3.3. The primary roles of Ca2+ and
H2O2 signaling
Intracellular Ca2+ increases are a feature of all
plant stress responses, and heat stress is no exception (Finka et al.,
2012; Saidi et al., 2011). Ca2+ increases were found
to be required for the induction of HSPs through
Ca2+/calmodulin-dependent kinases and for the
acquisition of heat tolerance (H.-T. Liu et al., 2003, 2007). It has
been reasoned that Ca2+ channels could function as
thermosensors at moderate temperature elevations and many attempts have
been made to identify such channels (Ding et al., 2020; Horvath et al.,
1998; Saidi et al., 2011).
Using Arabidopsis seedlings expressing the Ca2+reporter aequorin, Lenzoni and Knight (2019) were unable to detect a
heat-induced cytosolic Ca2+ response. Instead, they
found a Ca2+ increase in the chloroplast stroma
(Lenzoni & Knight, 2019). Temperatures of >35°C induced
rapid Ca2+ responses, with higher temperatures
provoking higher and faster peaks. The response was not influenced by
the rate of warming, but was determined by the absolute temperature. The
thylakoid membrane Ca2+ sensor CAS was required for
full induction of stromal Ca2+ in response to heat
(Fig. 3). CAS amplified the Ca2+ signal, but what
governs the initial signal is still unknown.
Cyclic nucleotide-gated channels (CNGCs) have also been shown to mediate
heat-induced cytosolic Ca2+ increases inPhyscomitrella patens and in Arabidopsis. A temperature
increase from 22°C to 28°C in moss quickly triggered a transient inward
electrical current, likely reflecting Ca2+ influx
through PpCNGCb (Finka et al., 2012; Saidi et al., 2009). Based on the
use of channel blockers and chelators, the heat-activated
Ca2+ current was attributed to CNGC6 in Arabidopsis
(Finka et al., 2012; F. Gao et al., 2012) (Fig. 4). CNGC6 is activated
by cAMP, which also increases upon heat stress. This has led to the
suggestion that an, as yet unidentified, adenylyl cyclase activity could
act as membrane-associated temperature sensor (Thomas et al., 2013).
Research on CNGC functions is complicated by the fact that CNGC subunits
make various combinations to form heterotetrameric channels. These
channels differ in ion conductance, physiological function and mode of
regulation, and mutations in CNGC subunit genes often lead to pleotropic
mutant phenotypes (Dietrich et al., 2020).
Identifying the primary heat-activated Ca2+ channel
remains a challenge. The animal heat-activated Transient Receptor
Potential channel, TRPV1, is a mechanosensitive and voltage-gated cation
channel (Benítez-Angeles et al., 2020). It likely responds to forces
transmitted via microtubules (Bavi et al., 2017; Prager-Khoutorsky et
al., 2014). Plants lack TRP channel homologs, but possess other
mechanosensitive channel types that may function in heat signaling,
including OSCA1 (reduced hyperosmolality-induced
[Ca2+] increase1), MCA (Mid1-complementing
activity), which was implicated in cold sensing (Mori et al., 2018), and
Small Conductance Mechanosensitive Ion Channel (MscS)-Like (MSL)
proteins (Ackermann & Stanislas, 2020).
Annexins are another class of membrane proteins that may play a role in
heat-induced Ca2+ signaling. Proteomic and forward
genetic approaches identified several Arabidopsis annexins that enhanced
heat- and oxidative stress-induced Ca2+ responses and
the expression of HSPs and HSFs (Liao et al., 2017; X. Wang et al.,
2015). In the presence of Ca2+, annexins bind to
anionic lipids, such as phosphatidic acid (PA) and phosphatidylserine
(PS) in the plasma membrane (Yadav et al., 2018). Heat stress rapidly
induces ANNEXIN1 (ANN1) membrane association, but this may be a
downstream effect of Ca2+ and/or PA accumulation (see
below), rather than a direct effect of temperature on ANN1.
In addition to Ca2+,
H2O2 levels at the plasma membrane also
rise quickly in response to heat stress, a process catalyzed by the
NADPH oxidase, RbohD . The plasma membrane
H2O2 signal is required for the heat
stress gene expression and the enhancement of heat tolerance (Suzuki et
al., 2012; Volkov et al., 2006). There is also accumulation of
H2O2 in chloroplasts and mitochondria
and this may provide additional priming signals (Sun & Guo, 2016). ANN1
is activated by H2O2 (Richards et al.,
2014) and so it may be that annexins function to co-ordinate
H2O2 and Ca2+ signals
under heat stress (Fig. 4).
RbhoD-derived H2O2 has also been shown
to accumulate in the apoplastic space in a self-propagating manner. This
results in an extracellular ROS wave travelling through the plant at a
speed of 8.4 cm/min (Miller et al., 2009; Suzuki et al., 2012). Local
heat stress triggered accumulation of
H2O2 can thereby lead to acclimation of
systemic tissues (Miller et al., 2009).
H2O2 and Ca2+ signals
are not unique to heat stress and it is presently unclear how these
signals lead to particular stress responses. It has been proposed that
the relative intensity or timing of these signals confers specificity to
the response (Gilroy et al., 2016).