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This report provides a brief review of key findings related to frost resistance in alpine woody plant species, summarizes data on their frost resistance, highlights the importance of freeze avoidance mechanisms, and indicates areas of future research. Freezing temperatures are possible throughout the whole growing period in the alpine life zone. Frost severity, comprised of both intensity and duration, becomes greater with increasing elevation and, there is also a greater probability, that small statured woody plants, may be insulated by snow cover. Several frost survival mechanisms have evolved in woody alpine plants in response to these environmental conditions. Examples of tolerance to extracellular freezing and freeze dehydration, life cycles that allow species to escape frost, and freeze avoidance mechanisms can all be found. Despite their specific adaption to the alpine environment, frost damage can occur in spring, while all alpine woody plants have a low risk of frost damage in winter. Experimental evidence indicates that premature deacclimation in Pinus cembra in the spring, and a limited ability of many species of alpine woody shrubs to rapidly reacclimate when they lose snow cover, resulting in reduced levels of frost resistance in the spring, may be particularly critical under the projected changes in climate. In this review, frost resistance and specific frost survival mechanisms of different organs (leaves, stems, vegetative and reproductive over-wintering buds, flowers, and fruits) and tissues are compared. The seasonal dynamics of frost resistance of leaves of trees, as opposed to woody shrubs, is also discussed. The ability of some tissues and organs to avoid freezing by supercooling, as visualized by high resolution infrared thermography, are also provided. Collectively, the report provides a review of the complex and diverse ways that woody plants survive in the frost dominated environment of the alpine life zone.

Main conclusionsIn contrast to Neltuma species, S. tamarugo exhibited higher stress tolerance, maintaining photosynthetic performance through enhanced gene expression and metabolites. Differentially accumulated metabolites include chlorophyll and carotenoids and accumulation of non-nitrogen osmoprotectants.Plant species have developed different adaptive strategies to live under extreme environmental conditions. Hypothetically, extremophyte species present a unique configuration of physiological functions that prioritize stress-tolerance mechanisms while carefully managing resource allocation for photosynthesis. This could be particularly challenging under a multi-stress environment, where the synthesis of multiple and sequential molecular mechanisms is induced. We explored this hypothesis in three phylogenetically related woody species co-occurring in the Atacama Desert, Strombocarpa tamarugo, Neltuma alba, and Neltuma chilensis, by analyzing their leaf dehydration and freezing tolerance and by characterizing their photosynthetic performance under natural growth conditions. Besides, the transcriptomic profiling, biochemical analyses of leaf pigments, and metabolite analysis by untargeted metabolomics were conducted to study gene expression and metabolomic landscape within this challenging multi-stress environment. S. tamarugo showed a higher photosynthetic capacity and leaf stress tolerance than the other species. In this species, a multifactorial response was observed, which involves high photochemical activity associated with a higher content of chlorophylls and β-carotene. The oxidative damage of the photosynthetic apparatus is probably attenuated by the synthesis of complex antioxidant molecules in the three species, but S. tamarugo showed the highest antioxidant capacity. Comparative transcriptomic and metabolomic analyses among the species showed the differential expression of genes involved in the biosynthetic pathways of key stress-related metabolites. Moreover, the synthesis of non-nitrogen osmoprotectant molecules, such as ciceritol and mannitol in S. tamarugo, would allow the nitrogen allocation to support its high photosynthetic capacity without compromising leaf dehydration tolerance and freezing stress avoidance.

Premise Physiological responses to temperature extremes are considered strong drivers of species’ demographic responses to climate variability. Plants are typically classified as either avoiders or tolerators in their freezing‐resistance mechanism, but a gradient of physiological‐threshold freezing responses may exist among individuals of a species. Moreover, adaptive significance of physiological freezing responses is poorly characterized, particularly under warming conditions that relax selection on cold hardiness. Methods Freezing responses were measured in winter and again for new foliage in spring for 14 populations of Artemisia tridentata collected throughout its range and planted in a warm common garden. The relationships of the freezing responses to survival were evaluated in the warm garden and in two colder gardens. Results Winter and spring freezing resistance were not correlated and appeared to be under differing selection regimes, as evident in correlations with different population climate of origin variables. All populations resisted considerably lower temperatures in winter than in spring, with populations from more continental climates showing narrower freezing safety margins (difference in temperatures at which ice‐nucleation occurs and 50% reduction in chlorophyll fluorescence occurs) in spring. Populations with greater winter freezing resistance had lower survivorship in the warmest garden, while populations with greater spring freezing resistance had lower survivorship in a colder garden. Conclusions These survivorship patterns relative to physiological thresholds suggest excess freezing resistance may incur a survival cost that likely relates to a trade‐off between carbon gain and freezing resistance during critical periods of moisture availability. This cost has implications for seed moved from cooler to warmer environments and for plants growing in warming environments.

High mountain ecosystems are subjected to frequent freeze–thaw events all-year round, consequently plants have developed freezing resistance mechanisms to cope with extreme low temperatures. Additionally, these events have also been correlated with the risk of cavitation so that plants need to adapt their water transport system. Information on freezing resistance and xylem vessel characteristics along elevation gradients is scarce for neotropical high elevation species. In this study we aboard two specific questions: 1. Are there intraspecific differences in freezing resistance between low and high elevation populations of Senecio formosus Kunth? 2. Could an increase in freeze–thaw cycle frequency and lower freezing temperatures at higher elevations determine differences in xylem conduit traits between low and high elevation S. formosus populations? We expect greater freezing resistance and a safer water transport system, mainly shaped by narrower tracheary elements in higher elevation populations compared to lower ones. Freezing resistance (avoidance and tolerance) and tracheary elements were studied in S. formosus at its lower (3100 m) and upper (4200 m) distributional limits in the Venezuelan paramo. Freezing resistance was determined through injury and freezing temperature determinations; whereas xylem conduit characteristics dealt with were: vessel element and tracheid diameters, % conducting area and vessel element density. S. formosus increased freezing resistance and presented narrower vessel element diameters under more extreme thermal conditions (4200 m). Increasing evidence of intraspecific plant trait variations under different environmental gradients will aid to determine the outcome of individual species and their effects on ecosystem functioning under a changing climate.

Thermal analyses of freezing events in hydrated lettuce (Lactuca sativaL.) seeds show a correlation between low temperature exotherms (LTEs) (evidence of ice crystal formation) and seed death. Yet, weather patterns common to the Northern Great Plains of North America regularly create conditions where non-dormant seeds of native plants hydrate with snow melt and are subsequently exposed to -30 °C or colder conditions. To determine if such weather patterns decimate dispersed seeds, we measured the effects of freezing on fully hydrated winterfat (Eurotia lanata(Pursh) Moq.) seeds harvested from the Northern Plains at two USA and one Canadian location. Survival of hydrated seeds to -30 °C at a cooling rate of 2.5 °C h-1was similar to that of seeds not subjected to cooling, even though both a high temperature exotherm (HTE) and an LTE were observed. Although the LTE was not related to winterfat seed survival, freeze-stressed seeds had reduced germination rates and reduced seedling vigour, particularly for the collection with the lightest seeds. The temperature of LTEs was similar among seed collections with a mean of -17.6 °C, but was warmer when the seeds were imbibed at 0 °C compared to 5, 10 or 20 °C. We found a significant correlation between the HTE and LTE temperatures. The difference and the correlation may be due to the higher moisture content of seeds imbibed at 0 °C. After pericarp removal, only one exotherm in the range of the LTE was observed. This was also true for the naked embryo. We conclude that an LTE indicates ice formation in the embryo, but that it does not signal the death of a winterfat seed.

Wintering Sasa senanensis, dwarf bamboo, is known to employ deep supercooling as the mechanism of cold hardiness in most of its tissues from leaves to rhizomes. The breakdown of supercooling in leaf blades has been shown to proceed in a random and scattered manner with a small piece of tissue surrounded by longitudinal and transverse veins serving as the unit of freezing. The unique cold hardiness mechanism of this plant was further characterized using current year leaf blades. Cold hardiness levels (LT20: the lethal temperature at which 20% of the leaf blades are injured) seasonally increased from August (-11°C) to December (-20°C). This coincided with the increases in supercooling capability of the leaf blades as expressed by the initiation temperature of low temperature exotherms (LTE) detected in differential thermal analyses (DTA). When leaf blades were stored at -5°C for 1-14 days, there was no nucleation of the supercooled tissue units either in summer or winter. However, only summer leaf blades suffered significant injury after prolonged supercooling of the tissue units. This may be a novel type of low temperature-induced injury in supercooled state at subfreezing temperatures. When winter leaf blades were maintained at the threshold temperature (-20°C), a longer storage period (1-7 days) increased lethal freezing of the supercooled tissue units. Within a wintering shoot, the second or third leaf blade from the top was most cold hardy and leaf blades at lower positions tended to suffer more injury due to lethal freezing of the supercooled units. LTE were shifted to higher temperatures (2-5°C) after a lethal freeze-thaw cycle. The results demonstrate that the tissue unit compartmentalized with longitudinal and transverse veins serves as the unit of supercooling and temperature- and time-dependent freezing of the units is lethal both in laboratory freeze tests and in the field. To establish such supercooling in the unit, structural ice barriers such as development of sclerenchyma and biochemical mechanisms to increase the stability of supercooling are considered important. These mechanisms are discussed in regard to ecological and physiological significance in winter survival.

The marble notothen, Notothenia rossii , is widely distributed around the waters of sub-Antarctic islands in the Southern Ocean and is exposed to different temperatures that range from −1.5 to 8 °C. This study investigates whether the different environmental conditions experienced by N. rossii at different latitudes in the Southern Ocean affect the levels of its blood serum antifreeze glycoprotein (AFGP). N. rossii specimens were collected from four localities, including the Ob’ Seamount in the Indian Ocean sector, and South Georgia Island, South Shetland Islands and Dallman Bay in the Atlantic Ocean sector. Serum AFGP activity was determined in terms of thermal hysteresis, i.e. the difference between the equilibrium melting and non-equilibrium freezing points (f.p.s.). Among the four populations, the Ob’ Seamount specimen had the lowest serum AFGP activity (0.44 °C) and concentration (4.88 mg/mL), and the highest non-equilibrium f.p. (−1.39 °C). These results are consistent with the warmer, ice-free waters around the Ob’ Seamount. The other three higher latitude populations have 2–3 times greater serum AFGP activity and concentration, and much lower non-equilibrium f.p.s. In contrast, the physiological profiles of serum AFGP size isoforms revealed that all N. rossii populations, including the Ob’ Seamount specimen, possess an extensive complements of AFGP proteins. Isoform variation was observed, especially in the large size isoforms (AFGPs 1–5), when compared to AFGP of the high Antarctic Dissostichus mawsoni. The lower levels of AFGP and the absence of some of the large isoforms are likely responsible for higher non-equilibrium f.p.s. of the Ob’ seamount specimen.

The effect of freezing temperatures on stem diameter was measured in the field and in climatic chambers using linear variable differential transformers (LVDT sensors). In acclimated stems, there was reversible stem shrinkage associated with freeze–thaw cycles. The maximum shrinkage correlated with stem diameter (thickness of the bark). The wood was responsible for only 15% of the shrinkage associated with a freeze event, and experiments with isolated bark showed that connection with the wood was not necessary for most of the freeze‐induced shrinkage to occur. Considering the amount of stem shrinkage associated with summer drought in walnut, the amount of contraction of the bark with freezing was actually much less than might be predicted by water relations theory. Reversible stem shrinkage occurred in living tissues, but not in autoclaved tissues. For the latter, swelling was observed with freezing and this swelling could be explained by the bark alone. Similar swelling was observed during September and October for non‐acclimated plants. Water was lost with each freeze–thaw cycle starting with the first, and freezing injury of the bark, with discoloration of tissues, was also observed in non‐acclimated plants. Given that the diameter fluctuation patterns were dramatically different for acclimated versus non‐acclimated plants, and for living versus autoclaved tissues, LVDT sensors could represent a novel, non‐invasive approach to testing cold hardiness.

Arabidopsis thaliana (L.) Heynh. has been described as a freezing-tolerant species based on freezing-resistance assays. Nonetheless, this type of experiment does not discriminate between freezing-tolerance and freezing-avoidance mechanisms. The purpose of this paper was to determine which of these two freezing-resistance mechanisms is responsible for freezing resistance in A. thaliana. This was achieved by comparing the thermal properties (ice-nucleation temperature and the freezing temperature) of leaves and the lethal temperature to 10, 50 and 90% of the plants (LT₁₀, LT₅₀, and LT₉₀, respectively). Two wild-type genotypes were used (Columbia and Ler) and their mutants (esk-1 and frs-1, respectively), which differ in their freezing resistance. This study's results indicated that the mutant esk-1, described as a freezing-tolerant species showed freezing tolerance only after a cold-acclimation period. The mutant frs-1, described as freezing sensitive, presented freezing avoidance. Both wild genotypes presented LT₅₀ similar to or higher than the ice-nucleation temperature. Thus, the main freezing-resistance mechanism for A. thaliana is avoidance of freezing by supercooling. No injury of the photosynthetic apparatus was shown by measuring the maximal photochemical efficiency (Fv/Fm) and pigments (chlorophyll and carotenoid) during cold acclimation in all genotypes. During cold acclimation, Columbia and esk-1 increased total soluble carbohydrates in leaves. esk-1 was the only genotype that presented freezing tolerance after cold acclimation. This feature could be related to an increase in sugar accumulation in the apoplast.

After a field survey of 1985 freeze damage in central Italy, a cross of a high quality cv. Ascolana tenera, with a resistant pollinator, 'Lecdno', was evaluated. Phenolic substance release in the ortet was correlated with resistance and was found variable to a degree that renders questionable its use in screening. Only an arbitrary regressive analysis was able to estimate the increase of resistance in the cross population. On twig samples, treatment with mannitol 15 mM increased freezing avoidance and calcium chelation completely removed this effect. Possibly calcium chelation and return sap composition are found euristically as determinants of freezing avoidance. Cultural practices might be relevant variables in context