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 6A). In contrast, UWO 241 exhibited significantly higher
activity for both enzymes under control and all stress treatments
relative to SAG 49.72 (Figure. 6). 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 10.0 ±
4.7 and 12.3 ± 5.71 mM ascorbate under control and high salinity
conditions, respectively (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) which was not located (Table S2).
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 S2).