[Hill reaction and photophosphorylation of isolated chloroplasts in relation to water content : II. Removal of water by CaCl2].

1. Isolated chloroplasts from leaves of spinach and beets were dehydrated by drying for 3 hours in vacuo over CaCl2 at +2°C in the absence and in the presence of different substances. After rehydration ferricyanide reduction, cyclic photophosphorylation with PSM as cofactor, noncyclic photophosphorylation and the level of free SH groups were investigated. Furthermore, the quantity of water bound under the conditions of the test by the chloroplast lamellae and by the different substances was determined. 2. Isolated chloroplasts, which were dehydrated for 3 hours over CaCl2 lost 98-99% of their water content. Under these conditions a sharp increase of SH groups occurred indicating protein denaturation. In addition Hill reaction and photophosphorylation were inactivated. The presence of sugars, soluble proteins and polypeptides during dehydration protected chloroplasts, fully or in part, against denaturation. At low concentrations of the protective substances preservation increased more or less linearly with increasing concentration. Inorganic and organic salts could not prevent the destruction of the system during dehydration. On the contrary, salts abolish the protection afforded by sugars. More sugar was required to give protection for photophosphorylation than for the electron transfer reactions of the Hill reaction. Uncoupling of photophosphorylation from electron transport therefore precedes the destruction of electron transfer due to dehydration. In principle, cyclic and noncyclic photophosphorylation showed the same behaviour. - In spinach and bett leaves, the critical limit for the dehydration of the protoplasmic structures seemed to be nearly 10-15% of the total water content. Removal of the "critical" water leads to injury. 3. The protective action of sugars and, at least in part, of peptone and bovine albumin may be explained by their ability to retain water during the drying. Under specified conditions 1 mol of sucrose binds twice as much water as the same amount of glucose. On a molar basis sucrose is twice as effective as glucose in protecting the Hill reaction. On the other hand it is also possible that sugars protect the sensitive proteins directly and specifically. - Accumulation of ions, even though these may bind as much water as neutral solutes such as sugars, is destructive. 4. No change in the SH content of the chloroplasts was obtained during dehydration in the presence of very small amounts of sugar, which is not sufficient to protect Hill reaction and photophosphorylation. In the absence of sugar a considerable increase in SH groups is observed on drying. No obvious correlation exists between the liberation of SH groups and the inactivation of Hill reaction and photophosphorylation. 5. The results demonstrate that plants resistant to high dehydration can increase their desiccation resistance through mobilisation of sugars and soluble proteins during the water loss. These substances can protect the sensitive protein structures during the dehydration. 6. The results obtained when isolated chloroplats were dehydrated with CaCl2 are consistent with those obtained in freezing experiments. In other words, the response of chloroplasts to dehydration is identical whatever the mode of dehydration is. The findings explain the similarities between frost and drought resistance observed by many different authors.