Photorespiration is an essential process of phototropic organisms caused by the limited ability of rubisco to distinguish between CO2 and O2. To understand the metabolic flux through the photorespiratory pathway, we combined a mass spectrometry-based approach with a shift experiment from elevated CO2 (3000 ppm) to ambient CO2 (390 ppm). Here, we describe a protocol for quantifying photorespiratory intermediates, starting from plant cultivation through extraction and evaluation.

Modulation of photoassimilate export from the chloroplast is essential for controlling the distribution of fixed carbon in the cell and maintaining optimum photosynthetic rates. In this study, we identified chloroplast TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR 2 (CreTPT2) and CreTPT3 in the green alga Chlamydomonas (Chlamydomonas reinhardtii), which exhibit similar substrate specificities but whose encoding genes are differentially expressed over the diurnal cycle. We focused mostly on CreTPT3 because of its high level of expression and the severe phenotype exhibited by tpt3 relative to tpt2 mutants. Null mutants for CreTPT3 had a pleiotropic phenotype that affected growth, photosynthetic activities, metabolite profiles, carbon partitioning, and organelle-specific accumulation of H2O2. These analyses demonstrated that CreTPT3 is a dominant conduit on the chloroplast envelope for the transport of photoassimilates. In addition, CreTPT3 can serve as a safety valve that moves excess reductant out of the chloroplast and appears to be essential for preventing cells from experiencing oxidative stress and accumulating reactive oxygen species, even under low/moderate light intensities. Finally, our studies indicate subfunctionalization of the TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR (CreTPT) transporters and suggest that there are differences in managing the export of photoassimilates from the chloroplasts of Chlamydomonas and vascular plants.

28 Modulation of the rates of export of photoassimilates from the chloroplast is essential for 29 maintaining optimum photosynthetic rates. Additionally, the export of carbon metabolites from 30 the chloroplast can serve in the transduction of chloroplast signals to the nucleus in response to 31 environmental stresses. In this study we characterized a major chloroplast translocator of fixed 32 carbon, the triose phosphate/phosphate translocator 3 (CreTPT3), of the green alga 33 Chlamydomonas reinhardtii. This translocator is expressed at high levels, was shown to be on the 34 chloroplast envelope membrane and preferentially exchanges DHAP and 3-PGA for Pi based on 35 an in vitro yeast liposome uptake assay. Null mutants for CreTPT3, generated by CRISPR-Cas9, 36 exhibited a pleiotropic phenotype that impacted the growth, the rate of photosynthetic electron 37 transport, metabolite profiles, carbon partitioning, and organelle-specific accumulation of H2O2. 38 The severe phenotype of the tpt3 mutant demonstrates that this transporter is a dominant conduit 39 on the chloroplast envelope for the transport of photosynthetically fixed carbon. In addition, 40 CreTPT3 can serve as a safety valve that moves excess reductant out of the chloroplast which helps 41 stabilize the redox state of the plastid. Finally, we demonstrated that CreTPT3 is essential for 42 preventing the cells from experiencing oxidative stress by sustaining a high rate of photosynthetic 43 electron transport as the cells acclimate to high light. Our studies reveal important features about 44 the localization and activity of CreTPT3, its importance in controlling metabolite partitioning, and 45 suggest differences in the export of photoassimilates from the chloroplasts of Chlamydomonas and 46 vascular plants. 47 48

SignificancePhotorespiration is essential for photosynthesis in an oxygen-containing atmosphere. By mass flow, photorespiration is exceeded only by photosynthetic carbon assimilation. Photorespiration, initiated by the oxygenation reaction of Rubisco, is a major constraint on the photosynthetic efficiency of C3 plants and consequently on crop yield. Mitigating the negative effects of photorespiration holds potential for yield increases and contributes to achieving food and energy security for a growing population. This work presents a synthetic bypass to natural photorespiration (i.e., the conversion of photorespiratory glycolate into a C4 compound via a recently discovered microbial glycolate assimilation pathway, the β-hydroxyaspartate cycle [BHAC]). Simultaneous expression of four enzymes of microbial origin in the land plant modelArabidopsis thalianaenables efficient glycolate conversion into BHAC products.

Peroxisomes are eukaryotic organelles that are essential for growth and development. They are highly metabolically active and house many biochemical reactions, including lipid metabolism and synthesis of signaling molecules. Most of these metabolic pathways are shared with other compartments, such as Endoplasmic reticulum (ER), mitochondria, and plastids. Peroxisomes, in common with all other cellular organelles are dependent on a wide range of cofactors, such as adenosine 5′-triphosphate (ATP), Coenzyme A (CoA), and nicotinamide adenine dinucleotide (NAD). The availability of the peroxisomal cofactor pool controls peroxisome function. The levels of these cofactors available for peroxisomal metabolism is determined by the balance between synthesis, import, export, binding, and degradation. Since the final steps of cofactor synthesis are thought to be located in the cytosol, cofactors must be imported into peroxisomes. This review gives an overview about our current knowledge of the permeability of the peroxisomal membrane with the focus on ATP, CoA, and NAD. Several members of the mitochondrial carrier family are located in peroxisomes, catalyzing the transfer of these organic cofactors across the peroxisomal membrane. Most of the functions of these peroxisomal cofactor transporters are known from studies in yeast, humans, and plants. Parallels and differences between the transporters in the different organisms are discussed here.

AbstractPlant peroxisomes are unique subcellular organelles which play an indispensable role in several key metabolic pathways, including fatty acid β‐oxidation, photorespiration, and degradation of reactive oxygen species. The compartmentalization of metabolic pathways into peroxisomes is a strategy for organizing the metabolic network and improving pathway efficiency. An important prerequisite, however, is the exchange of metabolites between peroxisomes and other cell compartments. Since the first studies in the 1970s scientists contributed to understanding how solutes enter or leave this organelle. This review gives an overview about our current knowledge of the solute permeability of peroxisomal membranes described in plants, yeast, mammals and other eukaryotes. In general, peroxisomes contain in their bilayer membrane specific transporters for hydrophobic fatty acids (ABC transporter) and large cofactor molecules (carrier for ATP, NAD and CoA). Smaller solutes with molecular masses below 300–400 Da, like the organic acids malate, oxaloacetate, and 2‐oxoglutarate, are shuttled via non‐selective channels across the peroxisomal membrane. In comparison to yeast, human, mammals and other eukaryotes, the function of these known peroxisomal transporters and channels in plants are discussed in this review.