A method for cellular fractionation of Chiamydomonas reinhardii, SAG 11-32/b, and isolation of intact chloroplasts from synchronized cells of the alga is described. The procedure for ceil fractionation comprises essentially four steps: (1) protoplast production with autolysine; (2) lysis of the protoplasts with digitonin; (3) aggregation of broken protoplasts; and (4) separation of organelles by differential centrifugations.Replacing the differential centrifugations (step 4) by Percoll cushion centrifugations yields intact chloroplasts. Starting with 100 milliliters of an algal culture containing 3000 micrograms chlorophyll, intact chloroplasts with 100 to 200 micrograms of chlorophyll can be isolated. Envelope integrity is about 90% (ferricyanide assay). Examination of the chloroplasts by electron microscopy and marker enzyme activities indicated some mitochondrial and cytoplasmic contamination.The biochemistry and physiology of unicellular green algae have been studied intensively because these algae can be maintained easily under laboratory conditions. Cultures can be synchronized (21) and some strains grow heterotrophically as well as autotrophically and mixotrophically. Many similarities with the metabolism of higher plants have been established. The pathway of photosynthetic CO2 reduction was primarily elucidated with unicellular green algae. Enzymes of the glyoxylate cycle and glycolate metabolism, previously found in higher plants, could also be measured in algae, proving the general occurrence of these pathways in plants. Although it was possible to relate these pathways and cycles to cellular organelles in higher plants, very few investigations are reported on the compartmentation of metabolism in unicellular green algae. The first detailed study of localization of enzymes in a unicellular alga of the Chlamydomonas type was made by Kombrink and Wober (16) who were able to demonstrate the activity of starch-metabolizing enzymes in chloroplasts of Dunaliella marina. In their investigation, they used a new method involving DEAE-dextran for cell lysis.One reason for the difficulties in cell fractionation of unicellular

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Cultivars of barley (Hordeum vulgare L.) were tested for germination sensitivity to progressively higher concentrations of salt, mannitol, and betaine. The three solutes were equally inhibitory at equal osmotic potential, but there was a consistent difference in osmotic sensitivity between two cultivars, CM‐67 and Briggs (Briggs was the most sensitive). There was no difference between the two cultivars in salt or water uptake from salt solutions during imbibition. Brief presoaking in water did not improve salt resistance, indicating that a hydration‐dependent decrease in membrane permeability is not involved in salt tolerance. The calcium content of Briggs was higher than CM‐67. These results suggest that salt inhibits barley germination primarily by osmotic effects, and that salt influx during imbibition does not play a role in this inhibition. A hypothesis regarding salt effects on germination is discussed.

The mesocarp tissue of ripening avocado fruits was studied by freeze fracture, thin section, and scanning electron microscopy. CO 2 and ethylene production by individual fruit were monitored, and samples were analyzed at several stages of the ripening process. The tissue is composed primarily of large, isodiametric, lipid-containing parenchyma cells. At maturity these cells contain the normal complement of plant cell organelles, and all membranes appear intact. When ripening begins, several changes in the ultrastructure occur. The most obvious changes are a loosening and eventual breakdown of the cell wall, and swelling and vesiculation of the rough endoplasmic reticulum. In freeze fracture replicas a significant increase in the number of intramembranous particles in the EF face of the plasmamembrane was observed at the climacteric peak. In postclimacteric, soft fruit the particle density of the EF face of the plasmamembrane decreased to the density observed in the membrane of preclimacteric cells. All of the organelles and membranes appear whole and intact whether examined by thin section, freeze fracture, or scanning electron microscopy. However, the cell walls in postclimacteric fruit have almost completely disappeared. These results indicate that the ripening process per se in avocados does not involve a complete loss of compartmentalization nor a breakdown of organelle and membrane integrity. It may, however, lead to these or similar senescence changes as a result of the loss of the cell walls. The variations in particle density of the plasmamembrane during ripening may reflect one or more of several structural, compositional, or functional membrane phenomena, and this aspect of ripening warrants further study.

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