Freezing Temperatures and Biomembranes

The manner in which chilling affects membrane fluidity applies in principle to frost as well, but to a more severe extent. Measurements of fluidity by means of electron spin resonance or fluorescence polarisation show primarily the stiffening effect which a high protein/lipid ratio has on the fluidity of biomembranes at low temperatures. Particularly protein-rich biomembranes, such as those of the thylakoids, reduce the protein/lipid ratio during frost hardening. In addi tion, a desaturation of the fatty acids in the membrane lipids is often observed. More details in this regard will be given in Chapter

It is important to realise that the freeze-desic-cation of frost-hardened tissues is inevitably accompanied by a more or less pronounced shrinkage of the protoplast.

As the biomembrane cannot expand or shrink like a balloon would [the intrinsic elastic flexibility of a biomembrane should not exceed 23% (Wolfe and Steponkus 1983)], shrinking processes are often associated with a removal of lipids from the membrane and expansion processes with the insertion of lipids. During reversible freeze-desiccation, this material must be deposited in such a way as to be immediately available upon thawing (Fig. 1.3.15).

A quite similar problem occurs during plas-molysis. Steponkus et al. (1983) showed with isolated protoplasts from rye mesophyll cells that damage to frost-sensitive cells occurs during thawing, because the amount of material available for re-incorporation is no longer sufficient for the "areal growth" of the plasma membrane. The resulting lysis of the cells was called "expansion-induced lysis". Protoplasts of frost-hardy winter rye leaves did not show this phenomenon. They do not deposit the material removed from the membrane during contraction within the cell, but deposit it at the external side of the membrane where it is not accessible to lipases. The different behaviour of the lipids depends on the degree of their unsaturation: Frost-sensitive plasma membranes of protoplasts can be artificially "hardened" if they are treated with a surplus of unsaturated lipids (in the experiments phosphatidylcholine with one- or two-fold unsaturated fatty acids was used) and a partial exchange of the membrane lipids takes place. Even though protoplasts are an excellent in vitro system for the study of many processes, they can only provide answers to some partial aspects of frost hardening in biomembranes.

In considering the freezing of cellular water, it is very important to regard the involvement of the cell wall in stabilising the protoplast. The freezing of cell water has often been compared to plasmolysis - a very different process. During freezing, the cellular fluid must crystallise in the intercellular spaces, as otherwise biomembranes bordering the ice crystals would disintegrate, because the hydrophobic interactions of the lipids which stabilise the membranes require the presence of liquid water. This also means that no ice

Fig. 1.3.15. Behaviour of isolated protoplasts from rye leaves in isotonic and hypertonic solutions. Protoplasts were isolated from leaves of frost-sensitive (A, B) and frost-hardened (C, D) rye plants and incubated in isotonic medium (A, C,G) and in a medium with a concentration double that of the isotonic medium (B, D, H). The outer surface of the non-hardened protoplasts remained almost smooth on shrinking (B), i.e. lipids excluded from the membrane were displaced to the interior of the protoplast. Protoplasts from frost-hardened cells deposited their excluded lipids in extrusions of various shapes (E) on the outside of the plasma membrane when they shrank, where they cannot be degraded by endogenous lipases (D). The transmission electron micrographs (G, H) also show the shrinking (compare the size of the vacuoles in G and H) and the lipid extrusions (arrow) which are strongly contrasted by osmium staining (Os). The bar corresponds in each case to 5 |.im. E An enlarged section of a protoplast from frost-hardened leaves in hypertonic medium. F The surface of a protoplast from non-hardened leaves in hypertonic medium, after fusion of the protoplast with liposomes consisting of dilino-leoylphosphatidylcholine. Protoplasts which deposit lipids internally on shrinking burst upon swelling in isotonic medium, because the lipids are degraded by phospholipases. Lipids which are deposited externally can be reintegrated into the membrane upon swelling of the protoplast. The bar corresponds to 1 jim. (After Steponkus et al. 1983, 1988)

may be formed between the plasma membrane and the cell wall. This space, which in the case of plasmolysis is filled with the plasmolyticum, would have to fill with air during the extracellular freezing of cellular liquid if the protoplast were to withdraw from the cell wall. The air would have to enter through the pores of the cell wall. These pores are very small (about 4 nm diameter in the primary wall), and they are filled with water due to the high matrix potential of the cell wall. It would require a suction of more than 80 MPa to suck air through the cell wall pores (Zhu and Beck 1991). Such high suction has never been observed in living cells, however. The protoplast is therefore not normally able to detach itself from the cell wall during the exogenous freezing of the cellular liquid of the protoplast, and the entire cell must collapse upon the dehydration resulting from freezing (Fig. 1.3.16).

Cells which are not particularly rigid, e.g. those of the mesophyll, indeed wrinkle and fold under these conditions (freezing cytorrhysis). Since the cell wall itself cannot shrink, the plasma membrane must not reduce its surface significantly. The problem as to the extra- or intracellular deposition of membrane material de-

| Fig. 1.3.16. A Plasmolysis and freezing of mesophyll cells from a leaf of Pachysandra terminalis. During plasmolysis (/), the plasmolyticum intrudes into the space between the cell wall and the protoplast, and the cell wall is relaxed. During freezing (3) the protoplast remains attached to the cell wall despite the export of cell fluids into the intercellular spaces, because the cell wall is air-tight in the swollen state. The cell wall dents or buckles, depending on the extent of the freeze-desiccation. Since crystallisation takes place extracellularly, no ice is formed between the cell wall and the protoplast. B: a-d Spongy parenchyma of a leaf of Pachysandra terminalis. a Surface view of the spongy parenchyma following removal of the lower epidermis; chloroplasts can be clearly seen in the cells, b Spongy parenchyma after taking up neutral red through the leaf stalk. Chi Chloroplasts. d The same section of the spongy parenchyma at -12°C (l,-l3 indicate the same intercellular spaces). Deposition of colourless ice (£) can be clearly recognised within the intercellular spaces, c Spongy parenchyma was stained with neutral red and then killed before cooling to -12°C. The ice crystals are not restricted to the intercellular spaces, and they contain drops of coloured cell sap (Z). Note also the change in the neutral red colour resulting from acidification as a consequence of killing the tissue. (Photos J.J. Zhu)

scribed above is thus of hardly any consequence in this regard. Freezing plasmolysis does not occur even in rigid cells which are tightly integrated into a tissue if the cells are to survive the freezing (see Chap. 1,3.6.3).

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