Role of asparagine in the photorespiratory nitrogen metabolism of pea leaves

In pea leaves, much of the metabolism of imported asparagine is by transamination. This activity was previously shown to be localized in the peroxisomes, suggesting a possible connection between asparagine and photorespiratory nitrogen metabolism. This was investigated by examination of the transfer of ¹⁵N from the amino group of asparagine, supplied via the transpiration stream, in fully expanded pea leaves. Label was transferred to aspartate, glutamate, alanine, glycine, serine, ammonia, and glutamine (amide group). Under low oxygen (1.8%), or in the presence of α-hydroxy-2-pyridine methanesulfonic acid (an inhibitor of glycolate oxidase, a step in the photorespiratory formation of glyoxylate), there was a substantial (60-80%) decrease in transfer of label to glycine, serine, ammonia, and glutamine. Addition of isonicotinyl hydrazide (an inhibitor of formation of serine from glycine) caused a 70% decrease in transfer of asparagine amino nitrogen to serine, ammonia, and glutamine, while a 4-fold increase in labeling of glycine was observed. The results demonstrate the involvement of asparagine in photorespiration, and show that photorespiratory nitrogen metabolism is not a closed cyclic process.     

Genetic manipulation of glycine decarboxylation

The glycine-serine interconversion, catalysed by glycine decarboxylase and serine hydroxymethyltransferase, is an important reaction of primary metabolism in all organisms including plants, by providing one-carbon units for many biosynthetic reactions. In plants, in addition, it is an integral part of the photorespiratory metabolic pathway and produces large amounts of photorespiratory CO(2) within mitochondria. Although controversial, there is significant evidence that this process, by the relocation of glycine decarboxylase within the leaves from the mesophyll to the bundle-sheath, contributed to the evolution of C(4) photosynthesis. In this review, some aspects of current knowledge about glycine decarboxylase and serine hydroxymethyltransferase and the role of these enzymes in metabolism, about the corresponding genes and their expression as well as about mutants and anti-sense plants related to these genes or processes will be summarized and discussed. From a comparison of the available information about the number and organization of GDC and SHMT genes in the genomes of Arabidopsis thaliana and Oryza sativa it appears that these and, possibly, other genes related to photorespiration, are similarly organized even in only very distantly related angiosperms.     

水稻叶片生育过程中RubisCO活性与光合、光呼吸的关系

水稻生育过程中,ＲｕＢＰ羧化酶活性与光合速率、ＲｕＢＰ加氧酶活性与光呼吸速率、ＲｕＢＰ羧化酶活性与加氢酶活性以及光合速率与光呼吸速率之间是相关的。籼型品种与粳型品种间酶活性的高低及光合、光呼吸速率的高低基本一致,籼型三系杂交稻（Ｆ１）无明显的光合优势。酶的羧化活性的高低只在一定范围内与光合速率的高低平行。在正常生育条件下,酶蛋白的数量不是水稻光合速率的限制因子。     

Aminotransfer from Alanine and Glutamate to Glycine and Serine during Photorespiration in Oat Leaves

¹⁵N-Labeled glutamate and alanine were used to examine the photorespiratory nitrogen metabolism in oat (Avena sativa L.) leaf slices. Glutamate and alanine supply amino groups for glycine formation during photorespiration. The nitrogen flux from alanine to glycine was estimated to be 3 times higher than that from glutamate. It is concluded from these results that alanine is a direct and important amino donor for photorespiratory glycine formation in oat leaves. The ¹⁵N labeling of serine was almost as high as that of glycine during the initial period of the labeling experiments. Thereafter, the ratio of ¹⁵N label in serine to ¹⁵N label in glycine declined substantially.     

Comparison of the effectiveness of glycolic Acid and glycine as substrates for photorespiration

Considerable evidence exists that the carboxyl-carbon atom of glycolic acid is the primary source of the CO₂ produced during photorespiration by leaves of many species of plants, including tobacco. Experiments were conducted to determine whether glyoxylate or glycine, both products of glycolic acid metabolism, is the more immediate precursor of photorespiratory CO₂.     

Photorespiratory metabolism of glyoxylate and formate in glycine-accumulating mutants of barley and Amaranthus edulis

Glycine-accumulating mutants of barley (Hordeum vulgare L.) and Amaranthus edulis (Speg.), which lack the ability to decarboxylate glycine by glycine decarboxylase (GDC; EC 2.1.2.10), were used to study the significance of an alternative photorespiratory pathway of serine formation. In the normal photorespiratory pathway, 5,10-methylenetetrahydrofolate is formed in the reaction catalysed by GDC and transferred to serine by serine hydroxymethyltransferase. In an alternative pathway, glyoxylate could be decarboxylated to formate and formate could be converted into 5,10-methylenetetrahydrofolate in the C1-tetrahydrofolate synthase pathway. In contrast to wild-type plants, the mutants showed a light-dependent accumulation of glyoxylate and formate, which was suppressed by elevated (0.7%) CO₂ concentrations. After growth in air, the activity and amount of 10-formyltetrahydrofolate synthetase (FTHF synthetase; EC 6.3.4.4), the first enzyme of the conversion of formate into 5,10-methylenetetrahydrofolate, were increased in the mutants compared to the wild types. A similar increase in FTHF synthetase could be induced by incubating leaves of wild-type plants with glycine under illumination, but not in the dark. Experiments with ¹⁴C showed that the barley mutants incorporated [¹⁴C]formate and [2-¹⁴C]glycollate into serine. Together, the accumulation of glyoxylate and formate under photorespiratory conditions, the increase in FTHF synthetase and the ability to utilise formate and glycollate for the formation of serine indicate that the mutants are able partially to compensate for the lack of GDC activity by bypassing the normal photorespiratory pathway. Received: 14 August 1998 / Accepted: 30 September 1998     

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.     

Evidence for the presence of photorespiration in desiccation-sensitive leaves of the C4 'resurrection' plant Sporobolus stapfianus during dehydration stress

The possible role of photorespiration as a general stress protectionmechanism, and in C₄ plant metabolism, is controversial. Inparticular, the potential involvement of photorespiration inthe acquisition of desiccation tolerance in ‘resurrection’plants is unknown. An investigation was carried out into whetherphotorespiration is present in leaves of the C₄ resurrectionplant Sporobolus stapfianus Gandoger (Poaceae) and whether itfunctions as a mechanism of stress resistance in the desiccation-tolerantyounger leaves (YL) of this plant. It is shown that the enzymesinvolved in the photorespiratory pathway maintain their activityuntil 88% relative water content (RWC) in both YL and desiccation-sensitiveolder leaves (OL). In subsequent stages of dehydration stress,the enzymatic activity declined similarly in both YL and OL.The content of the phorespiratory metabolite, serine, and ethanolamine,a direct product of serine decarboxylation, is higher in theearly stages of dehydration (88% RWC) in OL, suggesting a transientlyenhanced photorespiratory activity in these leaves. This wasconfirmed by simultaneous gas exchange and fluorescence measurements,showing suppression of the electron transport rate in OL exposedto non-photorespiratory conditions (2% O₂) at 85% RWC. It isconcluded that a higher photorespiratory electron transportoccurs in desiccation-sensitive OL, and it is therefore proposedthat the capacity to scavenge excess electrons through photorespirationdoes not contribute to protect leaves of the desiccation-tolerantYL of S. stapfianus during the stress. Key words: Ethanolamine, glycine, photorespiratory enzymes, photosynthesis, poikilohydric plant, serine Received 5 June 2007; Revised 3 September 2007 Accepted 17 September 2007     

The biochemistry and cell biology of photorespiration

Since the discovery of RuBP oxygenase activity more than a decade ago, our understanding of the sequence of metabolic events associated with photorespiratory activity in C₃ plants has matured considerably. A coherent model of photorespiratory metabolism has been substantiated by a wide variety of experimental approaches and most photorespiratory phenomena can be satisfactorily explained. By contrast, the issues pertaining to the regulation of photorespiratory activity by genetic or chemical means have not been resolved. In addition, the wealth of biochemical, physiological, and genetic information available about photorespiratory metabolism offers as yet unexploited opportunities to investigate the complex cellular processes which organize and coordinate a series of reactions in three distinct metabolic compartments in leaf cells.     

Stereochemical determination of carbon partitioning between photosynthesis and photorespiration in C3 plants: use of (3R)-D-[3-3H1, 3-14C]glyceric acid

When (3R)-D-[3-3H1,3-14C]glyceric acid is supplied in tracer amounts to illuminated tobacco leaf discs, the acid penetrates to the chloroplasts without loss of 3H, and is phosphorylated there. Subsequent metabolism associated with the reductive photosynthetic cycle fully conserves 3H. Oxidation of ribulose bisphosphate (RuBP) by RuBP carboxylase-oxygenase (EC 4.1.1.39) results in the formation of (2R)-[2-3H1, 14C]glycolic acid which, on oxidation by glycolate oxidase (EC 1.1.3.1), releases 3H to water. Loss of 3H from the combined photosynthetic and photorespiratory systems is, therefore, associated with the oxidative photorespiratory loop. Assuming steady-state conditions and a basic metabolic model, the fraction of RuBP oxidized and the photorespiratory carbon flux relative to gross or net CO2 fixation can be calculated from the fraction of supplied 3H retained in the triose phosphates exported from the chloroplasts. This retention can be determined from the 3H:14C ratio for glucose obtained from isolated sucrose. The dependence of 3H retention upon O2 and CO2 concentrations can be deduced by assuming simple competitive kinetics for RuBP carboxylase-oxygenase. The experimental results confirmed the stereochemical assumptions made. Under conditions of negligible photorespiration 3H retention was essentially complete. The change in 3H retention with O2 and CO2 concentrations were investigated. For leaf discs (upper surface up) in normal air, it was estimated that 39% of the RuBP was oxidized, 32% of the fixed CO2 was photorespired, and the photorespiration rate was 46% of the net photosynthetic CO2 fixation rate. These are minimal estimates, as it is assumed that the only source of photorespired CO2 is glycine decarboxylation.     

A possible role for abscisic Acid in regulation of photosynthetic and photorespiratory carbon metabolism in barley leaves

The influence of abscisic acid (ABA) on carbon metabolism, rate of photorespiration, and the activity of the photorespiratory enzymes ribulose bisphosphate oxygenase and glycolate oxidase in 7-day-old barley seedlings (Hordeum vulgare L. var. Alfa) was investigated. Plants treated with ABA had enhanced incorporation of labeled carbon from ¹⁴CO₂ into glycolic acid, glycine, and serine, while ¹⁴C incorporation into 3-phosphoglyceric acid and sugarphosphate esters was depressed. Parallel with this effect, treated plants showed a rise in activity of RuBP oxygenase and glycolic acid oxidase. The rate of photorespiration was increased twofold by ABA treatment at IO⁻⁶ molar while the CO₂-compensation point increased 46% and stomatal resistance increased more than twofold over control plants.     

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.     

Evolution of enzymes involved in the photorespiratory 2-phosphoglycolate cycle from cyanobacteria via algae toward plants

The photorespiratory pathway was shown to be essential for organisms performing oxygenic photosynthesis, cyanobacteria, algae, and plants, in the present day O(2)-containing atmosphere. The identification of a plant-like 2-phosphoglycolate cycle in cyanobacteria indicated that not only genes of oxygenic photosynthesis but also genes encoding photorespiratory enzymes were endosymbiotically conveyed from ancient cyanobacteria to eukaryotic oxygenic phototrophs. Here, we investigated the origin of the photorespiratory pathway in photosynthetic eukaryotes by phylogenetic analysis. We found that a mixture of photorespiratory enzymes of either cyanobacterial or α-proteobacterial origin is present in algae and higher plants. Three enzymes in eukaryotic phototrophs clustered closely with cyanobacterial homologs: glycolate oxidase, glycerate kinase, and hydroxypyruvate reductase. On the other hand, the mitochondrial enzymes of the photorespiratory cycle in algae and plants, glycine decarboxylase subunits and serine hydroxymethyltransferase, evolved from proteobacteria. Other than most genes for proteins of the photosynthetic machinery, nearly all enzymes involved in the 2-phosphogylcolate metabolism coexist in the genomes of cyanobacteria and heterotrophic bacteria.     

The role of Pi recycling processes during photosynthesis in phosphate-deficient bean plants

Bean plants (Phaseolus vulgaris L. cv. Zlota Saxa) were grownon complete (control plants) and phosphate-deficient (low-Pplants) culture solutions for 17 d. Phosphate deficiency markedlyreduced leaf growth, but only slightly decreased the photosynthesisrate. The intensity of reactions releasing inorganic ortho-phosphateduring photosynthesis was examined. In the leaves of low-P plantsthe pools of photorespiratory metabolites (glycolate and glycine+serine)were markedly increased. At the same time synthesis of solublesugars from intermediates of glycolic acid cycle was probablyenhanced. In low-P leaves the phosphoenolpyruvate carboxylaseactivity and malate synthesis were increased. Phosphoenolpyruvateand malate were effectively used for amino acid synthesis. Bothaspartate and alanine accumulation was twice higher in low-Pleaves. It was found that no enhancement in starch and sucrosesynthesis rate takes place in phosphate deficient bean leaves.Modifications of photosynthetic metabolism observed under moderatephosphate deficiency facilitate plants acclimation to low-Pconditions by enhancement of P₁ recirculation during glycolicand phosphoenolpyruvate metabolism. Key words: Phosphate deficiency, photosynthesis, photorespiration, phosphoenolpyruvate carboxylase     

Source of glycolate and cyclic changes in photosynthetic and photorespiratory activity during the development of barley leaves

¹⁴CO₂ assimilation, RuBP earboxylase and PEP carboxylase activities show cyclic changes during the development of barley leaves. Cyclic changes, but in phase opposition with respect to carboxylating enzymes, are shown by RuBP oxygenase, phosphoglycolate phosphatase, glycolate oxidase and nitrate reductase activities. The oxygenase function of RuBP carboxylase appears to be the primary source of glycolate in young leaves, whereas in old ones glycolate could be supplied from some source in addition to RuBP oxygenase activity.     

Photorespiratory nitrogen cycle – A critical evaluation

The photorespiratory nitrogen cycle proposed by Keys et al. (Nature 275: 741–743, 1978) involved formation of glycine by transamination of glyoxylate in the peroxisomes utilizing glutamate. Subsequently, glycine is oxidized to ammonia, serine and CO₂ in the mitochondria. The ammonia is reassimilated via the GS/GOGAT pathway generating glutamate. In this article, experimental evidence which suggests the occurrence of alternative mechanisms of glycolate and serine synthesis as well as of CO₂ and ammonia evolution is discussed. The problem of utilization of NADH coupled to ATP synthesis during photosynthesis is still unresolved, which complicates the glycine oxidation reaction in light. Further, factors are presented that determine the availability of amino donors in the peroxisomes and of amino acids viz., glycine, serine and glutamate for the operation of the photorespiratory N cycle. Recent evidence regarding the role of formate arising out of the reaction of glyoxylate with H₂O₂ in the regulation of photosynthetic electron flow in the Hill reaction, as well as of photorespiratory substrates functioning as carbon sources for the citric acid cycle in the light or for export to the growing tissues, suggests that the role of photo-respiration in plant metabolism needs to be reexamined.     

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     

13C nuclear magnetic resonance detection of interactions of serine hydroxymethyltransferase with C1-tetrahydrofolate synthase and glycine decarboxylase complex activities in Arabidopsis.

In C3 plants, serine synthesis is associated with photorespiratory glycine metabolism involving the tetrahydrofolate (THF)-dependent activities of the glycine decarboxylase complex (GDC) and serine hydroxymethyl transferase (SHMT). Alternatively, THF-dependent serine synthesis can occur via the C1-THF synthase/SHMT pathway. We used 13C nuclear magnetic resonance to examine serine biosynthesis by these two pathways in Arabidopsis thaliana (L.) Heynh. Columbia wild type. We confirmed the tight coupling of the GDC/ SHMT system and observed directly in a higher plant the flux of formate through the C1-THF synthase/SHMT system. The accumulation of 13C-enriched serine over 24 h from the GDC/SHMT activities was 4-fold greater than that from C1-THF synthase/SHMT activities. Our experiments strongly suggest that the two pathways operate independently in Arabidopsis. Plants exposed to methotrexate and sulfanilamide, powerful inhibitors of THF biosynthesis, reduced serine synthesis by both pathways. The results suggest that continuous supply of THF is essential to maintain high rates of serine metabolism. Nuclear magnetic resonance is a powerful tool for the examination of THF-mediated metabolism in its natural cellular environment.     

The use of 3-amino-1,2,4-triazole to investigate the short-term effects of oxygen toxicity on carbon assimilation by Pisum sativum seedlings

To study the in vivo short-term effect of hydrogen peroxide on plant metabolism, 2 mol m^?3 3-amino-1,2,4-triazole, a catalase inhibitor, was applied through the transpiration stream to Pisum sativum seedlings, and gas exchange characteristics, ascorbate peroxidase, glutathione reductase and catalase activities, and levels of hydrogen peroxide and formate were determined. Carbon dioxide assimilation rates were inhibited after the addition of aminotriazole: photorespiratory conditions exacerbated this inhibition. Carbon dioxide response curves showed that aminotriazole reduced both the RuBP regeneration rate and the efficiency of the carboxylation reaction of Rubisco. Catalase activity was completely inhibited 200 min after the application of this inhibitor, but no concomitant increase in H₂O₂ concentration was found. Under enhanced photorespiratory conditions, H₂O₂ concentrations increased. This suggests that under normal environmental conditions hydrogen peroxide is metabolized via alternative mechanisms. The aminotriazole treatment had no effect on the ascotbate peroxidase and glutathione reductase activities, but caused a substantial increase in the formate pool size. These results suggest that hydrogen peroxide is metabolized by reacting with glyoxylate to produce formate and CO₂. The increased production of formate may reduce the flow of carbon through the normal photorespiratory pathway and may also be used anaplerotically as a precursor of products of 1-C metabolism other than serine. This would prevent the return of photorespiratory carbon to the RPP pathway, leading to a smaller RuBP pool size which would in turn result in a decrease in carboxylation conductance (carboxylation efficiency) and regeneration rate of RuBP.     

Fixation of O(2) during Photorespiration: Kinetic and Steady-State Studies of the Photorespiratory Carbon Oxidation Cycle with Intact Leaves and Isolated Chloroplasts of C(3) Plants

Mass spectrometric techniques were used to trace the incorporation of [¹⁸O]oxygen into metabolites of the photorespiratory pathway. Glycolate, glycine, and serine extracted from leaves of the C₃ plants, Spinacia oleracea L., Atriplex hastata, and Helianthus annuus which had been exposed to [¹⁸O]oxygen at the CO₂ compensation point were heavily labeled with ¹⁸O. In each case one, and only one of the carboxyl oxygens was labeled. The abundance of ¹⁸O in this oxygen of glycolate reached 50 to 70% of that of the oxygen provided after only 5 to 10 seconds exposure to [¹⁸O]oxygen. Glycine and serine attained the same final enrichment after 40 and 180 seconds, respectively. This confirms that glycine and serine are synthesized from glycolate.

The labeling of photorespiratory intermediates in intact leaves reached a mean of 59% of that of the oxygen provided in the feedings. This indicates that at least 59% of the glycolate photorespired is synthesized with the fixation of molecular oxygen. This estimate is certainly conservative owing to the dilution of labeled oxygen at the site of glycolate synthesis by photosynthetic oxygen. We examined the yield of ¹⁸O in glycolate synthesized in vitro by isolated intact spinach chloroplasts in a system which permitted direct sampling of the isotopic composition of the oxygen at the site of synthesis. The isotopic enrichment of glycolate from such experiments was 90 to 95% of that of the oxygen present during the incubation.

The carboxyl oxygens of 3-phosphoglycerate also became labeled with ¹⁸O in 20- and 40-minute feedings with [¹⁸O]oxygen to intact leaves at the CO₂ compensation point. Control experiments indicated that this label was probably due to direct synthesis of 3-phosphoglycerate from glycolate during photorespiration. The mean enrichment of 3-phosphoglycerate was 14 ± 4% of that of glycine or serine, its precursors of the photorespiratory pathway, in 10 separate feeding experiments. It is argued that this constant dilution of label indicates a constant stoichiometric balance between photorespiratory and photosynthetic sources of 3-phosphoglycerate at the CO₂ compensation point.

Oxygen uptake sufficient to account for about half of the rate of ¹⁸O fixation into glycine in the intact leaves was observed with intact spinach chloroplasts. Oxygen uptake and production by intact leaves at the CO₂ compensation point indicate about 1.9 oxygen exchanged per glycolate photorespired. The fixation of molecular oxygen into glycolate plus the peroxisomal oxidation of glycolate to glyoxylate and the mitochondrial conversion of glycine to serine can account for up to 1.75 oxygen taken up per glycolate.

These studies provide new evidence which supports the current formulation of the pathway of photorespiration and its relation to photosynthetic metabolism. The experiments described also suggest new approaches using stable isotope techniques to study the rate of photorespiration and the balance between photorespiration and photosynthesis in vivo.