Ascorbate Pathway
Glutathione reductase (GR) activity was measured using a glutathione
reductase assay kit based on NADPH oxidations (Kit 703202, Cayman
Chemicals, Ann Arbor). Mid-log phase cultures (~3-10 x
106 cells) were collected by centrifugation and
resuspended in GR assay buffer (50 mM potassium phosphate, 1 mM EDTA, pH
7.5). Cells were lysed by 4 x 30 s beadbeating cycles. Twenty
microliters of lysed cell supernatant was mixed with 100 μL GR assay
buffer and 20 μL oxidized glutathione. The reactions were initiated with
50 μL NADPH, and oxidation of NADPH was measured kinetically over 10 min
at 340 nm at an assay temperature of 25° C. Activity was
calculated by ΔA340min-1mg-1 protein using an NADPH
extinction coefficient of 0.00373 μM-1.
APX activity was measured according to (Venisse, Gullner, & Brisset,
2001) with some modifications. Sample extracts were prepared as
described for the GR activity assay. Ten microliters of supernatant was
added to 190 µL of reaction buffer (50 mM potassium phosphate buffer, pH
7.8), supplemented with 0.5 mM ascorbic acid and 0.1 mM hydrogen
peroxide. Oxidation of ascorbate was monitored spectrophotometrically as
a decrease A290 (extinction coefficient 0.00168
μM-1) over 10 min to determine APX activity (Venisse
et al., 2001).
For ascorbate quantitation, 25 mL of culture (6 to 7 x
107 total cells) was pelleted and washed once in HPLC
grade H2O. Pellets were resuspended in an extracted in 2
mM ETA containing 5 mM dithiothreitol and 1% orthophosphoric acid.
Cells were broken using a bead beater (2 x 30s cycles) and samples were
centrifuged for 30 min at 19,000 x g and total cellular ascorbate levels
(ascorbate + dehydroascorbate) were determined using a commercial kit
(Ascorbate Assay Kit, Cayman Chemical. #700420).
The genome and transcriptome of UWO 241 were sequenced and assembled as
described before (Cvetkovska et al., 2019). These datasets were screened
for the presence of the genes encoding for enzymes of the AsA-GSH cycle.
Previously identified genes from the model alga Chlamydomonas
reinhardtii were obtained from the Phytozome database (v12, Joint
Genome Institute) and used as a query. Genomic sequences with a high
degree of identity (E-value cutoff 10-20) were
obtained and annotated using Geneious Prime (Biomatters Ltd, Auckland,
New Zealand).
The amino acid sequence was predicted based on the gene coding sequence,
and the identity of the enzyme was confirmed based on conserved motifs
(Pitsch Witsch, & Baier, 2010; Wu and Wang, 2019). Multiple sequence
alignments were performed using Clustal Omega (Sievers and Higgins,
2018), and protein localization was predicted using PSORT (Horton et
al., 2007) and PredAlgo (Tardif et al., 2012). Sequence data for the
UWO241 genes can be found in GenBank/EMBL database under accession
numbers listed in Table S2.