SummaryObservations have been made by phase contrast and fluorescence microscopy on chloroplasts both in living cells of higher plants and in an isolated state.In the living cell the chloroplast is often surrounded by a mobile jacket of material which resembles mitochondrial substance. Many chloroplasts retain this outer jacket after isolation.The behaviour of isolated chloroplasts in hypotonic sucrose solutions indicates that this jacket of mitochondria-like material is the only all-encompassing structure around the chloroplast. The jacket swells and ruptures in o· 1-0' 2M sucrose solution. At still lower sucrose concentrations the remaining chloroplast behaves as though composed of numerous individual osmotic units. Many small blebs appear around the margin of the chloroplast and with increasing dilution these swell further, often to a larger size than the original chloroplast. The inner surfaces of these swollen blebs can fuse, but the outer surfaces do not have this property.Grana are fairly resistant to swelling in hypotonic solutions and many are well preserved even in distilled water.A modelis proposed for the structure of a typical grana-containing chloroplast, and the swelling patterns which have been observed are interpreted in terms of this model. The chloroplast is depicted as a stack of flat sealed bags, each bag corresponding to a pair of stroma lamellae. The many blebs which form in sucrose concentrations below O· 1M are envisaged as individual swollen stroma bags. It is suggested that the chloroplast jacket is commonly represented as a double-layered outer membrane in electron.micrographs.

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1. Photosynthesis of leaf slices, mesophyll protoplasts, and intact chloroplasts of spinach was inhibited in hypertonic sorbitol solutions. Sorbitol could be replaced by other nonpenetrating osmotica such as sucrose or glycinebetaine. As a penetrating solute, ethyleneglycol was also inhibitory, but osmolarities required for inhibition of photosynthesis were considerably higher than in the case of non-penetrating osmotica.-2. With leaf slices and protoplasts, 50% inhibition by sorbitol was usually observed at osmotic potentials between 25 and 40 bar. With isolated intact chloroplasts, the osmotic potentials producing 50% inhibition varied considerably. Depending on the growth conditions of the plant material, 50% inhibition occurred between 14 and 40 bar. The integrity of the chloroplast envelope as measured by the accessibility of the thylakoid system for ferricyanide was not affected by osmotic stress.-3. Quantum requirements for CO2 assimilation and reduction of 3-phosphoglycerate or nitrite by intact chloroplasts increased under osmotic stress. The increase was larger for CO2 reduction than for reduction of 3-phosphoglycerate or nitrite.-4. In intact chloroplasts, electron transport to methylviologen was not much affected by osmotic stress. Basal electron transport was not stimulated, suggesting absence of uncoupling.-5. The increase in ATP/ADP ratios on illumination of intact chloroplasts was slower at an osmotic potential of 36 bar than at 11 bar.-6. The results indicate that inhibition of photosynthesis is not caused by the sensitivity of a single photosynthetic reaction to increased osmotic potentials. Rather, several reactions are sensitive to water stress. Osmotic stress acts on the photosynthetic apparatus mainly at the level of dark reactions and ATP synthesis, and much less on primary photoreactions or electron transport, between water and the primary oxidant of photosystem I.-7. The different sensitivity of chloroplasts to penetrating and non-penetrating solutes and the observed variability of chloroplast sensitivity to stress suggests that the reduction in water potential is not directly responsible for damage to the photosynthetic apparatus during osmotic stress. Rather, the composition of the chloroplasts appears to be a decisive factor which determines sensitivity or resistance to osmotic stress.

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