Cold hardiness abilities vary with the size of the land snail Cornu aspersum

The land snail Cornu aspersum (syn. Helix aspersa) living in Brittany (France) can be considered partially freezing tolerant as it possesses a low ability to supercool and a limited capacity to bear freezing of its body tissues. The absence of a marked cold hardiness strategy permits the emphasis of the role of parameters such as individual size or water mass (W(M)) contained by the organism. Adult snails (shell diameter 30-32 mm) had a supercooling ability, about 1-1.5 degrees C lower than that of immatures (shell diameter 12-20 mm) and survived longer to an exposure to -5 degrees C, with an Lt(50) comprised between 6.0 and 9.8 h against 2.6 to 4.2 h for immature snails. This better ability to bear freezing was explained by the faster dynamic of body ice formation observed in small individuals, which attained ice lethal quantity more rapidly. At the species level, large snails will then tend to be more tolerant to freezing and small ones to be freezing avoidant, a statement also observable at the phylum level.     

Stem diameter variations and cold hardiness in walnut trees

The effect of freezing temperatures on stem diameter was measured in the field and in climatic chambers using linear variable differential transformers (LVDT sensors). In acclimated stems, there was reversible stem shrinkage associated with freeze-thaw cycles. The maximum shrinkage correlated with stem diameter (thickness of the bark). The wood was responsible for only 15% of the shrinkage associated with a freeze event, and experiments with isolated bark showed that connection with the wood was not necessary for most of the freeze-induced shrinkage to occur. Considering the amount of stem shrinkage associated with summer drought in walnut, the amount of contraction of the bark with freezing was actually much less than might be predicted by water relations theory. Reversible stem shrinkage occurred in living tissues, but not in autoclaved tissues. For the latter, swelling was observed with freezing and this swelling could be explained by the bark alone. Similar swelling was observed during September and October for non-acclimated plants. Water was lost with each freeze-thaw cycle starting with the first, and freezing injury of the bark, with discoloration of tissues, was also observed in non-acclimated plants. Given that the diameter fluctuation patterns were dramatically different for acclimated versus non-acclimated plants, and for living versus autoclaved tissues, LVDT sensors could represent a novel, non-invasive approach to testing cold hardiness.     

Neutron scattering in the plane of membranes: structure of alamethicin pores.

A technique of neutron in-plane scattering for studying the structures of peptide pores in membranes is described. Alamethicin in the inserted state was prepared and undeuterated and deuterated dilauroyl phosphatidylcholine (DLPC) hydrated with D2O or H2O. Neutron in-plane scattering showed a strong dependence on deuteration, clearly indicating that water is a part of the high-order structure of inserted alamethicin. The data are consistent with the simple barrel-stave model originally proposed by Baumann and Mueller. The theoretical curves computed with this model at four different deuteration conditions agree with the data in all cases. Both the diameter of the water pore and the effective outside diameter of the channel are determined accurately. Alamethicin forms pores in a narrow range of size. In a given sample condition, > 70% of the peptide forms pores of n and n +/- 1 monomers. The pore size varies with hydration and with lipid. In DLPC, the pores are made of n = 8-9 monomers, with a water pore approximately 18 A in diameter and with an effective outside diameter of approximately 40 A. In diphytanoyl phosphatidylcholine, the pores are made of n approximately 11 monomers, with a water pore approximately 26 A in diameter, with an effective outside diameter of approximately 50 A.     

Nature of the water permeability increase induced by antidiuretic hormone (ADH) in toad urinary bladder and related tissues

In artificial lipid bilayer membranes, the ratio of the water permeability coefficient (Pd(water)) to the permeability coefficient of an arbitrary nonelectrolyte such as n-butyramide (Pd(n-butyramide)) remains relatively constant with changes in lipid composition and temperature, even though the individual Pd's increase more than 100- fold. I propose that this is a general rule that also holds for the lipid bilayers of cells and tissues, and that therefore if Pd(water)/Pd(solute greatly exceeds the value found for artifical lipid bilayers (where "solute" is a molecule, such as 1,6 hexanediol or n- butyramide, that crosses the cell membrane by a solubility-diffusion mechanism without the aid of a special transporting system), then water crosses the cell membrane via aqueous pores. Applying this criterion to the toad urinary bladder, we find that even in the unstimulated bladder, water probably crosses the luminal membrane primarily through small aqueous pores, and that this almost certainly the case after antidiuretic hormone (ADH) stimulation. I suggest that ADH stimulation ultimately leads either to formation (or enlargement) of pores, by the rearrangement of preexisting subunits, or to an unplugging of these pores.     

Role of membrane transport of water and glycerol in the freeze tolerance of the rice stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae)

Overwintering larvae of the rice stem borer, Chilo suppressalis accumulate glycerol and are freezing tolerant to about -25 degrees C. However, non-diapausing larvae cannot accumulate glycerol and are killed by freezing. We compared the extent of tissue damage, the effects of glycerol concentration, and the transport of glycerol and water in fat body tissues from these larvae at selected freezing temperatures. Tissues from overwintering larvae, but not non-diapausing larvae, survive when frozen at -20 degrees C with 0.25 M glycerol, but the protection afforded by glycerol is offset by the water-channel inhibitor mercuric chloride. Glycerol in higher concentration (0.75 M) affords some protection even to the fat body of non-diapausing larvae. Radiotracer assays of overwintering larvae show that water leaves the tissues during freezing while glycerol enters, and that mercuric chloride disrupts this process. Transport is also disrupted after lethal freezing at -35 degrees C. Therefore, membrane transport of water and glycerol is involved in the avoidance of freezing injury to fat body cells of the rice stem borer, apparently by mediating the replacement of water with glycerol in freezing-tolerant tissues.     

Function and immuno-localization of aquaporins in the Antarctic midge Belgica antarctica

Aquaporin (AQP) water channel proteins play key roles in water movement across cell membranes. Extending previous reports of cryoprotective functions in insects, this study examines roles of AQPs in response to dehydration, rehydration, and freezing, and their distribution in specific tissues of the Antarctic midge, Belgica antarctica (Diptera, Chironomidae). When AQPs were blocked using mercuric chloride, tissue dehydration tolerance increased in response to hypertonic challenge, and susceptibility to overhydration decreased in a hypotonic solution. Blocking AQPs decreased the ability of tissues from the midgut and Malpighian tubules to tolerate freezing, but only minimal changes were noted in cellular viability of the fat body. Immuno-localization revealed that a DRIP-like protein (a Drosophila aquaporin), AQP2- and AQP3 (aquaglyceroporin)-like proteins were present in most larval tissues. DRIP- and AQP2-like proteins were also present in the gut of adult midges, but AQP4-like protein was not detectable in any tissues we examined. Western blotting indicated that larval AQP2-like protein levels were increased in response to dehydration, rehydration and freezing, whereas, in adults DRIP-, AQP2-, and AQP3-like proteins were elevated by dehydration. These results imply a vital role for aquaporin/aquaglyceroporins in water relations and freezing tolerance in B. antarctica.     

DIFFUSION THROUGH STOMATES

Ting, Irwin P., and Walter E. Loomis. (Iowa State U., Ames.) Diffusion through stomates. Amer. Jour. Bot. 50(9): 866–872. Illus. 1963.—It is shown that the rule that diffusion through isolated, small pores is proportional to the diameter rather than the area of the pores is valid for pores of diameters as small as 20 μ, and that the curve extends to the origin at zero diameters, indicating that the law is effective throughout the range of stomatal sizes. Suggestions that an elliptical pore will be relatively more effective in diffusion than a circular one and that diffusion is concentrated at the periphery of the pore are not supported by experimental evidence and are physically improbable. Brown and Escombe's conclusion that there is no interference in the diffusion through the individual pores of a multiperforate membrane if the pores are spaced 10 diameters apart is not valid for diffusion through the stomates of a leaf. With pores of 200 μ and less spaced 10 diameters apart, interference increases rapidly with a smaller size and larger number of pores. As a result, the diffusion through a membrane with pores 19 μ in diameter and 190 μ apart was the same as that through a membrane with pores 132 μ in diameter and 1.32 mm apart, although the calculated capacity of the first membrane was 7 times that of the second. The diffusion of water vapor through multiperforate membranes with pores spaced 10 diameters apart has an apparent maximum of 65–70% of the diffusion through an open tube. Calculations of the effect of partial closing of stomates, using Verduin's equation for interference between pores, indicate that the theoretical diffusion capacity of 10 μ stomates spaced at 10 diameters would be increased several times by closing to an average diameter of 5 μ. This increase illustrates the dominant effect of interference in diffusion through small, closely spaced pores. Calculated diffusion through these stomates would not be decreased until they were more than 95% closed. It is concluded that stomatal opening will have no important effect on diffusion from or into a leaf until the stomates are essentially closed.     

Leakage of a trapped fluorescent marker from liposomes: effects of eutectic crystallization of NaCl and internal freezing.

Leakage of trapped carboxyfluorescein from DL-alpha-dipalmitoylphosphatidylcholine multilamellar liposomes (diameter 1-2 microns) in NaCl solutions was measured after rapid freezing to temperatures between -15 and -55 degrees C. Leakage was low after freezing between -15 and -35 degrees C, but increased steeply between -35 and -45 degrees C. From DSC measurements it was found that the increase in leakage was associated with two crystallization processes: Eutectic crystallization of NaCl and freezing of undercooled solvent trapped in the interior of the liposomes ("internal freezing"). Damage caused by the former process could effectively be prevented by small amounts of trehalose (1% less than or equal to w less than or equal to 1.5%). Trehalose in these concentration also decreased damage due to internal freezing, but to a minor degree. In addition to these damaging transitions, a time-dependent process was found to cause leakage from the liposomes at -25 degrees C. The association between leakage and thermal activity suggests that DSC supplements cryomicroscopy and leakage measurements in the characterization of cryostability of liposomes.     

Role of Pore Size Location in Determining Bacterial Activity during Predation by Protozoa in Soil

The predation of a luminescence-marked strain of Pseudomonas fluorescens by the soil ciliate Colpoda steinii was studied in soil microcosms. Bacterial cells were introduced in either small (neck diameter, <6 (mu)m) or intermediate-sized (neck diameter, 6 to 30 (mu)m) pores in the soil by inoculation at appropriate matric potentials, and ciliates were introduced into large pores (neck diameter, 30 to 60 (mu)m). Viable cell concentrations of bacteria introduced into intermediate-sized pores decreased at a greater rate than those in small pores, with reductions in bacterial populations being accompanied by an increase in viable cell numbers of the ciliate. The data indicate that the location of bacteria in small pores provides significant protection from predation. In the absence of C. steinii, the level of metabolic activity of the bacterial population, measured by luminometry, decreased at a greater rate than cell number, and the level of luminescence cell(sup-1) consequently decreased. The decrease in levels of luminescence indicates a loss of activity due to starvation. During predation by C. steinii, the level of the activity of cells introduced into small pores fell in a similar manner. The level of cell activity was, however, significantly greater for cells introduced into intermediate-sized pores, despite their greater susceptibility to predation. The data suggest that increased activity arises from a release of nutrients by the predator and the greater accessibility of bacteria to nutrients in larger pores. Nutrient amendment of microcosms resulted in increases in bacterial populations to sustained, higher levels, while levels of luminescence increased transiently. The predation of cells introduced into intermediate-sized pores was greater, and there was also evidence that the level of activity of surviving bacteria was greater for bacteria in intermediate-sized but not small pores.     

Arrest of membrane fusion events in mast cells by quick-freezing

We have used quick-freezing and freeze-fracture to study early stages of exocytosis in rat peritoneal mast cells. Mast cells briefly stimulated with 48/80 (a synthetic polycation and well-known histamine- releasing agent) at 22 degrees C displayed single, narrow-necked pores (some as small as 0.05 micrometer in diameter) joining single granules with the plasma membrane. Pores that had become as large as 0.1 micrometer in diameter were clearly etchable and thus represented aqueous channels connecting the granule interior with the extracellular space. Granules exhibiting pores usually did not have wide areas of contact with the plasma membrane, and clearings of intramembrane particles, seen in chemically fixed mast cells undergoing exocytosis, were not present on either plasma or granule membranes. Fusion of interior granules later in the secretory process also appeared to involve pores; granules were often joined by one pore or a group of 2-4 pores. Also found were groups of extremely small, etchable pores on granule membranes that may represent the earliest aqueous communication between fusing granules.     

Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells

In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion pore (Haller, T., J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. 1998. Proc. Natl. Acad. Sci. USA. 95:1579-1584). Thus, we have been able to monitor the dynamics of individual fusion pores up to hours in intact cells, and to calculate pore diameters using a diffusion model derived from Fick's law. After formation, fusion pores were arrested in a state impeding the release of vesicle contents, and expanded at irregular times thereafter. The expansion rate of initial pores and the probability of late expansions were increased by elevation of the cytoplasmic Ca2+ concentration. Consistently, content release correlated with the occurrence of Ca2+ oscillations in ATP-treated cells, and expanded fusion pores were detectable by EM. This study supports a new concept in exocytosis, implicating fusion pores in the regulation of content release for extended periods after initial formation.     

The mechanism of freezing injury in xylem of winter apple twigs

In acclimated winter twigs of Haralson apple (Pyrus Malus L.), a lag in temperature during cooling at a constant rate was observed at about −41 C by differential thermal analysis. The temperature at which this low temperature exotherm occurred was essentially unaffected by the cooling rate. During thawing there was no lag in temperature (endotherm) near the temperature at which the low temperature exotherm occurred, but upon subsequent refreezing the exotherm reappeared at a somewhat higher temperature when twigs were rewarmed to at least −5 C before refreezing. These observations indicate that a small fraction of water may remain unfrozen to as low as −42 C after freezing of the bulk water in stems. The low temperature exotherm was not present in twigs freeze-dried to a water content below 8.5% (per unit fresh weight), but it reappeared when twigs were rehydrated to 20% water. When freeze-dried twigs were ground to a fine powder prior to rehydration, no exotherm was observed. Previous work has shown that the low temperature exotherm arises from xylem and pith tissues, and that injury to living cells in these tissues invariably occurs only when twigs are cooled below, but not above the temperature of the low temperature exotherm. This study revealed that the low temperature exotherm resulted from the freezing of a water fraction, that the freezing of this water was independent of the freezing of the bulk water, that the exotherm was associated with some gross structural feature but not the viability of the tissue, and that injury to living cells in the xylem and pith was closely and perhaps causally related to the initial freezing of this water.     

Porin pores of mitochondrial outer membranes from high and low eukaryotic cells: biochemical and biophysical characterization

The mitochondrial porins from mammalian tissues and from low eukaryotic cells were purified with a high yield, and their biochemical and functional properties were investigated. When analyzed by SDS gel electrophoresis, all mammalian porins show a very similar apparent molecular mass (35-35.5 kDa). In contrast yeast and Paramecium porins have a molecular mass of 30 and 37 kDa, respectively. The peptide maps of mammalian porins are very similar although small differences are apparent between porins of different tissues of the same organism and also between those of the same tissue of different organisms. The peptide patterns of porins from yeast and Paramecium are completely different from those of mammalian porins. Antibodies raised against the rat liver porin cross-react with all the other mammalian porins but not with that of yeast. The incorporation of porins into artificial lipid bilayer membranes showed that they are able to form pores with approximately the same specific activity. The single-channel conductance is for all porins, except for that of Paramecium, about 4 nS in 1 M KCl, corresponding to an effective pore diameter of 1.7 nm. They are voltage-dependent and switch to substates at transmembrane potentials higher than 10 mV. The number of gating charges varies, however, for pores from different tissues, indicating a different sensitivity to the potential as a result of a possible different function.     

Visualization of Freezing Behaviors in Leaf and Flower Buds of Full-Moon Maple by Nuclear Magnetic Resonance Microscopy

1H-Nuclear magnetic resonance (NMR) microscopy was used to study the freezing behavior of wintering buds of full-moon maple (Acer japonicum Thunb.). The images obtained predominantly reflected the density of mobile (i.e. non-ice) protons from unfrozen water. A comparison of NMR images taken at different subfreezing temperatures revealed which tissues produced high- and low-temperature exotherms in differential thermal analyses. In leaf and lower buds of A. japonicum, the scales and stem bark tissues were already frozen by -7[deg]C, but the primordial inflorescence and terminal primordial shoots remained supercooled at -14[deg]C, and the lateral primordial shoots were unfrozen even at -21[deg]C. The freezing of these supercooled tissues was associated with their loss of viability. The size of the supercooled primordial shoots and inflorescences was gradually reduced with decreasing temperature, indicating extraorgan freezing in these tissues. During this process the formation of dark regions beneath the primordia and subsequent gradual darkening in the basal part of supercooled primordia were visible. As the lateral shoot primordia were cooled, the unfrozen area was considerably reduced. Since the lateral primordia remained viable down to -40[deg]C, with no detectable low-temperature exotherms, they probably underwent type I extraorgan freezing. Deep supercooling in the xylem was clearly imaged. NMR microscopy is a powerful tool for noninvasively visualizing harmonized freezing behaviors in complex plant organs.     

Indirect Measurement of Pore Size and Permeability in Scots Pine and Norway Spruce

Five experiments to investigate the water relationships of Scotspine and Norway spruce sapwood are described. Field exposureof stakes, and their moisture characteristic relating wood moisturecontent to the water potential of the surroundings showed thatspruce had a lower moisture content than pine under the sameconditions. A pore size distribution derived from the characteristicsshowed more large pores in spruce than in pine, but in sprucethere were also more small pores. The inference was that thelarge pores of spruce were readily accessible to water, allowinga fast rate of water uptake, as found in the wick action andswelling investigations, while the small pores were not accessible,resulting in a low rate of water uptake and a low rate of swelling.The possibility is briefly discussed that it is the pore sizedistribution of the two woods which determines their water relationships,and the difference in pore size which determines their treatability. Key words: Pinus sylvestris L, Picea abies (L) Karst, Pore size, Water uptake     

Response of Xylem Ray Parenchyma Cells of Red Osier Dogwood (Cornus sericea L.) to Freezing Stress (Microscopic Evidence of Protoplasm Contraction)

Freezing behavior of wood tissue of red osier dogwood (Cornus sericea L.) cannot be explained by current concepts of freezing resistance. Previous studies indicated that water in wood tissue presumably froze extracellularly. However, it was observed that xylem ray parenchyma cells within these tissues could survive temperatures as low as -80[deg]C and the walls of these cells did not collapse during freezing (S.R. Malone and E.N. Ashworth [1991] Plant Physiol 95: 871-881). This observation was unexpected and is inconsistent with the current hypothesis of cell response during freezing. Hence, the objective of our study was to further examine the mechanism of freezing resistance of wood tissue of red osier dogwood. We studied freezing stress response of xylem ray parenchyma cells of red osier dogwood using freeze substitution and transmission electron microscopy. Wood samples were collected in winter, spring, and summer of 1992. Specimens were cooled from 0[deg]C to -60[deg]C at 5[deg]C/h. Freezing stress did not affect the structural organization of wood tissue. However, the xylem ray parenchyma cells showed two unique responses to a freezing stress: protoplasm contraction and protoplasm fragmentation. Protoplasm contraction was evident at all freezing temperatures and in tissues collected at different times of the year. Cells with fragmented protoplasm, however, were noticed only in tissues collected in spring and summer. Protoplasm contraction in winter tissue occurred without apparent damage to the protoplasm. In contrast, protoplasm contraction in spring and summer tissues was accompanied by substantial damage. No evidence of intracellular ice formation was observed in parenchyma cells exposed to freezing stress. Differences in protoplasm contraction and appearance of cells with fragmented protoplasm likely indicated seasonal changes in cold hardiness of the wood tissue of red osier dogwood. We speculate that the appearance of fragmented protoplasm may indicate that cells are being injured by an alternative mechanism in spring and summer.     

Role of silicon in enhancing resistance to freezing stress in two contrasting winter wheat cultivars

The main objective of this study was to elucidate the roles of silicon (Si) in enhancing tolerance to freezing stress (?5 °C) in two contrasting wheat (Triticum aestivum L.) cultivars: i.e. cv. Yangmai No. 5, a freezing-susceptible cultivar and cv. Linmai No. 2, a freezing-tolerant cultivar. Shoot dry weight of the freezing-susceptible wheat was significantly lower under freezing stress than in controls, but increased significantly with Si amendment. The freezing treatment did not affect shoot dry weight of the freezing-tolerant cultivar. The leaf water content was considerably decreased by freezing stress in the freezing-susceptible cultivar, but was significantly increased by Si amendment. In contrast, freezing treatment did not significantly reduce leaf water content in the freezing-tolerant cultivar and Si played no role in water retention in this cultivar. The concentrations of H₂O₂ and free proline along with malondialdehyde (MDA) were progressively enhanced by freezing stress in the two wheat cultivars used, but were significantly suppressed by amendment with Si. The major antioxidant enzyme activities and non-enzymatic antioxidants (i.e. glutathione and ascorbic acid) in the leaves of freezing-stressed plants were decreased, but were stimulated significantly by the exogenous Si. The possible mechanisms for Si-enhanced freezing stress may be attributed to the higher antioxidant defense activity and lower lipid peroxidation through water retention in leaf tissues.     

A comparative study of diffusive and osmotic water permeation across bilayers composed of phospholipids with different head groups and fatty acyl chains.

Osmotic and diffusive water permeability coefficients Pf and Pd were measured for lipid vesicles of 100-250 nm diameter composed of a variety of phospholipids with different head groups and fatty acyl chains. Two different methods were applied: the H2O/D2O exchange technique for diffusive water flow, and the osmotic technique for water flux driven by an osmotic gradient. For phosphatidylcholines in the liquid-crystalline state at 70 degrees C, permeability constants Pd between 3.0 and 5.2.10(-4) cm/s and ratios Pf/Pd 7 and 23 were observed. The observation of a permeability maximum in the phase transition region and the fact that osmotically driven water flux is higher than diffusive water exchange suggest that water is diffusing through small transient pores arising from density fluctuations in the bilayers. The Pd values depend on the nature of the head group, on the chemical structure of the chains, and on the type of chain linkage. In the case of charged lipids, the ionic strength of the solution has a strong influence. For phosphatidylethanolamines, phosphatidic acids, and ether phosphatidylcholines, permeability constants Pd were considerably lower (2-4.10(-6) cm/s at 70 degrees C). For liquid-crystalline phosphatidylcholines, a strong reduction of Pd after addition of ethanol was observed (2-4.10(-6) cm/s at 70 degrees C). The experimental values are discussed in connection with different permeation models.     

Transient transcapillary exchange of water driven by osmotic forces in the heart

Osmotic transient responses in organ weight after changes in perfusate osmolarity have implied steric hindrance to small-molecule transcapillary exchange, but tracer methods do not. We obtained osmotic weight transient data in isolated, Ringer-perfused rabbit hearts with NaCl, urea, glucose, sucrose, raffinose, inulin, and albumin and analyzed the data with a new anatomically and physicochemically based model accounting for 1) transendothelial water flux, 2) two sizes of porous passages across the capillary wall, 3) axial intracapillary concentration gradients, and 4) water fluxes between myocytes and interstitium. During steady-state conditions approximately 28% of the transcapillary water flux going to form lymph was through the endothelial cell membranes [capillary hydraulic conductivity (Lp) = 1.8 +/- 0.6 x 10-8 cm. s-1. mmHg-1], presumably mainly through aquaporin channels. The interendothelial clefts (with Lp = 4.4 +/- 1.3 x 10-8 cm. s-1. mmHg-1) account for 67% of the water flux; clefts are so wide (equivalent pore radius was 7 +/- 0.2 nm, covering approximately 0.02% of the capillary surface area) that there is no apparent hindrance for molecules as large as raffinose. Infrequent large pores account for the remaining 5% of the flux. During osmotic transients due to 30 mM increases in concentrations of small solutes, the transendothelial water flux was in the opposite direction and almost 800 times as large and was entirely transendothelial because no solute gradient forms across the pores. During albumin transients, gradients persisted for long times because albumin does not permeate small pores; the water fluxes per milliosmolar osmolarity change were 200 times larger than steady-state water flux. The analysis completely reconciles data from osmotic transient, tracer dilution, and lymph sampling techniques.     

Calorimetric studies of the state of water in seed tissues

To understand the physical state of water in hydrating biological tissues, thermodynamic properties of water in cotyledons of pea and soybean with moisture contents ranging from 0.01 g H₂O/g dw to 1.0 g H₂O/g dw were studied using differential scanning calorimetry. The heat capacity of the tissues increased abruptly at moisture contents above 0.08 and 0.12 g H₂O/g dw for soybean and pea cotyledons, respectively. Melting transitions of water were observed at moisture contents >0.23 and 0.26 g H₂O/g dw for soybean and pea. However, freezing of water was not observed unless moisture contents exceeded 0.33-0.35 g H₂O/g dw. In both seed tissues, the temperatures of the freezing and melting varied with moisture content and showed hysterisis. The energy of the transition also varied with moisture content and was similar to the heats of fusion and crystallization of pure water only at moisture contents >0.54 and 0.58 gH₂O/g dw for soybean and pea seeds, respectively. The thermal properties of water change distinctly as seed moisture content changes: at least five states or water can be identified.