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Impact of Cold Stress |
By Dr.’s D. B. Fowler and A. E. Limin
Crop Development Centre |
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1. The
Environmental and Physiological Nature of Stress
Successful adaptation of a crop
species is dependent upon the programming of critical growth stages so that the
plant can capitalize on favorable weather periods
during the growing season. Plants have evolved a variety of adaptive mechanisms
that allow them to optimize growth and development while coping with
environmental stresses. These mechanisms include seed and bud dormancy,
photoperiod sensitivity, and low-temperature response. Seed dormancy delays
germination until after the embryo has gone through an after-ripening period.
The over-winter survival of buds of many temperate zone trees and shrubs is
dependent on a dormancy stage that starts in the late summer or early fall and
ends after exposure to an extended period of cold or increasing day length in
the spring. In addition to trees, many other dicots
and grasses have a photoperiod response that can advance or delay flowering. Vernalization is a requirement for growth at low
temperatures before a plant will flower. Most winter annual and biennial plants
have a vernalization requirement. Low-temperature
acclimation is an ability of plants to cold acclimate when exposed to gradually
decreasing temperatures below a specific threshold. This is the most common
mechanism that plants have evolved for adapting to low-temperature stress and
examples of plants with the capacity to cold harden can be found in most
species.
Types of Low Temperature Injury
There
are two types of injuries a plant can sustain through exposure to low
temperatures (Stushnoff et al. 1984). The first is chilling injury that occurs from
approximately 20 to 0oC. The
resultant injuries may include a variety of physiological disruptions in
germination, flower and fruit development, yield, and storage life. Minor
chilling stress at non-lethal temperatures is normally reversible. Exposure to
gradually decreasing temperatures above the critical range can also result in
hardening of plants that may reduce or eliminate injury during subsequent
exposure to low temperatures.
The
second type of injury is called freezing injury. This type of injury occurs
when the external temperature drops below the freezing point of water. Some varieties of plants that are susceptible
to chilling injury can be killed by the first touch of frost. At the other extreme,
many plants that are native to cold climates can survive extremely low
temperatures without injury (Levitt 1980). Plants may
experience intracellular freezing and/or extracellular
freezing. Intracellular freezing damages
the protoplasmic structure and the ice crystals kill the cell once they grow
large enough to be detected microscopically. In extracellular
freezing, the protoplasm of the plant becomes dehydrated because a water-vapor
deficit is created as cellular water is transferred to ice crystals forming in
the intercellular spaces. In some cases, water can remain liquid as low as -47oC
without nucleating and forming ice. When nucleation of this supercooled water does occur, intracellular ice forms
suddenly resulting in death of the plant.
Types of Plants
Plants can be
grouped into three different classes according to their low-temperature
tolerance (Stushnoff et al. 1984). The first group
includes frost tender plants that are sensitive to chilling injury and can be
killed by short periods of exposure to temperatures just below freezing. They
cannot tolerate ice in their tissues and readily exhibit frost injury symptoms
that include a water soaked flaccid appearance with
loss of turger followed by rapid drying upon exposure
to warm temperatures. Beans, corn, rice, and tomatoes are examples of plants in
this category.
Low-temperature
acclimation of plants in the second group allows them to tolerate the presence
of extracellular ice in their tissues. Their frost
resistance ranges from the broad-leafed summer annuals, which are killed at
temperatures slightly below freezing, to perennial grasses that can survive
exposure to -40oC. As temperatures decrease the outward migration of
intracellular water to the growing extracellular ice
crystal causes dehydration stress that will eventually result in irreversible
damage to the plasma membrane, which is the primary site of low-temperature
injury. If ice nucleation does not occur at -3 to -5oC, supercooling may result in intracellular freezing and death
of individual cells.
The
final group is made up of very cold hardy plants that are predominantly
temperate woody species. Like the plants in the previous group, their lower
limits of cold tolerance are dependent on the stage of acclimation, the rate
and degree of temperature decline, and the genetic capability of tissues to
accommodate extracellular freezing and the
accompanying dehydration stress. Deep supercooling
allows certain tissues in plants from this group to survive low temperatures
without the formation of extracellular ice. However,
the most cold hardy species do not rely on supercooling and can withstand temperatures of -196 oC.
2. Plant
Chilling Stress and Its Repercussions
Introduction
Most
crops of tropical origin as well as many of subtropical origin are sensitive to
chilling temperatures. This limits production areas and causes potential damage
during storage if they are exposed to low temperatures. The temperature below
which chill injury can occur varies with species and regions of origin, ranging
from 0 to 4oC for temperate fruits, 8oC for subtropical
fruits, and about 12oC for tropical fruits such as banana (
Physiological
age, seedling development, and pre-harvest climate can also influence chilling
sensitivity. Freshly imbibed seeds of chill-sensitive species tend to be very
sensitive, as does the pollen development stage. Fruits maturing at high
temperature are more susceptible than those maturing at lower temperatures.
Post-harvest storage at lower temperatures is commonly used to extend the
storage life of fruits and vegetables. Tropical and subtropical plants however
are often subject to physiological damage and loss of quality due to chill
injury under these storage conditions. The severity of injury to
chill-sensitive tissues tends to increase with decreasing temperatures and with
length of low-temperature exposure.
Chilling
has been found to change the entire metabolic system of the cell with some
processes recovering quickly and others only slowly. Chilling affects the
entire internal environment of each cell and each molecule within the cells.
Enzymatic reactions, substrate diffusion rates, and membrane transport
properties are all affected. Chilling injury is therefore likely a direct
consequence of these effects (Kratsch and Wise 2000).
Amelioration of
chilling injury
Avoidance: To avoid
chilling injury, planting dates can be altered though this is often difficult
because of its effect on later development of the plant. To overcome this
problem, cultivars have been bred for early vigor and maturity. In the case of
stored fruits and vegetables, maintenance of appropriate storage temperatures
is essential to avoid chilling injury. Investigations have also been undertaken
to examine synthetic plant growth regulators for the protection of chilling
sensitive crops (Li 1989).
Temperature conditioning: Low-temperature
‘hardening’ allowing tolerance to chilling temperatures appears to have little
effect although some sensitivity to ‘slight chilling’ can be reduced by
exposure to temperatures slightly above the chilling range. It also appears
that chilling injury to stored fruits and vegetables can be ameliorated by warm
temperatures if they are imposed before tissue degeneration becomes advanced.
Other treatments such as waxing, fungicides, hormones, and antioxidants have
produced variable results that have been dependent upon the species and
treatment conditions (
Duration: Ultrastructural-chilling
injury increases with time and with prolonged exposure the injury becomes
irreversible. It is therefore important to minimize the time of chilling
temperature exposure.
Relative humidity: High (100%)
relative humidity has been found to protect chloroplasts from chill injury, an
effect that is enhanced by darkness.
Theories of chilling injury
Early
research focused on chilling causing an imbalance in plant physiological
processes. Chilling was found to affect O2 evolution, organic acids,
sugars, polyphenols, phospholipids, protein, and ATP.
Research indicates that chilling stress in sensitive plants changes most
chemical entities. There is evidence of accumulation of toxins such as ethanol
and acetaldehyde. Although many altered processes involve key metabolites; it
is difficult to separate the critical chilling-sensitive metabolic processes
from those that are byproducts of metabolic disruptions or of ultrastructural breakdown. Ion leakage due to membrane
permeability changes has often been reported in chill sensitive plants. Phase
transition of the lipid portion of the cellular membranes has also received
considerable attention as the primary
response to chilling temperatures (
Ultrastructural changes: On an ultrastructural
level, several changes have been associated with chilling injury. Although
there are a number of variables affecting chill injury, the ultrastructural
symptoms are very similar across species. Ultrastructural
symptoms of chilling injury become evident before obvious physical symptoms are
visible. These include changes to chloroplasts, mitochondria and membranes
associated with these organelles and the vacuoles (Christiansen and St. John
1981). The symptoms include swelling and disorganization of the chloroplasts
and mitochondria, reduced size and number of starch granules, dilation of thylakoids and unstacking of grana, formation of small vesicles of chloroplast
peripheral reticulum, lipid droplet accumulation in chloroplasts, and
condensation of chromatin in the nucleus (Kratsch and
Wise 2000).
Chloroplasts are
the first and most severely affected organelle. Irradiance during chilling
greatly exacerbates the resulting injury. Chilled plants in darkness have been
found to remain green and, except for starch depletion, chloroplasts appear
normal. In the presence of light, however, chlorophyll becomes bleached, lipid
droplets accumulate, and thylakoids degenerate.
Mitochondria appear more resistant to chilling temperature but an immediate
effect of low temperature on chilling-sensitive species is a suppression of
mitochondrial activity. Electron micrographs of chilled sweet potato roots
revealed that the mitochondria had a swollen appearance due to the release of
phospholipids from the inner and outer membranes during storage at chilling
temperatures. The capacity to bind phospholipids was also greatly decreased.
Membrane permeability and phase
transition: Measures of solute leakage or ion permeability have provided
evidence of increased membrane permeability in response to chilling. The plasma
membrane is often considered the primary site of freezing injury and
electrolyte leakage. Early work indicated that plants originating in warm
climates tend to have more saturated fatty acids in their membrane lipids. More
recent work on mitochondrial membranes has shown that membranes do undergo a
physical phase transition from a flexible liquid-crystalline to a solid-gel
structure at 10 to 12oC, which coincides with the temperature
sensitivity range of species of tropical origin. Fruits of several apple
cultivars have been observed to undergo phase transition in the 3 to 10oC
range suggesting the same mechanism of chilling injury as found in tropical
species. The correlation between fatty acid composition and temperature induced
phase transition is, however, not precise. It may be that other membrane
components such as sterols also play a role.
It is possible
that the phase transition of cellular membranes could account for the entire
range of physiological and metabolic changes associated with chilling injury.
Increased membrane permeability could lead to an altered ion balance and also
to the ion leakage observed from chilling of sensitive tissues. Phase
transition could result in conformational changes in membrane bound enzymes and
account for the observed discontinuities in the function of many enzyme
systems. This may cause an imbalance between membrane bound and non-membrane
bound systems. Over time the cells inability to cope with increased
concentrations of metabolites could result in injury. Different tolerances to
these metabolites could explain why some cultivars are more resistant to damage
while still undergoing phase transition. Imbalances in metabolism, accumulation
of toxic compounds, and increased permeability could all be the result of
temperature-induced phase transition (
The
contribution of unsaturated fatty acids in cell membrane lipids has been
discussed for many years in relation to chilling sensitivity. Nishida and
Murata (1996) have shown that chilling injury can be manipulated by modulating
levels of unsaturation of fatty acids by the action
of acyl-lipid desaturases
and glycerol-3-phosphate acyltransferase.
Recently
the role of unsaturation of membrane lipids in
chilling tolerance and in response to low temperature has been reexamined using
mutant and transgenic lines (Nishida and Murata 1996). In this way unsaturated
fatty acids can be manipulated independent of temperature so that their
individual effects can be evaluated. Tobacco was transformed with squash and Arabidopsis phosphatidylglycerol
(PG) species found in thylakoid membranes. Squash has
low levels of cis-unsaturated
PG while Arabidopsis has relatively
high levels of cis-unsaturated
PG. It was found that tobacco transformed with squash PG was more chilling
sensitive and tobacco transformed with Arabidopsis
PG was the most chilling resistant, as measured by photosynthesis at 1oC
under strong illumination. These results indicate that chilling sensitivity can
be manipulated by altering the level of unsaturated PG in the chloroplasts.
These and other experiments have shown that unsaturation
of membrane lipids protect the photosystem II complex
from low-temperature photoinhibition by accelerating
recovery from the photoinhibited state. However, it
is likely that other factors such as accumulation of polyols
and amino acids, or their derivatives, contribute to chilling sensitivity in
plants. Some specific proteins may also be responsible for chilling tolerance.
Alteration
of intracellular pH: Yoshida et al. (1999)
noted that intracellular pH was, in part, actively controlled by H+-transport
from the cytoplasm to the vacuole catalyzed by H+-ATPase located on the vacuolar membrane in mung bean (Vigna
radiata L.),
which is a very chilling-sensitive species. The vacuolar H+-ATPase is extremely sensitive to low temperature and is
preferentially inactivated upon exposure to chilling temperatures. This
inactivation occurs much earlier than the symptoms of cell injury and the
decrease in enzyme activity associated with plasma membranes, endoplasmic
reticulum, and mitochondria. Cold-induced inactivation of H+-ATPase also occurs in chilling sensitive rice. Cold-induced
suppression of proton transport disrupts cytoplasmic
homeostasis and causes a change in the pH. The chilling sensitivity of cultured
mung bean cells changed markedly during the growth
cycle and a close relationship was found between sensitivity of the cells and
of H+-ATPase to the cold. Cold-induced
inactivation of the vacuolar H+-ATPase was
closely linked to acidification of the cytoplasm and the corresponding
alkalization of the vacuoles suggesting a passive release of H+ ions
across the vacuolar membrane. The susceptibility of vacuolar H+-ATPase to low temperature in vivo was found to be markedly
different between chilling-sensitive and chilling-resistant species. In
contrast to the H+-ATPases of
chilling-sensitive species like mung bean and kidney
bean (Phaseolus
vulgaris),
the H+-ATPases of the chilling-tolerant
species such as pea (Pisum
sativum)
and broad bean (Vicea
faba) were very
stable over long periods of low-temperature exposure. The molecular structures
of the 16 kDa proteolipids
from the two types of H+-ATPase appeared
to be very different. Low-temperature-induced pH reduction of the cytoplasm
caused by inactivation of vacuolar H+-ATPase
may therefore be the cause of extreme chilling-sensitivity.
Conclusion
Plants are uniquely adapted to their native environment through
developmental programming and the particular composition and conformation of
their molecular components is optimized within each species for maximum
competitive ability. These differences in adaptation result in the wide range
of cellular disturbances that have been observed when these plants are moved to
cooler environments. Changes in enzyme reactions, substrate diffusion rates,
membrane properties, and cytoplasmic pH affect the
entire metabolic system of cells subjected to chilling stress. The resulting
injury depends on the duration of exposure and on the individual species, or
variant, being observed.
3. The
Physiological and Agronomic Repercussions of Freezing Stress
Low-temperature response mechanisms
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Plants
have adapted two mechanisms to protect themselves from damage due to below
freezing temperatures. Supercooling is a low-temperature tolerance mechanism that
is usually associated with acclimated xylem parenchyma cells of moderately
hardy woody plants. When sources of ice nucleation are absent, pure water can supercool or remain unfrozen to its homogeneous nucleation
point of approximately -40oC. The initiation of freezing at the
limit of supercooling occurs suddenly and is
accompanied by an exotherm that can be detected by
thermal analyses of plant tissues. Plant tissues suffer irreversible damage
once ice nucleation of supercooled water occurs and
the distribution in nature of tree species with the ability to deep supercool is normally restricted to regions where winter
temperatures are warmer than -40oC (George et al. 1982).
The
second and most common low-temperature response mechanism is acclimation.
Low-temperature acclimation is a gradual process during which there are changes
in just about every measurable morphological, physiological, and biochemical
characteristic of the plant. These
changes are determined by genotype x environment interactions that are quite
complex and not clearly understood. They have
been studied most extensively in cereals where a wide range in genetic
potential and the availability of unique cytogenetic
stocks has allowed for novel approaches to investigations at the molecular and
whole plant level. Potential gene donors have been evaluated for use in interspecific transfers and the control of alien (donor
species) low-temperature gene expression has been studied in a variety of
backgrounds. A survey of the published research in these areas has allowed us to
construct a field validated winter survival model that successfully simulates
the over winter changes in low-temperature tolerance of a wide range of
genotypes (Fowler et al. 1999). Consequently, this review will
focus mainly on the genetic systems that winter cereals have evolved for
low-temperature adaptation, the regulation of these systems, and their complex
interaction with the environment.
Low-Temperature
Acclimation in Winter Cereals
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Figure
3. Relationship between average daily crown temperature (0 to 8oC) and
low-temperature tolerance of Norstar winter wheat
during acclimation at a constant temperature before vernalization
saturation (LT50 for Norstar
= - 24oC
when fully acclimated). |
When growth starts in the early
fall, winter cereal plants will not survive subfreezing temperatures much
better than spring cereal plants.
However, winter cereals grown under cool fall temperatures will cold
acclimate or 'harden off'. For example,
in Saskatchewan (western Canada), the minimum survival temperature for 'Norstar' winter wheat is normally near -3oC at
the beginning of September and -19oC or lower by the end of October
(Figure 1).
Under field conditions in western
temperatures at crown depth are above 9oC.
Both the cold acclimation process and winter survival require energy and this
period of warm temperature allows for the establishment of healthy vigorous
plants (Figure 2). Plants with
well-developed crowns before freeze-up are in the best position to withstand
the rigors of winter and regenerate roots and leaves in the spring. However,
plants that enter the winter with two to three leaves are usually not seriously
disadvantaged.
Cold acclimation of winter wheat plants begins once fall
temperatures drop below approximately 9oC. A translocatable
substance that promotes cold acclimation is not produced when winter wheat
plants are exposed to acclimating temperatures (Limin
and Fowler 1985). Consequently, the
cold-hardiness level of different plant parts, such as leaves, crowns and
roots, is dependent upon the temperature to which each part has been exposed.
Because the crown contains tissues that are necessary for plant survival, it is
the soil temperature at crown depth that determines critical cold-acclimation
rates.
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Figure
4. Relationship between average daily crown temperature (- 4 to 18oC)
and low-temperature tolerance of Norstar winter
wheat during dehardening at a constant temperature
(LT50 for Norstar = - 24oC when fully acclimated = day
0). |
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Plant growth slows considerably at
temperatures that promote cold acclimation. In the field, soil temperatures
gradually decrease as winter approaches and four to seven weeks at temperatures
below 9oC is usually required to fully cold-harden plants. Cold acclimation
during this period is dependent upon crown temperatures and the rate of
acclimation increases dramatically as temperature drop from 9 to 0oC
(Figure 3). Exposure of winter wheat crowns to soil temperatures above 9oC
during this period results in a rapid loss of cold hardiness. The rate of dehardening is dependent upon the temperature to which the
crown is exposed (Figure 4). At this
stage, plants that have been exposed to crown temperatures above 9oC
will resume cold acclimation once they return to temperatures below 9oC.
Winter
wheat normally does not realize its maximum cold hardiness potential until
after the soil is frozen in the late fall. In
Death of the crown tissue will result
if the soil temperature falls below the plants minimum survival temperature
(Figure 1). Exposure of winter cereal
plants to crown temperatures that are 2 to 3oC warmer than their
minimum survival temperature will cause immediate damage and a reduction in cold
hardiness (Fowler et al. 1999). Longer periods of exposure to temperatures
approaching the minimum survival temperature can quickly reduce the plant's
ability to tolerate cold stress. The expected LT50
for different exposure times (T) to constant temperature can be calculated from
Equation 1 (Fowler et al. 1999).
LT50(T) = LT50(0) + 5.72 + 1.53 *
ln(T) [Eq. 1]
Where T is
the number of days that plants are exposed to a constant low-temperature
stress.
LT50(0) is determined using a series of test temperatures
where the low-temperature stress is removed as soon as the crown samples are
exposed to a predetermined minimum temperature (day 0 in Figure 5). For example, fully acclimated Norstar winter wheat will normally survive to -24.0oC
in a controlled-freeze test where plant samples are gradually cooled at a rate
of 2 to 6oC hr-1 and removed as soon as they reach a
predetermined temperature (day 0 in Figure
5). However, two days
exposure to -17.2oC in a controlled environment will reduces the
minimum survival temperature of fully hardened Norstar
winter wheat from -24.0 to -17.2oC, a cold hardiness loss of 6.8oC.
Once
vernalization saturation is complete and the plant
enters the reproductive stage, it loses its ability to cold acclimate (Fowler et
al. 1996a) and it will start to deharden at
temperatures warmer than approximately -4oC (Figure 4). This means
that winter wheat will eventually completely deharden
once plant growth resumes in the spring (Figure 1, Fowler et al. 1999). Growth
rate and rate of dehardening are both temperature
dependent and because frozen soils warm slowly in the spring, several weeks of
warm air temperatures are required to re-establish and completely deharden winter cereal plants that have survived without
serious winter damage.
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