Impact of Cold Stress

By Dr.’s D. B. Fowler and A. E. Limin Crop Development Centre University of Saskatchewan Saskatoon, Saskatchewan, S7N 5A8 Canada

The Nature of Stress Plant Chilling Stress The Repercussions of Stress

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 0^oC. 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 -47^oC 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 -40^oC. 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 -5^oC, 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 4^oC for temperate fruits, 8^oC for subtropical fruits, and about 12^oC for tropical fruits such as banana (Lyons 1973). Amongst the highest volume world food crops, maize (Zea mays) and rice (Oryza sativa) are sensitive to chilling temperatures. Their growth and development can be adversely effected by temperatures below 10^oC resulting in yield loss or crop failure. Christiansen and St. John (1981) estimated annual losses of $60 million to the cotton industry due to chilling temperature immediately following field planting. Chilling during the seedling stage in cotton can reduce plant height, delay flowering and adversely affect yield and lint quality. Seedlings can also suffer water stress and leaf desiccation at chilling temperatures, floral initiation is inhibited at 7^oC and seed set is inhibited at 15^oC. Other crops suffering stand loss, delayed maturity, and reduced yield as a result of chilling after planting include soybean (Glycine max L.), lima bean (Phaseolus lunatus L.), cucurbits (Cucurbita sp.), tomato (Lycopersicon esculentum Mill.) pepper Capsicum annuum L.), eggplant (Solanum melongena L.), okra (Abelmoschus esculentus L.), and various cereal crops.

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 (Lyons 1973).

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 (Lyons 1973).

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 12^oC, 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 10^oC 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 (Lyons 1973).

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. Lyons (1973) proposed that temperature induced phase transition of membrane lipids may play a primary role in chilling sensitivity of plants. Continued exposure to chilling temperatures would result in phase separated membranes becoming incapable of maintaining ionic gradients resulting in metabolic disruption and eventual cell death. A positive correlation has been found between chilling sensitivity of herbaceous plants and the level of saturated and trans-monounsaturated molecular species of phosphatidylglycerol (also termed high-melting-point molecular species) in thylakoid membranes. However, there is still a question of how directly these high-melting-point molecular species relate to chilling sensitivity in plants. Growth at low temperature generally increases the degree of unsaturation of membrane lipids, which compensates for the decrease in fluidity caused by the lower temperature. This increased unsaturation is also correlated with the sustained activity of membrane-bound enzymes at low temperature. The unsaturation of membrane lipids is therefore considered critical for the functioning of biological membranes and the survival of plant cells at low temperature. However, since low temperature is also known to induce or alter the expression level of a large number of genes it is not clear if the association between membrane lipid unsaturation and chilling tolerance is a cause or effect relationship.

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 1^oC 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

Figure 1. Changes in cold hardiness of Norstar winter wheat for the period September to May. The primary factors responsible for these changes are shown at the bottom of the graph.

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 -40^oC. 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 -40^oC (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

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 -3^oC at the beginning of September and -19^oC or lower by the end of October (Figure 1).

Under field conditions in western Canada, eight to 12 weeks of fall growth is usually required for the full development of cold hardiness in winter cereals. The first four to five weeks is a period of active growth that takes place when average daily soil

temperatures at crown depth are above 9^oC. 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 9^oC. 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.

Figure 4. Relationship between average daily crown temperature (- 4 to 18^oC) and low-temperature tolerance of Norstar winter wheat during dehardening at a constant temperature (LT50 for Norstar = - 24^oC when fully acclimated = day 0).

Figure 5. The effect of exposure to a constant temperature on low-temperature tolerance of Norstar winter wheat (Norstar LT50 = - 24^oC when fully acclimated at day 0).

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 9^oC 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 0^oC (Figure 3). Exposure of winter wheat crowns to soil temperatures above 9^oC 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 9^oC will resume cold acclimation once they return to temperatures below 9^oC.

Winter wheat normally does not realize its maximum cold hardiness potential until after the soil is frozen in the late fall. In Saskatchewan, full acclimation is usually achieved by the middle to the end of November (Figure 1). Once cold acclimation has been completed, winter wheat can maintain a high level of cold hardiness provided crown temperatures remain below freezing. In the fall, winter wheat will cold acclimate when exposed to crown temperatures colder than 9^oC. However, prolonged exposure of acclimated plants to winter temperatures above freezing results in the transition of the plant from the vegetative to the reproductive phase and a gradual loss of cold hardiness (see the next section for more details on these changes). The warmer the crown temperature during the winter, the shorter the period that maximum levels of cold hardiness can be maintained and, once started, the more rapid the rate of decline in cold hardiness.

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 3^oC 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 -24.0^oC in a controlled-freeze test where plant samples are gradually cooled at a rate of 2 to 6^oC hr^-1 and removed as soon as they reach a predetermined temperature (day 0 in Figure 5). However, two days exposure to -17.2^oC in a controlled environment will reduces the minimum survival temperature of fully hardened Norstar winter wheat from -24.0 to -17.2^oC, a cold hardiness loss of 6.8^oC.

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 -4^oC (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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