1. With respect to the stressor cold, a distinction is made between damage arising at temperatures above the freezing point (damage through chilling in subtropical and tropical plants) and damage occurring upon frost and the subsequent formation of ice in the plant. Ice formation results in freeze-desiccation of tissues and cells, and frost damage is often damage due to dehydration. The freezing of water within the cell (intracellular ice formation) is always lethal (the exception is the artificial "vitrification" employed to conserve organisms).

2. Cold damage is primarily damage to membranes. This applies to chilling as well as to frost. If the proportion of high melting point lipids or the protein/lipid ratio is too high, large membrane domains are immobilised and their functions are lost when the temperature is lowered. Membrane damage is also caused by severe freeze-desiccation and the resulting accumulation of charged solutes (e.g. salt ions) at membrane surfaces.

3. Tropical and subtropical crop plants usually cannot harden to cold, or only to a very small extent. Metabolic imbalances and disturbances of cellular metabolism occur due to the different temperature tolerances of various meta bolic reactions, even at temperatures not yet low enough to account for membrane damage. The inactivation of ion pumps, particularly of those associated with the energy supply or the homeostasis of membrane potentials, is the major cause of the damage referred to as being due to membrane "leakiness".

4. Energisation of the photosynthetic apparatus when photosynthetic metabolism is disturbed leads to the formation of reactive oxygen species (ROS) which cause further damage, particularly through the formation of radicals in the biomembranes. This then exceeds the capacity of the plant's well-developed detoxication system to intercept the ROS and the reactive products which they subsequently form. The photoinhibition observed during the over-energisation of photosystem II, particularly in connection with chilling, is mainly triggered by ROS. ROS are not formed only in the context of photosynthesis, but also during dysfunction of the respiratory chain and in other reactions in which molecular oxygen participates. ROS detoxification systems occur, on the one hand, in membranes, e.g. as evidenced by tocopherol and xanthophylls in the photosynthetic membranes. In the cytosol or in the stroma of the chloroplasts, ascorbate and glutathione are the main compounds involved in the detoxification of ROS, but flavonoids also play a role.

5. Frost causes stress not only because of the low temperatures involved, but additionally due to (freeze-)desiccation. Therefore, the exact cause of the incurred damage cannot always be determined unequivocally. Freezing of tissue and cell water must always occur outside the cells in the intercellular spaces. Water must thus exit from the cells into the intercellular spaces prior to freezing. Cold inactivation of pumps in the plasma membrane could be a reason for this phenomenon. Ice formation in the intercellular spaces requires a nucleation trigger. Bacteria are often found on the epidermal surface of the plant, but also in the substomatal intercellular spaces, which act as nucleation triggers.

6. Bacteria which are active in nucleation, the so-called INA bacteria, possess "nucleators" consisting of aggregates of INPs (ice-nucleating proteins), whose molecular construction favours the fixation of water clusters into ice matrix-like structures and thus reduces the supercooling required for crystallisation. Nu-cleation-active proteins have also been ex tracted from the intercellular spaces of plant tissues, but their molecular effect has not yet been clarified. Ice formation, which takes place at relatively mild freezing temperatures, reduces the danger of the sudden intercellular freezing of cell water which takes place when supercooling has progressed too far. On the other hand, there are many plants that are frost-tolerant but not tolerant to freezing. Avoidance of nucleation is the only means of surviving frost for these plants (which include many crop plants).

7. The desiccation of cells stemming from the freezing of cellular water is enormous even upon moderate frost. As a rule, more than 80% of tissue water is already frozen at temperatures of about -10 °C. Freezing ceases when the water potential of the (extracellular) ice, which depends on the (freezing) temperature, equals the water potential of the cell. This is called "equilibrium freezing", whereby ideal and non-ideal equilibrium freezing are distinguished. In ideal equilibrium freezing, the water potential of the freeze-desiccated cell is determined solely by its osmotic potential. In non-ideal equilibrium freezing, a negative wall pressure (suction) which arises during the desiccation of the cell augments the water potential of the cell, so that more water remains in the liquid state than would be expected from the osmotic potential. The plasma membrane must always remain in contact with the cell wall in an intact cell during freeze-desiccation, as the matrix potential of the cell wall prevents the penetration of air from the intercellular spaces. Shrinkage of the cell volume caused by freeze-desiccation therefore leads to (reversible) deformation of the whole cell and even to wrinkling of the cell wall (freezing cytorrhysis).

8. Extreme freeze-desiccation leads to the breakdown of biomembranes, as the ordering of hydrophobic interactions between membrane lipids requires the presence of liquid water. Membrane breakdown is further stimulated by high concentrations of charged solutes, which can drastically alter the membrane potential.

9. The degree of frost hardening depends not only on the composition of the lipids (and the protein/lipid ratio) of the biomembranes, but also on the effectiveness of so-called cryopro-tectants. These are low molecular weight solutes such as many carbohydrates, which take over the membrane-stabilising role of water molecules during freeze-desiccation, and at the same time prevent the accumulation of ions at the membrane surfaces. In addition to low molecular weight cryoprotectants, frost-hardy plants also synthesise proteins which protect membranes and other proteins. These are, for example, proteins from the family of the dehydrins, which associate protectively with membranes during dehydration. The formation of such proteins is a good example of cross-protection, i.e. of a multiple protective system, the formation of which is triggered by one particular stressor but then also hardens the plant against other stressors (e.g. frost, drought and high salt concentrations). The synthesis of such proteins may also be induced by the plant hormone abscisic acid.

10. The phenomenon of cross-protection suggests the existence of a molecular "master switch". Indeed, in addition to the presence of plant hormones, there appears to be a higher hierarchical level of genes, the products of which activate a whole series of further genes. The products of these lower-level genes contribute to stress management either directly or indirectly (e.g. via the synthesis of low molecular weight osmolytes - here cryoprotective solutes). Such a system is called a "regulon". Finding such master-switch genes would be of particular interest with regard to increasing the stress tolerance of plants by genetic transformation. It seems more feasible at present, however, to transform plants by introducing several genes from the lower hierarchical level into a crop plant (Holmberg and Bülow 1998). Candidate genes for transforming plants with a master switch would be, for example, those encoding transcription activators such as DREB 1A, B, C and others (see Chap. 1.6: "proline"). These genes are high up in the cascade hierarchy for drought tolerance (Sarhan and Danyluk 1998).

11. So-called antifreeze proteins are well known in the animal kingdom, and have recently also been found in plants. They impede crystallisation of water and hinder the growth of ice crystals, thus delaying freezing, but not thawing. They are, therefore, also called THPs (thermal hysteresis proteins). The molecular structures and mechanisms of action of some of these proteins are known. They attach to ice crystals and turn their hydrophobic side towards the aqueous medium, thus making it difficult for other water molecules to associate with the ice. The formation of many, small ice crystals instead of fewer large ones is the consequence.

12. Perennial plants from moderate and higher latitudes exhibit the phenomenon of seasonal frost hardening and dehardening. Frost hardening is accompanied at the molecular level by desaturation of membrane lipids, reduction of the protein/lipid ratios in biomembranes, and the synthesis of cryoprotective and ROS-de-toxifying compounds and possibly also of proteins which promote or hinder ice nucleation. Plants have only a low photosynthetic capacity in the frost-hardened state. This is predominantly due to the degradation of the photosystems in the thylakoid membranes, but it also significantly reduces the danger of over-energisation in the frozen state. Arctic and tropical high mountain plants are permanently frost-hardy. The latter have developed various mechanisms to avoid freezing in accordance with the daily-frost climate, such as insulation and heat storage which can be of a permanent (cloaking of the stem) or transient (night buds) nature. Such mechanisms only work, however, because the nightly frost periods last, at most, for only a few hours before the tropical sun raises the temperature above the freezing point again.

13. Frost drought comprises a stress situation for the entire plant, for which there is no avoidance response except leaf shedding and also no specific tolerance. Frost drought results from the transpiration of above-ground plant parts when water cannot be taken up from the frozen soil. Frost-hardened plant organs can survive this situation only as long as the mechanisms for damage avoidance are not overtaxed.


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