Plant Physiol. (1984) 76, 201-2040032-0889/84/76/020 1/04/$0l .00/0

Freezing Behavior of Water in Small Pores and the PossibleRole in the Freezing of Plant Tissues

Received for publication January 16, 1984 and in revised form May 24, 1984

EDWARD N. ASHWORTH* AND FRED B. ABELESUnited States Department ofAgriculture, Agricultural Research Service, Appalachian Fruit ResearchStation, Route 2, Box 45, Kearneysville, West Virginia 25430

ABSTRACT

Two model systems were used to study the freezing of water in smalldiameter pores. Water in pores having a dmeter of less than 100anometers froze at lower temperatures than bulk water. Data obtained

with a range of pore sizes were consistent with predicted values based onequations developed by Mazur (1965 Ann NY Acad Sci 125: 658-676),and Homshaw (1980 J Soil Sd 31: 399-414). The addition of soluteslowered the freezing point of water in small pores. We propose that thefreezing behavior of water in small pores may account for some of thefreezing patterns observed in plant tissues. In tissues where cells aretightly packed, share common walls, and lack intercelhllar spaces, thepresence of water in cell wall microcapiaries would alter the freezingtemperature of tissue water, impede the spread of ice, and facilitatesupercooing.

Water in small diameter pores will freeze at sub-zero temper-atures (2, 5, 9). This principle has been applied to studying thefreezing ofwater in soils and building materials. Both the freezingtemperature and the spread of ice were influenced by the sizeand distribution of small diameter pores (2, 5-7). Calorimetryhas been used to determine the melting point of water in porousmaterials so that the porosity and pore size distribution can beestimated (5-7). Possible effects of small diameter pores on thefreezing of water in plant tissues have received little attention.Mazur (9) discussed this principle and related it to the preventionof ice propagation through cell membrane pores and into theprotoplasm. George (4) recently observed that melting begannear -30C in water saturated sycamore heartwood. He specu-lated that this fraction of water may be located in cell wallmicrocapillaries and discussed the possible role of the microcap-illaries in limiting the spread of ice in these tissues. In this paperwe utilized two model systems to study the freezing of water insmall diameter pores. We propose that this phenomenon mayaccount for some of the freezing patterns observed in planttissues.

MATERIALS AND METHODS

Experiments with Polycarbonate Membranes. Freezing pointdepression experiments were performed using a modified Boydenchamber (Bio-Rad Labs).' The width of the acrylic block was

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reduced to 16 mm to facilitate temperature equilibration betweenthe chamber and cooling bath. The chamber was fitted with 13-mm diameter polycarbonate filters (Nuclepore Corporation,Pleasanton, CA 94566) or aluminum discs. The chamber wascooled in a 30% ethanol bath held at - 12C. Chamber temper-atures were monitored using copper-constantan thermocouplesand a recording potentiometer. The experimental apparatus isillustrated in Figure 1. Chamber 1 was designated as the icedonor chamber and was filled with a suspension of an icenucleating strain of Erwinia herbicola (Lohnis) Dye, isolate 26(106 cells/ml). Nuclepore filters or aluminum discs separated thedonor from the second chamber. Thermocouples were insertedinto both sides of the modified Boyden chamber which was thenlowered into the - 12C bath. Freezing was initiated in the donorchamber between 0 and -1C. The temperature at which freezingwas initiated in the second chamber was recorded. Individualexperiments required 15 min and were repeated three times.Values reported are the mean plus or minus the standard devia-tion.

Experiments with Controlled Pore Glass. Controlled pore glassparticles (Sigma Chemical Co.) were obtained with a range ofnanometer pore diameters (7.5 ± 6%, 16 ± 12%, 33 ± 8%, 73± 9%, 149 ± 6%, and 300 ± 9%). Glass particles were uniformlysized (80-120 mesh) by the manufacturer. Both the particles andthe pores were irregularly shaped as observed using scanningelectron microscopy (data not presented). Dry glass particleswere placed into modified Beem capsules (Ladd Research Indus-tries, Inc., Burlington, VT 05402) (100 psl) and saturated witheither deionized water or dilute aqueous solutions.The freezing of water within saturated controlled pore glass

particles was characterized using DTA.2 The technique was amodification of that described by Quamme et al. (11). Thejunction of a 40-gauge copper-constantan thermocouple wassubmerged into the slurry of glass particles. Excess solution wasremoved using a micropipette. Dry controlled pore glass wasused as a reference. The output of the thermojunctions wasmonitored with a strip chart recorder (0.5 mv/full scale). Sampleswere placed into glass test tubes which were fitted into holesbored into an aluminum block. The aluminum block was placedinto a -70C deep freeze. Block temperature and cooling rateswere controlled using a resistance heater and temperature pro-grammer.

RESULTS

Experiments with Polycarbonate Filters. The effect of filterpore size on the growth of ice crystals is presented in Table I.Data obtained with aluminum foil represents a control, anddemonstrates that when physically separated, water in the second

2Abbreviation: DTA, differential thermal analysis.201

ASHWORTH AND ABELES

MODIFIED BOYDEN CHAMBER

TSiEMCLLS DELRIN RETAINING4 ) / ~~~PLUJG

BATHICE DONOR

DELRIN PLUG _ FILTERaWE2

S U E R ~~~~~COOLING

+1cmL(XI

FIG. 1. Illustration of the modified Boyden chamber used to studythe effect of filter pore size on ice propagation.

Table I. Effect ofPore Size ofNuclepore Filters on the Spread ofIce

Extent of Supercooling

Pore Diameter in Chamber 2

nm °C± SD0, (aluminum disk) -4.7 ± 0.315 -4.0 ± 1.0

100 -0.8 ± 1.01000 0±0

TEMPERATURE, *CFIG. 2. DTA of water saturated gass particles with 7.5 nm diameter

pores. Sample was cooled at IO C/h.

chamber would supercool to -4.7°C before freezing. When alu-minum foil was replaced with filters having increasing pore sizes,the difference in freezing temperature between the donor andreceiver chambers was reduced. The freezing point of water in15 and 100 nm pores was lower than that of the bulk solution.Therefore, filters with these pore sizes prevented ice from prop-agating into the second chamber until sample temperature wasreduced. Filters with 1000 nm diameter pores did not impedethe spread of ice.

Experiments with Controlled Pore Glass. DTA of water satu-rated, controlled pore glass particles detected a large exotherminitiated between -5 and -10C (Fig. 2). This exotherm corre-sponds to the freezing of bulk water surrounding the glass parti-cles. The temperature at which this first exotherm was initiatedwas not influenced by the size of the pores within the glassparticles. However, when glass particles with small internal poreswere frozen, a shoulder on the first exotherm or a distinct secondexotherm was observed (Fig. 2). A broad exotherm between -9and -15°C was detected in particles having 7.5 nm dianieterpores (Fig. 2). To distinguish whether the second exothermresulted from the freezing of supercooled water or a depressionof melting point, a series of experiments were conducted. DTA

"Jo

o- 2LLJ(/)0ALI

-30 -20 -10

TEMPERATURE, *C

0

FIG. 3. DTA of water saturated glass particles with 7.5 nm diameterpores. Sample was warmed at lOC/h.

PORE DIAMETER, nm

FIG. 4. Relationship between pore diameter and melting point. Ex-perimental values obtained using controlled pore glass and polycarbonatemembrane filters were compared to values predicted by equations devel-oped by Mazur (9) and Homshaw (5).

Table II. Freezing Point Depression ofDilute Aqueous Solutions in 7.Snm Diameter Pores

Water in 7.5 nm pores fioze between -10 and -15°C.Solute Conc.

0.2M 0.4M 0.8M

Glycine -II to -16 -10 to -17 -12 to -19Glucose -II to -18 -12 to -19 -18 to -25Sucrose -10 to -18 -2 to -19 -16 to -28KCIa -i2 to-18 -3 to-21, -17 to-25,

-26 to -30 -25 to -28

*Two low-temperature exotherms were observed with the 0.4 and 0.8M KCI solutions. The latter appears to be the result of the freezing of aeutectic mixture within the pores. The eutectic point of KCI in bulksolution is -II1C.

of saturated 7.5 nm diameter controlled pore glass detected twodistinct endotherms during thawing (Fig. 3). Melting points of-IOC and OC were observed. In addition, when saturated 7.5nm diameter controlled pore glass which had been frozen to-40C was warmed to -1OC and subsequently recooled, anexotherm between -10 and -15°C was detet*ed (data not pre-sented).' The fraction of water in the 7.5 nm diameter pores wasalso stable at sub-zero temperatures. When samples were cooledto - °OC, the freezing of bulk water was detected. However,water within the 7.5 nm diameter pores remained liquid for 24h at -10C and would not freeze until subsequently cooled. Thecombined evidence confirms that the second exotherm was dueto the freezing of water with a lower melting point than the bulk

THAWING SATURATED MLASSPARTICLES WITH 7.5 rvm P0ES

202 -PI-a-nt Physiol. Vol. 76, 1984

FREEZING OF WATER IN SMALL PORES

" LLJ.-2zLLIRaLLI WLL LLJLL O.0

-30 -2 -10

TEMPERATUJRE, *Cu

FIG. 5. DTA of saturated glass particles with 7.5 nm diameter pores.

Glass particles were saturated with either water, 0.8 M glucose, or 0.8 MKCa. Samples were warmed at 10°C/h.

water and was not the result of a supercooling phenomenon.A relationship between pore diameter and melting point was

observed (Fig. 4). The melting point ofwater within 300 and 149nm diameter pores was 0°C. Pores with diameters less than 73nm reduced the melting point. The observed melting pointdepressions due to small diameter pores compared favorablywith predicted values (Fig. 4). Predicted values were generatedby equations developed by Homshaw (5) and Mazur (9). Mazurderived the equation AT = -300 cos Olr (9). Where r = radiusof the pore in A and 0 is the angle ofthe meniscus within a pore.To generate the predicted values, 0 was set equal to 0 (cos 0 =

1), a value which would result from complete wetting of thepores. This assumption was supported by the observation that1% (v/v) Tween 20 had no effect on freezing behavior. Theequation developed by Homshaw (5) was AT = -47/r, where r= the pore radius in nanometers. Based on the calculated values(Fig. 4), a large effect of pore diameter on melting point wouldbe expected in pores smaller than 7 nm.The addition of solutes further depressed the freezing point of

water in small pores (Table II). Dissolved solutes lowered thetemperature and changed the shape ofthe second exotherm (datanot presented) and altered the melting curve during warming(Fig. 5). The effect of solutes on melting point was greater in thepores than in the bulk solution. Equal molar concentrations ofglycine, glucose, sucrose, and KCI had different effects on thefreezing point of water in small pores (Table II; Fig. 5).

DISCUSSION

Experiments with polycarbonate membranes and controlledpore glass illustrated the freezing behavior of water in smalldiameter pores. A reduction in freezing point was observed inpores having a diameter of 100 nm or less. The experimentalvalues were comparable to predicted ones (5, 9). Pores withdiameters of less than 6 nm were not available for this study.However, the equations developed by Mazur (9) and Homshaw(5) predict a sharp decline in freezing point at pore diametersbelow 6 nm. The addition of solutes further reduced the freezingpoint of water in small pores.

Ice formation is generally initiated extracellularly when planttissues are exposed to sub-zero temperatures (8). As ice crystalsform in the intercellular spaces, a vapor pressure gradient isestablished between the ice-water mixture in the intercellularspaces and the water within the cells. To attain equilibrium,water effluxes from the cells and moves into the intercellularspace. Further cooling results in the continued growth of icecrystals in the intercellular space and a decline in cell watercontent.Many plant tissues have been observed to supercool to -40C

(1, 3, 1 1). A feature of tissues which exhibit deep supercooling istightly packed cells with common cell walls. In these tissues,

FIG. 6. Model to illustrate the effect of microcapillaries on the spreadof ice in plant tissues.

large intercellular spaces are absent and the extracellular waterwould be in cell wall microcapillaries. These capillaries form acontinuous network between the microfibrils and the moleculesof the cell wall matrix (10). Both the size and shape of themicrocapillaries are probably irregular. Several different tech-niques have been used to estimate the size of the cell wall voidvolume, and the average diameter of the cell wall microcapillar-ies. The results indicate that voids equivalent to capillaries with4 nm diameter are present (10). Therefore, one would predictthat water in these capillaries would freeze between -15 and-25°C (Fig. 4). The freezing of dilute aqueous solutions withinthese capillaries would be at lower temperatures. These predic-tions are based on generalizations of cell wall structure. Unfor-tunately, data on the size of microcapillaries in cell walls ofspecies which deep supercool is not available.The presence of microcapillaries in cell walls would not only

depress the freezing point of water in the intercellular spaces butwould impede the spread of ice throughout the tissue. Ice wouldpropagate through the largest diameter voids. However, its spreadwould be limited by the smallest diameter within a given capil-lary. Due to this effect, the presence of small diameter poreswould facilitate the supercooling of water in large volumes. Asimple model can illustrate this point (Fig. 6). A cavity with adiameter of the order of microns is connected to the bulk phaseby a small diameter capillary. Even if ice is present in the bulkphase, water in the cavity would remain as a supercooled liquid.Under these conditions, water in the cavity would freeze if thetemperature dropped below the melting point of water in theconnecting microcapillary. This would permit ice to propagatethrough the capillary and nucleate the supercooled water withinthe cavity. If the diameter of the microcapillary was sufficientlysmall, the water in the cavity would supercool until it wasnucleated by heterogeneous ice nuclei present within the cavityor by homogeneous ice nucleation at -38°C. Upon warming,the melting point of water within the cavity would be independ-ent of the melting point of the water within the pore. Therefore,only the water within the microcapillaries would melt appreciablybelow 0°C. DTA of this model system would reveal a largeexotherm associated with the freezing of the bulk phase and alow temperature exotherm corresponding to the freezing ofwaterin the cavity and microcapillary. Upon warming the modelsystem, only the small fraction of water in the microcapillarywould melt at low temperatures. A single large endotherm wouldbe observed at OC whicfi would correspond to the thawing ofice in both the cavity and the bulk phase. We believe thatanalogous situations may exist in some plant tissues (i.e. thexylem parenchyma cells of hardwood tree species, and dormantflower bud primordia of several woody species). The presence ofwater within cell wall microcapillaries would alter the freezing

CONTROL

0.8 M GLUCOSE

0.8 M KC -

_ ^

203

204 ASHWORTH

behavior of tissue water and impede the spread of ice.

Acknowledgments-The authors wish to thank Glen Davis and Kevin Weaverfor technical assistance, Wilbur Hershberger for conducting the scanning electronmicroscopy, Dixie Gaynor for help in preparing the manuscript, and Gary Lightnerand Dale Dillow for help in preparing the graphics. Kevin Weaver was supportedby a Research Apprenticeship for Minority High Schoolers.

LITERATURE CITED

1. BURKE MJ, LV GUSrA, HA QUAMME, CJ WEISER, PH Li 1976 Freezing andinjury in plants. Annu Rev Plant Physiol 27: 507-528

2. EVERET DH 1961 The thermodynamics of frost damage to porous solids.Trans Faraday Soc 57: 1541-1551

3. GEORGE MF, MJ BURKE 1977 Cold hardiness and deep supercooling in xylemof shagbark hickory. Plant Physiol 59: 319-325

4. GEORGE MF 1983 Freezing avoidance by deep supercooling in woody plantxylem: preliminary data on the importance of cell wall porosity. In DD

AND ABELES Plant Physiol. Vol. 76, 1984

Randall, DG Blevins, RL Larson, BJ Rapp, eds, Current Topics in PlantBiochemistry and Physiology. University of Missouri Prss, pp 84-95

5. HOMSHAW LG 1980 Freezing and melting temperature hys sis of water inporous materials: Application to the study of pore form. J Soil Sci 31: 399-414

6. HOMSHAW LG 1980 High resolution heat flow DSC: Application to study ofphase trnsitions and pore size distribution in saturted porous materials. JTherm Anal 19: 215-233

7. HOMSHAW LG 1981 Supercooling and pore size distribution in water-saturatedporous materials: Application to study of pore form. J Colloid Interface Sci84:141-148

8. LEvrrr J 1980 Responses ofPlants to Environmental Stresses, Vol 1. AcademicPress, New York

9. MAZUR P 1965 The role of cell membranes in the freezing of yeast and othersingle oells. Ann NY Acad Sci 125: 658-676

10. PRESTON RD 1974 The Physical Biology of Plant Cell Walls. Chapman andHall, London

11. QUAMmE HA, C STUSHNOFF, Cl WEISR 1972 The reationship of exothermsto cold injury in apple stem tissues. J Am Soc Hortic Sci 97: 608-613