4.. Conclusions and future challenges
Clearly, our knowledge of thermosensory systems of plants has greatly expanded in the past decade. Important discoveries in ambient temperature signaling include the identification of photo/thermal sensors, an RNA switch and self-coalescence of ELF3 through its prion-like domain. These three systems unambiguously translate high ambient temperatures into altered gene expression. The reprogramming of development by these factors assists plants to avoid damaging high temperatures. In Arabidopsis, the inhibition of phyB activity by warm temperatures has been shown in detail. It is however still unknown if other photoreceptors function in a similar way. Phototropin plays a role in low temperature signalling in Physcomitrella but it is not known whether this temperature-dependent activity also stretches to warm temperatures. Other photoreceptors undergo thermal reversion and so could conceptually also function in warm temperature sensing, but this remains to be demonstrated. The finding that PIF7 RNA translation is enhanced at warm temperatures opens the possibility that other RNAs act in a similar way. Indeed, the authors of the PIF7 study also show that HSF2 RNA may also be regulated through a comparable mechanism (Chung et al., 2020). The finding that ELF3 contains a prion-like domain that undergoes temperature-dependent coalescence likely has effects beyond just evening complex transcriptional repression. ELF3 acts as a scaffold protein for large protein complexes (Huang et al., 2016) and directly binds to PIF4 to inhibit its transcriptional activity (Nieto et al., 2015). Both of these functions are likely inhibited at warm temperatures.
Upon moderate temperature increases, plants trigger a heat stress response for acclimation, but the sensing mechanism is still largely unknown. Rather than unfolded proteins, the increased membrane fluidity at high temperatures is speculated to be a molecular basis of sensing. While no fluidity sensor has been found in plants, evidence is accumulating for thermosensory mechanisms based on heat-induced phase changes in lipids and proteins.
Under heat stress, thylakoid membranes locally undergo transition to non-bilayer, HII phases, which are essential for heat acclimation, since they compartmentalize and activate the enzymes of the xanthophyll cycle. At the plasma membrane, microdomains are formed containing lipids in a liquid-ordered phase. These domains harbor potential signaling lipids and proteins, including RbohD, which is activated in response to heat stress. Formation of microdomains and HII phases could constitute a basis for thermosensitive regulation of enzymes. Simultaneously, they provide potential avenues for the rapid trafficking of lipids between phases, in order to preserve membrane integrity under heat stress. For the latter function, a membrane fluidity sensor would thus not be required.
The regulation of ELF3 has unveiled a novel type of ambient temperature sensing mechanism, based on liquid-liquid phase separation (Jung et al., 2020). Under heat stress, the formation of spherical, condensed liquid phases within a bulk dilute phase can be triggered by the coalescence of proteins through their intrinsically disordered, prion-like domains. The resulting liquid droplets, also called membraneless organelles, constitute compartments that can contain proteins with associated regulatory functions. The reversible process of droplet formation is highly temperature sensitive. Could liquid-liquid phase separation also function, at higher temperatures, in the activation of the heat stress response? Such a function was recently proposed for the yeast RNA-binding protein, Pab1 (poly(A)-binding protein), which displayed self-coalescence and phase separation upon a shift to a temperature that induces the heat-shock response (Riback et al., 2017). The extreme thermosensitivity of this process was quantified using the temperature coefficient Q 10, the ratio of biological properties measured 10°C apart. With a Q 10 of 350, it exceeds by far any other known biological thermosensory process. This indicates the potential of liquid-liquid phase separation of proteins as a thermosensing mechanism. The sharp threshold temperature above which phase separation is triggered, which is determined by the amino acid side chains in the prion-like domain, allows for precise temperature-dependent regulation of responses. Pab1 was speculated to activate the heat stress response by sequestering a negative regulator of HSF in liquid droplets, calling into question the requirement of unfolded proteins for activation (Riback et al., 2017). It seems plausible that similar regulation governs heat stress responses in plants.
The stress-induced clustering of proteins into membrane microdomains could trigger liquid-liquid phase separation in the adjacent cytosol. This could result in coupled lipid and liquid compartments, that assemble selected response components, allowing for specific channeling of sensory signals to downstream responses (Jaillais & Ott, 2020). Similarly, plasma membrane organization could respond to changes in the cell wall, which may also adopt different biophysical states dependent on temperature (Wu et al., 2018). As yet, such potential interactions are unexplored territory.
Identifying plant proteins that could act as thermosensors through liquid-liquid phase separation will be challenging. Previously, heat stress was found to induce relocalization of splicing factors with disordered domains, e.g. serine/arginine-rich protein SR45, into enlarged nuclear speckles (Ali et al., 2003; Reddy et al., 2012), which could underlie alternative splicing of pre-mRNAs. Many of the, approximately, 500 proteins in plants with predicted prion-like domains are transcription factors with potential roles in temperature signalling. There are prion-like domains in HSFA1b, and several PIFs, auxin response factors and ABRE-binding factors (Chakrabortee et al., 2016). Investigating the effect of temperature on the coalescence of these factors in vitro could yield interesting results.
Thermosensing appears to be a highly distributed capacity, based on a range of mechanisms which are only just beginning to come to light. Most strikingly, the temperature-dependent behavior of phyB, the PIF7 RNA hairpin, and both lipid and liquid phase separations, provides an impressive spectrum of potential heat sensing and responding modes, essential for plants to acclimate and survive.