[Studies on the efflux of malate from the vacuoles of the assimilating cells in Bryophyllum and the possible effects of this process on Crassulacean acid metabolism]

Abstract

Kinetic studies on the release of [(14)C] malate into unlabelled buffer in tissue slices of Bryophyllum leaves labelled by (14)CO2 dark fixation showed a curve characterized by three phases. According to literature, these phases indicate malate efflux from free space, cytoplasm and vacuoles. From the curves obtained it could be estimated that the cytoplasmatic pool of [(14)C] malate after (14)CO2 dark fixation is higher in "acidified" tissue (i.e. high malate content) than in "deacidified" tissue (i.e. low malate content). Efflux of [(14)C] malate from the vacuoles is also higher in "acidified" tissue. It increases when the malate solution enclosed in the vacuoles becomes more concentrated. This could be demonstrated in experiments in which water was extracted from the labelled tissue by raising the osmotic potential of the buffers in which the tissue slices were suspended. The increase of [(14)C] malate efflux from the vacuoles followed a sigmoid curve when plotted against the osmotic potential of the washing buffer, i.e. agaisnt the degree of dehydratation of the tissue.The osmotic potential of the buffer in which leaf tissue of Bryophyllum was suspended also had an effect on the distribution of radiocarbon among the metabolites when the tissue was allowed to fix (14)CO2 in the light. In deacidified tissue the incorporation of (14)C into malate was inhibited whereas label found in carbohydrates (starch + sucrose) remained nearly unchanged when the osmotic potential of the buffer increased up to 12 atm. This effect is explained in terms of inhibition of PEP-carboxylase by a growing cytoplasmic malate pool, which is caused by the increasing malate efflux from the vacuole and by retarded malate flux from cytoplasm into vacuole under these conditions. However, in acidified tissue labelling of malate was already low with no osmotic stress, and no further inhibition of malate synthesis could be observed when the osmotic potential of the buffer was increased.Label found in starch after (14)CO2-fixation decreased in the light under osmotic stress, with more label being transferred into sucrose. This effect could be interpreted as osmoregulation which forces the cells of the leaf tissue to produce osmotically effective substances to balance the higher osmotic potential of the buffer.