HSP protein accumulation in UWO241 and C. reinhardtii
Next, we investigated whether HSP expression trends are mirrored at the protein level using antibodies for several major HSPs in UWO241 andC. reinhardtii . In the first set of experiments, we cultured both species at a range of temperatures, including the highest temperature that can support growth (4-17°C for UWO241; 22-37°C for C. reinhardtii ). The cytosolic HSP70A, HSP90A, and the chloroplast CPN60A exhibited higher protein levels in UWO241 when compared to C. reinhardtii at most culturing temperatures (Figure 9). HSP70B accumulated at comparable levels in both species, especially at higher growth temperatures, while HSP90C was lower in UWO241 in comparison toC. reinhardtii (Figure 9, Supplementary Figure S6). The steady-state culturing temperature had only a minor effect on HSP accumulation, with four of the tested HSPs (CPN60A, HSP70A, HSP90A, HSP90C) accumulating at slightly higher levels at 4°C when compared to higher growth temperatures in UWO241 (Figure 9, Supplementary Figure S6). To test whether the high accumulation of HSPs is due to growth at low temperatures regardless of the species, we also cultured C. reinhardtii at 15°C and 10°C. We compared low temperature HSP accumulation to that at optimal growth conditions (28°C) and to cultures exposed to heat shock (42°C) for 6 hours. Algal cultures that were acclimated to 10°C and 15°C accumulated HSPs at much higher levels than those acclimated to 28°C and at comparable levels to those exposed to heat shock at 42°C (Figure 10). These data suggest that accumulation of high HSP levels could be an adaptive strategy for low temperature growth in green algae.
HSP gene expression and protein accumulation is strongly induced by heat stress in C. reinhardtii (Mühlhaus et al. 2011; Schmollinger et al. 2013). To test whether the same is true for an obligate psychrophile, we exposed UWO241 cultures grown at 4°C, 10°C and 15°C to heat stress (24°C) for 6 hours. We applied an equivalent treatment to C. reinhardtii grown at low (10°C), optimal (28°C) and high (37°C) temperature by exposing the cultures to heat shock (42°C) for 6 hours. Most C. reinhardtii HSPs increased in abundance during heat shock, including the HSF1 transcription factor, regardless of the initial culturing temperature (Figure 11). HSP accumulation was induced even in cultures acclimated to 10°C, which accumulated high HSP amounts prior to the heat shock (Figure 6b, Figure 7). In contrast, heat shock had only a very minor effect on HSP accumulation in UWO241, and HSP accumulation did not significantly increase with heat stress, regardless of the initial culturing temperature (Figure 7b, 7d). We could not detect HSF1 in UWO241, either due to low antibody specificity or very low protein levels. Thus, HSP accumulation is significantly induced by heat stress in the mesophile but not appreciably in the psychrophile.