Ice Management and Freeze Dehydration of Plant Cells

Plants, in contrast to many other organisms, cannot run away but need to master the full impact of environmental stresses where they are rooted. Hence, they have excellently learned to cope with abiotic stresses. Still, plant recruitment, survival, productivity and geographic distribution can be significantly filtered by environmental stressors. Besides water availability the occurrence, frequency and severity of frost events is the major factor in this respect. However, there remain substantial gaps in our understanding about low temperature injury and frost survival of plant cells, particularly about the subcellular changes that occur during freezing of plant cells. One of the apparent changes during freezing is the transformation of liquid water to ice. While ice susceptible plant cells get immediately killed upon ice formation, ice tolerant plant cells readily survive exposure to extracellular ice masses in their tissue. Extracellular ice withdraws cellular water which is a temperature dependent process. Freeze dehydration is, additionally, influenced by cell wall rigidity, a factor which is less well understood. Upon extracellular ice formation ice tolerant plant cells either tolerate freeze dehydration that in extreme causes non-lethal freezing cythorrhysis (cells collapse and loose reversibly approximately 80% of cellular water) or survive by supercooling. In supercooled cells water remains liquid inside the cells but below a certain temperature threshold cells freeze intracellularly. The necessary subcellular changes allowing a cell to freeze dehydrate or supercool are not known. By employment of improved high-resolution biophysical and cell biological techniques subcellular changes involved in the extent, velocity and dynamic of freeze dehydration and in supercooling but also in frost damage shall be studied in 14 plant species possessing diverse cell wall properties. Chemical components and structural features of cell walls will be investigated as attributes of additionally measured elastic properties to relate them to the peculiar species-specific cryo-behaviour. Results will have implications for applications related to cryo- and lyo-preservation and may provide new strategies to bioengineer increased freezing tolerance in commercially important plants. In an eco- physiological perspective the cryo-behaviour of cells shall be related to the frost demand (frequency and severity) at natural growing sites. Results on cryo-behaviour of plant cells are particularly timely as climate change will sensitively affect frost survival of plants. At first sight this may sound counter intuitive. However, warmer winters affect plant phenology exposing them unprepared to erratic weather patterns. Devastating spring frost damages in the US (2007) may be a foretaste. Results on cryo-behaviour of plant cells will yield a solid basis for the understanding of structural, cytological and physiological mechanisms allowing plant cells to escape frost damage and eventually to survive freezing temperatures in a future climate.

Unlike many other organisms, plants can survive ice formation in their tissues. How this is possible, is still poorly understood. There is general agreement that only extracellular ice formation can be survived, intracellular ice formation kills the cells immediately. For the first time, the unambiguous distinction between ice and liquid water in frozen leaves became possible with the development of a cryo-microscope using reflected polarized light. The study focused on structurally different photosynthetically active cells, found in algae to trees. Cell wall stiffness turned out to be a key for freeze dehydration. In conifer needles, stiff cell walls produce negative tension and prevent freeze dehydration. When frozen, ice is confined to the vascular cylinder. Ice-free photosynthetic tissue enables free gas exchange. At treeline, where frosts last for more than 6 months, this seems indispensable. In potato leaves, cells tolerate ice under moderate freezing (>-3C) and for a limited duration (10-80 min). The cells stay supercooled and are killed by intracellular ice formation. This has implications for potato breeding the strategy must be frost avoidance. While supercooling potato cells get killed by intracellular ice formation, this could not be observed in conifer mesophyll cells, for which the cause of frost damage remains an enigma. Opposed to supercooling cells, the majority of 14 tested species showed freeze dehydration and cytorrhysis upon ice formation. Besides cell wall properties, cell dimensions, relative area of intercellulars and squared thickness-to-span- ratio were important factors. The nival plant Ranunculus glacialis is an example where cells are cytorrhysed upon freezing. Huge ice masses form in the intercellular spaces, exclusively in the spongy parenchyma. The cell walls are rich in pectin and triglycerides occur, particularly in the spongy parenchyma. The role of triglycerides for extracellular ice mass growth warrants further investigation. Additionally, a new technical approach was developed to fix frozen leaves for electron microscopy. This gave unprecedented insights. Conspicuous ultrastructural changes in extracellularly frozen cells ranged from aggregation and fusion of mitochondria into three-dimensional networks to signs of degradation and appearance of autophagic structures. It is believed that these changes allow the maintenance of respiration during freezing. Autophagy and degenerative processes may indicate recycling of injured cytoplasmic constituents, which appears important to maintain cellular metabolism during freezing. In a warming climate, also frost survival is a challenge for plants. This may sound counterintuitive at first glance. However, warmer winters affect plant phenology, exposing them unprepared to erratic weather patterns. Recent and repeated devastating spring frost damages to crops are a foretaste. Improving knowledge about cryo-behavior of plant cells, as obtained here, is a prerequisite for understanding cytological and physiological mechanisms that allow escaping frost damage. The findings offer valuable clues for improvement of freezing stress tolerance in crops.