Photorespiratory C2 Cycle Research Articles

Assessing phosphoglycolate phosphatase activity in C3 cereals: unveiling insights into photosynthesis and stress response

The article sheds new light on the biochemical and molecular mechanisms that connect plant respiration, photosynthesis, and photorespiration. The activity of the main enzyme of the photorespiratory C2 cycle, a major pathway of plant primary metabolism 2-Phosphoglycolate (2PG) phosphatase (PGLP; EC 3.1.3.18) was studied in the leaves of wheat (Triticum aestivum L. and Triticum durum L.) and barley (Hordeum vulgare L.) genotypes that differ in drought tolerance and belong to the C3 pathway of photosynthesis. The highest PGLP activity was detected in Barakatli 95, Gobustan, Dayanatli and Garabag 22 genotypes. The obtained results revealed that Gobustan and Garabagh 22 genotypes can be cultivated in rainfed and semi-arid regions, and can be used as a starting material for creating stress-tolerant varieties in practical breeding programs. Keywords: Drought stress, 2-Phosphoglycolate phosphatase (PGLP), C3 cereals, plant breeding

Salicylic acid enhances the resistance of Neoporphyra haitanensis to pathogen Vibrio mediterranei 117-T6

The economically important seaweed Porphyra are often infected by various diseases during sea farming. As one of the most important phytohormones in disease resistance, the protective effect of salicylic acid (SA) on Neoporphyra haitanensis during Vibrio mediterranei 117-T6 infection was investigated by biochemical methods and transcriptomic profile analysis. The addition of exogenous SA at a moderate concentration alleviated disease caused by the pathogenic bacteria. SA enhanced the photosynthetic activity of thalli by upregulating genes of the photosynthetic system along with genes associated with carbon fixation. A moderate release of H2O2 stimulated by SA was observed, which may be due to the upregulation of the photorespiratory C2 cycle and the inhibition of catalase activity. SA treatment enhanced the activity and transcription of antioxidant enzymes in the glutathione-ascorbate (GSH-AsA) cycle involved in balancing the redox system. In addition, the levels of multiple carotenoids were increased in N. haitanensis thalli, and genes involved in their synthesis were upregulated. The results suggest that exogenous SA may enhance the defense of N. haitanensis against pathogen attack by the release of H2O2. However, to avoid oxidative stress, alga can establish redox balance by activating the GSH-AsA cycle and by regulating the generation of carotenoids. In summary, this study indicates that N. haitanensis may have evolved mechanisms to respond to SA stimulation and protect against invading pathogens. Our research can provide valuable information for the farming of healthy N. haitanensis.

Genome-Scale Fluxome of Synechococcus elongatus UTEX 2973 Using Transient 13C-Labeling Data.

Synechococcus elongatus UTEX 2973 (Synechococcus 2973) has the shortest reported doubling time (2.1 h) among cyanobacteria, making it a promising platform for the solar-based production of biochemicals. In this meta-analysis, its intracellular flux distribution was recomputed using genome-scale isotopic nonstationary 13C-metabolic flux analysis given the labeling dynamics of 13 metabolites reported in an earlier study. To achieve this, a genome-scale mapping model, namely imSyu593, was constructed using the imSyn617 mapping model for Synechocystis sp. PCC 6803 (Synechocystis 6803) as the starting point encompassing 593 reactions. The flux elucidation revealed nearly complete conversion (greater than 96%) of the assimilated carbon into biomass in Synechococcus 2973. In contrast, Synechocystis 6803 achieves complete conversion of only 86% of the assimilated carbon. This high biomass yield was enabled by the reincorporation of the fixed carbons lost in anabolic and photorespiratory pathways in conjunction with flux rerouting through a nondecarboxylating reaction such as phosphoketolase. This reincorporation of lost CO2 sustains a higher flux through the photorespiratory C2 cycle that fully meets the glycine and serine demands for growth. In accordance with the high carbon efficiency drive, acetyl-coenzyme A was entirely produced using the carbon-efficient phosphoketolase pathway. Comparison of the Synechococcus 2973 flux map with that of Synechocystis 6803 revealed differences in the use of Calvin cycle and photorespiratory pathway reactions. The two species used different reactions for the synthesis of metabolites such as fructose-6-phosphate, glycine, sedoheptulose-7-phosphate, and Ser. These findings allude to a highly carbon-efficient metabolism alongside the fast carbon uptake rate in Synechococcus 2973, which explains its faster growth rate.

Targeted Knockdown of GDCH in Rice Leads to a Photorespiratory-Deficient Phenotype Useful as a Building Block for C4 Rice.

The glycine decarboxylase complex (GDC) plays a critical role in the photorespiratory C2 cycle of C3 species by recovering carbon following the oxygenation reaction of ribulose-1,5-bisphosphate carboxylase/oxygenase. Loss of GDC from mesophyll cells (MCs) is considered a key early step in the evolution of C4 photosynthesis. To assess the impact of preferentially reducing GDC in rice MCs, we decreased the abundance of OsGDCH (Os10g37180) using an artificial microRNA (amiRNA) driven by a promoter that preferentially drives expression in MCs. GDC H- and P-proteins were undetectable in leaves of gdch lines. Plants exhibited a photorespiratory-deficient phenotype with stunted growth, accelerated leaf senescence, reduced chlorophyll, soluble protein and sugars, and increased glycine accumulation in leaves. Gas exchange measurements indicated an impaired ability to regenerate ribulose 1,5-bisphosphate in photorespiratory conditions. In addition, MCs of gdch lines exhibited a significant reduction in chloroplast area and coverage of the cell wall when grown in air, traits that occur during the later stages of C4 evolution. The presence of these two traits important for C4 photosynthesis and the non-lethal, down-regulation of the photorespiratory C2 cycle positively contribute to efforts to produce a C4 rice prototype.

The emerging role of photorespiration and non‐photorespiratory peroxisomal metabolism in pathogen defence

Photorespiration represents one of the major highways of primary plant metabolism and is the most prominent example of metabolic cell organelle integration, since the pathway requires the concerted action of plastidial, peroxisomal, mitochondrial and cytosolic enzymes and organellar transport proteins. Oxygenation of ribulose-1,5-bisphosphate by Rubisco leads to the formation of large amounts of 2-phosphoglycolate, which are recycled to 3-phosphoglycerate by the photorespiratory C2 cycle, concomitant with stoichiometric production rates of H2 O2 in peroxisomes. Apart from its significance for agricultural productivity, a secondary function of photorespiration in pathogen defence has emerged only recently. Here, we summarise literature data supporting the crosstalk between photorespiration and pathogen defence and perform a meta-expression analysis of photorespiratory genes during pathogen attack. Moreover, we screened Arabidopsis proteins newly predicted using machine learning methods to be targeted to peroxisomes, the central H2 O2 -producing organelle of photorespiration, for homologues of known pathogen defence proteins and analysed their expression during pathogen infection. The analyses further support the idea that photorespiration and non-photorespiratory peroxisomal metabolism play multi-faceted roles in pathogen defence beyond metabolism of reactive oxygen species.

Evolution of the biochemistry of the photorespiratory C2 cycle

Oxygenic photosynthesis would not be possible without photorespiration in the present day O2 -rich atmosphere. It is now generally accepted that cyanobacteria-like prokaryotes first evolved oxygenic photosynthesis, which was later conveyed via endosymbiosis into a eukaryotic host, which then gave rise to the different groups of algae and streptophytes. For photosynthetic CO2 fixation, all these organisms use RubisCO, which catalyses both the carboxylation and the oxygenation of ribulose 1,5-bisphosphate. One of the reaction products of the oxygenase reaction, 2-phosphoglycolate (2PG), represents the starting point of the photorespiratory C2 cycle, which is considered largely responsible for recapturing organic carbon via conversion to the Calvin-Benson cycle (CBC) intermediate 3-phosphoglycerate, thereby detoxifying critical intermediates. Here we discuss possible scenarios for the evolution of this process toward the well-defined 2PG metabolism in extant plants. While the origin of the C2 cycle core enzymes can be clearly dated back towards the different endosymbiotic events, the evolutionary scenario that allowed the compartmentalised high flux photorespiratory cycle is uncertain, but probably occurred early during the algal radiation. The change in atmospheric CO2 /O2 ratios promoting the acquisition of different modes for inorganic carbon concentration mechanisms, as well as the evolutionary specialisation of peroxisomes, clearly had a dramatic impact on further aspects of land plant photorespiration.

Current methods for estimating the rate of photorespiration in leaves

Photorespiration is a process that competes with photosynthesis, in which Rubisco oxygenates, instead of carboxylates, its substrate ribulose 1,5-bisphosphate. The photorespiratory metabolism associated with the recovery of 3-phosphoglycerate is energetically costly and results in the release of previously fixed CO2. The ability to quantify photorespiration is gaining importance as a tool to help improve plant productivity in order to meet the increasing global food demand. In recent years, substantial progress has been made in the methods used to measure photorespiration. Current techniques are able to measure multiple aspects of photorespiration at different points along the photorespiratory C2 cycle. Six different methods used to estimate photorespiration are reviewed, and their advantages and disadvantages discussed.

Recent developments in photorespiration research

Photorespiration is the light-dependent release of CO(2) initiated by Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) during oxygenic photosynthesis. It occurs during the biochemical reactions of the photorespiratory C(2) cycle, which is an ancillary metabolic process that allows photosynthesis to occur in oxygen-containing environments. Recent research has identified the genes for many plant photorespiratory enzymes, allowing precise functional analyses by reverse genetics. Similar studies with cyanobacteria disclosed the evolutionary origin of photorespiratory metabolism in these ancestors of plastids.

Regulation of Plant Glycine Decarboxylase byS-Nitrosylation and Glutathionylation

Mitochondria play an essential role in nitric oxide (NO) signal transduction in plants. Using the biotin-switch method in conjunction with nano-liquid chromatography and mass spectrometry, we identified 11 candidate proteins that were S-nitrosylated and/or glutathionylated in mitochondria of Arabidopsis (Arabidopsis thaliana) leaves. These included glycine decarboxylase complex (GDC), a key enzyme of the photorespiratory C(2) cycle in C3 plants. GDC activity was inhibited by S-nitrosoglutathione due to S-nitrosylation/S-glutathionylation of several cysteine residues. Gas-exchange measurements demonstrated that the bacterial elicitor harpin, a strong inducer of reactive oxygen species and NO, inhibits GDC activity. Furthermore, an inhibitor of GDC, aminoacetonitrile, was able to mimic mitochondrial depolarization, hydrogen peroxide production, and cell death in response to stress or harpin treatment of cultured Arabidopsis cells. These findings indicate that the mitochondrial photorespiratory system is involved in the regulation of NO signal transduction in Arabidopsis.

The photorespiratory glycolate metabolism is essential for cyanobacteria and might have been conveyed endosymbiontically to plants

Photorespiratory 2-phosphoglycolate (2PG) metabolism is essential for photosynthesis in higher plants but thought to be superfluous in cyanobacteria because of their ability to concentrate CO(2) internally and thereby inhibit photorespiration. Here, we show that 3 routes for 2PG metabolism are present in the model cyanobacterium Synechocystis sp. strain PCC 6803. In addition to the photorespiratory C2 cycle characterized in plants, this cyanobacterium also possesses the bacterial glycerate pathway and is able to completely decarboxylate glyoxylate via oxalate. A triple mutant with defects in all 3 routes of 2PG metabolism exhibited a high-CO(2)-requiring (HCR) phenotype. All these catabolic routes start with glyoxylate, which can be synthesized by 2 different forms of glycolate dehydrogenase (GlcD). Mutants defective in one or both GlcD proteins accumulated glycolate under high CO(2) level and the double mutant DeltaglcD1/DeltaglcD2 was unable to grow under low CO(2). The HCR phenotype of both the double and the triple mutant could not be attributed to a significantly reduced affinity to CO(2), such as in other cyanobacterial HCR mutants defective in the CO(2)-concentrating mechanism (CCM). These unexpected findings of an HCR phenotype in the presence of an active CCM indicate that 2PG metabolism is essential for the viability of all organisms that perform oxygenic photosynthesis, including cyanobacteria and C3 plants, at ambient CO(2) conditions. These data and phylogenetic analyses suggest cyanobacteria as the evolutionary origin not only of oxygenic photosynthesis but also of an ancient photorespiratory 2PG metabolism.

Long-term response toward inorganic carbon limitation in wild type and glycolate turnover mutants of the cyanobacterium Synechocystis sp. strain PCC 6803.

Concerted changes in the transcriptional pattern and physiological traits that result from long-term (here defined as up to 24 h) limitation of inorganic carbon (C(i)) have been investigated for the cyanobacterium Synechocystis sp. strain PCC 6803. Results from reverse transcription-polymerase chain reaction and genome-wide DNA microarray analyses indicated stable up-regulation of genes for inducible CO(2) and HCO(3)(-) uptake systems and of the rfb cluster that encodes enzymes involved in outer cell wall polysaccharide synthesis. Coordinated up-regulation of photosystem I genes was further found and supported by a higher photosystem I content and activity under low C(i) (LC) conditions. Bacterial-type glycerate pathway genes were induced by LC conditions, in contrast to the genes for the plant-like photorespiratory C2 cycle. Down-regulation was observed for nitrate assimilation genes and surprisingly also for almost all carboxysomal proteins. However, for the latter the observed elongation of the half-life time of the large subunit of Rubisco protein may render compensation. Mutants defective in glycolate turnover (DeltaglcD and DeltagcvT) showed some transcriptional changes under high C(i) conditions that are characteristic for LC conditions in wild-type cells, like a modest down-regulation of carboxysomal genes. Properties under LC conditions were comparable to LC wild type, including the strong response of genes encoding inducible high-affinity C(i) uptake systems. Electron microscopy revealed a conspicuous increase in number of carboxysomes per cell in mutant DeltaglcD already under high C(i) conditions. These data indicate that an increased level of photorespiratory intermediates may affect carboxysomal components but does not intervene with the expression of majority of LC inducible genes.

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.

The photorespiratory Arabidopsis shm1 mutant is deficient in SHM1.

Mitochondrial serine hydroxymethyltransferase (SHMT), combined with glycine decarboxylase, catalyzes an essential sequence of the photorespiratory C2 cycle, namely, the conversion of two molecules of glycine into one molecule each of CO2, NH4+, and serine. The Arabidopsis (Arabidopsis thaliana) mutant shm (now designated shm1-1) is defective in mitochondrial SHMT activity and displays a lethal photorespiratory phenotype when grown at ambient CO2, but is virtually unaffected at elevated CO2. The Arabidopsis genome harbors seven putative SHM genes, two of which (SHM1 and SHM2) feature predicted mitochondrial targeting signals. We have mapped shm1-1 to the position of the SHM1 gene (At4g37930). The mutation is due to a G --> A transition at the 5' splice site of intron 6 of SHM1, causing aberrant splicing and a premature termination of translation. A T-DNA insertion allele of SHM1, shm1-2, and the F1 progeny of a genetic cross between shm1-1 and shm1-2 displayed the same conditional lethal phenotype as shm1-1. Expression of wild-type SHM1 under the control of either the cauliflower mosaic virus 35S or the SHM1 promoter in shm1-1 abrogated the photorespiratory phenotype of the shm mutant, whereas overexpression of SHM2 or expression of SHM1 under the control of the SHM2 promoter did not rescue the mutant phenotype. Promoter-beta-glucuronidase analyses revealed that SHM1 is predominantly expressed in leaves, whereas SHM2 is mainly transcribed in the shoot apical meristem and roots. Our findings establish SHM1 as the defective gene in the Arabidopsis shm1-1 mutant.

Glycine Metabolism in Intact Leaves by in Vivo 13C and 15N Labeling

Solid-state (13)C NMR measurements of intact soybean leaves labeled by (13)CO(2) (at subambient concentrations) show that excess glycine from the photorespiratory C(2) cycle (i.e. glycine not part of the production of glycerate in support of photosynthesis) is either fully decarboxylated or inserted as (13)C-labeled glycyl residues in proteins. This (13)C incorporation in leaf protein, which is uniformly (15)N labeled by (15)NH(4)(15)NO(3), occurs as soon as 2 min after the start of (13)CO(2) labeling. In those leaves with lower levels of available nitrogen (as measured by leaf nitrate and glutamine-glutamate concentrations), the excess glycine is used primarily as glycyl residues in protein.

D-GLYCERATE 3-KINASE, the Last Unknown Enzyme in the Photorespiratory Cycle in Arabidopsis, Belongs to a Novel Kinase Family

D-GLYCERATE 3-KINASE (GLYK; EC 2.7.1.31) catalyzes the concluding reaction of the photorespiratory C2 cycle, an indispensable ancillary metabolic pathway to the photosynthetic C3 cycle that enables land plants to grow in an oxygen-containing atmosphere. Except for GLYK, all other enzymes that contribute to the C2 cycle are known by their primary structures, and the encoding genes have been identified. We have purified and partially sequenced this yet missing enzyme from Arabidopsis thaliana and identified it as a putative kinase-annotated single-copy gene At1g80380. The exclusive catalytic properties of the gene product were confirmed after heterologous expression in Escherichia coli. Arabidopsis T-DNA insertional knockout mutants show no GLYK activity and are not viable in normal air; however, they grow under elevated CO2, providing direct evidence of the obligatory nature of the ultimate step of the C2 cycle. The newly identified GLYK is both structurally and phylogenetically distinct from known glycerate kinases from bacteria and animals. Orthologous enzymes are present in other plants, fungi, and some cyanobacteria. The metabolic context of GLYK activity in fungi and cyanobacteria remains to be investigated.

Evolution of Photosynthetic Metabolism in Terrestrial Plants

Two significant events in the evolution of terrestrial plants are associated with the oxygenation reaction of Rubisco. First, land plants are derived from the evolutionary line of green algae (the Charophytes) that first evolved leaf-type peroxisomes with a high-capacity photorespiratory C2 cycle. Most algae lack or have a limited capacity for photorespiration, and instead excrete excessive levels of photorespiratory metabolites into the external medium. Excretion of photorespiratory intermediates is not an option for terrestrial plants, and an efficient C2 cycle is required to prevent oxygenation products from accumulating to toxic levels. The colonization of land by just the Charophycean line, and its timing, may result from the evolution of novel photorespiratory metabolism to minimize the consequences of RuBP oxygenation. Second, C4 plant evolution likely required atmospheric conditions favoring high rates of photorespiration in C3 plants. During most of the history of land plants, atmospheric CO2 levels were too high to allow for elevated rates of photorespiration. In the past 40 million years, CO2 levels fell low enough such that photorespiration became an important limitation for C3 photosynthesis. Little evidence exists for C4 plants prior to the CO2 decline, but there is sound evidence for C4 photosynthesis beginning 20-30 million years ago. Aridity likely influenced C4 plant evolution by promoting the origin of drought-adapted features in C3 plants such as enlarged bundle sheath cells. These features in turn may have facilitated the development of a photorespiratory CO2 concentrating system, which is considered the first step in the evolution of C4 photosynthesis.

The structural properties of plant peroxisomes and their metabolic significance.

Plant peroxisomes can be isolated by Percoll density gradient centrifugation at high purity and metabolic competence as well as in relatively large quantities. According to biochemical and electrophysiological analyses, plant peroxisomes have recently been shown to differ from other cell organelles in essential structural properties. Unlike mitochondria or plastids, compartmentalization of plant peroxisomal metabolism is in major parts not caused by a boundary function of the membrane but is primarily due to the specific structure of the protein matrix. The enzymes of the photorespiratory C2 cycle of leaf peroxisomes are arranged as multienzyme complexes that allow efficient metabolic channelling with high flux rates and minimum leakage of reactive oxygen species from the organelle. Transfer of metabolites, such as carboxylates, proceeds across the peroxisomal membrane via a porin-like channel, which represents a relatively unspecific but highly efficient transport system. Because all variants of peroxisomes, which all contain only a single boundary membrane, are confronted with the task of transporting a large group of metabolites while preventing the escape of reactive intermediates, it is reasonable to speculate that the unique compartmentalization feature of leaf peroxisomes also applies to peroxisomes from fungi and mammals.