Hsp70 is not a sensitive indicator of thermal limitation in Gadus morhua

The levels of heat‐shock proteins of the 70 kDa family (Hsp70s) were measured in different soft tissues of Atlantic cod Gadus morhua from different locations and after exposure to various thermal conditions: acute temperature increments (1° C day⁻¹), mid‐term (73 days at 4–15° C) and long‐term thermal acclimation (278 days at 8–15° C), and seasonal and latitudinal temperature variations (field samples). Tissue specific distribution patterns of Hsp70s were observed: liver > gills > red blood cells > brain > white muscle. Thus, different tissues may have required different levels of protection by Hsp70s, and possibly this was related to the rate of protein synthesis. There were no differences in tissue Hsp70s between Arctic cod populations (Arctic, i.e . Barents and White Seas, Norwegian coast, and North or Baltic Seas). No changes in Hsp70s levels were observed in response to temperature variation of any intensity (acute fluctuation or seasonal and latitudinal) within the range of physiological temperatures (4–15° C) in wild and laboratory Atlantic cod. This confirms previous observations that changes in Hsp70 caused by such temperature variation are often small in fishes. Probably, the constitutive level of Hsp70s in Atlantic cod was high enough to overcome potentially harmful effects of temperature variations within the physiological range. A suppressing effect of high temperature (15° C) has already been observed at a systematic level (as reduced rate of somatic growth), whereas it is not reflected in modified Hsp70s. Therefore, Hsp70s apparently played a secondary role in defining thermal tolerance limits in Atlantic cod. These conclusions are in line with a recent concept of thermal tolerance which indicated that the first line of thermal limitation in the cold and warm is a loss in aerobic scope.     

Hsp101 is necessary for heat tolerance but dispensable for development and germination in the absence of stress

Hsp101 is a molecular chaperone that is required for the development of thermotolerance in plants and other organisms. We report that Arabidopsis thaliana Hsp101 is also regulated during seed development in the absence of stress, in a pattern similar to that seen for LEA proteins and small Hsps; protein accumulates during mid-maturation and is stored in the dry seed. Two new alleles of the locus encoding Hsp101 (HOT1) were isolated from Arabidopsis T-DNA mutant populations. One allele, hot1-3, contains an insertion within the second exon and is null for Hsp101 protein expression. Despite the complete absence of Hsp101 protein, plant growth and development, as well as seed germination, are normal, demonstrating that Hsp101 chaperone activity is not essential in the absence of stress. In thermotolerance assays hot1-3 shows a similar, though somewhat more severe, phenotype to the previously described missense allele hot1-1, revealing that the hot1-1 mutation is also close to null for protein activity. The second new mutant allele, hot1-2, has an insertion in the promoter 101 bp 5' to the putative TATA element. During heat stress the hot1-2 mutant produces normal levels of protein in hypocotyls and 10-day-old seedlings, and it is wild type for thermotolerance at these stages. Thus this mutation has not disrupted the minimal promoter sequence required for heat regulation of Hsp101. The hot1-2 mutant also expresses Hsp101 in seeds, but at a tenfold reduced level, resulting in reduced thermotolerance of germinating seeds and underscoring the importance of Hsp101 to seed stress tolerance.     

Primary and secondary induction requirements for flowering of Festuca rubra

Root and shoot temperatures were varied independently to determine the importance of root temperature during cold acclimation. Spinach ( Spinacia oleracea L. cvs Harbin and Bloomsdale) plants were subjected to 20/20°C. 20/5°C, 5/20°C, and 5/5°C (shoot/root) temperature treatments. Leaf freezing tolerance, water potential, stomatal resistance, osmotic potential, and water content were measured at 0.25. 1.25, 3.25, and 7.25 days of treatment. There was no change in freezing tolerance or the water relations of the 20/20°C treated plants during the course of the experiment. Freezing tolerance was increased by the 5°C shoot temperature treatments, but was not enhanced by water stress induced by the low root temperature. Leaf water potential and water content decreased and stomatal resistance increased within 6 h in the 20/5°C plants. By day 3, osmotic potential began decreasing in the 20/5°C plants. Leaf water content, osmotic potential, and water potential decreased more gradually in plants grown with 5°C shoot temperature, irrespective of root temperature. Decreased water content and osmotic potential were not correlated with increased freezing tolerance as reported for other herbaceous crop plants.     

Arabidopsis thaliana ecotype Cvi shows an increased tolerance to photo-oxidative stress and contains a new chloroplastic copper/zinc superoxide dismutase isoenzyme.

A new chloroplastic Cu/Zn-superoxide dismutase (SOD) isoenzyme was identified in Arabidopsis thaliana ecotype Cvi. Genetic analyses indicated that the new isoenzyme was encoded by a Cvi-specific allele of Csd2 that was named Csd2-2. Paraquat treatments of A. thaliana ecotypes Ler and Cvi resulted in higher levels of chloroplastic Cu/Zn-SOD activity in Cvi, suggesting that the Cvi isoenzyme has a higher stability and/or turnover rate than the Ler variant under photo-oxidative conditions. In addition, Cvi showed a higher tolerance to paraquat treatments. Hybrid plant populations expressing Csd2-2 also exhibited an increased tolerance, suggesting that the Cvi isoenzyme is one of the factors that contribute to a better fitness in photo-oxidative stress conditions.     

Induction of freezing tolerance in potato (Solanum commersonü) suspension cultured cells

The induction of freezing tolerance by abscisic acid (ABA) or cold treatment in suspension cultured cells of Solanum commersonii was studied. Both ABA (50–100 μ M ) at 23°C and low temperature (4°C) increased freezing tolerance in cultured Solanum commersonii cells from a LT₅₀ (freezing temperature at which 50% cells were killed) of —5°C (control) to —11.5°C in 2 days. Cold-induced freezing tolerance reached its maximum at 2 days and remained constant throughout the cold acclimation period of 11 days. The freezing tolerance induced by ABA, however, showed a rapid decline 2 to 5 days after initiation of ABA treatments. Addition of ABA (100 μ M ) to the culture medium at the inception of low temperature treatment did not enhance freezing tolerance of the cells beyond the level attainable by either treatment singly. Poly(A⁺)-RNA was isolated from the respective treatments, translated in a rabbit reticulocyte lysate cell free system, and the translation products were resolved by two dimensional polyacrylamide gel electrophoresis (ID-PAGE). Analysis of the in vitro translated products revealed changes in the abundance of approximately 26 products (encoding for polypeptides with M, of 14 to 69 kDa and pl of 4.90 to 6.60) in ABA-treated cells 12 h after treatment, and 20 (encoding for polypeptides with M_r of 12 to 69 kDa, with pl of 4.80 to 6.42) in cells exposed to 4°C for 12 h. There were only 5 novel translation products observed when the ABA-treated cells reached the highest level of freezing tolerance (2 days after the initiation of ABA treatment). Changes in translatable RNA populations during the induction of freezing tolerance in cells treated with either ABA or low temperature are discussed.     

Freezing and desiccation tolerance of imbibed canola seed

Depending on the environmental conditions, imbibed seeds survive subzero temperatures either by supercooling or by tolerating freezing-induced desiccation. We investigated what the predominant survival mechanism is in freezing canola ( Brassica napus cv. Quest) and concluded that it depends on the cooling rate. Seeds cooled at 3°C h⁻¹ or faster supercooled, whereas seeds cooled over a 4-day period to −12°C and then cooled at 3°C h⁻¹ to−40°C did not display low temperature exotherms. Both differential thermal analysis and nuclear magnetic resonance (NMR) spectroscopy confirmed that imbibed canola seeds undergo freezing-induced desiccation at slow cooling rates. The freezing tolerance of imbibed canola seed (LT₅₀) was determined by slowly cooling to −12°C for 48 h, followed with cooling at 3°C h⁻¹ to −40°C, or by holding at a constant −6°C (LD₅₀). For both tests, the loss in freezing tolerance of imbibed seeds was a function of time and temperature of imbibition. Freezing tolerance was rapidly lost after radicle emergence. Seeds imbibed in 100 μ M abscisic acid (ABA), particularly at 2°C, lost freezing tolerance at a slower rate compared with water-imbibed seeds. Seeds imbibed in water either at 23°C for 16 h, or 8°C for 6 days, or 2°C for 6 days were not germinable after storage at −6°C for 10 days. Seeds imbibed in ABA at 23°C for 24 h, or 8°C for 8 days, or 2°C for 15 days were highly germinable after 40 days at a constant −6°C. Desiccation injury induced at a high temperature (60°C), as with injury induced by freezing, was found to be a function of imbibition temperature and time.     

Intergenerational acclimation in aphid overwintering

Abstract.  1. When first instar nymphs and adults of the grain aphid Sitobion avenae (Fabricius) (Hemiptera: Aphidiae) were maintained in long-term cultures (>6 months) at 20 °C and 10 °C, the LT₅₀ decreased from −8 and −8.8 °C to −16.0 and −13.5 °C, respectively.
2. When aphids from the 20 °C culture were transferred to 10 °C, there was a progressive increase in cold tolerance through three successive generations. Transfer of newly moulted pre-reproductive adults reared at 10 °C for three generations back to 20 °C resulted in a rapid loss of cold hardiness in their nymphal offspring.
3. In all generations reared at 10 °C, first born nymphs were more cold hardy than those born later in the birth sequence. The LT₅₀ of nymphs produced on the first day of reproduction in the first, second and third generations maintained at 10 °C were −14.8, −17.0 and −16.6 °C, respectively. Thereafter, nymphal cold hardiness decreased over the subsequent 14 days of reproduction in each generation at 10 °C with mean LT₅₀ values of −10.3, −12.6 and −14.8 °C, respectively. By contrast, the cold tolerance of first born nymphs of aphids reared continuously at 20 °C did not differ in comparison with later born siblings. The LT₅₀ of adult aphids was also unaffected by ageing.
4. The ecological relevance of these findings is discussed in relation to the overwintering survival of aphids such as S. avenae .     

Climate warming will reduce growth and survival of Scots pine except in the far north

Tree growth and survival were assessed in 283 populations of Scots pine ( Pinus sylvestris L.) originating from a broad geographic range and grown at 90 common-garden experimental sites across Europe, and in 101 populations grown at 14 sites in North America. Growth and survival were analysed in response to climatic transfer distance, the difference in mean annual temperature (MAT) between the site and the population origin. Differences among populations at each site, and across sites for regional groups of populations, were related to climate transfer distance, but in opposite ways in the northern vs. southern parts of the species range. Climate transfers equivalent to warming by 1–4 °C markedly increased the survival of populations in northern Europe (≥ 62°N, < 2 °C MAT) and modestly increased height growth ≥ 57°N but decreased survival at < 62°N and modestly decreased height growth at < 54°N latitude in Europe. Thus, even modest climate warming will likely influence Scots pine survival and growth, but in distinct ways in different parts of the species range.     

A Bacillus subtilis gene-encoding protein homologous to eukaryotic SMC motor protein is necessary for chromosome partition

We have analysed the function of a gene of Bacillus subtilis , the product of which shows significant homology with eukaryotic SMC proteins essential for chromosome condensation and segregation. Two mutant strains were constructed; in one, the expression was under the control of the inducible spac promoter (conditional null) and, in the other, the gene was disrupted by insertion (disrupted null). Both could form colonies at 23°C but not at 37°C in the absence of the expression of the Smc protein, indicating that the B. subtilis smc gene was essential for cell growth at higher temperatures. Microscopic examination revealed the formation of anucleate and elongated cells and diffusion of nucleoids within the elongated cells in the disrupted null mutant grown at 23°C and in the conditional null mutant grown in low concentrations of IPTG at 37°C. In addition, immunofluorescence microscopy showed that subcellular localization of the Spo0J partition protein was irregular in the smc disrupted null mutant, compared with bipolar localization in wild-type cells. These results indicate that the B. subtilis smc gene is essential for chromosome partition. The role of B. subtilis Smc protein in chromosome partition is discussed.     

Temperature and chemical shocks induce chilling tolerance in germinating Cucumis sativus (cv. Poinsett 76) seeds

Roots of 24-h-old germinated cucumber ( Cucumis sativus cv. Poinsett 76) seeds were subjected to thermal and chemical stresses, equilibrated at 25°C for 2 h and chilled at 2.5°C for 96 h. The germinated seeds were then held at 25°C for 72 h after they were chilled and the elongation of the primary root was used as a measure of chilling tolerance. Control roots elongated from an initial length of 0.2 cm to a final length of 6.3 cm at the end of 72 h. while chilled roots elongated to a final length of only 0.4 to 0.6 cm. Exposure to 0.4 M ethanol for 4 h or to 40°C for 1 h induced substantial chilling tolerance and the roots had a final length of 4.1 and 3.1 cm. respectively. Exposure to 7.5°C for 3 h conferred less chilling tolerance (elongation to 1.4 cm). while exposure to other chemicals (i.e. aqueous solutions of Ca(NO₃)₂, mannitol. methanol and NaCl) produced less, though still significant increases in chilling tolerance. A more severe chilling treatment of 144 h at 2.5°C was required to consistently induce elevated rates of ion leakage. Only the heat and the ethanol shock treatments significantly reduced chilling-induced ion leakage. Inclusion of the protein synthesis inhibitor cycloheximide negated the protective effects of these shock treatments. It appears that de novo protein synthesis is required for induction of chilling tolerance by a variety of chemical and thermal shock treatments.     

Elevated temperature reduces the respiratory scope of coral reef fishes

The capacity for marine fishes to perform aerobically (aerobic scope) is predicted to control their thermal tolerance and, thus, the impact that rapid climate change will have on their populations. We tested the effect of increased water temperatures on the resting and maximum rates of oxygen consumption in five common coral reef fishes at Lizard Island on the northern Great Barrier Reef, Australia. All species exhibited a decline in aerobic capacity at elevated water temperatures (31, 32 or 33 °C) compared with controls (29 °C); however, the response was much stronger in two cardinalfishes, Ostorhinchus cyanosoma and O. doederleini , compared with three damselfishes, Dascyllus anuarus, Chromis atripectoralis and Acanthochromis polyacanthus . Aerobic scope of the two cardinalfishes was reduced by nearly half at 31 °C compared with 29 °C, and virtually all capacity for additional oxygen uptake was exhausted by 33 °C. In contrast, the three damselfishes retained over half their aerobic scope at 33 °C. Such differences in thermal tolerance between species, and possibly families, suggest that the community structure of reef fish assemblages might change significantly as ocean temperatures increase. Populations of thermally tolerant species are likely to persist at higher temperatures, but populations of thermally sensitive species could decline on low-latitude reefs if individual performance falls below levels needed to sustain viable populations.     

The effect of photoperiod and temperature on growth and frost resistance of winter rye root systems

Root growth, development and frost resistance were examined in winter rye ( Secale cereale L. cv. Puma) plants grown under 6 combinations of temperature and photoperiod (20/16°C or 5/3°C, day/night; 8, 16- or 24-h days). Overall root system growth is influenced by the interaction of temperature and photoperiod. Maximum shoot growth occurs at a 24-h photoperiod in 20°C plants and at a 16-h photoperiod in 5°C plants, and is correlated in both treatments with a high root:shoot ratio. Frost resistance of rye roots is affected by short photoperiods in 2 ways. First, short photoperiod and low temperature delay production of new adventitious roots so that newly developing roots are not exposed to freezing temperatures. Second, short photoperiod alone can induce several degrees of frost tolerance in existing roots during the lag phase of growth. Low temperature alone does not decrease the rate of dry weight accumulation in rye root systems, but cold temperature does retard developmental processes within the roots. Rye roots grown at 5°C develop first order lateral roots, differentiate metaxylem vessels and suberize endodermal cell walls more slowly than roots grown at 20°C.     

Geographical variation in Japan in egg development of the mayfly, Ephoron shigae (Ephemeroptera: Polymitarcyidae)

1. The effect of temperature on embryonic development was compared in four populations, two bisexual and two unisexual, of Ephoron shigae , including one each near the northern and southern periphery of the species range in Japan.
2. Eggs from every population were chilled at 4, 8 or 12 °C for diapause development after 50 days at 20 °C for pre-diapause development (experiment I). Some eggs hatched during chilling at 8 °C or 12 °C, whereas no eggs hatched at 4 °C. The rate of hatching in a given condition of chilling was higher for the eggs from warmer winter environments.
3. Chilling at 4 or 8 °C effectively facilitated diapause development. Chilling at 12 °C was, in general, not so effective, but relatively effective for the eggs from warmer winter environments.
4. Eggs were incubated at 8, 12, 15 or 20 °C after chilling at 4 °C to examine the effect of temperature on post-diapause development (experiment II). The eggs incubated at higher temperature after chilling hatched quicker and more synchronously and had higher hatching success.
5. The relationship between temperature and the days required for hatching after chilling was well described by the power function. There was no significant difference in the slope of the regression lines (i.e. temperature dependency) among local populations. However, a longer time was required for hatching at a given temperature for the population from the colder winter environment.
6. There was no detectable difference in the observed intraspecific variations between unisexual and bisexual populations.     

Effects of temperature and delayed initial feeding on the survival and growth of Japanese flounder larvae

The effects of the timing of initial feeding (0, 1, 2, 3 and 4 days after yolk exhaustion) and temperature (15, 18 and 21° C) on the point‐of‐no‐return (PNR), survival and growth of laboratory‐reared Japanese flounder Paralichthys olivaceus larvae were studied under controlled conditions. The larvae reached PNR on 7·7, 5·2 and 4·2 days‐post‐hatching (dph) at 15, 18 and 21° C, respectively. At each temperature, larval growth did not differ significantly among the delayed initial feedings 1 day before PNR but decreased significantly in larvae first fed after that. In the treatments where initial feeding was equally delayed, larvae grew significantly faster at 18 and 21° C than at 15° C. The larvae survived apparently better at 15 and 18° C than at 21° C when initial feeding was equally delayed. At each temperature, survival of the larvae first fed before PNR did not differ noticeably, while delayed initial feeding after that apparently reduced their survival. These results indicated that there existed a negatively temperature‐dependent PNR in the Japanese flounder larvae. Survival and growth of the larvae strongly depended on temperature as well as the timing of initial feeding. High temperature accelerated the yolk exhaustion and growth of the larvae and thus reduced their starvation tolerance and survival. To avoid potential starvation mortality and obtain good growth, the Japanese flounder larvae must establish successful initial feeding within 2 days after yolk exhaustion at 15° C and within 1 day at both 18 and 21° C.     

Remarkable longevity and stress resistance of nematode PI3K-null mutants

The great majority of lifespan-augmenting mutations were discovered in the nematode Caenorhabditis elegans . In particular, genetic disruption of insulin-like signaling extends longevity 1.5- to 3-fold in the nematode, and to lesser degrees in other taxa, including fruit flies and mice. C. elegans strains bearing homozygous nonsense mutations in the age-1 gene, which encodes the class-I phosphatidylinositol 3-kinase catalytic subunit (PI3K_CS), produce progeny that were thought to undergo obligatory developmental arrest. We now find that, after prolonged developmental times at 15–20 °C, they mature into extremely long-lived adults with near-normal feeding rates and motility. They survive to a median of 145–190 days at 20 °C, with nearly 10-fold extension of both median and maximum adult lifespan relative to N2DRM, a long-lived wild-type stock into which the null mutant was outcrossed. PI3K-null adults, although a little less thermotolerant, are considerably more resistant to oxidative and electrophilic stresses than worms bearing normal or less long-lived alleles. Their unprecedented factorial gains in survival, under both normal and toxic environments, are attributed to elimination of residual and maternally contributed PI3K_CS or its products, and consequent modification of kinase signaling cascades.     

Differential expression of the CBF pathway and cell cycle-related genes in Arabidopsis accessions in response to chronic low-temperature exposure

Low, non-freezing temperatures are a major factor limiting growth and development of vegetation in cold climates. Activation of the C-repeat binding factor (CBF) regulatory pathway by acute cold treatment is important for cold acclimation and freezing tolerance in Arabidopsis thaliana ; however, the potential role of this pathway in response to chronic cold treatment has been less well characterised. We studied long-term (chronic) effects of low, non-freezing temperatures on the expression of CBF pathway genes ( CBF2/3 , COR15a , RD29A ) and cell cycle-related genes ( CDKA;1 , CYCD2;1 , CYCB1;1 ) in roots of accessions from habitats differing in growing season temperatures. Elongation rates of primary roots at 21 and 10 °C were not significantly correlated with average growing season temperatures, indicating that there is no ecotypic differentiation for these traits. Measurements of mRNA accumulation in roots of seven accessions showed that expression of CBF2/3 , COR15a and RD29A is induced by both acute cold treatment (2–24 h at 4 °C) and chronic cold treatment (5–6 weeks at 10 °C), while CYCB1;1 is only induced by chronic cold treatment. RD29A and COR15a mRNA levels were correlated (P < 0.05) with the rate of root elongation in the cold for three high-altitude accessions relative to the common laboratory stain, Col-0. Our results are consistent with the hypothesis that induction of CBF2/3 , COR15a , RD29A and CYCB1;1 is a physiological response to cold that, in the case of RD29A and COR15a , may be important for root growth at low temperatures.     

Acetic Acid, Ethanol and Steam Effects on the Growth of Botrytis cinerea in vitro and Combination of Steam and Modified Atmosphere Packaging to Control Decay in Kiwifruit

The effects of acetic acid fumigation, ethanol fumigation, and steam heat treatment on growth of Botrytis cinerea in vitro were investigated. The effect of steam heat treatments in combination with modified atmosphere packaging (MAP) on Botrytis decay development on 'Hayward' kiwifruit was also studied. The fungus was grown in Petri dishes on potato dextrose agar. Ethanol fumigation with 100  μ l/l for 3 or 6 min, or 200  μ l/l for 6 min enhanced the growth of B. cinerea . The effect of acetic acid on growth of B. cinerea was time and dosage-dependent. Fumigation with 1  μ l/l for 6 min, 2  μ l/l for 3 min, and 4  μ l/l for 3 min promoted radial growth of the fungus when compared to the growth of the untreated control. Fumigation with 2  μ l/l for 6 min delayed the growth of the fungus for the first 6 days, while fumigation with 6  μ l/l for 3 min delayed the growth of the fungus after the sixth day. Fumigation with 4 or 6  μ l/l acetic acid for 6 min, and 8  μ l/l acetic acid for 3 or 6 min resulted in complete inhibition of fungal growth. Steam heat treatment at 45°C for 6 min, and at 48, 51, and 54°C for 3 or 6 min completely inhibited fungal growth in vitro . Furthermore, steam treatments at 47, 50, and 53°C for 3 or 6 min completely inhibited decay at the stem end of kiwifruit kept at 10°C in MAP for 12 days. However, none of the steam treatments inhibited decay in wounds on the surface of the fruit kept in MAP.     

Characterization of a novel fibroblast‐like cell line from rainbow trout and responses to sublethal anoxia

A novel fibroblast‐like cell line RTHDF was established from hypodermal connective tissue of rainbow trout Oncorhynchus mykiss and telomerase activity was demonstrated early and late in cell line development. When RTHDF cells were exposed to bioenergetic stress, i.e . anoxia, activation of the stress activated member of the mitogen‐activated protein kinase family, p38^MAPK and induction of heat shock protein (Hsp70) were evident. The time‐course of the p38^MAPK activation and the induction of Hsp70 expression in RTHDF were studied in response to chemically induced anoxia. p38^MAPK was activated rapidly, with maximal activity after 10 min of anoxia. Hsp70 was induced after 30 min of anoxia, followed by overnight recovery in growth medium at 21° C. Using the p38^MAPK‐specific inhibitor SB203580, the enhanced expression of Hsp70 occurred independently of p38^MAPK activation in RTHDF. These data suggests that RTHDF can be useful in studying biochemical responses of teleost cells to environmental stress.