pHSP70 expression increases after preconditioning
To unveil the molecular mechanisms underlying acquired higher thermal resilience in preconditioned corals, we first analyzed the early hyperthermal stress response of PC and NPC corals by RT-qPCR. We expected lively cell signaling reflected in rapid changes in gene expression to occur in corals during the first 24h of acute heat stress. To increase the chance of capturing these changes, we sampled PC and NPC corals (n = 10 per conditioning) exposed to 32ºC at 0, 1, 3, 6, 12, 24, 48, and 72 hours. To exclude the influence of natural circadian gene expression oscillation, control samples (NPC corals at ambient temperature) were sampled as well. We expressed gene expression level relatively to control at time 0. Considering the qPCR calculation method and the fact that gene expression changes are very dynamic and can happen in only a very short time window with no regards to previous or future levels, we analyzed the results independently for every timepoint using one-way ANOVA.
Heat shock proteins (HSP) are considered first-line stress responders, protecting other proteins from misassembling during extreme conditions. In our system, the expression of pHSP70 was very heterogeneous and colony-specific (Fig. 2A). We observed a small increase inHSP70 gene expression in NPC corals compared to control after heat stress. This result supports previous experiments that showed high variability in basal expression of HSP proteins depending on colony depth (Poli, Fabbri, Goffredo, Airi, & Franzellitti, 2017). Corals living in shallow waters (up to 3m) have high basal pHSP70expression and do not increase it significantly during heat shock, suggesting that their molecular guard mechanisms stay on alert all the time. Pocillopora corals for our experiment were collected from shallow waters (1-4m), which potentially clarifies why we did not detect changes in pHSP70 expression in NPC corals. Nevertheless, after preconditioning, pHSP70 gene expression increased at two timepoints - 3h (p =0.053) and 24h (p = 0.070) compared to NPC corals. The function of HSP in preconditioning and acclimatization remains disputed. Palumbi et al. observed alterations in HSP family gene expression after acclimatization (Palumbi et al., 2014), but others detected only limited (Bay & Palumbi, 2015) or no (Bellantuono et al., 2012) differences. Considering the dynamics of HSP70 expression, detection of its alterations is largely dependent on the experimental design and different observations might be attributed to different sampling times.
Transcription of HSP is regulated by HSF1 transcription factor (Pirkkala, Nykänen, & Sistonen, 2001). In our experiment, pHSF1 basal expression level was higher in non-stressed PC corals (p = 0.000) but after the beginning of heat stress, the levels of PC and NPC corals aligned until day 3 when we observed significant decrease of HSF1 expression in NPC corals (p = 0.001) (Figure S1). The expression of pHSF1 does not correlate with the expression of HSP70, which is not much surprising since changes in gene expression may be caused by many other factors such as transcription factor localization, promoter accessibility etc.