Deletion of any of the core enzymes of the photorespiratory cycle, one of the major pathways of plant primary metabolism, results in severe air-sensitivity of the respective mutants. The peroxisomal enzyme hydroxypyruvate reductase (HPR1) represents the only exception to this rule. This indicates the presence of extraperoxisomal reactions of photorespiratory hydroxypyruvate metabolism. We have identified a second hydroxypyruvate reductase, HPR2, and present genetic and biochemical evidence that the enzyme provides a cytosolic bypass to the photorespiratory core cycle in Arabidopsis thaliana. Deletion of HPR2 results in elevated levels of hydroxypyruvate and other metabolites in leaves. Photosynthetic gas exchange is slightly altered, especially under long-day conditions. Otherwise, the mutant closely resembles wild-type plants. The combined deletion of both HPR1 and HPR2, however, results in distinct air-sensitivity and a dramatic reduction in photosynthetic performance. These results suggest that photorespiratory metabolism is not confined to chloroplasts, peroxisomes, and mitochondria but also extends to the cytosol. The extent to which cytosolic reactions contribute to the operation of the photorespiratory cycle in varying natural environments is not yet known, but it might be dynamically regulated by the availability of NADH in the context of peroxisomal redox homeostasis.

The mitochondrial multienzyme glycine decarboxylase (GDC) catalyzes the tetrahydrofolate-dependent catabolism of glycine to 5,10-methylene-tetrahydrofolate and the side products NADH, CO 2 , and NH 3 . This reaction forms part of the photorespiratory cycle and contributes to one-carbon metabolism. While the important role of GDC for these two metabolic pathways is well established, the existence of bypassing reactions has also been suggested. Therefore, it is not clear to what extent GDC is obligatory for these processes. Here, we report on features of individual and combined T-DNA insertion mutants for one of the GDC subunits, P protein, which is encoded by two genes in Arabidopsis (Arabidopsis thaliana). The individual knockout of either of these two genes does not significantly alter metabolism and photosynthetic performance indicating functional redundancy. In contrast, the double mutant does not develop beyond the cotyledon stage in air enriched with 0.9% CO 2 . Rosette leaves do not appear and the seedlings do not survive for longer than about 3 to 4 weeks under these nonphotorespiratory conditions. This feature distinguishes the GDC-lacking double mutant from all other known photorespiratory mutants and provides evidence for the nonreplaceable function of GDC in vital metabolic processes other than photorespiration.The mitochondrial multienzyme complex Gly decarboxylase (GDC) contributes to the two strategically important metabolic pathways of (1) photorespiration in all photosynthesizing organs and (2) one-carbon metabolism in all biosynthetically active tissues. In each of these two metabolic contexts, GDC closely cooperates with a second mitochondrial enzyme, Ser hydroxymethyltransferase (SHM), in the conversion of Gly to Ser. In the course of the tetrahydrofolate (THF)-dependent GDC reaction cycle comprising three individual reactions, CO 2 and NH 3 are released, and NAD 1 becomes reduced to NADH. The remaining methylene moiety becomes attached to THF to produce the one-carbon donor compound 5,10-methylene-THF (CH 2 -THF). SHM subsequently synthesizes Ser from CH 2 -THF and a second molecule of Gly in a fully reversible reaction (Douce et al., 2001;Hanson and Roje, 2001).The combined GDC/SHM reaction represents the mitochondrial part of the photorespiratory C 2 cycle, which occurs in all photosynthesizing tissues of C 3 plants, extends over three cellular compartments, and converts Rubisco-generated 2-phosphoglycolate into the Calvin cycle metabolite 3-phosphoglycerate (Tolbert, 1997;Douce and Neuburger, 1999). The importance of GDC and SHM for photorespiration becomes apparent from the fact that all as yet-reported mutants and antisense plants show strong metabolic disturbations in normal air, but grow well in the nonphotorespiratory conditions of approximately 1% CO 2 (Somerville and

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