Two alanine aminotranferases link mitochondrial glycolate oxidation to the major photorespiratory pathway in Arabidopsis and rice

The major photorespiratory pathway in higher plants is distributed over chloroplasts, mitochondria, and peroxisomes. In this pathway, glycolate oxidation takes place in peroxisomes. It was previously suggested that a mitochondrial glycolate dehydrogenase (GlcDH) that was conserved from green algae lacking leaf-type peroxisomes contributes to photorespiration in Arabidopsis thaliana. Here, the identification of two Arabidopsis mitochondrial alanine:glyoxylate aminotransferases (ALAATs) that link glycolate oxidation to glycine formation are described. By this reaction, the mitochondrial side pathway produces glycine from glyoxylate that can be used in the glycine decarboxylase (GCD) reaction of the major pathway. RNA interference (RNAi) suppression of mitochondrial ALAAT did not result in major changes in metabolite pools under standard conditions or enhanced photorespiratroy flux, respectively. However, RNAi lines showed reduced photorespiratory CO(2) release and a lower CO(2) compensation point. Mitochondria isolated from RNAi lines are incapable of converting glycolate to CO(2), whereas simultaneous overexpression of GlcDH and ALAATs in transiently transformed tobacco leaves enhances glycolate conversion. Furthermore, analyses of rice mitochondria suggest that the side pathway for glycolate oxidation and glycine formation is conserved in monocotyledoneous plants. It is concluded that the photorespiratory pathway from green algae has been functionally conserved in higher plants.     

Cyanobacterial lactate oxidases serve as essential partners in N2 fixation and evolved into photorespiratory glycolate oxidases in plants

Glycolate oxidase (GOX) is an essential enzyme involved in photorespiratory metabolism in plants. In cyanobacteria and green algae, the corresponding reaction is catalyzed by glycolate dehydrogenases (GlcD). The genomes of N(2)-fixing cyanobacteria, such as Nostoc PCC 7120 and green algae, appear to harbor genes for both GlcD and GOX proteins. The GOX-like proteins from Nostoc (No-LOX) and from Chlamydomonas reinhardtii showed high L-lactate oxidase (LOX) and low GOX activities, whereas glycolate was the preferred substrate of the phylogenetically related At-GOX2 from Arabidopsis thaliana. Changing the active site of No-LOX to that of At-GOX2 by site-specific mutagenesis reversed the LOX/GOX activity ratio of No-LOX. Despite its low GOX activity, No-LOX overexpression decreased the accumulation of toxic glycolate in a cyanobacterial photorespiratory mutant and restored its ability to grow in air. A LOX-deficient Nostoc mutant grew normally in nitrate-containing medium but died under N(2)-fixing conditions. Cultivation under low oxygen rescued this lethal phenotype, indicating that N(2) fixation was more sensitive to O(2) in the Δlox Nostoc mutant than in the wild type. We propose that LOX primarily serves as an O(2)-scavenging enzyme to protect nitrogenase in extant N(2)-fixing cyanobacteria, whereas in plants it has evolved into GOX, responsible for glycolate oxidation during photorespiration.     

Photorespiration in diatoms. Mitochondrial glycolate dehydrogenase in clyindrotheca fusiformis and Nitzschia alba.

Glycolate dehydrogenase activity has been localized in the mitochondria of two marine diatoms. Polarographic data and difference spectra show that the enzyme is linked indirectly to oxygen via the electron transport system. The results presented indicate that the system responsible for the oxidation of glycolic acid in the diatom has evolved along lines distinctly different from glycolate oxidation in higher plants.     

Spindle-shaped archaeal viruses evolved from rod-shaped ancestors to package a larger genome

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Light regulation of leaf mitochondrial pyruvate dehydrogenase complex : role of photorespiratory carbon metabolism

Light-dependent inactivation of mitochondrial pyruvate dehydrogenase complex (mtPDC) in pea (Pisum sativum L.) leaves was further characterized, and this phenomenon was extended to several monocot and dicot species. The light-dependent inactivation of mtPDC in vivo was rapidly reversed in the dark, even after prolonged illumination. The mtPDC can be efficiently cycled through the inactivated-reactivated status by rapid light-dark cycling. Light-dependent inactivation of mtPDC was shown to be suppressed by inhibitors of photorespiratory carbon metabolism, including 2-pyridylhydroxymethane sulfonate, isonicotinic acid hydrazide, and aminoacetonitrile, and by an inhibitor of photosynthesis, 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Glycine fed to pea leaf strips in the dark yielded partially inactivated leaf mtPDC, and this inactivation was blocked by inhibitors of glycine oxidation. It is concluded that the photorespiratory glycine to serine conversion that occurs in C₃ leaf mitochondria can provide the NADH to drive oxidative phosphorylation and subsequent inactivation of mtPDC. Glycine oxidation also produces ammonium ion, which has been shown to enhance the inactivation of mtPDC in vitro by stimulating the pyruvate dehydrogenase kinase that catalyzes the phosphorylation (inactivation) of the mtPDC. Thus, light-dependent, photorespiration-stimulated inactivation of the mtPDC can regulate carbon entry into the Krebs cycle during C₃ photosynthesis.     

Control of photorespiratory glycolate metabolism in an oxygen-resistant mutant of Chlorella sorokiniana

Under a gas atmosphere of 99% O2/1% CO2, wild-type cells of Chlorella sorokiniana excreted 12% of their dry weight as glycolate during photolithotrophic growth, whereas mutant cells excreted glycolate at only 3% of the cellular dry weight. The observed difference in glycolate excretion by the two cell types appears to be due to a different capacity for the metabolism of glycolate, rather than to a different glycolate formation rate. This was concluded from experiments in which the metabolism of glycolate via the glycine-serine pathway was inhibited by the addition of isoniazid. Under such conditions, glycolate excretion rates for both cell types were identical. The mutant appeared to have significantly higher specific activities of glycine decarboxylase, serine hydroxymethyltransferase, serine-glyoxylate aminotransferase, glycerate kinase, and phosphoglycolate phosphatase than did the wild type. The specific activities of D-ribulose-1,5-bisphosphate carboxylase/oxygenase, glycolate dehydrogenase, glyoxylate-aminotransferase, and hydroxypyruvate reductase were the same for wild-type and mutant cells. The internal pool sizes of ammonia and amino acids increased in wild-type cells grown under high-oxygen concentrations but were hardly affected by high oxygen tensions in the mutant cells. Our results indicate that, under the growth conditions applied, the decarboxylation of glycine becomes the rate-limiting step of the glycine-serine pathway for the wild-type cells of C. sorokiniana.     

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.     

Intracellular localization of glycolate dehydrogenase in a blue-green alga

Glycolate dehydrogenase activity was detected in cell-free extracts of Oscillatoria sp. prepared by osmotic lysis of spheroplasts in 0.05 m potassium phosphate buffer, pH 7.5, containing 0.3 m mannitol. Most of the enzyme activity was found in a particulate fraction and localized in the photosynthetic lamellae after centrifugation in a discontinuous sucrose density gradient. Enzyme activity was detected in this fraction both in the presence and absence of the artificial electron acceptor 2,6-dichlorophenolindophenol (DPIP) and a low rate of O(2) uptake was detected in this lamellar fraction. Activity was lost from the lamellar fraction by repeated washing or by treatment with 0.005% Triton X-100 and the solubilized enzyme activity was DPIP-dependent. The data indicate that both glycolate dehydrogenase and its natural electron acceptor are bound to the photosynthetic lamellae in vivo. In contrast, catalase activity was found in the soluble cytoplasmic fraction.     

The occurrence of glycolate dehydrogenase and glycolate oxidase in green plants: an evolutionary survey

Homogenates of various lower land plants, aquatic angiosperms, and green algae were assayed for glycolate oxidase, a peroxisomal enzyme present in green leaves of higher plants, and for glycolate dehydrogenase, a functionally analogous enzyme characteristic of certain green algae. Green tissues of all lower land plants examined (including mosses, liverworts, ferns, and fern allies), as well as three freshwater aquatic angiosperms, contained an enzyme resembling glycolate oxidase, in that it oxidized l- but not d-lactate in addition to glycolate, and was insensitive to 2 mm cyanide. Many of the green algae (including Chlorella vulgaris, previously claimed to have glycolate oxidase) contained an enzyme resembling glycolate dehydrogenase, in that it oxidized d- but not l-lactate, and was inhibited by 2 mm cyanide. Other green algae had activity characteristic of glycolate oxidase and, accordingly, showed a substantial glycolate-dependent O₂ uptake. It is pointed out that this distribution pattern of glycolate oxidase and glycolate dehydrogenase among the green plants may have phylogenetic significance.     

No unique mitochondrial translation products in respiratory chain-linked NADH dehydrogenase

Antibody prepared against beef heart mitochondrial NADH dehydrogenase immunoprecipitated 26 polypeptides from detergent solubilized beef heart mitochondria. All 26 polypeptides co-migrated with those present in the dehydrogenase antigen when resolved side by side on sodium dodecyl sulfateurea polyacrylamide gels. From mixed rat liver-[³⁵S]methionine pulsed hepatoma mitochondria the antibody immunoprecipitated 24 stained liver polypeptides and 19 radio-labelled hepatoma polypeptides. The translation of three of the labelled polypeptides was resistent to inhibition by cycloheximide, indicating these are translated on mitochondrial ribosomes. These same polypeptides, however, wre previously identified as cytochrome c oxidase subunits; and, apparently, non-specifically co-precipitate with dehydrogenase associated polypeptides. We conclude that there are no mitochondrially translated polypeptides specifically associated with NADH dehydrogenase.     

Nuclear glutamate dehydrogenase: unique antigenic determinants and determinants common to the mitochondrial enzyme

An antiserum against glutamate dehydrogenase from ox liver nuclei precipitates both the nuclear and the mitochondrial enzymes, with different equivalence zones. The antibodies of this serum have been fractionated by means of an immunoadsorbent to which mitochondrial glutamate dehydrogenase is covalently linked. After the affinity chromatography, the unretained antibodies had virtually lost the ability to precipitate the mitochondrial enzyme, whereas the retained portion, after elution, precipitated both glutamate dehydrogenases. These findings suggest that nuclear glutamate dehydrogenase contains specific antigenic determinants as well as determinants common to the mitochondrial enzyme, and that only the antibodies against the latter determinants have been selectively removed by the affinity chromatography.     

Thioredoxin h2 contributes to the redox regulation of mitochondrial photorespiratory metabolism

Thioredoxins (TRXs) are important proteins involved in redox regulation of metabolism. In plants, it has been shown that the mitochondrial metabolism is regulated by the mitochondrial TRX system. However, the functional significance of TRX h2, which is found at both cytosol and mitochondria, remains unclear. Arabidopsis plants lacking TRX h2 showed delayed seed germination and reduced respiration alongside impaired stomatal and mesophyll conductance, without impacting photosynthesis under ambient O₂ conditions. However, an increase in the stoichiometry of photorespiratory CO₂ release was found during O₂-dependent gas exchange measurements in trxh2 mutants. Metabolite profiling of trxh2 leaves revealed alterations in key metabolites of photorespiration and in several metabolites involved in respiration and amino acid metabolism. Decreased abundance of serine hydroxymethyltransferase and glycine decarboxylase (GDC) H and L subunits as well as reduced NADH/NAD⁺ ratios were also observed in trxh2 mutants. We further demonstrated that the redox status of GDC-L is altered in trxh2 mutants in vivo and that recombinant TRX h2 can deactivate GDC-L in vitro, indicating that this protein is redox regulated by the TRX system. Collectively, our results demonstrate that TRX h2 plays an important role in the redox regulation of mitochondrial photorespiratory metabolism.     

Improvement of biomass accumulation of potato plants by transformation of cyanobacterial photorespiratory glycolate catabolism pathway genes

Transgenic potato (Solanum tuberosum L. cv. Desiree) plants expressing components of a novel cyanobacterial photorespiratory glycolate catabolism pathway were developed. Transgenic plant expressing glcD1 (glycolate dehydrogenase I) gene was referred to as synGDH and transgenic plants expressing gcl (glyoxylate carboligase) and tsr (tartronic semialdehyde reductase) genes simultaneously were designated as synGT. Both synGDH and synGT plants showed stable gene transformation, integration and expression. Enhanced glyoxylate contents in synGDH plants were detected as compared to synGT and non-transgenic (NT) plants. Phenotypic evaluation revealed that synGDH plants accumulated 11 % higher dry weight, while, tuber weight was 38 and 16 % higher than NT and synGT, respectively. Upon challenging the plants in high temperature and high light conditions synGDH plants maintained higher Fv/Fm and showed less bleaching of chlorophyll as compared to synGT and NT plants. These results indicate that genetic transformation of complete pathway in one plant holds promising outcomes in terms of biomass accumulation to meet future needs for food and energy.     

NADH dehydrogenase of Corynebacterium glutamicum. Purification of an NADH dehydrogenase II homolog able to oxidize NADPH

NADPH oxidase activity, in addition to NADH oxidase activity, has been shown to be present in the respiratory chain of Corynebacterium glutamicum. In this study, we tried to purify NADPH oxidase and NADH dehydrogenase activities from the membranes of C. glutamicum. Both the enzyme activities were simultaneously purified in the same fraction, and the purified enzyme was shown to be a single polypeptide of 55 kDa. The N-terminal sequence of the enzyme was consistent with the sequence deduced from the NADH dehydrogenase gene of C. glutamicum, which has been sequenced and shown to be a homolog of NADH dehydrogenase II. In addition to high NADH-ubiquinone-1 oxidoreductase activity at neutral pH, the purified enzyme showed relatively high NADPH oxidase and NADPH-ubiquinone-1 oxidoreductase activities at acidic pH. Thus, NADH dehydrogenase of C. glutamicum was shown to be rather unique in having a relatively high reactivity toward NADPH.     

Cytochemical localization of glycolate dehydrogenase in mitochondria of chlamydomonas

Mildly disrupted cells of Chlamydomonas reinhardi Dangeard were incubated in a reaction medium containing glycolate, ferricyanide, and cupric ions, and then processed for electron microscopy. As a result of the cytochemical treatment, an electron opaque product was deposited specifically in the outer compartment of mitochondria; other cellular components, including microbodies, did not accumulate stain. Incubation with d-lactate yielded similar results, while treatment with l-lactate produced only a weak reaction. Oxamate, which inhibits glycolate dehydrogenase activity in cell-free extracts, also inhibited the cytochemical reaction. These findings demonstrate in situ that glycolate dehydrogenase is localized in mitochondria, and thus corroborate similar conclusions reached on the basis of enzymic studies of isolated algal organelles.     

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.     

Localization of glycolate dehydrogenase in two species of Dunaliella

The occurrence of glycolate oxidase in addition to glycolate dehydrogenase in Dunaliella salina and D. primolecta, as reported in the literature, could not be confirmed. Both species were demonstrated to possess only glycolate dehydrogenase. After separation of organelles by gradient centrifugation, glycolate dehydrogenase along with hydroxypyruvate reductase was found exclusively in the mitochondria. Thus the peroxisomes from Dunaliella are not of the leaf-type: because of their content of catalase, uricase and hydroxyacyl-CoA dehydrogenase they appear to be of the same type as in Eremosphaera and other chlorophycean algae. No activity of glycolate dehydrogenase was found in the chloroplast fraction when the 2,6-dichlorophenol-indophenol test was used.This work was supported by the Deutsche Forschungsgemeinschaft.