Genetic analysis of frost hardiness traits in tuber-bearingSolanum species

The inheritance of frost hardiness and cold acclimation potential traits was studied in three segregating populations derived from a cross betweenSolanum commersonii Dun. PI 243503 (cmm) andSolanum cardiophyllum Lindl., PI 184762 (cph), two parental genotypes with contrasting frost hardiness and cold acclimation potential. The levels of frost hardiness and cold acclimation potential were expressed as the LT₅₀, the temperature at which 50% of the cells in leaf discs were killed, as measured by the ion leakage method, following a controlled freeze test There was considerable variation in both frost hardiness and cold acclimation potential in all three segregating populations (F₁ F₁ xcmm, and F₁ xcph). Frost hardiness and cold acclimation potential were not correlated, suggesting that these two traits are under independent genetic control. The analysis of generation means indicated that the variation for both traits could be best explained by an additive-dominance model, with additive gene effects the most important Broad-sense heritability was 0.73 and 0.74 in the F₁ population, for frost hardiness and cold acclimation potential, respectively, and was 0.85 for either trait in the F₁ xcmm population, indicating that these two traits are highly inheritable. Our results suggest that it should be possible to incorporate the frost hardiness and cold acclimation traits from S.commersonii into cultivated potato species.     

Maximal aerobic performance of deer mice in combined cold and exercise challenges

In nature, animals frequently need to deal with several physiological challenges simultaneously. We examined thermoregulatory performance (body temperature stability) and maximal oxygen consumption of deer mice (Peromyscus maniculatus) during intense exercise at room temperature, acute cold exposure, and exercise during cold exposure. Results with exercise and cold exposure alone were consistent with previous studies: there was little difference between maximal metabolism elicited by exercise alone or cold exposure alone in warm-acclimated mice; after cold acclimation (9 weeks at 5 °C), maximal exercise metabolism did not change but maximum thermogenic capacity increased by >60%. Warm acclimated animals did not increase maximal oxygen consumption when exercise was combined with moderate cold (0 °C) and had decreased maximal oxygen consumption when exercise was combined with severe cold (–16 °C). Combined cold and exercise also decreased thermoregulatory performance and exercise endurance time. Cold acclimation improved thermoregulatory performance in combined cold and exercise, and there was also a slight increase in endurance. However, as for warm-acclimated animals, maximal exercise metabolism did not increase at low temperatures. We interpret these results as an indication of competition between thermoregulatory and locomotor effectors (brown adipose tissue and skeletal muscle) under the combined challenges of cold exposure and maximal exercise, with priority given to the locomotor function.Abbreviations BAT brown adipose tissue - T _b body temperature - O ₂ rate of oxygen consumption - O ₂ max maximal O₂ in exercise - O ₂ sum maximal O₂ during cold exposure Communicated by G. Heldmaier     

Responses of antioxidant enzymes to cold and high light are not correlated to freezing tolerance in natural accessions of Arabidopsis thaliana

Low temperatures and high light cause imbalances in primary and secondary reactions of photosynthesis, and thus can result in oxidative stress. Plants employ a range of low‐molecular weight antioxidants and antioxidant enzymes to prevent oxidative damage, and antioxidant defence is considered an important component of stress tolerance. To figure out whether oxidative stress and antioxidant defence are key factors defining the different cold acclimation capacities of natural accessions of the model plant Arabidopsis thaliana, we investigated hydrogen peroxide (H₂O₂) production, antioxidant enzyme activity and lipid peroxidation during a time course of cold treatment and exposure to high light in four differentially cold‐tolerant natural accessions of Arabidopsis (C24, Nd, Rsch, Te) that span the European distribution range of the species. All accessions except Rsch (from Russia) had elevated H₂O₂ in the cold, indicating that production of reactive oxygen species is part of the cold response in Arabidopsis. Glutathione reductase activity increased in all but Rsch, while ascorbate peroxidase and superoxide dismutase were unchanged and catalase decreased in all but Rsch. Under high light, the Scandinavian accession Te had elevated levels of H₂O₂. Te appeared most sensitive to oxidative stress, having higher malondialdehyde (MDA) levels in the cold and under high light, while only high light caused elevated MDA in the other accessions. Although the most freezing‐tolerant, Te had the highest sensitivity to oxidative stress. No correlation was found between freezing tolerance and activity of antioxidant enzymes in the four accessions investigated, arguing against a key role for antioxidant defence in the differential cold acclimation capacities of Arabidopsis accessions.     

Inheritance of cold shock tolerance in hybrids of Drosophila virilis and Drosophila lummei

The genetic basis of the difference in cold shock tolerance between the southern temperate Drosophila virilis and its boreal relative D. lummei is studied. After adult eclosion, the parental stocks, reciprocal F₁ and backcross hybrids were pretreated for eight days at 18°C or at 6°C. The cold shock used consisted of fast cooling to-10°C and exposure to this temperature for varying lengths of time. D. lummei tolerated such exposure for 40–50% longer than did D. virilis (100–135% after acclimation). Reciprocal F₁ females, differing only in their maternal cytoplasm deviated significantly from each other, and the reciprocal F₁ males even more so, the contribution of the X chromosome being three to four times that of the cytoplasm. The cold resistance scores of the hybrid males were more extreme than those of the parental stocks. Autosomally heterozygous males with the X chromosome and cytoplasm of virilis were the weakest flies studied. The reciprocal males (X chromosome and cytoplasm of lummei) survived better than the parental lummei stock. The reciprocal differences decreased after cold temperature acclimation. The roles of the four major autosomes were analyzed by backcrossing the reciprocal F₁ males with females of the virilis marker stock. The third chromosome of lummei as heterozygous contributed most to cold tolerance, while the other autosomes had a rather weak effect in the opposite direction (virilis homozygotes survived better), which disappeared after acclimation at 6°C. Some of the cold susceptibility of F₁ hybrids disappeared in chromosomally identical backcross flies, indicating complex cytoplasmchromosomal interactions.     

Thermoregulatory changes in the cold induced by physical training in humans

It has previously been demonstrated that metabolic heat production (M˙) during cold exposure at rest was related to maximal oxygen uptake (O_2max). Consequently, an increase in O_2max could allow an increase M˙ in the cold. The aim of the present study was therefore to test this hypothesis. Eight male volunteers undertook interval training (periods of 25% V˙O_2max of 30-s duration and 110% V˙O_2max of 60-s duration until exhaustion, five times a week over 8 weeks) to increase V˙O_2max. Both before and after this physical training, they were subjected to a 10^∘, 5^∘ and 1^∘C 2-h cold air test in a climatic chamber. During the cold exposure, rectal temperature (T _re), tympanic temperature (T _ty), mean skin temperature () and M˙ were measured as well as the time to onset of shivering (t) and body temperatures () at t. The results showed that physical training involved an increase in O_2max (14%–15%, P < 0.05). During the cold exposure, T _re was higher after training both at 10^∘,5^∘ and 1^∘C (P < 0.05) whereas were not significantly changed. However, an increase in the sensitivity of the thermoregulatory system was attested by a decreased t at higher These slight physiological changes found after training were not related to the increases in V˙O_2max. In conclusion, this study demonstrated that interval training induced slight thermoregulatory changes unrelated to changes in V˙O_2max and it suggested that M˙ during cold exposure could be related mainly to the level of V˙O_2max observed before training, since increases in V˙O_2max did not modify M˙. Accepted: 8 April 1998     

Phenotypic flexibility in passerine birds: Seasonal variation of aerobic enzyme activities in skeletal muscle

Improved winter cold tolerance is widespread among small passerines resident in cold climates and is generally associated with elevated summit metabolic rate (M_sum=maximum thermoregulatory metabolic rate) and improved shivering endurance with increased reliance on lipids as fuel. Elevated M_sum and improved cold tolerance may result from greater metabolic intensity, due to mass-specific increase in oxidative enzyme capacity, or increase in the masses of thermogenic tissues. To examine the mechanisms underlying winter increases in M_sum, we investigated seasonal changes in mass-specific and total activities of the key aerobic enzymes citrate synthase (CS) and β-hydroxyacyl CoA-dehydrogenase (HOAD) in pectoralis, supracoracoideus and mixed leg muscles of three resident passerine species, black-capped chickadee (Poecile atricapillus), house sparrow (Passer domesticus), and white-breasted nuthatch (Sitta carolinensis). Activities of CS were generally higher in winter than in summer muscles for chickadees and house sparrows, but not nuthatches. Mass-specific HOAD activity was significantly elevated in winter relative to summer in all muscles for chickadees, but did not vary significantly with season for sparrows or nuthatches, except for sparrow leg muscle. These results suggest that modulation of substrate flux and cellular aerobic capacity in muscle contribute to seasonal metabolic flexibility in some species and tissues, but such changes play varying roles among small passerines resident in cold climates.     

THERMOREGULATORY EFFECT IN HUMANS OF SUPPRESSED ENDOGENOUS MELATONIN BY PRE-SLEEP BRIGHT-LIGHT EXPOSURE IN A COLD ENVIRONMENT

This study investigated the physiological function of suppressed melatonin through thermoregulation in a cold environment. Interactions between thermoregulation directly affected by exposure to a cold environment and indirectly affected by endogenous melatonin suppression by bright-light exposure were examined. Ten male subjects were exposed to two different illumination intensities (30 and 5000 lux) for 4.5?h, and two different ambient temperatures (15 and 27°C) for 2?h before sleep under dark and thermoneutral conditions. Salivary melatonin level was suppressed by bright light (p?<?0.001), although the ambient temperature condition had no significant effect on melatonin. During sleep, significant effects of pre-sleep exposure to a cold ambient temperature (p?<?0.001) and bright light (p?<?0.01) on rectal temperature (T_re) were observed. Pre-sleep, bright-light exposure led to an attenuated fall in T_re during sleep. Moreover, T_re dropped more precipitously after cold exposure than thermoneutral conditions (cold: ?0.54?±?0.07°C/h; thermoneutral: ?0.16?±?0.03°C/h; p?<?0.001). Pre-sleep, bright-light exposure delayed the nadir time of T_re under thermoneutral conditions (p?<?0.05), while cold exposure masked the circadian rhythm with a precipitous decrease in T_re. A significant correlation between the T_re nadir and melatonin level (r?=??0.774, p?<?0.05) indicated that inter-individual differences with higher melatonin levels lead to a reduction in T_re after cold exposure. These results suggest that suppressed endogenous melatonin inhibits the downregulation of the body temperature set-point during sleep. (Author correspondence: ishibasi@design.kyushu-u.ac.jp)     

Comparative proteomic and metabolomic analyses reveal mechanisms of improved cold stress tolerance in bermudagrass (Cynodon dactylon (L.) Pers.) by exogenous calcium

As an important second messenger, calcium is involved in plant cold stress response, including chilling (<20 °C) and freezing (<0 °C). In this study, exogenous application of calcium chloride (CaCl₂) improved both chilling and freezing stress tolerances, while ethylene glycol‐bis‐(β‐aminoethyl) ether‐N,N,N,N‐tetraacetic acid (EGTA) reversed CaCl₂ effects in bermudagrass (Cynodon dactylon (L.) Pers.). Physiological analyses showed that CaCl₂ treatment alleviated the reactive oxygen species (ROS) burst and cell damage triggered by chilling stress, via activating antioxidant enzymes, non‐enzymatic glutathione antioxidant pool, while EGTA treatment had the opposite effects. Additionally, comparative proteomic analysis identified 51 differentially expressed proteins that were enriched in redox, tricarboxylicacid cycle, glycolysis, photosynthesis, oxidative pentose phosphate pathway, and amino acid metabolisms. Consistently, 42 metabolites including amino acids, organic acids, sugars, and sugar alcohols were regulated by CaCl₂ treatment under control and cold stress conditions, further confirming the common modulation of CaCl₂ treatment in carbon metabolites and amino acid metabolism. Taken together, this study reported first evidence of the essential and protective roles of endogenous and exogenous calcium in bermudagrass response to cold stress, partially via activation of the antioxidants and modulation of several differentially expressed proteins and metabolic homeostasis in the process of cold acclimation.     

Factors affecting the daily rhythm of body temperature of captive mouse lemurs (<Emphasis Type="Italic">Microcebus murinus</Emphasis>)

Microcebus murinus, a small nocturnal Malagasy primate, exhibits adaptive energy-saving strategies such as daily hypothermia and gregarious patterns during diurnal rest. To determine whether ambient temperature (T_a), food restriction and nest sharing can modify the daily body temperature (T_b) rhythm, T_b was recorded by telemetry during winter in six males exposed to different ambient temperatures (T_a=25, 20, 15°C) and/or to a total food restriction for 3 days depending on social condition (isolated versus pair-grouped). At 25°C, the daily rhythm of T_b was characterized by high T_b values during the night and lower values during the day. Exposure to cold significantly decreased minimal T_b values and lengthened the daily hypothermia. Under food restriction, minimal T_b values were also markedly lowered. The combination of food restriction and cold induced further increases in duration and depth of torpor bouts, minimal T_b reaching a level just above T_a. Although it influenced daily hypothermia less than environmental factors, nest sharing modified effects of cold and food restriction previously observed by lengthening duration of torpor but without increasing its depth. In response to external conditions, mouse lemurs may thus adjust their energy expenditures through daily modifications of both the duration and the depth of torpor.     

Osmotin induces cold protection in olive trees by affecting programmed cell death and cytoskeleton organization

Osmotin is a pathogenesis-related protein exhibiting cryoprotective functions. Our aim was to understand whether it is involved in the cold acclimation of the olive tree (Olea europaea L.), a frost-sensitive species lacking dormancy. We exposed olive trees expressing tobacco osmotin gene under the 35S promoter (35S:osm) [in the same manner as wild type (wt) plants] to cold shocks in the presence/absence of cold acclimation, and monitored changes in programmed cell death (PCD), cytoskeleton, and calcium ([Ca²⁺]_c) signalling. In the wt, osmotin was immunolocalized only in cold-acclimated plants, and in the tissues showing PCD. In the 35S:osm clones, the protein was detected also in the non-acclimated plants, and always in the tissues exhibiting PCD. In the non-acclimated wt protoplasts exposed to cold shock, a transient decrease in phallotoxin signal suggests a temporary disassembly of F-actin, a transient increase occurred instead in 35S:osm protoplasts exposed to the same shock. Transient increases in [Ca²⁺]_c were observed only in the wt protoplasts. However, when F-actin was depolymerized by cytochalasin or latrunculin, and microtubules by colchicine, increase in [Ca²⁺]_c also occurred in the 35S:osm protoplasts. Successive cold shocks caused transient rises in [Ca²⁺]_c and transient decreases in the phallotoxin signal in wt protoplasts. No change occurred in [Ca²⁺]_c occurred in the 35S:osm protoplasts. The phallotoxin signal transiently increased at the first shock, but did not change after the subsequent shocks, and an overall signal reduction occurred with shock repetition. Following acclimation, no cold shock-induced change in [Ca²⁺]_c levels and F-actin signal occurred either in wt or 35S:osm protoplasts. The results show that osmotin is positively involved in the acclimation-related PCD, in blocking the cold-induced calcium signalling, and in affecting cytoskeleton in response to cold stimuli.     

Low temperature regulates sucrose-phosphate synthase activity in Colobanthus quitensis (Kunth) Bartl. by decreasing its sensitivity to Pi and increased activation by glucose-6-phosphate

Colobanthus quitensis (Kunth) Bartl. is widely distributed from Mexico to the Antarctic. C. quitensis is a freezing resistant species that accumulates sucrose in response to cold. We tested the hypothesis that low temperature modifies the kinetic properties of C. quitensis sucrose phosphate synthase (SPS) to increase its activity and ability to synthesize sucrose during cold acclimation. Cold acclimation caused a fourfold increment in sucrose concentration and a 100% increase in SPS activity, without changes in the level of SPS protein. Cold acclimation did not affect the optimal temperature and pH for SPS activity. However, it caused a tenfold increase in the inhibition constant (K _i) for inorganic phosphate (Pi) calculated as a function of fructose-6-phosphate (Fruc-6-P). SPS from cold acclimated plants also exhibited a higher reduction of its Michaelis constant (K _m) for glucose-6-phosphate (Gluc-6-P) with respect to non-acclimated plants. We suggest that the increase in C. quitensis SPS K _i for Pi and the increase in activation by Gluc-6-P in response to cold keep SPS activated, leading to high sucrose accumulation. This may be an important adaptation that allows efficient accumulation of sucrose during the harsh Antarctic summer.     

The acclimation of photosynthesis and respiration to temperature in the C3–C4 intermediate Salsola divaricata: induction of high respiratory CO2 release under low temperature

Photosynthesis in C₃–C₄ intermediates reduces carbon loss by photorespiration through refixing photorespired CO₂ within bundle sheath cells. This is beneficial under warm temperatures where rates of photorespiration are high; however, it is unknown how photosynthesis in C₃–C₄ plants acclimates to growth under cold conditions. Therefore, the cold tolerance of the C₃–C₄ Salsola divaricata was tested to determine whether it reverts to C₃ photosynthesis when grown under low temperatures. Plants were grown under cold (15/10 °C), moderate (25/18 °C) or hot (35/25 °C) day/night temperatures and analysed to determine how photosynthesis, respiration and C₃–C₄ features acclimate to these growth conditions. The CO₂ compensation point and net rates of CO₂ assimilation in cold‐grown plants changed dramatically when measured in response to temperature. However, this was not due to the loss of C₃–C₄ intermediacy, but rather to a large increase in mitochondrial respiration supported primarily by the non‐phosphorylating alternative oxidative pathway (AOP) and, to a lesser degree, the cytochrome oxidative pathway (COP). The increase in respiration and AOP capacity in cold‐grown plants likely protects against reactive oxygen species (ROS) in mitochondria and photodamage in chloroplasts by consuming excess reductant via the alternative mitochondrial respiratory electron transport chain.     

Two forms of sustained xanthophyll cycle-dependent energy dissipation in overwintering Euonymus kiautschovicus

Seasonal differences in PSII efficiency (F_v/F_m), the conversion state of the xanthophyll cycle (Z + A)/ (V + A + Z), and leaf adenylate status were investigated in Euonymus kiautschovicus. On very cold days in winter, F_v/F_m assessed directly in the field remained low and Z + A high throughout day and night in both sun and shade leaves. Pre-dawn transfer of leaves from subfreezing temperatures in the field to room temperature revealed that recovery (increases in F_v/F_m and conversion of Z + A to violaxanthin) consisted of one, rapid phase in shade leaves, whereas in sun leaves a rapid phase was followed by a slow phase requiring days. The pre-dawn ATP/ADP ratio, as well as that determined at midday, was similar when comparing overwintering leaves with those sampled in the summer, although pre-dawn levels of ATP + ADP were elevated in all leaves during winter relative to summer. After a natural transition to warmer days during the winter, pre-dawn F_v/F_m and Z + A in shade leaves had returned to values typical for summer, whereas in sun leaves F_v/F_m and Z + A levels remained intermediate between the cold day in winter and the summer day. Thus two distinct forms of sustained (Z + A)-dependent energy dissipation were identified based upon their differing characteristics. The form that was sustained on cold days but relaxed rapidly upon warming occurred in all leaves and may result from maintenance of a low lumenal pH responsible for the nocturnal engagement of (Z + A)-dependent thermal dissipation exclusively on very cold days in the winter. The form that was sustained even upon warming and correlated with slow Z + A to violaxanthin conversion occurred only in sun leaves and may represent a sustained engagement of (Z + A)-dependent energy dissipation associated with an altered PSII protein composition. In the latter, warm-sustained form, uncoupler or cycloheximide infiltration had no effect on the slow phase of recovery, but lincomycin infiltration inhibited the slow increase in F_v/F_m and the conversion of Z + A to violaxanthin.     

Cold adaptive potential of chironomids overwintering in a glacial stream

Insects inhabiting cold streams must either tolerate or avoid freezing to survive. The present study reports the strategy adopted by fourth‐instar larvae of two chironomid species [Pseudodiamesa branickii (Nowicki) and Diamesa cinerella (Meigen)] overwintering in a glacial stream (in the Italian Alps). The cold adaptive potential of both species under acute cold stress is investigated down to –30 °C. Supercooling points, lower lethal temperatures (LLTs), haemolymph thermal hysteresis, whole body content of sugars and polyols, and the expression of heat shock protein (HSP) genes (hsc70 and hsp70) expression are estimated. Comparable thermal hysteresis (> 2 °C) is measured in the two species, both of which accumulate glucose and sucrose as the main cryoprotectants. According to the supercooling points (= –6.37 and –6.85 °C, respectively) and LLT₁₀₀ (= –16.2 and –14.7 °C, respectively), P. branickii and D. cinerella can both be considered as freeze tolerant. However, the cumulative proportion of individual freezing values and the LLT₅₀ (–9.14 and –6.13 °C, respectively) suggest that P. branickii is more cold hardy than D. cinerella, whereas the gene expression data (i.e. an absence of up‐regulation of hsp70 in D. cinerella) suggest that D. cinerella is more cold hardy than P. branickii. These findings are discussed in relation to the validity of the different metabolic indicators for defining the level of cold hardiness of a species, even in relation to its cold stenothermy. The results are also discussed in relation to climate warming, which represents a serious threat for species from glacier‐fed streams.     

Uncoupling protein1 mRNA, mitochondrial GTP-binding, and T4 5′-deiodinase of brown adipose tissue in euthermic Daurian ground squirrel during cold exposure

Regulation of thermogenic activity and uncoupling protein1 (UCP1) expression in brown adipose tissue (BAT) were studied in euthermic Daurian ground squirrel after acute and chronic cold exposure at 4°C. The UCP1 concentration was indirectly determined by titration with its specific ligand [³H]-labeled GTP, and Ucp1 mRNA was detected by using a [³²P]-labeled antisense oligonucleotide probe. Both acute and chronic cold exposure stimulated up-regulation of Ucp1 mRNA. Although UCP1 concentration is not significantly increased after 24 h of cold exposure, it is markedly elevated by 75% in squirrels after 4-week cold adaptation compared with controls raised at 22°C. Changes in T₄ 5′-deiodinase activity were closely associated with variations of Ucp1 mRNA level. Ucp1 gene expression is significantly affected by cold exposure in BAT from euthermic Daurian ground squirrels. In addition, the activation of T₄ 5′-deiodinase may be an important regulatory factor in cold-induced Ucp1 expression.