DNA intermediates of meiotic recombination in synchronous S. pombe at optimal temperature

Crossovers formed by recombination between homologous chromosomes are important for proper homolog segregation during meiosis and for generation of genetic diversity. Optimal molecular analysis of DNA intermediates of recombination requires synchronous cultures. We previously described a mutant, pat1-as2, of the fission yeast Schizosaccharomyces pombe that undergoes synchronous meiosis at 25°C when an ATP analog is added to the culture. Here, we compare recombination intermediates in pat1-as2 at 25°C with those in the widely used pat1-114 temperature-sensitive mutant at 34°C, a temperature higher than optimal. DNA double-strand breaks at most hotspots are similarly abundant in the two conditions but, remarkably, a few hotspots are distinctly deficient at 25°C. In both conditions, Holliday junctions at DNA break hotspots form more frequently between sister chromatids than between homologs, but a novel species, perhaps arising from invasion by only one end of broken DNA, is more readily observed at 25°C. Our results confirm the validity of previous assays of recombination intermediates in S. pombe and provide new information on the mechanism of meiotic recombination.     

Temperature-Sensitive Yeast Mutants Defective in Meiotic Recombination and Replication

A system is described for isolating temperature-sensitive mutants of Saccharomyces cerevisiae with defects in early meiotic events. We used an otherwise haploid strain disomic (n+1) for chromosome III, and heteroallelic at the leucine-2 locus. Meiotic development was initiated by exposure of the strain to acetate sporulation medium, and monitored by the appearance of leucine-independent intragenic recombinants. Mutant isolation was based on the recovery of thermally induced defects in recombination. The temperature-sensitive characteristic was included to allow eventual characterizations of the temporal period during meiosis when each gene performs its essential function. Following mutagenesis with either ethyl methane sulfonate or nitrosoguanidine individual clones were tested at 34° and 24° for acetate-induced recombination. Starting with 2700 clones, derived from cells that survived mutagenic treatment, we isolated 48 strains with thermally induced lesions in recombination. In the majority of mutants premeiotic replication occurred normally, or nearly normally, at the restrictive temperature, indicating that the meiotic cycle was initiated and that there was a defect in an event required for intragenic recombination. We also detected mutants where the thermally induced lesion in recombination resulted from temperature-sensitive premeiotic DNA synthesis.     

Differential Sensitivities and the Target of Heat-Induced Recombination at the Base of the X Chromosome of DROSOPHILA MELANOGASTER

The effect of heat on two small adjacent segments at the base of the X chromosome was examined. Recombination in the two segments delimited by recessive lethals was measured after treatment with 30° and 34°.Both segments were sensitive at 30°, while only the proximal one responded to 34° treatment. When the same segments were studied in structural heterozygotes (for deletions) the relative increase in recombination was greater, suggesting that heat exerts its effect on the "pairing" rather than the "exchange" components of crossing over. The effect of c (3) G/+ on the same segments in both homozygous and heterozygous structurals was studied. The results indicate that this meiotic mutant mediates its effect on a step different than that affected by heat.     

Heat Shock Proteins in Tobacco Cell Suspension during Growth Cycle

Tobacco (Nicotiana tabacum L. cv Wisconsin 38) cells grown in suspension culture at 26°C produce heat shock proteins (HSPs) when exposed to elevated temperature of 34 to 42°C. At 34 and 38°C, synthesis of normal proteins is maintained while HSPs are expressed within 30 minutes after initiation of the shock. At 42°C, HSPs are still expressed but normal proteins are made at a reduced rate or not at all. Exposure of cells to 38°C allows for a full expression of HSPs without inhibition of the synthesis of normal proteins. Induced synthesis of HSPs at 38°C is maximal 1 to 2 hours after elevation of temperature and diminishes thereafter through at least 6 hours. Cells growing asynchronously in the logarithmic phase of growth produce HSPs at a much higher rate than those in the stationary phase. The ability to synthesize HSPs disappears about one generation time before the cells reach a growth plateau.     

Isolation, Complementation, and Initial Characterization of Temperature-Sensitive Mutants of the Baculovirus Autographa californica Nuclear Polyhedrosis Virus

Sixteen temperature-sensitive mutants of Autographa californica nuclear polyhedrosis virus were isolated. Several interesting phenotypes were observed. A large proportion of the mutants were unable to form polyhedral occlusion bodies (polyhedra) at the nonpermissive temperature (32.5°C). At 32.5°C, one mutant formed plaques in which the cells lacked polyhedra. Another mutant type was defective in the production of progeny extracellular nonoccluded virus and produced a “plaque” consisting of only a single cell containing polyhedra at 32.5°C. One mutant was defective in plaque formation, progeny nonoccluded virus formation, and polyhedra formation at 32.5°C. Several mutants produced nonoccluded virus but failed to produce plaques or polyhedra at 32.5°C. Other phenotypes were also distinguished. Complementation analyses, performed by either measuring the increase in extracellular nonoccluded virus formation or by observing polyhedra formation in mixed infections at 32.5°C, indicated the presence of 15 complementation groups. A high frequency of recombination was observed. Four of the mutants were found to be host dependent in their temperature sensitivity for polyhedra formation.     

Temperature Compensation of Circadian Period Length in Clock Mutants of Neurospora crassa

Temperature compensation of circadian period length in 12 clock mutants of Neurospora crassa has been examined at temperatures between 16 and 34°C. In the wild-type strain, below 30°C (the “breakpoint” temperature), the clock is well-compensated (Q₁₀ = 1), while above 30°C, the clock is less well-compensated (Q₁₀ = 1.3). For mutants at the frq locus, mutations that shorten the circadian period length (frq-1, frq-2, frq-4, and frq-6) do not alter this temperature compensation response. In long period frq mutants (frq-3, frq-7, frq-8), however, the breakpoint temperature is lowered, and the longer the period length of the mutants the lower the breakpoint temperature. Long period mutants at other loci exhibit other types of alterations in temperature compensation—e.g. chr is well-compensated even above 30°C, while prd-3 has a Q₁₀ significantly less than 1 below 30°C. Prd-4, a short period mutant, has several breakpoint temperatures. Among four double mutants examined, the only unusual interaction between the individual mutations occurred with chr prd, which had an unusually low Q₁₀ value of 0.86 below 27°C. There was no correlation between circadian period length and growth rate. These strains should be useful tools to test models for the temperature compensation mechanism.     

Enhanced Gene Conversion and Postmeiotic Segregation in Pachytene-Arrested SACCHAROMYCES CEREVISIAE

Previous study has demonstrated that incubation of yeast cells of strain AP-1 in sporulation medium at 36° permits them to begin meiosis but that they become arrested at pachytene and undergo enhanced intragenic recombination between ade2 heteroalleles. Tetrad analysis was undertaken to characterize the altered program of meiotic recombination more widely. In one set of experiments, pachytene-arrested cells were permitted to resume sporulation upon transfer to the permissive temperature. In the resulting asci, both postmeiotic segregation and gene conversion were increased several-fold at a number of loci relative to unarrested controls, whereas reciprocal recombination increased two- to threefold. Another set of experiments analyzed the genetic consequences of inducing the pachytene-arrested cells to revert directly to mitotic growth without completion of meiosis. The appearance of homozygous sectors from heterozygous markers revealed that these cells had become committed to appreciable recombination but that reciprocal exchange was less frequent than in normal asci. Taken together, the data indicate that pachytene arrest rendered the cells committed to enhanced recombination upon resumption of sporulation but that most of the crossing over did not occur until release from the arrest. —The genetic basis of pachytene arrest by AP-1 was investigated by mating each of its parents with progeny of strain Y55, which is able to sporulate at 36°. Both of these diploids sporulated at 36°, and asci from the one studied further exhibited 2:2 segregation of the sporulation defect, indicating that pachytene arrest is dependent on a recessive, temperature-sensitive allele at a chromosomal locus.     

Temperature-dependent phase behavior of phosphatidylglycerols from chilling-sensitive and chilling-resistant plants

Seven major lipid classes were isolated from leaves of chilling-sensitive and chilling-resistant plants, and the temperature-dependent phase behaviors of their aqueous dispersions were studied by a fluorescence polarization method using trans-parinaric acid and its methyl ester. Phosphatidylglycerols from the chilling-sensitive plants went from the liquid crystalline state into the phase separation state at about 30°C in 100 mm NaCl and at about 40°C in 5 mm MgCl₂. In contrast, phosphatidylglycerols from the chilling-resistant plants went into the phase separation state at a much lower temperature. The other classes of lipids remained in the liquid crystalline state at all temperatures between 5°C and 40°C regardless of the chilling sensitivity of the plants, except sulfoquinovosyl diacylglycerol from sponge cucumber in which phase separation seemed to begin at about 15°C. Compositions and positional distributions of fatty acids of the lipids suggest that the phosphatidylglycerols from the chilling-sensitive plants, but no other lipids, contained large proportions of molecular species which undergo phase transition at room temperature or above. The thermotropic phase behaviors and the fatty acid compositions suggest that, among the major lipid classes from leaves of the chilling-sensitive plants, only phosphatidylglycerol can induce a phase transition. Since a major part of this lipid in leaves originates from the chloroplasts, phase transition probably occurs in the chloroplast membranes.     

Cultured Ovules as Models for Cotton Fiber Development under Low Temperatures

Cotton fibers (Gossypium hirsutum L.) developing in vitro responded to cyclic temperature change similarly to those of field-grown plants under diumal temperature fluctuations. Absolute temperatures and rates of temperature change were similar under both conditions. In vitro fibers exhibited a “growth ring” for each time the temperature cycled to 22 or 15°C. Rings were rarely detected when the low point was 28°C. The rings seemed to correspond to alternating regions of high and low cellulose accumulation. Fibers developed in vitro under 34°C/22°C cycling developed similarly to constant 34°C controls, but 34°C/22°C and 34°C/15°C cycling caused delayed onset and prolonged periods of elongation and secondary wall thickening. Control fiber length and weight were finally achieved under 34°C/22°C cycling, but both parameters were reduced at the end of the experiment under 34°C/15°C cycling. Fibers developed under all conditions had equal bundle tensile strength. These results demonstrate that: (a) cool temperature effects on fiber development are at least partly fiber/ovule-specific events; they do not depend on whole-plant physiology; and (b) cultured ovules are valid models for research on the regulation of the field cool temperature response.     

Strong Costs and Benefits of Winter Acclimatization in Drosophila melanogaster

Studies on thermal acclimation in insects are often performed on animals acclimated in the laboratory under conditions that are not ecologically relevant. Costs and benefits of acclimation responses under such conditions may not reflect costs and benefits in natural populations subjected to daily and seasonal temperature fluctuations. Here we estimated costs and benefits in thermal tolerance limits in relation to winter acclimatization of Drosophila melanogaster. We sampled flies from a natural habitat during winter in Denmark (field flies) and compared heat and cold tolerance of these to that of flies collected from the same natural population, but acclimated to 25 °C or 13 °C in the laboratory (laboratory flies). We further obtained thermal performance curves for egg-to-adult viability of field and laboratory (25 °C) flies, to estimate possible cross-generational effects of acclimation. We found much higher cold tolerance and a lowered heat tolerance in field flies compared to laboratory flies reared at 25 °C. Flies reared in the laboratory at 13 °C exhibited the same thermal cost-benefit relations as the winter acclimatized flies. We also found a cost of winter acclimatization in terms of decreased egg-to-adult viability at high temperatures of eggs laid by winter acclimatized flies. Based on our findings we suggest that winter acclimatization in nature can induce strong benefits in terms of increased cold tolerance. These benefits can be reproduced in the laboratory under ecologically relevant rearing and testing conditions, and should be incorporated in species distribution modelling. Winter acclimatization also leads to decreased heat tolerance. This may create a mismatch between acclimation responses and the thermal environment, e.g. if temperatures suddenly increase during spring, under current and expected more variable future climatic conditions.     

Effects of cycling temperatures on fiber metabolism in cultured cotton ovules

The effects of temperature on rates of cellulose synthesis, respiration, and long-term glucose uptake were investigated using cultured cotton ovules (Gossypium hirsutum L. cv Acala SJ1). Ovules were cultured either at constant 34°C or under cycling temperatures (12 h at 34°C/12 h at 15-40°C). Rates of respiration and cellulose synthesis at various temperatures were determined on day 21 during the stage of secondary wall synthesis by feeding cultured ovules with [¹⁴C]glucose. Respiration increased between 18 and approximately 34°C, then remained constant up to 40°C. In contrast, the rate of cellulose synthesis increased above 18°C, reached a plateau between about 28 and 37°C, and then decreased at 40°C. Therefore, the optimum temperature for rapid and metabolically efficient cellulose synthesis in Acala SJ1 is near 28°C. In ovules cycled to 15°C, respiration recovered to the control rate immediately upon rewarming to 34°C, but the rate of cellulose synthesis did not fully recover for several hours. These data indicate that cellulose synthesis and respiration respond differently to cool temperatures. The long-term uptake of glucose, which is the carbon source in the culture medium, increased as the low temperature in the cycle increased between 15 and 28°C. However, glucose uptake did not increase in cultures grown constantly at 34°C compared to those cycled at 34/28°C. These observations are consistent with previous observations on the responses of fiber elongation and weight gain to cycling temperatures in vitro and in the field.     

Heat Stress Responses in Cultured Plant Cells : Heat Tolerance Induced by Heat Shock versus Elevated Growing Temperature

Using cultured pear (Pyrus communis cv Bartlett) cells, heat tolerance induced by heat shock was compared to that developed during growth at high temperature. After growth at 22°C, cells exposed to 38°C for 20 minutes (heat shock) showed maximum increased tolerance within 6 hours. Cells grown at 30°C developed maximum heat tolerance after 5 to 6 days; this maximum was well below that induced by heat shock. Heat shock-induced tolerance was fully retained at 22°C for 2 days and was only partly lost after 4 days. However, pear cells acclimated at 30°C lost all acquired heat tolerance 1 to 2 days after transfer to 22°C. In addition, cells which had been heat-acclimated by growth at 30°C showed an additional increase in heat tolerance in response to 39°C heat shock. The most striking difference between heat shock and high growth temperature effects on heat tolerance was revealed when tolerance was determined using viability tests based on different cell functions. Growth at 30°C produced a general hardening, i.e. increased heat tolerance was observed with all three viability tests. In contrast, significantly increased tolerance of heat-shocked cells was observed only with the culture regrowth test. The two types of treatment evoke different mechanisms of heat acclimation.     

Photosynthesis at High Temperature in Tuber-Bearing Solanum Species : A Comparison between Accessions of Contrasting Heat Tolerance

Differences in the photosynthetic performance between pairs of heat tolerant (HT) and heat sensitive (HS) accessions of tuber-bearing Solanum species were measured at 40 °C, after treating plants at 40/30 °C. After 1 to 9 days of heat treatment, both HT and HS accessions showed progressive inhibitory effects, primarily decreased rates of CO₂ fixation, and loss of leaf chlorophyll. These effects were most pronounced in the HS accessions. Stomatal conductivity and internal CO₂ concentrations were lower for both accessions at 40 °C especially for the HS accessions, suggesting that at ambient CO₂ concentrations, stomatal conductance was limiting CO₂ availability at the higher temperature. In the HT accessions, stomatal limitations were largely attributed to differences in vapor pressure deficit between 25° and 40 °C, while the HS accessions exhibited significant nonstomatal limitations. The young expanding leaves of the HS accession showed some HT characteristics, while the oldest leaves showed severe senescence symptoms after 9 days at 40/30 °C. The data suggest that differences in heat sensitivity between HT and HS accessions are the result of accelerated senescence, chlorophyll loss, reduced stomatal conductance, and inhibition of dark reactions at high temperature.     

Growth Temperature-Induced Alterations in the Thermotropic Properties of Nerium oleander Membrane Lipids

The temperature boundary for phase separation of membrane lipids extracted from Nerium oleander leaves was determined by analysis of spin label motion using electron spin resonance spectroscopy and by analysis of polarization of fluorescence from the probe, trans-parinaric acid. A discontinuity of the temperature coefficient for spin label motion, and for trans-parinaric acid fluorescence was detected at 7°C and −3°C with membrane lipids from plants grown at 45°C/32°C (day/night) and 20°C/15°C, respectively. This change was associated with a sharp increase in the polarization of fluorescence from trans-parinaric acid indicating that significant domains of solid lipid form below 7°C or −3°C in these preparations but not above these temperatures. In addition, spin label motion indicated that the lipids of plants grown at low temperatures are more fluid than those of plants grown at higher temperatures.

A change in the molecular ordering of lipids was also detected by analysis of the separation of the hyperfine extrema of electron spin resonance spectra. This occurred at 2°C and 33°C with lipids from the high and low temperature grown plants, respectively. According to previous interpretation of spin label data the change at 29°C (or 33°C) would have indicated the temperature for the initiation of the phase separation process, and the change at 7°C (or −3°C) its completion. Because of the present results, however, this interpretation needs to be modified.

Differences in the physical properties of membrane lipids of plants grown at the hot or cool temperatures correlate with differences in the physiological characteristics of plants and with changes in the fatty acid composition of the corresponding membrane lipids. Environmentally induced modification of membrane lipids could thus account, in part, for the apparently beneficial adjustments of physiological properties of this plant when grown in these regimes.

     

Accumulation of heat shock proteins in field-grown cotton

Cotton (Gossypium hirsutum L.) plants grown under field water deficits exhibited an 80 to 85% reduction in leaf area index, plant height, and dry matter accumulation compared with irrigated controls. Midday photosynthetic rates of dryland plants decreased 2-fold, and canopy temperatures increased to 40°C at 80 days after planting compared with canopy temperatures of 30°C for irrigated plants. Leaves from dryland plants which had exhibited canopy temperatures of 40°C for several weeks accumulated stainable levels of polypeptides with apparent molecular weights of 100, 94, 89, 75, 60, 58, 37, and 21 kilodaltons. These polypeptides did not accumulate in leaves from irrigated plants.

Addition of [³⁵S]methionine to leaves of growth chamber-grown cotton plants and subsequent incubation at 40°C for 3 hours radiolabeled polypeptides with molecular weights similar to those that accumulate in dryland cotton leaves. These data suggest that the proteins which accumulate in water-stressed cotton leaves at elevated temperatures (40°C) are heat shock proteins and that these proteins can accumulate to substantial levels in field-stressed plants.

     

Modeling the Recovery of Heat-Treated Bacillus licheniformis Ad978 and Bacillus weihenstephanensis KBAB4 Spores at Suboptimal Temperature and pH Using Growth Limits

The apparent heat resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis were produced at 45°C and 20°C. B. weihenstephanensis spores were then heat treated at 85°C, 90°C, and 95°C, and B. licheniformis spores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis and 15°C to 60°C for B. licheniformis) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory.     

Temperature response of photosynthesis in different drug and fiber varieties of Cannabis sativa L.

The temperature response on gas and water vapour exchange characteristics of three medicinal drug type (HP Mexican, MX and W1) and four industrial fiber type (Felinq 34, Kompolty, Zolo 11 and Zolo 15) varieties of Cannabis sativa, originally from different agro-climatic zones worldwide, were studied. Among the drug type varieties, optimum temperature for photosynthesis (T_opt) was observed in the range of 30–35 °C in high potency Mexican HPM whereas, it was in the range of 25–30 °C in W1. A comparatively lower value (25 °C) for T_opt was observed in MX. Among fiber type varieties, T_opt was around 30 °C in Zolo 11 and Zolo 15 whereas, it was near 25 °C in Felinq 34 and Kompolty. Varieties having higher maximum photosynthesis (P_{N max}) had higher chlorophyll content as compared to those having lower P_{N max}. Differences in water use efficiency (WUE) were also observed within and among the drug and fiber type plants. However, differences became less pronounced at higher temperatures. Both stomatal and mesophyll components seem to be responsible for the temperature dependence of photosynthesis (P_N) in this species, however, their magnitude varied with the variety. In general, a two fold increase in dark respiration with increase in temperature (from 20 °C to 40 °C) was observed in all the varieties. However, a greater increase was associated with the variety having higher rate of photosynthesis, indicating a strong association between photosynthetic and respiratory rates. The results provide a valuable indication regarding variations in temperature dependence of P_N in different varieties of Cannabis sativa L.     

A role for Caenorhabditis elegans chromatin-associated protein HIM-17 in the proliferation vs. meiotic entry decision

Chromatin-associated protein HIM-17 was previously shown to function in the chromosomal events of meiotic prophase. Here we report an additional role for HIM-17 in regulating the balance between germ cell proliferation and meiotic development. A cryptic function for HIM-17 in promoting meiotic entry and/or inhibiting proliferation was revealed by defects in germline organization in him-17 mutants grown at high temperature (25°) and by a synthetic tumorous germline phenotype in glp-1(ar202); him-17 mutants at 15°.     

Understanding the Physiological Responses of a Tropical Crop (Capsicum chinense Jacq.) at High Temperature

Temperature is one of the main environmental factors involved in global warming and has been found to have a direct effect on plants. However, few studies have investigated the effect of higher temperature on tropical crops. We therefore performed an experiment with a tropical crop of Habanero pepper (Capsicum Chinense Jacq.). Three growth chambers were used, each with 30 Habanero pepper plants. Chambers were maintained at a diurnal maximum air temperature (DMT) of 30 (chamber 1), 35 (chamber 2) and 40°C (chamber 3). Each contained plants from seedling to fruiting stage. Physiological response to variation in DMT was evaluated for each stage over the course of five months. The results showed that both leaf area and dry mass of Habanero pepper plants did not exhibit significant differences in juvenile and flowering phenophases. However, in the fruiting stage, the leaf area and dry mass of plants grown at 40°C DMT were 51 and 58% lower than plants at 30°C DMT respectively. Meanwhile, an increase in diurnal air temperature raised both stomatal conductance and transpiration rate, causing an increase in temperature deficit (air temperature – leaf temperature). Thus, leaf temperature decreased by 5°C, allowing a higher CO₂ assimilation rate in plants at diurnal maximum air temperature (40°C). However, in CO₂ measurements when leaf temperature was set at 40°C, physiological parameters decreased due to an increase in stomatal limitation. We conclude that the thermal optimum range in a tropical crop such as Habanero pepper is between 30 and 35°C (leaf temperature, not air temperature). In this range, gas exchange through stomata is probably optimal. Also, the air temperature–leaf temperature relationship helps to explain how temperature keeps the major physiological processes of Habanero pepper healthy under experimental conditions.     

Control of Lignin Peroxidase Production by Phanerochaete chrysosporium INA-12 by Temperature Shifting

There are two temperature optima connected with lignin peroxidase synthesis by Phanerochaete chrysosporium INA-12. One, at 37°C, is for the mycelium-growing phase; the other, at 30°C, is for the lignin peroxidase-producing phase. One of six extracellular proteins with ligninase activity increased when cultures were grown at 30°C for the entire fermentation period or when cultures were grown at 37°C for the first 2 days of incubation and then shifted to 30°C, compared with the activity of control cultures grown at 37°C for the entire fermentation period. The unsaturation of fatty acid (Δ/mole) of P. chrysosporium INA-12 mycelium decreased from 1.25 to 1.03 when the growth temperature was shifted from 20 to 40°C.