Mechanisms for tolerating freeze-thaw stress of two evergreen chaparral species: Rhus ovata and Malosma laurina (Anacardiaceae)

The response to freeze-thaw stress was examined for two co-occurring evergreen species, Malosma laurina and Rhus ovata. Laboratory and field experiments on adults and seedlings were made in the spring and winter in 1996 and again on adults in 2003 and 2004. Laboratory and field results indicated that the stem xylem for adults of M. laurina and R. ovata were similarly susceptible to freezing-induced cavitation (percentage loss of conductivity = 92 ± 2.6% for R. ovata and 90 ± 4.2% for M. laurina at ≤ -6°C). In contrast, leaves of M. laurina were more susceptible to freezing injury than leaves of R. ovata. Among seedlings in the field, leaves of M. laurina exhibited freezing injury at -4°C and total shoot mortality at -7.2°C, whereas co-occurring seedlings of R. ovata were uninjured. Surprisingly, R. ovata tolerates high levels of freezing-induced xylem embolism in the field, an apparently rare condition among evergreen plants. Rhus ovata avoids desiccation when xylem embolism is high by exhibiting low minimum leaf conductance compared to M. laurina. These results suggest a link between minimum leaf conductance and stem hydraulics as a mechanism permitting the persistence of an evergreen leaf habit in freezing environments.     

Cold tolerance and supercooling capacity in overwintering adults of elm leaf beetle Xanthogaleruca luteola (Coleoptera: Chrysomelidae)

Elm leaf beetle, Xanthogaleruca luteola (Muller) is one of the key pests of elm trees all over the world, and survives winter in reproductive diapause in sheltered locations. Seasonal variation of whole body supercooling points (SCPs), LT50 (temperature at which 50% of the test individuals die) and survival rate after exposure to subzero temperatures were determined in field collected adults during October 2008 to May 2009 and October 2009 to May 2010. The SCP of adults decreased significantly from October (median=-13.8°C) to January (median=-20.7°C) in first year, relatively similar results was observed in the second year. The lowest LT50 was observed in overwintering adults collected in January (-16.81°C) in the first year and December (-15.59°C) in the second year. Mortality at -15°C for 24 h was >70% in early autumn in both years whereas it decreased to lower than 45% in early winter, the highest mortality (100%) was observed in adults collected in May in both years. Cold acclimated adults (30 d, 5°C) in November 2008 exhibited significantly higher SCP (-12.21±0.64°C) than nonacclimated adults (-15.57±1.35°C). A 30-d exposure to 5°C caused >20% mortality in November, while <9% mortality was observed in adults collected in December and January 2008. Overwintering adults died upon freezing and the lower lethal temperatures were within the range of SCP, indicating that X. luteola is a freeze intolerant insect.     

Xylem dysfunction caused by water stress and freezing in two species of co-occurring chaparral shrubs

Water transport from the roots to leaves in chaparral shrubs of California occurs through xylem vessels and tracheids. The formation of gas bubbles in xylem can block water transport (gas embolism), leading to shoot dieback. Two environmental factors that cause gas embolism formation in xylem conduits are drought and freezing air temperatures. We compared the differential vulnerabilities of Rhus laurina and Ceanothus megacarpus, co-dominant shrub species in the coastal regions of the Santa Monica Mountains of southern California, to both water stress-induced and freezing-induced embolism of their xylem. Rhus laurina has relatively large xylem vessel diameters, a deep root system, and a large basal burl from which it vigorously resprouts after wildfire or freezing injury. In contrast, Ceanothus megacarpus has small-diameter vessels, a shallow root system, no basal burl and is a non-sprouter after shoot removal by wildfire. We found that R. laurina became 50% embolized at a water stress of –3 MPa and 100% embolized by a freeze–thaw cycle at all hydration levels. In contrast, C. megacarpus became 50% embolized at a water stress of –9 MPa and 100% embolized by freeze–thaw events only at water potentials lower than –3 MPa. Reducing thaw rates from 0·8 °C min^?1 to 0·08 °C min^?1 (the normal thaw rate measured in situ) had no effect on embolism formation in R. laurina but significantly reduced embolism occurrence in well-hydrated C. megacarpus (embolism reduced from 74 to 35%). These results were consistent with the theory of gas bubble formation and dissolution in xylem sap. They also agree with field observations of differential shoot dieback in these two species after a natural freeze–thaw event in the Santa Monica Mountains.     

Ecologically relevant measures of the physiological tolerance of light brown apple moth, Epiphyas postvittana, to high temperature extremes

Invasive ectothermic species are limited in their geographic range expansion primarily by their capacity to withstand temperature extremes. Epiphyas postvittana is a highly polyphagous invasive leafroller that was discovered in California in 2006. To predict its potential range and future response to climate change, high temperature tolerance of this species was determined for all life stages and larval instars. Using the static method to estimate high temperature tolerance with response to probing as an endpoint, the mean time leading to 50% mortality (LT(50)) ranged from 45 to 187h at 32.3°C, 34 to 68h at 36°C, 11 to 21h at 38°C, and 1.2 to 5.6h at 40.4°C. There was no clear pattern in the relative tolerance of the life stages across the range of temperatures tested. For pupae and adults, gender did not influence the LT(50) values at any of the temperatures tested. For the larval instars, LT(50) values increased with increasing larval instar at the highest three temperatures while this trend was reversed for the lowest temperature (32.3°C). An analysis of LT(50) values obtained from acute responses to probing compared to subsequent survival to adult emergence, showed that chronic mortality severely affected all larval instars at three out of the four constant temperatures and resulted in 64-85% reduction in LT(50) values. No difference in acute and chronic mortality was found for exposure of the egg stage to high temperatures. These findings have important implications for predicting thermal limits and range expansions of insect species, since upper thermal tolerance could readily be overestimated from the use of ad hoc rather than ecologically relevant endpoint measurements such as survival to adult emergence.     

Cold tolerance and freeze-induced glucose accumulation in three terrestrial slugs

Cold tolerance and metabolic responses to freezing of three slug species common in Scandinavia (Arion ater, Arion rufus and Arion lusitanicus) are reported. Autumn collected slugs were cold acclimated in the laboratory and subjected to freezing conditions simulating likely winter temperatures in their habitat. Slugs spontaneously froze at about -4 °C when cooled under dry conditions, but freezing of body fluids was readily induced at -1 °C when in contact with external ice crystals. All three species survived freezing for 2 days at -1 °C, and some A. rufus and A. lusitanicus also survived freezing at -2 °C. (1)H NMR spectroscopy revealed that freezing of body fluids resulted in accumulation of lactate, succinate and glucose. Accumulation of lactate and succinate indicates that ATP production occurred via fermentative pathways, which is likely a result of oxygen depletion in frozen tissues. Glucose increased from about 6 to 22 μg/mg dry tissue upon freezing in A. rufus, but less so in A. ater and A. lusitanicus. Glucose may thus act as a cryoprotectant in these slugs, although the concentrations are not as high as reported for other freeze tolerant invertebrates.     

The effect of newt toxin on an invasive snail

Hydrobiologia - Invasive species are well documented to impact native species where they are introduced. In the Santa Monica Mountains, a native species of amphibian, the California newt (Taricha...     

Using Arabidopsis thaliana as a model to study subzero acclimation in small grains

The suitability of using Arabidopsis as a model plant to investigate freezing tolerance was evaluated by observing similarities to winter cereals in tissue damage following controlled freezing and determining the extent to which Arabidopsis undergoes subzero-acclimation. Plants were grown and frozen under controlled conditions and percent survival was evaluated by observing re-growth after freezing. Paraffin embedded sections of plants were triple stained and observed under light microscopy. Histological observations of plants taken 1 week after freezing showed damage analogous to winter cereals in the vascular tissue of roots and leaf axels but no damage to meristematic regions. The LT(50) of non-acclimated Arabidopsis decreased from about -6 degrees C to a minimum of about -13 degrees C after 7 days of cold-acclimation at 3 degrees C. After exposing cold-acclimated plants to -3 degrees C for 3 days (subzero-acclimation) the LT(50) was lowered an additional 3 degrees C. Defining the underlying mechanisms of subzero-acclimation in Arabidopsis may provide an experimental platform to help understand winter hardiness in economically important crop species. However, distinctive histological differences in crown anatomy between Arabidopsis and winter cereals must be taken into account to avoid misleading conclusions on the nature of winter hardiness in winter cereals.     

Cold resistance in Antarctic angiosperms

Deschampsia antarctica Desv. (Poaceae) and Colobanthus quitensis (Kunth) Bartl. (Cariophyllaceae) are the only two vascular plants that have colonized the Maritime Antarctic. The primary purpose of the present work was to determine cold resistance mechanisms in these two Antarctic plants. This was achieved by comparing thermal properties of leaves and the lethal freezing temperature to 50% of the tissue (LT50). The grass D. antarctica was able to tolerate freezing to a lower temperature than C. quitensis. The main freezing resistance mechanism for C. quitensis is supercooling. Thus, the grass is mainly a freezing‐tolerant species, while C. quitensis avoids freezing. D. antarctica cold acclimated; thus, reducing its LT50. C. quitensis showed little cold‐acclimation capacity. Because day length is highly variable in the Antarctic, the effect of day length on freezing tolerance, growth, various soluble carbohydrates, starch, and proline contents in leaves of D. antarctica growing in the laboratory under cold‐acclimation conditions was studied. During the cold‐acclimation treatment, the LT50 was lowered more effectively under long day (21/3 h light/dark) and medium day (16/8) light periods than under a short day period (8/16). The longer the day length treatment, the faster the growth rate for both acclimated and non‐acclimated plants. Similarly, the longer the day treatment during cold acclimation, the higher the sucrose content (up to 7‐fold with respect to non‐acclimated control values). Oligo and polyfructans accumulated significantly during cold acclimation only with the medium day length treatment. Oligofructans accounted for more than 80% of total fructans. The degrees of polymerization were mostly between 3 and 10. C. quitensis under cold acclimation accumulated a similar amount of sucrose than D. antarctica, but no fructans were detected. The suggestion that survival of Antarctic plants in the Antarctic could be at least partially explained by accumulation of these substances is discussed.     

The effect of cold acclimation and deacclimation on cold tolerance,trehalose and free amino acid levels in Sitophilus granarius and Cryptolestes ferrugineus (Coleoptera)

Canadian and French laboratory strains of Sitophilus granarius (L.) and Cryptolestes ferrugineus (Stephens) were cold acclimated by placing adults at 15, 10 and 5 degrees C successively for 2wk at each temperature before deacclimating them for 1wk at 30 degrees C. Unacclimated S. granarius had an LT(50) (lethal time for 50% of the population) of 12days at 0 degrees C compared with 40days after the full cold acclimation. At -10 degrees C, unacclimated C. ferrugineus had an LT(50) of 1.4days compared with 24days after the full acclimation. Cold acclimation was lost within a week after returning insects to 30 degrees C. Trehalose, as well as the amino acids proline, asparagine, glutamic acid and lysine were higher in cold acclimated insects for both species. For S. granarius, glutamine was higher in cold acclimated insects and isoleucine, ethanolamine and phosphoethanolamine, a precursor of phospholipids, were lower in cold acclimated insects. For C. ferrugineus, alanine, aspartic acid, threonine, valine, isoleucine, leucine, phenylalanine and phosphoethanolamine were higher in cold acclimated insects. For both species tyrosine was lower in cold acclimated insects. There were small but significant differences between Canadian and French strains of S. granarius, with the Canadian strain being more cold hardy and having higher levels of trehalose. There were small but significant differences between male and female S. granarius, with males being more cold hardy and having higher levels of proline, asparagine and glutamic acid. In conclusion, high levels of trehalose and proline were correlated with cold tolerance, as seen in several other insects. However, correlation does not prove that these compounds are responsible for cold tolerance, and we outline further tests that could demonstrate a causal relationship between trehalose and proline and cold tolerance.     

The effects of thermal acclimation on immature mortality in the Queensland fruit fly Bactrocera tryoni and the light brown apple moth Epiphyas postvittana at a lethal temperature

Mortality data for non-acclimated and acclimated 3rd instar larvae and mid-term eggs of Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) were obtained after immersing in hot water at 46 °C. Acclimation consisted of holding the larvae and eggs at 35 °C for 20 and 11 h respectively just prior to heat-treatment. The median lethal time (LT50) for acclimated larvae was found to be 6.9 min compared to 2.5 min for non-acclimated larvae. LT99.999 for acclimated larvae was 20.9 min compared to 8.7 min for non-acclimated larvae. LT50 for acclimated eggs was 5.0 min compared to 2.4 min for non-acclimated eggs. LT99.999 for acclimated eggs was 26.0 min compared to 6.6 min for non-acclimated eggs. For 3rd instar larvae, most acclimation effect on mortality had occurred by 8 h. A notable residual response was present 20 h after acclimation had occurred, reducing mortality at 46 °C for 4.5 min by roughly 25%. Mortality data at 46 °C were also obtained for non-acclimated and acclimated late instar larvae of Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae). With this species, LT50 for acclimated larvae was 2.5 min compared to 1.1 min for non-acclimated larvae. LT99.999 for acclimated larvae was 9.5 min compared to 4.6 min for non-acclimated larvae.     

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.     

Seed dispersal and seedling establishment of Rhus trichocarpa promoted by a crow (Corvus macrorhynchos) on a volcano in Japan

The interaction between frugivorous birds and trees producing bird-dispersed seeds in devastated areas has been considered to be weak, owing to the paucity of avifauna and/or food resources for birds. Here, we present evidence that strong interactions between birds and plants may promote the enlargement of tree distribution on harsh environments. The summit of Mount Koma, northern Japan, was denuded by the 1929 volcanic eruption. Vegetation cover gradually decreases from the bottom (secondary forest) to the top (bareground) of the mountain. We recorded 48 bird species in the four seasons of 2001, along a 5-km line transect on the southwestern slope of the mountain. Birds faeces collected along the transect contained seeds of more than 14 plant taxa. Five of the 14 taxa were bird-dispersal tree species ( Rhus trichocarpa , Sorbus commixta , Prunus ssiori , Prunus maximowiczii and Prunus sargentii ) and were established in the summit area. Most faeces were derived from Corvus spp. (mostly C . macrorhynchos ) and Turdus naumanni . In particular, the seeds of R . trichocarpa were found mostly from the faeces of Corvus spp. and the seeds of Gaultheria miqueriana , a shrub species, were only from T . naumanni . Rhus trichocarpa retained fruits on the canopy at all times of the year, and crows could feed on them even when food resources were poor in winter. Rhus trichocarpa seedlings established well near rock at higher elevation, while they occurred mostly under the larch canopy of larches at lower elevation. Crows mostly utilized tree canopies and rocks as perches in respective habitats. Therefore, seedlings should be abundant in specific habitats at different elevations. Size-class distribution of seedlings suggested that seedling mortality was lower at higher elevation where open sites were more abundant. These findings indicate that strong mutual advantages for C . macrorhynchos and R . trichocarpa on denuded areas play an important role on revegetation.     

Ground nesting in the paper wasp <Emphasis Type="Italic">Polistes aurifer</Emphasis> (Hymenoptera,Vespidae)

Summary Polistes paper wasp species vary in their nest site selection, with some nesting in relatively exposed areas and others in protected cavities. Locating the nest sites of cavity dwelling species in natural habitat can prove difficult, and most behavioral studies on these species are therefore conducted using human-built structures. Since Hungerford and Williams (1912) and Rau (1929) noted the location of several P. fuscatus (Fabricius) nests in rodent burrows, there have been no published accounts of ground nesting Polistes species. I report the occurrence of a large, dense cluster of P. aurifer (Saussure) nests located within cracks in the dried soil of the Santa Monica Mountains of southern California, USA.Received 8 April 2003; revised 10 October 2003; accepted 31 October 2003.     

Regulation of proline and ethylene levels in rape seedlings for freezing tolerance

This study was aimed to investigate the possibility of regulating free proline content and ethylene production in the resistant to abiotic stress cv. ‘Hornet H’ and the tolerant to stress cv. ‘Sunday’ of winter rapeseed seedlings by pretreatment with exogenous L-proline and L-glutamine in non-acclimated and cold-acclimated seedlings in relation to freezing tolerance. The ratio of proline content in acclimated (at 4°C) versus non-acclimated (18°C) ‘Hornet H’ seedlings increased 2.12-fold and in ‘Sunday’ seedlings 1.95-fold. Exogenously applied, proline and glutamine produced a positive effect on free proline content in both cold-acclimated and non-acclimated seedlings. At a temperature of -1°C the proline content significantly increased in non-acclimated and especially in cold-acclimated seedlings. At an intensified freezing temperature (?3°C, ?5°C, ?7°C), the proline content decreased in comparison with that at ?1°C, but glutamine, especially proline, in cold-acclimated seedlings takes part in free proline level increase and in seedlings’ resistance to freezing. Ethylene production increased in cold-acclimated conditions and under the effect of exogenous proline and glutamine. In freezing conditions, ethylene production decreased, but in cold-acclimated seedlings and under pretreatment of proline and glutamine the ethylene synthesis was intensive. Thus, free proline content and ethylene production increase in cold-acclimated winter rapeseed seedlings and under pretreatment with glutamine and especially with proline. Free proline is involved in the response to cold stress, and its level may be an indicator of cold-hardening and freezing tolerance, but the role of ethylene in the regulation of cold tolerance remains not quite clear.     

Thermal tolerance of Limnoperna fortunei to gradual temperature increase and its applications for biofouling control in industrial and power plants

The acute upper lethal temperature (AULT) at different rates of increase was evaluated as a tool for the design of cheaper and environmentally friendlier control strategies for the invasive bivalve Limnoperna fortunei. Survivorship of 6 ± 2 mm and 20 ± 2 mm mussels acclimated to 12, 23 and 28 ° C and subjected to different heating rates (1 ° C per 5, 15 and 30 min) was estimated in the laboratory. The temperatures required to kill 50% (LT(50)) and 100% (SM(100)) of the mussels, and the mean death temperature (MDT) varied between 42.2 and 51 ° C over 54 experiments. Heating rates significantly (p < 0.001) affected LT(50), SM(100), and MDT. AULT was not affected by mussel size and acclimation temperatures. Limnoperna appears to be more resistant to high temperatures than Dreissena polymorpha, a mussel invasive in the USA and Europe. Lethal temperatures of L. fortunei are within the current thermal operational industrial capacities, suggesting that heat treatment is a viable alternative for controlling its fouling in utility systems.     

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.     

Influence of soil moisture on the nodulation of post fire seedlings of Ceanothus spp. growing in the Santa Monica Mountains of Southern California

During the dry season (early May through September of 1994), following a fall 1993 wildfire, a survey of seedling nodulation was conducted at several sites in the Santa Monica Mountains of Southern California. Seedlings of Ceanothus spinosus, C. megacarpus, C. oliganthus , and C. cuneatus were manually excavated. During this period, only 12 of the 182 seedlings excavated were nodulated, and all of the nodulated seedlings were found in the relatively moist clay soils of a stream bank. No nodules were observed on the 170 seedlings excavated from the drier sites. An irrigation experiment was established in midsummer to assess whether water stress inhibits nodulation of post-fire Ceanothus seedlings. Four plots with numerous seedlings of C. cuneatus and C. spinosus were irrigated with distilled water and monitored over a 9-week period. There was a significant increase in nodulation frequency, water potential, stomatal conductance, transpiration, shoot elongation, and photosynthetic rate of irrigated seedlings compared with adjacent controls. Although these data support the hypothesis that water stress inhibits nodulation. it is unclear whether this is because of an effect of soil moisture on the nodulation capacity of the soils (i.e. on the size and physiological state of the soil Frankia population) or to a host plant response to drought which might prevent actinorhizal root infection and/or nodule development.     

海洋桡足类的热耐受性

为了探明热排放对近海生态的影响,选用我国东海近海主要桡足类,采用热升温实验方法对其半致死温度进行研究.结果表明,不同生物在相同适温条件下和同种生物在不同适温条件下的热耐受能力均存在差异.自然适应水温为13.5 ℃,中华哲水蚤(Calanus sinicus)和细巧华哲水蚤(Sinocalanus tenellus)的24 h半致死温度值分别为26.9 ℃和25.4 ℃;自然适应水温为14.2 ℃,中华异水蚤(Acartiella sinensis)和近缘大眼剑水蚤(Corycaeus affinis)的24 h半致死温度值分别为26.7 ℃和30.5 ℃;自然适应水温为28.0 ℃,背针胸刺水蚤(Centropages dorsispinatus)、强额拟哲水蚤(Paracalanus crassirostris)、刺尾纺锤水蚤(Acartia spinicauda)和尖额真猛水蚤(Euterpina acutifrons)的24 h半致死温度值分别为34.0 ℃、34.3 ℃、35.7 ℃和36.0 ℃.细巧华哲水蚤在自然适应水温分别为13.5 ℃和23.5 ℃下的24 h半致死温度值为25.4 ℃和33.0 ℃.     

Freezing sensitivity in the sfr4 mutant of Arabidopsis is due to low sugar content and is manifested by loss of osmotic responsiveness

Protoplasts were tested to determine whether the freezing sensitivity of the sfr4 (sensitive to freezing) mutant of Arabidopsis was due to the mutant's deficiency in soluble sugars after cold acclimation. When grown under nonacclimated conditions, sfr4 protoplasts possessed freezing tolerance similar to that of wild type, with the temperature at which 50% of protoplasts are injured (LT(50)) of -4.5 degrees C. In both wild-type and sfr4 protoplasts, expansion-induced lysis was the predominant lesion between -2 degrees C and -4 degrees C, but its incidence was low (approximately 10%); below -5 degrees C, loss of osmotic responsiveness (LOR) was the predominant lesion. After cold acclimation, the LT(50) was decreased to only -5.6 degrees C for sfr4 protoplasts, compared with -9.1 degrees C for wild-type protoplasts. Although expansion-induced lysis was precluded in both types of protoplasts, the sfr4 protoplasts remained susceptible to LOR. After incubation of seedlings in Suc solution in the dark at 2 degrees C, freezing tolerance and the incidence of freeze-induced lesions in sfr4 protoplasts were examined. The freezing tolerance of isolated protoplasts (LT(50) of -9 degrees C) and the incidence of LOR were now similar for wild type and sfr4. These results indicate that the freezing sensitivity of cold-acclimated sfr4 is due to its continued susceptibility to LOR (associated with lyotropic formation of the hexagonal II phase) and associated with the low sugar content of its cells.     

Evergreen alpine shrubs have high freezing resistance in spring,irrespective of snowmelt timing and exposure to frost: an investigation from the Snowy Mountains,Australia

Over winter, alpine plants are protected from low-temperature extremes by a blanket of snow. Climate change predictions indicate an overall reduction in snowpack and an earlier thaw; a situation which could expose the tips of shrubs which extend above the snowpack to freezing events in early spring, and cause foliar frost damage during the onset of physiological activity. We assessed the photosynthetic responses of freezing-damaged shrub leaves from an assay of freezing temperatures in the Snowy Mountains in south-eastern Australia, using chlorophyll fluorometery ex situ. We sampled leaves that were exposed early during the spring thaw and leaves that were buried in snow for up to two extra weeks, from four evergreen shrub species at monthly intervals following the period of snowmelt. Freezing resistance (estimated from LT₅₀) was poorest at the earliest spring sampling time, in both exposed above-snow and protected below-snow foliage in all species. Protected foliage in early spring had lower freezing resistance than exposed foliage, but not significantly so. By the third sampling time, freezing resistance was significantly better in the lower protected foliage (LT₅₀ of ? 14) compared with the upper exposed foliage (LT₅₀ of ? 10) in one species. Over the course of spring, freezing resistance improved significantly in all species, with LT₅₀ values of between ? 10 and ? 15 °C by the third sampling time, which is lower than the minimum air temperatures recorded at that time (> ? 5 °C). The results indicate that the dominant evergreen shrub species in this area may only be susceptible to freezing events very early in spring, before a period of frost-hardening occurs after snowmelt. Later in spring, these alpine shrubs appear frost hardy, thus further perpetuating the positive feedbacks surrounding shrub expansion in alpine areas.