Short-term photoinhibitory assays
Given the differential responses to long-term stress between the psychrophile and mesophile, we compared sensitivity and 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 tested sensitivity of both organisms to low temperature versus high light 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% decrease in FV/FM in UWO 241, while LT caused a 32% decrease in FV/FM in SAG 49.72 (Figure 8a). Conversely, LT and HL treatment cause only a moderate ( <10%) decrease in FV/FM in UWO 241 and SAG 49.72, respectively. In addition, HL-treatment of UWO 241 resulted in almost a complete loss of qL which was accompanied by an increase in ɸNPQ from 0.07 to 0.47. LT-treated SAG 49.72 exhibited only a 67% reduction in qL. Both strains exhibited comparable increases in ɸNPQ in response to the LT treatment (Figure 8b).
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 8a). The recovery was most dramatic in HL-treated UWO 241 which exhibited a rapid rate of recovery within the first 15 mins (Figure 8a). Strain- and treatment-specific differences were observed at the level of recovery of qL and ɸNPQ. Surprisingly, despite restoration of FV/FM to pre-treatment values, HL-treated cells of UWO 241 were unable to restore qL to initial levels: qL remained close to zero even after 2 hours of recovery (Figure 8b). Similarly, ɸNPQ did not relax in HL-treated UWO 241 during the recovery phase (Figure 8c). In contrast, both strains exhibited similar trends in relaxation of ɸNPQ following LT treatment (Figure 8c).
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 9). The mesophile SAG 49.72 exhibited significantly higher levels of O2- relative to pre-treated cells after 1 hr of short-term HL or LT treatment. Conversely, UWO 241 exhibited no significant change in ROS levels after the short-term stress treatments (Figure 9).