A Cytosolic Pathway for the Conversion of Hydroxypyruvate to Glycerate during Photorespiration in Arabidopsis W Stefan Timm, a Adriano Nunes-Nesi, b Tiit Pa ¨ rnik, c Katja Morgenthal, b Stefanie Wienkoop, b Olav Keerberg, c Wolfram Weckwerth, b Leszek A. Kleczkowski, d Alisdair R. Fernie, b and Hermann Bauwe a,1 a University of Rostock, BioScience Institute, Plant Physiology Department, D-18051 Rostock, Germany b Max Planck Institute of Molecular Plant Physiology, D-14476 Potsdam-Golm, Germany c Estonian University of Life Sciences, Institute of Agricultural and Environmental Sciences, EE-51014 Tartu, Estonia d University of Umea ˚ , Plant Physiology Department, SE-901 87 Umea ˚ , Sweden 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. INTRODUCTION Photorespiration represents one of the major pathways of plant primary metabolism. In terms of mass flow, it actually constitutes the second most important process in the land-based biosphere, exceeded only by photosynthesis. Carbon dioxide losses related to this process can be very high and are further elevated by warmer temperatures and drought, hence reducing the yields of important crops (Tolbert, 1997; Wingler et al., 2000). The core of the photorespiratory cycle, as revealed by the extensive biochemical analysis of wild-type and mutant plants (Lorimer and Andrews, 1981; Tolbert, 1997; Somerville, 2001), comprises at least eight individual enzymatic reactions distrib- uted over three different types of organelles, namely chloro- plasts, peroxisomes, and mitochondria (shown in Supplemental Figure 1 online). The cycle starts in the chloroplast with the synthesis of 2-phosphoglycolate from the Calvin cycle interme- diate ribulose 1,5-bisphosphate and oxygen by ribulose-1,5- bisphosphate carboxylase/oxygenase. In the course of the last two reactions of the cycle, catalyzed by the peroxisomal enzyme hydroxypyruvate reductase (HPR1; EC 1.1.1.29) and the plas- tidial glycerate 3-kinase, the intermediate compound hydroxy- pyruvate becomes converted to glycerate and eventually to another Calvin cycle intermediate, 3-phosphoglycerate. Al- though much of this appears as well-established textbook sci- ence, considerable gaps still remain concerning our knowledge of the cellular biology and biochemistry of photorespiration. One problem that is as yet only poorly resolved, and which is the focus of this article, concerns the possibility of multiple pathways for the conversion of hydroxypyruvate to glycerate. From earlier biochemical studies, there is much evidence that this reaction is exclusively catalyzed by HPR1 (Tolbert et al., 1970; Yu and Huang, 1986; Heupel et al., 1991). This enzyme is assumed to operate as part of a peroxisomal multienzyme complex, preventing equilibration of hydroxypyruvate with the cytosol (Reumann, 2000). However, HPR1 might not be the only enzyme that reduces photorespiratory hydroxypyruvate. This can be presumed from specific properties of a barley (Hordeum vulgare) mutant with severely reduced activities of HPR1 (Murray et al., 1989). In contrast with the typical lack of vitality exhibited when other photorespiratory mutants are grown in air, the pho- tosynthetic performance of this mutant was not greatly affected. Therefore, it was hypothesized that a cytosolic hydroxypyruvate reductase (HPR2; EC 1.1.1.81) (Kleczkowski and Randall, 1988) could provide a bypass in this mutant and also serve as an overflow mechanism for the utilization of hydroxypyruvate leaked from peroxisomes under conditions of maximum photorespira- tion in wild-type plants. The precise genetic defect in this mutant, unfortunately, has not yet been identified, and current EST databases do not exclude the presence of multiple HPR1 genes in barley (http://pgrc.ipk-gatersleben.de/cr-est/). Therefore, it is 1 Address correspondence to hermann.bauwe@uni-rostock.de. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Hermann Bauwe (hermann.bauwe@uni-rostock.de). W Online version contains Web-only data. www.plantcell.org/cgi/doi/10.1105/tpc.108.062265 The Plant Cell, Vol. 20: 2848–2859, October 2008, www.plantcell.org ã 2008 American Society of Plant Biologists