**1. Introduction**

Photorespiration begins with the fixation of O2 to ribulose-1,5-bisphosphate by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) leading to the formation of one molecule of 2-phosphoglycolate (2PG) and one molecule of 3-phosphoglycerate (3PGA). 2PG is then metabolized to produce 3PGA by the photorespiratory cycle which occurs in four subcellular compartments (chloroplasts, peroxisomes, mitochondria and cytosol), and involves eight core enzymes and several transporters [1]. Photorespiration has a negative impact on plant yield since it limits photosynthetic CO2 assimilation due to competition at the RuBisCO active site, and it releases assimilated carbon and nitrogen as CO2 and ammonium that have to be either reassimilated at an energetic cost or lost. This has led to efforts to minimize the negative effects of photorespiration to improve plant yield by producing plants containing a chloroplastic bypass to metabolize photorespiratory glycolate (the most recent examples being [2,3]). However, photorespiratory glycolate produced in the chloroplast from toxic 2PG [4] is normally metabolized in peroxisomes by glycolate oxidase (GOX), a flavin mononucleotide (FMN) containing enzyme that catalyzes the transformation of glycolate to glyoxylate

with the production of hydrogen peroxide [5]. This enzyme evolved from a bacterial lactate oxidase and it is a member of the α-hydroxy-acid oxidase superfamily [6]. In *Arabidopsis*, there are five GOX-related genes: *At3g14420*, *At3g14415*, *At4g18360*, *At3g14130* and *At3g14150* (encoding *At*GOX1, *At*GOX2, *At*GOX3, *At*HAOX1 and *At*HAOX2, respectively). According to transcriptomic analyses, *At3g14415* (*AtGOX2*) and *At3g14420* (*AtGOX1*) are highly expressed in leaves and they represent the major GOX isoforms in *Arabidopsis thaliana* [7]. *AtGOX3* is mainly expressed in senescing leaves and roots, where it has been proposed to also function as a lactate oxidase and thus play a role in lactate metabolism [8]. *AtHAOX1* and *AtHAOX2* are expressed in seeds and encode proteins preferring medium- and long-chain hydroxyl acids as substrates [6]. Knock-out mutants of each Arabidopsis *GOX* gene do not exhibit a photorespiratory growth phenotype although they were all more sensitive to *Pseudomonas syringae* and to ozone [9,10]. A photorespiratory phenotype in air that was reversed by elevated CO2 (3000 ppm) was observed however in an Arabidopsis artificial miRNA GOX line (*amiRgox1*/*2*) with both *AtGOX1* and *AtGOX2* knocked-down and only 5%–10% of wild-type Arabidopsis leaf GOX activity [7]. The transfer of *amiRgox1*/*2* plants from high CO2 to ambient air led to a 700-fold accumulation of glycolate and a reduced carbon allocation to sugars, organic acids and amino acids that induced an early senescence of older leaves [7]. Even though *At*GOX1 and *At*GOX2 showed a redundant photorespiratory function, *At*GOX1 appeared to have a more predominant role in photorespiration since it attenuated the phenotype of a *cat2* mutant [11]. The importance of GOX has been observed also in tobacco, rice and even maize (a C4-plant), where a reduction of GOX activity in RNAi, antisense or mutant lines led to delayed growth in air associated with a decrease of net CO2 assimilation rate [12–15].

Since photorespiration interacts with several metabolic processes including photosynthesis, nitrogen metabolism, respiration, C1 metabolism as well as H2O2 production by GOX [16], it might be expected that the photorespiratory cycle would be coordinated with these processes and modulated according to metabolic needs and perhaps environmental cues. Even though photorespiration has been widely studied over the last few decades, regulation of this C2-cycle and its enzymes is still poorly understood. It has been proposed that serine acts as a signal to regulate the expression of photorespiratory genes [17]. Several peroxisomal photorespiratory enzymes have been associated with putative post-translational modifications such as ubiquitination, nitration, persulfidation, and acetylation (for a review see [18]). Indeed, the activity of pea GOX was found to be inhibited by S-nitrosylation [19] as was Arabidopsis glycine decarboxylase [20]. The glycerate kinase of maize was shown to be redox-regulated by thioredoxin f; however, this was not the case for the Arabidopsis enzyme [21]. Arabidopsis mitochondrial glycine decarboxylase L-protein activity was found to be redox regulated by thioredoxin [22,23]. Protein phosphorylation could be another post-translational mechanism involved in the regulation of the photorespiratory cycle as phosphoproteomics studies have identified a number of phosphopeptides associated with all but one of the core photorespiratory enzymes [24]. Concerning photorespiratory *At*GOX1 and *At*GOX2, several phosphorylation sites (T4, T155, T158, S212, T265 and T355) have been reported (see Table 1).

In this study, the consequences of T4, T158, S212 and T265 phosphorylation on *At*GOX1 and *At*GOX2 enzymatic activities and kinetic properties were analysed using purified recombinant wild-type, phospho-dead and phosphorylation-mimetic GOX proteins. This was also carried out to test the equivalent residues (T5, T159, S213 and T266) of maize photorespiratory GOX (*Zm*GO1) [15]. Our results are discussed in terms of the possible consequences of each GOX phosphorylation on enzyme structure and function and in response to environmental stresses and future avenues to explore to better understand photorespiratory GOX phosphorylation are proposed.
