An Overview of Biomembrane Functions in Plant Responses to High-Temperature Stress

1The Fifth Assessment Report of the IPCC showed that the increase in the global average surface temperature from 1880 to 2012 reached 0.85°C, and the last three decades in particular were persistently warmer than any preceding decades. Moreover, the report predicted that the extreme annual daily maximum temperature would be elevated by approximately 1–3°C by the mid-21st century. Although warming has been observed to benefit crop production in some high-latitude regions, the increasing threat of climate change has had negative impacts on crop yields over a wide range of areas, and the detrimental effects are expected to be aggravated in the next century. In addition, secondary disasters that accompany high temperatures, such as drought and soil salinization, could trigger dramatic crop loss worldwide and even lead to global food shortages in the future. In fact, regardless of the climatological extremes, plants, being sessile, are inevitably exposed to a wide range of temperature stresses, including daily and/or seasonal temperature fluctuations that may reach dozens of degrees. Each plant species naturally exhibits its own optimal temperature range for growth and reproduction, and thus, extreme variations can rapidly exert a thermodynamic influence on the intracellular macromolecules (nucleic acids and proteins) and substructures of plant cells. For instance, a temperature of 5°C above optimal growing conditions can induce a series of changes in the organization of cell compartments, including the organelles and cytoskeleton, as well as the membrane microdomains necessary for plant survival at high temperatures. Plants have evolved different adaptation mechanisms to withstand adverse conditions for optimal growth, the most common of which involve the activation of stress-induced gene expression related to the homeostatic adjustment of plant metabolism and development. Therefore, breeding efforts focused on the transformation of these genes as supported by genetic approaches have been employed to efficiently improve plant adaptability to extreme temperatures. In particular, heat shock transcription factors and heat shock proteins (HSFs/HSPs) have been demonstrated to constitute a master regulatory pathway involved in the response to heat stress based on half a century of research, and some of these genes have since been applied in sustainable agriculture. Nevertheless, in considering the mechanisms underlying the high-temperature stress response, one should not ignore how plants first quickly perceive and translate the stress into intracellular response signaling. In this respect, cell membranes, which constitute the barrier to the external environment and separate the cytosol into several microcompartments, are well suited for sensing stress and providing a “crosstalk" interface. Subtle alterations in lipids may affect various properties of the membrane, including its fluidity, thickness, permeability, and packing, and thus affect the clustering of important membrane proteins, which are sensitive to ambient conditions. However, unlike in animal cells, membrane structures in plant cells possessing a cell wall may display an enormous and specific variety of components and functions. Combined with the diversity of plant responses to adverse environments, this complexity is such that the majority of the underlying thermotolerance mechanisms in plants remain unclear. The various challenges remaining in this field will be addressed in the following open questions.

2In this review, we provide an overview of the currently known factors and/or mechanisms that can affect the states and properties of the plasma membrane (PM) and thylakoid membrane as well as the endoplasmic reticulum system (ER) in responses to and in tolerance of high-temperature stress in land plants (largely in reference to Arabidopsis thaliana). Some critical perspectives on the roles of membranes in these processes will also be presented, and we will discuss membrane-stress responses under heat stress within different plant taxa, including several psychrophiles, mesophiles, and thermophiles in the plant kingdom (as classified by;; Table 1).

3Membranes serve as a selectively permeable barrier, and they are primarily composed of proteins and lipids in moving mosaics. Lipids with a polar head group and diverse types of long hydrophobic tails spontaneously form two leaflets in aqueous environments due to their amphipathic properties. Lipid species can be divided into three primary classes by chemical structure, namely, glycerolipids, sphingolipids and sterols. Among the abundant glycerolipids, phospholipids predominantly occur in the PM and mitochondrial envelope, while galactolipids primarily constitute the thylakoid membrane. Phospholipids contain two fatty acyl chains and a variable polar head group assembled to the glycerol backbone. Although phospholipids are usually sorted by their polar head groups, the fatty acid moieties greatly control their physicochemical properties. The number and position of C–C bonds within hydrophobic acyl tails and the length of the carbon chains are thought to be key factors in determining the membrane fluidity. Moreover, the lipid composition of each leaflet reflects another membrane property (asymmetry). Lipids are selectively embedded in membrane bilayers; phosphatidylcholine (PC), glycolipids and sphingomyelin are predominantly enriched on the exoplasmic face of the PM and the lumenal side of internal organelles, while phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) are primarily found on the cytoplasmic side; cholesterol shows preferential accumulation on the exoplasmic leaflet, although it can move freely between the two leaflets. Additionally, some minor phospholipids, such as phosphatidic acid (PA), phosphatidylinositol-4-monophosphate (PIP) and phosphatidylinositol-4,5-biphosphate (PIP2), are also enriched on the cytoplasmic leaflet.

4The fluid mosaic hypothesis proposed by indicated that lipids formed a discontinuous, fluid bilayer in which proteins were embedded via specific interactions with lipids. Today, evidence regarding the structure and function of membranes inspires us to consider the biomembrane as a more complex and exquisite device. Different regions of membranes with defined lipids and clusters of proteins are not always equal, forming discrete platforms with varying sizes and traits in the plane of the membrane (which are denominated as membrane microdomains) (;;;;; Figure 1). Historically, lipid rafts enriched in sphingolipids, sterols and glycosylphosphatidylinositol (GPI)-anchored proteins were thought to be equivalent to membrane microdomains or detergent-insoluble membranes (treated with 1% Triton X-100 at 4°C;;; ). Over the past decade, this idea has come to be considered controversial, if all of the above concepts reflect the authentic membrane substructure. With the development of experimental approaches, the microdomains of membranes can now be distinguished at multiple scales (reviewed by ). Nanoscale domains based on lipid rafts (2–100 nm) are incorporated into larger microdomains (e.g., raft platforms) via the interactions between specific lipids and proteins; both cytoskeleton and cell wall components prop up the largest units, which are denominated as the membrane compartments (40–300 nm), depending on the cytoskeletal and cell wall restriction of the lateral diffusion of membrane proteins. In contrast to lipid rafts with a highly dynamic nature in mammalian cells, special lateral membrane compartments in plant cells are more stable. Substantial evidence supports the notion that the microdomain acts as a harbor in which cellular signaling is clustered by the interactions between membrane components (including previously isolated channels, receptors, other signaling complexes and specific lipids) during many physiological processes, such as biotic and abiotic stress responses, membrane transport and polarized plant growth. An ROS-generating enzyme known as respiratory burst oxidase homolog D (RBOHD) has been suggested to localize to membrane microdomains (;; Figure 1) and could be activated by a heat-induced cytosolic calcium increase to immediately produce an oxidative burst. However, numerous microdomain-associated proteins remain to be elucidated in plants. Notably, increasing evidence from mammals has illustrated that several HSPs localize to PM rafts via interactions with specific lipids (reviewed by ). These HSPs include abundant HSP70, HSP90 and other small HSPs (e.g., HSP27), the functions of which have been suggested to include the recruitment of signaling proteins to the PM, the folding of membrane proteins, assisting polypeptides in translocation across the membrane bilayer, and rapidly remodeling and protecting the PM under cellular stresses. Although the mechanism controlling the interactions between HSPs and membrane lipids and proteins is less well known, it is speculated that the structure of lipid rafts is crucial for HSPs to perform their individual functions in cell responses. Whether HSPs in plants exhibit similar behaviors at PM microdomains has not yet been explored.

5Plants that survive under extreme temperatures must first maintain constant membrane fluidity and integrity, which requires dynamic changes in the membrane composition. Early studies have shown that the ratio of saturated and unsaturated fatty acids differs greatly between lipid species and tissues. In addition, the saturated and monounsaturated fatty acid contents increase in response to elevated temperatures, while the proportion of polyunsaturated fatty acids can be increased by a sudden drop in temperature. Temperature is the most important contributing factor in regulating the unsaturation degree of fatty acid chains, and thus, it has a large impact on the properties of membrane bilayers. The numbers and sites of double bonds within fatty acid chains are modified by desaturases via an oxygen-dependent reaction. In plants, the fatty acid desaturase (FAD) family, which plays an important role in adaptation to high-temperature stress, has been studied extensively. Among the eight members of the Arabidopsis FAD family (FAD1 to FAD8), FAD2 and FAD3 are located in the ER membrane, while the others are plasmid-specific desaturases (;; Figure 1). Although the synthesis and unsaturation of the major lipids are performed in the chloroplasts or ER in higher plants, acyl chain modifications of lipids embedded in the PM rely on regulation by various desaturases and on lipid transfer. Ambient stresses, such as temperature fluctuations, are perceived by the PM and/or membranous organelles and can affect FAD activities at both the transcriptional and post-transcriptional levels, thereby altering the membrane fluidity. Recently, increasing evidence has shown that FAD2 and FAD3 are unstable and less enriched at elevated temperatures. The seedlings of the fad2 mutant are unable to elongate their stems at 12°C and die at 6°C; similarly, the lethal temperature for fad1 is 2°C. Interestingly, the dwarf fad2 plant has been found to exhibit enhanced reactive oxygen species (ROS) and salicylic acid (SA) signaling, which are considered to regulate the heat stress response. Additionally, other fad mutants, such as fad5, show higher growth rates depending on the thermal conditions. FAD3, FAD7, and FAD8 have all been demonstrated to confer high-temperature instability, and three transgenic lines have exhibited higher levels of trienoic fatty acids in response to extreme temperatures. In fact, the varying amounts of unsaturated fatty acids incorporated into the major glycerolipids in fad mutants alter the composition and properties of their membranes, which may be the reason why their cells can sense and react to high temperatures. Confusingly, however, the mechanism through which FADs rapidly react to sudden temperature fluctuations and upstream factors with temperature sensitivity to regulate the activities of FADs remains unclear in plants.

6It is not only the chemical constitution of lipid membrane bilayers that is closely associated with the temperature; the multiple types of molecular motion by lipids (including rotation around their own axes, lateral diffusion within a monolayer and flip-flopping between the two monolayers) involved in the physical properties of the membrane are also highly temperature dependent. The transbilayer translocation of lipids is crucial for maintaining lipid asymmetry, which is necessary for membrane stability and the production of transport vesicles; translocation is also related to the demands of cell growth as well as cellular responses to physiological stresses. For instance, PS acts as a common type of phospholipid that is inserted in the membranes, and the flipping of the PS under stress is considered to be an important “eat-me” signal for macrophages in eukaryotes. In general, the asymmetrical distribution of lipids can be achieved through the actions of three protein families, scramblases, ABC transporters and P4-ATPases, which are found in plants and other organisms. The first group of proteins catalyzes the ATP-free bidirectional movement of lipids, whereas most ABC transporters shift lipids from the cytoplasmic to the exoplasmic leaflet (flopping), and P4-ATPases do just the opposite (flipping) (; Figure 1). The implications of scramblases in abiotic stress responses in the plant kingdom remain unclear, but there are a number of reports on the involvement of the other two types of floppase/flippase in responding to environmental challenges, including extreme temperatures. ABC transporters in A. thaliana belong to a large protein family with 129 members. Among these transporters, a previous study showed that reducing the functions of plasmid-specific ABC proteins TGD1, 2, 3, and 4 can effectively suppress the low-temperature-induced phenotype of the vte2 mutant. Earlier research indicated that the transcription of a homolog (TUR2) of the yeast ABC transporter gene PDR5 was induced by low temperatures in Spirodela polyrrhiza. found that the expression of ABC1 was specifically induced under a combination of saline and heat shock stresses in Suaeda salsa. Moreover, a subsequent report suggested that similar to other abiotic-stress-related genes, ABC transporters were abundantly enriched during cold acclimation in the grapevine Vitis amurensis. Likewise, 12 P4-ATPases in the Arabidopsis genome designated ALA1 to ALA12 were shown to perform inward lipid translocation, and the evidence obtained to date demonstrates that the functions of the proteins in this group are all affected to varying degrees by temperature fluctuations. For instance, ALA1 was shown to play a crucial role in chilling tolerance, while ALA3 regulates vegetative and reproductive growth in a temperature-dependent manner. ALA6 and ALA7 determine the fitness of pollen by regulating adaptations to daily temperature swings, and ALA6 has been verified to directly participate in thermotolerance. In addition, a recent study suggested that ALA10 contributed to regulating the activities of FAD2/FAD3 to control galactolipid synthesis in leaf development, and this positive regulation is susceptible to chilling stress. Nevertheless, solid evidence for the involvement of lipid translocation in temperature sensing has yet to be obtained.

7Temperature is a physical cue, and it directly alters the fluidity of biomembranes and the remodeling of membrane microdomains. This may have comprehensive effects not only on the membrane composition but also on specific interactions between lipids and proteins such as ion channels and kinases, subsequently altering the conformation and function of proteins and triggering particular signal transduction pathways (;; Figure 1). Cells remodel and reestablish membrane fluidity to cope with a change in the ambient temperature via the real-time control of lipid saturation and fatty acid length. Above all, subtle changes in membrane fluidity should be promptly perceived by PM sensors. Although studies have indicated that there are several ion channels that function as temperature sensors in animal cells, such as the transient receptor potential cation channel subfamily V (TRPV) and cyclic nucleotide-gated channels (CNGCs;; ), the PM thermosensor in plants long remained unidentified until the existence of primary sensors for heat stress was revealed in the PM of Physcomitrella patens. However, the membrane fluidizer benzyl alcohol can mimic the effects of high temperatures on the PM calcium-dependent heat shock response (HSR); conversely, the rigidifying agent dimethyl sulfoxide can decrease the heat-activated expression of HSPs, which also supports the conclusion that heat-mediated calcium channels are probably regulated by membrane fluidity. Further evidence of the role of these calcium channels as “thermosensors” in plants is needed. In particular, it is poorly understood how these calcium channels perceive and distinguish among different types of high-temperature stress (e.g., basal heat shock, warm priming/acclimation before heat shock, gradual temperature increases, hot-day/cold-night, or short-term and long-term heat stress).

8One of the earliest identified plant responses to elevated temperatures was a specific transient Ca2+ influx from the extracellular matrix to the cytoplasm. The effective suppression of the HSR pathway in higher plants by both calcium chelators and channel blockers suggested that an inward flux of Ca2+ and the ensuing signaling could play critical roles in the heat stress response. The presence of a calcium spike within seconds of thermal treatment in most model plants strongly indicates that heat-sensitive calcium-permeable channels are present in the PM and can transiently open or close during the thermotolerance process. Over 40 putative calcium channels located in the PM in the A. thaliana genome are regarded as the candidate heat sensors. indicated that cyclic nucleotide-gated ion channel 6 (CNGC6) might mediate the heat-activated Ca2+ influx and the expression of HSPs in A. thaliana during plant adaptive responses to heat (Figure 1). Additionally, CNGC6 was also activated by cytosolic cyclic adenosine monophosphate (cAMP), the concentration of which could be rapidly increased within 2 min after heat shock. The binding of free Ca2+ ions to calmodulins (CaMs) has been demonstrated to trigger PM calcium signal cascades, which can activate the HSF and HSP networks. In A. thaliana, AtCaM3 is primarily involved in the activation of transcription factors comprising HSFs, multiprotein bridging factor 1c (MBF1c), WRKY DNA-binding domain superfamily protein (WRKY39) and dehydration-responsive element binding protein (DREB) (;;; Figure 1). MBF1c has been demonstrated to regulate the expression of over 30 heat-related transcripts, including HSFB2a and HSFB2b as well as DREB2A, which is upstream of HSFA3. WRKY39 facilitates the jasmonic acid and SA signaling pathways under heat stress. With respect to the HSFs and the HSF-HSP signaling pathway, the numerous efforts aimed at understanding these proteins began as early as 1960s and 1970s and will therefore not be addressed in this review (see reviews from;;; ). AtCaM3 binds to the Arabidopsis CaM-binding protein kinase 3 (AtCBK3) to promote the phosphorylation of HSFA1a via the activation of a heat-induced transient Ca2+ increase, while calmodulin-dependent phosphatase 7 (PP7) mediates the dephosphorylation of HSFA1a in a Ca2+-dependent manner.

9Hydrogen peroxide (H2O2) is considered to be the other crucial second messenger when plants are exposed to elevated temperatures, because a rapid accumulation of H2O2 can occur within several minutes. The PM-located protein RBOHD is involved in the generation of H2O2 and is directly activated by an increase in cytosolic calcium and/or the phosphorylation of a calcium-dependent protein kinase (CDPK;; Figure 1). The downstream signal cascades of RBOHD also comprise certain HSFs, mitogen-activated protein kinases (MAPKs) and MBF1c, which are involved in HSR (,;; Figure 1). For example, HSFA4a might be a sensor of the H2O2 signaling, while HSFA5 serves as a repressor of this pathway. MAPKs can affect the expression and activity of catalase (CAT) to control the ROS levels while conversely regulating the expression of RBOHD in various genera.

10In addition, studies have shown that more than 1000 lipids are engaged in stress-induced cell signaling, a number far exceeding that of structural lipids. Lipids themselves appear to be versatile, acting not only as structural components but also as signaling mediators. One well-known example is the obvious rapid increases in PA and PIP2 observed under heat stress, the synthesis of which are catalyzed by PM-localized phospholipase D (PLD) and phosphatidylinositol-4-phosphate 5-kinase (PIPK), respectively (; Figure 1). Heat-induced changes in membrane fluidity might activate phospholipases and kinases on the PM, including PLD and PIPK. Additionally, PIP2 is hydrolyzed by membrane-bound PLCs and releases diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3), which increases rapidly within 3 min after heat shock in Arabidopsis. IP3, which is generally considered a classic second messenger that is phosphorylated into IP6, is implicated in the regulation of intracellular calcium stores (; Figure 1). However, an IP6 receptor has yet to be identified in plants. Furthermore, PIP2 itself also plays an important role in biotic and abiotic stress-signaling transduction that are related, for example, to membrane stability, the cytoskeletal order and the budding of secretory vesicles. PA is a structural lipid, and it acts as a key coordinator of the PLC signaling pathway in plants. Arabidopsis AtPLC3 and AtPLC9 are associated with the regulation of small HSP (sHSP) expression during the thermotolerance process by the same mechanisms.

11A growth temperature that is higher or lower than the optimum range is defined as heat or cold stress. Although this concept is related to different plants, in general, cold-sensitive species can be harmed at temperatures below 15°C, while cold-tolerant plants may survive at temperatures close to 5°C. As temperature effects, the cold signaling responses involve similar stages as those observed in plants exposed to heat. For instance, the cold first has an influence on the membrane fluidity via fatty acid saturation. In contrast to heat stress, low temperatures cause changes in the physical state of the membrane bilayer from a fluid-crystalline phase to a solid-gel phase, accompanied by decreased permeability, which is referred to as the membrane rigidity. Incremental membrane rigidity can also alter the activities of some channels, kinases and G-protein-associated receptors on the PM and thus rapidly induce increases in intracellular calcium, PA, and ROS similar to those in heat-treated cells. However, differences between these two types of temperature stress arise in the subsequent signal cascades. The increased PA, which can be produced by activated PLD under cold conditions, can trigger ROS production in cells and may be involved in the ABA-dependent responses under cold stress. In addition, a Ca2+ signature can be decoded by many calcium sensors, among which the CaM-binding transcription activator (CAMTA3) has been shown to bind to the promoters of C-repeat-binding factors (CBF2/DREB1C) and induce their gene expression. The MAPK cascades are also implicated in the up-regulation of CBF/DREB1s transcription factors during cold stress signaling, including that of MPK3, MPK4, and MPK6 in Arabidopsis. CBF-dependent cold-responsive (COR) gene expression is considered to be a central regulatory pathway in the cold stress response, which is regulated by ICEs (inducers of CBF expression; ). The numbers and categories of COR genes are diverse; some of their products have been found to maintain cell membrane structures, activate ROS-scavenging enzymes, and promote the accumulation of osmolytes when plants are exposed to cold stress. Interestingly, identified 130 CORs that were similarly induced by warm temperatures according to analyses in CBF-overexpressing plants. Furthermore, HSFC1 positively regulates COR gene expression independent of the CBF2 signaling pathway under freezing conditions. Thus, the different types of temperature (heat or cold)-induced signal transduction share an affinity with each other in terms of both molecular components and signaling pathways, which may start at temperature-altered membrane fluidity and induce responsive gene expression to maintain cell integrity and function. Additionally, similar to studies on HSR, the specific plant sensors for the cold stress response and the mechanisms by which the sensors perceive cold signaling remain to be further elucidated.

12The chloroplast and mitochondrion are membranous organelles in which carbon metabolism and energy exchange occur. Due to the ROS [including H2O2, superoxide anions (O2∙-), hydroxyl radicals (•OH), and singlet oxygen (1O2)] that are produced in the electron transport chain and the accompanying metabolic energy distribution during photosynthesis and respiration, chloroplast and mitochondrion components are the major targets of oxidative damage in plants, especially in the presence of other environmental stresses, such as high temperatures. Moreover, because both the chloroplast and mitochondrion are semiautonomous organelles regulated by the nucleus, the communication of organelle stresses to the nucleus and the modulation of the expression of stress-related genes, which are defined as retrograde signaling pathways, are considered to be extremely significant during physiological challenges, including heat stress (; Figure 2).

13In chloroplasts, the photosynthetic apparatus primarily consists of the thylakoid membrane; all the components related to photon capture, electron transfer and energy exchange are found in different compartments of the thylakoid, and its lipid composition and physical state therefore appear to be particularly important for the photosynthetic system. Unlike the PM, the thylakoid membrane is composed of highly unsaturated glyceroglycolipids containing considerable amounts of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) as well as small amounts of phosphatidylglycerol (PG) and sulfoquinovosyldiacylglycerol (SQDG). Hence, polyunsaturated fatty acids unavoidably become a site of attack by lipid peroxyl radicals under heat stress if the fatty acid moiety is near the QB site on the PSII electron acceptor side, suggesting that the fluidity of the thylakoid membrane is important for PSII function (;;; Figure 2). Moreover, both 1O2 formation on the electron acceptor side and •OH production on the electron donor side result from the inactivation of PSII electron transport when plants are exposed to heat stress. For instance, high temperatures efficiently shift the midpoint redox potential of QA/QA-, which leads to the suppression of electron transfer from QA to QB on the electron acceptor side of PSII. In the green alga Chlamydomonas reinhardtii, 1O2 formation as initiated by heat-induced lipid peroxidation, in which lipoxygenase plays the primary role, has been demonstrated. Lipid peroxidation products also have a pernicious effect on PSII proteins via oxidative modification, protein cleavage and irreversible protein aggregation. Studies have shown that the reaction center binding protein D1 can be cleaved in spinach thylakoid membranes under heat stress and that lipid peroxidation occurs in spinach thylakoids, and 1O2 is subsequently produced, which in turn causes D1 protein degradation via interactions with the protein. In addition, heat-induced lipid peroxidation can facilitate the denaturation and destabilization of PsbO (PsbP and PsbQ in spinach PSII) binding to the PSII core complex and promote manganese ion release from the Mn4O5Ca cluster, which has been demonstrated to be the main reason for the inactivation of electron transport on the donor side of PSII. In maintaining the chloroplast membrane under heat stress, a remarkable role is played by the vesicle-inducing protein in plastid 1 (VIPP1), which localizes to the chloroplast envelope and thylakoid membrane. VIPP1 was recently indicated to be a GTPase involved in maintaining the integrity of photosynthetic membranes via an effect on membrane remodeling when plants were subjected to stresses.

14Chloroplast retrograde signaling can be initiated by the accumulation of the chlorophyll intermediate Mg-protoporphyrin IX (Mg-ProtoIX, belonging to tetrapyrroles), as shown by gun (genome uncoupled) mutants, which exhibit impaired communication from the chloroplast to the nucleus, controlling photosynthesis-associated nuclear gene (PhANG) expression (;;;;;;;; Figure 2). GUN1, which encodes a unique plastid-localized pentatricopeptide repeat (PPR) protein, has been shown to be required to transmit stress signaling to the nuclear transcription factor ABA insensitive 4 (ABI4; ). Additionally, ABI4 is considered to be a central regulator that is involved in many essential environmental signaling pathways and in the developmental as well as central metabolic processes of cells or organelles (reviewed by ). Findings in Chlamydomonas reinhardtii have further indicated that Mg-ProtoIX transiently promotes the expression of heat shock protein 70 (HSP70), which may localize to both the plastid and cytoplasm. In addition, nuclear gene transcriptional studies on the above green alga showed that more than half of tetrapyrrole-interrelated genes were also activated by heat stress. Moreover, the chloroplast ribosomal protein S1 (RPS1) was reported to activate the expression of HSFA2, which is considered to regulate heat-stress-responsive genes during heat acclimation.

15In the mitochondria, a type of phospholipid known as cardiolipin (CL), which has a high percentage of polyunsaturated fatty acids, is embedded in the mitochondrial membrane, where lipid peroxidation occurs (;; Figure 2). Damaged CL strongly decreases the activity of cytochrome c oxidase (CCO) under stress. Moreover, in A. thaliana, cardiolipin synthase (CLS) is required for mitochondrial morphogenesis and the response to high-temperature stress. The physical state of the mitochondrial membrane is also crucial; the depolarization of the mitochondrial membrane by the protonophore CCCP has been shown to inhibit ROS generation under thermal treatment. Additionally, the mitochondrion is one of the key compartments in ROS generation, especially when the rate of the electron transport chain has been decreased by environmental stresses. ROS negatively affect both lipids and proteins. The products of protein oxidation consist mostly of tryptophan and products of carbonylation and fragmentation. For example, some Krebs cycle enzymes, other redox enzymes, and HSPs as well as Mn superoxide dismutase (Mn-SOD) and the voltage-dependent anion channel from rice leaf mitochondria, can be carbonylated under stress (; Figure 2).

16The mitochondrial retrograde signaling pathway also shows a relationship with ROS generation and accumulation (;; Figure 2). When electron transport is disrupted by abiotic stresses, the increased expression of nuclear-encoded alternative oxidase 1 (AOX1) regulates the ROS level to trigger retrograde responses in plants. The AOX1A promoter sequence is one of the targets of ABI4 in A. thaliana; ABI4 is involved in mitochondrion-to-nucleus retrograde signaling as well as that in chloroplasts. Because mitochondrial complexes I and III are the primary sites of ROS generation, a mutation in complex I can promote ROS accumulation and decrease the expression of COR genes. Mitochondrion-derived DEXH box RNA helicases or PPR proteins also induce ROS accumulation and are involved in ABA responses.

17The roles of Ca2+ and HSPs in the regulation of organelle signaling under high-temperature stress should not be ignored. Earlier studies showed that chloroplasts contained high total Ca2+concentrations, while the potential of the plant inner mitochondrial membrane may affect the level of cytosolic Ca2+ and, thus, the expression of heat-responsive genes. However, further studies are needed in this field.

18The HSPs exhibit a chaperone function, folding polypeptides into a mature state or preventing misfolded proteins from aggregation, and they have been found to accumulate in cells under thermal stress. Because most proteins in chloroplasts and mitochondria are encoded by nuclear genes, and the polypeptides are exported from the nucleus to the organelles, HSPs are correspondingly required for the protein conformational changes during these processes. HSP70 acts upstream of other HSPs and cooperates with other chaperones in protein folding, refolding and disaggregation. Chloroplast stromal HSP70 was found to be involved in PSII repair under stress and to be affected by the redox state of the chloroplast. Another HSP, HSP90, more likely serves as a regulator of signal transfer to the nucleus, including the modulation of photosynthesis-related genes and stress-related genes, although it can also stabilize unfolded proteins. The sHSPs are chaperones that show high diversity among species and tissues and can prevent improper protein aggregation and partially unfold under stress conditions. Several lines of evidence have suggested that a small chloroplast HSP, HSP21, is involved in plant adaptations to heat stress and oxidative stress. A recent study demonstrated that HSP21 could bind to and stabilize PSII core proteins D1 and D2 under heat stress, which was activated by the GUN5-associated retrograde signaling pathway. GUN5 encodes an Mg chelatase that can insert Mg2+ into the protoporphyrin ring during tetrapyrrole biosynthesis, which regulates ROS-induced gene expression. Furthermore, HSP21 was demonstrated to interact with another protein, plastid transcriptionally active 5 (pTAC5), which is required for plastid-encoded RNA polymerase (PEP)-dependent transcription, and the latter protein plays a crucial role in plastid development under heat stress.

19The ER is the largest endomembrane system, and it is responsible for protein synthesis, folding, modification and export as well as lipid metabolism. More than a third of the total proteins pass through the secretory pathway via the ER lumen. Therefore, ER homeostasis is highly susceptible to environmental cues, inevitably resulting in the production of misfolded proteins or the accumulation of unfolded proteins, which are also known as ER stress. The mechanism underlying the response to ER stress at the transcriptional and translational levels is referred to as the unfolded protein response (UPR), which includes the detection of unfolded or misfolded proteins; the activation of chaperones, foldases and pertinent factors; and the reduction of the secreted and membrane protein contents. If the UPR is overextended and ER homeostasis cannot be sustained, cells will initiate programmed death. In addition to aberrant proteins, instability of phospholipid metabolism also leads to ER stress and activates the UPR. Studies on yeast and mammalian cells revealed that an imbalanced PC to PE ratio is a crucial cause of ER stress. However, the link between lipid functions and the ER stress response is still poorly understood in plants. verified that Arabidopsis glycerolipid homeostasis might be sustained at the post-transcriptional level under ER stress. Additionally, a study on maize illustrated that ER stress could affect the enzyme activity of diacylglycerol kinase (DGA), choline phosphate cytidylyltransferase, phosphatidylinositol-4-kinase, and PIPK.

20Nascent proteins require molecular chaperones to prevent their aggregation and assist in their folding into functional conformations. Binding protein (BiP), an HSP70, is a common chaperone in the ER that is recruited by the DNAJ protein ERdj3 to bind unfolded proteins (; Figure 1). Other chaperones known as foldases have also been described in several previous reviews.

21Plants harbor two primary signaling pathways that have been reported to sense ER stress. One is aided by membrane-associated transcription factors (the basic leucine zipper transcription factors bZIP17 and bZIP28), and the other involves an RNA splicing factor (the inositol-requiring enzyme IRE1) (; Figure 1). The bZIP28 can specifically sense heat-triggered ER stress via BiP, after which it translocated into the Golgi, and its TF domain on the cytosol side can be cleaved by proteolytic processing. The TF portion subsequently enters the nucleus to activate the expression of stress-related genes. In addition, IRE1 is activated by interaction with unfolded proteins and then recognizes and splices the mRNA of bZIP60, after which the splicing products enter the nucleus to trigger the expression of UPR genes (;; Figure 1). Heat stress has been validated as a direct and efficient factor that influences the nuclear relocation of bZIP28 and the mRNA splicing of bZIP60.

22Temperature fluctuation is an unusual ambient signal that causes concurrent alterations in multiple cellular compartments, including the PM, cytosol, energy-associated organelles and chromatin. In fact, there is widespread support for the notion that an increase in PM fluidity as induced by high temperatures first activates stress-signaling pathways (reviewed by ). However, how should the concept of “primary heat sensors” be understood? If the macromolecules in plant cells are defined as thermosensors that are able to not only precisely perceive various temperature changes but also differentially trigger specific signaling pathways, the most feasible strategy might be to first divert ambient temperature signals into different cell signaling patterns before they are perceived by sensors. From this perspective, the specific role of the membrane might be to serve as both a thermometer and a stress encoder. Additionally, subtle changes in the lipid composition of fatty acid chains or head groups can alter both the membrane properties and the interaction with specific proteins and even the membrane substructures (microdomains), which are highly sensitive to environmental perturbation; in this context, the membrane clearly meets the requirement for encoding stress. Of course, more evidence must be obtained and evaluated to dissect the mechanisms of membrane-associated stress sensing and signaling. One important reason why it is so difficult to study these mechanisms is that membrane structures in plant cells possessing a cell wall exhibit an enormous variety of membrane components with diverse localizations, interactions, and functions. The conventional methods for the investigation of membrane proteins have obvious limitations (e.g., specific protein associations can be disrupted by detergents;; ). The other reason is that the available studies on lipids and relevant enzymes are not equivalent to the attention paid to the proteins involved in signal transduction, despite the fact that membrane lipids show the best fit for the response to various environmental cues among these molecules. Fortunately, the direct visualization of the distribution of membrane constituents that rely on fluorescence microscopy approaches makes it possible to detect the dynamic interaction and distribution of PM substructures (reviewed by;; ). In addition, based on improvements in the sensitivity and accuracy of mass spectrometry and chromatography as well as microarray transcriptome analyses, it is possible to obtain knowledge of the entire lipidome and the complicated regulatory network at a certain time point in an individual tissue type under stresses.

23Furthermore, numerous vesicles in cells are also an important part of the membrane system whose formation and trafficking in plants have been shown to be involved in membrane repair under abiotic and biotic stresses (reviewed by;; ). However, much about the underlying mechanism of communication between extra-membrane and membranous organelles remains to be elucidated. Moreover, heat stress is inevitably associated with various other stresses during actual crop production. For example, high temperatures are often accompanied by high light, which can accelerate the plant injury inflicted by drought and salinity stress in inland regions. Elevated temperatures have also been shown to promote the infection of plant cells by pathogens and cause damage to SA defense. The identification and elucidation of these “crosstalk” mechanisms seem precisely the task of the day.

24In short, although studies on the underlying mechanisms of plant adaptation to high temperature and the engineering of crop plants with higher thermotolerance are making steady progress, many questions are still open and need to be further addressed. Important issues include the following:

25Are other primary thermosensors present in the PM or other intracellular compartments that synergistically trigger multiple and/or specific responses?

26How is the signaling that is triggered by various forms, durations and intensities of high-temperature stress transmitted?

27What precise role do membrane microdomains play in the plant HR?

28How can the dynamic remodeling of membranes under various heat stresses be recorded or mimicked?

29How do second messengers transmit multiple signals to downstream components of HS signal transduction chains?

30How do the UPR in chloroplasts, the cytosol and the ER coordinately and collaboratively function in protein homeostasis?

31How does the PM contact other membranous organelles under elevated temperatures?

32How does the lipid metabolism in chloroplasts and the ER participate in membrane sensing and remodeling during the rapid HR?

33Apart from HSF-HSP responses, are there other HSR effectors that switch the comprehensive responses on/off in plant cells?

34How is heat stress sensing distinguished from and integrated with the sensing of other stresses?

35YN prepared and wrote this manuscript. YX contributed by organizing and reviewing a major part of the manuscript.

36The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.