Morphologically detectable protein (intramembrane particles) and cholesterol (filipin labelling) in the membranes of autophagic vacuoles and lysosomes were studied in mouse hepatocytes using thin-section and freeze-fracture electron microscopy. Both isolated autophagic vacuoles and lysosomes, and intact tissue blocks were used due to the facts (i) that lysosomes are difficult to recognize in freeze-fracture replicas of intact hepatocytes, and (i) that filipin penetration into the tissue blocks is unsatisfactory. Intramembrane particle density was low in the membranes of early autophagic vacuoles (defined as round-shaped vacuoles in which an inner membrane parallel with the outer limiting membrane was clearly visible). The lysosomal membranes contained considerably more intramembrane particles. Particle-rich lysosomes or other vesicles were observed to fuse with the early autophagic vacuoles. The membranes of nascent autophagic vacuoles with morphologically intact contents were usually not labelled by filipin, whereas the membranes of all other autophagic vacuoles and lysosomes were heavily labelled. The increased cholesterol in the membranes of slightly older autophagic vacuoles is presumably derived from cholesterol-rich lysosomes or other vesicles fusing with the vacuoles and from the degrading organelles inside the autophagic vacuoles.

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Protoplasts were enzymically isolated from 2‐week‐old non‐acclimated rye (Secale cereale L. cv. Puma) seedlings. They were resuspended in isotonic sorbitol with different concentrations (0–10%) of dimethyl sulfoxide (DMSO). The survival of the protoplasts frozen in isotonic sorbitol solutions declined at temperatures below the freezing point with the LT50 being ‐8°C. Addition of DMSO to the osmoticum increased survival at freezing temperatures. The optimum concentration of DMSO was 4% and lowered the LT50 to ‐19°C. Freeze‐fracture studies of the plasma membrane revealed aparticulate lipid lamellae at ‐4°C, but the first appearance of lateral phase separations, striations and inverted cylindrical micelles (hexagonal11‐type structures) occurred at ‐6°C. At lower temperatures, ‐8 and ‐10°C, the occurrence of nonbilayer structures became more common. The addition of DMSO decreased the incidence of the ultrastructural changes. With 2 or 4% DMSO, non‐bilayer structures were not observed at temperatures above ‐10°C. Instead, striations and H11‐type structures were observed at ‐ 15 and ‐20°C.

Terminal buds of Pinus silvestris L. containing microsporangiate strobilus primordia were collected once a month throughout the winter. The electron microscopic studies indicated that in October and December, the cells of the strobili contained a large number of vacuoles, a portion of which was supposedly autophagic, and stacked rough endoplasmic reticulum. By February, the amount of these had decreased, and instead, a large population of dense bodies was visible. Additional phenomena, characteristic at this state, were the occurrences of highly uneven contours of the plasmalemma and of inclusions of various kinds between the plasmalemma and the cell wall. In March, autolysis was visible in a portion of cells outside the sporangia. In the sporangia the ground cytoplasm was thin but the number of organelles was increasing. In the April collections, cell divisions were visible. The amount of protein per dry weight increased during the winter reaching a peak in February. The activity of RNases, having optima of pH 5.0 and pH 7.5, was measured in two successive years. Both series showed a period of high activity during the middle of the winter. The exact timing of this period depended on the year in question. On the basis of these observations, the dormant period of the microsporangiate strobili of the Seotch pine is divided into three sub-periods. It is also suggested that the definition of dormancy of these structures should include a mentioning of alterations in the metabolical machinery of the cells.

Seasonal fluctuations in storage lipids in the cushion plant Diapensia lapponico, growing in Northern Finland (70"N 27"E). were studied by microscopy and chemical analysis. Lipid bodies in the mesophyll cells were stained with Sudan Black for quantitative observation by light microscope. Electron microscope observations were made using aldehyde prefixed and osmium tetroxide postfixed sections of leaf blades. Thin layer and gas capillary chromatographic techniques were used to analyse total lipids and total fatty acids in green shoots of Diapensia. Free sugars and starch were extracted scparately and determined by the anthrone method. A mesophyll cell was characterized by a large lipid body (storage lipid) in summer hut by several small spherules in winter. Total surface area of the cross-sectioned lipid globules was at its lowest from April to September; the maximal value was in March.The amount of total lipids in the leafy tops of D. lapponica was 91-200 mg g-' dry weight. Values were lowest at the end of June, when the total carbohydrate level was at its highest. Accordingly, the decrease in the total lipid level in the early growing season, when new leaves were developing, can be attributed primarily to the increase in the level of carbohydrates, particularly starch. The amount of total fatty acids varied from 21 to 30 mg g-' dry weight. The level increased in the early growing season and remained elevated throughout the summer. Like the total lipids, the total fatty acids arc derived from structurally different parts of the sclerophyllous leaves, including the well-developed cuticle and epicuticular wax layer. The discrepancies in the results obtained from microscopic and chemical analyses arc discussed.

Pihakaski, K. 1988. Seasonal changes in the chloroplast ultrastructure of Diapensia lapponica. -Nord. J. Bot. 8: 361-367. Copenhagen. ISSN 0107-055X.Leaf specimens of evergreen Diapensia lapponica were collected monthly in Northern Finland (ca 70' ") in order to study seasonal changes in the ultrastructure of the chloroplasts of the palisade mesophyll. The volume fraction of chloroplasts per cell was lowest in summer and increased towards autumn and winter. However, the relative size seemed to be higher in summer than in other seasons. Lengtwwidth (LW) ratio was calculated as an index of the chloroplast shape. The shape varied from elongated (usually concavo-convex in profile), with an UW ratio from 1.9 to 2.5 in summer, to roundish or irregular (UW ratio from 1.2 to 1.4) in midwinter. In autumn the chloroplasts were most elongated (LW ratio 2.6). The starch content was highest at the end of June when it constituted 38% of the volume of chloroplast. It decreased from then till November and was absent during the rest of the winter. Changes in the thylakoid system involved an increase in the number of partitions from an average of three in early summer to 11 in autumn and a decrease to M again towards winter. In spring it was up to 7 again. The large grana and high portion of stroma thylakoids observed in late August -September (the period of rhythmic light) are consistent with the high photosynthesis activity reported previously in D . lapponica in laboratory conditions.