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).