Pestic. Sci. 1978, 9, 173-1 83

Lipid Changes in Plants at Temperatures Inducing Chill-hardiness"

John M. Wilson

School of Plant Biology, University College of North Wales, Bangor

(Manuscript received 5 April 1977)

Phase transitions in the membrane lipids of chill-sensitive plants at 12°C and below are considered to result in an increase in the activation energy of membrane-bound enzymes and membrane permeability, leading to cell death. In agreement with this hypothesis, chill-hardening at 12°C results in an increase in the degree of unsaturation of the fatty acids associated with the membrane phospholipids of Phaseolus vulgaris and Gossypium hirsutum leaves. However, drought-hardening P . vulgaris leaves at 25°C is as effective in preventing chilling injury as chill-hardening at 12°C but drought- hardening produces no increase in the degree of unsaturation of the membrane fatty acids suggesting that lipid phase changes are not the primary cause of chilling injury. Furthermore, a re-examination of the conditions under which water and electrolytes leak from P . vulgaris leaves indicates that phase changes in the membrane lipids of the plasmalemma and other organelles at 5°C are only of minor importance in deter- mining the extent and speed of chilling injury. The primary cause of chilling injury to P . vulgaris leaves on transfer from 25T, 85 % r.h. to 5"C, 85 % r.h. is leaf dehydration due to the opening of the stomata at a time when the permeability of the roots to water is low. The primary factor inducing hardening against chilling injury in P . vulgaris is a water stress and not low temperature per se. Phase transitions in the membrane lipids of a chill-resistant species have now been detected within the same temperature range as those reported for chill-sensitive species. Therefore, the ability of temperate species to withstand chilling injury cannot be explained solely on the maintenance of a high degree of unsaturation of the membrane fatty acids.

1. Introduction

We live on a planet that can be described as predominantly cold because over two-thirds of the earth's land area experiences freezing (sub-zero) or chilling (0 to 10°C) temperatures at some period during the year. Therefore, low temperature is probably the most important ecological factor limiting the survival of wild and domesticated plants at the polar limits of their distribution. Only the relatively narrow band of land on either side of the earth's equator remains frost free throughout the year. However, many tropical and sub-tropical species are injured when their leaves are subjected to temperatures in the 0 to 10°C range. As these lethal temperatures are well above the freezing point and therefore distinct from frost killing this damage is usually referred to as chilling injury.l This inability of the cell to maintain itself as a functional unit at low temperatures imposes a major barrier to the polar migration of tropical plants.

In recent years research on chilling injury has been prompted by the problems of transporting tropical produce to the temperate regions. The chill-sensitivity of tropical fruits such as bananas requires that the cargo temperature should not fall below 12 to 15°C and yet not rise to ambient or above as the fruits would ripen too rapidly. The development of a method for lowering the storage temperature of tropical fruits below 10°C without injury would be of major economic advantage in

'I Presented at a meeting Physical mechanisms inuolred in plant growth regulation on 1 March 1977, organised by the Physicochemical and Biophysical Panel (Pesticides Group), Society of Chemical Industry.

0031-613X/78/04oO-o173 502.00 0 1978 Society of Chemical Industry

173

174 J. M. Wilson

prolonging the shelf and storage life of tropical produce. Chilling injury is also of economic import- ance in the cultivation of crops such as cotton in the Texas High Plains of the USA which are subject to occasional chilling periods in the Spring. Horticulturalists in this country are also familiar with the problems of chilling injury to crops of cucumbers and tomatoes when the air temperature falls below 10°C.

2. Hardening 2.1. Chill-hardening Broadly speaking, chill-sensitive plants can be divided into two categories based on their sensitivity to chilling, their ability to harden against chilling injury and on whether chilling injury can be prevented on direct transfer from 25 to 5"C, by maintaining a saturated (100% r.h.) atmosphere (Table 1). Category 2 plants, for example Phaseolus vulgaris, Gossypium hirsutum and Cucurnis sativus, can be chill-hardened by a 4-day acclimatisationperiodat 12°C which prevents chilling injury for up to 9 days on transfer to 5"C, 85 % r.h.2, Furthermore, drought-hardening these species by withholding water from the roots so that the leaves wilt over a 4-day period has been shown to be as effective as chill-hardening in preventing chilling injury.4 Injury to the leaves of these three species can also be prevented for up to 9 days on direct transfer from 25 to 5°C by enclosing the plant inside a polythene bag, thus maintaining a saturated (100 % r.h.) atmosphere.

Table 1. The division of chill-sensitive species into two categories based on their sensitivity to chilling, their ability to chill and drought-harden against chilling injury at 5"C, 85 % r.h., and on whether chilling injury can be delayed

on direct transfer from 25 to 5°C by maintaining a saturated (100% r.h.) atmosphere

Category 1. e.g. Episcia reptans, Nautilocalyx lynchii (a) Extremely chill-sensitive species which show injured spots after only 2 h at 5°C. (b) These plants cannot be chill-hardened at 12°C. 85% r.h. o r drought-hardened at 25"C, 40% r.h. to with-

stand chilling injury at 5°C. Even prolonged periods of acclimatisation at 15°C result in little increase in chill-tolerance.

(c) Maintaining a saturated atmosphere at 5°C does not delay the onset of injury.

Category 2. e.g. Phaseolus oulgaris, Cucumis satiuus, Gossypium hirsutum (a) Less chill-sensitive species usually incurring severe leaf injury after 24 h at 5"C, 85 % r.h. (b) Chill-hardening and drought-hardening can protect the leaves against chilling injury at S T , 85% r.h. for up

to 9 days in P . oulgaris. (c) Maintaining a saturated atmosphere at 5°C can prevent chilling-injury for up to 9 days on direct transfer

from 25 to 5°C.

In contrast, Category 1 species such as the extremely chill-sensitive Episcia reptans and Nautilo- calyx lynchii cannot be chill- or drought-hardened to withstand chilling injury. Even a prolonged period of acclimatisation of several months in a cool well-ventilated greenhouse at 15°C results in little increase in chill-tolerance. Tropical fruits also possess little ability to harden against chilling injury. Attempts at hardening sweet potatoes have not been successful in reducing chilling injury5 and hardening of cucumbers is only effective against slight chilling.6 The leaves of E. reptans show injured spots after only a few hours at 5°C and the onset of injury cannot be delayed by maintaining a saturated (100% r.h.) atmosphere at 5°C.

2.2. Prevention of lipid phase transitions by hardening A role for lipids and fatty acids in the prevention of chilling injury is suggested by increases in the degree of unsaturation and often the weight of lipid during the acclimatisation of plants, as well as poikilotherrnic and homeothermic animals, to low temperatures (Table 2). Research performed on the chill-sensitivity of tropical fruits and roots has also indicated that the site of chilling injury lies in the lipid region of the cellular membranes. Lyons et al.7 investigated the rate of succinate oxidation of mitochondria isolated from chill-sensitive and chill-resistant fruits, roots and buds at different temperatures. They detected an increase in the slope of Arrhenius plots of succinate oxidation in the

Lipid changes in plants at low temperatures 175

Table 2. Increases in the unsaturation of fatty acids during the acclimatisation of organisms to chilling and freezing temperatures

Acclimatisation Fatty acid or lipid temperature

Species fraction analysed ("C) Reference

Plants: Escherichia coli Anacystis nidulans Cyanidium caldarium Chlorella sorokina Medicago satiua Triticum aestiuitm Populus euramericana Hordeum uulgare Gossypium hirsutrrm Phaseolus uulgaris

Animals : Crustacean plankton Calliphora erythrocephala

Lampito mauritti (earthworm)

Carassius airratus (goldfish)

Rana esculenta (frog)

(blow-fly)

Mesocricetus auratiis

Rangifer tarandus (hamster)

(reindeer)

Total fatty acid Total fatty acid Phospholipid and glycolipid Total fatty acid Total fatty acid of the roots Total fatty acid of seedlings Phospholipid of stem bark Phospholipid of the leaves Phospholipid of the leaves Phospholipid of the leaves

Total fatty acid Phospholipid

Phospholipid

Total mitochondria1

Phospholipid and neutral fatty acid

lipid of liver and adipose tissue

Total fatty acids of sub-cutaneous fat

Total fatty acids of marrow fat

10.0 26.0 20.0 22.0

-2 .5 2 . 0 0 . 0

12.0 12.0 12.0

2 . 8 12.0

20.0

10.0

7 . 0

6 . 0

12.0

31 32 33 34 35 36 37

3 3 3

38 39

40

41

42

43

44

chill-sensitive tissues at approximately 12°C. Similar increases in slope and therefore activation energy were not detected in the mitochondria isolated from chill-resistant species at temperatures below 12°C. Fatty acid analyses of the mitochondria showed that those from the chill-resistant plants contained a greater percentage of unsaturated fatty acids than the mitochondria from chill- sensitive tissues. This study suggested that the fatty acids of chill-sensitive species undergo a temperature dependent change of phase from a liquid-crystalline to a solid gel state at a critical temperature within the chilling range (Figure 1). At 25°C the fatty acid chains are in a flexed and fluid state whereas at 5°C the fatty acids solidify and their packing becomes rigid and ordered. Similar transitions were not considered to occur in chill-resistant plants at temperatures in the 0 to 10°C range because their cell membranes contain a higher proportion of unsaturated fatty acid which reduces the transition temperature to below 0°C. This conclusion was supported by the investigations of Lyons and Asmundsons on the solidification temperatures of mixtures of saturated and unsaturated fatty acids. Plant cell membranes usually contain at least 70% of their fatty acids in the unsaturated form and Lyons and Asmundson showed that, at this concentration, a 10% in- crease in unsaturation could lower the solidification temperature by 20°C. In agreement with this theory Wilson3 reported increases of 5 to 12% in the degree of unsaturation of the fatty acids asso- ciated with the phospholipids of P. vulgaris and G. hirsuturn leaves during chill-hardening at 12°C. Phase transitions in chill-sensitive plants at 12°C and below are thought to result in chilling injury by increasing the activation energy of membrane-bound enzymes and the permeability of mem- branes to water and electrolytes.

12

176

Polor heod of the phospholipids

J. M. Wilson

SOLID GEL

I -

-Fatty acid toils of the phospholipids

Protem

Phose temp. 5°C tronsltlon Chilltng 1

I lncreosed membrane

I Increase In E, of membrane -bound permeablllty enzymes

Imbalance 1 of // Water loss 1 ond ion

metobolwn leokoge

t t

PROLONGED CHILLING

Loss of cellular comportmentatlon due to membrane permeoblllty and dehydrotlon.

t

increased

Breakdown of the cellulor membranes due to phosphollpose ond goloctolipase o c t ~ v ~ t y ond llpld peroxldotlon.

Loss of reverslblllty of the phase change, t t

INJURY AND DEATH OF THE LEAVES

Figure 1. A schematic pathway of the events leading to chilling injury in leaves according to the phase change theory. (The + and - signs indicate the ionic residues of the proteins).

3. Consequences of lipid phase transitions

3.1. Increased activation energies Further experimental support for the idea that lipid phase transitions are responsible for increases in the activation energy of membrane-bound enzymes from chill-sensitive plants at 5°C has been obtained from studies of non-membrane bound enzymes and membranes depleted of lipid by detergent. Raisong detected no increase in the activation energy of non-membrane bound enzymes such as monoamine oxidase and malic dehydrogenase from chill-sensitive tissues at 5°C. Although these enzymes were isolated from chill-sensitive animal tissues it is considered that non-membrane bound enzymes in plant cells behave in a similar manner at low temperature. In addition, Raison et af.10 have shown that the increase in the activation energy of membrane-bound enzymes such as succinic dehydrogenase in sweet potato mitochondria at 15°C can be abolished by treating the mitochondria with detergent.

Increases in the activation energy of NAD reduction by the chlorplasts of P. vulgaris leaves11 and impaired phosphorylation in C. hirsutum leaves12 at chilling temperatures have also been attributed to lipid phase transitions. It is suggested that these changes lead to the degeneration of the chloro- plast structure and a level of ATP which would be insufficient to maintain the metabolic integrity

Lipid changes in plants at low temperatures 177

of the cytoplasm. Furthermore, increases in the activation energy of membrane-bound enzymes may lead to chilling injury by producing metabolic imbalances with non-membrane bound systems such as glycolysis. A reduction in the tricarboxylic acid cycle activity below 12°C due to a lipid phase transition without a similar reduction in the rate of glycolysis might lead to the accumulation of the end products of glycolysis (i.e. ethanol and acetaldehyde) to toxic levels, resulting in cell death. In agreement with this theory Murata13 detected an increase in the ethanol and pyruvate content of chilled banana pulp and Wilson1* found an increase in the ethanol level of E. reptuns leaves at 5°C.

Other research workers have not been able to attribute increases in the activation energy of membrane-bound enzymes to lipid phase transitions at chilling temperatures. Increasing the degree of unsaturation of rat liver mitochondria by feeding the rats on a highly unsaturated fatty acid diet did not lower the temperature at which the phase transition occurred.15 In addition, the chill-harden- ing of cotton plants only partially prevents the rapid decrease in the respiration rate of root mito- chondria at 5"C.16

3.2. Increased membrane permeability Physical changes in membrane structure such as the liquid-crystalline to gel transition at chilling temperatures can be expected to alter membrane permeability. An increase in membrane perme- ability would be expected to accompany the lipid phase transition due to (a) a decrease in membrane thickness, (b) changes in the structure of the hydrocarbon chains important for diffusion across the membrane or (c) changes in the arrangement of the polar head groups important for the entry of permeants into the membrane.'7 In addition it is thought that 'cracks' or 'channels' may appear in the membrane at low temperatures due to the solidification of the lipid, thereby increasing mem- brane permeability.

The majority of studies on membrane permeability at chilling temperatures have measured permeability by the rate of electrolyte leakage from chill-sensitive tissues. Lieberman et d . 1 8 were the first to show that the rate of leakage of ions, mainly potassium, from sweet potato discs was increased at 7.5"C. In addition, Christiansenlg and GuinnzO detected an accelerated rate of leakage of electrolytes, proteins and carbohydrates from chilled cotton roots and cotyledons. Katz and Rein- holdz1 detected an increase in the permeability of Coleus petioles at 0°C by measuring the conduct- ivity of sections of petiole with electrodes inserted into the tissue. However, in all these studies, the rate of leakage only became significantly greater after many hours at the chilling temperature and this argues against any rapid rise in permeability which can be attributed to lipid phase transitions.

More recent studies by Wright and Simonz2 and Wilson4 have investigated the leakiness of chilled leaves of C. sutivus and P . vulgaris by measuring the rate of electrolyte leakage from leaves immersed in distilled water. Leaves of P . vulgaris leak electrolytes if they are chilled in air for 24 h at 5"C, 85 % r.h. so that the leaves wilt before they are transferred to distilled water at 25 or 5°C. Figure 2 shows that the rate of leakage is almost twice as fast from leaves chilled for 24 h at 5"C, 85 % r.h. if they are immersed in distilled water at 25°C compared to water at 5°C. This may be due to a heat shock on transfer from 5 to 25°C. In contrast, the control leaves transferred directly from 25°C to water at 25 and 5°C show a very slow rate of electrolyte leakage. This result is contrary to the phase change theory. If the leakage of electrolytes from the leaves chilled in air at 5"C, 85 % r.h. is due to a phase transition in the membrane lipids then we would expect the leaves to leak electrolytes when transferred directly from 25°C to water at 5°C (i.e. without chilling in air). However, Figure 2 shows that the leakage of electrolytes from unchilled leaves of P . vulgaris transferred directly to water at 5°C is very slow and that leakage is faster from the control leaves transferred directly to water at 25°C-the opposite effect to that predicted by the phase change theory.

Therefore, in leaves, considerable water loss and membrane damage must occur during chilling in air at 5"C, 85% r.h. for the leaves to lose their electrolytes rapidly when transferred to water. Although studies on the rates of leakage of electrolytes are useful in assessing the degree of damage to chilled tissues it is not possible to relate this increased leakiness directly to a phase transition in the membrane lipids at low temperatures. For example, leaves of the extremely chill-sensitive species, E. reptuns, transferred directly from 25°C to water at 5°C leak electrolytes rapidly (Figure 3) but visible signs of cell death accompany this rise in conductivity of the water so that it is not

J. M. Wilson

A

Hours after immersion in water

I40

I20

I00

- ln

=t 80 % .- - ? .- " < 60 0 V

40

20

P /

/ /

F' /

/ /

d /

/

a 6 12 18 24 Hours ofler immersion in water

Figure 2. Leakage of electrolytes from leaves of Phaseolus aulguris chilled at 5"C, S 5 % r.h. for 24 h ( A); in comparison to unchilled leaves ( 0) placed in water at 25°C (-), or 5°C (- - -). ( B) Denotes leak- age from either chill- or drought-hardened leaves Wilson.4 which had not been chilled at 5"C, S 5 % r.h. before immersion in water at 25°C. Based on Wilson.J

Figure 3. Leakage of electrolytes from leaves of Episciu repruns transferred directly from 25"C, S 5 % r.h. to water at 25°C (-), or 5°C (- - -). Total quantity of electrolytes in leaves= 175 pS. Based on

possible to differentiate whether leakage is an immediate effect due to a phase transition in the membrane or whether leakage is a secondary effect due to cell death.

The evidence for an increase in membrane permeability as a result of lipid phase transitions is weak and examples of decreased permeability can be found. It is known that the rate of water absorbtion by the roots is reduced more at low temperatures in chill-sensitive than chill-resistant species.23 This may be due to a decreased permeability of the root cells as a result of a lipid phase transition as well as an increase in the viscosity of water at low temperature. Furthermore, vesicles of Escherichia coli pre-loaded with labelled proline at high temperatures did not lose their radio- activity when incubated at temperatures below the tran~ition.2~

Therefore, it is not clear whether the fatty acid composition (ratio of unsaturated/saturated) of the membrane lipids in higher plants has any dominant role in determining the physical state and phase change in response to decreasing temperature or to what extent other components such as c h ~ l e s t e r o l ~ ~ influence the physical state and presence or absence of the phase change. Investigations on the changes in the fatty acid composition of P . vulgaris leaves during chill and drought-hardening have indicated that the degree of unsaturation of the membrane lipids is not related to their chill- tolerance.

4. The cause of chilling injury to Phaseolus vulgaris leaves In agreement with the phase change theory the degree of unsaturation of the fatty acids associated with the membrane lipids of P. vulgaris and G. hirsutum leaves increases during hardening at 12°C. The figures in Table 3 show that the degree of unsaturation of phosphatidyl choline increases by approximately 10 % during chill-hardening. Similar increases in unsaturation occurred in the other phospholipids analysed but no increase in the degree of unsaturation of the glycolipids was detected.4 However, it has been demonstrated that leaves of P . vulgaris can be drought-hardened over a 4-day period at 25°C by withholding water from the roots until the leaves wilt and that these drought- hardened leaves are as resistant to chilling injury as leaves chill-hardened for 4 days at 12°C. During drought-hardening at 25°C there is no increase in the degree of unsaturation of the phospholipids

Lipid changes in plants at Low temperatures 179

Table 3. Changes in % fatty acid composition of phosphatidyl choline from leaves of Phaseolus vulgaris during chill-hardening at 12"C, 85 % r.h. and drought-hardening at 25"C,

40% r.h.a

Fatty acid composition of phosphatidyl choline (%)

Chill-hardened at 12"C, Drought-hardened at 25T, Fatty acidb Control 85 % r.h. for 4 days 40 % r.h. for 4 days _ _ ~ _ _ _

14:O 3.8 2.1 5.3 16:O 20.4 12.8 24.3 16: 1 0 .9 1 .o 0.1 16:2 0 .9 1 . 1 0 .4 18:O 6 .5 4 . 3 6 .2 18:l 4 .0 3 .5 2 .0 18:2 27.5 40.0 24.1 18:3 36.0 35.2 37.0

Total % unsaturated 69.3 80.8 64.2 fatty acid

Based on Wi l~on .~

bonds in the molecule. b The numbers shown are the ratios of the number of carbon atoms to the number of double

or glycolipids. Table 3 shows that there is a slight decrease in the total proportion of unsaturated fatty acid associated with phosphatidyl choline during drought-hardening so that according to the phase change theory we would expect the drought-hardened plants to be more chill-sensitive and not chill-resistant.

The phase change theory is also unable to account for the prevention of chilling injury to leaves of P. vulgaris by enclosing the plant inside a polythene bag before transfer to 5°C. Plants maintained in a saturated (100% r.h.) atmosphere in this manner can withstand direct transfer from 25 to 5°C for up to 9 days.4 During this period the leaves do not wilt or leak electrolytes. If lipid phase transitions resulted in an increase in the permeability of the plasmalemma of the leaf cells at 5°C then we would expect the cells to wilt on transfer to 5"C, 100% r.h., as the turgor pressure of the cell would facili- tate the loss of water and electrolytes. In addition, the prevention of chilling injury simply by enclosure in a polythene bag indicates that metabolic changes such as impaired phosphorylation12 and NAD reduction11 can only play a minor role in the development of chilling injury as the leaves can function without visible damage for up to 9 days at 5"C, 100% r.h.

A re-examination of ATP supply in chilled leaves of P. vulgaris showed that the level of ATP increased in leaves chilled at 5"C, 100% r.h. over a 9 day period and that the level of ATP only decreased in the wilted and damaged leaves chilled at 5"C, 85 % r.h. (Figure 4). Although the leaves chilled at 85% r.h. were approximately 50% injured after 24 h the level of ATP had decreased by less than 33 %, indicating that a fall in ATP supply is not the cause of cell death at 5°C. It appears that the impaired phosphorylation of cotton leaves at 5°C reported by Stewart and Guinn12 is attributable to the effects of water stress and not low temperature per se.

Chill-hardening of P. vulgaris leaves at 12°C is not effective if the plants are maintained at 100% r.h.4 Although the degree of unsaturation of the phospholipids increased during the Cday enclosure at 12"C, 100% r.h. there was no increase in chill-tolerance indicating that chill-tolerance is not dependent on a highly unsaturated fatty acid composition.

It has been demonstrated that the primary cause of chilling injury to P. vukuris leaves at 5"C, 85% r.h., is water loss due to the rapid opening of the stomata at a time when the permeability of the roots to water is low.4 The opening of the stomata after 2 h at 5"C, 85% r.h., (Figure 5 ) is surprising as the leaf is wilted and in most plants the stomata close in the early stages of water stress before visible wilting occurs. The replacement of the water lost by evapotranspiration from the leaf

180 J. M. Wilson

10.00 2l 12.00 14.00 16.00 18.00 20.00 22.00

\

0 1 2 3 4 5 6 7 8 9

Doys chllled G i 5°C Time of doy

Figure 4. Changes in the level of ATP in Phaseolits oulgaris leaves during chilling at 5"C, 85% r.h. ( e), and 5"C, 100% r.h. ( 0).

Figure 5. Changes in stomatal aperture on trans- ferring entire plants OfPhnseolus oulgaris directly from 2 5 T , 8 5 % r.h. to (a) S T , 8 5 % r.h. (A), (b) 12°C. 85% r.h. ( e), compared to the controls maintained at 25°C. 85 % r.h. ( 0). Arrow shows start of night period. Based on Wilson.4

0.4 - - L g 0.1 - I JZ

E

$ 0.04 5: n

- u

L

0.02 B

0.01

0.1

0.01 - 34 35 36

I04/Root temperature ( K l)

Figure 6. Arrhenius plots of the effect of root temperature on the rate of water absorption by fhaseolus vulgaris plants grown at 25"C, 85 % r.h. ( A), chill-hardened at 12"C, 85 % r.h. ( e), and drought-hardened at 25°C. 40% r.h. ( 0). (a) Shows the rate o f water uptake plus exudation under 50 cmHg vacuum and (b) the rate of exudation alone. Each point represents the average value f rom at least five plants. Based on Wilson.?

is prevented by the low permeability of the roots to water at 5°C (Figure 6) resulting in rapid leaf dehydration and injury. Hence, the severity of chilling injury depends on a synergistic effect between stomatal opening and reduced permeability of the roots to water at 5°C.

5. The chill-hardening mechanism

Chill-hardening at 12"C, 85 % r.h. prevents leaf dehydration by conditioning the stomata so that they close on transfer to 5"C, 85 % r.h. (Figure 7). Similarly, drought-hardening causes stomatal closure and the stomata remain closed on transfer to 5"C, 85% r.h. The most important factor in the

Lipid changes in plants at low temperatures 181

i 2

10

- 0 c

Figure 7. Changes in stomatal aperture of Phuseolus oulgaris plants hardened at 1 2 T , 85 % r.h. ( a), and ineffectively hardened at 1 2 T , 100% r.h. (by enclosure in a polythene bag) ( W), on chilling at 5"C, 8 5 % r.h. compared to the controls maintained at 2 5 T , 85 % r.h. ( 0). Arrow shows start of night period. Based on Wilson.4

4

2

10 I 1 I I 1 I I

I0 12.00 14.00 16.00 18.00 20.00 22.00

Time of day

prevention of chilling injury to P. vulgaris during chill- and drought-hardening is the closure of the stomata. This can be demonstrated by spraying the leaves of plants grown at 25°C with 100 p~

abscisic acid which causes stomatal closure within 24 h. On transfer to 5"C, 85% r.h. the sprayed leaves do not wilt as the stomata remain closed and injury is prevented for approximately 2 days by which time the effectiveness of the abscisic acid has decreased.

With the above information it is possible to explain the mechanism of chill-hardening. At 12"C, 85 % r.h. the plant experiences a water stress (as shown by the temporary wilting of the leaves) due to the opening of the stomata (Figure 5) and a decrease in the permeability of the roots to water (Figure 6). However, at the inteLmediate temperature of 12°C the stress is not severe enough to result in damage and the wilting vanishes after 12 h. Similarly, during drought-hardening the water stress is imposed simply by withholding water from the roots under conditions of high evapo- transpiration so that the leaves wilt. In contrast, plants maintained at 12"C, 100% r.h. do not harden because they experience no water stress. Even though the stomata are open under these conditions no water can be lost from the leaf so that the stomata remain fully open on transfer to 5"C, 85 % r.h. (Figure 7). Therefore, enclosure in polythene bags is not a method which can be used to lower the hardening temperature below 12°C.

The correlation between chill- and drought-hardening has shown that an intermediate tempera- ture of 12°C is not essential for hardening. Therefore, a water stress and not low temperatureper se is the primary factor inducing hardening against chilling injury in P. vulgaris. Phase transitions in the membrane lipids can only be of minor importance in the development of chilling injury since it has not been possible to demonstrate that they increase membrane permeability at 5°C. Furthermore it appears that increases in the activation energy of membrane-bound enzymes at low temperature may only be important in the development of chilling injury after 9 days at 5"C, 100% r.h.

The death of P. vulgaris leaves after 9 days at 5"C, 100% r.h. is probably due to several factors. It is well known that photosynthesis is more sensitive to low temperature than respiration is so that starvation of plant tissue may occur. Translocation is inhibited in chill-sensitive species at 5"CZ6 which may lead to the starvation on non-photosynthetic parts of the plant and the accumulation of starch in the chloroplast may further inhibit photosynthesis. Photo-oxidation of chlorophyll and membrane lipids has been shown to occur in chill-sensitive species maintained at high light intensities at 5°C.27

6. Lipid phase transitions in chill-resistant species Recent research on the acclimatisation of temperate wheat (Triticum aestivum) to withstand frost- injury has also questioned the importance of lipid phase changes in the development of chilling injury to tropical species. Miller et al.28 have investigated the occurrence of lipid phase transitions in

182 J. M. Wilson

wheat mitochondria by electron-spin resonance (ESR). From the spectra obtained by ESR they calculated a relative motion parameter which provides a measure of molecular restraint within the membrane. Using this technique Miller et al. have detected three temperature-dependent changes in the slope of Arrhenius plots of the motion parameter as the temperature is decreased. These phase transitions occur at approximately 38,20 and 8°C in both the frost hardy Kharkov wheat and the unhardy Capelle wheat grown at 24°C. Hardening these 2 varieties against frost by a period of growth at 2°C increased the proportion of unsaturated fatty acid associated with the mito- chondrial phospholipids by approximately 15 %. In spite of this increase in unsaturation, frost- hardening at 2°C did not lower the temperature at which the phase transitions occurred. Further- more, Arrhenius plots of the respiration rate of mitochondria isolated from both hardened and unhardened wheat roots have shown breaks at approximately 10 to 12°C. The detection of lipid phase transitions and breaks in Arrhenius plots in wheat tissues within the same temperature range as those reported for chill-sensitive species casts further doubt on the importance of these changes in the development of chilling injury to tropical species.

7. Protein changes at chilling temperatures

Increases in the activation energy of enzyme reactions at 12°C and below in chill-sensitive plants may be the result of decreased protein stability due to ‘cold denaturation’ and inactivation of the enzyme. BrandtsZ9 has listed several examples of low temperature denaturation of proteins and he speculates that it is the weakening of the hydrophobic bonds that causes denaturation at low temperature. The minimum temperature for growth of a species could therefore reflect the tempera- ture at which the enzyme most sensitive to low temperature undergoes cold denaturation. Injury may be accelerated by the irreversible aggregation of denatured enzyme due to the oxidation of thiol groups and the formation of strong sulphur-sulphur bonds between protein molecules.30

8. Conclusion

Chilling injury to hardenable species such as P. vulgaris cannot be solely attributed to a phase transition in the membrane lipids and the degree of unsaturation of the fatty acids is not a reliable measure of chill-tolerance. Increases in unsaturation of the fatty acids in these plants at low tempera- tures cannot be directly related to the prevention of chilling injury and may only be a general re- sponse to the low temperature growth conditions. The increase in the linoleic acid content in P. vulguris leaves during growth at 12°C is probably the result of altered desaturase activity. Protein denaturation and changes in lipid-protein interactions may have an important role in enzyme and membrane stability at chilling temperatures, especially in extremely chill-sensitive species such as E. reptuns which cannot be hardened to withstand chilling injury.

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