Short-term photoinhibitory assays
We compared the sensitivity and the recovery capacity of UWO 241 and SAG 49.72 to low temperature- and high light-induced photoinhibition by monitoring changes in the PSII photochemistry parameter, FV/FM. First, we conducted photoinhibitory assays at variable incubation temperatures (2oC, 8oC, 20oC) and irradiances (50 µmol m-2 s-1, 300 µmol m-2 s-1, 1000 µmol m-2 s-1). Decline in FV/FM was recorded at various time points over 3 h (Figure S4). At 8oC and 20oC incubation temperatures, UWO 241 exhibited higher sensitivity to both medium and high light treatments, relative to SAG 49.72. However, when the assay temperature was decreased to 2oC, SAG 49.72 exhibited higher photoinhibition under low light, while both strains exhibited comparable sensitivity to medium or high light treatment (Figure S4). Thus, for the recovery assays, we choose to test samples for 1 hr under two photoinhibitory treatments: i) low temperature (LT, 2oC) and ii) high light (HL, 300 µmol m-2 s-1). In agreement with our results from the preliminary assays, UWO 241 and SAG 49.72 exhibited differential sensitivity to HL and LT treatments, respectively. 1 hr of HL-treatment resulted in 62% loss of FV/FM in UWO 241, while LT caused a 32 % reduction in FV/FM in SAG 49.72 (Figure 7a). LT and HL treatment cause <10% reduction in FV/FM in UWO 241 and SAG 49.72, respectively. In addition, HL-treatment of UWO 241 resulted in almost complete reduction in qL which was accompanied by a 7-fold increase in NPQ. LT-treated SAG 49.72 exhibited only a 67% reduction in qL. Both strains exhibited comparable increases in NPQ in response to the LT treatment (Figure 7b).
Despite the strain differences in sensitivity to short-term photooxidative stress, both organisms exhibited full recovery to initial FV/FM levels within 2 h (Figure 7a). The recovery was most dramatic in HL-treated UWO 241 which exhibited a rapid rate of recovery within the first 15 mins (Figure 7A). Strain- and treatment-specific differences were observed at the level of recovery of qL and NPQ. HL-treated cells of UWO 241 were unable to restore to initial qL levels, and qL remained close to zero even 2 hours of recovery (Figure 7b). Similarly, NPQ also remained high in HL-treated UWO 241 during the recovery phase (Figure 7c). In contrast, both strains exhibited similar trends in relaxation of NPQ following LT treatment (Figure 7c).
Last, we monitored whether short-term HL- or LT-treatments were accompanied by accumulation of an ROS, using the dye NBT as a semi-quantitative measurement of O2-(Figure 8). The mesophile SAG 49.72 produced higher levels of O2- relative to pre-treated cells under both HL and LT treatments. In marked contrast, UWO 241 maintained very low levels of O2- under either short-term HL or LT stress (Figure 8).