Freezing Resistance of plants
According to Levitt (1972,1980) plants have evolved two types of
survival strategies: avoidance and tolerance. Gusta et al. (1983)
concluded that in dormant plant material: a) a prolonged exposure to a
temperature range of -30 to -40 oC causes a decrease of the LTE to
temperatures below that of the homogeneous nucleation point, down to
-55 oC; or that b) a prolonged exposure to -30 oC to -40 oC causes an
extracellular crystallization of all freezable supercooled water
fractions, thereby forcing the LTE to escape from the freezing
profile. These conclusions imply that certain ray parenchyma cells
exhibit tolerance to freezing-induced dehydration stresses, but not
freezing avoidance, i.e. the maintenance of all celular water in the
supercooled state during cooling. In contrast, other studies showed
that supercooled water fractions are stable, even when the stem is
partly frozen. With these two possible strategies in mind, we consider
the mechanism of deep supercooling of cells in a strict sense, a state
in which all cellular water remains at a supercooled stage during
cooling. Hence, the definition of supercooling excludes all conditions
in which parts of the cellular water migrate to extracellular space
during cooling.
Freezing avoidance on a whole plant scale means that
there is no ice crystal formation on any tissues. This type of
avoidance is of a physical nature, i.e. determined by the presence,
location and properties of ice nucleators. Avoidance on a cell or
tissue scale, however, occurs even in the presence of intercellular
ice. Consequently, the difference between freezing avoidance and
freezing tolerance is related to the level of organization. On the
cellular scale, freezing avoidance may occur through dehydration and
freezing point depression: on the tissue and whole plant scale,
crystallization of cellular water in intercellular spaces should be
denoted as freezing tolerance.
Freezing avoidance
Two main types of freezing avoidance are exhibited by plant
material: 1) desiccation of plant tissues and 2) supercooling.
Desiccation of plant tissues
This type of freezing avoidance is exhibited by
plants or plant parts such as bryophytes, dried tubers and rice and
wheat seeds, which can sustain losses of tissue water to about 20% on
a fresh weight basis without loosing vialbility. The remaining tissue
water consists of fractions which are so tightly bound to cell
structures that it is impossible for the water to become crystallized
even when cooled down to -196 oC. In an imbibed or hydrated state,
extraorgan freezing takes place and the freezing temperature varies
with water content and cooling rate.
Supercooling
Supercooling can be separated into three
categories, depending upon its extent and duration: a) mild
supercooling (-0.6 to -15 oC) exhibited for a limited period of time;
b) supercooling at temperatures between -16 to -25 oC which can be
maintained for long time periods; or c) deep and persistent
supercooling down to or below the homogeneous nucleation point.
a) Mild transient supercooling
Most plant parts exhibit mild transient
supercooling in the subzero temperature range before extracellular ice
crystal formation takes place. In nature, extracellular freezing often
begins after mild supercooling in the temperature range of -0.6 to -4
oC. However, in a study of ice crystal formation in stem sections of
Veronica persica, Pisum sativum, Vicia faba, Vicia sativa and
Buxus microphylla var. japonica subjected to artificial
freezing conditions, the temperature range for induction of ice
nucleation was shown to vary between -5 to -15 oC. Supercooling varies
with the tissue moisture content and the applied colling rate, the
moisture content of the epicuticular surface, cell size and presence
or absence of intercellular spaces, plant size and exposure time, age
of plant material, water content, the physical surroundings such as
wind, agitation and the presence of plant-derived ice nuclei and
INA-bacteria. Therefore, supercooling is unpredictable with regard to
duration and extent. Reports and conclusions about supercooling and
its impact on survival are often ambiguous and must therefore be
carefully interpreted in relation to the circumstances.
b) Persistent supercooling
It takes place in plants or parts of plants which
not only exhibit supercooling between -16 to -25 oC but also may
maintain it for a long time. Specific characteristics of tissues and
cells exclude freezing stress, for example the small size of cells
with barriers against the spread of ice. Tightly packed cells and
reduced intercellular spaces might also be characteristics which allow
extensive supercooling in cold acclimated and non-acclimated plant
material. Plants native in the subtropical, tropical mountain and warm
temperate regions exhibit such supercooling.
Deep
persistent supercooling
It is favoured by loss of water during cold
acclimation, by small cells, certain cell wall properties and
structure of the pit membrane.
In the absence of heterogeneous ice nuclei, droplets of pure water can
be supercooled to -38.3 oC. This temperature is called the homogeneous
ice nucleation temperature of water. Results of research during the
past decades have substantiated the opinion the ray parenchyma cells
of certain species behave similarly to isolated droplets of pure
water, i.e. freezing occurs as either single units or in groups of
cells in the range of the homogeneous nucleation point. Death of xylem
ray parenchyma cells by crystallization of the supercooled
intracellular water fractions has been linked with the appearance of
low temperature exotherms. It has to be emphasized that, together with
the supercooling of ray parenchyma, other adjacent tissues (cortex)
survive by tolerance to freeze-induced dehydration stresses. Despite
the fact that cortical cells may be more frost resistant than
parenchyma cells, stem parts are killed when ray parenchyma cells are
exposed to temperatures resulting in LTE.
It has
been sugested that deep supercooling is a survival mechanism which is
associated with the distribution of woody species and the annual
minimum isotherm of -40 oC. The presence and degree of supercooling
varies with species and season.
Freezing tolerance
Freezing tolerance at cell level can be separated
into two mechanisms: a) avoidance of freeze-induced cellular
dehydration, i.e. by lowering the freezing point, b) tolerance of
freeze-induced cellular dehydration, which singly or in cooperation
determine the survival of plants subject to extracellular ice
formation.
Avoidance of freeze-induced dehydration
On a cell scale, avoidance of freeze-induced
dehydration can be accomplished by accumulation of solutes in the cell
sap. This increases osmotic potential of the cellular solutes and, in
turn, the depression of the freezing point. The depression of the
freezing point enables plant material to persist in a supercooled
state even at low subzero temperatures. The literature summarized by
Levitt (1980) and Sakai & Larcher (1987) provides evidence that plants
exhibit avoidance to freeze-induced dehydration throughout the year,
but to a varying extent. The importance of this survival mechanism
should not be underestimated, especially during active growth when
tolerance to freeze-induced dehydration stress is at its lowest level.
Application of the nuclear magnetic resonance (NMR) technique to plant
material has indicated that the behaviour of cellular solution is
similar to that of an ideal salt solution. This means that
supercooling of cells in the presence of extracellular ice will be in
proportion to the depression of freezing point of the cell sap.
Accumulation of soluble sugars in the vacuoles improves the
water-holding capacity of the cell and enables avoidance of stresses
caused through freeze-induced dehydration of cells. Accumulation of
other solutes will also enchance the avoidance of freeze-induced
dehydration. A solute which accumulates must be non-toxic at high
concentration.
As the osmolality of the cell sap of
plants is usually below 1 molar, it has been suggested that the
depression of the freezing point of the cell sap is limited to the
range of -1 to -2 oC. The freezing point depression is not necessarily
the temperature at which freezing will take place in the plant. Levitt
(1980) assumed that depression of the freezing point can be extended
to -4 oC. The efficiency of different solutes in enchancing the
freezing point depression is often difficult to determine, because
these compounds not only affect the freezing point by increasing the
osmolality, but also protect membranes, probably through stabilizing
the spatial structure of the membrane. This may occur by interaction
with the membrane phospholipids, leading to improved freezing
tolerance.
During cold acclimation, plants increase their
frost resistance by lowering the osmotic potential of the cell sap.
This alteration of the osmotic potential is an important adaptation to
environmental stress conditions such as low temperatures and shortage
of water. The most frequent soluble osmolyte is sucrose, but amount of
glucose, fructose and raffinoce also increase markedly in some
species.
Tolerance to freeze-induced dehydration
stress
The difference in freezing resistance between
growing and cold acclimated plants can not solely be attributed to the
avoidance mechanism, because in most conditions this is limited to
only a few degrees. Consequently, the increased frost resistance
during cold acclimation is attributable to enchanced tolerance to
freeze-induced dehydration of cells. This implies that the mechanisms
involved in the tolerance to freeze-induced dehydration stress are one
of the most essential components for plant survival in the temperate
regions., especially in regions with frequent untimely frosts. For
survival of cell dehydration caused by freezing, adequate amounts of
cryoprotective solutes with specific properties have to be
accumulated.
Changes in the composition and structure of cell
membranes take place during cold acclimation. Existing cryoprotectants
include low-molecular-weight solutes such as various types of sugars,
sugar alcohols, free amino acids, organic acids and soluble proteins.
Sucrose is one of the most essential osmolytes and cryoprotectants.
Cryoprotectants change the osmolality of the cell and thereby prevent
the deleterious effects caused by the accumulation of electrolytes.
Further, cryoprotectants stabilize cells by interacting with membranes
at certain loci. Their presence may help to stabilize the protein
structure even at a low water content.
Co-operation between the mechanisms of avoidance
and tolerance to freeze-induced dehydration stress
These two mechanisms will operate simultaneously in
plants, as it can be assumed that solutes which alter the osmolality
also have cryoprotective properties. In cold-acclimated cabbage
plants, both toleracne and avoidance of freeze-induced dehydration
stresses are enhanced. More recent studies of red osier dogwood (Cornus
stolonifera), further substantiated this conclusion. In contract,
studies by Pisek (1950) and Chen et al. (1976) have shown that
tolerance to freezing, but not avoidance of freeze-induced dehydration
was increased by cold acclimation. These contradictory findings may
reflect variations in species-specific properties or in grown
conditions which have not yet been understood.
