Leaf Respiration and Atp Levels at Chilling Temperatures

Leaf Respiration and Atp Levels at Chilling TemperaturesAuthor(s): J. M. WilsonSource: New Phytologist, Vol. 80, No. 2 (Mar., 1978), pp. 325-334Published by: Wiley on behalf of the New Phytologist TrustStable URL: http://www.jstor.org/stable/2433506 .

Accessed: 14/06/2014 20:12

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp

.JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact [email protected].

.

Wiley and New Phytologist Trust are collaborating with JSTOR to digitize, preserve and extend access to NewPhytologist.

http://www.jstor.org

This content downloaded from 185.2.32.21 on Sat, 14 Jun 2014 20:12:36 PMAll use subject to JSTOR Terms and Conditions

http://www.jstor.org/action/showPublisher?publisherCode=black

http://www.jstor.org/action/showPublisher?publisherCode=npt

http://www.jstor.org/stable/2433506?origin=JSTOR-pdf

http://www.jstor.org/page/info/about/policies/terms.jsp


New Phytol. (1978) 80, 325-334.

LEAF RESPIRATION AND ATP LEVELS AT CHILLING TEMPERATURES

By J. M. WILSON

School of Plant Biology, University College of North Wales, Bangor, Gwynedd LLS 7 2UW, U.K.

(Received 6 September 1977)

SUMMARY

In the extremely chill-sensitive species Episcia reptans the dark respiration rate of leaves after 80 min chilling' at 50C was three times greater than in leaves maintained at 250C. The rate and size of the respiratory increase depended on the chilling temperature and the growth conditions prior to chilling and was at a maximum at the same time as the onset of visible injury. The addition of 2,4-dinitrophenol and potassium cyanide to the leaves of E. reptans accelerated the rate of oxygen uptake at 5?C. In contrast, there was no increase in the respiration rate of the leaves of the less chill-sensitive species Phaseolus vulgaris after prolonged chilling at 50C.

A reduction in ATP supply below that necessary to maintain the metabolic integrity of the cytoplasm is not considered to be important in the development of chilling-injury to Episcia reptans or Phaseolus vulgaris as the ATP level decreased only after the onset of visible leaf injury. Furthermore, the increase in ATP level on chilling P. vulgaris leaves which had been either chill-hardened or maintained in a saturated atmosphere on transfer from 25 to 50C (Wilson, 1976) demonstrated that leaf injury and water stress are the primary cause of the decline in ATP level during chilling and not low temperature per se.

INTR,ODUCTION

Broadly speaking, chill-sensitive plants can be divided into two categories based on their sensitivity to chilling, their ability to chill- or drought-harden against chilling-injury and on whether injury can be prevented by maintaining a saturated (1 00% R.H.) atmosphere around the leaf (Wilson, 1976). Phaseolus vulgaris and Gossypium hirsutum are examples of category two species which can be chill- and drought-hardened to withstand chilling-injury and injury can be prevented on direct transfer from 25?C to 50C by maintaining a saturated atmosphere around the leaf. In contrast, the extremely chill-sensitive species Episcia reptans (category one) cannot be chill- or drought-hardened to withstand chilling-injury, nor can injury be prevented by maintaining a saturated atmosphere around the leaf.

The importance of water loss in relation to chilling-injury in the leaves of category two species such as Phaseolus vulgaris has recently been emphasized by Wilson (1976). However, the cause of chilling-injury to the more chill-sensitive Episcia reptans has been more difficult to elucidate. Although changes in respiratory behaviour have been widely investigated in relation to chilling-injury in fruits (Eaks and Morris, 1956) there have been comparatively few studies of changes in the respiration rate of chill-sensitive leaves at 50C, especially in very sensitive leaves such as E. reptans. In cucumber fruits, Eaks and Morris detected a

0028-646X/78/0300-0325 $02.00 ? 1978 Blackwell Scientific Publications 325



326 J. M. WILSON

doubling of the respiration rate after 8 days at 50C and this increase conincided with the onset and development of chilling-injury. This was followed by a decline in the respiration rate at the time of the general death of the tissue. Although Eaks and Morris were unable to explain the cause of the respiratory increase Creencia and Bramlage (1971) have shown, using the uncoupling agent 2,4-dinitrophenol, that the respiratory increase in Zea mays leaves after prolonged chilling for 24 to 48 h at 0.30C is partly due to the uncoupling of oxidative phosphorylation at chilling temperatures.

The effects of changes in respiration rate at 50C on ATP supply was also examined as Stewart and Guinn (1969) suggested that chilling-injury to Gossypium hirsutum (a category two species) may be due to low temperature inhibition of phosphorylation. They suggested that this inhibition may lead to a decrease in ATP supply below that necessary to maintain the metabolic integrity of the cytoplasm, thus causing injury. However, Stewart and Guinn did not separate the effects of low temperature from those of water stress at 50C. Wilson (1976) has demonstrated that water stress and chillinginjury to Phaseolus vulgaris leaves at 50C can be prevented for up to 9 days by enclosing the plant inside a polythene bag thus maintaining a saturated atmosphere around the leaves. Chill-hardening over a 4-day-period at 120C, 85% R.H. also prevents water loss and chilling-injury on subsequent transfer to 50C, 85% R.H. Therefore, this paper examines whether the reported decrease in ATP supply during chilling is due to water loss and leaf injury at 50C or low temperature per se. In addition, changes in the ATP level of Episcia reptans at 50C were followed as the removal of water stress at 50C does not prevent injury to this species.

MATERIALS AND METHODS

Growth and hardening conditions Plants of Phaseolus vulgaris, L. cv. Canadian Wonder were grown, hardened and chilled

under the growth cabinet conditions described previously (Wilson, 1976). Plants of Episcia reptans, Mart. were grown from cuttings for 8-12 weeks before use under summer glasshouse conditions of 250C to 350C and watered by an overhead mist spray to keep the leaves wet. E. reptans can only be grown successfully under conditions of high humidity. An attempt at hardening E. reptans against chilling-injury was made by progressively lowering the temperature from 250C to 130C by holding the plants under growth cabinet conditions for 5 days at each of the following temperatures 200C, 17.50C, 150C, 140C and 130C before transfer to 5?C.

Measurements of ATP level were made on leaves chilled under growth cabinet conditions in darkness so as to be comparable with the respiration measurements on leaf discs chilled in the dark in the Gilson respirometer. Plants were held for 12 h in the dark at 250C before transfer to 50C in darkness. However, the yellowing of Phaseolus vulgaris leaves held in the dark for long periods necessitated the prolonged chilling of the leaves at 50C, 100% R.H. and the prolonged chilling of the chill-hardened leaves to be performed under illuminated conditions.

Respiration measurements For these, chilling took place in a Gilson respirometer with a bath maintained at the

desired low temperature. Fifteen freshly cut leaf discs, each 1 cm in diameter, were placed in a Warburg flask with 1 ml of distilled water. The centre well contained 2 N KOH and a filter paper wick and the flasks were covered in aluminium foil to exclude light. Carbon dioxide



Leaf respiration and A TP levels 327

evolution was measured by the difference method using flasks containing no KOH in the centre well. The tissue samples were equilibrated for 30 min at each temperature before making measurements over the next 5 h. Tissue samples were run in triplicate. The effects of 1 mM 2,4-dinitrophenol (DNP) and 10 mM potassium cyanide (KSCN) on the respiration rate of Episcia reptans leaves at 50C was investigated by allowing the leaves to soak in the solutions for 80 min at 250C before chilling.

ATP, ADP and AMP measurem ents

Extraction. At each harvest the leaf tissue was immersed quickly in liquid nitrogen and transferred to a freeze drier. The dried tissue was then ground to a fine powder and stored at -200C over anhydrous CaCl2. 25 mg of the powdered leaf was added quickly to 5 ml of boiling distilled water for 1 min. The extract was then cooled rapidly in ice and centrifuged at 30,000 g for 15 min at 40C. The supernatant was used directly for assay.

ATP measurements. ATP was measured by the luciferin-luciferase technique as described by Bottomley and Stewart (1976) using an SL 30 liquid scintillation counter to measure the luminescence of the reaction. All reagents and standards were prepared in the same manner as Bottomley and Stewart and used for the assay within 2 h of preparation since the storage of ATP standards and experimental samples, even at 0?C, resulted in the loss of ATP.

Measurement of ADP and AMP. These measurements were performed using the method of Lin and Hanson (1974) and involved the conversion of ADP and AMP to ATP. The conversion of ADP to ATP used a coupled system of the following composition in a total volume of 0.6 ml. 25 ,umoles of Hepes buffer, pH 7.4; 1 pmole phosphoenolpyruvate; 8 units pyruvic kinase; 0.2 ml of extract or ADP standard. The mixture was incubated with shaking at 300C for 30 min and the total ATP estimated as described by Bottomley and Stewart (1976).

The conversion of AMP to ATP used a coupled system of the following composition in a total volume of 0.6 ml. 25 Mmoles Hepes buffer, pH 7.4; 1 pmole phosphoenolpyruvate; 8 units pyruvic kinase; 20 units adenyl kinase; 0.2 ml of extract or AMP standard and the analysis carried out as described above.

RESULTS

Respiration rates of Episcia reptans leaves Fig. l(a) shows that after only 80 min at 50C the oxygen uptake rate of E. reptans leaves

is three times higher than that of controls maintained at 250C. The rate of carbon dioxide production also increased to a maximum after 80 min at 50C but only to a maximum value of one third of the rate of oxygen uptake (Fig. lb). The respiratory peak after 80 min at 50C coincided with the first visible signs of the onset of chilling-injury. The early symptoms of chilling-injury in this species are the appearance of dark water-soaked patches on the leaf accompanied by a loss of leaf turgor. Plants chilled for 80 min at 50C showed few signs of injury on return to 250C. However, more prolonged chilling resulted in a gradual decline in respiration rate and an increase in leaf necrosis until after 5 h at 50C none of the leaves re- covered on subsequent transfer to 250C (Table 1).



328 J. M. WILSON

Chilling at temperatures higher than 5?C delayed the onset of the respiratory rise and reduced its height (Fig. 1 a). In leaves chilled at 100C the respiration rate was maximal after 100 min and peak height was reduced by one-fifth in comparison to leaves chilled at 5?C. The first visible signs of chiUling-injury at 10?C also occurred at approximately the same time as maximum respiration rate. The extreme chill-sensitivity of E. reptans is demonstrated by the 90% leaf injury incurred after 5 h at the relatively high chilling temperature of 10?C (Table 1). At 11 C there was an initial decrease in respiration rate followed by a gradual increase to the level of the control leaves maintained at 250C after 160 min (Fig. la). The development of chilling-injury was also slow at 11?C, the leaves incurring only 20% injury after 5 h chilling (Table 1), which indicates that 1 1?C is near the upper temperature limit for chilling-injury in this species. An upper temperature limit of 11-120C for chilling-injury in E. reptans is supported by the absence of any increase in the respiration rate of leaves held for 5 h at 12.50C (Fig. 1 a) and the development of only 5% leaf injury on transfer to 250C (Table 1). On transfer to 12.50C the respiration rate decreased to approximately one third of the rate of the control leaves at 250C and remained at this level over the 5 h period. However, prolonged chilling over several days at 12-15oC can cause chilling-injury to E. reptans (Wilson and Crawford, 1974).

30 - (a) 3*0 ( b)

25 A/A 25

T 2-0 -2.0 -

1 15 - 1.5 -

0 A

0 0- C) I

0 -5 -A-<'-0?5-

0 40 80 120 160 200 240 280 0 40 80 120 160 200 240 28(

Minutes chilling

Fig. 1. (a) Oxygen uptake and (b) carbon dioxide evolution in the dark by leaf discs of Episcia reptans chilled ( ) at 5'C (-), 10?C (a) and 11'C (X) in comparison to unchilled (----) leaf discs at 12.5?C (A) and 25?C (o).

Table 1. The development of chilling-injury in leaves of Episcia reptans

Values shown are a visual estimate of the percentage of the leaf area which was necrotic after 5 h chilling and 2 days

recovery at 25'C

Treatment % of leaf necrotic

Direct transfer from 25?C to 5?C 100 Direct transfer from 25'C to 10?C 90 Direct transfer from 25?C to 1 1'C 20 Direct transfer from 25?C to 12.5?C 5 Gradual transfer from 25?C to 130C

over 25 days before chilling at 5oC 100




In contrast to E. reptans there was no increase in the respiration rate of Phaseolus vulgaris leaves at 50C. At 50C the respiration rate of P. vulgaris leaves is approximately one twentieth of the rate of the control leaves at 250C and remained constant over a prolonged chilling treatment lasting 12 h.

It was considered that the respiratory increase in Episcia reptans leaves at chilling temperatures may provide a useful indicator of the speed of development of chilling-injury. Although earlier attempts at hardening E. reptans by a prolonged period of growth at 1 50C showed little increase in chill-tolerance (Wilson and Crawford, 1974) it was decided to in- vestigate whether gradually lowering the temperature from 250C to 130C over a 25-day period reduced the rate of the respiratory increase on transfer to 50C. Fig. 2 shows that plants treated in this manner have a slower increase in oxygen uptake and carbon dioxide evolution reaching a maximum value 4 h after the start of chilling, and a reduced peak

3 0 -

A

2-5- A t

2-0

(A~~ 0 ONJ 1-5*/0 o A

E X-- --A-_- 0 80 160 240 320 400 480

Minutes chilling

Fig. 2. The oxygen uptake (-) and carbon dioxide evolution (----) rates of leaf discs of Episcia reptans transferred directly from 25?C to 5?C (A) in comparison to plants slowly lowered from 25?C to 13?C over a 25-day period before transfer to 5?C (A).

height in comparison to plants transferred directly from 250C to 50C. However, it was not possible to correlate this delay in oxygen uptake and carbon dioxide evolution with a decrease in the rate of chilling-injury as plants gradually lowerd to 130C appeared very yellow and unhealthy at the start of the chilling treatment and were injured to the same extent on transfer to 50C as plants transferred directly from 250C to 50C (Table 1).

The effects of metabolic inhibitors on E. reptans leaves To determine whether the respiratory increase in E. reptans at 5?C was due to the un-

coupling of oxidative phosphorylation the effects of the uncoupling agent 2,4-DNP on the rate of oxygen uptake and carbon dioxide evolution at 50C were investigated. Fig. 3 shows that at 250C treating the leaves with 1 mM DNP doubled the oxygen uptake rate. However, transferring the DNP treated leaf discs to 5?C did not lessen the effect of DNP, as would be expected if chilling caused the uncoupling of oxidative phosphorylation. Fig. 3 shows that chilling the DNP treated leaf discs resulted in an extremely rapid three-fold increase in oxygen uptake within 40 min of the start of chilling whilst in the untreated controls at 50C the oxygen uptake rate did not attain its maximum value until 80 min after the start of chilling.



330 J. M. WILSON

The sensitivity of the leaves to KCN was also investigated. Fig. 3 shows that at 250C a 10 mM solution of KCN resulted in a 20%o reduction in respiration rate. However, at 50C the oxygen uptake rate of the KCN-treated leaves was accelerated in the same manner as the DNP-treated leaves (Fig. 3). A seven-fold increase in the oxygen uptake rate of KCN-treated leaves occurred within 40 min of the start of chilling.

Fig. 3 also shows that the rapid increase in the rate of oxygen uptake of the DNP and KCN-treated leaf discs at 50C is not accompanied by a higher rate of carbon dioxide production, although the maximum rate of carbon dioxide production occurred earlier, after only

4-5 -

4-0 250C 50C

3 30 0 600

-o ~~~~~~~~A / ~~~~~0

2-5 - 500

O2-0 - 400 E

0 - .5 -S < 300

A A~~~~~~~ 0-5 - 1~~~~~~~~~~ 00

0- Y)~~~~~~~~~~~~F 0

= ~ ~ ~ ~ ~ E

0 40 80 120 160 200 240 280 0 4 8 12 16 20 24

Minutes Hours chilled

Fig. 3. Fig. 4.

Fig. 3. The effects of 1 mM DNP (-) and 10 mM KCN (o) on the oxygen uptake (-) and carbon dioxide evolution (----) rates of Episcia reptans leaf discs at 5?C in comparison to untreated, control discs at 50C (A).

Fig. 4. Changes in the levels of ATP (-), ADP (o) and AMP (A) in the leaves of Episcia reptans during chilling in the dark at 5?C. Each point is the mean of three replicates.

40 minutes, in the DNP and KCN-treated discs then in the untreated discs. It was not possible to measure the rates of carbon dioxide production between 0 and 40 min after the start of chilling as this period was needed for the equilibration of the Gilson respirometer to 50C.

ATP, ADP and AMP levels in E. reptans leaves Although chilling leaves of E. reptans for 5 h at 50C produced 10%o leaf injury on return

to 250C there was no rapid decrease in ATP level during the first 5 h chilling. Fig. 4 shows that the ATP, ADP and AMP levels fell by only 25% after 5 h chilling. Prolonged chilling between 5 and 24 h resulted in a further gradual decline in ATP level and a stabilization of the ADP and AMP levels (Fig. 4).




A TP, ADP and AMP levels in Phaseolus vulgaris leaves Fig. 5(a) shows that chilling P. vulgaris leaves in the dark at 50C, 85% R.H. resulted in a

doubling of the ATP and ADP levels over the first 12 h and a rapid fall in the AMP level. In this experiment prolonged chilling from 12 to 48 h caused 40% leaf necrosis on return to 250C. Associated with the development of this injury between 12 and 48 h chilling there was a rapid decrease in ATP and ADP levels without any rise in AMP level (Fig. 5a). The development of chilling-injury in Episcia reptans and Phaseolus vulgaris leaves is therefore associated with a loss of adenosine phosphates.

Maintaining a saturated atmosphere on direct transfer from 25 to 50C has been shown to prevent chilling-injury to P. vulgaris for up to 9 days (Wilson, 1976). The level of ATP in

1200 - (a) 1200 - (b)

1000 0 1000

0~~~~~~ < 600 600

<X 400 4000

200! *\- \ 200 -

0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9

Days chilled

Fig. 5. Changes in the levels of ATP (-), ADP (o), and AMP (A) in the leaves of Phaseolus vulgaris, (a) during the development of chilling-injury and water loss on direct transfer from 250C to 50C, 85% R.H. and (b) during the prevention of chilling-injury and water loss by maintaining a saturated (100% R.H.) atmosphere around the leaves on transfer from 25?C to 50C. Each point is the mean of three replicates.

T 1200

1000 _

E)800 e |

< 600

C)400p

0:1\ 200 -

E I I I _ 0 1 2 3 4 5 6 7 8 9 10 11

Days hardening Days chilling 12?C, 85% R.H. 5?C, 85% R.H.

Fig. 6. Changes in the levels of ATP (*), ADP (o), and AMP (A) in the leaves of Phaseolus vulgaris during 4 days chill hardening at 120C, 85% R.H. in the light and on subsequent chilling at 5?C, 85% R.H. Each point is the mean of 3 replicates.



332 J. M. WILSON

the leaves chilled at 5?C, 100% R.H. doubled during the first 24 h chilling and was associated with a rapid decrease in AMP level (Fig. 5b). The levels of ATP and ADP did not fall on prolonged chilling between 1 and 6 days as injury was prevented by the maintenance of a saturated atmosphere around the leaf. Only after 7-8 days at 50C, 100% R.H. did the ATP and ADP levels decrease and this fall coincided with the first visible signs of injury to the leaves.

Chill-hardening for 4 days at 120C, 85% R.H. can prevent injury for up to 9 days at 50C, 85% R.H. (Wilson, 1976). Fig. 6 shows that transfer from 250C to 120C, 85% R.H. resulted in a gradual increase in ATP and ADP levels over the four day hardening period and a rapid decrease in AMP level. Subsequent transfer of the chill-hardened plants to 50C, 85% R.H. resulted in a further increase in ATP level but there was a fall in ADP level and the AMP remained at the same low level as at the end of the chill-hardening period.

DISCUSSION

The rapid rise in the respiration rate of Episcia reptans leaves and the onset of visible injury within 80 min of the start of chilling at 50C demonstrates the extreme chill-sensitivity of this species. The general pattern of respiratory disturbance in E. reptans leaves is similar to chilled cucumber fruits showing an increase in rate with the onset of injury followed by a decline in respiration rate at the time of the general death of the tissue (Eaks and Morris, 1956). However, in cucumber fruits the respiration rate increased much more slowly at 50C reaching a maximum value only after 8 days chilling. The speed and size of the respiratory increase in E. reptans leaves depends on their physiological condition before chilling (Fig. 2) as well as the chilling temperature so that the size of the respiratory increase is not a reliable method of estimating the chill-sensitivity of this species. In contrast to E. reptans the leaves of Phaseolus vulgaris (category 2) showed no increase in respiration rate at 50C. However, Creencia and Bramlage (1971) detected a 50% increase in the respiration rate of Zea mays leaf segments on prolonged chilling of 24-48 h at the very low chilling temperature of 0.30C. Nevertheless, the marked difference in respiratory behaviour between Phaseolus vulgaris and Episcia reptans at 50C emphasizes the very different nature of chilling-injury in these species and further supports the division of chill-sensitive species into two categories (Wilson, 1976).

The cause of the increase in oxygen uptake by E. reptans leaves at 50C was investigated using the metabolic inhibitors DNP and KCN and by following the changes in ATP, ADP and AMP levels. The rapid acceleration of respiration by the addition of DNP at 50C showed that this method could not be used to determine whether any part of the respiratory increase was due to the uncoupling of oxidative phosphorylation. The marked increase in the rate of oxygen uptake in the DNP-treated leaf discs of E. reptans at 50C is not accompanied by a higher rate of carbon dioxide production which indicates that the respiratory increase cannot be attributed to the uncoupling of oxidative phosphorylation at low temperature. However, the increased rate of carbon dioxide production by E. reptans leaves at 50C, although three times smaller than the increase in oxygen consumption, does indicate that part of the respiratory increase may be due to an increase in the rate of mitochondrial respiration. A respiratory quotient of 0.33 for E. reptans leaves at 50C is highly abnormal and may be caused by the peroxidation of membrane lipids.

A reduced ATP supply below that necessary to maintain the metabolic integrity of the cytoplasm at 50C is unlikely to be the cause of chilling-injury to E. reptans. Fig. 4 shows




that there is no rapid decrease in ATP level preceeding the onset of visible signs of chilling- injury in E. reptans leaves at 50C. The significance of changes in the ATP level in relation to chilling-injury in Phaseolus vulgaris was also investigated. The decrease in ATP level during the development of chilling-injury between 12 and 48 h chilling at 50C, 85% R.H. (Fig. 5a) is similar to that reported by Stewart and Guinn (1969) for cotton leaves chilled under conditions of water stress. However, these authors failed to detect any increase in ATP level during the early stages of chilling. An examination of the effects of the removal of water stress at 50C in P. vulgaris by maintaining a saturated atmosphere around the leaves also showed that the ATP and ADP levels increased rapidly during the first 12 h chilling and remained at this high level for up to 7 or 8 days by which time visible signs of leaf injury appeared (Fig. 5b). Similarly, the prevention of chilling-injury by hardening at 120C, 85% R.H. also prevented water loss and leaf injury on transfer to 50C, 85% R.H. and the ATP level remained high over many days chilling (Fig. 6). Therefore, water stress and subsequent leaf injury during chilling at 50C, 85% R.H. is the cause of the decrease in ATP level in P. vulgaris leaves and not low temperature per se. A reduction in ATP level on chilling leaves of P. vulgaris cannot be shown to be important in the development of chilling-injury as there is no rapid decrease in ATP level before the onset of visible leaf injury. The primary cause of chilling-injury to P. vulgaris leaves is leaf dehydration (Wilson, 1976). Increases in ATP level in P. vulgaris leaves at 40C may be due to the cold sensitivity of ATP-ase which is readily inactivated at low temperatures (Penefsky and Warner, 1965).

It is suggested that the cause of chilling-injury to Episcia reptans is a rapid change in the permeability of the cell membranes at 50C due to either protein denaturation (Brandts, 1967), lipid phase transitions (Lyons, 1972), changes in lipid-protein interactions (Yamaki and Uritani, 1974) or a combination of these events. This rapid change in permeability would account for the speed of chilling-injury to E. reptans and the rapid rise in respiration rate due to the loss of cell compartmentation which would allow enzymes increased access to substrates. Peroxidation of the membrane lipids either by autocatalysis or catalysed by lipoxygenase (Holden, 1970) and the aggregation of denatured proteins would result in irreversible damage to the cell membranes and leaf injury. The addition of DNP or KCN to E. reptans leaves on chilling is thought to increase the rate of oxygen uptake by decreasing membrane stability at low temperatures.

REFERENCES

BOTTOMLEY, P. J. & STEWART, W. 0. P. (1976). The measurement and signifilcance of ATP pools in filamentous blue-green algae. Br. phycol. J. 11,69.

BRANDTS, J. F. (1967). Heat effects on proteins and enzymes. In: Thermobiology (Ed. by A. H. Rose). Academic Press, London.

CREENCIA, R. P. & BRAMLAGE, W. J. (1971). Reversibility of chilling-injury to corn seedlings. PI. Physiol., Baltimore, 47, 389.

EAKS, I. L. & MORRIS, L. L. (1956). Respiration of cucumber fruits associated with physiological injury at chilling temperatures. PL Physiol., Lancaster, 31, 308.

HOLDEN, M. (1970). Lipoxidase activity in leaves. Phytochemistry, 9, 507. JONES, P. C. T. (1970). The effect of light, temperature, and anaesthetics on ATP levels in the leaves of

Chenopodium rubrum and Phaseolus vulgaris. J. exp. Bot. 21, 58. LIN, W. & HANSON, J. B. (1974). Phosphate absorption rates and ATP concentration in corn root tissue.

PI Physiol., Baltimore, 54, 250. LYONS, J. M. (1972). Phase transitions and the control of cellular metabolism at low temperatures.

Cryobiology, 9, 341. PENEFSKY, H. S. & WARNER, R. C. (1965). Partial resolution of the enzymes catalysing oxidative

phosphorylation. VI. Studies on the inactivation of mitochondrial adenosine triphosphatase. J. biol. Chem. 250, 4694.



334 J. M. WILSON

STEWART, J. MC D. & GUINN, G. (1969). Chilling-injury and changes in adenosine triphosphate of cotton seedlings. Pi. Physiol., Lancaster, 44, 605.

WILSON, J. M. (1976). The mechanism of chill- and drought-hardening of Phaseolus vulgaris leaves. New Phytol. 76,257.

WILSON, J. M. & CRAWFORD, R. M. M. (1974). The acclimatization of plants to chilling temperatures in relation to the fatty-acid composition of leaf polar lipids. New Phytol. 73, 805.

YAMAKI, S. & URITANI, I. (1974). Mechanism of chilling-injury in sweet potato. XI. Irreversibility of physiological deterioration. PI. Cell Physiol. 15, 385.



Documents

Leaf Respiration and Atp Levels at Chilling Temperatures