Foliar freezing resistance of Australian alpine plants over the growing season

We assessed the freezing resistance of leaves ex situ of 25 Australian alpine plant species. We compared the freezing resistance of forb, graminoid and shrub species from three alpine summits of different altitudes; from a low altitude site just above treeline, to a fully alpine tundra site. Foliar freezing resistance (LT₅₀) in spring varied from ?5.9°C to ?18.7°C and standardized LT₅₀ values within species were significantly related to site altitude. Additionally, when comparing all the species in the study, freezing resistance was significantly related to site; the LT₅₀ of samples from a low‐altitude summit (1696 m) were significantly lower than those of samples from mid‐ (1805 m) and high‐altitude (1860 m) summits. The LT₅₀ of juvenile foliage did not differ significantly from that of adult foliage. Shrubs were highly resistant to freezing. At the highest summit, we examined the course of seasonal freezing resistance from early summer to early autumn across three alpine plant communities that differed in the time of natural snowmelt; from sheltered (snowpatch) to exposed (open heath). No differences in freezing resistance over the growing season were detected for exposed or sheltered communities and there were no consistent trends indicating frost hardening over the growing season. Overall, the common Australian alpine species we investigated appear well adapted to freezing conditions throughout the snow‐free growing season. We have no evidence to suggest that freezing temperatures soon after snowmelt in spring are especially damaging to the alpine plants at these summits.     

Increased spring freezing vulnerability for alpine shrubs under early snowmelt

Alpine dwarf shrub communities are phenologically linked with snowmelt timing, so early spring exposure may increase risk of freezing damage during early development, and consequently reduce seasonal growth. We examined whether environmental factors (duration of snow cover, elevation) influenced size and the vulnerability of shrubs to spring freezing along elevational gradients and snow microhabitats by modelling the past frequency of spring freezing events. We sampled biomass and measured the size of Salix herbacea, Vaccinium myrtillus, Vaccinium uliginosum and Loiseleuria procumbens in late spring. Leaves were exposed to freezing temperatures to determine the temperature at which 50 % of specimens are killed for each species and sampling site. By linking site snowmelt and temperatures to long-term climate measurements, we extrapolated the frequency of spring freezing events at each elevation, snow microhabitat and per species over 37 years. Snowmelt timing was significantly driven by microhabitat effects, but was independent of elevation. Shrub growth was neither enhanced nor reduced by earlier snowmelt, but decreased with elevation. Freezing resistance was strongly species dependent, and did not differ along the elevation or snowmelt gradient. Microclimate extrapolation suggested that potentially lethal freezing events (in May and June) occurred for three of the four species examined. Freezing events never occurred on late snow beds, and increased in frequency with earlier snowmelt and higher elevation. Extrapolated freezing events showed a slight, non-significant increase over the 37-year record. We suggest that earlier snowmelt does not enhance growth in four dominant alpine shrubs, but increases the risk of lethal spring freezing exposure for less freezing-resistant species.     

Drought increases the freezing resistance of high-elevation plants of the Central Chilean Andes

Freezing temperatures and summer droughts shape plant life in Mediterranean high-elevation habitats. Thus, the impacts of climate change on plant survival for these species could be quite different to those from mesic mountains. We exposed 12 alpine species to experimental irrigation and warming in the Central Chilean Andes to assess whether irrigation decreases freezing resistance, irrigation influences freezing resistance when plants are exposed to warming, and to assess the relative importance of irrigation and temperature in controlling plant freezing resistance. Freezing resistance was determined as the freezing temperature that produced 50 % photoinactivation [lethal temperature (LT₅₀)] and the freezing point (FP). In seven out of 12 high-Andean species, LT₅₀ of drought-exposed plants was on average 3.5 K lower than that of irrigated plants. In contrast, most species did not show differences in FP. Warming changed the effect of irrigation on LT₅₀. Depending on species, warming was found to have (1) no effect, (2) to increase, or (3) to decrease the irrigation effect on LT₅₀. However, the effect size of irrigation on LT₅₀ was greater than that of warming for almost all species. The effect of irrigation on FP was slightly changed by warming and was sometimes in disagreement with LT₅₀ responses. Our data show that drought increases the freezing resistance of high-Andean plant species as a general plant response. Although freezing resistance increases depended on species-specific traits, our results show that warmer and moister growing seasons due to climate change will seriously threaten plant survival and persistence of these and other alpine species in dry mountains.     

How endangered is sexual reproduction of high-mountain plants by summer frosts? Frost resistance, frequency of frost events and risk assessment

In temperate-zone mountains, summer frosts usually occur during unpredictable cold spells with snow-falls. Earlier studies have shown that vegetative aboveground organs of most high-mountain plants tolerate extracellular ice in the active state. However, little is known about the impact of frost on reproductive development and reproductive success. In common plant species from the European Alps (Cerastium uniflorum, Loiseleuria procumbens, Ranunculus glacialis, Rhododendron ferrugineum, Saxifraga bryoides, S. moschata, S. caesia), differing in growth form, altitudinal distribution and phenology, frost resistance of reproductive and vegetative shoots was assessed in different reproductive stages. Intact plants were exposed to simulated night frosts between ?2 and ?14 °C in temperature-controlled freezers. Nucleation temperatures, freezing damage and subsequent reproductive success (fruit and seed set, seed germination) were determined. During all reproductive stages, reproductive shoots were significantly less frost resistant than vegetative shoots (mean difference for LT₅₀ ?4.2 ± 2.7 K). In most species, reproductive shoots were ice tolerant before bolting and during fruiting (mean LT₅₀ ?7 and ?5.7 °C), but were ice sensitive during bolting and anthesis (mean LT₅₀ around ?4 °C). Only R. glacialis remained ice tolerant during all reproductive stages. Frost injury in reproductive shoots usually led to full fruit loss. Reproductive success of frost-treated but undamaged shoots did not differ significantly from control values. Assessing the frost damage risk on the basis of summer frost frequency and frost resistance shows that, in the alpine zone, low-statured species are rarely endangered as long as they are protected by snow. The situation is different in the subnival and nival zone, where frost-sensitive reproductive shoots may become frost damaged even when covered by snow. Unprotected individuals are at high risk of suffering from frost damage, particularly at higher elevations. It appears that ice tolerance in reproductive structures is an advantage but not an absolute precondition for colonizing high altitudes with frequent frost events.     

No increase in alpine snowbed productivity in response to experimental lengthening of the growing season

Climate change effects on snow cover and thermic regime in alpine tundra might lead to a longer growing season, but could also increase risks to plants from spring frost events. Alpine snowbeds, i.e. alpine tundra from late snowmelt sites, might be particularly susceptible to such climatic changes. Snowbed communities were grown in large monoliths for two consecutive years, under different manipulated snow cover treatments, to test for effects of early (E) and late (L) snowmelt on dominant species growth, plant functional traits, leaf area index (LAI) and aboveground productivity. Spring snow cover was reduced to assess the sensitivity of snowbed alpine species to severe early frost events, and dominant species freezing temperatures were measured. Aboveground biomass, productivity, LAI and dominant species growth did not increase significantly in E compared to L treatments, indicating inability to respond to an extended growing season. Edapho‐climatic conditions could not account for these results, suggesting that developmental constraints are important in controlling snowbed plant growth. Impaired productivity was only detected when harsher and more frequent frost events were experimentally induced by early snowmelt. These conditions exposed plants to spring frosts, reaching temperatures consistent with the estimated freezing points of the dominant species (～?10 °C). We conclude that weak plasticity in phenological response and potential detrimental effects of early frosts explain why alpine tundra from snowbeds is not expected to benefit from increased growing season length.     

Soil biogeochemistry during the early spring in low arctic mesic tundra and the impacts of deepened snow and enhanced nitrogen availability

Air temperature freeze–thaw cycles often occur during the early spring period directly after snowmelt and before budbreak in low arctic tundra. This early spring period may be associated with nitrogen (N) and carbon (C) loss from soils as leachate or as trace gases, due to the detrimental impact of soil freeze–thaw cycles and a developing active layer on soil microorganisms. We measured soil and microbial pools of C and N in early spring during a period of fluctuating air temperature (ranging from ?4 to +10°C) and in midsummer, in low arctic birch hummock tundra. In addition we measured N₂O, CH₄ and CO₂ production in the early spring. All of these biogeochemical variables were also measured in long-term snowfence (deepened snow) and N-addition plots to characterize climate-change related controls on these variables. Microbial and soil solution pools of C and N, and trace gas production varied among the five early spring sample dates, but only marginally and no more than among sample dates in midsummer. N-addition greatly elevated N₂O fluxes, indicating that although denitrifiers were present their activity during early spring was strongly limited by N-availability, but otherwise trace gas production was very low in early spring. The later thaw, warmer winter and colder spring soil temperatures resulting from deepened snow did not significantly alter N pools or rates in early spring. Together, our results indicate strong stability in microbial and soil solution C and N pool sizes in the early spring period just after snowmelt when soil temperatures are close to 0°C (?1.5 to +5°C). A review of annual temperature records from this and other sites suggests that soil freeze–thaw cycles are probably infrequent in mesic tundra in early spring. We suggest that future studies concerned with temperature controls on soil and microbial biogeochemistry should focus not on soil freeze–thaw cycles per se, but on the rapid and often stepped increases in soil temperature that occur under the thawing snowpack.     

Exposure to subfreezing temperature and a freeze-thaw cycle affect freezing tolerance of winter wheat in saturated soil

Winter wheat is sown in the autumn and harvested the following summer, necessitating the ability to survive subfreezing temperatures for several months. Autumn months in wheat-growing regions typically experience significant rainfall and several days or weeks of mild subfreezing temperatures at night, followed by above-freezing temperatures in the day. Hence, the wheat plants usually are first exposed to potentially damaging subfreezing temperatures when they have high moisture content, are growing in very wet soil, and have been exposed to freeze-thaw cycles for a period of time. These conditions are conducive to freezing stresses and plant responses that are different from those that occur under lower moisture conditions without freeze-thaw cycles. This study was conducted to investigate the impact of mild subfreezing temperature and a freeze-thaw cycle on the ability of 22 winter wheat cultivars to tolerate freezing in saturated soil. Seedlings that had been acclimated at +4°C for 5 weeks in saturated soil were frozen to potentially damaging temperatures under three treatment conditions: (1) without any subzero pre-freezing treatment; (2) with a 16-h period at ?3°C prior to freezing to potentially damaging temperatures; and (3) with a freeze-thaw cycle of ?3°C for 24 h followed by +4°C for 24 h, followed by a 16-h period at ?3°C prior to freezing to potentially damaging temperatures. In general, plants that had been exposed to the freeze-thaw cycle survived significantly more frequently than plants frozen under the other two treatments. Plants that had been exposed to 16 h at ?3° (without the freeze-thaw cycle) before freezing to potentially damaging temperatures survived significantly more frequently than plants that were frozen to potentially damaging temperatures without a subzero pre-freezing treatment. These results indicated that cold-acclimated wheat plants actively acclimate to freezing stress while exposed to mild subfreezing temperatures, and further acclimate when allowed to thaw at +4°C for 24 h. The cultivar Norstar had the lowest LT₅₀ (temperature predicted to be lethal to 50% of the plants) of the 22 cultivars when frozen with either of the subzero pre-freezing treatments, but several cultivars had lower LT₅₀ scores than Norstar when frozen without a subzero pre-freezing treatment. We conclude it may be possible to improve winterhardiness of wheat grown in saturated soil by combining the ability to effectively respond to mild subzero pre-freezing temperatures with a greater ability to withstand freezing to damaging temperatures without a subzero pre-freezing exposure.     

Dehydrin, alcohol dehydrogenase, and central metabolite levels are associated with cold tolerance in diploid strawberry (Fragaria spp.)

The use of artificial freezing tests, identification of biomarkers linked to or directly involved in the low-temperature tolerance processes, could prove useful in applied strawberry breeding. This study was conducted to identify genotypes of diploid strawberry that differ in their tolerance to low-temperature stress and to investigate whether a set of candidate proteins and metabolites correlate with the level of tolerance. 17 Fragaria vesca, 2 F. nilgerrensis, 2 F. nubicola, and 1 F. pentaphylla genotypes were evaluated for low-temperature tolerance. Estimates of temperatures where 50 % of the plants survived (LT₅₀) ranged from ?4.7 to ?12.0 °C between the genotypes. Among the F. vesca genotypes, the LT₅₀ varied from ?7.7 °C to ?12.0 °C. Among the most tolerant were three F. vesca ssp. bracteata genotypes (FDP821, NCGR424, and NCGR502), while a F. vesca ssp. californica genotype (FDP817) was the least tolerant (LT₅₀ ?7.7 °C). Alcohol dehydrogenase (ADH), total dehydrin expression, and content of central metabolism constituents were assayed in select plants acclimated at 2 °C. The LT₅₀ estimates and the expression of ADH and total dehydrins were highly correlated (r _adh = ?0.87, r _dehyd = ?0.82). Compounds related to the citric acid cycle were quantified in the leaves during acclimation. While several sugars and acids were significantly correlated to the LT₅₀ estimates early in the acclimation period, only galactinol proved to be a good LT₅₀ predictor after 28 days of acclimation (r _galact = 0.79). It is concluded that ADH, dehydrins, and galactinol show great potential to serve as biomarkers for cold tolerance in diploid strawberry.     

Freezing resistance of high-elevation plant species is not related to their height or growth-form in the Central Chilean Andes

The level and mechanism of resistance to damage from freezing temperatures is related to the ambient temperatures experienced by plants. High-elevation habitats exhibit a thermal gradient in the air–soil profile, exposing ground-level species to more rigorous thermal microhabitats than well aboveground species. Such gradients could explain an inverse relationship between freezing temperature damage (LT₅₀) and plant height, such that leaves of smaller plants tolerate freezing conditions, while leaves of taller plants avoid them. However, microclimatic data have not been included in previous studies evaluating those patterns. On the other hand, a proposed trade-off between plant functional traits and freezing resistance suggest that leaves of growth-forms with higher intensity of growth are less freezing resistant than those with lower growth intensity. In this work, we determined whether the ability to resist freezing temperatures is related to plant height or growth-form in 37 high-elevation species from the Central Chilean Andes. We proposed that (1) plants with contrasting heights are exposed to different thermal conditions during freezing events, (2) the level and the mechanism of freezing resistance are inversely related to plant height, and that (3) the level of freezing resistance varied between plants with low- and high-growth intensities. We found that high-Andean species of different heights are exposed to different thermal conditions depending on the distance from the ground. However, neither level nor mechanisms of freezing resistance were related to plant height. Leaves of both short and tall plants showed similar LT₅₀ and their main freezing resistance mechanism was tolerance. Moreover, leaves of growth-forms with high- and low-growth intensity resisted similar freezing temperatures. Our results are discussed in relation to environmental conditions that characterize the Central Chilean Andes.     

Cold tolerance,water relations and accumulation of osmolytes in <Emphasis Type="Italic">Bituminaria bituminosa</Emphasis>

We determined the cold (freezing) tolerance of five Spanish populations of the perennial shrub Bituminaria bituminosa (L.) C.H. Stirton (Fabaceae), as the temperature at which 50 % of leaf electrolytes are released (LT₅₀) using leaves of field-grown plants, obtained in two winters and one spring. The freezing tolerance was greater in winter and reflected the minimum temperatures at the original sites from which the populations were obtained. Tolerance in vitro was related to osmotic adjustment in the leaves; more negative osmotic potential values and more positive pressure potential values (MPa) were associated with greater tolerance. Tolerance and osmotic potential were not related to leaf cation contents but to leaf amino acids, soluble sugar and proline contents.     

Summer frost resistance and freezing patterns measured in situ in leaves of major alpine plant growth forms in relation to their upper distribution boundary

Summer frost resistance and ice nucleation temperatures for 33 alpine plant species were measured in situ to avoid the shortcomings of laboratory tests. Species were selected to investigate the relationship between plant stature and upper distribution boundary, and frost resistance and freezing patterns. The species tested in situ were on average 1.1 K (± 0.2, SE) frost hardier than in laboratory tests. Frost resistance (LT₅₀) ranged from ?4.5 to ?14.6 °C and appeared insufficient to protect against air temperature minima, corroborating reports of natural frost damage. All species tolerated extracellular ice formation (recorded at ?1.9 ± 0.2 °C; E1). Initial frost damage occurred at average temperatures 4.9 K below E1. In 64% of the species a second exotherm (E2) and frost damage were recorded between ?3.7 and ?9.4 °C. In the highest ranging species E2 was not detectable. Frost resistance increased with increasing upper distribution boundary (0.4 K per 100 m), corresponding well with the altitudinal decrease in air temperature minima. No relationship between plant stature and frost resistance was found. Graminoids were significantly frost hardier than other growth forms. Frost survival at high altitudes will depend not only on altitudinal increase in frost resistance but also on freezing avoidance strategies, snow cover protection and a high recuperation capacity.     

Heat tolerance of early developmental stages of glacier foreland species in the growth chamber and in the field

In glacier forelands, seeds readily germinate, however, a high proportion of seedlings die shortly after their appearance. We hypothesized that besides drought, frost and missing safe sites, heat on the ground surface could be one of the major threats for seedlings. The heat strain in different ground strata was assessed from 2007 to 2010. The heat tolerance (LT₅₀) of eleven alpine species from different successional stages was tested considering imbibed (G1) and germinated seeds (G2) as well as seedlings (G3). Additionally, the heat hardening capacity of seedlings was determined in the field. Across all species, LT₅₀ decreased significantly by 9 K from G1 (55 °C) to G3 (46 °C), similarly in all species of the successional stages. Field-grown seedlings had mostly an increased LT₅₀ (2K). Intraspecifically, LT₅₀ of seedlings varied between 40.6 and 52.5 °C. Along the chronosequence, LT₅₀ in G1 was similar, but was higher in G2 and G3 of early successional species. The highest temperatures occurred at 0–0.5 cm in air (mean/absolute maximum: 42.6/54.1 °C) posing a significant heat injury risk for seedlings when under water shortage transpirational cooling is prevented. Below small stones (0–0.5 cm), maxima were 4 K lower, indicating heat safer microsites. Maxima >30 °C occurred at 32.3, >40 °C at 6.2 %. Interannually, 2010 was the hottest year with heat exceeding LT₅₀ at all microsites (0–0.5 cm). Temperature maxima on sandy surfaces were lower than on microsites with gravel (diameter <5–10 mm). The hot summer of 2010 may be a small foretaste of in future more severe and frequent heat waves. Ground surface temperature maxima at the pioneer stage are already now critical for heat survival and may partly explain the high seedling mortality recognized on recently deglaciated terrain.     

Deepened snow increases late thaw biogeochemical pulses in mesic low arctic tundra

Pulses of plant-available nutrients to the soil solution are expected to occur during the dynamic winter–spring transition in arctic tundra. Our aims were to quantify the magnitude of these potential nutrient pulses, to understand the sensitivity of these pulses to winter conditions, and to characterize and integrate the environmental and biogeochemical dynamics of this period. To test the hypotheses that snow depth, temperature and soil water—and not snow nutrient content—are important controls on winter and spring biogeochemistry, we sampled soil from under ambient and deepened snow every 3 days from late winter to spring, in addition to the snowpack at the start of thaw. Soil and microbial biogeochemical dynamics were divided into distinct phases that correlated with steps in soil temperature and soil water. Soil solution and microbial pools of C, N and P fluctuated with strong peaks and declines throughout the thaw, especially under deepened snow. Snowpack nutrient accumulation was negligible relative to these biogeochemical peaks. All nutrient and microbial peaks declined simultaneously at the end of snowmelt and so this decline was delayed by 15 days under deepened snow. The timing of these nutrient pulses is critical for plant species nutrient availability and landscape nutrient budgets. This detailed and statistically-based characterisation of the winter–spring transition in terms of environmental and biogeochemical variables should provide a useful foundation for future biogeochemical process-based studies of thaw, and indicate that spring thaw and possibly growing season biogeochemical dynamics are sensitive to present and future variability in winter snow depth.     

Impact of snowpack decrease on net nitrogen mineralization and nitrification in forest soil of northern Japan

Winter climate change is an important environmental driver that alters the biogeochemical processes of forest soils. The decrease in snowpack amplifies soil freeze–thaw cycles and decreases the snowmelt water supply to soil. This study examined how snow decrease affects nitrogen (N) mineralization and nitrification in forest soil in northern Japan by conducting an in situ experimental snowpack manipulation experiment and a laboratory incubation of soil with different moisture, temperature and freeze–thaw magnitudes. For the incubation studies, surface mineral soil (0–10 cm) was collected from a cool-temperate natural mixed forest and incubated using the resin core method during the winter. In the field, there were two treatments: 50 and 100 % snow removal and control plots. The increase in the soil freeze–thaw cycle increased net N mineralization and marginally decreased the net nitrification in soil. The dissolved organic carbon (DOC) and DOC/DON ratio in soil increased with the decrease in snowpack especially during the snow melt period. These results suggested that the change in substrate quality by the increase in freeze–thaw cycles caused the significant enhancement of microbial ammonium production in soil. The lower soil moisture and higher gross immobilization of inorganic N by soil microbes may be maintaining the slow net nitrification and low nitrate leaching in freeze–thaw cycles with less snowpack. The results indicate that winter climate change would strongly impact N biogeochemistry through the increase in ammonium availability in soil for plants and microbes, whereas it would be unlikely that nitrate loss from surface soil would be enhanced.     

Reduced early growing season freezing resistance in alpine treeline plants under elevated atmospheric CO2

The frequency of freezing events during the early growing season and the vulnerability to freezing of plants in European high‐altitude environments could increase under future atmospheric and climate change. We tested early growing season freezing sensitivity in 10 species, from four plant functional types (PFTs) spanning three plant growth forms (PGFs), from a long‐term in situ CO₂ enrichment (566 vs. 370 ppm) and 2‐year soil warming (+4 K) experiment at treeline in the Swiss Alps (Stillberg, Davos). By additionally tracking plant phenology, we distinguished indirect phenology‐driven CO₂ and warming effects from direct physiology‐related effects on freezing sensitivity. The freezing damage threshold (lethal temperature 50) under ambient conditions of the 10 treeline species spanned from ?6.7±0.3 °C (Larix decidua) to ?9.9±0.6 °C (Vaccinium gaultherioides). PFT, but not PGF, explained a significant amount of this interspecific variation. Long‐term exposure to elevated CO₂ led to greater freezing sensitivity in multiple species but did not influence phenology, implying that physiological changes caused by CO₂ enrichment were responsible for the effect. The elevated CO₂ effect on freezing resistance was significant in leaves of Larix, Vaccinium myrtillus, and Gentiana punctata and marginally significant in leaves of Homogyne alpina and Avenella flexuosa. No significant CO₂ effect was found in new shoots of Empetrum hermaphroditum or in leaves of Pinus uncinata, Leontodon helveticus, Melampyrum pratense, and V. gaultherioides. Soil warming led to advanced leaf expansion and reduced freezing resistance in V. myrtillus only, whereas Avenella showed greater freezing resistance when exposed to warming. No effect of soil warming was found in any of the other species. Effects of elevated CO₂ and soil warming on freezing sensitivity were not consistent within PFTs or PGFs, suggesting that any future shifts in plant community composition due to increased damage from freezing events will likely occur at the individual species level.     

Efficacy of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against Thaumetopoea pityocampa Shiff. (Lepidoptera: Thaumatopoeidae)

Abstract

In this study, Metarhizium anisopliae TR 106 and Beauveria bassiana TR 217 was tested against fourth instar larvae of Thaumetopoea pityocampa. The LT₅₀ and LT₉₀ of 1?×?10⁶ concentration of M. anisopliae against T. pityocampa were 3.60 and 4.11 for direct application, while these were 2.87 and 3.60?days, respectively in leaves application. The LT₅₀ and LT₉₀ of the 1?×?10⁸ concentration of the same isolate were 2.50 and 2.95?days for direct application, and 2.98 and 3.74?days for leaves application. The LT₅₀ of insect and leaves application for 1?×?10⁶ of B. bassiana were 3.75 and 3.49?days, respectively. The LT₉₀ of same concentration for insect application was 4.48?days, while LT₉₀ for leaves application was 4.63?days. Similarly, LT₅₀ of insect and leaves application for 1?×?10⁸ of B. bassiana were 3.03 and 3.31?days, while LT₉₀ were 3.68 and 4.29?days, respectively. Approximate 100% mycosis was observed in all treatments.     

Cold-induced changes of enzymes,proline, carbohydrates,and chlorophyll in wheat

Quantitative changes in total leaf soluble proteins, proline, carbohydrate content, chlorophyll fluorescence, guaiacol peroxidase (POD) and catalase (CAT) activities were determined in a less cold-hardy (LCH) spring cv. Kohdasht (LT₅₀ = −6°C), a semi cold-hardy (SCH) facultative cv. Azar 2 (LT₅₀ = −15°C), and a cold-hardy (CH) winter cv. Norstar (LT₅₀ = −26°C) of wheat (Triticum aestivum L.) exposed to 4°C for 9 weeks. Seedlings were grown in a controlled growth room for 14 days at 20°C and then transferred to 4°C (experimental day 0) for 63 days (cold treatment); otherwise they were maintained continuously at 20°C (control treatment). The samples were harvested 0, 2, 21, 28, 42, and 63 days after exposure to 4°C. The results showed significant low temperature (LT)-induced accumulation of total soluble proteins, proline, and carbohydrates and elevation in activities of CAT and POD in leaves of SCH and CH winter cultivars rather than in LCH spring cultivar. In contrast, the chlorophyll fluorescence (F _v/F _m) declined during LT treatment irrespective of cultivar. The results suggest that developmental traits such as vernalization requirement of wheat affects on cold-tolerance expression system of plants.     

Freezing cytorrhysis and critical temperature thresholds for photosystem II in the peat moss Sphagnum capillifolium

Leaflets of Sphagnum capillifolium were exposed to temperatures from ?5°C to +60°C under controlled conditions while mounted on a microscope stage. The resultant cytological response to these temperature treatments was successfully monitored using a light and fluorescence microscope. In addition to the observable cytological changes during freezing cytorrhysis and heat exposure on the leaflets, the concomitant critical temperature thresholds for inactivation of photosystem II (PS II) were studied using a micro fibre optic and a chlorophyll fluorometer mounted to the microscope stage. Chlorophyllous cells of S. capillifolium showed extended freezing cytorrhysis immediately after ice nucleation at ?1.1°C in the water in which the leaflets were submersed during the measurement. The occurrence of freezing cytorrhysis, which was visually manifested by cell shrinkage, was highly dynamic and was completed within 2 s. A total reduction of the mean projected diameter of the chloroplast containing area during freezing cytorrhysis from 8.9 to 3.8 μm indicates a cell volume reduction of approximately ?82%. Simultaneous measurement of chlorophyll fluorescence of PS II was possible even through the frozen water in which the leaf samples were submersed. Freezing cytorrhysis was accompanied by a sudden rise of basic chlorophyll fluorescence. The critical freezing temperature threshold of PS II was identical to the ice nucleation temperature (?1.1°C). This is significantly above the temperature threshold at which frost damage to S. capillifolium leaflets occurs (?16.1°C; LT₅₀) which is higher than observed in most higher plants from the European Alps during summer. High temperature thresholds of PS II were 44.5°C which is significantly below the heat tolerance of chlorophyllous cells (49.9°C; LT₅₀). It is demonstrated that light and fluorescence microscopic techniques combined with simultaneous chlorophyll fluorescence measurements may act as a useful tool to study heat, low temperature, and ice-encasement effects on the cellular structure and primary photosynthetic processes of intact 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.