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).