14.6.1 Physics and physiology

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Figure 14.20   A notional cooling curve for an aqueous solution. As heat is extracted steadily, solution temperature falls below 0 °C, and water molecules, now in an unstable state, supercool to around -5 °C. A nucleating event occurs at temperature TN, and heat is released as ice forms (latent heat of fusion), resulting in a sudden increase in temperature to TF. The extent of freezing-point depression (0 – TN °C) also serves as a measure of osmotic pressure. (Original sketch courtesy M.C. Ball)

Ice melts at 0°C, and an equilibrium mixture of ice and water has traditionally provided a temperature reference for thermocouples. Ice thawing in pure water will maintain a temperature of 0°C, but if instead of allowing ice to thaw, heat is extracted steadily from a body of water, then ice does not reform at 0°C (Figure 14.20). Instead, the water will remain liquid, and will supercool until some nucleation event occurs. A tiny particle of ice, or even vibration in the presence of dust particles, is usually sufficient to trigger ice formation together with an abrupt release of heat (latent heat of fusion). In Figure 14.20, TFTN represents supercooling, and the degree to which TF is less than zero (freezing-point depression) relates to the amount of solute present in solution. Osmotic pressure, vapour-pressure depression and elevation of boiling point are similarly related to the amount of solute present (referred to collectively as colligative properties of solutions).

Highly purified water can be supercooled to about –40°C, and ice will form spontaneously, but such conditions do not apply in plants because water is not absolutely pure. Instead tissue water is in contact with cell surfaces and invariably holds solutes in solution and colloids in suspension which aids ice nucleation.

During a frost episode in nature, plants experience a sequence of events similar to that summarised in Figure 14.20. Tissue water supercools, and cell sap freezing point is depressed by osmotically active material. Moreover, plants are also equipped with ice nucleating agents that can be of either plant or bacterial origin (e.g. Pseudomonas syringae). As with solutions, formation of ice in plants is accompanied by a release of heat. This exothermic response can be detected by sensitive infrared thermography, and has been used to trace ice propagation during a freezing event in leaves and shoots (Wisniewski et al. 1997).

When plants experience a frost, ice initially forms within intercellular spaces (apoplasm) where solute concentration is least. Water potential in that region is immediately lowered and water molecules migrate from symplasm to apoplasm across plasmalemma membranes and towards regions of ice crystallisation. Water potential in the apoplasm will decrease by about 1.2 MPa per degree below 0°C, so that an apoplasm at –4°C will have a water potential of around –4.8 MPa (i.e. equivalent to an osmotic pressure twice that of seawater), which will dehydrate the symplasm. As one positive trade-off, the symplasm is at least less likely to freeze thanks to solute concentration. In addition, plasmalemma membranes discourage entry of ice crystals that might otherwise seed ice crystal for-mation within the symplasm. Nevertheless, partial dehydration does perturb cell biochemistry due to concentration of metabolites, and the accompanying shrinkage of cells and organelles generates structural tensions.

In frost-sensitive material, cell disruption follows the course outlined above, which unfolds over about 3°C on a cooling curve. Membrane integrity might be lost at around –4°C as apoplasm ice formation ruptures membranes. Intracellular freezing ensues at around –7°C and is inevitably lethal due to the combined effects of membrane injury, symplasm dehydration and protein denaturation.

Tissue damage following freezing can be demonstrated by a loss of differential permeability, metabolite leakage and failure to achieve either plasmolysis or deplasmolysis. Solutions bathing frozen/thawed tissue thus show a sudden increase in electrical conductivity according to freezing damage, and that value then serves as a reliable assay for comparative frost tolerance. For example, population screening based on metabolite leakage has enabled breeding for improved frost tolerance in Eucalyptus nitens for plantation forestry (Raymond et al. 1992).

Leaves on temperate plants often need to accommodate ice formation, and Rhododendron provides such an example. Frozen leaves appear wilted, but regain their normal turgid appearance following thawing. Camellia leaves behave similarly. They take on a semi-transparent appearance when frozen, but recover without damage when thawed. In both cases, their altered physical appearance at low temperature is due to formation of a frostblaze, that is, a lens of ice crystals that forms between layers of tissue that are readily cleaved. Ice localised in this way is rendered harmless, and is lost easily on thawing.

Frost hardiness is a dynamic and composite property of plant cells involving cell size, wall thickness, osmotic pressure of cell sap and membrane properties, all of which can feature in either delaying onset or diminishing adverse consequences of ice formation (Steponkus et al. 1993; Wolfe and Bryant 1992). In any plant, organs that are growing rapidly are frost sensitive, and this is especially the case with early spring growth. Frost hardiness is thus least during the growing season, but increases during autumn and reaches a peak in winter. Clearly, this is an adaptive feature of perennial plants that is attuned to seasonal necessity. Moreover, the extent of this hardening process is heritable, and requires low temperature for onset and maintenance. Return to milder conditions can lead to dehardening with disastrous consequences for horticulture when an early spring is followed by a deep freeze. Frost prevention then becomes crucial.