Photorespiratory metabolism and nodule function: behavior of Lotus japonicus mutants deficient in plastid glutamine synthetase

Two photorespiratory mutants of Lotus japonicus deficient in plastid glutamine synthetase (GS(2)) were examined for their capacity to establish symbiotic association with Mesorhizobium loti bacteria. Biosynthetic glutamine synthetase (GS) activity was reduced by around 40% in crude nodule extracts from mutant plants as compared with the wild type (WT). Western blot analysis further confirmed the lack of GS(2) polypeptide in mutant nodules. The decrease in GS activity affected the nodular carbon metabolism under high CO(2) (suppressed photorespiration) conditions, although mutant plants were able to form nodules and fix atmospheric nitrogen. However, when WT and mutant plants were transferred to an ordinary air atmosphere (photorespiratory active conditions) the nodulation process and nitrogen fixation were substantially affected, particularly in mutant plants. The number and fresh weight of mutant nodules as well as acetylene reduction activity showed a strong inhibition compared with WT plants. Optical microscopy studies from mutant plant nodules revealed the anticipated senescence phenotype linked to an important reduction in starch and sucrose levels. These results show that, in Lotus japonicus, photorespiration and, particularly, GS(2) deficiency result in profound limitations in carbon metabolism that affect the nodulation process and nitrogen fixation.     

The phosphorylated pathway of serine biosynthesis links plant growth with nitrogen metabolism

Because it is the precursor for various essential cellular components, the amino acid serine is indispensable for every living organism. In plants, serine is synthesized by two major pathways: photorespiration and the phosphorylated pathway of serine biosynthesis (PPSB). However, the importance of these pathways in providing serine for plant development is not fully understood. In this study, we examine the relative contributions of photorespiration and PPSB to providing serine for growth and metabolism in the C3 model plant Arabidopsis thaliana. Our analyses of cell proliferation and elongation reveal that PPSB-derived serine is indispensable for plant growth and its loss cannot be compensated by photorespiratory serine biosynthesis. Using isotope labeling, we show that PPSB-deficiency impairs the synthesis of proteins and purine nucleotides in plants. Furthermore, deficiency in PPSB-mediated serine biosynthesis leads to a strong accumulation of metabolites related to nitrogen metabolism. This result corroborates ¹⁵N-isotope labeling in which we observed an increased enrichment in labeled amino acids in PPSB-deficient plants. Expression studies indicate that elevated ammonium uptake and higher glutamine synthetase/glutamine oxoglutarate aminotransferase (GS/GOGAT) activity causes this phenotype. Metabolic analyses further show that elevated nitrogen assimilation and reduced amino acid turnover into proteins and nucleotides are the most likely driving forces for changes in respiratory metabolism and amino acid catabolism in PPSB-deficient plants. Accordingly, we conclude that even though photorespiration generates high amounts of serine in plants, PPSB-derived serine is more important for plant growth and its deficiency triggers the induction of nitrogen assimilation, most likely as an amino acid starvation response.

The phosphorylated pathway of serine biosynthesis is required to synthesize serine for plant growth; and its deficiency triggers an amino acid starvation response by inducing nitrogen assimilation.     

Effects of tabtoxin on nitrogen metabolism

Tabtoxin is a chlorosis-inducing toxin produced by the plant pathogenic bacterium Pseudomonas syringae pv. tabaci. Previous studies have indicated that tabtoxin inhibits glutamine synthetase (EC 6.3.1.2) in vitro. We report here that tabtoxin also inhibits glutamine synthetase in vivo. The main evidence was that assimilation of exogenous ¹⁵NH₃ into Asparagus sprengeri protein was rapidly inhibited in isolated cells exposed to tabtoxin. This was associated with an equivalent decline in glutamine synthetase activity in extracts of these cells and the accumulation of extracellular ammonia. Glutamine synthetase was also inhibited in leaves of Nicotiana tabacum L. cv. White Burley treated with tabtoxin and the affected tissue accumulated ammonia and became chlorotic. However, the development of symptoms and accumulation of ammonia was suppressed when the leaves were held in air containing 1% CO₂ to reduce photorespiration. This indicates that the chlorotic symptom did not result from the inhibition of nitrogen assimilation but was a consequence of the interruption of the photorespiratory nitrogen cycle.     

Overexpression of cytosolic glutamine synthetase. Relation to nitrogen,light, and photorespiration

In plants, ammonium released during photorespiration exceeds primary nitrogen assimilation by as much as 10-fold. Analysis of photorespiratory mutants indicates that photorespiratory ammonium released in mitochondria is reassimilated in the chloroplast by a chloroplastic isoenzyme of glutamine synthetase (GS2), the predominant GS isoform in leaves of Solanaceous species including tobacco (Nicotiana tabacum). By contrast, cytosolic GS1 is expressed in the vasculature of several species including tobacco. Here, we report the effects on growth and photorespiration of overexpressing a cytosolic GS1 isoenzyme in leaf mesophyll cells of tobacco. The plants, which ectopically overexpress cytosolic GS1 in leaves, display a light-dependent improved growth phenotype under nitrogen-limiting and nitrogen-non-limiting conditions. Improved growth was evidenced by increases in fresh weight, dry weight, and leaf soluble protein. Because the improved growth phenotype was dependent on light, this suggested that the ectopic expression of cytosolic GS1 in leaves may act via photosynthetic/photorespiratory process. The ectopic overexpression of cytosolic GS1 in tobacco leaves resulted in a 6- to 7-fold decrease in levels of free ammonium in leaves. Thus, the overexpression of cytosolic GS1 in leaf mesophyll cells seems to provide an alternate route to chloroplastic GS2 for the assimilation of photorespiratory ammonium. The cytosolic GS1 transgenic plants also exhibit an increase in the CO(2) photorespiratory burst and an increase in levels of photorespiratory intermediates, suggesting changes in photorespiration. Because the GS1 transgenic plants have an unaltered CO(2) compensation point, this may reflect an accompanying increase in photosynthetic capacity. Together, these results provide new insights into the possible mechanisms responsible for the improved growth phenotype of cytosolic GS1 overexpressing plants. Our studies provide further support for the notion that the ectopic overexpression of genes for cytosolic GS1 can potentially be used to affect increases in nitrogen use efficiency in transgenic crop plants.     

A study of photorespiratory ammonia production in the C4 plant Amaranthus edulis, using mutants with altered photosynthetic capacities

Previous studies have indicated that the rate of photorespiration in C₄ plants is low or negligible. In this study, wild-type and mutant leaves of the C₄ plant Amaranthus edulis were treated with the glutamine synthetase inhibitor, phosphinothricin and the glycine decarboxylase inhibitor, aminoacetonitrile, at different concentrations of CO₂. The time course of ammonia accumulation in leaves of the wild type was compared with a mutant lacking phosphoenolpyruvate carboxylase activity (EC 4.1.1.31), and with three different mutants that accumulated glycine. The increase in the concentration of ammonia in the leaves, stimulated by the treatments was used as a measurement of the rate of photorespiration in C₄ plants. The application of glutamine and glycine maintained the rate of photorespiratory ammonia production for a longer period in the wild type, and increased the rate in a mutant lacking phosphoenolpyruvate carboxylase suggesting that there was a lack of amino donors in these plants. The calculated rate of photorespiration in Amaranthus edulis wild-type leaves when the supply of amino donors was enough to maintain the photorespiratory nitrogen flow, accounted for approximately 6% of the total net photosynthetic CO₂ assimilation rate. In a mutant lacking phosphoenolpyruvate carboxylase, however, this rate increased to 48%, when glutamine was fed to the leaf, a value higher than that found in some C₃ plants. In mutants of Amaranthus edulis that accumulated glycine, the rate of photorespiration was reduced to 3% of the total net CO₂ assimilation rate. The rate of ammonia produced during photorespiration was 60% of the total produced by all metabolic reactions in the leaves. The data suggests that photorespiration is an active process in C₄ plants, which can play an important role in photosynthetic metabolism in these plants.     

Isolation of photorespiratory mutants from Lotus japonicus deficient in glutamine synthetase

A mutagenesis programme using ethyl methanesulphonate (EMS) was carried out on Lotus japonicus (Regel) Larsen cv. Gifu in order to isolate photorespiratory mutants in this model legume. These mutants were able to grow in a CO₂-enriched atmosphere [0.7% (v/v) CO₂] but showed stress symptoms when transferred to air. Among them, three mutants displayed low levels of glutamine synthetase (GS; EC 6.3.1.2) activity in leaves. The mutants accumulated ammonium in leaves upon transfer from 0.7% (v/v) CO₂ to air. F₁ plants of back crosses to wild type were viable in air and F₂ populations segregated 3 : 1 (viable in air : air-sensitive) indicative of a single Mendelian recessive trait. Complementation tests showed that the three mutants obtained were allelic. Chromatography on DEAE-Sephacel used to separate the cytosolic and plastidic GS isoenzymes together with immunological data showed that: (1) mutants were specifically affected in the plastidic GS isoform, and (2) in L. japonicus the plastidic GS isoform eluted at lower ionic strength than the cytosolic isoform, contrary to what happens in most plants. The plastidic GS isoform present in roots of wild type L. japonicus was also absent in roots of the mutants, indicating that this plastidic isoform from roots was encoded by the same gene than the GS isoform expressed in leaf tissue. Viability of mutant plants in high-CO₂ conditions indicates that plastidic GS is not essentially required for primary ammonium assimilation. Nevertheless, mutant plants did not grow as well as wild type plants in high-CO₂ conditions.     

Antisense repression reveals a crucial role of the plastidic 2-oxoglutarate/malate translocator DiT1 at the interface between carbon and nitrogen metabolism

Ammonia assimilation by the plastidic glutamine synthetase/glutamate synthase system requires 2-oxoglutarate (2-OG) as a carbon precursor. Plastids depend on 2-OG import from the cytosol. A plastidic dicarboxylate translocator 1-[2-OG/malate translocator (DiT1)] has been identified and its substrate specificity and kinetic constants have been analyzed in vitro. However, the role of DiT1 in intact plants and its significance for ammonia assimilation remained uncertain. Here, to study the role of DiT1 in intact plants, its expression was antisense-repressed in transgenic tobacco plants. This resulted in a reduced transport capacity for 2-OG across the plastid envelope membrane. In consequence, allocation of carbon precursors to amino acid synthesis was impaired, organic acids accumulated and protein content, photosynthetic capacity and sugar pools in leaves were strongly decreased. The phenotype was consistent with a role of DIT1 in both, primary ammonia assimilation and the re-assimilation of ammonia resulting from the photorespiratory carbon cycle. Unexpectedly, the in situ rate of nitrate reduction was extremely low in alpha-DiT1 leaves, although nitrate reductase (NR) expression and activity remained high. We hypothesize that this discrepancy between extractable NR activity and in situ nitrate reduction is due to substrate limitation of NR. These findings and the severe phenotype of the antisense plants point to a crucial role of DiT1 at the interface between carbon and nitrogen metabolism.     

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.     

The supply of reducing power for nitrite reduction in plastids of seedling pea roots (Pisum sativum L.)

Plastids were separated from extracts of pea (Pisum sativum L.) roots by sucrose-density-gradient centrifugation. The incubation of roots of intact pea seedlings in solutions containing 10 mM KNO₃ resulted in increased plastid activity of nitrite reductase and to a lesser extent glutamine synthetase. There were also substantial increases in the activity of glucose-6-phosphate and 6-phosphogluconate dehydrogenases. No other plastid-located enzymes of nitrate assimilation or carbohydrate oxidation showed evidence of increased activity in response to the induction of nitrate assimilation. Studies with [1-¹⁴C]-and [6-¹⁴C]glucose indicated that there was an increased flow of carbon through the plastid-located pentose-phosphate pathway concurrent with the induction of nitrate assimilation. It is suggested that there is a close interaction through the supply and demand for reductant between the pathway of nitrite assimilation and the pentose-phosphate pathway located in the plastid.     

Assimilation of Nitrogen in Mutants Lacking Enzymes of the Glutamate Synthase Cycle

¹⁵N labelling was used to investigate the pathway of nitrogenassimilation in photorespiratory mutants of barley (Hordeumvulgare cv. Maris Mink), in which the leaves have low levelsof glutamine synthetase (GS) or glutamate synthase, key enzymesof ammonia assimilation. These plants grew normally when maintainedin high CO₂, but the deletions were lethal when photorespirationwas initiated by transfer to air. Enzyme levels in roots weremuch less affected, compared to leaves, and assimilation oflabelled nitrate into amino acids of the root showed very littledifference between wild type and mutants. Organic nitrogen wasexported from roots in the xylem sap mainly as glutamine, levelsof which were somewhat reduced in the GS-deficient mutant andenhanced in the glutamate synthase deficient mutant. In theleaf, the major effect was seen in the glutamatesynthase mutant,which had an extremely limited capacity to utilize the importedglutamine and amino acid synthesis was greatlyrestricted. Thiswas confirmed by the supply of [¹⁵N]-glutamine directly to leaves.Leaves of the GS-deficient mutant assimilatedammonia at about75% the rate found for the wild type, and this was almost completelyeliminated by addition of the inhibitormethionine sulphoximine.Root enzymes, together with residual levels of the deleted enzymesin the leaves, have sufficient capacityfor ammonia assimilation,through the glutamate synthase cycle, to provide adequate inputof nitrogen for normal growth of themutants, if photorespiratoryammonia production is suppressed. Key words: Hordeum vulgare, ¹⁵N, glutamine synthetase, glutamate synthase, ammonia assimilation     

Inorganic nitrogen assimilation in plants of Austrlian rainforest communities

In a study of the plant communities of two Australian rainforests, it was found that pioner species had high levels of nitrate reductase (EC 1.6.6.1) and were predominantly leaf nitrate assimilators. Under- and over-storey species had low levels of shoot and root nitrate reductase activity, and many of them showed little capacity for nitrate reduction even when nitrate ions were freely available. Although closed-forest species have lower levels of nitrate reductase than those of gaps and forest margins, their total nitrogen contents were similar, suggesting the former utilize nitrogen sources other than nitrate ions. Glutamine synthetase (EC 6.3.1.2) was present in the leaves of all species examined. In the leaves of pioneer species the chloroplastic isoform of glutamine synthetase predominted, while in most of the species typical of closed-forest the cytosolic isoform accounted for at least 40% of total leaf activity. Low levels of chloroplastic glutamine synthetase were correlated with a low capacity for leaf nitrate reduction, and both are characteristic of many species that regenerate and grow for some time in shade. Low levels of chloroplastic glutamine synthetase imply that, in some of these woody plants, photorespiratory ammonia is re-assimilated via cytosolic glutamine synthetase.     

Effect of Photorespiratory C2 Acids on CO2 Assimilation,PS II Photochemistry and the Xanthophyll Cycle in Maize

The photorespiration cycle plays an important role in avoiding carbon drainage from the Calvin cycle and in protecting plants from photoinhibition. The role of photorespiration is frequently underestimated in C(4) plants, since these are characterized by low photorespiration rates. The aim of this work was to study the relationship between CO(2) assimilation, PS II photochemistry and the xanthophyll cycle when the photorespiratory cycle is disrupted in Zea mays L. To this end, the photorespiration inhibitor phosphinothricin (PPT) was applied individually or together with the photorespiratory C(2) acids, glycolate and glyoxylate to maize leaves. Application of PPT alone led to the inhibition of CO(2) assimilation. Moreover, feeding with glycolate or glyoxylate enhanced the effect of PPT on CO(2) assimilation. Our results confirm that the avoidance of the accumulation of the photorespiratory metabolites glycolate, glyoxylate or phosphoglycolate, is of vital importance for coordinated functioning between the glycolate pathway and CO(2) assimilation. Relatively early changes in PS II photochemistry also took place when the photorespiratory cycle was interrupted. Thus, fluorescence photochemical quenching (qP) was slightly reduced (10%) due to the application of PPT together with glycolate or glyoxylate. A decrease in the efficiency of excitation-energy capture by open PS II reaction centres (F'v/F'm) and an increase in thermal energy dissipation (non-photochemical quenching, NPQ) were also measured. These observations are consistent with a limitation of activity of the Calvin cycle and a subsequent lower demand for reduction equivalents. The increase in NPQ is discussed on the basis of changes in the xanthophyll cycle in maize, which seem to provide a limited protective role to avoid photoinhibition when the glycolate pathway is blocked. We conclude that C(2) photorespiratory acids can act as physiological regulators between the photorespiratory pathway and the Calvin cycle in maize.     

Refixation of photorespiratory ammonia and the role of alanine in photorespiration: Studies with15N

Summary Labelling experiments with¹⁵N glutamate and¹⁵N alanine were conducted using slices from oat leaves to investigate photorespiratory nitrogen metabolism. It is concluded from the labelling kinetics of glutamine that the refixation of photorespiratory ammonia primarily occurs by glutamine synthetase in the chloroplast. The labelling kinetics of glutamine with¹⁵N glutamate indicate that the chloroplastic and cytoplasmic glutamate pools do not exchange easily in oat leaf cells. Alanine was shown to be an important amino donor for photorespiratory glycine formation. This result is discussed with regard to a possible role of alanine in photorespiration. A modification to the scheme of photorespiratory nitrogen metabolism is proposed.     

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     

Plant peroxisomes respire in the light: some gaps of the photorespiratory C2 cycle have become filled--others remain

The most prominent role of peroxisomes in photosynthetic plant tissues is their participation in photorespiration, a process also known as the oxidative C2 cycle or the oxidative photosynthetic carbon cycle. Photorespiration is an essential process in land plants, as evident from the conditionally lethal phenotype of mutants deficient in enzymes or transport proteins involved in this pathway. The oxidative C2 cycle is a salvage pathway for phosphoglycolate, the product of the oxygenase activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), to the Calvin cycle intermediate phosphoglycerate. The pathway is highly compartmentalized and involves reactions in chloroplasts, peroxisomes, and mitochondria. The H2O2-producing enzyme glycolate oxidase, catalase, and several aminotransferases of the photorespiratory cycle are located in peroxisomes, with catalase representing the major constituent of the peroxisomal matrix in photosynthetic tissues. Although photorespiration is of major importance for photosynthesis, the identification of the enzymes involved in this process has only recently been completed. Only little is known about the metabolite transporters for the exchange of photorespiratory intermediates between peroxisomes and the other organelles involved, and about the regulation of the photorespiratory pathway. This review highlights recent developments in understanding photorespiration and identifies remaining gaps in our knowledge of this important metabolic pathway.     

Analysis of glutamate homeostasis by overexpression of Fd-GOGAT gene in Arabidopsis thaliana

Glutamate plays a central role in nitrogen flow and serves as a nitrogen donor for the production of amino acids. In plants, some amino acids work as buffers: during photorespiration, ammonium derived from the conversion of glycine to serine is promptly reassimilated into glutamate by the glutamine synthetase (GS-2)/ferredoxin-dependent glutamate synthase (Fd-GOGAT) cycle. The glutamate concentration is relatively stable compared with those of other amino acids under environmental changes. The few studies dealing with glutamate homeostasis have but all used knockouts or mutants of these enzymes. Here, we generated Fd-GOGAT (GLU1)-overexpressing Arabidopsis plants to analyze changes in the amino acid pool caused by glutamate overproduction under different ammonium conditions controlled by CO₂ concentration, light intensity and nitrate concentration. Under photorespiratory conditions with sufficient ammonium supply, aspartate increased and glutamine and glycine decreased, but glutamate barely changed. Under non-photorespiratory conditions, however, glutamate and most other amino acids increased. These results suggest that the synthesized glutamate is promptly converted into other amino acids, especially aspartate. In addition, ammonium supply by photorespiration does not limit glutamate biosynthesis, but glutamine and glycine are important. This study will contribute to the understanding of glutamate homeostasis in plants.