Freezing thylakoids during

Figure 3. Inactivation of thylakoids during freezing at various low temperatures as a function of time. Washed thylakoids were suspended in a solution containing 50 mM sucrose as a cryoprotectant and 20 mM sodium phenylpyru-vate as a cryotoxic solute. The suspensions were rapidly frozen and thawed. After thawing, photophosphorylation was determined. For experimental conditions, see notes in legend for Fig. 2...

Cations. During freezing, thylakoids suffer more damage in the presence of LiCl than in the presence of isosmolar concentrations of NaCl (Figure 4). For different alkali metal chlorides, damage decreased... 

Figure 5. Inactivation of thylakoids during freezing in the presence of chlorides of different divalent and monovalent metals. Washed thylakoids were suspended before freezing to —20°C in a solution containing 0.1 M sucrose as cryoprotectant ana various chlorides at concentrations indicated on the abscissa. After thawing, the activity of cyclic photophosphorylation was measured. For experimental conditions, see legend for Fig. 2.

Little is known concerning mechanisms by which these proteins prevent inactivation of thylakoids during freezing, but they somehow contribute to membrane stabilization. They act with some specificity, since cryoprotective proteins from spinach not only fail to protect red blood cells during freezing but are actually injurious. 

In contrast to these salts, sodium succinate increases thylakoid damage during freezing if present as the only solute. As will be discussed later, the situation becomes more complicated if significant concentrations of other solutes are also present with sodium succinate during freezing. Membrane inactivation by high concentrations of sodium succinate occurs even at 0°C (21). 

Salts of weak organic acids that are soluble in lipids are also injurious to thylakoids. Examples are the salts of phenylpyruvic add (Figure 3) and caprylic acid (58). These salts, even if present at very low concentrations, cause extensive membrane inactivation during freezing, if cryo-protectants are absent. At 0°C, moderate concentrations of these salts will slowly inactivate thylakoids. 

Amino Acids. As is true of organic acids, amino acids can either prevent inactivation of thylakoids by freezing or they can aggravate the situation. Some of them, for instance glycine, serine, glutamate, or aspartate, promote injury if present as the only major solutes during freezing. However, the same amino acids can be protective if certain other solutes are also present (58). The reason for this behavior, which is also observed with succinate, will be considered later. 

Proline, threonine, or y-aminobutyric acid can protect thylakoids against inactivation during freezing. Amino acids with apolar side chains such as phenylalanine, leucine, or valine always contribute to thylakoid inactivation during freezing. 

Protein Release in Relation to Loss of Membrane Function. When thylakoids are frozen in the presence of sucrose, membrane function is preserved. If a cryotoxic salt such as NaCl is also present, retention of membrane functionality during freezing depends on the ratio of sucrose to salt (5). Loss of cyclic photophosphorylation is the most sensitive parameter of membrane inactivation. Photophosphorylation is largely lost before significant protein release from the membranes can be detected (Figure 8). Since photophosphorylation requires membranes with un-... 

It is appropriate to consider the toxicities of different ions toward membranes. During freezing of thylakoids, anion toxicity decreases in the order 1 > Br > N03 > CT > F > acetate. This is reminiscent of the Hofmeister lyotropic power series, which was originally observed with regard to denaturation of euglobulins, then for blood clotting, then for... 

Figure 12 shows preservation of membrane function during freezing of thylakoids in the presence of different concentrations of sodium caprylate or sodium phenylpyruvate. Other solutes present in the system... 

Decrease in variable fluorescence in the presence of FeCN indicates a net loss of PSII-Qg-nonreducing centers at RT during high light exposure of the leaves. Concomitantly there occurred a decrease in the density of EFu particles in freeze fracture replicas of the thylakoids. These observations are consistent with the hypothesis that PSIIB-Qg-nonreducing centers from stroma thylakoid membranes are used to replace the photoinhibited PSII centers in grana partitions (1). 

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