The ancestors of diatoms evolved a unique mitochondrial dehydrogenase to oxidize photorespiratory glycolate

Like other oxygenic photosynthetic organisms, diatoms produce glycolate, a toxic intermediate, as a consequence of the oxygenase activity of Rubisco. Diatoms can remove glycolate through excretion and through oxidation as part of the photorespiratory pathway. The diatom Phaeodactylum tricornutum encodes two proteins suggested to be involved in glycolate metabolism: PtGO1 and PtGO2. We found that these proteins differ substantially from the sequences of experimentally characterized proteins responsible for glycolate oxidation in other species, glycolate oxidase (GOX) and glycolate dehydrogenase. We show that PtGO1 and PtGO2 are the only sequences of P. tricornutum homologous to GOX. Our phylogenetic analyses indicate that the ancestors of diatoms acquired PtGO1 during the proposed first secondary endosymbiosis with a chlorophyte alga, which may have previously obtained this gene from proteobacteria. In contrast, PtGO2 is orthologous to an uncharacterized protein in Galdieria sulphuraria, consistent with its acquisition during the secondary endosymbiosis with a red alga that gave rise to the current plastid. The analysis of amino acid residues at conserved positions suggests that PtGO2, which localizes to peroxisomes, may use substrates other than glycolate, explaining the lack of GOX activity we observe in vitro. Instead, PtGO1, while only very distantly related to previously characterized GOX proteins, evolved glycolate-oxidizing activity, as demonstrated by in gel activity assays and mass spectrometry analysis. PtGO1 localizes to mitochondria, consistent with previous suggestions that photorespiration in diatoms proceeds in these organelles. We conclude that the ancestors of diatoms evolved a unique alternative to oxidize photorespiratory glycolate: a mitochondrial dehydrogenase homologous to GOX able to use electron acceptors other than O₂.     

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.     

Arabidopsis SHMT1, a serine hydroxymethyltransferase that functions in the photorespiratory pathway influences resistance to biotic and abiotic stress

We found that a recessive mutation, shmt1-1 , causes aberrant regulation of cell death resulting in chlorotic and necrotic lesion formation under a variety of environmental conditions. Salicylic acid-inducible genes and genes involved in H₂O₂ detoxification were expressed constitutively in shmt1-1 plants in direct correlation with the severity of the lesions. The shmt1-1 mutants were more susceptible than control plants to infection with biotrophic and necrotrophic pathogens, developing severe infection symptoms in a high percentage of infected leaves. In addition, mutants carrying shmt1-1 or a loss-of-function shmt1-2 allele, were smaller and showed a greater loss of chlorophyll and greater accumulation of H₂O₂ than wild-type plants when subjected to salt stress. SHMT1 was map-based cloned and found to encode a serine hydroxymetyltransferase (SHMT1) involved in the photorespiratory pathway. Our results indicate that this enzymatic activity plays a critical role in controlling the cell damage provoked by abiotic stresses such as high light and salt and in restricting pathogen-induced cell death, supporting the notion that photorespiration forms part of the dissipatory mechanisms of plants to minimize production of reactive oxygen species (ROS) at the chloroplast and to mitigate oxidative damage. Moreover, results shown here indicate that whereas production of ROS is an essential component of the hypersensitive defense response, the excessive accumulation of these toxic compounds impairs cell death containment and counteracts the effectiveness of the plant defenses to restrict pathogen infection.     

Mitochondrial glycolate oxidation contributes to photorespiration in higher plants

The oxidation of glycolate to glyoxylate is an important reaction step in photorespiration. Land plants and charophycean green algae oxidize glycolate in the peroxisome using oxygen as a co-factor, whereas chlorophycean green algae use a mitochondrial glycolate dehydrogenase (GDH) with organic co-factors. Previous analyses revealed the existence of a GDH in the mitochondria of Arabidopsis thaliana (AtGDH). In this study, the contribution of AtGDH to photorespiration was characterized. Both RNA abundance and mitochondrial GDH activity were up-regulated under photorespiratory growth conditions. Labelling experiments indicated that glycolate oxidation in mitochondrial extracts is coupled to CO(2) release. This effect could be enhanced by adding co-factors for aminotransferases, but is inhibited by the addition of glycine. T-DNA insertion lines for AtGDH show a drastic reduction in mitochondrial GDH activity and CO(2) release from glycolate. Furthermore, photorespiration is reduced in these mutant lines compared with the wild type, as revealed by determination of the post-illumination CO(2) burst and the glycine/serine ratio under photorespiratory growth conditions. The data show that mitochondrial glycolate oxidation contributes to photorespiration in higher plants. This indicates the conservation of chlorophycean photorespiration in streptophytes despite the evolution of leaf-type peroxisomes.     

Evidence for a disease-resistance pathway in rice similar to the NPR1-mediated signaling pathway in Arabidopsis

The Arabidopsis NPR1/NIM1 gene is a key regulator of systemic acquired resistance (SAR). Over-expression of NPR1 leads to enhanced resistance in Arabidopsis. To investigate the role of NPR1 in monocots, we over-expressed the Arabidopsis NPR1 in rice and challenged the transgenic plants with Xanthomonas oryzae pv. oryzae (Xoo), the rice bacterial blight pathogen. The transgenic plants displayed enhanced resistance to Xoo. RNA blot hybridization indicates that enhanced resistance requires expression of NPR1 mRNA above a threshold level in rice. To identify components mediating the resistance controlled by NPR1, we used NPR1 as bait in a yeast two-hybrid screen. We isolated four cDNA clones encoding rice NPR1 interactors (named rTGA2.1, rTGA2.2, rTGA2.3 and rLG2) belonging to the bZIP family. rTGA2.1, rTGA2.2 and rTGA2.3 share 75, 76 and 78% identity with Arabidopsis TGA2, respectively. In contrast, rLG2 shares highest identity (81%) to the maize liguleless (LG2) gene product, which is involved in establishing the leaf blade-sheath boundary. The interaction of NPR1 with the rice bZIP proteins in yeast was impaired by the npr1-1 and npr1-2 mutations, but not by the nim1-4 mutation. The NPR1-rTGA2.1 interaction was confirmed by an in vitro pull-down experiment. In gel mobility shift assays, rTGA2.1 binds to the rice RCH10 promoter and to a cis-element required sequence-specifically for salicylic acid responsiveness. This is the first demonstration that the Arabidopsis NPR1 gene can enhance disease resistance in a monocot plant. These results also suggest that monocot and dicot plants share a conserved signal transduction pathway controlling NPR1-mediated resistance.     

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.     

Identification and characterization of the major mitochondrial Fe transporter in rice

The uptake, translocation and compartmentalization of Fe are essential for plant cell function and life cycle. Despite rapid progress in our understanding of Fe homeostasis in plants, Fe transport from the cytoplasm to mitochondria was, until recently, poorly understood. The screening of 3,993 mutant lines for symptoms of Fe deficiency resulted in the identification and characterization of a major mitochondrial Fe transporter (MIT) in rice. MIT was found to localize to mitochondria and to complement the growth of a yeast strain defective in mitochondrial Fe transport. The knockout of MIT resulted in a lethal phenotype, and in knock-down plants, several agronomic characteristics were compromised, such as plant height, average number of tillers, days to flower, fertility and yield. Changes in the expression of genes involved in Fe transport suggested a disturbance of cellular Fe transport. Furthermore, the mitochondrial Fe concentration and the activity of the mitochondrial Fe-S enzyme aconitase were significantly reduced compared with wild-type plants. The identification of MIT is a significant advance in the field of plant Fe nutrition and should facilitate the cloning of paralogs from other plant species.Key words: aconitase, iron, mitochondria, mitochondrial iron transporter, Oryza sativaIron (Fe) is an integral cofactor for several proteins, as its redox state can be readily changed. In plants, Fe is essential for chlorophyll biosynthesis and the synthesis of heme. In mitochondria, including those of plants, Fe is essential for electron transport as well as the synthesis of heme and cytoplasmic Fe-sulfur (Fe-S) cluster-containing proteins.^1–3 Thus, when the mitochondrial Fe supply is limited, the metabolic and respiratory activities of this organelle are impaired, and the supply of Fe-S proteins to the cytoplasm is disrupted.^4,5 However, excess Fe is toxic due to the production of reactive oxygen species (ROS). In mitochondria, where free ROS are generated as a side reaction of electron transport, excess Fe can be particularly harmful.The synthesis of Fe chelators, the regulation of Fe-associated enzymes, and the properties of transporters involved in the absorption and translocation of Fe are well described in references ^6–11; however, the protein responsible for transporting Fe into the mitochondria of plants was only recently identified. We screened a rice T-DNA library consisting of 3,993 independent lines for typical symptoms of Fe deficiency despite the presence of Fe-sufficient conditions. Screening resulted in the identification of a line harboring a T-DNA insertion in the second exon of the mitochondrial Fe transporter (MIT).¹² MIT belongs to the mitochondrial solute carrier (MSC) family, whose members localize to the mitochondrial inner membrane and transport a wide range of substrates, including Fe.¹³ The yeast (Mrs3-Mrs4 ¹⁴) and zebrafish (mitoferrin¹⁵) mitochondrial Fe transporters also belong to the MSC family. Given that the rice genome contains several members of the MSC family, it would have been difficult to identify the mitochondrial Fe transporter through homology search; thus, a screening approach was used. In addition to MIT in rice, we identified putative mitochondrial Fe transporters in Arabidopsis, maize, barley, oat, grapes and castor bean (Fig. 1). Interestingly, unlike yeast, zebra fish and Arabidopsis, rice has only one MIT gene.Open in a separate windowFigure 1Phylogeny of putative plant mitochondrial Fe transporters. ZmMIT 1 (Zea mays): AC {"type":"entrez-protein","attrs":{"text":"F87311","term_id":"25288411","term_text":"pir||F87311"}}F87311; ZmMIT 2: AC {"type":"entrez-nucleotide","attrs":{"text":"G39656","term_id":"3358865","term_text":"G39656"}}G39656; VvMIT (Vitis vinifera): ACA {"type":"entrez-protein","attrs":{"text":"O21380","term_id":"75277206","term_text":"O21380"}}O21380; AtMIT 1: {"type":"entrez-protein","attrs":{"text":"NP_172184","term_id":"15222270","term_text":"NP_172184"}}NP_172184; AtMIT 2: {"type":"entrez-protein","attrs":{"text":"NP_180577","term_id":"15227718","term_text":"NP_180577"}}NP_180577; HVMIT (Hordeum vulgare): {"type":"entrez-protein","attrs":{"text":"BAJ88514","term_id":"326522937","term_text":"BAJ88514"}}BAJ88514; SbMIT (Sorghum bicolor): {"type":"entrez-protein","attrs":{"text":"XP_002468019","term_id":"1205924179","term_text":"XP_002468019"}}XP_002468019; Rc (Ricinus communis): {"type":"entrez-protein","attrs":{"text":"XP_002516353","term_id":"255550607","term_text":"XP_002516353"}}XP_002516353.MIT knock-out results in a lethal phenotype, with defects in several growth characteristics also observed in heterozygous plants. Hydroponically grown MIT knock-down (mit-2) plants accumulated 41 and 51% more Fe than wild-type (WT) plants in their roots and shoots, respectively, and their mitochondrial Fe content was lower. Changes in gene expression, determined through microarray analysis, were consistent with a disruption of cellular Fe homeostasis in mit-2 plants. These results suggested that changes in mitochondrial Fe alter cellular Fe homeostasis. The growth of mit-2 plants was significantly weaker than that of WT plants in terms of chlorophyll content as well as the length and dry weight of the roots and shoots. Furthermore, compared with WT plants, there was a decrease in the activity of the Fe-S protein aconitase in the roots (data not shown), shoots and purified mitochondria of mit-2 plants, suggesting significant effects on Fe-S cluster synthesis in the knock-down plants.¹² The growth of mit-2 plants in soil was also significantly compromised in terms of average number of tillers, plant height, delayed flowering and fertility, such that the yield of mit-2 plants was only 34% of that obtained with WT plants.MIT localized to mitochondria and in yeast was able to complement the growth defect of the Δmrs3Δmrs4 mutant, confirming the protein''s function as a mitochondrial Fe transporter. To understand the role of MIT during germination and seed development in rice, the MIT promoter was used to drive the expression of β-glucuronidase (GUS). GUS was expressed during germination and at all stages of seed development. Gene expression was also observed in the callus, around the basal part of the leaf sheath, and in all leaf tissues except vascular tissue (Fig. 2). A search of the microarray database (RiceXPro;¹⁶ ricexpro.dna.affrc.go.jp/) revealed that MIT expression is not diurnally regulated in the leaf blade, roots or stem. Instead, steady-state expression was observed in those tissues as well as in the anther, ovary, embryo, endosperm and pistil. In the leaf blade, expression slightly decreased from 27 to 76 days after transplantation (DAT) and then increased at 125 DAT (Fig. 3A).Open in a separate windowFigure 2MIT promoter-driven GUS expression. (A) Callus; (B) longitudinal section at the basal part of the leaf sheath; (C–E) leaf transverse sections.Open in a separate windowFigure 3Expression of MIT during the different growth stages of rice. (A) Leaf blade: 27, 76 and 125 days after transplantation (DAT); (B) root: 27 and 76 DAT ; (C) stem: 83 and 90 DAT ; Normalized signal intensity for MIT, derived from spatiotemporal profiling of various tissues and organs (RXP_0001) generated through ricexpro.dna.affrc.go.jp/RXP_0003/index.php. Error bars represent the SD. n = 3. Dotted line: samples collected at midnight; continuous line: samples collected at noon.As Fe is involved in vital mitochondrial processes,^1–3 it is not surprising that a mutation in the mitochondrial Fe transporter is lethal. In zebrafish, a mutation in the mitoferrin gene results in severely hypochromic erythrocytes,¹⁵ whereas defective mitochondrial Fe homeostasis in Arabidopsis is lethal.¹⁷ Moreover, knock-out plants for the mitochondrially synthesized Fe-S cluster exporter exhibit chlorosis.¹⁸It is still not clear in which form Fe is transported by MIT. Arabidopsis ferric chelate reductase 7 (FRO7) localizes to chloroplasts, where it is important for Fe homeostasis.¹⁹ The two members of the Arabidopsis FRO family localize to mitochondria,²⁰ raising the possibility that, as in the chloroplast, Fe(III) is reduced to Fe²⁺ before being transported to mitochondria. However, rice has only two FRO family genes, and in silico analysis revealed that neither localizes to mitochondria.²¹ Thus, the redox state of Fe transported to rice mitochondria remains to be determined.The lethal phenotype of MIT knockout plants and the significantly impaired growth of mit-2 plants highlight the importance of mitochondrial Fe transport in plant growth and development. Not only is the identification of MIT a significant advance in understanding cellular Fe homeostasis in rice, but it may also lead to improved Fe content in this essential grain. Moreover, it will facilitate the identification of mitochondrial Fe transporters in other plant species.     

Integrated temporal regulation of the photorespiratory pathway. Circadian regulation of two Arabidopsis genes encoding serine hydroxymethyltransferase

The photorespiratory pathway is comprised of enzymes localized within three distinct cellular compartments: chloroplasts, peroxisomes, and mitochondria. Photorespiratory enzymes are encoded by nuclear genes, translated in the cytosol, and targeted into these distinct subcellular compartments. One likely means by which to regulate the expression of the genes encoding photorespiratory enzymes is coordinated temporal control. We have previously shown in Arabidopsis that a circadian clock regulates the expression of the nuclear genes encoding both chloroplastic (Rubisco small subunit and Rubisco activase) and peroxisomal (catalase) components of the photorespiratory pathway. To determine whether a circadian clock also regulates the expression of genes encoding mitochondrial components of the photorespiratory pathway, we characterized a family of Arabidopsis serine hydroxymethyltransferase (SHM) genes. We examined mRNA accumulation for two of these family members, including one probable photorespiratory gene (SHM1) and a second gene expressed maximally in roots (SHM4), and show that both exhibit circadian oscillations in mRNA abundance that are in phase with those described for other photorespiratory genes. In addition, we show that SHM1 mRNA accumulates in light-grown seedlings, although this response is probably an indirect consequence of the induction of photosynthesis and photorespiration by illumination.     

A glycolate dehydrogenase in the mitochondria of Arabidopsis thaliana

The fixation of molecular O2 by the oxygenase activity of Rubisco leads to the formation of phosphoglycolate in the chloroplast that is further metabolized in the process of photorespiration. The initial step of this pathway is the oxidation of glycolate to glyoxylate. Whereas in higher plants this reaction takes place in peroxisomes and is dependent on oxygen as a co-factor, most algae oxidize glycolate in the mitochondria using organic co-factors. The identification and characterization of a novel glycolate dehydrogenase in Arabidopsis thaliana is reported here. The enzyme is dependent on organic co-factors and resembles algal glycolate dehydrogenases in its enzymatic properties. Mutants of E. coli incapable of glycolate oxidation can be complemented by overexpression of the Arabidopsis open reading frame. The corresponding RNA accumulates preferentially in illuminated leaves, but was also found in other tissues investigated. A fusion of the N-terminal part of the Arabidopsis glycolate dehydrogenase to red fluorescent protein accumulates in mitochondria when overexpressed in the homologous system. Based on these results it is proposed that the basic photorespiratory system of algae is conserved in higher plants.     

Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana

We introduced the Escherichia coli glycolate catabolic pathway into Arabidopsis thaliana chloroplasts to reduce the loss of fixed carbon and nitrogen that occurs in C(3) plants when phosphoglycolate, an inevitable by-product of photosynthesis, is recycled by photorespiration. Using step-wise nuclear transformation with five chloroplast-targeted bacterial genes encoding glycolate dehydrogenase, glyoxylate carboligase and tartronic semialdehyde reductase, we generated plants in which chloroplastic glycolate is converted directly to glycerate. This reduces, but does not eliminate, flux of photorespiratory metabolites through peroxisomes and mitochondria. Transgenic plants grew faster, produced more shoot and root biomass, and contained more soluble sugars, reflecting reduced photorespiration and enhanced photosynthesis that correlated with an increased chloroplastic CO(2) concentration in the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase. These effects are evident after overexpression of the three subunits of glycolate dehydrogenase, but enhanced by introducing the complete bacterial glycolate catabolic pathway. Diverting chloroplastic glycolate from photorespiration may improve the productivity of crops with C(3) photosynthesis.     

Regulation by amino acids of photorespiratory ammonia and glycolate release from ankistrodesmus in the presence of methionine sulfoximine

Methionine sulfoximine induced release of ammonia from illuminated cells of Ankistrodesmus braunii (Naegeli) Brunnth, in normal air, but less in air enriched to 3% CO₂. In normal air, methionine sulfoximine also induced glycolate release. Addition of either glutamate, glycine, or serine suppressed glycolate release, whereas glutamate and glycine at the same time stimulated ammonia release. The results indicate that inhibition of glutamine synthetase and thereby inhibition of photorespiratory nitrogen cycling restricts the sink capacity for glycolate in the photorespiratory carbon cycle. An external supply of glutamate, glycine, or serine seems to stimulate glyoxylate transamination and thus partly restores the sink capacity. Calculations of total glycolate formation rates in air from glycolate and ammonia release rates in the presence of methionine sulfoximine and glutamate revealed values of approximately 20 micromoles glycolate per milligram chlorophyll per hour on the average. Similar calculations led to an estimated rate of photorespiratory ammonia release in air, in the absence of methionine sulfoximine, of about 10 micromoles per milligram chlorophyll per hour on the average, a value comparable to the primary nitrogen assimilation rate of 8 micromoles per milligram chlorophyll per hour.     

The glycolate pathway and photosynthetic competence in euglena

The development of glycolate pathway enzymes has been determined in relation to photosynthetic competence during the regreening of Euglena cultures. Phosphoglycolate phosphatase and glycolate dehydrogenase rapidly reached maximal levels of activity but the complete development of ribulose 1,5-diphosphate carboxylase and concomitant photosynthetic carbon dioxide fixation were not attained until 72 hours of illumination. Specific inhibitors of protein synthesis showed that the formation of ribulose 1,5-diphosphate carboxylase in both division-synchronized and regreening cultures was prevented by both cycloheximide and d-threo-chloramphenicol, whereas phosphoglycolate phosphatase formation was only inhibited by d-threo-chloramphenicol but not by l-threo-chloramphenicol or cycloheximide. Since cycloheximide prevented ribulose diphosphate carboxylase synthesis and photosynthetic carbon dioxide fixation without affecting phosphoglycolate phosphatase synthesis during regreening, it was concluded that photosynthetic competence was not necessary for the development of the glycolate pathway enzymes. The inhibition of phosphoglycolate phosphatase synthesis by d-threo-chloramphenicol but not by l-threo-chloramphenicol or cycloheximide shows that the enzyme was synthesized exclusively on chloroplast ribosomes, whereas protein synthesis on both chloroplast and cytoplasmic ribosomes was required for the formation of ribulose 1,5-diphosphate carboxylase. Although light is required for the development of both Calvin cycle and glycolate pathway enzymes during regreening it is concluded that the two pathways are not coordinately regulated.     

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.     

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.     

Identification of the photorespiratory 2-phosphoglycolate phosphatase, PGLP1, in Arabidopsis

The chloroplastidal enzyme 2-phosphoglycolate phosphatase (PGLP), PGLP1, catalyzes the first reaction of the photorespiratory C(2) cycle, a major pathway of plant primary metabolism. Thirteen potential PGLP genes are annotated in the Arabidopsis (Arabidopsis thaliana) genome; however, none of these genes has been functionally characterized, and the gene encoding the photorespiratory PGLP is not known. Here, we report on the identification of the PGLP1 gene in a higher plant and provide functional evidence for a second, nonphotorespiratory PGLP, PGLP2. Two candidate genes, At5g36700 (AtPGLP1) and At5g47760 (AtPGLP2), were selected by sequence similarity to known PGLPs from microorganisms. The two encoded proteins were overexpressed in Escherichia coli and both show PGLP activity. T-DNA knockout of one of these genes, At5g36700, results in very low leaf PGLP activity. The mutant is unviable in normal air but grows well in air enriched with 0.9% CO(2). In contrast, deletion of At5g47760 does not result in a visible phenotype, and leaf PGLP activity is unaltered. Sequencing of genomic DNA from another PGLP-deficient mutant revealed a combined missense and missplicing point mutation in At5g36700. These combined data establish At5g36700 as the gene encoding the photorespiratory PGLP, PGLP1.