Calcium-Dependent Freezing Tolerance in Arabidopsis Involves Membrane Resealing via Synaptotagmin SYT1

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.     

Freeze-Induced Membrane Ultrastructural Alterations in Rye (Secale cereale) Leaves

Freezing injury in protoplasts isolated from leaves of nonaccli-mated rye (Secale cereale cv Puma) is associated with the formation of the inverted hexagonal (HII) phase. However, in protoplasts from cold-acclimated rye, injury is associated with the occurrence of localized deviations in the fracture plane, a lesion referred to as the "fracture-jump lesion." To establish that these ultrastructural consequences of freezing are not unique to protoplasts, we have examined the manifestations of freezing injury in leaves of non-acclimated and cold-acclimated rye by freeze-fracture electron microscopy. At -10[deg]C, injury in nonacclimated leaves was manifested by the appearance of aparticulate domains in the plasma membrane, aparticulate lamellae subtending the plasma membrane, and by the frequent occurrence of the HII phase. The HII phase was not observed in leaves of cold-acclimated rye frozen to -35[deg]C. Rather, injury was associated with the occurrence of the fracture-jump lesion between the plasma membrane and closely appressed cytoplasmic membranes. Studies of the time dependence of HII phase formation in nonacclimated leaves indicated that freeze-induced dehydration requires longer times in leaves than in isolated protoplasts. These results demonstrate that the freeze-induced formation of the HII phase in nonacclimated rye and the fracture-jump lesion in cold-acclimated rye are not unique to protoplasts but also occur in the leaves from which the protoplasts are isolated.     

Effect of microtubule stabilization on the freezing tolerance of mesophyll cells of spinach

Freezing, dehydration, and supercooling cause microtubules in mesophyll cells of spinach (Spinacia oleracea L. cv Bloomsdale) to depolymerize (ME Bartolo, JV Carter, Plant Physiol [1991] 97: 175-181). The objective of this study was to determine whether the LT₅₀ (lethal temperature: the freezing temperature at which 50% of the tissue is killed) of spinach leaf tissue can be changed by diminishing the extent of microtubule depolymerization in response to freezing. Also examined was how tolerance to the components of extracellular freezing, low temperature and dehydration, is affected by microtubule stabilization. Leaf sections of nonacclimated and cold-acclimated spinach were treated with 20 micromolar taxol, a microtubule-stabilizing compound, prior to freezing, supercooling, or dehydration. Taxol stabilized microtubules against depolymerization in cells subjected to these stresses. When pretreated with taxol both nonacclimated and cold-acclimated cells exhibited increased injury during freezing and dehydration. In contrast, supercooling did not injure cells with taxol-stabilized microtubules. Electrolyte leakage, visual appearance of the cells, or a microtubule repolymerization assay were used to assess injury. As leaves were cold-acclimated beyond the normal period of 2 weeks taxol had less of an effect on cell survival during freezing. In leaves acclimated for up to 2 weeks, stabilizing microtubules with taxol resulted in death at a higher freezing temperature. At certain stages of cold acclimation, it appears that if microtubule depolymerization does not occur during a freeze-thaw cycle the plant cell will be killed at a higher temperature than if microtubule depolymerization proceeds normally. An alternative explanation of these results is that taxol may generate abnormal microtubules, and connections between microtubules and the plasma membrane, such that normal cellular responses to freeze-induced dehydration and subsequent rehydration are blocked, with resultant enhanced freezing injury.     

Cold Acclimation of Arabidopsis thaliana (Effect on Plasma Membrane Lipid Composition and Freeze-Induced Lesions)

Maximum freezing tolerance of Arabidopsis thaliana L. Heyn (Columbia) was attained after 1 week of cold acclimation at 2[deg]C. During this time, there were significant changes in both the lipid composition of the plasma membrane and the freeze-induced lesions that were associated with injury. The proportion of phospholipids increased from 46.8 to 57.1 mol% of the total lipids with little change in the proportions of the phospholipid classes. Although the proportion of di-unsaturated species of phosphatidylcholine and phosphatidylethanolamine increased, mono-unsaturated species were still the preponderant species. The proportion of cerebrosides decreased from 7.3 to 4.3 mol% with only small changes in the proportions of the various molecular species. The proportion of free sterols decreased from 37.7 to 31.2 mol%, but there were only small changes in the proportions of sterylglucosides and acylated sterylglucosides. Freezing tolerance of protoplasts isolated from either nonacclimated or cold-acclimated leaves was similar to that of leaves from which the protoplasts were isolated (-3.5[deg]C for nonacclimated leaves; -10[deg]C for cold-acclimated leaves). In protoplasts isolated from nonacclimated leaves, the incidence of expansion-induced lysis was [less than or equal to]10% at any subzero temperature. Instead, freezing injury was associated with formation of the hexagonal II phase in the plasma membrane and subtending lamellae. In protoplasts isolated from cold-acclimated leaves, neither expansion-induced lysis nor freeze-induced formation of the hexagonal II phase occurred. Instead, injury was associated with the "fracture-jump lesion," which is manifested as localized deviations of the plasma membrane fracture plane to subtending lamellae. The relationship between the freeze-induced lesions and alterations in the lipid composition of the plasma membrane during cold acclimation is discussed.     

Responses of the plasma membrane to low temperatures

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.     

Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation of wheat

Expression of the acidic dehydrin gene wcor410 was found to be associated with the development of freezing tolerance in several Gramineae species. This gene is part of a family of three homologous members, wcor410, wcor410b, and wcor410c, that have been mapped to the long arms of the homologous group 6 chromosomes of hexaploid wheat. To gain insight into the function of this gene family, antibodies were raised against the WCOR410 protein and affinity purified to eliminate cross-reactivity with the WCS120 dehydrin-like protein of wheat. Protein gel blot analyses showed that the accumulation of WCOR410 proteins correlates well with the capacity of each cultivar to cold acclimate and develop freezing tolerance. Immunoelectron microscope analyses revealed that these proteins accumulate in the vicinity of the plasma membrane of cells in the sensitive vascular transition area where freeze-induced dehydration is likely to be more severe. Biochemical fractionation experiments indicated that WCOR410 is a peripheral protein and not an integral membrane protein. These results provide direct evidence that a subtype of the dehydrin family accumulates near the plasma membrane. The properties, abundance, and localization of these proteins suggest that they are involved in the cryoprotection of the plasma membrane against freezing or dehydration stress. We propose that WCOR410 plays a role in preventing the destabilization of the plasma membrane that occurs during dehydrative conditions.     

A Comparison of Freezing Injury in Oat and Rye: Two Cereals at the Extremes of Freezing Tolerance

A detailed analysis of cold acclimation of a winter rye (Secale cereale L. cv Puma), a winter oat (Avena sativa L. cv Kanota), and a spring oat cultivar (Ogle) revealed that freezing injury of leaves of nonacclimated seedlings occurred at -2[deg]C in both the winter and spring cultivars of oat but did not occur in winter rye leaves until after freezing at -4[deg]C. The maximum freezing tolerance was attained in all cultivars after 4 weeks of cold acclimation, and the temperature at which 50% electrolyte leakage occurred decreased to -8[deg]C for spring oat, -10[deg]C for winter oat, and -21[deg]C for winter rye. In protoplasts isolated from leaves of nonacclimated spring oat, expansion-induced lysis was the predominant form of injury over the range of -2 to -4[deg]C. At temperatures lower than -4[deg]C, loss of osmotic responsiveness, which was associated with the formation of the hexagonal II phase in the plasma membrane and subtending lamellae, was the predominant form of injury. In protoplasts isolated from leaves of cold-acclimated oat, loss of osmotic responsiveness was the predominant form of injury at all injurious temperatures; however, the hexagonal II phase was not observed. Rather, injury was associated with the occurrence of localized deviations of the plasma membrane fracture plane to closely appressed lamellae, which we refer to as the "fracture-jump lesion." Although the freeze-induced lesions in the plasma membrane of protoplasts of spring oat were identical with those reported previously for protoplasts of winter rye, they occurred at significantly higher temperatures that correspond to the lethal freezing temperature.     

Freezing Characteristics of Rigid Plant Tissues (Development of Cell Tension during Extracellular Freezing)

The freezing characteristics and development of cell tension during extracellular freezing were examined in supercooling stem tissues of riverbank grapes (Vitis riparia) and cold-hardened leaves of live oak (Quercus virginiana) and mountain cranberry (Vaccinium vitis-idaea). Dormant stem xylem and pith tissues of river-bank grapes were resistant to freeze-induced dehydration above the homogeneous nucleation temperature, and they developed cell tension reaching a maximum of 27 MPa. Similarly, extracellular freezing induced cell tension in the leaves of live oak and mountain cranberry. Maximum cell tension in the leaves of live oak was 16.8 MPa and 8.3 MPa in the leaves of mountain cranberry. Following peak tensions in the leaves, a decline in the pressure was observed with progressive freezing. The results suggest that resistance to cell deformation during extracellular freezing due to cell-wall rigidity can lead to reduced cell dehydration and increased cell tension. A relationship to predict freezing behavior in plant tissues based on cell rigidity is presented. Based on cell-water relations and ice nucleation rates, cell-wall rigidity has been shown to effect the freezing characteristics of plant tissues, including freeze-induced dehydration, supercooling, and homogeneous nucleation temperatures.     

Freeze/thaw-induced destabilization of the plasma membrane and the effects of cold acclimation

Disruption of the plasma membrane is a primary cause of freezing injury. In this review, the mechanisms of injury resulting from freeze-induced cell dehydration are presented, including destabilization of the plasma membrane resulting from (a) freeze/thaw-induced osmotic excursions and (b) lyotropic phase transitions in the plasma membrane lipids. Cold acclimation dramatically alters the behavior of the plasma membrane during a freeze/thaw cycle—increasing the tolerance to osmotic excursions and decreasing the propensity for dehydration-induced lamellar to hexagonal-II phase transitions. Evidence for a casual relationship between the increased cryostability of the plasma membrane and alterations in the lipid composition is reviewed.     

Thylakoid membrane stability in drought-tolerant and drought-sensitive plants

The stress stability of membranes from two drought-tolerant plants (Craterostigma plantagineum andCeterach officinarum) was compared with that of a drought-sensitive plant (Spinacia oleracea) in model experiments. Thylakoids from these plants were exposed to excessive sugar or salt concentrations or to freezing. All stresses caused loss of membrane function as indicated by the loss of cyclic photophosphorylation or the inability of the membranes to maintain a large proton gradient in the light. However, loss of membrane functions caused by osmotic dehydration in the presence of sugars was reversible. Irreversible membrane damage during freezing or exposure to salt was attributed mainly to chaotropic solute effects. The sensitivity to different stresses was comparable in thylakoid membranes from tolerant and sensitive plants indicating that the stress tolerance of a plant can hardly be attributed to specific membrane structures which would increase membrane stability. Levels of membrane-compatible solutes such as sugars or amino acids, among them proline, were much higher in the drought-tolerant plants than in spinach. Isolated thylakoids suspended in solutions containing an excess of sugars remained functional after dehydration by freeze-drying. This indicates that membrane-compatible solutes are important in preventing membrane damage during dehydration of poikilohydric plants.Abbreviation BSA bovine serum albumin     

Comparison of freezing tolerance in cultured plant cells and their respective protoplasts

The possibility that the plant cell wall influences the severity of freezing injury was examined by comparing the freeze stress response of intact cells and protoplasts from four different suspension cultures. In no case did the intact cells suffer more injury than the respective wall-less protoplasts, showing that mechanical strain imposed by the cell wall during freeze-thaw stress is not a major determinant of injury. For three of the four species studied, cells from which the wall was removed showed significantly greater freezing injury, indicating that the plant cell wall may have a protective role. Other researchers have suggested that cell wall rigidity may minimize freezing injury by slowing freeze-induced loss of cell water. We found that decreased enzyme digestibility (perhaps indicating greater rigidity) of cell walls accompanied cold acclimation in various tissues. These results provide impetus to research which will characterize low-temperature-induced cell wall modification in cold acclimating tissues.     

Freezing sensitivity in the sfr4 mutant of Arabidopsis is due to low sugar content and is manifested by loss of osmotic responsiveness

Protoplasts were tested to determine whether the freezing sensitivity of the sfr4 (sensitive to freezing) mutant of Arabidopsis was due to the mutant's deficiency in soluble sugars after cold acclimation. When grown under nonacclimated conditions, sfr4 protoplasts possessed freezing tolerance similar to that of wild type, with the temperature at which 50% of protoplasts are injured (LT(50)) of -4.5 degrees C. In both wild-type and sfr4 protoplasts, expansion-induced lysis was the predominant lesion between -2 degrees C and -4 degrees C, but its incidence was low (approximately 10%); below -5 degrees C, loss of osmotic responsiveness (LOR) was the predominant lesion. After cold acclimation, the LT(50) was decreased to only -5.6 degrees C for sfr4 protoplasts, compared with -9.1 degrees C for wild-type protoplasts. Although expansion-induced lysis was precluded in both types of protoplasts, the sfr4 protoplasts remained susceptible to LOR. After incubation of seedlings in Suc solution in the dark at 2 degrees C, freezing tolerance and the incidence of freeze-induced lesions in sfr4 protoplasts were examined. The freezing tolerance of isolated protoplasts (LT(50) of -9 degrees C) and the incidence of LOR were now similar for wild type and sfr4. These results indicate that the freezing sensitivity of cold-acclimated sfr4 is due to its continued susceptibility to LOR (associated with lyotropic formation of the hexagonal II phase) and associated with the low sugar content of its cells.     

Increased ethylene synthesis enhances chilling tolerance in tomato

Freezing of nonacclimated protoplasts close to lethal temperatures induces alterations in the macromolecular organization of the plasma membrane but the significance of these structural changes in freezing injury is still uncertain. We therefore cooled non-acclimated protoplasts isolated from cultivars of winter rye ( Secale cereale L.) to two sub-zero temperatures using two different cooling rates and analyzed freeze-induced plasma membrane changes by freeze-fracture electron microscopy. When a high cooling rate was used a lipid phase transition was observed in 34% of the total membrane fracture faces of the protoplasts, while with a slow cooling rate it occurred only to a very small extent. Smooth, aparticulate lamellae were approximately three times more frequent at low than at high cooling rate. Lipid phase transition from lamellar to hexagonal_II (H_II) phase occurred at high cooling rate more frequently at −10°C than at −30°C in three cultivars. The results suggest that the greatly increased proportion of phase transition from bilayer to non-bilayer phase is an artifact caused by too fast a cooling rate of protoplasts. Furthermore, lateral phase separation of the plasma membrane with segregation of intramembrane particles and the appearance of membrane associated stacks of lipid lamellae, may cause cellular death by retarding the flow of intracellular water towards extracellular ice crystals formed during freezing.     

Spin-label Studies of Membranes in Rye Protoplasts during Extracellular Freezing

Protoplasts isolated from epicotyls of nonhardened winter rye seedlings were spin-labeled with the N-oxyl-4-4-dimethyloxazolidine derivatives of 5-ketostearic (5NS) and 16-ketostearic (16NS) acids. Spectra of the membrane-bound labels showed motional broadening with a rotational correlation time of 1.5 × 10⁻⁸ second for 5NS and 1.5 × 10⁻¹⁰ second for 16NS at 0 C. A procedure was developed to follow membrane changes in these protoplasts during extracellular freezing. With freezing, molecular motion of 5NS, but not of 16NS, spin probes was restricted. The increase in molecular order near the hydrated end of the membrane did not result from lowered temperatures inasmuch as no such change was observed in supercooled samples. These changes are probably due to dehydration of protoplast membranes during extracellular freezing. Similar results were obtained with multilayered egg yolk lecithin and are consistent with previous observations of changes in lecithin multibilayers during dehydration. Such alterations in membrane order might lead to irreversible membrane damage during extracellular freezing of plant cells.     

Vacuolar Membrane Lesions Induced by a Freeze-Thaw Cycle in Protoplasts Isolated from Deacclimated Tubers of Jerusalem Artichoke (Helianthus tuberosus L.)

The processes of freezing injury in Jerusalem artichoke (Helianthustuberosus L.) tubers were studied using protoplasts isolatedfrom cold-acclimated and deacclimated tubers. Prior to freezing,protoplasts were preloaded with 10 µM fluorescein diacetate(FDA) in an isotonic sorbitol solution. After freeze-thawingat various temperatures, cell viability was evaluated undera fluorescence microscope. In cold-acclimated tubers, more than80% of protoplasts survived freezing to – 20°C. Bycontrast, in deacclimated tubers, the cell survival abruptlydeclined after freezing to temperatures below – 5°C.Thus, freezing tolerance differed significantly between protoplastsisolated from cold-acclimated and deacclimated tubers. Two distincttypes of cell injury, which were caused by either damage toplasma membrane (cell-lysis type) or by damage to the vacuolarmembrane (abnormal-staining type), were observed, dependingon the cold hardiness and freezing temperature. In the cellsof the abnormal-staining type, shrinkage of the central vacuolarspace and simultaneous acidification of the cytoplasmic spacewere characteristically observed immediately before completecell-rehydra-tion during thawing. The decrease in freezing toleranceof protoplasts after deacclimation was suggested to be due mainlyto destabilization of the vacuolar membrane by freeze-induceddehydration stress. ¹Contribution no. 3945 from The Institute of Low TemperatureScience, Hokkaido University. This research was supported inpart by the grant from Japan Society for the Promotion of Science(JSPS-RFTF 96L00602) ²Present address: Tohoku National Agricultural Experiment Station, Morioka, Iwate, 020-01 Japan     

A freezing-sensitive mutant of Arabidopsis, frs1, is a new aba3 allele

To investigate the molecular mechanisms controlling the process of cold acclimation and to identify genes involved in plant freezing tolerance, mutations that impaired the cold acclimation capability of Arabidopsis thaliana (L.) Heynh. were screened for. A new mutation, frs1 (freezing sensitive 1), that reduced both the constitutive freezing tolerance as well as the freezing tolerance of Arabidopsis after cold acclimation was characterized. This mutation also produced a wilty phenotype and excessive water loss. Plants with the frs1 mutation recovered their wild-type phenotype, their capability to tolerate freezing temperatures and their capability to retain water after an exogenous abscisic acid (ABA) treatment. Measurements of ABA revealed that frs1 mutants were ABA deficient, and complementation tests indicated that frs1 mutation was a new allele of the ABA3 locus showing that a mutation in this locus leads to an impairment of freezing tolerance. These results constitute the first report showing that a mutation in ABA3 leads to an impairment of freezing tolerance, and not only strengthen the conclusion that ABA is required for full development of freezing tolerance in cold-acclimated plants, but also demonstrate that ABA mediates the constitutive freezing tolerance of Arabidopsis. Gene expression in frs1 mutants was altered in response to dehydration, suggesting that freezing tolerance in Arabidopsis depends on ABA-regulated proteins that allow plants to survive the challenges imposed by subzero temperatures, mainly freeze-induced cellular dehydration. Received: 16 December 1999 / Accepted: 31 March 2000     

Freezing behavior of free protoplasts of winter rye

Free protoplasts prepared from the epicotyls of nonhardened rye seedlings were subjected to fast and slow freezing on a microscope-adapted thermoelectric stage. During rapid freezing to ?12 °C, ice formation occurred inside the protoplasts causing lethal disruption of cell and membrane organization. Under slow freezing to ?12 °C, ice formation occurred outside the protoplast with accompanying dehydration and contraction of the protoplast. Complete rehydration and recovery of the protoplasts occurred upon thawing after slow freezing. Free protoplasts therefore afford a new system for the study of mechanisms of plant cell freezing injury and resistance free of the complications presented by a cell wall.     

Dynamics of membrane exchange of the plasma membrane and the lysis of isolated protoplasts during rapid expansions in area

Summary The plasma membrane of protoplasts isolated from rye leaves (Secale cereale L. cv. Puma) can withstand a maximum elastic stretching of about 2%. Larger area expansions involve the incorporation of new material into the membrane. The dynamics of this process during expansion from isotonic solutions and the probable frequency of lysis have been measured as a function of membrane tension in populations of protoplasts isolated from both cold-acclimated and nonacclimated plants. To a first approximation, both increase exponentially with tension. An analytical solution is reported for the membrane tension as a function of time during an arbitrary expansion in area.     

Phylogenetic analyses in cornus substantiate ancestry of xylem supercooling freezing behavior and reveal lineage of desiccation related proteins

The response of woody plant tissues to freezing temperature has evolved into two distinct behaviors: an avoidance strategy, in which intracellular water supercools, and a freeze-tolerance strategy, where cells tolerate the loss of water to extracellular ice. Although both strategies involve extracellular ice formation, supercooling cells are thought to resist freeze-induced dehydration. Dehydrin proteins, which accumulate during cold acclimation in numerous herbaceous and woody plants, have been speculated to provide, among other things, protection from desiccative extracellular ice formation. Here we use Cornus as a model system to provide the first phylogenetic characterization of xylem freezing behavior and dehydrin-like proteins. Our data suggest that both freezing behavior and the accumulation of dehydrin-like proteins in Cornus are lineage related; supercooling and nonaccumulation of dehydrin-like proteins are ancestral within the genus. The nonsupercooling strategy evolved within the blue- or white-fruited subgroup where representative species exhibit high levels of freeze tolerance. Within the blue- or white-fruited lineage, a single origin of dehydrin-like proteins was documented and displayed a trend for size increase in molecular mass. Phylogenetic analyses revealed that an early divergent group of red-fruited supercooling dogwoods lack a similar protein. Dehydrin-like proteins were limited to neither nonsupercooling species nor to those that possess extreme freeze tolerance.     

Strategies for exploration of freeze responsive gene expression: advances in vertebrate freeze tolerance

Winter survival for many cold-blooded species involves freeze tolerance, the capacity to endure the freezing of a high percentage of total body water as extracellular ice. The wood frog (Rana sylvatica) is the primary model animal used for studies of vertebrate freeze tolerance and current studies in my lab are focused on the freeze-induced changes in gene expression that support freezing survival. Using cDNA library screening, we have documented the freeze-induced up-regulation of a number of genes in wood frogs including both identifiable genes (fibrinogen, ATP/ADP translocase, and mitochondrial inorganic phosphate carrier) and novel proteins (FR10, FR47, and Li16). All three novel proteins share in common the presence of hydrophobic regions that may indicate that they have an association with membranes, but apart from that each shows unique tissue distribution patterns, stimulation by different signal transduction pathways and responses to two of the component stresses of freezing, anoxia, and dehydration. The new application of cDNA array screening technology is opening up a whole new world of possibilities in the search for molecular mechanisms that underlie freezing survival. Array screening of hearts from control versus frozen frogs hints at the up-regulation of adenosine receptor signaling for the possible mediation of metabolic rate suppression, hypoxia inducible factor mediated adjustments of anaerobic metabolism, natriuretic peptide regulation of fluid dynamics, enhanced glucose transporter capacity for cryoprotectant accumulation, defenses against the accumulation of advanced glycation end products, and improved antioxidant defenses as novel parts of natural freeze tolerance that remain to be explored.