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.