1.1. The Origin and Significance of Photorespiration

Photorespiration is the process of light-dependent uptake of molecular oxygen (O₂) concomitant with release of carbon dioxide (CO₂) from organic compounds. The gas exchange resembles respiration and is the reverse of photosynthesis where CO₂ is fixed and O₂ released. As shown in Figure 1, the entrance reactions to both photosynthesis and photorespiration are catalyzed by the same enzyme: Ribulose-1,5-bisphosphate-carboxylase/oxygenase (RUBISCO, EC 4.1.1.39). Carbon fixation results in the formation of two molecules 3-phosphoglycerate (PGA) that are integrated into the Calvin cycle ultimately to form sugars. During oxygen fixation, one molecule of PGA and one molecule of 2-phosphoglycolate are formed. The latter is converted back to PGA in the photorespiratory cycle. The pathway requires energy (ATP) and reducing (NAD(P)H) equivalents. This is in great part due to the release of CO₂ and ammonia (NH₃) that have to be refixed (for details see chapter 2).

RUBISCO evolved already early in biotic evolution about 3 billion years ago probably from enzymes involved in sulfur metabolism (Tabita et al., 2007), but is until today the enzyme accounting for the vast amount of net CO₂ fixation from the atmosphere. At the time of RUBISCO evolution, the only source of molecular oxygen in the atmosphere was probably photolysis of water by UV light and concentrations were 10^-14 below present concentrations (Buick, 2008). At the same time, CO₂ concentrations were at least 100-fold higher than today (Kasting and Howard, 2006; Kasting and Ono, 2006). Initial RUBISCO enzymes were probably extremely bad in discriminating CO₂ and O₂ (Tabita et al., 2007; Badger and Bek, 2008) due to the absence of evolutionary pressure. With the advent of oxygenic photosynthesis in cyanobacteria, huge amounts of CO₂ were fixed into biomass that in part sedimented and did not return into the global carbon cycle. Concomitantly, equimolar amounts of O₂ were released into the atmosphere, because water was used as the reductant for the photosynthetic electron transport chain (Xiong and Bauer, 2002). Cyanobacteria, the subsequently evolving algae and particularly land plants (Igamberdiev and Lea, 2006) were so successful in doing this that O₂ became the second most prominent gas in today's atmosphere and CO₂ is extremely scarce. Selection pressure induced some improvement in RUBISCO's specificity for CO₂ that might not be further optimized without slowing down catalytic rates (Tcherkez et al., 2006): The more the structure of the bound CO₂ molecule resembles a carboxylate group, the better it will be discriminated from O₂, but the worse the first intermediate of the condensation can be cleaved into the end products of the reaction. Therefore, O₂ uptake by RUBISCO and the subsequent metabolism of the reaction product in the photorespiratory pathway is the second highest metabolite flux in plants behind photosynthesis with flux rates around 25 % of the photosynthetic rate at 25 °C (Sharkey, 1988). Photorespiratory fluxes are even higher under hot and dry growth conditions mainly for three reasons:

Under such conditions, the leaf internal CO_{2 concentration may approach the apparent CO2 compensation point where CO2} uptake by photosynthesis equals CO₂ release by photorespiration and respiration. This finally results in an inability of the plant to grow autotrophically (Sage, 2004).

Figure 1:

Schematic overview of photosynthesis and photorespiration.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) catalyzes both CO₂ and O₂ fixation. The product of CO₂ fixation is phosphoglycerate (P-glycerate) that enters the Calvin cycle. During oxygenation, equimolar amounts of P-glycerate and phosphoglycolate (P-glycolate) are formed. P-glycolate is recycled to P-glycerate in the photorespiratory pathway. In this reaction cascade, reducing equivalents (NAD(P)H) and energy equivalents are consumed. Ammonia (NH₃) and CO₂ are released and have to be refixed. Under current atmospheric gas concentrations and moderate environmental conditions, approximately each fourth reaction catalyzed by RUBISCO is an oxygenase reaction.

1.2. Photorespiration: An Early Case Study for Arabidopsis Genetics

In the early 1970s, Arabidopsis was introduced as a plant model for genetic analyses. Before, the genetics of other plants such as barley or maize had been studied extensively, but a plant with features of a model organism was required: These features included a small genome size, efficient inbreeding, a small space required for growth and a short generation time (Redei, 1975). The necessity of a genetic approach in photorespiration research and the benefits of the Arabidopsis system were early recognized by Chris and Shauna Somerville in William Ogren's laboratory. At that time, there were still ongoing debates whether the substrate for photorespiration was produced by the oxygenase activity of RUBISCO and which biochemical reaction resulted in the release of CO₂. The conflicting scientific hypotheses are excellently reviewed in Ogren (2003). The Somervilles' approach was to chemically mutagenize Arabidopsis seeds and to grow the M2 generation derived from this mutagenesis at 1 % CO₂ (see Figure 2). The rate of photorespiration was very low under these conditions, thus, mutants in the pathway were expected to grow normally. At seedling stage, all plants showing growth defects or chlorosis were discarded to remove mutants with developmental defects apart from photorespiration. The remaining plants were shifted to normal air with a CO₂ concentration of approximately 0.03 %. Plants that developed chlorosis under these conditions were identified as potential mutants in components of the photorespiratory pathway. This was again verified by shifting plants back to 1 % CO₂ where photorespiratory mutants should recover from growth inhibition. Using this strategy, they identified “dozens” of plants with an inheritable conditional chlorotic phenotype at ambient CO₂ concentrations (Somerville, 2001). Genetic mapping and map-based isolation of the mutated gene (Jander et al., 2002; Peters et al., 2003) was not straightforward in Arabidopsis at these times, thus, enzymatic assays, metabolite profiles and gas exchange measurements were used to characterize the nature of the genetic lesion causing oxygen sensitivity. The first published mutant carried a mutation in phosphoglycolate phosphatase (PGLP, Somerville and Ogren, 1979) which indicated that phosphoglycolate is indeed the source substance for photorespiration. Together with earlier results that molecular oxygen is a competitive inhibitor of CO₂ fixation and that RUBISCO can form phosphoglycolate from ribulose-1,5 bisphosphate and O₂ (Bowes et al., 1971; Bowes and Ogren, 1972), this result unequivocally linked photorespiration to the oxygenase activity of RUBISCO. Shortly after, analysis of a mutant in mitochondrial serine hydroxymethyltransferase (SHM) defined the major biochemical reaction resulting in photorespiratory CO₂ release (Somerville and Ogren, 1981). Mutants deficient in chloroplastic glutamate-oxoglutarate aminotransferase (GLU, Somerville and Ogren, 1980b) and a chloroplast dicarboxylate transporter (DIT, Somerville and Ogren, 1983) confirmed the earlier observations by Keys et al. (1978) that ammonia released during photorespiration is refixed in the chloroplast. These and other mutants are described in more detail in chapter 2. This screen is one of the first examples where the power of Arabidopsis as a tool for plant genetic studies was proven. However, a similar screen performed shortly later in barley identified beside mutants known from Arabidopsis additional mutants in catalase (CAT, Kendall et al., 1983) and glutamine synthetase (GS, Wallsgrove et al., 1987) that have not been recovered in the Arabidopsis screens. Mutants for some of the major photorespiratory genes were never identified. Today, we know that this was in part due to genetic redundancy, e.g. glycolate oxidase (GO) is encoded by multiple genes (Reumann, 2004). Other genes are also required for growth under non-photorespiratory conditions (Engel et al., 2007) or alternative pathways can partially replace the deleted activity (Niessen et al., 2007; Timm et al., 2008). The latter case is discussed in more detail in chapter 4. In summary, the mutant approach clarified many open questions about the major photorespiratory pathway in Arabidopsis. It is now evident that most of the components are shared and that photorespiration is required by all oxygenic photosynthetic organisms (see chapter 5).

Figure 2:

Screening for photorespiratory mutants of Arabidopsis.

A segregating M2 population is grown at a high CO₂ concentration that suppresses photorespiration. Stunted or chlorotic plants at this stage probably carry defects in genes unrelated to photorespiration and are discarded. Healthy plants that become chlorotic after the population was shifted to atmospheric CO₂ concentrations are candidates for photorespiratory mutants. These mutants are rescued by shifting plants back to a high CO₂ concentration.

The work on photorespiration during these years was motivated by the expectation that a reduction of photorespiration could significantly improve plant productivity (Zelitch and Day, 1973; Kelly and Latzko, 1976). However, attempts to identify second-site mutations that could alleviate the deleterious effects of a disrupted photorespiratory pathway were never successful (Somerville and Ogren, 1982b). Metabolic engineering approaches now indicate that deviation of some of the glycolate from photorespiration into novel pathways might indeed enhance biomass production and possibly yield (see chapter 6). Moreover, numerous studies describe photorespiration as a beneficial pathway that supports growth under stress conditions (Wingler et al., 2000) or that provides metabolites for other basal metabolic pathways of the plant (Novitskaya et al., 2002). These positive aspects of photorespiration are exemplified in chapter 3.

2.1. Enzymatic Reactions in the Major Photorespiratory Pathway

2.1.1. Dephosphorylation of 2-Phosphoglycolate

2-Phosphoglycolate phosphatase (PGLP, EC 3.1.3.18) catalyzes the entry reaction of photorespiration, dephosphorylation of phosphoglycolate formed by the oxygenase activity of RUBISCO to glycolate. Knock-outs of this gene requiring high CO₂ for survival have been identified in both the Arabidopsis and barley mutant screens (see chapter 1) indicating that the functional PGLP is encoded by a single gene. The Arabidopsis mutant CS119 revealed oxygen-sensitive photosynthesis, strong accumulation of phosphoglycolate and absence of PGLP activity (Somerville and Ogren, 1979). The responsible gene has been recently identified by Schwarte and Bauwe (2007) using a candidate gene approach. T-DNA insertion lines for two (At5g36700 encoding AtPGLP1 and At5g47760 encoding AtPGLP2) of a total of 13 annotated potential Pglp genes in Arabidopsis were selected based on homology of the deduced protein to the afore identified Chlamydomonas PGLP. Only one of the two lines showed the typical conditional lethal phenotype. When this gene was sequenced in mutant CS119 lacking PGLP activity, a splice site mutation was identified that resulted in the formation of non-functional transcripts. Thus, At5g36700 encodes the photorespiratory PGLP in Arabidopsis.

Figure 3.

The major photorespiratory pathway in Arabidopsis.

Overview of the major photorespiratory pathway in Arabidopsis. For details and chemical structures, see text. RuBP, ribulose-1,5-bisphosphate; RUBISCO, RuBP carboxylase/oxygenase; PGLP, phosphoglycolate phosphatase; GO, glycolate oxidase; CAT, catalase; GGAT, glyoxylate:glutamate aminotransferase; SGAT, serine:glyoxylate aminotransferase; DIT1, dicarboxylate transporter 1; DIT2, dicarboxylate transporter 2; GLD, glycine decarboxylase; SHM, serine hydroxymethyltransferase; HPR, hydroxypyruvate reductase; GLYK, glycerate kinase; GS, glutamine synthetase; Fd-GOGAT, glutamine:oxoglutarate aminotransferase; THF, tetrahydrofolate; CH₂-THF, 5, 10-methylene-THF. The stoichiometry of the reactions is not included.

There is relatively little known about the regulation of PGLP in Arabidopsis. The gene is expressed in leaves and green parts of flowers. Transcription of the gene is induced by light and repressed by osmotic stress or low nitrate availability (Foyer et al., 2009). Biochemical analyses have been performed with purified enzyme form spinach, tobacco, Chlamydomonas and cyanobacteria. These analyses revealed an usual requirement for both cations such as Mg²⁺ and anions such as Cl^- for enzymatic activity (Husic and Tolbert, 1984; Norman and Colman, 1991). It was suggested that the phosphate moiety of phosphoglycolate is transferred to the protein and that the Cl^- ion is required for hydrolysis of the reaction intermediate (Seal and Rose, 1987). Whereas the spinach enzyme was allosterically inhibited by ribose-5-phosphate (Husic and Tolbert, 1984), activation by the same compound was described for the Chlamydomonas enzyme in another study (Mamedov et al., 2001).

2.1.2. Glycolate metabolism in the peroxisome

Glycolate oxidase (GO, EC 1.1.3.15) belongs to the class of flavindependent 2-hydroxy acid oxidases and catalyzes the oxidation of glycolate to glyoxylate. Molecular oxygen is used as a co-substrate and, thus, H₂O₂ is produced by the enzyme. The Go gene family in Arabidopsis contains 5 members of which three are potentially localized in the peroxisome (Reumann, 2004; Reumann et al., 2007), Two of these genes (At3g14415 and At3g14420) are transcriptionally co-regulated with other photorespiratory genes with regard to tissue-specific expression as well as positive regulation by light and nitrogen (Reumann and Weber, 2006; Foyer et al., 2009). Probably because of this genetic redundancy, no mutants were identified in the classical Arabidopsis and barley screens (Somerville and Ogren, 1982b; Blackwell et al., 1988). In tobacco and rice, lines with reduced GO activity were generated by co-suppression (Yamaguchi and Nishimura, 2000) and inducible antisense expression (Xu et al., 2009). These studies revealed a positive and linear correlation between GO-activity and the net photosynthetic rate. Once photosynthetic rates fell below 40% of control plants, photoinhibition occurred indicating that GO can exert a strong regulatory effect on photosynthesis, possibly through a reduction in RUBISCO activation state (Xu et al., 2009). Beside metabolic regulation, GO activity is also induced by pathogen attacks. This is probably due to the formation of H₂O₂ and its importance as a second messenger in plant defense reactions (Taler et al., 2004, see also chapter 3).

H₂O₂ produced by the glycolate oxidase reaction is disproportionated to H₂O and O₂ by catalase (CAT, EC 1.11.1.6) in order to prevent accumulation of reactive oxygen species. In Arabidopsis, three catalase genes are known, but only one gene (Cat2, At4g35090) encodes the major enzyme involved in photorespiration. In ambient air, a mutation in Cat2 caused severely decreased rosette biomass, intracellular redox perturbation and activation of oxidative signaling pathways. These effects were absent when mutant plants were grown at high CO₂ concentrations (Queval et al., 2007). Cat2 gene expression is light-stimulated and clock-regulated with an induction in the early morning (Zhong et al., 1997; Queval et al., 2007).

The next reaction step in the photorespiratory pathway is the transamination of glyoxylate to glycine by glutamate:glyoxylate aminotransferase (GGAT, EC 2.6.1.4). In Arabidopsis, two loci encode peroxisomal glutamate:glyoxylate aminotransferases (At1g23310 encoding GGAT1 and At1g70580 encoding GGAT2) (Igarashi et al., 2003; Liepman and Olsen, 2003; Igarashi et al., 2006). Ggat1 knock-outs show a weak photorespiratory phenotype and survive in air at moderate light intensities. Residual GGAT activity can be detected in mutant leaf extracts (Igarashi et al., 2003). Furthermore, both genes exhibit a differential diurnal expression pattern. Whereas Ggat2 is induced at the beginning of the photoperiod, Ggat1 is rather repressed. However, GGAT activity in leaves remains constant during the day (Igarashi et al., 2006) suggesting that both loci contribute to the total leaf GGAT content. Ggat1 is not only highly expressed in leaves but also in roots, indicating that its function is not restricted to photorespiration.

2.1.3. Mitochondrial conversion of glycine to serine

The combined reaction of glycine decarboxylase (GLD) and serine hydroxymethyltransferase (SHM) represents the mitochondrial part of the major photorespiratory pathway. Glycine is decarboxylated and deaminated by the multienzyme complex glycine decarboxylase yielding CO₂, NH₃, NADH and 5, 10-methylenetetrahydrofolate. The latter is used by SHM as a substrate for the transfer of an activated C1 moiety to another molecule of glycine resulting in the formation of serine.

The GLD reaction involves four different proteins (Figure 4). P-protein (EC 1.4.4.2) is a pyridoxal-5-phosphate-containing homodimer, that catalyzes the actual glycine decarboxylation reaction (Bauwe and Kolukisaoglu, 2003). The remaining amino methylene part of glycine is transferred to the distal sulphur atom of the oxidized lipoamide arm of H-protein (Douce et al., 2001). H-protein is not an enzyme component of the complex but a cofactor that interacts with all three enzymes of the complex. The amino methylene group bound to the H-protein is further metabolized by the monomeric aminomethyltransferase T-protein (EC 2.1.2.10) (Douce and Neuburger, 1999). During this reaction, NH₃ is released and a methylene group is transferred to tetrahydrofolate resulting in the formation of 5,10-methylene tetrahydrofolate (CH₂-THF) (Bauwe and Kolukisaoglu, 2003). Concomitantly, the lipoamide arm of H-protein becomes fully reduced and needs to be re-oxidized for the next reaction cycle. This reaction is catalyzed by L-protein, a homodimeric dihydrolipoamide dehydrogenase (EC 1.8.1.4). Electrons are first transferred to the co-factor FAD that is re-oxidized by NAD⁺ resulting in the formation of NADH (Bauwe and Kolukisaoglu, 2003). The four proteins are present in unequal molar amounts in the complex. For pea, a subunit stoichiometry of 4P:27H:9T:2L was observed (Oliver et al., 1990), but the functional relevance of this composition of the complex is still elusive. In Arabidopsis, the four proteins are encoded by eight genes, two genes each for P-protein (At4g33010 and At2g26080) and L-protein (At3g17240 and At1g48030), three genes for H-protein (At2g35370, At2g35120 and At1g32470) and one gene for T-protein (At1g11860) (Bauwe and Kolukisaoglu, 2003). Most of these genes are strongly light regulated and some are repressed by low nitrogen concentrations, like most other genes contributing to the photorespiratory pathway (Foyer et al., 2009 and  http://www.genevestigator.com).

The classical Arabidopsis mutant gld1 showing a photorespiratory phenotype related to loss of function of the GLD complex (Somerville and Ogren, 1982a) was characterized on the molecular level by Ewald et al. (2007). The mutant is deficient in a 3-oxoacyl-(acyl-carrier-protein) synthase (At2g04540, Mt-kas1) that is required for lipoylation of H-protein, Some residual lipoylation by an independent second pathway might be sufficient to rescue the phenotype under non-photorespiratory conditions. In contrast, the combined knock-out of the two genes encoding P-protein is lethal under all growth conditions (Engel et al., 2007) suggesting that the GLD reaction is not only essential for photorespiration, but also for the synthesis of activated C1 moieties required for nucleic acid and amino acid metabolism (Cossins and Chen, 1997)

Figure 4:

The glycine decarboxylase complex in Arabidopsis.

Glycine cleavage by the glycine decarboxylase (GLD) complex. GLD consists of P-, H-, T- and L-protein. THF = tetrahydrofolate; CH₂-THF = 5,10-methylene THF. Glycine binds to the pyridoxalphosphate co-factor (not shown) of P-protein, is decarboxylated and transferred to H-protein. T-protein releases NH₃ and transfers the methylene group to THF. L-protein catalyzes the re-oxidation of H-protein. The subunit stoichiometry is not shown and the size of the symbols does not imply the size of the proteins. For further details, see main text.

The photorespiratory SHM enzyme (EC. 2.1.2.1) is encoded by a single gene (At4g37930) in Arabidopsis. A knock-out of this gene is lethal at ambient CO₂ levels, but mutants grow normally at high CO₂ (Somerville and Ogren, 1981; Voll et al., 2006). The mRNA abundance is positively regulated by light and the circadian clock, and the gene is not expressed in roots (McClung et al., 2000). Recently, a second class of mutants deficient in mitochondrial SHM activity was identified (Jamai et al., 2009). A mutation in Glu1 (At5g04140) which encodes ferredoxin-dependent glutamine:oxoglutarate aminotransferase (Fd-GOGAT (TAIR abbreviation: GLU); EC 1.4.7.1) reduced mitochondrial SHM activity. It was known before that this protein is involved in ammonia re-assimilation in the chloroplast during photorespiration (Coschigano et al., 1998, see also below), but the new results indicate that Fd-GOGAT is targeted not only to the chloroplast but also to the mitochondria where it physically interacts with SHM. This interaction is necessary for photorespiratory SHM activity (Jamai et al., 2009).

An additional level of regulation of the GLD/SHM complex was recently identified by characterization of plants that were unable to convert 10-formyl-THF, an intermediate of C1 metabolism (Hanson and Roje, 2001), to formate and THF (Collakova et al., 2008). In these plants, the isomer 5-formyl-THF accumulated and inhibited photorespiration resulting in retarded growth and glycine accumulation. Thus, 5-formyl-THF might act as a physiological regulator of GLD/SHM activity in vivo.

2.1.4. Formation of glycerate in the peroxisome

Back in the peroxisome, serine is deaminated to hydroxypyruvate by serine:glyoxylate aminotransferase (SGAT (TAIR abbreviation: AGT), EC 2.6.1.45). The amino group is used for the concomitant amination of glyoxylate to glycine. This is important for the stoichiometry of photorespiration, because only one amino donor for the GGAT reaction is recycled during NH₃ re-assimilation (see below), but two glycine are required for the mitochondrial formation of serine. In Arabidopsis, SGAT is encoded by a single gene (At2g13360) and its deletion results in plants that are unviable under normal atmospheric conditions (Somerville and Ogren, 1980a; Liepman and Olsen, 2001). A similar phenotype was also described for a barley SGAT mutant (Murray et al., 1987). The Arabidopsis gene is induced by light and repressed by low nitrogen like most of the other photorespiratory genes (Foyer et al., 2009), but transcripts are also detected in flowers and siliques apart from leaves (Liepman and Olsen, 2001).

Hydroxypyruvate resulting from the SGAT reaction is reduced to glycerate by hydroxypyruvate reductase (HPR, EC 1.1.1.29). In Arabidopsis, the peroxisomal reaction is catalyzed by HPR1 (At1g68010) using NADH as a co-factor (Givan and Kleczkowski, 1992). The Hpr1 gene shows similar regulatory properties like Sgat with positive light regulation and repression under low nitrogen conditions (Foyer et al., 2009). Reducing equivalents for the reaction are probably imported into the peroxisome by a malate/oxaloacetate shuttle and released by a peroxisomal malate dehydrogenase (pMDH, Cousins et al., 2008). However, Arabidopsis mutants with deletions in the genes encoding HPR1 (Timm et al., 2008) and pMDH (Pracharoenwattana et al., 2007) as well as barley HPR mutants (Murray et al., 1989) do not show drastic photorespiratory phenotypes. These data suggest the existence of alternative pathways for the conversion of hydroxypyruvate to glycerate or other metabolites that are further discussed in chapter 4.

2.1.5. Regeneration of phosphoglycerate

The chloroplastic D-glycerate 3-kinase (GLYK, EC 2.7.1.31) catalyzes the phosphorylation of glycerate to 3-phosphoglycerate with ATP as a co-substrate. As no mutants were identified in the classical screens, the gene was instead isolated by purification of the enzymatic activity from Arabidopsis leaf extracts and partial sequencing of the purified protein (Boldt et al., 2005). GLYK is encoded by a single copy gene (At1g80380). Knock-out of this gene results in a conditionally lethal photorespiratory phenotype. The enzyme is the founder of a structurally and phylogenetically novel protein family that is different from known glycerate kinases of non-photosynthetic organisms (Boldt et al., 2005), albeit sequence homologues are also found in yeast (Bartsch et al., 2008). Transcriptional regulation of the Glyk gene is different from most of the other photorespiratory genes. Particularly, Glyk gene expression is not activated by light (Foyer et al., 2009).

2.1.6. Ammonia re-assimilation

During the GLD reaction, one-quarter of the carbon that entered the photorespiratory cycle and an equimolar amount of NH₃ are released. Re-assimilation of NH₃ takes place in the chloroplast through the sequential action of two enzymes, glutamine synthetase (GS; EC 6.1.3.2) and ferredoxin-dependent glutamine:oxoglutarate aminotransferase (Fd-GOGAT (TAIR abbreviation: GLU); EC 1.4.7.1) (Keys, 2006). NADH-GOGAT (EC 1.4.1.14), that is additionally found in chloroplasts, does not play a significant role in photorespiration (Coschigano et al., 1998).

The net reaction of the GS/Fd-GOGAT cycle is the animation of oxoglutarate to glutamate consuming one ATP and two reduced ferredoxin.

First, GS catalyses the amination of glutamate to glutamine which requires one ATP. GS mutants have not been identified in Arabidopsis, but in barley (Wallsgrove et al., 1987). The barley mutant showed the typical requirement for elevated CO₂ concentrations emphasizing the major contribution of photorespiration to plant NH₃ production and re-assimilation (Keys, 2006). Six Arabidopsis genes encode GS enzymes, but only GS2 (encoded by At5g35630) is targeted to the chloroplast (Peterman and Goodman, 1991). Transcriptional regulation of the gene for GS2 is similar to most photorespiratory genes including light induction and repression by low nitrogen (Peterman and Goodman, 1991; Foyer et al., 2009).

Fd-GOGAT uses the glutamine formed by GS as an amino donor for the transamination of oxoglutarate. Two molecules of glutamate are the product of this reaction. Whereas one glutamate is used as a substrate for the next NH₃ assimilation reaction by GS, the second is transferred to the peroxisome for transamination of glyoxylate to glycine. Arabidopsis contains two expressed genes for Fd-GOGAT (Glu1 (At5g04140) and Glu2 (At2g41220)), but only mutation of the Glu1 gene resulted in a photorespiratory phenotype (Coschigano et al., 1998). Glu1 mRNA shows highest abundance in leaves and is specifically induced by light and sucrose. In contrast, Glu2 is only slightly expressed in leaves, but the mRNA preferentially accumulates in roots (Coschigano et al., 1998). Although the photorespiratory nitrogen cycle is closed in theory as all the re-fixed NH₃ is required for transamination of glyoxylate, some excess NH₃ might escape fixation in the GS/Fd-GOGAT cycle. There is physiological evidence that formation of carbamoyl phosphate and ultimately arginine provides an alternative route for NH₃ re-assimilation (Potel et al., 2009).

As described above, Fd-GOGAT has been shown to be dual targeted to the chloroplast and the mitochondrion (Jamai et al., 2009). Mitochondrial localization is seemingly not important for NH₃ re-assimilation, but rather for regulation of SHM activity. However, dual targeting has also been suggested for GS2 based on the localization of overexpressed fusions of the protein to green fluorescent protein (Taira et al., 2004). The presence of both enzymes would imply that photorespiratory NH₃ could also be directly re-assimilated in the mitochondrion. On the other hand, neither Fd-GOGAT nor GS have been identified in proteome analyses of the mitochondrial matrix (Eubel et al., 2007; Jamai et al., 2009). This at least implies that the proteins are scarce in the mitochondrion which would again support a regulatory instead of a catalytic role. Further research is necessary to clarify the role of these dual targeting events in photorespiration.

2.2. Transport of Photorespiratory Intermediates

Due to the complex biochemistry of photorespiration and the involvement of three different compartments, numerous transport steps over membranes are necessary for function of the pathway. However, most transporters have not been identified or even biochemically characterized in detail. The available information on the transporters for photorespiratory intermediates is summarized below. Data on the oxaloacetate/malate shuttles in the membranes of all organelles that are indirectly involved in photorespiration by transferring reducing equivalents during photorespiration (Reumann and Weber, 2006) are not included.

2.3. Energy Balance Sheet

Photorespiration imposes costs on plant energy metabolism. The oxygenase reaction of RUBISCO does not result in energy or carbon gain for the plant. The ribulose-1,5-bisphosphate molecule for this reaction is therefore “wasted”. Our calculation of the energy costs is based on the assumption that this molecule of ribulose-1,5-bisphosphate has to be completely regenerated to reconstitute the “status quo ante”, i.e. the metabolic state of the Calvin cycle before oxygen fixation. Table 1 summarizes these costs. Possible energetic costs of transport processes are unknown.

Table 1.

The energy balance sheet of the oxygenase reaction of RUBISCO and photorespiration.

Based on these calculations, what is the energy burden of the oxygenase reaction on carbon fixation? RUBISCO highly prefers CO₂ to O₂ as a substrate with a specificity factor of approximately 80 for land plants (Jordan and Ogren, 1981) although this varies between species and is dependent on temperature (Ku and Edwards, 1977a; Jordan and Ogren, 1984). However, atmospheric concentrations of O₂ are much higher than CO₂ concentrations (250 µM and 8 µM, respectively, at 25 °C) resulting in an oxygen surplus of 31-fold. Therefore, approximately each fourth reaction of RUBISCO is an oxygenase instead of a carboxylase reaction under moderate growth conditions (Ehleringer et al., 1991). Costs for each carbon fixation reaction are 3 ATP and 2 NADPH, or 8 ATP in total if reducing equivalents are converted to energy equivalents as described above. Statistically, the costs for one oxygenation reaction have to be allocated to 3 carboxylations, i.e. 4.1 ATP per carboxylation. This results in extra energy costs of about 50 % for photosynthesis caused by the oxygenase activity of RUBISCO. This calculated number is comparable to the measured about 40–50 % increase of photosynthetic rates in Arabidopsis and other C3 plants in atmospheres containing low O₂ concentrations (Hesketh, 1967; Kebeish et al., 2007).

3.1. Photorespiration Removes Toxic Metabolic Intermediates

Photorespiration is the only pathway in the plant for phosphoglycolate metabolism that would otherwise accumulate. Phosphoglycolate has been shown to strongly inhibit triose phosphate isomerase from pea leaves at concentrations in the low micromolar range (Anderson, 1971). This would interfere with the regeneration of ribulose-1,5-bisphosphate in the Calvin cycle. In addition, phosphoglycolate inhibits the chloroplast phosphofructokinase (Kelly and Latzko, 1976) and, by this, the replenishment of Calvin cycle intermediates from the breakdown of transitory starch. This dual inhibitory function is probably responsible for the low photosynthetic rate of mutants accumulating phosphoglycolate (Somerville and Ogren, 1979).

To our knowledge, no direct inhibitory function has been described for glycolate, the product of phosphoglycolate dephosphorylation, albeit glycolate accumulation has been observed in many photorespiratory mutants. Glycolate is further converted to glyoxylate and this compound is again a strong inhibitor of photosynthesis. Campbell and Ogren (1990) and Chastain and Ogren (1989) reported that micromolar concentrations of glyoxylate inhibited activation of RUBISCO in isolated organelles and in vivo. However, inhibition of the activase enzyme (Campbell and Ogren, 1990) or purified RUBISCO (Cook et al., 1985) required much higher concentrations. Furthermore, glyoxylate was inefficient in rendering the chloroplast envelope permeable to protons as observed for other weak acids before (Flügge et al., 1980). Thus, glyoxylate negatively impacts on the activation state of RUBISCO by a yet unidentified mechanism. The importance of this mechanism is exemplified by the presence of glyoxylate reductase in the cytosol and the chloroplast that might function in protecting the photosynthetic machinery from excess glyoxylate leaking from the peroxisomes (Givan and Kleczkowski, 1992; Allan et al., 2009). Conversely, glyoxylate may be instrumental in reducing RUBISCO activity and, by this, also phosphoglycolate formation under conditions where photorespiration cannot cope with the rates of RUBISCO oxygenase activity. This might be of physiological importance under low nitrogen supply when the transamination reactions converting glyoxylate to glycine are limiting photorespiratory flux. The glyoxylate overflow would simply stop both photosynthesis and photorespiration under such emergency conditions. Alternative mechanisms to metabolize glyoxylate are also discussed explicitly in chapter 4. Taken together, photorespiration is important for the removal of toxic intermediates, but surprisingly little is known about the regulatory function of these intermediates in coordinating photorespiration with other pathways of plant basal metabolism.

3.2. Photorespiration Protects from Photoinhibition

Under stress conditions, such as drought, cold, or high light, NADPH production in the light reactions of photosynthesis often exceeds the demand of the Calvin cycle for reducing power. In that case, energy is dissipated as heat or electrons are transferred from different complexes in the electron transport chain to other acceptor molecules than NADPH. If the acceptor molecule is O₂, the reaction results in the formation of reactive oxygen species (ROS, reviewed in Murata et al., 2007). Because of their high reactivity, ROS unspecifically oxidize proteins and lipids. Moreover, ROS also interfere with recycling of specific components of the electron transport chain by inhibiting translation of new proteins (Nishiyama et al., 2004). The chloroplast contains several mechanisms to detoxify ROS. There are compounds that scavenge ROS non-enzymatically like carotenoids and flavonoids, and enzymes, like ascorbate peroxidase, superoxide dismutase and catalase, that detoxify ROS in partly complex pathways (reviewed in Apel and Hirt, 2004). A welcome effect of these pathways is that they ultimately consume reducing equivalents and by this contribute to the provision of fresh NADP+, the terminal electron acceptor of the photosynthetic electron transport chain. Alternatively, excess light energy can be mitigated by the xanthophyll cycle that dissipates the energy of absorbed photons into heat (Yamamoto et al., 1962; Davison et al., 2002). Also photorespiration can act as an electron sink especially under stress conditions (Wingler et al., 2000) by consuming reducing equivalents during the refixation of released ammonia and by exporting reduced components from the chloroplast to the mitochondrion (Igamberdiev and Lea, 2002). This might even result in situations where the mitochondrion becomes over-reduced instead of the chloroplast because of the huge amounts of NADH generated by glycine decarboxylation. To allow for high fluxes through photorespiration under such conditions, mitochondrial electron transport might be uncoupled from ATP synthesis by uncoupling proteins (UCPs) that catalyse proton conductance through the inner mitochondrial membrane and dissipate the energy of the mitochondrial proton gradient as heat (Sweetlove et al., 2006). The importance of photorespiration in protection from photoinhibition was directly tested by Kozaki and Takeba (1996) by measuring the rates of photoinhibition in plants with enhanced or diminished capacities for photorespiration caused by manipulation of the levels of glutamine synthetase (GS). Plants with an improved capacity for photorespiration because of increased GS levels were more tolerant to high light stress. Accordingly, photorespiratory mutants of Arabidopsis showed enhanced photoinhibition (Takahashi et al., 2007). In this study, the authors provide evidence that photoinhibition was rather caused by suppression of the repair of photosystem II (PSII) than by an increase in PSII damage.

Any mechanism that uses photorespiration as a pressure relief valve for reducing power from the chloroplast would ultimately run out of the O₂ acceptor molecule ribulose-1,5-bisphosphate because of the net carbon loss associated with photorespiration. Replenishment of O₂ acceptor molecules is probably brought about by starch breakdown (Weise et al., 2006). As a consequence, the chloroplast stroma is seemingly less reduced under conditions of high photorespiration than during maximum photosynthesis (Weise et al., 2006). Still, the relative importance of photorespiratory export of reducing equivalents among the multitude of different processes that protect plants from photoinhibition is a matter of ongoing debates and might strongly depend on the conditions (cf. Osmond et al., 1997). Beside its function as an export mechanism for reducing power, photorespiration is also directly involved in the synthesis of sufficient amounts of glycine that is required to cover the strongly enhanced demands for glutathione during stress (Noctor et al., 1998; Noctor et al., 1999).

3.3. Photorespiration Supports Plant Defense Reactions

H₂O₂ is known to be a signaling molecule in plants involved in both biotic and abiotic stress responses. Because of its participation in multiple pathways, H₂O₂ is especially suitable for mediating crosstalk between the different resistance mechanisms (reviewed in Neill et al., 2002). During pathogen attack, H₂O₂ has multiple roles in the so called oxidative burst. On the one hand, it triggers the plant defense response system, including cell wall strengthening and activation of phytoalexin biosynthesis (Wu et al., 1997). On the other hand, H₂O₂ can damage the pathogen by its reactive potential. Ultimately, H₂O₂ triggers the hypersensitive response and the attacked cell eventually undergoes programmed cell death (Heath, 2000). As photorespiration is a major source of H₂O₂ in plants, an involvement in resistance reactions was often discussed (Chamnongpol et al., 1998). This was recently confirmed by the identification of “enzymatic resistance” genes in melon (Taler et al., 2004). Efficient resistance of a wild melon line to the oomycete Pseudoperonospora cubensis was shown to be caused by constitutively enhanced expression of two genes encoding peroxisomal glyoxylate aminotransferases. A mechanism was suggested, in which the elevated activity of these enzymes positively feeds back on glycolate oxidase, thereby enhancing the capacity for H₂O₂ release.

3.4. Photorespiration is Intimately Integrated Into Primary Metabolism

Photorespiration is obviously tightly integrated into plant primary metabolism. Most photorespiratory intermediates are also part of other metabolic pathways and photorespiration significantly contributes to the synthesis of several amino acids (Novitskaya et al., 2002). Photorespiration connects the metabolic compartments of the cell and is therefore ideally suited to transport information about the energetic state between these compartments (Nunes-Nesi et al., 2008). The conversion of glycine to serine in the mitochondrion is probably essential to supply serine for the cytosolic production of C1-compounds that are in turn required for biosynthetic processes such as methionine synthesis (Mouillon et al., 1999; Engel et al., 2007). Beside this, specific roles have been suggested for photorespiration under certain growth conditions:

Sucrose and starch are the major sinks for photosynthetic products in leaves. Under conditions of high light and carbon availability, synthesis of these sink compounds might not keep pace with production of phosphorylated intermediates in the Calvin cycle. This results in an imbalance of phosphate release during synthesis of sucrose or starch and phosphate use during the synthesis of Calvin cycle intermediates that may ultimately limit photosynthesis (Harley and Sharkey, 1991). Under such conditions, fixation of oxygen by RUBISCO and the subsequent reactions of photorespiration might provide the cell with an additional method for the synthesis of sink products such as glycine and serine instead of sucrose and starch (Harley and Sharkey, 1991). This is consistent with the finding that source leaves start to export glycine and serine instead of sucrose to sink leaves under conditions of high photorespiration (Madore and Grodzinski, 1984).

Another connection of photorespiration and nitrogen metabolism was observed by Rachmilevitch et al. (2004). They found that nitrate assimilation was inhibited in an atmosphere containing 2% O₂ where photorespiration is repressed. They proposed a mechanism how photorespiration provides reducing equivalents for cytoplasmic nitrate reductase: The reducing equivalents are exported from the chloroplast into the cytoplasm via the malate/oxaloacetate shuttle to provide reducing power for peroxisomal hydroxypyruvate reduction. Part of these reducing equivalents might then be diverted for nitrate reduction. Reducing equivalents for peroxisomal reactions can alternatively be provided by mitochondria that synthesize the required amounts of NADH during photorespiration (Raghavendra et al., 1998). However, there is evidence that part of the mitochondrial reducing equivalents may rather be used for ATP synthesis (Krömer and Heldt, 1991). The relative importance of these processes requires detailed investigation.

4.1. Possible Alternative Pathways in the Chloroplast

An endogenous pathway for glycolate metabolism in the chloroplast was proposed by Goyal and Tolbert (1996). Using biochemical analyses, they demonstrated the existence of a salicylhydroxamic acid (SHAM)-inhibited light-dependent glycolate-quinone oxidoreductase system that is associated with thylakoid membranes of the chloroplasts of both algae and spinach. By this system, electrons from glycolate oxidation could be transferred to the photosynthetic electron transport chain and used for ATP synthesis. In this scenario, glyoxylate would be reduced back to glycolate by the plastidal glyoxylate reductase (Givan and Kleczkowski, 1992). This would provide an alternative sink for excess electrons under stress conditions (Allan et al., 2009, see also chapter 3). The presence of a glycolate-oxidizing enzyme in chloroplasts of Arabidopsis was recently confirmed by Kebeish et al. (2007). However, the cycle of glycolate oxidation and glyoxylate reduction does not consume glycolate and is therefore not suitable as an alternative pathway for the conversion of the products of RUBISCO's oxygenase activity. Instead, glyoxylate coming from plastidal glycolate oxidation might be further oxidized to CO₂ in the chloroplast (Kisaki and Tolbert, 1969; Zelitch, 1972; Oliver, 1981, dotted grey pathway in Figure 5). Slow CO₂ release from glyoxylate in chloroplasts from a set of plant species was shown to be stimulated by light and oxygen (Kisaki and Tolbert, 1969; Zelitch, 1972). Although glycolate was not converted by chloroplasts to CO₂ in these studies, later analysis in Arabidopsis also revealed coupling of glycolate oxidation and CO₂ release (Kebeish et al., 2007). Conversion of glyoxylate to CO₂ might be catalyzed by plastidal pyruvate decarboxylase that also accepts glyoxylate as a substrate (Davies and Corbett, 1969). Alternatively, glyoxylate can react non-enzymatically with H₂O₂ or O₂- to formate (Elstner and Heupel, 1973; Halliwell and Butt, 1974, see also chapter 4.2). Such reactive oxygen species are produced in the chloroplast especially under stress conditions (see chapter 3). Formate could be further oxidized to CO₂ by a formate dehydrogenase that is dual-targeted to mitochondria and chloroplasts in Arabidopsis (Olson et al., 2000; Herman et al., 2002). Together, this would result in a low-capacity pathway for glycolate oxidation in the chloroplast that is more energy-efficient than the major photorespiratory pathway and that enhances plastidal CO₂ concentrations. However, ribulose-1,5-bisphosphate is not recycled in these reactions which may result in a depletion of CO₂ acceptor molecules at higher fluxes.

Figure 5:

Possible alternative photorespiratory pathways in Arabidopsis.

Overview of the major photorespiratory pathway (black) and four possible alternative photorespiratory pathways (dotted grey, green, red, and dashed grey) in Arabidopsis that are described in the main text. RuBP, ribulose-1,5-bisphosphate; RUBISCO, RuBP carboxylase/oxygenase; PGLP, phosphoglycolate phosphatase; GO, glycolate oxidase; CAT, catalase; GGAT, glyoxylate:glutamate aminotransferase; SGAT, serine:glyoxylate aminotransferase; DIT1, dicarboxylate transporter 1; DIT2, dicarboxylate transporter 2; GLD, glycine decarboxylase; SHM, serine hydroxymethyltransferase; HPR, hydroxypyruvate reductase; GLYK, glycerate kinase; GS, glutamine synthetase; Fd-GOGAT, glutamine:oxoglutarate aminotransferase; GlyDH, glycolate dehydrogenase; AT, aminotransferase; FDH, formate dehydrogenase; FTHFS, 10-formyl-THF-synthetase;CYC, 5,10-methenyl-THF-cyclohydrolase;DHY, 5,10-methenyl-THF-dehydrogenase;THF, tetrahydrofolate; HCO-THF, formyl-THF; CH₃-THF, methenyl-THF; CH₂-THF, methylene-THF. The stoichiometry of the reactions is not included.

4.2. Possible Alternative Pathways in the Peroxisome

The presence of an alternative pathway for glyoxylate conversion in the peroxisome that does not involve transamination to glycine was suggested by the characterization of Arabidopsis mutants deficient in SHM (Somerville and Ogren, 1981). Although accumulating high amounts of glycine, these mutants still released CO₂ from photorespiratory intermediates. The responsible pathway might involve the non-enzymatic decarboxylation of glyoxylate to formate using H₂O₂ as an oxidizing agent (Oliver, 1981; Igamberdiev and Lea, 2002). Alternatively, catalase can also act as a peroxidase in the oxidation of glyoxylate (Grodzinski, 1978). Peroxisomal glyoxylate decarboxylation could play a physiological role especially under conditions of low nitrogen supply where transamination is inhibited (Somerville and Ogren, 1982b; Singh et al., 1986; Wingler et al., 1999).

Formate resulting from glyoxylate decarboxylation might act a C1 donor in the formation of serine. Based on studies of barley and Amaranthus mutants lacking GLD activity, Wingler et al. (1999) suggested that the C1-tetrahydrofolate (THF) synthase pathway converts photorespiratory formate to 5,10-methylene-THF (green pathway in Figure 5). The enzymes involved in plants are a monofunctional 10-formyl-THF-synthetase (FTHF synthetase, EC 6.3.4.3) and a bifunctional 5,10-methenyl-THF-cyclohydrolase:5,10-methylene-THF dehydrogenase (CYC-DHY, EC 3.5.4.9 and EC 1.5.1.5). FTHF synthetase catalyses the synthesis of 10-formyl-THF, which is converted into 5,10-methenyl-THF by CYC and reduced to 5,10-methylene-THF by DHY. 5,10-methylene-THF is used by SHM to synthesize serine from glycine (Prabhu et al., 1996; Hanson and Roje, 2001). In the barley and Amaranthus GLD mutants, evidence for function of this pathway in photorespiration was provided by the increase in FTHF-synthetase, the accumulation of glyoxylate and formate under photorespiratory conditions, and the ability of the mutants to utilize formate and glycolate for the formation of serine (Wingler et al., 1999). Similar flux analyses were later on also performed with Arabidopsis mutants deficient in GLD activity that confirmed the described results (Li et al., 2003). However, the enzymes catalyzing the conversion of formate to 5,10-methylene-THF have been poorly characterized in Arabidopsis and the subcellular localization is unknown. Enzyme activities have been shown to be mainly associated with the cytosolic fraction in pea leaves (Chen et al., 1997), but isoenzymes are also present in the mitochondrion and the chloroplast (Hanson and Roje, 2001; Christensen and MacKenzie, 2006). In animals and yeast, partially redundant pathways exist in the cytoplasm and the mitochondrion (Piper et al., 2000; Christensen and MacKenzie, 2006). Folate transporters have been identified in the Arabidopsis chloroplast membrane (Bedhomme et al., 2005) and animal mitochondria (McCarthy et al., 2004). The pathway for the conversion of formate resulting from peroxisomal glyoxylate decarboxylation was therefore placed by default in the cytoplasm in Figure 5 (green pathway).

4.3. Possible Alternative Pathways in the Mitochondrion

Metabolic profiling of Go antisense lines in rice (see chapter 2) revealed the expected accumulation of glycolate, but downstream metabolites such as glycine and serine were not reduced in concentration. This confirmed that GO-catalyzed glycolate oxidation is the dominant pathway for glycolate conversion, but also implied the existence of alternative pathways for glyoxylate production (Xu et al., 2009). Moreover, Ggat1 knockout lines (Igarashi et al., 2003, see also chapter 2) showed only weak photorespiratory phenotypes that might not be fully explained by the residual low activity of GGAT2 suggesting the existence of alternative pathways for the conversion of glycolate to glycine.

Leaf-type peroxisomes are absent in chlorophytes, the second big lineage of green algae, that subsumes the classes Chlorophyceae, Ulvophyceae, and Trebouxiophyceae (Frederick et al., 1973). Beside secreting glycolate from the cell, this group uses a mitochondrial glycolate dehydrogenase (GlyDH, EC 1.1.99.14) for the production of glyoxylate that most probably transfers electrons to the respiratory electron transport chain (Paul and Volcani, 1976). Analyses in the trebouxiophycean species Eremosphaera viridis revealed that the subsequent metabolism is similar to photorespiration in higher plants, but that all reactions are located in mitochondria and that the capacity of this pathway for glycolate conversion is lower compared to the major pathway in higher plants (Stabenau and Winkler, 2005). The importance of the mitochondrial GlyDH in chlorophytes has been demonstrated by the characterization of a Chlamydomonas strain carrying a plasmid insertion in the corresponding gene. The mutation is conditionally lethal and the strain is only capable of growing at elevated CO₂ concentrations (Nakamura et al., 2005, see also chapter 5).

In charophytes, the nearest relatives to land plants, glycolate oxidation was shifted from the mitochondrion to the peroxisome (Stabenau and Winkler, 2005), most probably because the specific activity of GlyDH is low and allows for low flux rates only. The peroxisomal GO does not show significant homology to GlyDH on the protein level and differs in important enzymatic properties: GlyDH enzymes use D-lactate as an alternative substrate, whereas GO preferentially oxidizes L-lactate. This allows for a precise discrimination of both activities.

Studies in Arabidopsis questioned the clear differentiation of glycolate metabolism in higher plants and chlorophytes (Bari et al., 2004). An enzyme with homology to bacterial and algal GlyDH enzymes (At5g06580) was shown to be targeted to the mitochondrion. The recombinant enzyme complemented Escherichia coli mutants deficient in GlyDH activity and oxidized glycolate to glyoxylate in vitro. Knock-out of At5g06580 drastically reduced the capacity of isolated Arabidopsis mitochondria to convert glycolate to CO₂ and reduced metabolite flux through photorespiration (Niessen et al., 2007). Inhibitor studies suggested that a mitochondrial alanine:glyoxylate aminotransferase might further convert glyoxylate resulting from the GlyDH reaction to glycine (Niessen et al., 2007, red pathway in Figure 5). Alternatively, this reaction might also be catalyzed by mitochondrial γ-aminobutyrate transaminase that has been shown to accept glyoxylate as a substrate. The accumulation ofγ-aminobutyrate during stress is a well documented phenomenon and would provide enough amino donor for the transamination of glyoxylate (Clark et al., 2009). By any of the aminotransferase reactions discussed, mitochondrial glycolate oxidation can be linked to the GLD/SHM complex of the major pathway.

The main advantage of the mitochondrial pathway is the direct coupling of glycolate oxidation to the electron transport chain that allows to recover energy equivalents (Paul and Volcani, 1976; Stabenau and Winkler, 2005). The combination with the high capacity major pathway in a single cell provides the plant with an optimized adaptability to changes in environmental conditions. However, neither knock-out of GlyDH nor γ-aminobutyrate transaminase resulted in an obvious phenotype under normal growth conditions (Niessen et al., 2007; Clark et al., 2009). In addition, the view that Arabidopsis GlyDH is involved in photorespiration was recently challenged by characterization of the enzymatic properties (Engqvist et al., 2009). In vitro data revealed a 2000-fold higher preference for D-lactate compared to glycolate due to a very low catalytic rate with the latter substrate. The enzyme was necessary for growth of Arabidopsis on media containing high concentrations of D-lactate, but not of glycolate. However, glycolate might have been detoxified by the peroxisomal glycolate oxidase in such a setup and this would mask a possible supplementary function of GlyDH activity. Further research is necessary to explain the conflicting data sets. As At5g06580 is expressed in all plant organs, the function of the enzyme might also differ between roots and leaves.

4.4. Possible Alternative Pathway in the Cytosol

As specified in chapter 2, Hpr1 encodes the peroxisomal hydroxypyruvate reductase involved in the major photorespiratory pathway. However, knock-out in Arabidopsis (Timm et al., 2008) or barley (Murray et al., 1989) did not result in severe photorespiratory phenotypes. It was therefore speculated that cytosolic HPR2 (Kleczkowski et al., 1991) could provide a bypass for the peroxisomal reaction (dashed grey pathway in Figure 5). A double mutant with T-DNA insertions in the genes encoding both HPR1 and HPR2 was recently characterized by Timm et al. (2008). The double knock-out showed the typical air-sensitivity and drastically reduced photosynthetic performance of photorespiratory mutants. Thus, the cytosolic pathway seemed to have the capacity to fully replace the peroxisomal pathway at least under moderate growth conditions. However, metabolic profiling and gas exchange revealed clearly stronger perturbances of photosynthesis and basal metabolism in the HPR1 compared to the HPR2 mutant (Timm et al., 2008). This suggests that peroxisomal HPR1 plays a dominant role under moderate growth conditions whereas HPR2 provides an overflow mechanism for the utilization of excess hydroxypyruvate leaking from peroxisomes under conditions of very high photorespiratory flow. Such a function in protection and/or improvement of the energy balance under non-standard growth conditions might be a joint feature of all alternative pathways and explain why these pathways have been conserved in evolution.