14.4 - Chilling injury

Original Author: Susan E Hetherington, University of Queensland, with update by David Brummell, Plant and Food Research, New Zealand

Plants may develop physiological disorders when exposed to low but non-freezing temperatures. The German plant physiologist Molisch suggested the term ‘chilling injury’ as long ago as 1897 to describe this phenomenon. Symptoms of chilling injury can differ widely between species, but usually develop rapidly in plants native to tropical and subtropical climates and almost imperceptibly slowly in plants originating in cool temperate climates. Within the range of chilling temperatures, that is, from the temperature of the freezing point of the plant tissue up to about 13°C, the rate at which chilling injury develops intensifies with decreasing temperature and increasing duration.

Throughout history, people around the world have collected plants on their travels and taken them to other countries and continents. When tropical and subtropical plants collected from low altitudes have been taken to temperate climates they have had to be housed in protective structures for all or part of the year. In the case of Citrus species introduced into northern Europe in the sixteenth and seventeenth centuries from warmer southern climates, summer temperatures in countries such as France and Germany were mild enough to allow these trees to be grown outside in the summer. At other times of the year, however, potted trees were moved into buildings known as orangeries for protection against the low temperatures. Similarly, during the late seventeenth century, a time when a great number of exotic chilling-sensitive palms, trees and foliage plants were introduced into temperate countries by plant collectors operating in the tropics and subtropics, these plants had to be protected from exposure to low temperatures at all times. Chilling-sensitive foliage plants such as Episcia spp., which are native to the Amazon basin, are killed within 30 min of being exposed to 1°C. Survival of such highly sensitive plants necessitated year-round protection from cold. This requirement was met by the invention of the heated glasshouse in the 1880s.

In modern agriculture, many species are cultivated outside their original microclimate. For example, avocado (Persea americana Mill.) was taken from tropical highlands in Mexico and is now grown in the temperate North Island of New Zealand and in inland Australia where nights are cold. When the new location has temperature minima below those of the region in which the plant evolved, problems of chilling injury can arise.

14.4.1 - Symptoms of chilling injury


Figure 14.17. Cavendish Williams bananas harvested at the hard green stage from the same banana hand were either stored at 22°C for 11 d (non-chilled) or placed at 4°C for 7 d (chilled) before transfer to 22°C for 4 d. Compared to the non-chilled bananas, which gradually turned from green to yellow as they ripened, the chilled bananas failed to yellow and instead developed extensive peel blackening due to cell death. Slight peel blackening was evident when the bananas were removed from the 4°C treatment but greatly intensified at 22°C. To maintain the postharvest quality of Williams bananas, a crop which is worth approximately $180 million per annum to the Queensland economy, marketing authorities stipulate that the produce must not be cooled below 13°C during fruit storage, and for optimal fruit condition it should be kept in the temperature range 14–21°C. During the ripening of green bananas in the commercial ripening rooms at the Brisbane fresh produce market, the lowest temperature the fruit is allowed to equilibrate to is 14.5°C (Photograph courtesy S.E. Hetherington)

With annual crops, the time of greatest risk is likely to be early in a growth season, and especially during seedling establishment. Chilling injury to seedlings can show up as necrotic lesions on the young roots and shoots, with slowed growth and increased susceptibility to disease attack, and even death. Crops adversely affected by low temperatures during the establishment period take longer to mature and this in turn may mean that they are at risk to chilling temperatures towards the end of the growing season.

Not only does chilling exposure retard the growth and maturation of crops, but chilling damage to fresh produce during postharvest storage is also of economic importance (Figure 14.17, and see Section 11.6.5). Chilling injury is a particular problem with fresh fruit, vegetables and flowers, because storage at temperatures low enough to retard tissue respiration is still the most effective postharvest method for extending the shelf life of produce. Even in produce-handling industries, there is often insufficient appreciation of requirements and behaviour of individual crops or even specific cultivars, and losses ensue. The time taken for symptoms to develop varies greatly and is influenced by a number of factors including genotype, cultivar, stage of maturity and preharvest growth conditions. For example, with fruit stored at 1–2°C, it takes several months for chilling injury to develop in apples as a brown discolouration of the cortex, several weeks for the flesh of peaches to become mealy in texture, a number of days for avocados to show areas of grey discolouration in the flesh, and only a few hours for cucumbers to display tissue breakdown in the mesocarp. Obviously storage at 0–2°C is an excellent method for extending the storage life of apples, is moderately useful for peaches, but disastrous for cucumbers. Avocados are better kept at a higher storage temperature; the recommendation for extended storage of avocados is 6°C. Even this temperature is too low for tomato, another sub-tropical species susceptible to chilling injury. Ripe tomatoes should be stored cool, at 12°C or above, and not at refrigerator temperature.

Chilling injury becomes apparent in a variety of ways (Table 14.1) that vary with species and tissue. Visible symptoms are outcomes of physiological disorders, and may develop slowly during the actual chilling period, to be expressed more rapidly once the tissue is returned to warmer, non-chilling conditions.

In defining the physiological basis of chilling injury, loss of membrane integrity emerges as a major symptom and much research has been directed to elucidating the chemical and physical nature of lipoprotein membranes of species having different climatic origins. Dr John Raison and other scientists at the former CSIRO Division of Food Research in Sydney provided evidence that physical changes occur in membranes of chilling-susceptible plants during low-temperature exposure. They suggested that the molecular ordering of membrane lipids is altered in the temperature range where chilling effects become apparent. In particular, lipid composition appears to determine how membranes respond to low temperatures. Tropical species tend to have lipids with a higher proportion of saturated fatty acids (these are fatty acids such as palmitic acid which lack double bonds in their structure and therefore have higher melting points), while cool-climate plants tend to have more unsaturated fatty acids such as oleic acid. However, a consistent pattern of differences in lipid membrane composition between chilling-susceptible and chilling-resistant plants has yet to emerge and additional factors are likely to be involved. The physical nature of cell membranes remains an important point for research into chilling injury, but as yet no single physiological factor has been linked with plant susceptibility to chilling injury.

14.4.2 - Quantifying chilling injury


Figure 14.18. Changes with time in in vivo chlorophyll a fluorescence induction kinetics in response to chilling. In chilling studies, chlorophyll fluorescence has commonly been measured as the rate of rise in fluorescence yield induced by illuminating dark-adapted tissue (the FR value). In isolated chloroplasts, a decrease in photosystem II activity is correlated with a decrease in FR (Smillie and Nott 1979). This figure shows the progressive decline in FR in a trifoliate bean (Phaseolus vulgaris L. cv. Canadian Wonder) leaflet chilled at 0°C in darkness. The first measurement of FR was made after allowing 30 min at 0°C for temperature equilibration of the leaflet. The measurement was repeated on the same area of the leaflet at the times indicated on the figure. The longer the time of` chilling, the greater the degree of chilling injury, and the slower the rate of rise of FR. The greater the chilling sensitivity of a cultivar, the shorter the time taken for a 50% decrease in FR (Original data courtesy R.M. Smillie)

One chilling response is a loss of membrane integrity. This loss has been measured by the extent of electrolyte leakage from cut pieces of chilled tissue. Other physiological methods used to quantify chilling injury include determinations of chlorophyll in seedlings kept at different temperatures, uptake of amino acids into pieces of chilled tissue, comparisons of fruit ripening rates and post-chilling measurements of plant growth and survival.

Pollen development is particularly sensitive to chilling temperatures, and assessments of pollen quality and anther length have been used to select specific genotypes of rice, tomato and other plants showing improved flower fertility under chilling conditions. However, though improved flower resistance to cold conditions is a desirable end-product in its own right, pollen resistance does not appear to be genetically linked with resistance of vegetative tissue to chilling stress.

A particularly versatile physiological method for following chilling stress in photosynthetic tissues makes use of in vivo chlorophyll a fluorescence. When plants become chill injured, fluorescence yield decreases (Figure 14.18) in response to effects of chilling on the photosynthetic system (Smillie and Nott 1979). The time taken for a 50% decrease in fluorescence has been used to compare the relative chilling tolerances of different species and cultivars. Using chilled maize seedlings the extent of the decrease in fluorescence has been positively correlated with physiological and visual symptoms of chilling injury (Hetherington and Öquist 1988).

14.4.3 - Ranges of chilling tolerance


Figure 14.19. Increasing chilling tolerance of wild species of potato and tomato with increasing altitude implies an adaptation to that location. Each point represents a different species of Solanum (●), or variant of Lycopersicum hirsutum (O), originally collected at the altitude indicated in the graph and grown under similar field conditions at sea level. Chilling tolerance determined by the chlorophyll fluorescence method. (Original data courtesy R.M. Smillie)

Plants are commonly reported in scientific literature as being either chilling sensitive or chilling tolerant. This can be a misleading simplification, because in practice when a range of plants are compared for chilling tolerance there is an almost continuous gradient of tolerance between the two extremes (Table 14.2). Tolerance of an individual species is likely to be related to the lowest prevailing temperature in the original habitat of that particular species.

Progressive changes in chilling tolerance have also been documented in closely related plants naturally distributed over latitudinal or altitudinal clines. Growth at high altitudes and also at high latitudes represents a selection pressure for cold tolerance. Wild species of potato restricted to ecological niches within narrow spans of altitude in the Andean mountains of Peru and Ecuador provide a good example of how chilling tolerance changes along an altitudinal cline. Variants of wild tomato (Lycopersicum hirsutum L.) collected in the same regions as the potatoes behaved similarly, with chilling tolerance increasing with altitude (Figure 14.19).

14.4.4 - Chill hardening


Figure 14.20. The ability to survive chilling stress is increased in chill-hardened maize seedlings. Unhardened, hardened and dehardened seedlings (see text) were chilled at 1°C for 3 d and then placed at 20/15°C for 3 d to allow symptoms of chilling injury to develop. (Based on Hetherington and Öquist, 1988)

Differences between species imply a genetic basis to variation in chilling tolerance, and this adaptive response includes a further element, namely acclimation. Tolerance shown by individuals of a particular species can be increased by exposing plants to progressively lower but only marginally chilling temperatures. This process is variously called ‘acclimation’, ‘hardening’ or ‘conditioning’. Chill hardening of plants appears to bring about changes in their metabolism, including an increase in unsaturation of membrane lipids, and allows plants to withstand subsequent and more severe stress. Such enhanced tolerance is generally lost within a few days of returning to warmer regimes, a process called ‘dehardening’.

Maize seedlings provide an example of this reversible process (Figure 14.20). Hardening has a dramatic effect on survival of seedlings subsequently exposed to a chilling stress of 1°C for 3 d. Seedlings were first hardened for 4 d in a 15°C day, 5°C night regime. Dehardening was achieved by returning the hardened plants to the original growth regime (20°C day, 15°C for 2 d). Chilling tolerance monitored by chlorophyll fluorescence measurements increased three-fold as a result of this hardening process.

In conclusion, chilling injury can occur in the field and in postharvest storage, especially when crop or horticultural species have been introduced from warmer climates. Produce losses due to chilling injury are frequently overlooked because symptom expression often takes several days to develop after the produce is returned to a non-chilling environment. A combination of better management and introduction of chilling-tolerant genotypes can reduce postharvest losses.

Further Reading:

Hetherington SE, Öquist G (1988) Physiol Plant 72: 241-247

Hetheringon SE, Smillie RM, Hardacre AK, Eagles HA (1983) Using chlorophyll fluorescence in vivo to measure the chilling tolerance of different populations of maize. Aust J Plant Physiol 10: 247-256

Smillie RM, Nott R (1979) Assay of chilling injury in leaves of alpine, temperate and tropical plants. Aust J Plant Physiol 6: 135-141