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Nuclear magnetic resonance of water in cold acclimating red osier dogwood stem.

The pulsed and continuous-wave nuclear magnetic resonance of water in cold-acclimating red osier dogwood (Cornus stolonifera Michx) stem showed reduced relaxation times and increased line width. The reduction of relaxation times suggests an over-all restriction in the motional characteristics of the water. The increased line width is not related to a molecular property of the water, but is useful in estimating the initiation of cold acclimation. Biphasic relaxation characteristics may be related to partitioning of the water at the cellular level. The liquid water content of the stem was a weak function of temperature between -25 and -55 C, corresponding to approximately 0.15 gram of water per gram of dry stem. The quantity of unfrozen water at subfreezing temperatures was not strongly dependent on the degree of cold acclimation. It is concluded that the ability of dogwood to survive low temperatures depends on its ability to tolerate diminished quantities of liquid water.

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A Nuclear Magnetic Resonance Study of Water in Cold-acclimating Cereals.

Continuous wave nuclear magnetic resonance (NMR) studies indicated that the line width of the water absorption peak (Deltav(1/2)) from crowns of winter and spring wheat (Triticum aestivum L.) increased during cold acclimation. There was a negative correlation between Deltav(1/2) and crown water content, and both of these parameters were correlated with the lowest survival temperature at which 50% or more of the crowns were not killed by freezing (LT(50)). Regression analyses indicated that Deltav(1/2) and water content account for similar variability in LT(50). Slow dehydration of unacclimated winter wheat crowns by artificial means resulted in similarly correlated changes in water content and Deltav(1/2). Rapid dehydration of unacclimated crowns reduced water content but did not influence Deltav(1/2). The incubation of unacclimated winter wheat crowns in a sucrose medium reduced water content and increased Deltav(1/2). The increase in Deltav(1/2) appears to be dependent in part on a reduction in water content and an increase in solutes.Longitudinal (T(1)) and transverse (T(2)) relaxation times of water protons in cereals at different stages of cold acclimation were measured using pulse NMR methods. The T(1) and T(2) signals each demonstrated the existence of two populations of water, one with a short and one with a long relaxation time. During the first 3 weeks of acclimation, the long T(2) decreased significantly in winter-hardy cereals, and did not change in a spring wheat until the 5th week of hardening. There was no change in the long T(1) until the 3rd week of hardening for the winter cereals and until the 7th week of hardening for the spring wheat. No simple relationship could be established between T(1) or T(2) and cold hardiness. Neither continuous wave or pulsed NMR spectroscopy can be used as a diagnostic tool in predicting the cold hardiness of winter wheats. An increase in Deltav(1/2) or a reduction in relaxation times does not provide evidence for ordering of the bulk of the cell water.

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Acclimation of photosynthesis to low leaf water potentials.

Photosynthesis is reduced at low leaf water potentials (Psi(l)) but repeated water deficits can decrease this reduction, resulting in photosynthetic acclimation. The contribution of the stomata and the chloroplasts to this acclimation is unknown. We evaluated stomatal and chloroplast contributions when soil-grown sunflower (Helianthus annuus L.) plants were subjected to water deficit pretreatments for 2 weeks. The relationship between photosynthesis and Psi(l), determined from gas-exchange and isopiestic thermocouple psychometry, was shifted 3 to 4 bars towards lower Psi(l), in pretreated plants. Leaf diffusive resistance was similarly affected. Chloroplast activity, demonstrated in situ with measurements of quantum yield and the capacity to fix CO(2) at all partial pressures of CO(2), and in vitro by photosystem II activity of isolated organelles, was inhibited at low Psi(l) but less in pretreated plants than in control plants. The magnitude of this inhibition indicated that decreases in chloroplast activity contributed more than closure of stomata both to losses in photosynthesis and to the acclimation of photosynthesis to low Psi(l).

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Chloroplast Phospholipid Molecular Species Alterations during Low Temperature Acclimation in Dunaliella.

The alterations in chloroplast phospholipid acyl chain composition and phospholipid molecular species composition of Dunaliella salina (UTEX 1644) were monitored during acclimation to low temperature. Chlorophyll fluorescence yield, an indicator of chloroplast membrane stability, was used as a physical means of following the acclimation process.Minor alterations in phospholipid acyl chain composition were evident within 36 hours of shifting the cells from 30 to 12 degrees C. Between 36 and 60 hours, pronounced changes in the acyl chain composition of phosphatidylglycerol (PG) were observed. Changes in the acyl chain composition of phosphatidylcholine (PC) did not occur until sometime after 60 hours.Alterations in the phospholipid molecular species during acclimation were also examined. The pattern of change observed in PC molecular species, namely a decrease in species having one saturated chain (16:0) paired with a C(18) acyl chain and a concomitant increase in species having two unsaturated C(18) acyl chains, suggests that molecular species changes augment fatty acid compositional changes as a mean of adapting to low temperature. The molecular species of PG were found to change abruptly between 36 and 60 hours following a shift to low temperature. During this time, a dramatic alteration in the threshold temperature of thermal denaturation of the photosynthetic apparatus, as measured by chlorophyll fluorescence, also occurred. Lipid compositional changes other than those associated with PG were negligible during this time. This strongly suggests that a correlation exists between the molecular species composition of PG and the thermal stability of the photosynthetic membrane.

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Recovery of turgor by wilted, excised cabbage leaves in the absence of water uptake : a new factor in drought acclimation.

Cabbage leaves excised from a fully turgid plant wilt within 20 minutes to 2 hours (depending on plant age) with a loss of about 10% relative water content (RWC). If droughted for 2 to 4 days in a high relative humidity leaf chamber, they may acclimate, recovering their turgor without the absorption of water, in fact at a loss of 15 to 25% RWC. This turgor recovery in the absence of water uptake occurs only if (a) the rate of water loss is slow enough (about 1-5% RWC per day after the first 24 hours drought loss of about 15% RWC), (b) if the leaves are no longer growing actively. Osmotic adjustment accompanies the turgor adjustment, but cannot be the cause in the absence of water uptake. The recovery of turgor by wilted cabbage leaves in the absence of water uptake cannot be explained by (a) transfer of reserve water from apoplast to symplast either from the cell walls or from the vessel lumens by cavitation or (b) metabolic loss of dry matter and gain of water. It can be explained by a contraction of the cell walls around the partially dehydrated protoplasts, until they regain their elastic extensibility. These proposed cell wall changes during drought acclimation are therefore the opposite of those occurring during growth. This hypothesis therefore explains the long recognized inverse relation between growth and acclimation. Two predictions of this hypothesis were tested and substantiated.

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High-temperature sensitivity and its acclimation for photosynthetic electron transport reactions of desert succulents.

Photosynthetic electron transport reactions of succulent plants from hot deserts are able to tolerate extremely high temperatures and to acclimate to seasonal increases in temperature. In this study, we report the influence of relatively long, in vivo, high-temperature treatments on electron transport reactions for two desert succulents, Agave deserti and Opuntia ficus-indica, species which can tolerate 60 degrees C. Whole chain electron transport averaged 3 degrees C more sensitive to a 1-hour high-temperature treatment than did PSII (Photosystem II) which in turn averaged 3 degrees C more sensitive than did PSI. For plants maintained at day/night air temperatures of 30 degrees C/20 degrees C, treatment at 50 degrees C caused these reactions to be inhibited an average of 39% during the first hour, an additional 31% during the next 4 hours, and 100% by 12 hours. Upon shifting the plants from 30 degrees C/20 degrees C to 45 degrees C/35 degrees C, the high temperatures where activity was inhibited 50% increased 3 degrees C to 8 degrees C for the three electron transport reactions, the half-times for acclimation averaging 5 days for A. deserti and 4 days for O. ficus-indica. For the 45 degrees C/35 degrees C plants treated at 60 degrees C for 1 hour, PSI activity was reduced by 54% for A. deserti and 36% for O. ficus-indica. Acclimation leads to a toleration of very high temperatures without substantial disruption of electron transport for these desert succulents, facilitating their survival in hot deserts. Indeed, the electron transport reactions of these species tolerate longer periods at higher temperatures than any other vascular plant so far reported.

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Cell Wall and Extensin mRNA Changes during Cold Acclimation of Pea Seedlings.

During exposure to 2 degrees C, pea (Pisum sativum) seedlings cold acclimated to a killing temperature of -6 degrees C. Associated with this increase in freezing resistance was an increase in the weight of cell walls and changes in wall composition. Arabinosyl content increased by 100%, while other cell wall glycosyl residues and cellulose increased by about 20%. The cell wall hydroxyproline content increased by 80%. Arabinose and hydroxyproline are both major components of the structural cell wall glycoprotein, extensin. The increase in these components indicates that the level of extensin in the cell wall increases during cold acclimation. Northern blot analysis, using the pDC5A1 genomic clone as a probe, revealed a more than three-fold increase in total extensin mRNA during exposure to cold temperature. Specific extensin transcripts of 6.0, 4.5, 3.5, 2.6, 2.3, 1.8, and 1.5 kilobases were identified. Those at 6.0, 2.6, and 1.5 kilobases were especially promoted by low temperature treatment. The rise in extensin during cold acclimation may be regulated, at least in part, at the gene level. The possible structural role of this protein in freezing protection is discussed.

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Acclimation of Photosynthetic Light Reactions during Induction of Inorganic Carbon Accumulation in the Green Alga Chlamydomonas reinhardtii.

Cells of the unicellular green algae Chlamydomonas reinhardtii were grown in high dissolved inorganic carbon (DIC) concentrations (supplied with 50 milliliters per liter CO(2)[g]) and transferred to low DIC concentrations (supplied with </= 100 microliters per liter CO(2)[g]). Immediately after transfer from high to low DIC the emission of photosystem II related chlorophyll a fluorescence was substantially quenched. It is hypothesized that the suddenly induced inorganic carbon limitation of photosynthesis resulted in a phosphorylation of LHCII, leading to the subsequent state 1 to state 2 transition. After 2 hours of low-DIC acclimation, 77 K fluorescence measurements revealed an increase in the fluorescence emitted from photosystem I, due to direct excitation, suggesting a change in photosystem II/photosystem I stoichiometry or an increased light harvesting capacity of photosystem I. After 5 to 6 hours of acclimation a considerable increase in spillover from photosystem II to photosystem I was observed. These adjustments of the photosynthetic light reactions reached steady-state after about 12 hours of low DIC treatment. The quencher of fluorescence could be removed by 5 minutes of dark treatment followed by 5 minutes of weak light treatment, of any of four different light qualities. It is hypothesized that this restoration of fluorescence was due to a state 2 to state 1 transition in low-DIC acclimated cells. A decreased ratio of violaxanthin to zeaxanthin was also observed in 12 hour low DIC treated cells, compared with high DIC grown cells. This ratio was not coupled to the level of fluorescence quenching. The role of different processes during the induction of a DIC accumulating mechanism is discussed.

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Changes in Osmotic Pressure and Mucilage during Low-Temperature Acclimation of Opuntia ficus-indica.

Opuntia ficus-indica, a Crassulacean acid metabolism plant cultivated for its fruits and cladodes, was used to examine chemical and physiological events accompanying low-temperature acclimation. Changes in osmotic pressure, water content, low molecular weight solutes, and extracellular mucilage were monitored in the photosynthetic chlorenchyma and the water-storage parenchyma when plants maintained at day/night air temperatures of 30/20 degrees C were shifted to 10/0 degrees C. An increase in osmotic pressure of 0.13 megapascal occurred after 13 days at 10/0 degrees C. Synthesis of glucose, fructose, and glycerol accounted for most of the observed increase in osmotic pressure during the low-temperature acclimation. Extracellular mucilage and the relative apoplastic water content increased by 24 and 10%, respectively, during exposure to low temperatures. These increases apparently favor the extracellular nucleation of ice closer to the equilibrium freezing temperature for plants at 10/0 degrees C, which could make the cellular dehydration more gradual and less damaging. Nuclear magnetic resonance studies helped elucidate the cellular processes during ice formation, such as those revealed by changes in the relaxation times of two water fractions in the chlorenchyma. The latter results suggested a restricted mobility of intracellular water and an increased mobility of extracellular water for plants at 10/0 degrees C compared with those at 30/20 degrees C. Increased mobility of extracellular water could facilitate extracellular ice growth and thus delay the potentially lethal intracellular freezing during low-temperature acclimation.

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Group 3 LEA Gene HVA1 Regulation by Cold Acclimation and Deacclimation in Two Barley Cultivars with Varying Freeze Resistance.

The level of expression of the group 3 late embryogenesis abundant abscisic acid-regulated gene (HVA1) to cold treatment has been studied in winter barley (Hordeum vulgare) seedling tissue. The cDNA clone (pHVA1) encoding this late embryogenesis abundant protein was used as a hybridization probe to detect the corresponding mRNA. Expression of the HVA1 gene was determined after the tissue had been subjected to a regimen of 2 degrees C exposure (cold acclimation), followed by a return to 25 degrees C growth conditions (deacclimation). Accumulation of HVA1 mRNA occurred upon cold acclimation of the tissue and disappeared as early as 2 hours after exposure to deacclimation conditions. A comparison of the response to cold acclimation and deacclimation was made between seedling tissue of a freeze-resistant and less freeze-resistant cultivar. In both cultivars, the HVA1 gene was expressed and modulated by cold treatment. Within 2 hours of deacclimation HVA1 mRNA was no longer detectable in either cultivar independently of freeze resistance. The level of expression of HVA1 appeared to be greater in the less freeze-resistant cultivar (Winter Malt).

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Cloning, characterization, and expression of a cDNA encoding a 50-kilodalton protein specifically induced by cold acclimation in wheat.

We have isolated, sequenced, and expressed a cold-specific cDNA clone, Wcs120, that specifically hybridizes to a major mRNA species of approximately 1650 nucleotides from cold-acclimated wheat (Triticum aestivum L.). The accumulation of this mRNA was induced in less than 24 hours of cold treatment, and remained at a high steady-state level during the entire period of cold acclimation in the two freezing-tolerant genotypes of wheat tested. The expression of Wcs120 was transient in a less-tolerant genotype even though the genomic organization of the Wcs120 and the relative copy number were the same in the three genotypes. The mRNA level decreased rapidly during deacclimation and was not induced by heat shock, drought, or abscisic acid. The Wcs120 cDNA contains a long open reading frame encoding a protein of 390 amino acids. The encoded protein is boiling stable, highly hydrophilic, and has a compositional bias for glycine (26.7%), threonine (16.7%), and histidine (10.8%), although cysteine, phenylalanine, and tryptophan were absent. The WCS120 protein contains two repeated domains. Domain A has the consensus amino acid sequence GEKKGVMENIKEKLPGGHGDHQQ, which is repeated 6 times, whereas domain B has the sequence TGGTYGQQGHTGTT, which is repeated 11 times. The two domains were also found in barley dehydrins and rice abscisic acid-induced protein families. The expression of this cDNA in Escherichia coli, using the T(7) RNA polymerase promoter, produced a protein of 50 kilodaltons with an isoelectric point of 7.3, and this product comigrated with a major protein synthesized in vivo and in vitro during cold acclimation.

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DNA microarray analysis of cyanobacterial gene expression during acclimation to high light.

DNA microarrays bearing nearly all of the genes of the unicellular cyanobacterium Synechocystis sp PCC 6803 were used to examine the temporal program of gene expression during acclimation from low to high light intensity. A complete pattern is provided of gene expression during acclimation of a photosynthetic organism to changing light intensity. More than 160 responsive genes were identified and classified into distinct sets. Genes involved in light absorption and photochemical reactions were downregulated within 15 min of exposure to high light intensity, whereas those associated with CO(2) fixation and protection from photoinhibition were upregulated. Changes in the expression of genes involved in replication, transcription, and translation, which were induced to support cellular proliferation, occurred later. Several unidentified open reading frames were induced or repressed. The possible involvement of these genes in the acclimation to high light conditions is discussed.

Adaptation, Physiological↗

Heterogeneity of plant mitochondrial responses underpinning respiratory acclimation to the cold in Arabidopsis thaliana leaves.

In this study, we investigated whether changes in mitochondrial abundance, ultrastructure and activity are involved in the respiratory cold acclimation response in leaves of the cold-hardy plant Arabidopsis thaliana. Confocal microscopy [using plants with green fluorescence protein (GFP) targeted to the mitochondria] and transmission electron microscopy (TEM) were used to visualize changes in mitochondrial morphology, abundance and ultrastructure. Measurements of respiratory flux in isolated mitochondria and intact leaf tissue were also made. Warm-grown (WG, 25/ 20 degrees C day/night), 3-week cold-treated (CT) and cold-developed (CD) leaves were sampled. Although CT leaves exhibited some evidence of acclimation (as evidenced by higher rates of respiration at moderate measurement temperatures), it was only the CD leaves that were able to re-establish respiratory flux within the cold. Associated with the recovery of respiratory flux in the CD leaves were: (1) an increase in the total volume of mitochondria per unit volume of tissue in epidermal cells; (2) an increase in the ratio of cristae to matrix within mesophyll cell mitochondria; and (3) an increase in the capacity of the energy-producing cytochrome pathway in mitochondria isolated from whole leaf homogenates. Regardless of growth temperature, we found that contrasting cell types exhibited distinct differences in mitochondrial ultrastructure, morphology and abundance. Collectively, our data demonstrated the diversity and tissue-specific nature of mitochondrial responses that underpin respiratory acclimation to the cold, and revealed the heterogeneity of mitochondrial structure and abundance that exists within leaves.

Adaptation, Physiological↗

High temperature acclimation of C4 photosynthesis is linked to changes in photosynthetic biochemistry.

With average global temperatures predicted to increase over the next century, it is important to understand the extent and mechanisms of C4 photosynthetic acclimation to modest increases in growth temperature. To this end, we compared the photosynthetic responses of two C4 grasses (Panicum coloratum and Cenchrus ciliaris) and one C4 dicot (Flaveria bidentis) to growth at moderate (25/20 degrees C, day/night) or high (35/30 degrees C, day/night) temperatures. In all three C4 species, CO2 assimilation rates (A) underwent significant thermal acclimation, such that when compared at growth temperatures, A increased less than what would be expected given the strong response of A to short-term changes in leaf temperature. Thermal photosynthetic acclimation was further manifested by an increase in the temperature optima of A, and a decrease in leaf nitrogen content and leaf mass per area in the high- relative to the moderate-temperature-grown plants. Reduced photosynthetic capacity at the higher growth temperature was underpinned by selective changes in photosynthetic components. Plants grown at the higher temperature had lower amounts of ribulose-1,5-bisphosphate carboxylase/oxygenase and cytochrome f and activity of carbonic anhydrase. The activities of photosystem II (PSII) and phosphoenolpyruvate carboxylase were not affected by growth temperature. Chlorophyll fluorescence measurements of F. bidentis showed a corresponding decrease in the quantum yield of PSII (phi(PSII)) and an increase in non-photochemical quenching (phi(NPQ)). It is concluded that through these biochemical changes, C4 plants maintain the balance between the various photosynthetic components at each growth temperature, despite the differing temperature dependence of each process. As such, at higher temperatures photosynthetic nitrogen use efficiency increases more than A. Our results suggest C4 plants will show only modest changes in photosynthetic rates in response to changes in growth temperature, such as those expected within or between seasons, or the warming anticipated as a result of global climate change.

Adaptation, Physiological↗

Cold acclimation increases physiological responsiveness to intraventricular injection of beta-endorphin in pentobarbital anaesthetized rats. II. Metabolic function.

beta-endorphin administered intraventricularly into pentobarbital anaesthetized rats led to a reduction in rectal temperature and a dose dependent biphasic response in metabolic rate with increase at low doses (7-10 nmol/kg) and decrease at high doses (30-300 nmol/kg) in rats acclimated to 22 degrees. These metabolic responses to beta-endorphin were enhanced both in magnitude and in duration in rats acclimated to 5 degrees for 3 weeks, indicating an increased responsiveness to beta-endorphin. Naloxone antagonized the metabolic effects of beta-endorphin in rats acclimated to both 22 degrees and 5 degrees, indicating the metabolic effects of beta-endorphin were via the naloxone sensitive opiate receptors.

Acclimatization↗

Circulatory changes in cold-acclimation and cold stress.

1. The cardiovascular changes of conscious cold-acclimated (CA) and warm-acclimated (WA) rats during exposure to 5 degrees C or 28 degrees C were studied. 2. The cardiac output, heart rate and stroke volume of Ca rats exposed to 5 degrees C and of WA rats during cold stress were significantly greater, and their calculated total peripheral resistance significantly less than those of WA rats ats exposed to 28 degrees C. These results show that circulatory changes participate in cold acclimation and cold stress. The circulatory changes in the two conditions were compared and the mechanism of the observed differences were discussed. 3. CA rats exposed to 28 degrees C showed a striking decrease of oxygen consumption and arterio-venous O2 difference, but significant circulatory changes were decreased heart rate and cardiac index only, indicating that the response was mainly metabolic.

Acclimatization↗

Effects of noradrenaline and carbachol on temperature regulation of cold-stressed and cold-acclimated rats.

1 Noradrenaline (20 micrograms) and carbachol (1 microgram) injected into the anterior hypothalamus of rats at an ambient temperature of 23 degrees C evoked significant falls in core temperature and increases in tail temperature. 2 When rats were cold-stressed (4 degrees C for 90 min) or cold-acclimated (4 degrees C for 4 weeks) and the above amine injections repeated, only carbachol evoked significant falls in core temperature and neither amine increased tail temperature. 3 Central injections of noradrenaline and carbachol also evoked increases in plasma glucose concentrations but not plasma non-esterified fatty acid (NEFA) concentrations in control, acutely cold-stressed and cold-acclimated rats. 4 Although concentrations of plasma glucose and blood lactate of rats were unaffected by cold exposure to 4 degrees C for 1 to 28 days, glucose oxidation rate of both cold-stressed and cold-acclimated rats was significantly greater than in rats at 23 degrees C. Concentrations of plasma NEFA were increased after 1 to 28 days of cold exposure.

Acclimatization↗

Progressive enhancement in the secretory functions of the digestive system of the rat in the course of cold acclimation.

1. The secretory function of the exocrine pancreas and the stomach have been studied in the course of cold acclimation of rats that had been fed at an ambient temperature of 1 degree C in a climatic room. 2. The secretory responses of pancreatic enzymes evoked by continuous infusion of pancreozymin (PZ, 2-5 mu./kg. hr) and a rapid single injection of PZ (1.7 mu./kg) reached a maximum in the group of rats fed at 1 degree C for 4 weeks, and fell to the control levels after 8 weeks. The increase in the flow of pancreatic juice evoked by single injection of PZ was maximal at 4 weeks and slightly decreased after 8 weeks. 3. The insulin (3-0 i.u./kg) evoked secretion of pancreatic enzymes gradually increased after cold exposure, reached a maximum at 4 weeks and fell to the control levels after 8 weeks. The flow of pancreatic juice after insulin injection was almost the same in every group throughout the course of cold exposure. 4. The ratio of amylase to the total amount of the protein in the pancreatic juice decreased abruptly, in contrast to an increase in the ratio of protease in the process of cold acclimation. The change in the ratio of enzyme activity in the pancreatic juice may reflect parallel changes in enzyme activity in the exocrine pancreas. 5. The gastric secretion in response to insulin and bile secretion in the group fed at 1 degree C for 7 weeks was significantly higher than that in the control group. 6. It was thus concluded that the secretory activities of digestive system were enhanced by prolonged cold exposure and then returned to control level, and that the activites of the pancreatic enzymes were altered in the process of cold acclimation in rats.

Acclimatization↗