Supercooling and heterogeneous nucleation of freezing in tissues of tender plants

16, 74-77

CRYOBIOLOGY

(1979)

Supercooling

and Heterogeneous Nucleation in Tissues of Tender Plants H. MARCELLOS

Agricultural

Research

Centre,

AND

R.M.B.

MATERIALS

AND

March

22,

METHODS

1978;

accepted

June

2,

74 OOll-2240/79/010074-04$02.00/0 Copyright All rights

0 1979 by Academic Press, Inc. of reproduction in any form reserved.

N.S.W.

2340, Australia

number of frost tender plants and simultaneously cool them while observing and recording the temperatures at which they froze. The plant species used were spring wheat (T&cum aestivum L.), potato (Solarium tuberosum L.), lucerne or alfalfa (Medicago sativa) french bean (Phaseolus vulgaris L. ), field pea (Pisum sativum L.), and broad bean ( Vicia faba L.). Plants were raised in a greenhouse under 15 hr photoperiod, and 2O/lO”C day/ night temperature, and were sampled during active growth to provide groups of stem pieces 2 cm in length. Tests were carried out in a small freezing chamber at cooling rates in the range 1 to 1.5”C per minute. A cooling run involved 24 tissue pieces of one cultivar or species and lasted 15 to 20 min; it was concluded when all tissue segments had frozen. For sample mounting, 24 thermocouples of 0.25 mm gauge copper-constantan wires, were arranged upright in a grid through a 15 cm square acrylic stage located in the center of the chamber. These were lightly coated with epoxy resin, and then with paraffin. A canopy made from thin polyethylene film was supported above the stage to intercept any ice crystals that might dislodge from the chamber roof. Conditions within the chamber precluded the formation of dew on samples since water vapor was transferred to its walls at -25°C; relative humidity was in the range 70 to 80%.

The approach adopted in this study was to take groups of tissue segments from a Received 1978.

W. V. SINGLE

944, Tamworth

The ability of tissues of many plants to undergo supercooling appears widespread, especially under conditions when external sources of ice nucleation, such as frost, are absent. This occurs because cells do not contain effective nucleators of intracellular water (3, 11) and extracellular sites for nucleation may also be ineffective, especially at warm temperatures near zero. The few papers dealing with this subject point to a varying nature and efficacy of nucleators in tissues (2, 5-8). A result of the above in nature is that external sources of nucleation are believed to initiate freezing in extracellular water. this does not always occur, However, especially under the conditions of short duration spring and autumn frosts often encountered by tender crop plants. Limited supercooling has been reported to afford protection to olive leaves (9), eucalyptus leaves ( 13), azalea buds (4), corn leaves ( 1)) and young ears of winter cereals ( 12). Factors affecting the stability of supercooled water in frost tender plants have received little attention. The purpose of the work reported here was to study the variation in supercooling and ice nucleation in tissues from a range of crop plants.

of Freezing

NUCLEATION

AND

FREEZING

Tissue samples were mounted so that the thermocouple junction was inserted from one end approximately 1 cm into the hollow lumen as in wheat stems, or into the pith of solid stems after puncturing with an alcohol and flame cleaned, paraffin coated dissecting needle. The freezing of a population of 1 mm diameter drops of distilled water was also investigated to provide a reference for comparing the behavior of biological samples. Drops were placed on paraffin in contact with the thermocouples using a small stainless steel syringe. The temperature of each sample was monitored on a potentiometric recorder and its freezing point taken at the initiation of the exotherm produced on freezing. Several tests ruled out the possibility of artifacts due to the thermocouples, sample size, sample mounting, and tissue injury. Refreezing samples did not alter the mean freezing temperature, showing that tissue injury was not important. There were no differences in mean freezing temperatures between samples adhered to the thermocouples with paraffin, and those impaled. There was no effect of sample size which was varied from 3 to 20 cm in length.

l

O\

OF TENDER

. water

l

l

\\o ,

-20

-16

-12

ItMPtRATIJRE

FIG. 1. Differential samples.

\ O0

4.

rapeseed

nucleus

1

Temperatures

Plant

of Plant Samples Freezing

temperature (“C)

MGLIl

Standard deviation

Broad bean French bean Field pea Lucerne (alfalfa) Rapeseed cv. Torch cv. Midas cv. Sinus cv. Rapid0 Potato cv. 2501 Wheat cv. Gamut cv. Timgalcn

- 5.6 - 7.1 - 7.2 - 10.3 - 10.6 - 11.5 -11.6 -11.4 -11.7 - 13.2 - 12.8 - 14.4

1.99 0.96 0.76 1.98 1.54 0.83 1.43 1.64 1.85 1.34 1.04 1.23

Distilled

- 17..5

2.a

water

A number of freezing runs were carried out for each plant species, and the results pooled since tests for heterogeneity of variance, and difference between means were not significant. A differential nucleus spectrum was derived for each species or cultivar according to the method of Vali (14). The spectrum is a plot of the minimum concentration k( 19) of nuclei per average sample active in a given temperature interval and is derived from the following equation -A In (1-

AN/N(~)),

where k( 0) is the minimum concentration of nuclei per average sample active in the temperature interval f3 --ho, AN is the number of samples freezing in 13-A8, N( 8 ) is the number of samples unfrozen at 0, and A is a constant that includes volume of sample and temperature interval.

‘.

Oftenchbean

75

TABLE Mean Freezing

k(e)=

3 v/t-eat

PLANTS

.%

RESULTS

.

-8

-4

0

( C)

spectra

for various

AND

DISCUSSION

Both the water and plant samples exhibited varying capacities for supercooling. Each collection of samples showed a spread of freezing temperatures that could be represented by a slightly skewed, unimodal

76

MARCELLOS

frequency distribution, consistent with patterns found by Kaku (7, 8). However, these distributions provide only qualitative information that is adequately summarized by means and standard deviations (Table I). The more objective nucleus spectra are presented for four cases in Fig. 1. These results show firstly that there appears to be some plant control over supercooling, and secondly, that the probability of nucleation increases exponentially, with increasing supercooling. In the case of water, the log-linear increase in nucleus concentration, or the probability of nucleation, as temperature was lowered was consistent with findings of others (15). The biological samples, although revealing a similar increase in nucleation with decreasing temperature, contained much more effective nucleating sites, and in all cases, froze at higher temperatures. Differences among cultivars in their nucleus concentrations were small, but variation among species was considerable (Table 2). For example, the samples of wheat contained virtually no nuclei active at -8”C, whereas French bean contained at least one nucleating site per sample active at that temperature, and would have little likelihood of supercooling below it. The sites and agents for nucleation, and therefore the reasons for variation in supercooling among tissues are unknown. Maki et al. (10) demonstrated that cultures of the bacterium Pseudomonas syringae isolated from decaying alder leaves could initiate freezing in supercooled aquebus drops at very warm temperatures (-1.8 to -36°C). The presence of these bacteria in corn leaves increases their frost sensitivity, possibly by reducing their chance for supercooling ( 1). However, whether the presence of these bacteria is involved in this study is not known. Other researchers have observed that nucleation occurs in the vascular tissues, presumably on loci on the structural elements of the tissue and not in

AND

SINGLE TABLE

2

Relationships Between Differential Nucleus Concentration, k (0) and Temperature, 0 Logk(e) =a-be Plant

Broad bean French bean Field pea Lucerne (alfalfa) Itapeseed cv. Torch cv. Midas cv. Sinus cv. Rzpido

EL:: cv. z.501 cv. Gamut cv. Timgalen Distilled

water

a

b

r

n

-1.405 -4.076 -5.352 -2.958 -5.084 -6.527 -4.467 -3.910 -3.382 -5.031 -6.416 -6.511

0.140 0.544 0.742 0.237 0.443 0.548 0.352 0.307 0.244 0.346 0.481 0.455

0.905 0.952 0.987 0.935 0.978 0.958 0.923 0.995 0.963 0.947 0.940 0.985

250 96 89 70 115 68 69 69 92 136 92 115

-4.603

0.195

0.983

150

Note : “n” is the number of observations ; “r” the correlation coefficient ; “a” and “b” are intercept and regression coefficient, respectively.

solution (7). Leaves and stems may also have different nucleus spectra (7, 8)) and nucleating efficiency in leaves can alter with season and maturity. The value of supercooling capability in contributing to frost hardiness in tender plants is conjectural. First there is no correlation between ability to supercool and hardiness or tolerance of extracellular freezing. For example, spring wheat and potato tissues could supercool to about -13 and -12°C respectively (Table l), well below their rated killing temperatures of about -8 and -2°C. This is consistent with observations (16) that differences in supercooling were apparent among unhardened seedlings of citrus varieties, but these were not satisfactory indices for varietal differences in cold hardiness rating. However, other implications may be considered. For example, freezing of tissues that can supercool to temperatures below those normally encountered during short duration mild frosts must be a result of external nucleation by surface ice. In the same plants, sensitive tissues such as floral primordia can also avoid freezing due to the presence

NUCLEATION

AND

FREEZING

of barriers against propagation of crystallization from stem and leaf (3, 12), provided they can supercool to temperatures below those normally encountered.

OF TENDER

4.

5. SUMMARY

The extent to which tissue pieces of several frost tender plant species could be supercooled in the absence of external sources of ice nucleation was determined in a small cold chamber. A considerable range among plant species was revealed in their ability to supercool, This could be expressed as a differential nucleus spectrum that derives the minimum concentration of nucleating sites within the samples. French bean (Phaseolus vulgaris L.) was found to contain nucleators effective in the range -4 to -8°C whereas spring wheat (Triticum aestivum L.) contained sites active between -8 and -16’C. The data indicate that these plant samples can supercool to temperatures below those normally injurious to them when they are frozen, ACKNOWLEDGMENTS We are indebted to Miss Sue Balfe for assistance in recording and processing data and to Mr. E. A. Roberts for statistical advice. This study was supported financially by the Wheat Industry Research Council of Australia. REFERENCES 1. Amy, D. C., Lindow, S. E., and Upper, C. D. Frost sensitivity of Zea mays increased by application of Pseudomonas syringae. Nature 262, 282-284 ( 1976). 2. Cary, J. W., and Mayland, H. F. Factors influencing freezing of supercooled water in tender plants. Agron. J. 62,715-719 ( 1970). 3. George, M. F., and Burke, M. J. The occurrence of deep supercooling in cold hardy

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

PLANTS

77

plants. Curr. Adu. Pl. Sci. 22, 349350 (1976). George, M. F., Burke, M. J., ad Weiser, C. J. Supercooling in overwintering azalea flower buds. Plant PhysioE. 54, 29-35 ( 1974). Kitaura, K, Supercooling and ice formation in mulberry trees. In “Cellular Injury and Resistance in Freezing Organisms” (E. Asahina, Ed.), pp. 143-156. The Inst. Low Temp. Sci., Hokkaido Univ., Sapporo, 1967. Kaku, S. Changes in supercooling and freezing process accompanying leaf maturation in Buxus. Plant Cell Physiol. 12, 147-155 (1971). Kaku, S. High ice nucleating ability in plant leaves. Plant Cell Physiol. 14, 1035-1038 (1973). Kaku, S. Analysis of freezing temperature distribution in plants. Cyobiology 12, 154159 ( 1975). Larcher, W. Das assimilation svermogen von Quercus ilex und Olea europea im winter. Ber. Deut. Bot. Ges. 72, 18 (1959). Maki, L. R., Galyan, E. L., Chang-Chien, M., and Caldwell, D. R. Ice nucleation by Pseudomonas syringae. Appl. Microbial. 28, 456-459 (1974). Mazur, P. The role of intracellular freezing in the death of cells cooled at supraoptimal rates. Cryobiology 14, 251-272 ( 1977). Single, W. V. Studies on frost injury to wheat. II. Ice formation within the plant. Amt. J. Agric. Res. 15, 869-875 ( 1964). Thomas, D. A., and Barber, H.-N. Studies on leaf characteristics of a cline of Eucalyptus urnigera from Mount Wellington, Tasmania. I. Water repellancy and the freezing of leaves. Aust. J. Bot. 22, 501-512 (1974). Vali, G. Quantitative evaluation of experimental results on the heterogeneous freezing nucleation of supercooled liquids. J. Atmos. Sci. 28, 402409 ( 1971) . Vali, G., and Stansbury, E. J. Time-dependant characteristics of the heterogeneous nucleation of ice. Can. 1. Phys. 44, 477-502 (1966). Yelenosky, G., and Horanic, G. Subcooling in wood of citrus seedlings. Cryobiology 5, 281-283.