Rapid increase of vacuolar volume in response to salt stress

Suspension-cultured cells of mangrove [Bruguiera sexangula (Lour.) Poir.] showed a rapid increase in vacuolar volume under salt stress, although there was no change in the cell volume. The rapid increase in the vacuolar volume was an active process, which followed the activation of the tonoplast H(+)-ATPase and the vacuolar acid phosphatase. The same phenomenon was observed in barley (Hordeum vulgare L. cv. Doriru) root meristematic cells under salt stress but not in pea ( Pisum sativum L.). Increases in vacuolar volume could potentially protect the cytoplasm by decreasing the cytoplasmic volume during the initial phases of salt stress.     

Functional linkage between phycobilisome and reaction center in two phycobilisome oxygen-evolving photosystem II preparations isolated from the thermophilic cyanobacterium Synechococcus sp

Photosystem II oxygen-evolving preparations with attached phycobilisomes were isolated from the thermophilic cyanobacterium Synechococcus sp. with beta-octylglucoside or digitonin. Fluorescence emission spectra of the two preparations determined at 77 K largely lacked a far red band which originates from photosystem I. The spectrum of the digitonin preparation was otherwise similar to that of intact cells, whereas the beta-octylglucoside preparation showed a pronounced band at 687 nm, which is considered to be emitted from phycobilisomes. The relative yield of phycobilin fluorescence was similar between the digitonin preparations and the cells but was considerably larger in the beta-octylglucoside preparations at room temperature. The quantum yield of ferricyanide photoreduction determined with light which is absorbed mainly by phycobiliproteins was 0.85 for the digitonin preparation and 0.57 for the beta-octylglucoside preparation. The results indicate that excitation energy is transferred from phycobilisomes to photosystem II reaction centers in the digitonin preparation as efficiently as in intact cells, while a significant portion of light energy harvested by phycobilisomes is not utilized by the primary photochemistry in the beta-octylglucoside preparation. Digitonin and beta-octylglucoside preparations had 65 and 48 chlorophyll a molecules per photosystem II reaction center, respectively. The beta-octylglucoside preparation contained twice as much phycocyanin and allophycocyanin per photosystem II reaction center as the digitonin preparation, which has a phycobiliprotein-to-photosystem II reaction center ratio very similar to that of cells. It is concluded that whereas the beta-octylglucoside preparation contains a considerable amount of free phycobilisomes, all phycobilisomes present in the digitonin preparation are physically and functionally linked to photosystem II reaction center complexes.     

Quantitative Determination of Changes in the Number and Size of Chloroplasts in Naturally Senescing Leaves of Rice Seedlings

Changes in the number and size of chloroplasts in senescingleaves of rice seedlings were determined. The method employedinvolves electron microscopic examination of large numbers ofcells and chloroplasts in the mesophyll of leaves at differentstages of senescence with the aid of a microcomputer. Analysisshowed that, once leaves had been fully expanded, the numberand size of the mesophyll cells remained unaltered throughoutthe course of senescence. By contrast, the quantity of chloroplastspresent in leaves decreased with advancing senescence. Whencompared with the newly expanded 6th leaves, the chloroplastnumber per unit area of mesophyll section was reduced by 40%and the mean cross section area of chloroplasts by 23% in theoldest leaves (3rd leaves) of seedlings. Chloroplasts occupied33% of the mesophyll section area in the 6th leaves and thepercentage decreased slightly in the 5th leaves and markedlyin lower leaves to reach 17% in the 3rd leaves. The rate ofoxygen evolution decreased approximately in parallel to thedecline in the chloroplast content. Thus, sequential decreasein the amount of chloroplasts is a main cause of loss of photosynthesisduring foliar senescence of rice seedlings. (Received May 31, 1989; Accepted October 17, 1989)     

Respiration-Dependent H+ Efflux from Intact Cells of Cyanidium caldarium

An active H⁺ efflux depending on respiration was found in anacidophilic unicellular alga, Cyanidium caldarium. Alkalizationof the medium due to passive H⁺ transport into the cells wasobserved when the respiratory activity was inhibited by addingrespiratory poisons, such as rotenone or antimycin A, or byintroducing pure nitrogen into the cell suspension. The extentof the H⁺ influx increased as the pH of the medium was loweredto 2.9, indicating that H⁺ leaks into the cells according tothe pH gradient across the plasma membrane. The medium pH whichhad increased under anaerobic condition returned to the originallevel with aeration of the cell suspension. This suggests thatan active H⁺ transport, related to respiration, pumps out theexcess H⁺ accumulated in the cells during anaerobic preincubation.The pH changes in the cell suspension were related to the intracellularATP level. From these results it was concluded that active H⁺efflux dependent upon oxidative phosphorylation functions inthe dark to maintain a constant intracellular pH against passiveH⁺ leakage through the plasma membrane. The light-induced H⁺ efflux and the respiration-dependent H⁺efflux were also compared in relation to the physiological roleof the active H⁺ efflux, especially with respect to the intracellularpH regulation in this alga. ¹The data in this paper are included in the Ph. D. dissertationsubmitted by M. Kura-Hotta to Tokyo Metropolitan University. (Received February 3, 1984; Accepted June 14, 1984)     

Light-induced H+ Efflux from Intact Cells of Cyanidium caldarium

Light-induced pH changes in suspensions of an acidophilic unicellularalga, Cyanidium caldarium Geitler, were studied as a functionof the pH of the medium. In the neutral pH region, alkalizationof the medium due to photosynthetic CO₂ uptake was observed.In the acidic pH region, illumination caused a significant decreasein the pH of the medium, indicating the efflux of H⁺ from thecells. Both the rate and extent of the pH decrease increasedas the pH of the medium was lowered to 3.0. The H⁺ efflux wasnot affected by 3-(3',4'-dichlorophenyl)-l,l-dimethylurea, butwas inhibited by phenylmercuric acetate. The fastest H⁺ effluxoccurred at 45°C, whereas its extent was almost constantfrom 25 to 50°C. The activity decreased at temperaturesabove 50°C and was inactivated completely at 60°C. Itsaction spectrum corresponded the spectrum for chlorophyll aabsorption. Results indicate that the light-induced H⁺ effluxis driven by photosystem I and is important in the maintenanceof the intracellular pH at the functional neutral region againsta steep pH gradient across the cell membrane. (Received May 6, 1981; Accepted August 8, 1981)     

Relationship between Photosynthesis and Chlorophyll Content during Leaf Senescence of Rice Seedlings

Photosynthetic oxygen evolution, chlorophyll contents and chlorophylla /b ratios of 3rd to 6th leaves of rice seedlings were measuredto examine whether or not inactivation of photosynthesis duringsenescence is related to loss of chlorophyll. Photosyntheticactivity decreased more rapidly than chlorophyll content duringleaf senescence; as a result, the lower the leaf position, thelower was the rate of oxygen evolution determined on the basisof chlorophyll. Chlorophyll ab ratio also decreased with advancingsenescence. Electrophoretic analysis revealed that the declinein chlorophyll ab ratio is due to more rapid degradation ofthe reaction center complexes than light-harvesting chlorophyllab proteins of photosystem II and that the photosystem I reactioncenter disappears in parallel with the inactivation of photosynthesis.A simple method was developed to estimate the amounts of chlorophylla associated with the reaction center complexes of the two photosystemsfrom the total chlorophyll contents and chlorophyll ab ratiosof leaves. Rates of oxygen evolution, determined on the basisof chlorophyll a bound to the reaction center complexes, remainedconstant throughout the course of senescence. Thus, inactivationof photosynthesis is closely related with loss of the reactioncenter complexes during leaf senescence of rice seedlings. Theresults suggest that loss of photosynthesis is mainly causedby loss of a functional unit of photosynthesis or by a decreasein the number of whole chloroplasts. (Received April 22, 1987; Accepted August 13, 1987)     

Effect of Intracellular ATP Levels on the Light-Induced H+ Efflux from Intact Cells of Cyanidium caldarium

The light-induced H⁺ efflux observed at acidic pH in Cyanidiumcells was shown to be an active H⁺ transport depending on theintracellular ATP produced by cyclic photo-phosphorylation.Triton X-100 was found to act as an effective uncoupler in intactCyanidium cells without collapsing the pH gradient across theplasma membrane. Triton X-100 at 0.015% significantly reducedthe intracellular ATP levels, stimulated the p-BQ, Hill reactionand completely inhibited the light-induced H⁺ efflux. Inhibitionof the H⁺ efflux by Triton X-100 correlated well with the depressionof the apparent rale of light-induced ATP synthesis as wellas the decrease in the intracellular ATP level in light. The light-induced H⁺ efflux was completely inhibited by diethylstilbestrol,a specific inhibitor of plasma membrane ATPase, without anychanges in the intracellular ATP level, thereby suggesting theparticipation of the plasma membrane ATPase in the light-inducedH⁺ efflux. ¹The data in this paper are included in the Ph. D. dissertationsubmitted by M. Kura-Hotta to Tokyo Metropolitan University. (Received February 3, 1984; Accepted June 14, 1984)     

Changes in Protein Content and in the Structure and Number of Chloroplasts during Leaf Senescence in Rice Seedlings

Two types of experiment were carried out to examine whetheror not the inactivation of photosynthesis is related to lossof chloroplasts during foliar senescence of rice seedlings.Levels of both soluble and insoluble leaf proteins decreasedduring senescence, the loss of the soluble proteins being fasterthan that of the insoluble ones. There was a good positive correlationbetween the rate of oxygen evolution and the level of solubleproteins. The inactivation of photosynthesis was also linearlyrelated to the loss of a major fraction of insoluble proteins.Thus, the loss of photosynthetic ability is ascribable to thedegradation of relevant proteins and enzymes during leaf senescence.Electron microscopy revealed that senescence caused the disorientationof the grana and stroma thylakoids, a decrease in the numberof starch granules, and an increase in the size and number ofplastoglobuli. Large grana consisting 20 to 30 thylakoids appearedin aged leaves. In addition to these changes in ultrastructure,there was a significant decrease in the size of chloroplasts.Furthermore, the number of chloroplasts in mesophyll cells wasalso notably reduced during senescence. Thus, the loss of leafproteins and inactivation of photosynthesis are both relatedto the decrease in the total mass of chloroplasts during senescenceof rice seedlings. ³Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113 Japan. (Received January 4, 1989; Accepted April 19, 1989)