AsA-GSH pathway
The enzymes APX and GR are key enzymes of an ROS detoxification pathway, the AsA-GSH cycle, catalyzing the first step of the pathway and regeneration of reduced glutathione, respectively (Noctor & Foyer, 1998). Enzymatic assays revealed low activity for either APX or GR in SAG 49.72, grown under control or HL, LT and HS stress conditions (Figure 7A). In contrast, UWO 241 exhibited significantly higher activity for both enzymes under control and all stress treatments relative to SAG 49.72 (Figure. 7). GR activity was highest in HS-UWO 241 cells, while APX activity was highest in LT-UWO 241 cells relative to controls. Last, AsA-GSH pathway substrate, ascorbate, was significantly higher in UWO 241 compared with values typically reported for other algae (Gest, Gautier, & Stevens, 2013). Total cellular ascorbate 9.61 ± 1.20 and 18.52 ± 1.90 mM ascorbate under control and high salinity conditions, respectively, compared with 0.93 ± in the mesophile,C. reinhardtii (Figure S2).
Screening of the UWO 241 genome and transcriptome revealed homologs for all genes involved in the AsA-GSH cycle, with the exception of monodehydroascorbate reductase (MDHAR) (Tables S2 and S3). The genome of UWO 241 encodes 5 genes identified as APX (APX1, APX2-A to –D), which share a high sequence similarity with homologous genes from other photosynthetic organisms and the presence of conserved motifs involved in APX catalytic function (Figure S3a). Four of these genes (APX2-A to –D) are found on the same contig in a head-to-tail orientation and share a high sequence similarity (83.1-93.3%), suggesting a recent gene duplication event (Figure S3b). This is in contrast with other green algae that typically encode one or two APX genes with confirmed APX activity (Pitsch et al., 2010; Gest et al., 2013). All other genes, including GR, were present as a single copy and shared a high sequence identity with homologous genes from C. reinhardtii (Table S3).