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.