Freezing of Isolated Thylakoid Membranes in Complex Media X. Interactions among Various Low Molecular Weight Cryoprotectants

Author： Santarius K.A.

Publisher： Academic Press

ISSN： 0011-2240

Source： Cryobiology, Vol.33, Iss.1, 1996-02, pp. : 118-126

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Abstract

Thylakoid membranes were isolated from spinach leaves ( Spinacia oleracea L.) and exposed to temperatures down to -30#°C in the presence of inorganic electrolytes and combinations of various potentially cryoprotective solutes. When mixtures of rapidly penetrating compounds such as ethylene glycol monomethyl ether, various glycols, methanol, and dimethyl sulfoxide are used, cryopreservation is presumably due to the colligative action of the solutes at any freezing temperature. Likewise, cryoprotection at relatively high subzero temperatures by combinations of nonpenetrating solutes such as sugars and polyols can be explained by this mechanism. In contrast, the additive action of nonpenetrating cryoagents at lower freezing temperatures appears to be achieved by interactions of the solutes with the membranes. When thylakoids were exposed to severe freezing in the presence of combinations of rapidly penetrating cryoagents and carbohydrates, the former may act either as cryoprotectants or as cryosensitizers. Very low concentrations of permeating solutes, which are not sufficient to impair the solute-membrane interactions of nonpenetrating cryoagents, insignificantly accelerate thylakoid protection, presumably due to a slight decrease of the electrolyte concentration in the residual unfrozen fraction. The increase in concentration of rapidly penetrating cryoagents during freezing causes dissociation of the chloroplast coupling factor, and hence the cryoprotective action of carbohydrates can be diminished or abolished. Under these conditions, the colligative action of the solutes plays the dominant role. Therefore, during severe freezing, cryoprotection is determined by the total amount of penetrating and nonpenetrating cryoagents in relation to the electrolyte concentration.