Studies on golden hamsters during cooling to and rewarming from body temperatures below 0 degrees C. II. Observations during and after resuscitation.
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Fifty-two lines of Drosophila melanogaster founded by single-pair population bottlenecks were used to study the effects of inbreeding and environmental stress on phenotypic variance, genetic variance and survivorship. Cold temperature and high density cause reduced survivorship, but these stresses do not cause repeatable changes in the phenotypic variance of most wing morphological traits. Wing area, however, does show increased phenotypic variance under both types of environmental stress. This increase is no greater in inbred than in outbred lines, showing that inbreeding does not increase the developmental effects of stress. Conversely, environmental stress does not increase the extent of inbreeding depression. Genetic variance is not correlated with environmental stress, although the amount of genetic variation varies significantly among environments and lines vary significantly in their response to environmental change. Drastic changes in the environment can cause changes in phenotypic and genetic variance, but not in a way reliably predicted by the notion of 'stress'.
The reduction of exogenous ferric iron by Listeria monocytogenes, a Gram-positive food-borne pathogen, was investigated. Using an assay incorporating the ferrous iron chelator ferrozine, we showed that intact cells of L. monocytogenes, when exposed to ferric iron, were able to rapidly reduce and solubilize the iron to the ferrous form. Reduction occurred only after direct contact between the bacteria and the iron source. A number of different ferric iron chelates, including transferrin and lactoferrin-bound iron, haemoglobin, ferritin, and iron complexed to siderophores, could be reduced. The ferric reductase activity was expressed by both reference strains and clinical isolates of L. monocytogenes and by all other species of Listeria, although significant quantitative differences were observed. In L. monocytogenes, the expression of ferric reductase was not affected by the growth phase of the bacteria nor by the presence or absence of iron in the growth medium. However, expression was greatly reduced in bacteria grown anaerobically and when cultured in media of reduced pH. In addition, bacteria grown at a cold temperature displayed greater ferric reductase activity than cells grown at higher temperatures. A surface-associated ferric reductase system may be one component of a general iron scavenging mechanism which can be used by Listeria growing in a variety of environments.
Ozone is the most harmful air pollutant for plant ecosystems in the Mediterranean and Alpine areas due to its biological and economic damage to crops and forests. In order to evaluate the relation between ozone exposure and vegetation injury under on-field conditions, suitable ozone monitoring techniques were investigated. In the framework of a 5-year research project aimed at ozone risk assessment on forests, both continuous analysers and passive samplers were employed during the summer seasons (1994-1998) in different sites of a wide mountain region (80 x 40 km2) on the southern slope of the European Alps. Continuous analysers allowed the recording of ozone hourly concentration means necessary both to calculate specific exposure indexes (such as AOT, SUM, W126) and to record daily time-courses. Passive samplers, even though supplied only weekly mean concentration values, made it possible to estimate the altitude concentration gradient useful to correct the altitude dependence of ozone concentrations to be inserted into exposure indexes. In-canopy ozone profiles were also determined by placing passive samplers at different heights inside the forest canopy. Vertical ozone soundings by means of tethered balloons (kytoons) allowed the measurement of the vertical concentration gradient above the forest canopy. They also revealed ozone reservoirs aloft and were useful to explain the ozone advection dynamic in mountain slopes where ground measurement proved to be inadequate. An intercomparison between passive (PASSAM, CH) and continuous measurements highlighted the necessity to accurately standardize all the exposure operations, particularly the pre- and postexposure conservation at cold temperature to avoid dye (DPE) activity. Advantages and disadvantages from each mentioned technique are discussed.
We show that inhomogeneous boundary conditions (BCs) in a distributed reaction-diffusion excitable system are a natural source of permanent perturbations that can induce wave trains, which can be characterized as mixed-mode temporal oscillations and, when a parameter is varied, admit a period-adding bifurcation. To that end we analyze: a pair of coupled excitable and oscillatory cells, a distributed FitzHugh-Nagumo model, and a distributed five-variable model that describes catalytic oxidation. The obtained results account for the recently reported experimental observations of mixed-mode oscillations showing a period-adding bifurcation during oxidation on a disk-shaped catalytic cloth with imposed cold temperature BC.
Spike-shaped structures are produced by light-driven ablation in very different contexts. Penitentes 1-4 m high are common on Andean glaciers, where their formation changes glacier dynamics and hydrology. Laser ablation can produce cones 10-100 microm high with a variety of proposed applications in materials science. We report the first laboratory generation of centimeter-scale snow and ice penitentes. Systematically varying conditions allows identification of the parameters controlling the formation of ablation structures. We demonstrate that penitente initiation and coarsening require cold temperatures, so that ablation leads to sublimation. Once penitentes have formed, further growth of height can occur by melting. The penitentes initially appear as small structures (3 mm high) and grow by coarsening to 1-5 cm high. Our results are an important step towards understanding ablation morphologies.
The U-box motif is a conserved domain found in the diverse isoforms of E3 ubiquitin ligase in eukaryotes. From water-stressed hot pepper (Capsicum annuum L. cv Pukang) plants, we isolated C. annuum putative U-box protein 1 (CaPUB1), which encodes a protein containing a single U-box motif in its N-terminal region. In vitro ubiquitination and site-directed mutagenesis assays revealed that CaPUB1 possessed E3 ubiquitin ligase activity and that the U-box motif was indeed essential for its enzyme activity. RNA gel-blot analysis showed that CaPUB1 mRNA was induced rapidly by a broad spectrum of abiotic stresses, including drought, high salinity, cold temperature, and mechanical wounding, but not in response to ethylene, abscisic acid, or a bacterial pathogen, suggesting its role in the early events in the abiotic-related defense response. Because transgenic work was extremely difficult in hot pepper, in this study we overexpressed CaPUB1 in Arabidopsis (Arabidopsis thaliana) to provide cellular information on the function of this gene in the development and plant responses to abiotic stresses. Transgenic Arabidopsis plants that constitutively expressed the CaPUB1 gene under the control of the cauliflower mosaic virus 35S promoter had markedly longer hypocotyls and roots and grew more rapidly than the wild type, leading to an early bolting phenotype. Microscopic analysis showed that 35S::CaPUB1 roots had increased numbers of small-sized cells, resulting in disordered, highly populated cell layers in the cortex, endodermis, and stele. In addition, CaPUB1-overexpressing plants displayed increased sensitivity to water stress and mild salinity. These results indicate that CaPUB1 is functional in Arabidopsis cells, thereby effectively altering cell and tissue growth and also the response to abiotic stresses. Comparative proteomic analysis showed that the level of RPN6 protein, a non-ATPase subunit of the 26S proteasome complex, was significantly reduced in 35SCaPUB1 seedlings as compared to the wild type. Pull-down and ubiquitination assays demonstrated that RPN6 interacted physically with CaPUB1 and was ubiquitinated in a CaPUB1-dependent manner in vitro. Although the physiological function of CaPUB1 is not yet clear, there are several possibilities for its involvement in a subset of physiological responses to counteract dehydration and high-salinity stresses in transgenic Arabidopsis seedlings.
Characterization of the expression of hsp90 genes of Brassica napus by northern blot analysis and immunoblotting showed that the hsp90 mRNA and protein are present in all B. napus tissues examined, albeit at different levels. High levels of hsp90 mRNA and protein were found in young and rapidly dividing tissues such as shoot apices and flower buds, suggesting that hsp90 may have an important role in plant growth and development. A significant increase in hsp90 mRNA levels was detected in seedlings exposed to 5[deg]C. The transcript levels reached a maximum within 1 d of cold treatment and remained elevated for the entire duration of cold treatment. The levels of hsp90 mRNA rapidly decreased to the level found in control plants upon return to 20[deg]C. The cold-induced accumulation of hsp90 mRNA closely resembles the expression of two previously identified cold-regulated genes of B. napus. We have also confirmed cold regulation of hsp90 mRNA in spinach (Spinacea oleracea). Our results suggest a role for hsp90 in adaptation to cold temperature stress.
The ability to control extracellular ice formation during freezing is critical to the survival of freezing-tolerant plants. Antifreeze proteins, which are proteins that have the ability to retard ice crystal growth, were recently identified as the most abundant apoplastic proteins in cold-acclimated winter rye (Secale cereale L.) leaves. In the experiments reported here, amino-terminal sequence comparisons, immuno-cross-reactions, and enzyme activity assays all indicated that these antifreeze proteins are similar to members of three classes of pathogenesis-related proteins, namely, endochitinases, endo-beta-1,3-glucanases, and thaumatin-like proteins. Apoplastic endochitinases and endo-beta-1,3-glucanases that were induced by pathogens in freezing-sensitive tobacco did not exhibit antifreeze activity. Our findings suggest that subtle structural differences may have evolved in the pathogenesis-related proteins that accumulate at cold temperatures in winter rye to confer upon these proteins the ability to bind to ice.
The seeds of six woody species of Oleaceae representing three genera, contain high concentrations of water-soluble glucosides, with major absorption maxima below 240 nanometers. In Fraxinus americana seeds three of these compounds, designated GL-3, GL-5, and GL-6, account for almost 10% of the dry weight. They are found in the endosperm and embryo but not in the pericarp. While the level of GL-5 is not particularly influenced by the physiological state of the embryo, that of GL-3 and GL-6 decreases as a result of germination and growth during a 10-day period. As the concentrations of GL-3 and GL-6 decrease, new ultraviolet-absorbing compounds are formed. The changes in the concentration of the ultraviolet-absorbing glucosides during cold temperature after-ripening, prior to germination, are small. When germination of dormant embryos is induced with gibberellic acid, the concentrations of GL-3 and GL-6 decrease in a manner similar to that observed with nondormant embryos. In the presence of abscisic acid no losses of GL-3 and GL-6 were observed. It is suggested that GL-3 and GL-6 fulfill some definite functions in the germination and growth of F. americana embryos, and that gibberellic acid and abscisic acid can exert a regulatory effect on the metabolism of these glucosides.
Following excision, etiolated epicotyl segments of Pisum sativum L. cv. Alaska exhibit a marked hyperpolarization of membrane potential which is followed by a linear accumulation of K(+) when segments are incubated in Higinbotham nutrient solution. Segments aged for several hours and then reexcised display only a slight depolarization of membrane potential and no delay in ion accumulation; thus, recovery from injury appears an unlikely explanation for these responses. Substances originating in either the plumule or the cotyledons do not seem to be directly involved in these "aging" responses. However, locally produced substances, such as ethylene, or substances originating in the roots have not been eliminated as causative factors. Cold temperatures and cycloheximide prolong the lag in K(+) accumulation indicating a metabolic explanation for the induced K(+) accumulation. However, similar specific activities of plasma membrane-bound ATPase were found in isolates from fresh and aged epicotyl segments. Reactivation of an ion transport mechanism, perhaps responsible for the osmotic control of growth in immature cells, is suggested as a possible explanation for the pattern of ion accumulation characteristic of excised pea epicotyl tissue.
When a plasma membrane-enriched fraction isolated from red beet (Beta vulgaris L.) was incubated in the presence of 40 micromolar [gamma-(32)P] ATP, 40 micromolar MgSO(4) at pH 6.5, a rapidly turning over phosphorylated protein was formed. Phosphorylation of the protein was substrate-specific for ATP, sensitive to diethylstilbestrol and vanadate, but insensitive to azide. When the dephosphorylation reaction was specifically studied, KCl was found to increase the turnover of the phosphorylated protein consistent with its stimulatory effect upon plasma membrane ATPase. The protein-bound phosphate was found to be most stable at a pH between 2 and 3 and under cold temperature, suggesting that the protein phosphate bond was an acyl-phosphate. When the phosphorylated protein was analyzed with lithium dodecyl sulfate gel electrophoresis, a labeled polypeptide with a molecular weight of about 100,000 daltons was observed. Phosphorylation of this polypeptide was rapidly turning over and Mg-dependent. It is concluded that the phosphorylation observed represents a reaction intermediate of the red beet plasma membrane ATPase.
The water transport properties of etiolated pea (Pisum sativum L.) internodes were studied using both dynamic and steady-state methods to determine (a) whether water transport through the growing tissue limits the rate of cell enlargement, and (b) whether auxin stimulates growth in part by increasing the hydraulic conductance of the growing tissue.Measurements using the pressure probe technique showed that the hydraulic conductivity of cortical cell membranes was the same for both slowly growing and auxin-induced rapidly growing cells (membrane hydraulic conductivity, about 1.5 x 10(-5) centimeters per second per bar). In a second technique which measured the rate of water movement through the entire pea internode, the half-time for radial water flow was about 60 seconds and was not altered by auxin application. These results indicate that auxin does not alter the hydraulic conductance of pea stem tissue, either at the cellular or the whole tissue level.Measurements of the turgor pressure of cortical cells, combined with osmotic pressure measurements of expressed cell sap, show that the water potential of growing pea stems was about -3 bars. When the growth rate was altered by various treatments, including decapitation, auxin application, cold temperature, and KCN treatment, the water potential was independent of the growth rate of the stem. We attribute the depression of the water potential in young pea stems to the presence of solutes in the cell wall free space of the tissue. This interpretation is supported by the results of infiltration and perfusion experiments.From the results of these dynamic and steady-state experiments, we conclude that the internal gradient in water potential (from the xylem to the epidermis) needed to sustain cell enlargement is small (no greater than 0.5 bar). Thus, the hydraulic conductance of the tissue is sufficiently large that it does not control or limit the rate of cell enlargement.
The role of ethylene in the senescence of detached rice leaves in relation to their changes in 1-aminocyclopropane-1-carboxylic acid (ACC) content and ethylene production was studied. In freshly excised rice leaf segments, ACC level and ethylene production rates were very low. Following incubation, the rates of ethylene production increased and reached a maximum in 12 h, and subsequently declined. The rise of ethylene production was associated with a 20- to 30-fold increase in ACC level.Ethylene seems to be involved in the regulation of the senescence of detached rice leaves. This conclusion was based on the observations that (a) maximum ethylene production preceded chlorophyll degradation, (b) ACC application promoted chlorophyll degradation, (c) inhibitors of ethylene production and ethylene action retarded chlorophyll degradation, and (d) various treatments such as light, cycloheximide, alpha,alpha-dipyridyl, Ni(2+), and cold temperature, which retarded chlorophyll degradation, also inhibited ethylene production.Abscisic acid promoted senescence but significantly decreased ethylene production, whereas benzyladenine retarded senescence but promoted ethylene production. This is interpreted to indicate that abscisic acid treatment increased the tissue sensitivity to ethylene, whereas benzyladenine treatment decreased it.