Michael Hodges

Photorespiration is an essential high flux metabolic pathway that is found in all oxygen-producing photosynthetic organisms. It is often viewed as a closed metabolic repair pathway that serves to detoxify 2-phosphoglycolic acid and to recycle carbon to fuel the Calvin-Benson cycle. However, this view is too simplistic since the photorespiratory cycle is known to interact with several primary metabolic pathways, including photosynthesis, nitrate assimilation, amino acid metabolism, C₁ metabolism and the Krebs (TCA) cycle. Here we will review recent advances in photorespiration research and discuss future priorities to better understand (i) the metabolic integration of the photorespiratory cycle within the complex network of plant primary metabolism and (ii) the importance of photorespiration in response to abiotic and biotic stresses.

Photorespiration is one of the major carbon metabolism pathways in oxygen-producing photosynthetic organisms. This pathway recycles 2-phosphoglycolate (2-PG), a toxic metabolite, to 3-phosphoglycerate when ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) uses oxygen instead of carbon dioxide. The photorespiratory cycle is in competition with photosynthetic CO₂ fixation and it is accompanied by carbon, nitrogen and energy losses. Thus, photorespiration has become a target to improve crop yields. Moreover, during the photorespiratory cycle intermediate metabolites that are toxic to Calvin–Benson cycle and RuBisCO activities, such as 2-PG, glycolate and glyoxylate, are produced. Thus, the presence of an efficient 2-PG/glycolate/glyoxylate ‘detoxification’ pathway is required to ensure normal development of photosynthetic organisms. Here we review our current knowledge concerning the enzymes that carry out the glycolate–glyoxylate metabolic steps of photorespiration from glycolate production in the chloroplasts to the synthesis of glycine in the peroxisomes. We describe the properties of the proteins involved in glycolate–glyoxylate metabolism in Archaeplastida and the phenotypes observed when knocking down/out these specific photorespiratory players. Advances in our understanding of the regulation of glycolate–glyoxylate metabolism are highlighted.

Metabolic and physiological analyses of Arabidopsis thaliana glycolate oxidase (GOX) mutant leaves were performed to understand the development of the photorespiratory phenotype after transfer from high CO₂ to air. We show that two Arabidopsis genes, GOX1 and GOX2, share a redundant photorespiratory role. Air-grown single gox1 and gox2 mutants grew normally and no significant differences in leaf metabolic levels and photosynthetic activities were found when compared with wild-type plants. To study the impact of a highly reduced GOX activity on plant metabolism, both GOX1 and GOX2 expression was knocked-down using an artificial miRNA strategy. Air-grown amiRgox1/2 plants with a residual 5% GOX activity exhibited a severe growth phenotype. When high-CO₂-grown adult plants were transferred to air, the photosynthetic activity of amiRgox1/2 was rapidly reduced to 50% of control levels, and a high non-photochemical chlorophyll fluorescence quenching was maintained. ¹³C-labeling revealed that daily assimilated carbon accumulated in glycolate, leading to reduced carbon allocation to sugars, organic acids, and amino acids. Such changes were not always mirrored in leaf total metabolite levels, since many soluble amino acids increased after transfer, while total soluble protein, RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), and chlorophyll amounts decreased in amiRgox1/2 plants. The senescence marker, SAG12, was induced only in amiRgox1/2 rosettes after transfer to air. The expression of maize photorespiratory GOX in amiRgox1/2 abolished all observed phenotypes. The results indicate that the inhibition of the photorespiratory cycle negatively impacts photosynthesis, alters carbon allocation, and leads to early senescence in old rosette leaves.

Summary: Metabolic and physiological analyses of glutamate:glyoxylate aminotransferase 1 (GGT1) mutants were performed at the global leaf scale to elucidate the mechanisms involved in their photorespiratory growth phenotype. Air‐grown ggt1 mutants showed retarded growth and development, that was not observed at high CO₂ (3000 μL L⁻¹). When compared to wild‐type (WT) plants, air‐grown ggt1 plants exhibited glyoxylate accumulation, global changes in amino acid amounts including a decrease in serine content, lower organic acid levels, and modified ATP/ADP and NADP⁺/NADPH ratios. When compared to WT plants, their net CO₂ assimilation rates (A_n) were 50% lower and this mirrored decreases in ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) contents. High CO₂‐grown ggt1 plants transferred to air revealed a rapid decrease of A_n and photosynthetic electron transfer rate while maintaining a high energetic state. Short‐term (a night period and 4 h of light) transferred ggt1 leaves accumulated glyoxylate and exhibited low serine contents, while other amino acid levels were not modified. RuBisCO content, activity and activation state were not altered after a short‐term transfer while the ATP/ADP ratio was lowered in ggt1 rosettes. However, plant growth and RuBisCO levels were both reduced in ggt1 leaves after a long‐term (12 days) acclimation to air from high CO₂ when compared to WT plants. The data are discussed with respect to a reduced photorespiratory carbon recycling in the mutants. It is proposed that the low A_n limits nitrogen‐assimilation, this decreases leaf RuBisCO content until plants attain a new homeostatic state that maintains a constant C/N balance and leads to smaller, slower growing plants.

Photorespiration allows the recycling of carbon atoms of 2‐phosphoglycolate produced by ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) oxygenase activity, as well as the removal of potentially toxic metabolites. The photorespiratory pathway takes place in the light, encompasses four cellular compartments and interacts with several other metabolic pathways and functions. Therefore, the regulation of this cycle is probably of paramount importance to plant metabolism, however, our current knowledge is poor. To rapidly respond to changing conditions, proteins undergo a number of different post‐translational modifications that include acetylation, methylation and ubiquitylation, but protein phosphorylation is probably the most common. The reversible covalent addition of a phosphate group to a specific amino acid residue allows the modulation of protein function, such as activity, subcellular localisation, capacity to interact with other proteins and stability. Recent data indicate that many photorespiratory enzymes can be phosphorylated, and thus it seems that the photorespiratory cycle is, in part, regulated by protein phosphorylation. In this review, the known phosphorylation sites of each Arabidopsis thaliana photorespiratory enzyme and several photorespiratory‐associated proteins are described and discussed. A brief account of phosphoproteomic protocols is also given since the published data compiled in this review are the fruit of this approach.

The PII protein is a signal integrator involved in the regulation of nitrogen metabolism in bacteria and plants. Upon sensing of cellular carbon and energy availability, PII conveys the signal by interacting with target proteins, thereby modulating their biological activity. Plant PII is located to plastids; therefore, to identify new PII target proteins, PII-affinity chromatography of soluble extracts from Arabidopsis leaf chloroplasts was performed. Several proteins were retained only when Mg-ATP was present in the binding medium and they were specifically released from the resin by application of a 2-oxoglutarate-containing elution buffer. Mass spectroscopy of SDS/PAGE-resolved protein bands identified the biotin carboxyl carrier protein subunits of the plastidial acetyl-CoA carboxylase (ACCase) and three other proteins containing a similar biotin/lipoyl-binding motif as putative PII targets. ACCase is a key enzyme initiating the synthesis of fatty acids in plastids. In in vitro reconstituted assays supplemented with exogenous ATP, recombinant Arabidopsis PII inhibited chloroplastic ACCase activity, and this was completely reversed in the presence of 2-oxoglutarate, pyruvate, or oxaloacetate. The inhibitory effect was PII-dose-dependent and appeared to be PII-specific because ACCase activity was not altered in the presence of other tested proteins. PII decreased the V _max of the ACCase reaction without altering the K _m for acetyl-CoA. These data show that PII function has evolved between bacterial and plant systems to control the carbon metabolism pathway of fatty acid synthesis in plastids.

NAD-dependent isocitrate dehydrogenase (IDH) is a tricarboxylic acid cycle enzyme that produces 2-oxoglutarate, an organic acid required by the glutamine synthetase/glutamate synthase cycle to assimilate ammonium. Three Arabidopsis (Arabidopsis thaliana) IDH mutants have been characterized, corresponding to an insertion into a different IDH gene (At5g03290, idhv; At4g35260, idhi; At2g17130, idhii). Analysis of IDH mRNA and protein show that each mutant lacks the corresponding gene products. Leaf IDH activity is reduced by 92%, 60%, and 43% for idhv, idhi, and idhii, respectively. These mutants do not have any developmental or growth phenotype and the reduction of IDH activity does not impact on NADP-dependent isocitrate dehydrogenase activity. Soil-grown mutants do not exhibit any alterations in daytime sucrose, glucose, fructose, citrate, ammonium, and total soluble amino acid levels. However, gas chromatography-mass spectrometry metabolic profiling analyses indicate that certain free amino acids are reduced in comparison to the wild type. These data suggest that IDH activity is not limiting for tricarboxylic acid cycle functioning and nitrogen assimilation. On the other hand, liquid culture-grown mutants give a reduced growth phenotype, a large increase in organic acid (citrate is increased 35-fold), hexose-phosphate, and sugar content, whereas ammonium and free amino acids are moderately increased with respect to wild-type cultures. However, no significant changes in 2-oxoglutarate levels were observed. Under these nonphysiological growth conditions, pyridine nucleotide levels remained relatively constant between the wild-type and the idhv line, although some small, but significant, alterations were measured in idhii (lower NADH and higher NADPH levels). On the other hand, soil-grown idhv plants exhibited a reduction in NAD and NADPH content.