Abstract
Low-temperature scanning electron microscopy was used to examine transverse fracture faces through cereal leaf pieces subjected to frost. Specimens were studied before and after sublimation of the ice. The position of extracellular ice in the leaf was inferred from the difference between the specimen before and after sublimation and from ridges and points which occurred in the extracellular ice during sublimation. Steps in the fracture surface indicated that the fracture plane passed through the extracellular ice crystals as well as through cells and also helped identify extracellular ice. The cells in controls were turgid and extracellular ice was absent. Leaf pieces from hardened rye were excised and frost-stressed to-3.3°,-21° and-72°C, cooling at 2–12°·h-1. Cell collapse and extracellular ice were evident at-3.3°C and increased considerably by-21° C. At-21° and-72°C the leaf pieces were mainly filled with extracellular ice and there were few remaining gas spaces. The epidermal and mesophyll cells were laterally flattened, perpendicular to their attachment to adjacent cells, and phloem and vascular sheath cells were more irregularly deformed. Leaf pieces from tender barley were cooled at 2°C·min-1 to-20° C; they were then mainly filled with extracellular ice, and the cells were highly collapsed as in the rye. In rye leaves frozen to-3.6° C before excision, ice crystals occurred in peri-vascular, sub-epidermal and intervening mesophyll spaces. In rye leaf pieces frozen to-3.3° C after excision or to-3.6° C before excision, mesophyll cells were partly collapsed even when not covered by ice, indicating that collapse of the cell wall, as well as the enclosed protoplast, was driven by dehydration. No gas or ice-filled spaces were found between wall and the enclosed protoplast. It is suggested that this can be explained without invoking chemical bonding between cell wall and plasma membrane: when the wall pores are filled by water, the pore size would reduce vapour pressure so making penetration of the wall by ice or gas less likely.
Similar content being viewed by others
Abbreviations
- SEM:
-
scanning electron microscopy
References
Asahina, E. (1978) Freezing processes and injury in plant cells. In: Plant cold hardiness and freezing stress, pp. 17–36, Li P.M., Sakai, A., eds. Academic Press, New York
Ashworth, E.N., Ables, F.B. (1984) Freezing behaviour of water in small pores and the possible role in the freezing of plant tissues. Plant Physiol. 76, 201–204
Ashworth, E.N., Echlin, P., Pearce, R.S., Hayes, T.L. (1988) Ice formation and tissue response in apple twigs. Plant Cell Environ, in press
Beck, E., Schulze, E.-D., Senser, M., Scheibe, R. (1984) Equilibrium freezing of leaf water and extracellular ice formation in Afroalpine “giant rosette” plants. Planta 162, 276–282
Childs, E.C. (1969) An introduction to the physical basis of soil water phenomena. Wiley, London
Davy, J.G., Branton, D. (1970) Subliming ice surfaces: Freezeetch electron microscopy. Science 168, 1216–1218
Gusta, L.V., Burke, M.J., Kapoor, A. (1975) Determination of unfrozen water in winter cereals at subfreezing temperatures. Plant Physiol. 56, 707–709
Idle, D.B. (1966) The photography of ice formation in plant tissue. Ann. Bot. 30, 199–206
Ishikawa, M., Sakai, A. (1981) Freezing avoidance mechanisms by supercooling in some Rhododendron flower buds with reference to water relations. Plant Cell Physiol. 22, 953–967
Ishikawa, M., Sakai, A. (1985) Extraorgan freezing in winter flower buds of Cornus officinalis Sieb. et Zucc. Plant Cell Environ. 8, 333–338
Jeffree, C.E., Read, N.D., Smith, J.A.C., Dale, J.E. (1987) Water droplets and ice deposits in leaf intercellular spaces: redistribution of water during cryofixation for scanning electron microscopy. Planta 172, 20–37
Johnson, R.P.C. (1968) Microfilaments in pores between frozen-etched sieve elements. Planta 81, 314–332
Kitaura, K. (1967) Freezing and injury of mulberry trees by late spring frost. Bull. Sericult. Exp. Stn. (Tokyo) 22, 202–323
Levitt, J. (1980) Responses of plants to environmental stress, vol. 1, 2nd edn. Academic Press, New York
Moor, H. (1964) Die Gefrier-Fixation lebender Zellen und ihre Anwendung in der Elektronenmikroskopie. Z. Zellforsch. 62, 546–580
Morris, G.J., Coulson, G.E., Engels, M. (1986) A cryomicroscospic study of Cylindrocystis brebissonii De Bary and two species of Micrasterias Ralfs (Conjugatophyceae, Chlorophyta) during freezing and thawing. J. Exp. Bot. 37, 842–856
Nei, T. (1978) Structure and function of frozen cells: Freezing patterns and post-thaw survival. J. Microsc. 112, 197–204
Olien, C.R., Smith, M.N. (1977) Ice adhesions in relation to freeze stress. Plant Physiol. 60, 499–503
Pearce, R.S. (1982) Ultrastructure of tall fescue (Festuca arundinacea Schreb. cv. S 170) cells fixed while exposed to lethal or non-lethal extracellular freezing. New Phytol. 92, 259–272
Pearce, R.S. (1985a) A freeze-fracture study of membranes of rapidly drought-stressed leaf bases of wheat. J. Exp. Bot. 36, 1209–1221
Pearce, R.S. (1985b) The membranes of slowly drought-stressed wheat seedlings: a freeze-fracture study. Planta 166, 1–14
Pearce, R.S., Beckett, A. (1987) Cell shape in leaves of drought-stressed barley examined by low temperature scanning electron microscopy. Ann. Bot. 59, 191–195
Pearce, R.S., Willison, J.H.M. (1985) Wheat tissues freezeetched during exposure to extracellular freezing: distribution of ice. Planta 163, 295–303
Preston, R.D. (1974) The physical biology of plant cell walls. Chapman and Hall, London
Read, N.D., Porter, R., Beckett, A. (1983) A comparison of preparative techniques for the examination of the external morphology of fungal material with the scanning electron microscope. Can. J. Bot. 61, 2059–2078
Sakai, A. (1979) Freezing avoidance mechanism of primordial shoots of conifer buds. Plant Cell Physiol. 20, 1381–1390
Sakai, A. (1982) Freezing tolerance of shoot and flower primorida of confierous buds by extraorgan freezing. Plant Cell Physiol. 23, 1219–1227
Sakai, A., Larcher, W. (1987) Frost survival of plants. Springer, Berlin
Schulz, D., Neidhart, H.V., Perner, E., Jaenicke, J., Sommer, J. (1973) Verbesserte Darstellung der Feinstruktur ruhender Pflanzenzellen. Protoplasma 78, 41–55
Staehelin, L.A., Bertaud, W.S. (1971) Temperature and contamination dependent freeze-etch images of frozen water and glycerol solutions. J. Ultrastruct. Res. 37, 146–168
Steponkus, P.L. (1981) Response to extreme temperatures. Cellular and sub-cellular bases. In: Encyclopedia of plant physiology. N.S., vol. 12A: Physiological plant ecology I. Responses to the physical environment, pp. 371–402, Lange, O.L., Nobel, P.S., Osmond, C.B., Ziegler, H., eds. Springer, Berlin
Steponkus, P.L., Evans, R.Y., Singh, J. (1982) Cryomicroscopy of isolated rye mesophyll cells. CryoLetters 3, 101–114
Stout, D.G. (1979) Plant plasma membrane permeability and slow freezing injury. Plant Cell Environ. 2, 273–275
Terumoto, I. (1960) Ice masses in root tissue of table beet. Low Temp. Sci. Ser. B 18, 39–42
Willison, J.H.M., Rowe, A.J. (1980) Replica, shadowing and freeze-etching techniques. North-Holland Publishing Co., Amsterdam
Withers, L.A., Davey, M.R. (1978) A fine-structural study of the freeze-preservation of plant tissue cultures. I. The frozen state. Protoplasma 94, 207–219
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Pearce, R.S. Extracellular ice and cell shape in frost-stressed cereal leaves: A low-temperature scanning-electron-microscopy study. Planta 175, 313–324 (1988). https://doi.org/10.1007/BF00396336
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00396336