhydroxymethylase, serine-glyoxylate aminotransferase, glutamate synthase, and chloroplast dicarboxylate transport activities, but inhibition did not occur in a glycolate-P phosphatase-deficient strain. Also, the photorespiration inhibitor aminoacetonitrile produced a decline in leaf and protoplast ribulose bisphosphate carboxylase activation level, but was without effect on intact chloroplasts. Fructose bisphosphatase, a light-activated enzyme which is strongly dependent on stromal pH and Mg²⁺ for regulation, was unaffected by conditions which caused inhibition of ribulose bisphosphate carboxylase. Thus, the mechanism of inhibition does not appear to involve changes in stromal Mg²⁺ and pH but rather is associated with metabolite flux through the photorespiratory pathway.