3.2. Regulation based on physical changes in the membrane
The membrane is the most thermally sensitive macromolecular structure in the cell (Balogh et al., 2013; Niu & Xiang, 2018). With increasing temperature, the rotational motion, lateral diffusion, and fatty acid disorder of the lipid bilayer increase, while the headgroup packing density decreases. These four parameters are different aspects of the commonly used term ’membrane fluidity’. Changes in membrane fluidity affect the folding, mobility, and activity of membrane proteins. These changes can have deleterious effects on cell functions, but at moderate levels can also serve as a basis for thermosensing. Plants, like other non-homeothermic organisms, actively maintain an almost constant membrane fluidity upon shifts in temperature (Higashi et al., 2015; Los & Murata, 2004). The increased fluidity under heat stress is counteracted by the incorporation of fluidity-decreasing saturated fatty acids, a process known as homeoviscous adaptation. In bacteria, membrane thickness is measured through the membrane protein DesK and in yeast, lipid packing density is measured by Mga2. These sensors support membrane homeostasis by the transcriptional activation of lipid desaturases when temperature drops (Ballweg et al., 2020; Covino et al., 2016; Cybulski et al., 2010).
In plants, such membrane property sensors have not been identified. Instead, heat was found to directly inhibit the activity of critical desaturases, simply by virtue of their heat-instability. The plastidial FAD8 enzyme is responsible for the synthesis of α-linolenic acid (18:3), a component of the main thylakoid lipid, monogalacosyldiacylglycerol (MGDG). FAD8 contains a labile autoregulatory domain, that destabilizes the protein upon a temperature shift from 22°C to 27°C (Matsuda et al., 2005) (Fig. 3). Reduced FAD8 stability resulted in decreased accumulation of 18:3 and reduced membrane fluidity. The importance of this is clear from the finding that mutants with low 18:3 content in their MGDG showed improved heat tolerance (Murakami, 2000). This may be because 18:3-MGDG is prone to oxidative damage and perturbs membranes. The ER desaturases FAD2 and FAD3 also display thermolability. Upon transfer to warm temperatures, FAD2 and FAD3 are targeted for ubiquitin-mediated or ER-associated degradation, respectively (O’Quin et al., 2010; Tang et al., 2005). Currently the mechanism by which these desaturases are inactivated upon heat stress is unknown; they may either be direct thermosensors or act downstream of temperature perception.
Adjustment of membrane fluidity through changes in membrane desaturation is a slow process and can take several takes days (Falcone et al., 2004). In the case of acute heat stress, alternative mechanisms are employed to secure bilayer integrity. These sense-and-respond mechanisms are based on heat-induced, biophysical changes in membrane properties. In thylakoid membranes, heat induces packing defects in the lipid headgroups. These defects provide a spatial cue for docking of proteins with membrane-protecting functions, such as sHSP (Heckathorn et al., 1998) and vesicle-inducing protein in plastids 1 (VIPP1, Fig. 3) (Theis et al., 2019; L. Zhang et al., 2016). The inducible association of these proteins with membranes likely follows the sensing of the membrane status through their amphipathic α-helices.
Acute heat also induces the aggregation of light-harvesting complex II (LHCII) proteins in the thylakoid membranes. MGDG is normally associated with LHCII, and upon aggregation of LHCII, excess MGDG gets extruded to the lumen (Jahns et al., 2009; Schaller et al., 2010) (Fig. 3). Due to MGDG’s non-bilayer propensity, extruded MGDG forms a so-called inverted hexagonal phase (HII) (Garab et al., 2017; Krumova et al., 2008). Thylakoid membranes are always close to HII phase transition and, as HII phases emerge, they must be controlled to avoid damage. HII phases are however key to chloroplast heat acclimation because when they emerge under stress, they recruit and activate the xanthophyll cycle enzyme, violaxanthin de-epoxidase (VDE). VDE synthesizes zeaxanthin which quenches excess excitation energy and enhances membrane stability. The HII phases serve the sequestering of excess MGDG and promote the diffusion of xanthophylls (Latowski et al., 2002) (Fig. 3).
Another membrane feature that can undergo rapid stress-induced modification are microdomains. Most lipids within a membrane exist in liquid-disordered phase, often envisioned as a two-dimensional fluid. Lipids can however also exist in the liquid-ordered phase known as nano- and microdomains (Jaillais & Ott, 2020; Saenz et al., 2012). Microdomains form coherent, dynamic platforms for proteins with functions in sensing, signaling, membrane integrity maintenance and transport. Even mild changes in temperature can result in altered microdomain fluidity and consequently, redistribution and modified activity of these proteins (Török et al., 2014). Based on studies of membranes and Molecular Dynamics simulation, microdomains are speculated to act as dynamic reservoirs of fluidity-decreasing lipids. Heat may trigger increased partitioning of these lipids from microdomains to the bulk fluid phase (Nickels et al., 2019). This simple buffering effect that can occur in complex membranes is based on thermodynamics of phase separation and could be far more responsive than the metabolic responses of homeoviscous adaptation (Ernst et al., 2018).
Some plasma membrane microdomains are tethered to the underlying cortical ER at so-called ER-plasma membrane contact sites (EPCSs) through synaptotagmins (SYT1 and SYT3) (Ruiz-Lopez et al., 2020). SYT1/3 are ER proteins that bind (via C2-domains) to phosphatidylinositolphosphate (PIP)-containing microdomains of the plasma membrane (Fig. 4). The close proximity of the two membranes allows for exchange/removal of detrimental lipids, e.g. diacylglycerol that is formed at the plasma membrane during phospholipase C (PLC) signaling (see below ). In yeast, EPCSs are important for plasma membrane integrity maintenance under heat stress (Collado et al., 2019), and they appear to function similarly under stresses in plants (Ruiz-Lopez et al., 2020; Yan et al., 2017).
The biophysical changes in membranes under heat stress can be sensed by altered protein activity and/or location. Moreover, the alternative lipid phases allow for prompt, thermosensitive responses, and thereby provide structural and functional flexibility that is of vital importance under heat stress. Notably, this suggests that homeoviscous adaptation does not necessarily involve a sensor of membrane fluidity. Whether fluidity sensing underlies other heat stress responses remains unknown. Many studies have attempted to probe the effect of membrane fluidization using pharmacological and genetic interventions, but it is becoming clear that these techniques have indirect effects on proteins and gene expression (Rütgers, Muranaka, Schulz-Raffelt, et al., 2017; Vu et al., 2019).