*2.1. Phosphopeptides and Phosphorylated Residues of Arabidopsis GOX1 and GOX2*

In the context of a possible regulation of photorespiratory GOX by protein phosphorylation, a first step was to retrieve potential phosphorylated residues from phosphopeptides associated with *At*GOX1 and *At*GOX2 from published phosphoproteomics studies and the PhosPhAt 4.0 database (http://phosphat.mpimp-golm.mpg.de/) (see Table 1). In this way, six phosphopeptides were found with six different phosphorylated residues; T4, T155, T158, S212, T265 phosphopeptides associated with both *At*GOX1 and *At*GOX2 and T355 that was in a peptide associated only with *At*GOX1 (Table 1).


**Table 1.** Phosphopeptides associated with *At*GOX1 and *At*GOX2.

The phosphorylated residue in each peptide is shown in bold with its position numbered according to *At*GOX1. Peptide location shows the position of each phosphopeptide with respect to the number of residues in *At*GOX1. <sup>1</sup> PhosPhAt 4.0: http://phosphat.mpimp-golm.mpg.de/.

All of these phosphorylated amino acids were conserved among *At*GOX1, *At*GOX2 and *At*GOX3 proteins while T4 and T158 were replaced by a valine in *At*HAOX1 and *At*HOAX2 (Figure S1). To analyse the conservation of these phosphorylated residues amongst (S)-2-hydroxy-acid oxidases (EC 1.1.3.15) of bacteria, cyanobacteria, algae, human and plants, we compared amino acid sequences of lactate oxidase (LOX) from *Aerococcus viridians, Lactococcus lactis, Nostoc* and *Chlamydomonas reinhardtii,* GOX from *Zea mays*, *Arabidopsis thaliana*, *Brassica napus*, *Vitis vinifera*, *Nicotiana benthamiana*, *Spinacia oleracea*, *Populus alba* and *Homo sapiens* and HAOX from *Arabidopsis thaliana* and *Homo sapiens* (Figure S2). The residues corresponding to T155, S212 and T265 of Arabidopsis GOX were conserved as either a serine or a threonine in all species (except for T265 in *Chlamydomonas reinhardtii* LOX, S212 in *Lactococcus* LOX and HAOX2 of *Homo sapiens,* T265 in *Chlamydomonas reinhardtii* LOX), whereas T4 was conserved only in the plant GOX proteins while T158 was conserved in all GOX proteins, human HAOX2, *Aerococcus viridians* LOX and *Lactococcus lactis* LOX (Figure S2). Based on these observations, it was decided to investigate further the phosphoregulation of photorespiratory GOX by characterising phospho-mimetic recombinant *At*GOX1 and *At*GOX2 as well as the C4-plant enzyme, *Zm*GO1 that had been shown to be important for growth in air (400 ppm CO2) [15]. Among the six phosphorylation sites, the highly conserved plant GOX residues, T4, T158, S212 and T265 of *At*GOX1 and *At*GOX2 were chosen for this study that corresponded to T5, T159, S213, T266 of *Zm*GO1 (Figure S2).
