The plasma membrane is considered to be the primary site of injury when plant cells are subjected to extracellular freezing. In order for plants or plant cells to acquire freezing tolerance, it is, thus, necessary that the plasma membrane increases its cryostability during freeze‐thaw excursion. During cold acclimation both under natural and artificial conditions, there are compositional, structural and functional changes occurring in the plasma membrane, many, if not all, of which ultimately contribute to increased stability of the plasma membrane under freezing conditions. In addition, changes in the cytosol or intracellular compartments also affect the cryobehaviour of the plasma membrane during freeze‐induced dehydration. Although many alterations occurring during cold acclimation influence the cryobehaviour of the plasma membrane comprehensively, recent advances in functional genomics approaches provide interesting information on the function of specific proteins for plasma membrane behaviour under freezing conditions.

Summary: Although enhancement of freezing tolerance in plants during cold acclimation is closely associated with an increase in the cryostability of plasma membrane, the molecular mechanism for the increased cryostability of plasma membrane is still to be elucidated. In Arabidopsis, enhanced freezing tolerance was detectable after cold acclimation at 2°C for as short as 1 day, and maximum freezing tolerance was attained after 1 week. To identify the plasma membrane proteins that change in quantity in response to cold acclimation, a highly purified plasma membrane fraction was isolated from leaves before and during cold acclimation, and the proteins in the fraction were separated with gel electrophoresis. We found that there were substantial changes in the protein profiles after as short as 1 day of cold acclimation. Subsequently, using matrix‐assisted laser desorption–ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS), we identified 38 proteins that changed in quantity during cold acclimation. The proteins that changed in quantity during the first day of cold acclimation include those that are associated with membrane repair by membrane fusion, protection of the membrane against osmotic stress, enhancement of CO₂ fixation, and proteolysis.

Plant freezing tolerance involves the prevention of lethal freeze-induced damage to the plasma membrane. We hypothesized that plant freezing tolerance involves membrane resealing, which, in animal cells, is accomplished by calcium-dependent exocytosis following mechanical disruption of the plasma membrane. In Arabidopsis thaliana protoplasts, extracellular calcium enhanced not only freezing tolerance but also tolerance to electroporation, which typically punctures the plasma membrane. However, calcium did not enhance survival when protoplasts were exposed to osmotic stress that mimicked freeze-induced dehydration. Calcium-dependent freezing tolerance was also detected with leaf sections in which ice crystals intruded into tissues. Interestingly, calcium-dependent freezing tolerance was inhibited by extracellular addition of an antibody against the cytosolic region of SYT1, a homolog of synaptotagmin known to be a calcium sensor that initiates exocytosis. This inhibition indicates that the puncture allowing the antibody to flow into the cytoplasm occurs during freeze/thawing. Thus, we propose that calcium-dependent freezing tolerance results from resealing of the punctured site. Protoplasts or leaf sections isolated from Arabidopsis SYT1-RNA interference (RNAi) plants lost calcium-dependent freezing tolerance, and intact SYT1-RNAi plants had lower freezing tolerance than control plants. Taken together, these findings suggest that calcium-dependent freezing tolerance results from membrane resealing and that this mechanism involves SYT1 function.

Microdomains in the plasma membrane (PM) have been proposed to be involved in many important cellular events in plant cells. To understand the role of PM microdomains in plant cold acclimation, we isolated the microdomains as detergent-resistant plasma membrane fractions (DRMs) from Arabidopsis seedlings and compared lipid and protein compositions before and after cold acclimation. The DRM was enriched in sterols and glucocerebrosides, and the proportion of free sterols in the DRM increased after cold acclimation. The protein-to-lipid ratio in the DRM was greater than that in the total PM fraction. The protein amount recovered in DRMs decreased gradually during cold acclimation. Cold acclimation further resulted in quantitative changes in DRM protein profiles. Subsequent mass spectrometry and Western blot analyses revealed that P-type H⁺-ATPases, aquaporins and endocytosis-related proteins increased and, conversely, tubulins, actins and V-type H⁺-ATPase subunits decreased in DRMs during cold acclimation. Functional categorization of cold-responsive proteins in DRMs suggests that plant PM microdomains function as platforms of membrane transport, membrane trafficking and cytoskeleton interaction. These comprehensive changes in microdomains may be associated with cold acclimation of Arabidopsis.

Acidification of intracellular compartments by the vacuolar-type H⁺-ATPases (VHA) is known to energize ion and metabolite transport, though cellular processes influenced by this activity are poorly understood. At least 26 VHA genes encode 12 subunits of the V₁V_o-ATPase complex in Arabidopsis, and how the expression, assembly, and activity of the pump are integrated into signaling networks that govern growth and adaptation are largely unknown. The role of multiple VHA-c genes encoding the 16-kD subunit of the membrane V_o sector was investigated. Expression of VHA-c1, monitored by promoter-driven β-glucuronidase (GUS) activity was responsive to light or dark in an organ-specific manner. VHA-c1 expression in expanding cotyledons, hypocotyls of etiolated seedlings, and elongation zone of roots supported a role for V-ATPase in cell enlargement. Mutants reduced in VHA-c1 transcript using dsRNA-mediated interference showed reduction in root growth relative to wild-type seedlings. In contrast, VHA-c3 promoter::GUS expression was undetectable in most organs of seedlings, but strong in the root cap. Interestingly, dsRNA-mediated mutants of vha-c3 also showed reduced root length and decreased tolerance to moderate salt stress. The results suggest that V-ATPase functions in the root cap influenced root growth. Expression of VHA-c1 and VHA-c3 in tissues with active membrane flow, including root cap, vascular strands, and floral style would support a model for participation of the V_o sector and V₁V_o-ATPase in membrane trafficking and fusion. Two VHA-c genes are thus differentially expressed to support growth in expanding cells and to supply increased demand for V-ATPase in cells with active exocytosis.

High-temperature-mediated adaptation in plant architecture is linked to the increased synthesis of the phytohormone auxin, which alters cellular auxin homeostasis. The auxin gradient, modulated by cellular auxin homeostasis, plays an important role in regulating the developmental fate of plant organs. Although the signaling mechanism that integrates auxin and high temperature is relatively well understood, the cellular auxin homeostasis mechanism under high temperature is largely unknown. Using the Arabidopsis thaliana root as a model, we demonstrate that under high temperature, roots counterbalance the elevated level of intracellular auxin by promoting shootward auxin efflux in a PIN-FORMED2 (PIN2)-dependent manner. Further analyses revealed that high temperature selectively promotes the retrieval of PIN2 from late endosomes and sorts them to the plasma membrane through an endosomal trafficking pathway dependent on SORTING NEXIN1. Our results demonstrate that recycling endosomal pathway plays an important role in facilitating plants adaptation to increased temperature.