Seasonal changes of ultrastructure were studied by electron microscopy and by determining the chlorophyll and starch content of the plastids. Young plastids of spruce (Picea abies (L.) Karst.) first function as amyloplasts which store reserve material for the growth of the young needles. Then they develop a normal thylakoid system and produce assimilation starch during the day. In autumn, starch synthesis ceases and the plastids group together. In winter they swell and their membrane system becomes disorganized and reduced. With increasing temperatures in spring the chloroplasts recover, but then they accumulate large amounts of starch, which is not broken down during the night or even during a dark period of several days. As in the previous year they now function as amyloplasts providing reserve material for the new shoot. In summer these plastids are again converted into typical chloroplasts. The same seasonal changes of structure and function could be observed in chloroplasts from 2- or 3-year old needles. Thus these changes represent cyclic processes, which repeat each year. Features of slow aging are superimposed on to these cycles.

The water potentials of frozen leaves of Afroalpine plants were measured psychrometrically in the field. Comparison of these potentials with the osmotic potentials of an expressed cellular sap and the water potentials of ice indicated almost ideal freezing behaviour and suggested equilibrium freezing. On the basis of the osmotic potentials of expressed cellular sap, the fractions of frozen cellular water which correspond to the measured water potentials of the frozen leaves could be determined (e.g. 74% at -3.0° C). The freezing points of leaves were found to be in the range between 0° C and -0.5° C, rendering evidence for freezing of almost pure water and thus confirming the conclusions drawn from the water-potential measurements. The leaves proved to be frost resistant down to temperatures between -5° C and -15° C, as depending on the species. They tolerated short supercooling periods which were necessary in order to start ice nucleation. Extracellular ice caps and ice crystals in the intercellular space were observed when cross sections of frozen leaves were investigated microscopically at subfreezing temperatures.

Hill reaction and noncyclic photophosphorylation of isolated class C chloroplasts of spruce (Picea abies (L.) Karst.), as well as (14)CO2 fixation by whole needles at constant laboratory conditions proceeded at high rates during spring and early summer, declined during late summer and autumn by about 60%, remained at this level during winter, and recovered quickly in early spring. During summer, the whole needles proved to be frost labile, since after exposure to-20°C and careful thawing, fast chlorophyll degradation occurred. In addition, only photosynthetically inactive chloroplasts could be isolated from those precooled needles. On the contrary, during winter the photochemical activities of plastids from freshly harvested needles did not differ from those of artificially frozen-thawed needles. When isolated spruce chloroplasts were exposed to the same subfreezing temperatures as the whole needles, no influence of freezing on the photochemical activities was observed, irrespective of whether the plastids were isolated from frost sensitive or frost hardened needles. It is concluded that frost damage to spruce chloroplasts is due to an attack of membrane toxic compounds or lytic enzymes which were liberated upon freezing from more labile compartments. Frost hardening of the chloroplasts, as determined by the stability of chlorophyll after exposure of the needles to low temperatures, as well as by the isolation of photosynthetically active chloroplasts from such precooled needles, appeared to depend at least on 2 processes: (i) an alteration of the composition of the photosynthetically active membranes and (ii) and additional stabilization of these membranes by protecting substances. The first process was indicated by a large increase (decrease) of the capability of isolated chloroplasts for PMS-mediated photophosphorylation which accompanied natural or artificial frost hardening (dehardening). Production of cryoprotecting compounds was suggested by a significant higher stability against NaCl observed with class C chloroplasts isolated from frost hardened needles as compared to that of plastids from frost labile material. The decrease of the capability for both, the ferricyanide dependent photoreactions of the plastids and the CO2 fixation by whole needles, which was observed during the frost hardening phase, cannot be due to freezing injuries; it rather appears to be a consequence of the frost hardening process.

The large leaf rosettes ofthe so-called 'giant rosette' plants which inhabit the alpine region of tropical mountains are composed of a great number of adult leaves surrounding a central cone of developing leaves. Upon onset of the nocturnal frost period the adult leaves nyctinastically bend inwards and form a night-bud around the central leaf bud. The insulating effect of the night-bud was analysed in four species: Setwcio keniodendrott, Senecio brassiea. Lobelia kettiensis and Lobelia telekii which grow on Mt Kenya (Kenya). Freezing is avoided by a delay of cooling which is sufficient until rewarming by the next day's sunshine. A consequence of this delay is that the temperature in the nocturnal bud remains higher than that of the outer leaves which are often stiffly frozen after cold nights. Only one freezing point was detected on the leaf temperature recordings. Depending on the water state, the freezing points were in the range -1 C to -4.2C. Sucrose, amounting to 38% ofthe leaf dry weight, may act as a cryoprotectant for the cell membranes. Erost hardiness of the leaves, as determined with a laboratory method, was sufficient to enable the plants to survive during the nocturnal frost temperatures as measured in the field.

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