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Low temperature molecular adaptation of the skeletal muscle sarco(endo)plasmic reticulum Ca2+-ATPase 1 (SERCA 1) in the wood frog (Rana sylvatica).

We have compared the primary sequence and enzymatic properties of the sarcoplasmic reticulum Ca(2+)-ATPases from a cold-tolerant frog Rana sylvatica with those of a closely related cold-intolerant frog, Rana clamitans. Sarcoplasmic reticulum isolated from leg muscles of both species contains a major protein ( approximately 100 kDa) that reacts with a monoclonal antibody against sarco(endo)plasmic reticulum Ca(2+)-ATPase type 1 (SERCA1). The apparent molecular mass of R. sylvatica SERCA1 is 115 kDa, whereas that of R. clamitans is 105 kDa. However, the deduced amino acid sequences obtained from cDNAs do not indicate a difference in molecular weight, thus suggesting post-translational protein modification of R. sylvatica SERCA1. Comparison of the temperature dependence of both ATP hydrolysis and Ca(2+) transport indicates that R. sylvatica SERCA1 exhibits significantly lower activation energy below 20 degrees C and an approximately 2-fold greater Ca(2+)-ATPase activity near 0 degrees C. Furthermore, R. sylvatica SERCA1 exhibits simple Michaelis-Menten kinetics with ATP and Ca(2+) as opposed to the two-site ATP kinetics and positive cooperativity with Ca(2+) observed for R. clamitans and mammalian SERCA1s. Cooperativity has been linked to protein-protein interaction in SERCA1, and this property may be altered in R. sylvatica SERCA1. Primary sequence comparison shows that R. sylvatica SERCA1 exhibits seven unique amino acid substitutions, three of which are in the ATP binding domain. We also report for the first time the presence of alternative splicing in the frog, resulting in isoforms SERCA1a and SERCA1b. Thus, it appears that the low temperature muscle contractility of R. sylvatica can be explained partially by significant functional and structural differences in SERCA1.

Adaptation, Physiological↗

Membrane lipid physiology and toxin catabolism underlie ethanol and acetic acid tolerance in Drosophila melanogaster.

Drosophila melanogaster has evolved the ability to tolerate and utilize high levels of ethanol and acetic acid encountered in its rotting-fruit niche. Investigation of this phenomenon has focused on ethanol catabolism, particularly by the enzyme alcohol dehydrogenase. Here we report that survival under ethanol and acetic acid stress in D. melanogaster from high- and low-latitude populations is an integrated consequence of toxin catabolism and alteration of physical properties of cellular membranes by ethanol. Metabolic detoxification contributed to differences in ethanol tolerance between populations and acclimation temperatures via changes in both alcohol dehydrogenase and acetyl-CoA synthetase mRNA expression and enzyme activity. Independent of changes in ethanol catabolism, rapid thermal shifts that change membrane fluidity had dramatic effects on ethanol tolerance. Cold temperature treatments upregulated phospholipid metabolism genes and enhanced acetic acid tolerance, consistent with the predicted effects of restoring membrane fluidity. Phospholipase D was expressed at high levels in all treatments that conferred enhanced ethanol tolerance, suggesting that this lipid-mediated signaling enzyme may enhance tolerance by sequestering ethanol in membranes as phophatidylethanol. These results reveal new candidate genes underlying toxin tolerance and membrane adaptation to temperature in Drosophila and provide insight into how interactions between these phenotypes may underlie the maintenance of latitudinal clines in ethanol tolerance.

Acetate-CoA Ligase↗

The OsLti6 genes encoding low-molecular-weight membrane proteins are differentially expressed in rice cultivars with contrasting sensitivity to low temperature.

Rice (Oryza sativa L.) is sensitive to chilling particularly at early stages of seedling establishment. Two closely related genes (OsLti6a, OsLti6b), which are induced by low temperature during seedling emergence were isolated from a cold tolerant temperate japonica rice cultivar. These genes are closely related to the Arabidopsis rare cold-inducible (RCI2) and barley low-temperature-inducible (BLT101) genes. Based on direct biochemical and indirect physiological evidence and similarity with a conserved protein domain in the Cluster of Orthologous Groups (COG) database (e.g., yeast PMP3), the rice genes belong to a class of low-molecular-weight hydrophobic proteins involved in maintaining the integrity of the plasma membrane during cold, dehydration and salt stress conditions. Both genes exhibit a genotype-specific expression signature characterized by early and late stress-inducible expression in tolerant and intolerant genotypes, respectively. The differences in temporal expression profiles are consistent with cultivar differences in cold-induced membrane leakiness and seedling vigor. The presence of CRT/DRE promoter cis-elements is consistent with the synchronized expression of OsLti6 genes with the C-repeat binding factor/drought responsive element-binding protein (CBF/DREB) transcriptional activator. The present results indicate that the Oslti6 genes are part of a battery of cold stress defense-related genes regulated by a common switch.

Abscisic Acid↗

Analysis of mRNAs that Accumulate in Response to Low Temperature Identifies a Thiol Protease Gene in Tomato.

We have studied the induction of gene expression at low temperature by cloning mRNAs that accumulate when unripe tomato (Lycopersicon esculentum) fruit are incubated at 4 degrees C. Two cloned mRNAs, C14 and C17, accumulate relatively rapidly in response to cold treatment, while a third, C19, displays a delayed response. Significant levels of these mRNAs were not detected during fruit ripening at normal temperature. We have analyzed gene expression at different temperatures and detect half-maximal accumulation of the C14 and C17 mRNAs at 16 degrees C and 11 degrees C, respectively, and have observed that sustained gene expression requires continuous cold treatment. Furthermore, the level of C14 and C17 gene expression in cold-tolerant (hybrid L. esculentum/Lycopersicon pimpinellifolium) fruit is different from that in cold-sensitive (L. esculentum) fruit. DNA sequence analysis indicates that the C14 mRNA encodes a polypeptide with a region that is homologous to the plant thiol proteases actinidin and papain and to the animal thiol protease cathepsin H. We conclude from these experiments that low temperature selectively induces the expression of specific genes and that one such gene encodes a thiol protease.

Journal Article↗

Following the heart: temperature and salinity effects on heart rate in native and invasive species of blue mussels (genus Mytilus).

The three species of blue mussels, Mytilus trossulus Gould 1850, M. edulis Linnaeus 1758 and M. galloprovincialis Lamarck 1819, have distinct global distribution patterns that are hypothesized to reflect differences in their tolerances of temperature and salinity. We examined effects on heart rate (beats min(-1)) of acute exposure and acclimation to different combinations of temperature and salinity to test this hypothesis and, in the context of the invasive success of M. galloprovincialis, to gain insights into the factors that may explain the replacement of the temperate Pacific native, M. trossulus, by this Mediterranean Sea invader along much of the California coast. Heart rate of M. trossulus was significantly higher than that of M. galloprovincialis, consistent with evolutionary adaptation to a lower habitat temperature (temperature compensation) in the former species. Heart rates of M. trossulus/M. galloprovincialis hybrids were intermediate between those of the parental species. Following acclimation to 14 degrees C and 21 degrees C, heart rates of all species exhibited partial compensation to temperature. Heart rate increased with rising temperature until a high temperature was reached at which point activity fell sharply, the high critical temperature (H(crit)). H(crit) increased with increasing acclimation temperature and differed among species in a pattern that reflected their probable evolutionary adaptation temperatures: M. galloprovincialis is more heat tolerant than the other two congeners. Ability to sustain heart function in the cold also reflected evolutionary history: M. trossulus is more cold tolerant than M. galloprovincialis. Heart rates for all three congeners decreased gradually in response to acute reductions in salinity until a low salinity (S(crit)) was reached at which heart rate dropped precipitously. S(crit) decreased with decreasing salinity of acclimation and was generally lowest for M. galloprovincialis. Mortality during acclimation under common garden conditions was greatest in M. trossulus and was highest at high acclimation temperatures and salinities. These intrinsic differences in basal heart rate, thermal and salinity responses, acclimatory capacity, and survivorship are discussed in the contexts of the species' biogeographic patterning and, for the invasive species M. galloprovincialis, the potential for further range expansion along the Pacific coast of North America.

Acclimatization↗

Characterization of a Ca2+-dependent protein kinase from rice root: differential response to cold and regulation by abscisic acid.

The Ca2+-dependent protein kinases (CDPKs) and abscisic acid (ABA) are known to be involved in low-temperature stress response. The focus of this study was to characterize the 45 kDa protein kinase identified in the crude extract of rice (Oryza sativa L.) seedling roots in response to cold (5 degrees C) stress. The activity of the 45 kDa protein kinase decreased at low temperature as evident by an in-gel kinase assay using histone III-S as a substrate. Also, the Ca2+-dependent activity of this protein kinase was suppressed by cold in the membrane fractions of the root. A general protein kinase inhibitor and Ca2+ chelator inhibited the activity of the 45 kDa protein kinase, suggesting that it was a plant CDPK. The 45 kDa CDPK identified was found to be independent of photosynthetic tissues such as the leaf and leaf sheath of rice seedlings, supporting a direct sensing mechanism in the roots of rice seedlings to cold stress. The suppressed activity of the 45 kDa CDPK was reverted by supplementing with 5 microM ABA under cold stress. The 45 kDa CDPK activity was stronger in the cold-tolerant variety of the 4 types tested than it was in the cold-sensitive one. These results suggest the involvement of endogenous ABA in regulating the activity of the 45 kDa CDPK in response to cold stress.

Abscisic Acid↗

Transcriptional expression of a Solanum sogarandium pGT::Dhn10 gene fusion in cucumber, and its correlation with chilling tolerance in transgenic seedlings.

The expression pattern of a Solanum sogarandinum pGT::Dhn10 gene fusion encoding a dehydrin DHN10 protein and the potential role of that protein in cold tolerance in cucumber were analysed in three T1transgenic lines. An accumulation of Dhn10 mRNA was detected in the leaves, cotyledons, hypocotyls and roots of the transgenic seedlings both under the control conditions and after a cold treatment at 6 degrees C for 24 h. This was confirmed by RT-PCR. However, no DHN10 protein was detected by the alkaline phosphatase-conjugated antibody. The transgenic lines exhibited different levels of chilling tolerance. The TCC5/1 line showed a significant increase in its chilling tolerance compared to the non-transgenic line. No chilling injury was observed when the cold hardened (6 degrees C, 24 h) TCC5/1 plants were subsequently exposed to a temperature of 2 degrees C for 6 h. The other two transgenic lines, TCC2/1 and TCC3/2, exhibited a comparable level of chilling tolerance to that of the non-transgenic control. The transgenic lines showed similar or significantly decreased freezing tolerance compared to the non-transgenic control, as evaluated by an electrolyte leakage test. We concluded that the S. sogarandnium GT promoter is functional in the chilling sensitive species Cucumis sativus L., and that the pGT::Dhn10 gene fusion is expressed at the transcriptional level.

Artificial Gene Fusion↗

Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants.

A rice gene encoding a calcium-dependent protein kinase (CDPK), OsCDPK7, was induced by cold and salt stresses. To elucidate the physiological function of OsCDPK7, we generated transgenic rice plants with altered levels of the protein. The extent of tolerance to cold and salt/drought stresses of these plants correlated well with the level of OsCDPK7 expression. Therefore, OsCDPK7 was shown to be a positive regulator commonly involved in the tolerance to both stresses in rice. Over-expression of OsCDPK7 enhanced induction of some stress-responsive genes in response to salinity/drought, but not to cold. Thus, it was suggested that the downstream pathways leading to the cold and salt/drought tolerance are different from each other. It seems likely that at least two distinct pathways commonly use a single CDPK, maintaining the signalling specificity through unknown post-translational regulation mechanisms. These results demonstrate that simple manipulation of CDPK activity has great potential with regard to plant improvement.

Adaptation, Physiological↗

Low temperature-induced systems failure in Escherichia coli: insights from rescue by cold-adapted chaperones.

The growth of Escherichia coli cells is impaired at temperatures below 21 degrees C and stops at 7.5 degrees C; however, growth of a transgenic strain producing the cold-adapted chaperones Cpn60 and Cpn10 from the psychrophilic bacterium Oleispira antarctica is good at low temperatures. The E. coli cpn(+) transgene offers a novel opportunity for examining the essential protein for cell viability at low temperatures. By screening a large-scale protein map (proteome) of cells of K-12 and its Cpn(+) transgene incubated at 4 degrees C, we identified 22 housekeeping proteins involved in systems failure of E. coli when confronted with low temperature. Through co-immunoprecipitation of Cpn60, Northern blot, and in vitro refolding, we systematically identified that protein-chaperone interactions are key determinants of their protein functions at low temperatures. Furthermore, chromosomal gene deletion experiments suggest that the mechanism of cold-induced systems failure in E. coli is cold-induced inactivation of the GroELS chaperonins and the resulting failure to refold cold-inactivated Dps, ClpB, DnaK and RpsB proteins. These findings: (1) indicate the potential importance of chaperones in cold sensitivity, cold adaptation and cold tolerance in cellular systems, and (2) suggest the identity of a few key cold-sensitive chaperone-interacting proteins that get inactivated and ultimately cause systems failure in E. coli cells at low temperatures.

Adaptation, Physiological↗

Effect of temperature on spore germination and vegetative cell growth of Clostridium botulinum.

Spore germination and vegetative growth of Clostridium botulinum type E strain VH at 2 to 50 degrees C were studied. At all of these temperatures, germination began immediately after the addition of the spores to the germination medium. Microscopic observations during germination revealed three types of spores: phase bright (ungerminated), phase variable (partially germinated), and phase dark (fully germinated). At all temperatures except 50 degrees C, there was a pronounced lag between the initial appearance of phase-variable spores and their eventual conversion to phase-dark spores. The number of partially germinated spores increased steadily, reaching 40 to 60% by 18 to 21 h of incubation. During this time, phase-dark, fully germinated spores developed slowly and did not exceed 28% in any of the samples. At 18 to 26 h of incubation, the rate of full germination increased abruptly four-fold. There was extensive and relatively rapid germination at 2 degrees C, the lowest temperature tested, yielding about 60% phase-variable spores by 18 h, which became phase-dark by 26 h of incubation. The optimum temperature for partial and full germination was consistently 9 degrees C. Germination at 50 degrees C was exceptionally rapid and was completed within 1 to 2 h, although 40% remained phase bright. Vegetative cells showed detectable growth at 6 to 41 degrees C, with a distinct optimum at 32.5 degrees C. No growth occurred at 50 degrees C, and only marginal growth was observed at 6 to 14 degrees C. The psychrophilic nature of the germination process coupled with the cold tolerance of vegetative growth appears to give C. botulinum type E an advantage in cold climates as well as in cold-stored foods.

Clostridium botulinum↗

Signal transduction leading to low-temperature tolerance in Arabidopsis thaliana.

Calcium is used by most cells to convert external signals into biochemical events within the cytosol. To detect the effects of cold stress, a gene encoding apoaequorin has been introduced into various cell types that, in the presence of coelenterazine, allows calcium levels to be monitored by the emission of blue light. All cell types respond to the cold by elevating calcium. This event is rapid after cold stress but then shows a slower kinetic response. The magnitude is dependent on both the rate and the final temperature to which cooling occurs. It would appear that calcium is transferred both from outside the cell and from the vacuole into the cytosol. The more rapid phase involves the former and the slower phase the latter. By studying mutant plants, it has been identified that an increase in intracellular calcium can activate the expression of transcription factors that control the subsequent transcription of a whole battery of genes which must be switched on to provide cold tolerance in the plant.

Acclimatization↗

Expression of the heat shock gene clpL of Streptococcus thermophilus is induced by both heat and cold shock.

BACKGROUND: Heat and cold shock response are normally considered as independent phenomena. A small amount of evidence suggests instead that interactions may exist between them in two Lactococcus strains. RESULTS: We show the occurrence of molecular relationships between the mechanisms of cold and heat adaptations in Streptococcus thermophilus, a lactic acid bacterium widely used in dairy fermentation, where it undergoes both types of stress. We observed that cryotolerance is increased when cells are pre-incubated at high temperature. In addition, the production of a protein, identified as ClpL, a member of the heat-shock ATPase family Clp A/B, is induced at both high and low temperature. A knock-out clpL mutant is deficient in both heat and cold tolerance. However lack of production of this protein does not abolish the positive effect of heat pre-treatment towards cryotolerance. CONCLUSION: Dual induction of ClpL by cold and heat exposure of cells and reduced tolerance to both temperature shocks in a clpL mutant indicates that the two stress responses are correlated in S. thermophilus. However this protein is not responsible by itself for cryotolerance of cells pre-treated at high temperature, indicating that ClpL is necessary for the two phenomena, but does not account by itself for the relationships between them.

Journal Article↗

Changes in gut and Malpighian tubule transport during seasonal acclimatization and freezing in the gall fly Eurosta solidaginis.

Since few studies have examined cold tolerance at the organ level in insects, our primary objective was to characterize the functional responses of the gut and Malpighian tubules (MT) to seasonal acclimatization, chilling and freezing in larvae of the goldenrod gall fly Eurosta solidaginis Fitch (Diptera, Tephritidae). From September to December, hemolymph osmolality (455-926 mOsmol kg l(-1)) and freezing tolerance increased markedly in field-collected larvae. Chlorophenol Red was readily transported into the lumen of the foregut, the posterior portion of the midgut, the ureter, the proximal region of the anterior pair of MT, and entire posterior pair of MT. Ouabain and KCN inhibited transport of Chlorophenol Red in the gut and MT. Transport was readily detected at 0 degrees C and the rate of transport was directly related to temperature. The rate of fluid transport by the MT decreased steadily from a monthly high in September (10.7+/-0.8 nl min(-1) for the anterior pair; 12.7+/-1.0 nl min(-1) for the posterior pair) until secretion was no longer detectable in December; this decrease parallels entry into diapause for this species. Even in larvae that died following freezing for 40 days at -20 degrees C, individual organ function was retained to a limited extent. Through the autumn, cholesterol concentrations in the hemolymph increased nearly fourfold. In contrast, the ratio of cholesterol to protein content (nmol mg l(-1)) in the MT membrane remained relatively constant (22 approximately 24 nmol mg l(-1) protein) during this period. Freezing of larvae for 20 days at -20 degrees C caused a significant decrease in cholesterol levels in the hemolymph and the MT membranes compared to unfrozen controls. These results suggest that cholesterol plays a role in seasonal cold hardening and freeze tolerance in insects.

Acclimatization↗

Heat loss during cold exposure in adult and aged C57BL/6J mice.

Metabolic heat production (MHP), colonic temperature (Tco), and nonevaporative (dry) heat loss were measured in ADULT and AGED C57BL/6J male mice during cold exposure. Dry heat loss was assessed as a differential temperature (Td) between incoming and outgoing air through the chamber for indirect calorimetry. The average Td during cold exposure normalized to surface area for ADULT mice was significantly higher than that for the AGED animals (0.0618 +/- 0.0003 degree C/cm2 and 0.0553 +/- 0.0005 degree C/cm2, respectively). Linear regression analysis showed that at the same Tco AGED mice showed lower values of Td normalized to surface area, indicating that at the same body temperature they were losing less heat than ADULT animals. It was concluded that age-related decline in cold tolerance in mice is not due to a lack of ability to reduce heat loss during cold exposure. On the contrary, AGED animals had lower heat loss in comparison with ADULT. We suggest that augmentation of heat conservation mechanisms is an adaptive response to diminishing cold-induced heat production.

Aging↗

Energy metabolism during cold exposure.

Recent advances on the influence of cold exposure on energy metabolism in animals and humans are summarized. Although the cold-induced enhancements in carbohydrate metabolism have been the focus of numerous studies, it was only recently that pieces of evidence from animal studies have suggested that cold exposure exerts an insulin-like effect on peripheral tissue glucose uptake, which appears to proceed primarily via insulin-independent pathways. Interestingly, this phenomenon was observed in insulin-sensitive tissues of warm- a well as cold-adapted rats. Whereas previous human studies have concentrated on the cold-induced changes in basal levels of hormones and metabolic substrates, recent work from our laboratory has demonstrated that exposure to cold at rest shifts substrate utilization from mainly lipids at thermal neutrality to carbohydrates, representing the main fuel for shivering thermogenesis. Further investigation has revealed that the marked increment in carbohydrate oxidation in cold-exposed humans is derived from a greater utilization of both circulating glucose and intramuscular glycogen. With respect to lipid metabolism, recent studies have shown that the cold-induced increase in lipid oxidation in man is fuelled primarily by the fatty acids released from white adipose tissue triglycerides (TG) and possibly intramuscular TG, not plasma TG. One practical application of this work on energy metabolism in the cold resides in the pharmacological approach to improve cold tolerance, where pharmacological agents that alter energy metabolism and substrate utilization could be used to enhance cold thermogenesis and produce warmer body temperatures.

Animals↗

Increased ACTH response to corticotropin-releasing factor in cold-adapted rats in vivo.

We have recently reported that chronically repeated restraint stress results in improved cold tolerance in rats via an increased activity of nonshivering thermogenesis, a characteristic metabolic change observed during cold adaptation, suggesting the presence of cross-adaptation between cold and stress. It is well established that the hypothalamic-pituitary-adrenal (HPA) axis is activated in various stress responses. In the present study, therefore, we examined whether cold adaptation would alter the adrenocorticotropic hormone (ACTH)-releasing state in vivo using freely moving, conscious rats chronically implanted with intra-atrial cannulas. There was no difference in the basal levels of plasma ACTH between warm control and cold-adapted rats. On the other hand, the ACTH response to the intravenous administration of corticotropin-releasing factor (CRF; 2 micrograms/animal) was significantly elevated in cold-adapted rats. However, the injection of 10 micrograms of CRF, which was considered as a dose to elicit the maximal ACTH response, resulted in similar ACTH release patterns between the two groups. These changes in the responsiveness of ACTH secretion have been observed in rats chronically exposed to stressful conditions. The results demonstrated in the present study, therefore, provide further evidence for our hypothesis that there may exist cross-adaptation between cold and nonthermal stress.

Adaptation, Physiological↗

Modulation of maximum thermogenesis by feeding in the white rat.

The present study investigated the availability of substrates as a limiting factor in acute cold exposure. By use of a self-control experimental design, thermogenesis was measured in rats at -10 degrees C under 21% O2-79% He following different feeding regimes. Overnight fasting resulted in the lowest total heat production (9,949 +/- 189 cal/120 min, mean +/- SE, n = 11), maximum rate of thermogenesis (1,320 +/- 23 cal/15 min), and final body temperature (31.0 +/- 0.3 degrees C). The values were significantly lower than those found after overnight rationing (10,630 +/- 231, 1,386 +/- 29, and 32.8 +/- 0.5, respectively) or ad libitum feeding (10,821 +/- 224, 1,395 +/- 27, and 33.5 +/- 0.3). Rats fasted overnight but fed intragastrically a 5-ml substrate mixture (3 kcal/ml) 60 min prior to cold exposure resulted in the highest values for all parameters (11,553 +/- 207, 1,488 +/- 23, and 33.7 +/- 0.4). The increases were not due to the specific dynamic action of food. Since the levels of maximum thermogenesis and cold tolerance can be modulated by feeding, it is evident that substrate availability limits thermogenesis in severe cold independent of respiratory-cardiovascular functions and cellular oxidative capabilities.

Animals↗

Use of commercial freezers to control cowpea weevil, Callosobruchus maculatus (Coleoptera: Bruchidae), in organic garbanzo beans.

One California processor of organic garbanzo beans (Cicer arietinum L.), unable to use chemical fumigants, relies on 30-d storage at -18 degrees C to disinfest product of the cowpea weevil, Callosobruchus maculatus (F). To determine whether the storage period may be shortened, the most cold-tolerant life stage of the cowpea weevil was identified. Laboratory studies showed that the egg stage was most tolerant to -18 degrees C and that adults were most susceptible. To examine the efficacy of cold storage disinfestation, bags of black-eyed peas, Vigna unguiculata (L.) Walp., infested with cowpea weevil eggs were buried within garbanzo bean bins placed in a commercial cold storage facility kept at approximately -18 degrees C and removed after 7, 14, and 21 d. Survival was highest in eggs located at the center of the bins and coincided with the slowest cooling rate. Although temperatures within the bins did not reach -18 degrees C until after 14-19 d, egg mortality was estimated to be >98% after just 7 d of exposure. Complete mortality of eggs occurred after 14 d of cold storage. A 2-wk treatment regimen may be sufficient for control of cowpea weevil in organic legumes.

Animals↗