Frost survival at temperatures lower than -38°C

Ecophysiological aspect Freezing temperatures are an important filter on plant recruitment, survival, productivity and geographic distribution and can have dramatic impacts on plants at cellular to ecosystem scales. Survival of ultra-low temperatures (-38C) by plant tissues is - in the current understanding - achieved by tolerance of extracellular ice and consequent freeze dehydration of cells. When during cooling the point is reached where all freeze able water has been extracted to extracellular ice, so called glassed cell solutions are supposed to form. In this state each further temperature drop is not assumed to exert any effect on such cells and some have even been shown to survive dipping in liquid nitrogen (-196C). From this no large inter-specific differences in maximum frost resistance (<-38C) should be expected. However, in the literature a distinct variation in maximum frost resistance between plant species is reported (-40 down to -196C). This discrepancy may be the result of inadequate freezing procedures as in many early investigations control of cooling and thawing was either not possible or very difficult to solve. Innovative aspect For testing of plants at temperatures lower than -38C under adequate temperature control even nowadays, only a few, expensive and not really appropriate commercial freezing systems are available. We, therefore, aim to develop and employ a new freezing equipment that is especially designed to frost test plants in an adequate frost treatment procedure (cooling and thawing at 2C/h) in the desired low temperature range down to -80C. The freezing chambers will be additionally designed to allow monitoring the formation, growth and point of maximum expansion of extracellular ice through an inspection window using an advanced microscopy technology. Climate change aspect Subalpine woody species of the European Alps survive frosts from -35 down to -90C in midwinter. Potential leaf temperature minima of -44C may pose a risk of frost damage during winter for some species. The risk of winter frost damage could dramatically increase in response to climate change for all species as increased temperatures (+4C) have been shown to significantly accelerate premature frost dehardening in Northern Hemisphere woody plants. Employing the new freezing equipment and by simulating in situ "warm spells" during winter with large infrared lamps we aim to assess the degree of this risk for woody subalpine species including timberline trees from the protection forests.

Freezing stress is one of the most important environmental constraints that globally limits the distribution of wild and crop plants and can cause significant yield losses in agriculture. The mechanisms of frost resistance, i.e. how living plant cells survive ice formation within the tissue and extremely low freezing temperatures down to -196C (liquid nitrogen), are only scarcely understood. Some plant tissue have the capability to supercool down to certain freezing temperatures but get then killed by intracellular ice formation, at least around -40C which is the homogenous ice nucleation temperature of water. Intracellular ice formation is in all cases lethal. Extracellular ice formation in intercellular spaces is tolerated by freezing tolerant plant species down to a certain freezing temperature. However, with decreasing freezing temperature the extracellular ice masses act increasingly dehydrating on cells. At a certain low freezing temperature all freeze able water is removed from the plant cell to the external ice masses and a glass-state is formed. Theoretically from this temperature onwards all cells should be able to withstand a further cooling even below - 40C without frost damage. However, the currently available frost resistance data are controversially in this respect as various degrees of frost resistance are reported, even below -40C. One point may be the fact that these frost resistance data were usually collected by simple and partially also divergent measurement procedures. In the present research project a novel field frost resistance testing system (LTFS) was developed that allows frost resistance investigations on plants directly at the natural growing site. Controlled temperature runs can be applied simulating natural frosts down to a freezing temperature of -70C. Four freezing chambers act in parallel. Applying the new method it could be shown that seasonal and species specific differences in frost resistance even at temperatures below -40C do exist. This means that even after complete freeze dehydration when cells are expected to be in the glassed state and below the homogenous ice nucleation point of water currently unknown factors take effect and yield different frost resistances. Tree species from the subalpine timberline such as the cembran pine show a maximum winter frost resistance lower than -70C which exceeds the naturally occurring low temperature minima by far. The results of the field frost experiments further show that some of the classical methods for the determination of frost resistance are inappropriate for the assessment of winter frost resistance. In this respect a rethinking is necessary. None of the subalpine plant species survived dipping into liquid nitrogen (-196C). In contrast this has been shown to be survived by boreal tree species. All species readily responded with frost hardening or dehardening respectively in response to artificial freezing or warming treatments conducted in the field. Out of this readiness an increased frost damage risk potential can be expected in the face of global warming.