Freezing of water in red-osier dogwood stems in relation to cold hardiness

Studies of stem water in red-osier dogwood (Cornus stolonifera Michx.) using nuclear magnetic resonance spectroscopy indicated that most freezing occurs at temperatures above −30 C in cold-hardy and tender stems. Hardy and tender stems had about the same amount of unfrozen water at −40 C (0.28 gram of water per gram dry weight). When hardy stems were slowly cooled below −20 C, the temperature below which little additional freezing occurs, they survived direct immersion in liquid N₂ (−196 C). Fully hardy samples not slowly precooled to at least −15 C did not survive direct immersion in liquid N₂. The results support the hypothesis that cooling rate is an unimportant factor in tissue survival at and below temperatures where there is little freezable water.     

Physiological and ultrastructural changes in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> as affected by changed GSH level and Cu excess

We investigated the responses of wild-type Arabidopsis thaliana plants to the excess of Cu under conditions of the changed intracellular level of reduced glutathione (GSH) after application of buthionine sulfoximine (BSO) or exogenous GSH to the nutrient solution. BSO (500 μM) decreased and exogenous GSH (500 μM) increased GSH level, while increasing Cu concentration (from 5 to 50 μM) resulted in a decreased GSH content in the roots, but in its increased content in the shoots. BSO did not affect plant growth in contrast to exogenous GSH and Cu, which significantly reduced plant fresh weight. Both Cu and BSO or GSH induced changes in the root structure and leaf chloroplasts ultrastructure. Cu did not induce phytochelatin accumulation. Application of BSO or exogenous GSH did not significantly affect either the GSH level in the Cu-treated plants (except for 50/50 and 50/500 μM/μM Cu/GSH treatments increasing intracellular GSH content in the roots) or Cu toxicity to plants. These results suggest that GSH is not directly involved in Cu detoxification and tolerance in A. thaliana, yet it influences the proper anatomical structure of plants.     

Role of silicon in enhancing resistance to freezing stress in two contrasting winter wheat cultivars

The main objective of this study was to elucidate the roles of silicon (Si) in enhancing tolerance to freezing stress (?5 °C) in two contrasting wheat (Triticum aestivum L.) cultivars: i.e. cv. Yangmai No. 5, a freezing-susceptible cultivar and cv. Linmai No. 2, a freezing-tolerant cultivar. Shoot dry weight of the freezing-susceptible wheat was significantly lower under freezing stress than in controls, but increased significantly with Si amendment. The freezing treatment did not affect shoot dry weight of the freezing-tolerant cultivar. The leaf water content was considerably decreased by freezing stress in the freezing-susceptible cultivar, but was significantly increased by Si amendment. In contrast, freezing treatment did not significantly reduce leaf water content in the freezing-tolerant cultivar and Si played no role in water retention in this cultivar. The concentrations of H₂O₂ and free proline along with malondialdehyde (MDA) were progressively enhanced by freezing stress in the two wheat cultivars used, but were significantly suppressed by amendment with Si. The major antioxidant enzyme activities and non-enzymatic antioxidants (i.e. glutathione and ascorbic acid) in the leaves of freezing-stressed plants were decreased, but were stimulated significantly by the exogenous Si. The possible mechanisms for Si-enhanced freezing stress may be attributed to the higher antioxidant defense activity and lower lipid peroxidation through water retention in leaf tissues.     

Freezing tolerance of citrus, spinach, and petunia leaf tissue : osmotic adjustment and sensitivity to freeze induced cellular dehydration

Seasonal variations in freezing tolerance, water content, water and osmotic potential, and levels of soluble sugars of leaves of field-grown Valencia orange (Citrus sinensis) trees were studied to determine the ability of citrus trees to cold acclimate under natural conditions. Controlled environmental studies of young potted citrus trees, spinach (Spinacia pleracea), and petunia (Petunia hybrids) were carried out to study the water relations during cold acclimation under less variable conditions. During the coolest weeks of the winter, leaf water content and osmotic potential of field-grown trees decreased about 20 to 25%, while soluble sugars increased by 100%. At the same time, freezing tolerance increased from lethal temperature for 50% (LT₅₀) of −2.8 to −3.8°C. In contrast, citrus leaves cold acclimated at a constant 10°C in growth chambers were freezing tolerant to about −6°C. The calculated freezing induced cellular dehydration at the LT₅₀ remained relatively constant for field-grown leaves throughout the year, but increased for leaves of plants cold acclimated at 10°C in a controlled environment. Spinach leaves cold acclimated at 5°C tolerated increased cellular dehydration compared to nonacclimated leaves. Cold acclimated petunia leaves increased in freezing tolerance by decreasing osmotic potential, but had no capacity to change cellular dehydration sensitivity. The result suggest that two cold acclimation mechanisms are involved in both citrus and spinach leaves and only one in petunia leaves. The common mechanism in all three species tested was a minor increase in tolerance (about −1°C) resulting from low temperature induced osmotic adjustment, and the second in citrus and spinach was a noncolligative mechanism that increased the cellular resistance to freeze hydration.     

SEASONALITY AND THERMAL ACCLIMATION OF REACTIVE OXYGEN METABOLISM IN FUCUS VESICULOSUS (PHAEOPHYCEAE)

The intertidal brown macroalga Fucus vesiculosus L. acclimates its defense against reactive oxygen in response to both (1) growth at different temperatures in laboratory culture and (2) seasonal changes in environmental conditions. Fucus vesiculosus was grown in seawater at 0° C, 20° C, and at 0° C with a 3-h daily emersion at −10° C. Algae grown at low temperature, both with and without freezing, produced less reactive oxygen after severe freezing stress than those grown at 20° C. These differences were correlated with growth temperature-induced changes in activities of superoxide dismutase (SOD), glutathione reductase, and ascorbate peroxidase. The contents of tocopherols increased with increased cultivation temperature, whereas the activity of catalase and the content of glutathione and ascorbate did not change. Growth at 0° C increased the resistance of photosynthesis to freezing and reduced photoinhibition in high light at 5° C; the latter effect was further increased in algae subject to daily freezing. These data suggest that elevated activity of reactive oxygen scavenging enzymes, especially SOD, increases the resistance to photoinhibition, at least at low temperature, as well as being important for freezing tolerance. Seasonal changes in reactive oxygen metabolism showed a similar pattern to those elicited by temperature in laboratory culture. Summer samples had lower activities of most reactive oxygen scavenging enzymes than algae collected in autumn and winter when water temperatures were lower. In contrast to the laboratory experiments, ascorbate content did change and was lower during the winter than summer, whereas the content of glutathione was not influenced by season. Overall, the data not only indicate that temperature plays an important role in the regulation of stress tolerance and reactive oxygen metabolism but also suggest that other factors are also involved.     

Freezing stress response in woody tissues observed using low-temperature scanning electron microscopy and freeze substitution techniques

The objective of the current research was to examine the response of woody plant tissues to freezing stress by using scanning electron microscopy (SEM). Nonsupercooling species red osier dogwood (Cornus stolonifera Michx.), weeping willow (Salix babylonica L.), and corkscrew willow (Salix matsudana Koidz. f. tortuosa Rehd.) survived freezing stress as low as −60°C. Cell collapse of ray parenchyma cells of these species was expected but did not occur. It was concluded that ray parenchyma cells of these species do not fit into either the supercooling or extracellular freezing classifications. Tissues from flowering dogwood (Cornus florida L.), apple (Malus domestica Borkh. cv “Starking III”), red oak (Quercus rubra L.), scarlet oak (Quercus coccinea Muench.), and red ash (Fraxinus pennsylvanica Marsh) were confirmed as supercooling species, and did not survive exposures below −40°C. Ray parenchyma cells of these species did not collapse in response to freezing stress, as was expected. Cell collapse along the margins of voids were observed in bark of all seven species. Voids were the result of extracellular ice crystals formed in the bark during exposure to freezing stress. Tissues prepared by freeze substitution techniques were found to be adequately preserved when compared to those prepared by conventional fixation and low temperature SEM techniques. A freezing protocol for imposing freezing stress at temperatures lower than experienced naturally in the area where the study was conducted was developed that produced responses comparable to those observed in specimens collected in the field during natural freezing events.     

Effect of cadmium and calcium treatments on phytochelatin and glutathione levels in citrus plants

Industry residues, phosphate fertilisers and wastewater as a source of irrigation have considerably increased levels of heavy metals in the soil, mainly cadmium (Cd²⁺). To test the effects of a calcium (Ca²⁺) treatment on Cd²⁺ accumulation and plant tolerance to this heavy metal, plants of two citrus genotypes, Cleopatra mandarin (CM) and Carrizo citrange (CC), were watered with increasing concentrations of Cd²⁺, and phytochelatin (PC) and glutathione (GSH) content were measured. Both genotypes were able to synthesise PCs in response to heavy metal intoxication, although CM seems to be a better Cd²⁺ excluder than CC. However, data indicate that CC plants had a higher capacity for regenerating GSH than CM plants. In this context, the effects of Ca²⁺ treatment on Cd²⁺ accumulation, plant survival and PC, GSH and oxidised glutathione (GSSG) content were assessed. Data indicate that treatment with Ca²⁺ had two positive effects on citrus physiology: it reduced Cd⁺² uptake into roots and also increased GSH content (even in the absence of Cd²⁺). Overall, the data indicate that although Cd²⁺ exclusion is a powerful mechanism to avoid heavy metal build‐up into photosynthetic organs, the capacity to maintain optimum GSH levels to feed PC biosynthesis could also be an important factor in stress tolerance.     

Survival of Cornus sericea L. stem cortical cells following immersion in liquid helium

Abstract Cold-acclimated stems of red-osier dogwood (Cornus sericea L.) were sampled in midwinter and early spring and subjected to the following low temperature treatments: (a)0 →?40 → 0°C; (b) 0 →?40 →? 196 → 0°C; (c) 0 →?40 →?196 →?269 →?196 → 0°C; (d) 0 →?40 →?269 →?196 → 0°C; (e) 0 →?196 → 0°C; (f) 0 →?269 →?196 →0°C. The cortical parenchyma cells of the outer stem layers survived exposure to ?269°C when pre-frozen to ?40°C and either transferred directly to ?269°C or to ?196°C and then to ?269°C (treatments c and d). Acclimated stems transferred to a greenhouse (22°C) 2 weeks prior to the low temperature treatments deacclimated and were not able to survive freezing to ?10°C. Cortical cells of stem samples taken in March, near the time when dogwood naturally deacclimates, survived ?196°C (treatment b), but not ?269°C (treatment cord). Thus, the freezing tolerance of dogwood varies seasonally from near ?10°C to below ?269°C.     

Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders

Although glutathione content in boar spermatozoa has been previously reported, the effect of reduced glutathione (GSH) on semen parameters and the fertilizing ability of boar spermatozoa after cryopreservation has never been evaluated. In this study, GSH content was determined in ejaculated boar spermatozoa before and after cryopreservation. Semen samples were centrifuged and GSH content in the resulting pellet monitored spectrophotometrically. The fertilizing ability of frozen-thawed boar sperm was also tested in vitro by incubating sperm with in vitro matured oocytes obtained from gilts. GSH content in fresh semen was 3.84 +/- 0.21 nM GSH/10(8) sperm. Following semen cryopreservation, there was a 32% decrease in GSH content (P < 0.0001). There were significant differences in sperm GSH content between different boars and after various preservation protocols (P = 0.0102 ). The effect of addition of GSH to the freezing and thawing extenders was also evaluated. Addition of 5 mM GSH to the freezing extender did not have a significant effect on standard semen parameters or sperm fertilizing ability after thawing. In contrast, when GSH was added to the thawing extender, a dose-dependent tendency to increase in sperm fertilizing ability was observed, although no differences were observed in standard semen parameters. In summary, (i) there was a loss in GSH content after cryopreservation of boar semen; (ii) addition of GSH to the freezing extender did not result in any improvement in either standard semen parameters or sperm fertilizing ability; and (iii) addition of GSH to the thawing extender resulted in a significant increase in sperm fertilizing ability. Nevertheless, future studies must conclude if this is the case for all boars. Furthermore, since addition of GSH to the thawing extender did not result in an improvement in standard semen parameters, this suggests that during the thawing process, GSH prevents damage of a sperm property that is critical in the fertilization process but that is not measured in the routine semen analysis.     

Seasonal changes in cold tolerance,water relations and accumulation of cations and compatible solutes in Atriplex halimus L.

We determined the cold (freezing) tolerance for field-grown plants of Atriplex halimus L. (Chenopodiaceae) in relation to plant ploidy level, leaf water relations and accumulation of osmolytes. Plants were grown at two sites in Murcia (Spain), having average minimum temperatures in the coldest month of 0.6 and 12.1 °C, respectively. LT₅₀ values derived from laboratory freezing tests, using leaves taken from the plants in early winter and in spring, showed greater tolerance for winter-harvested leaves; the acclimation was more pronounced at the cold-winter site. Cold tolerance was related positively with leaf K and/or Na accumulation. Analysis of compatible organic solutes (soluble sugars, total amino acids and quaternary ammonium compounds) showed that cold tolerance (measured both as LT₅₀ and as winter freezing damage in situ) was related most closely with leaf concentrations of soluble sugars. The leaf percentage dry matter content was related to both in vitro and in vivo tolerance, while tolerance in vitro was correlated also with the osmotic (potential ψ_s) and the relative water content. The two diploid (2n = 2x = 18) populations, from Spain, showed greater cold tolerance than the three tetraploid (2n = 4x = 36) populations, from North Africa and Syria, which may be related to the latter's greater cell size and consequent dilution of osmolytes. In this halophytic species, cold tolerance, like salinity and drought tolerance, seems to depend on osmotic adjustment, driven by vacuolar accumulation of K and Na and cytoplasmic accumulation of compatible solutes.     

Seasonal changes in cold tolerance,water relations and accumulation of cations and compatible solutes in Atriplex halimus L.

We determined the cold (freezing) tolerance for field-grown plants of Atriplex halimus L. (Chenopodiaceae) in relation to plant ploidy level, leaf water relations and accumulation of osmolytes. Plants were grown at two sites in Murcia (Spain), having average minimum temperatures in the coldest month of 0.6 and 12.1 °C, respectively. LT₅₀ values derived from laboratory freezing tests, using leaves taken from the plants in early winter and in spring, showed greater tolerance for winter-harvested leaves; the acclimation was more pronounced at the cold-winter site. Cold tolerance was related positively with leaf K and/or Na accumulation. Analysis of compatible organic solutes (soluble sugars, total amino acids and quaternary ammonium compounds) showed that cold tolerance (measured both as LT₅₀ and as winter freezing damage in situ) was related most closely with leaf concentrations of soluble sugars. The leaf percentage dry matter content was related to both in vitro and in vivo tolerance, while tolerance in vitro was correlated also with the osmotic (potential ψ_s) and the relative water content. The two diploid (2n = 2x = 18) populations, from Spain, showed greater cold tolerance than the three tetraploid (2n = 4x = 36) populations, from North Africa and Syria, which may be related to the latter's greater cell size and consequent dilution of osmolytes. In this halophytic species, cold tolerance, like salinity and drought tolerance, seems to depend on osmotic adjustment, driven by vacuolar accumulation of K and Na and cytoplasmic accumulation of compatible solutes.     

Glutathione metabolism in Urtica dioica in response to cadmium based oxidative stress

To investigate the antioxidative response of glutathione metabolism in Urtica dioica L. to a cadmium induced oxidative stress, activities of glutathione reductase (GR), glutathione-S-transferase (GST), and glutathione peroxidase (GSH-Px), content of reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation (LPO), and also accumulation of Fe, Zn, Mn, Cu besides Cd were determined in the roots, stems, and leaves of plants exposed to 0 (control), 0.045, and 0.09 mM CdCl₂ for 58 h. Whereas the Cd content continuously increased in all organs, the Fe, Zn, Mn, and Cu content decreased in dependence on the applied Cd concentration and incubation time. The Cd treatment resulted in increased GR and GST activities in all organs, however, GSH-Px activity was dependent on Cd concentration and plant organ. The GSH/GSSG ratio maintained above the control level in the stems at both Cd concentrations. The LPO was generally close to the control values in the roots and stems but it increased in the leaves especially at 0.09 mM Cd.     

Freezing avoidance by supercooling in Olea europaea cultivars: the role of apoplastic water,solute content and cell wall rigidity

Plants can avoid freezing damage by preventing extracellular ice formation below the equilibrium freezing temperature (supercooling). We used Olea europaea cultivars to assess which traits contribute to avoid ice nucleation at sub‐zero temperatures. Seasonal leaf water relations, non‐structural carbohydrates, nitrogen and tissue damage and ice nucleation temperatures in different plant parts were determined in five cultivars growing in the Patagonian cold desert. Ice seeding in roots occurred at higher temperatures than in stems and leaves. Leaves of cold acclimated cultivars supercooled down to ?13 °C, substantially lower than the minimum air temperatures observed in the study site. During winter, leaf ice nucleation and leaf freezing damage (LT₅₀) occurred at similar temperatures, typical of plant tissues that supercool. Higher leaf density and cell wall rigidity were observed during winter, consistent with a substantial acclimation to sub‐zero temperatures. Larger supercooling capacity and lower LT₅₀ were observed in cold‐acclimated cultivars with higher osmotically active solute content, higher tissue elastic adjustments and lower apoplastic water. Irreversible leaf damage was only observed in laboratory experiments at very low temperatures, but not in the field. A comparative analysis of closely related plants avoids phylogenetic independence bias in a comparative study of adaptations to survive low temperatures.     

Calcium-Dependent Freezing Tolerance in Arabidopsis Involves Membrane Resealing via Synaptotagmin SYT1

Plant freezing tolerance involves the prevention of lethal freeze-induced damage to the plasma membrane. We hypothesized that plant freezing tolerance involves membrane resealing, which, in animal cells, is accomplished by calcium-dependent exocytosis following mechanical disruption of the plasma membrane. In Arabidopsis thaliana protoplasts, extracellular calcium enhanced not only freezing tolerance but also tolerance to electroporation, which typically punctures the plasma membrane. However, calcium did not enhance survival when protoplasts were exposed to osmotic stress that mimicked freeze-induced dehydration. Calcium-dependent freezing tolerance was also detected with leaf sections in which ice crystals intruded into tissues. Interestingly, calcium-dependent freezing tolerance was inhibited by extracellular addition of an antibody against the cytosolic region of SYT1, a homolog of synaptotagmin known to be a calcium sensor that initiates exocytosis. This inhibition indicates that the puncture allowing the antibody to flow into the cytoplasm occurs during freeze/thawing. Thus, we propose that calcium-dependent freezing tolerance results from resealing of the punctured site. Protoplasts or leaf sections isolated from Arabidopsis SYT1-RNA interference (RNAi) plants lost calcium-dependent freezing tolerance, and intact SYT1-RNAi plants had lower freezing tolerance than control plants. Taken together, these findings suggest that calcium-dependent freezing tolerance results from membrane resealing and that this mechanism involves SYT1 function.     

Osmotic Adjustment and the Development of Freezing Resistance in Fragaria virginiana

Cold temperature acclimation in strawberry (Fragaria virginiana) leaves apparently involves the alteration of cellular osmotic properties. Alterations in leaf osmotic potential were closely correlated with alterations in soluble carbohydrate content of the leaf tissue and changing temperatures. Leaf starch content was inversely related to soluble carbohydrate levels, suggesting that starch is a partial source of osmoticum during osmotic adjustment associated with cold temperature stress. Free amino acid changes were more closely linked to senescence and growth processes while changes in ion content suggested a rapid mobilization of solutes at the onset of freezing temperatures. This was supported by changes in whole plant gradients in leaf osmotic potential before and after exposure to freezing temperatures. In terms of freezing resistance and the role of osmotic adjustment in the development of resistance, it was found that of all leaves undergoing osmotic adjustment only the younger leaves survived, suggesting an age-dependent component to freezing resistance in leaves. Freezing resistance appears to involve alterations in several cellular properties that act in concert to confer a hardy state of the tissue. Although osmotic adjustment may be an important component of the final combination of cellular properties, this study indicates that solute accumulation does not function alone to confer freezing resistance.     

Carbon monoxide enhances the chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated glutathione homeostasis

Both carbon monoxide (CO) and nitric oxide (NO) play fundamental roles in plant responses to environmental stress. Glutathione (GSH) homeostasis through the glutathione-ascorbate cycle regulates the cellular redox status and protects the plant from damage due to reactive oxygen species (ROS) or reactive nitrogen species (RNS). Most recalcitrant seeds are sensitive to chilling stress, but the roles of and cross talk among CO, NO, ROS, and GSH in recalcitrant seeds under low temperature are not well understood. Here, we report that the germination of recalcitrant Baccaurea ramiflora seeds shows sensitivity to chilling stress, but application of exogenous CO or NO markedly increased GSH accumulation, enhanced the activities of antioxidant enzymes involved in the glutathione-ascorbate cycle, decreased the content of H(2)O(2) and RNS, and improved the tolerance of seeds to low-temperature stress. Compared to orthodox seeds such as maize, only transient accumulation of CO and NO was induced and only a moderate increase in GSH was shown in the recalcitrant B. ramiflora seeds. Exogenous CO or NO treatment further increased the GSH accumulation and S-nitrosoglutathione reductase (GSNOR) activity in B. ramiflora seeds under chilling stress. In contrast, suppressing CO or NO generation, removing GSH, or blocking GSNOR activity resulted in increases in ROS and RNS and impaired the germination of CO- or NO-induced seeds under chilling stress. Based on these results, we propose that CO acts as a novel regulator to improve the tolerance of recalcitrant seeds to low temperatures through NO-mediated glutathione homeostasis.     

Lack of association between glutathione content and development of thermal tolerance in human fibroblasts

Thermal tolerance is a transient state of heat resistance occurring in cells and tissues after exposure to sublethal heat or certain chemicals. Although the mechanism of such resistance is unknown, it has been recently shown that preceding its development, cellular glutathione (GSH) levels rise. We have used a glutathione synthetase-deficient [GSH(-)] human fibroblast line to study the relationship between glutathione content and thermal tolerance. The GSH(-) cells had approximately 6% as much GSH as normal fibroblasts. Normal and GSH(-) fibroblasts showed similar survival after exposure to 45 degrees C. Exposure of normal fibroblasts to heat (45 degrees C for 15 min) led to a prompt rise in cellular GSH content as well as development of transient thermal tolerance. Similar treatment of GSH(-) fibroblasts produced no change in the very low GSH levels but was associated with a degree of thermal tolerance similar to that of normal cells. Thermal tolerance decayed more rapidly in GSH(-) cells than in normal fibroblasts. We conclude that the development of thermal tolerance in human fibroblasts is independent of GSH content.