Localization of phycoerythrin at the lumenal surface of the thylakoid membrane in Rhodomonas lens.

The thylakoids of cryptomonads are unique in that their lumens are filled with an electron-dense substance postulated to be phycobiliprotein. In this study, we used an antiserum against phycoerythrin (PE) 545 of Rhodomonas lens (gift of R. MacColl, New York State Department of Health, Albany, NY) and protein A-gold immunoelectron microscopy to localize this light-harvesting protein in cryptomonad cells. In sections of whole cells of R. lens labeled with anti-PE 545, the gold particles were not uniformly distributed over the dense thylakoid lumens as expected, but instead were preferentially localized either over or adjacent to the thylakoid membranes. A similar pattern of labeling was observed in cell sections labeled with two different antisera against PE 566 from Cryptomonas ovata. To determine whether PE is localized on the outer or inner side of the membrane, chloroplast fragments were isolated from cells fixed in dilute glutaraldehyde and labeled in vitro with anti-PE 545 followed by protein A-small gold. These thylakoid preparations were then fixed in glutaraldehyde followed by osmium tetroxide, embedded in Spurr, and sections were labeled with anti-PE 545 followed by protein A-large gold. Small gold particles were found only at the broken edges of the thylakoids, associated with the dense material on the lumenal surface of the membrane, whereas large gold particles were distributed along the entire length of the thylakoid membrane. We conclude that PE is located inside the thylakoids of R. lens in close association with the lumenal surface of the thylakoid membrane.

Effects of chloramphenicol on chloroplast and mitochondrial ultrastructure in Ochromonas danica.

The effect of chloramphenicol (CAP) on cell division and organelle ultrastructure was studied during light-induced chloroplast development in the Chrysophyte alga, Ochromonas danica. Since the growth rate of the CAP-treated cells is the same as that of the control cells for the first 12 hr in the light, CAP is presumed to be acting during that interval solely by inhibiting protein synthesis on chloroplast and mitochondrial ribosomes. CAP markedly inhibits chloroplast growth and differentiation. During the first 12 hr in the light, chlorophyll synthesis is inhibited by 93%, the formation of new thylakoid membranes is reduced by 91%, and the synthesis of chloroplast ribosomes is inhibited by 81%. Other chloroplast-associated abnormalities which occur during the first 12 hr and become more pronounced with extended CAP treatment are the presence of prolamellar bodies and of abnormal stacks of thylakoids, the proliferation of the perinuclear reticulum, and the accumulation of dense granular material between the chloroplast envelope and the chloroplast endoplasmic reticulum. CAP also causes a progressive loss of the mitochondrial cristae, which is paralleled by a decline in the growth rate of the cells, but it has no effect on the synthesis of mitochondrial ribosomes. We postulate that one or more chloroplast ribosomal proteins are synthesized on chloroplast ribosomes, whereas mitochondrial ribosomal proteins are synthesized on cytoplasmic ribosomes.

Autoradiographic evidence for many segregating DNA molecules in the chloroplast of Ochromonas danica.

Light-grown cells of Ochromonas danica, which contain a single chloroplast per cell, were labeled with [methyl-(3)H]thymidine for 3 h (0.36 generations) and the distribution of labeled DNA among the progeny chloroplasts was followed during exponential growth in unlabeled medium for a further 3.3 generations using light microscope autoradiography of serial sections of entire chloroplasts. Thymidine was specifically incorporated into DNA in both nuclei and chloroplasts. Essentially all the chloroplasts incorporated label in the 3-h labeling period, indicating that chloroplast DNA is synthesized throughout the cell cycle. Nuclear DNA has a more limited S period. Both chloroplast DNA and nuclear DNA are conserved during 3.3 generations. After 3.3 generations in unlabeled medium, grains per chloroplast followed a Poisson distribution indicating essentially equal labeling of all progeny chloroplasts. It is concluded that the average chloroplast in cells of Ochromonas growing exponentially in the light contains at least 10 segregating DNA molecules.

The route of entry of cytoplasmically synthesized proteins into chloroplasts of algae possessing chloroplast ER.

In 8 classes of algae, namely the Cryptophyceae, Raphidophyceae, Haptophyceae, Chrysophyceae, Bacillariophyceae, Xanthophyceae, Eustigmatophyceae and Phaeophyceae, the chloroplasts, in addition to being surrounded by a double-membraned chloroplast envelope, are also enclosed by a cisterna of endoplasmic reticulum called the chloroplast ER. Often this ER cisterna is continuous with the outher membrane of the nuclear envelope in such a manner that the nuclear envelope forms a part of the ER sac enclosing the chloroplast. In all these classes of algae except the Cryptophyceae, a regular network of tubules and vesicles, named the periplastidal reticulum, is present at a specific location between the chloroplast envelope and the chloroplast ER. In the Cryptophyceae, scattered vesicles are found between the chloroplast envelope and the chloroplast ER. Ribosomes which have been shown to be arranged to polysomes are found on the outer membrane of the chloroplast ER. It is proposed that nuclear-coded proteins which are destined for the chloroplast are synthesized on these polysomes, passing during synthesis into the lumen of the ER cisterna. Vesicles containing these proteins then pinch off the chloroplast ER and form the periplastidal reticulum. Vesicles containing these proteins then pinch off the chloroplast ER and form the periplastidal reticulum. Vesicles then fuse with the outer membrane of the chloroplast envelope thereby delivering their contents to the lumen of the chloroplast envelope. Proteins then cross the inner membrane of the chloroplast envelope in an as yet unknown manner. Experimental evidence for this hypothesis comes from studies on Ochromonas danica using chloramphenicol and spectinomycin, which inhibit protein synthesis on plastid ribosomes, and cycloheximide, which inhibits protein synthesis on cytoplasmic ribosomes. In cells of Ochromonas exposed to chloramphenicol or spectinomycin, the periplastidal reticulum proliferates markedly becoming several layers thick. Presumably this build up of periplastidal reticulum occurs because the transport of cytoplasmically synthesized plastid proteins is slowed down when protein synthesis in the chloroplast is inhibited. Conversely, when cells of Ochromonas are treated with cycloheximide, there is a reduction in the amount of periplastidal reticulum presumably because there are no cytoplasmically synthesized proteins to be transported into the chloroplast.

Ochromonas mitochondria contain a specific chloroplast protein.

Antibody raised against the small subunit of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] of Chlamydomonas reinhardtii labeled the mitochondria as well as the chloroplast of the chrysophyte alga Ochromonas danica in sections prepared for immunoelectron microscopy by the protein A-gold technique. The same antibody labeled the chloroplast but not the mitochondria of C. reinhardtii. A quantitative study of labeling in dark-grown, greening (32 hr light), and mature green cells of O. danica revealed that anti-small-subunit staining in the mitochondria increased progressively in the light as it does in the plastid. Antibody to the large subunit of the enzyme did not label the mitochondria of either O. danica or C. reinhardtii. In view of the recent demonstrations of homologous DNA sequences in the mitochondrial and chloroplast genomes of higher plants, we suggest that the DNA sequence coding for the small subunit has migrated to the mitochondria from nucleus or chloroplast and is expressed within the organelle.

Chloroplast replication in synchronously dividing Euglena gracilis.

Chloroplast replication was studied in Euglena gracilis Klebs, strain Z, synchronized by appropriate light-dark cycles. The chloroplasts divide synchronously, at the time of cytokinesis, but with a tighter synchrony than cell division itself. The chloroplasts within one cell are not noticeably better synchronized than those in the whole population. Chloroplast replication and cell division could not be separated by resetting the time of the light-dark cycle which induces the synchrony. These results are discussed for their implications concerning the mechanisms of integrating cell and plastid division.

Phycoerythrin is absent from the pyrenoid of Porphyridium cruentum: photosynthetic implications.

The thylakoid lamellae which traverse the pyrenoid of the unicellular red alga Porphyridium cruentum (Agardh) Nägeli appear to lack phycobilisomes. We have confirmed by immuno-electron microscopy that phycoerythrin (PE), an important structural component of the phycobilisomes of red algae, is absent from the pyrenoid. To characterize pyrenoid thylakoids further, electron-microscopic cytochemical methods were employed to detect photosystem activity. Photosystem (PS) I activity was demonstrated in both stromal and pyrenoid thylakoids by the photooxidation of 3,3'-diaminobenzidine. In contrast, the localization of photoreduced distyryl nitroblue tetrazolium demonstrated that PSII activity was restricted to stromal thylakoids. The observed partitioning of PE and PSII activity within the plastid may be related to another observation, that being the localization of nearly all ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) within the pyrenoid of this alga. It is possible that the pyrenoid of P. cruentum functions as a specific metabolic compartment where CO2 fixation is enhanced by the absence of photosynthetic O2 evolution.

Immunocytochemical Localization of Ribulose-1,5-Bisphosphate Carboxylase in the Pyrenoid and Thylakoid Region of the Chloroplast of Chlamydomonas reinhardtii.

The distribution of the large and small subunits of ribulose-1,5-bisphosphate carboxylase in the chloroplast of Chlamydomonas reinhardtii was studied by immunoelectron microscopy by labeling Lowicryl-embedded sections with antibody to each subunit followed by protein A-gold. In light-harvested synchronously dividing cells, antibodies to each subunit heavily labeled the pyrenoid, whereas the thylakoid region of the plastid was lightly labeled. By estimating the volume of each chloroplast compartment, it was determined that approximately 40% of the total small subunit in the plastid and 30% of the large subunit are localized in the thylakoid region, presumably in the stroma. In synchronously dividing cells exposed to an extended dark period, the amount of labeling of the pyrenoid region by antibody to the small subunit stayed constant, but the labeling of the thylakoid region decreased. In stationary phase cells, the proportion of the label over the pyrenoid is higher than in synchronously dividing cells suggesting that the pyrenoid may be a storage organelle.

Synchronous Growth and Plastid Replication in the Naturally Wall-less Alga Olisthodiscus luteus.

Olisthodiscus luteus is a unicellular biflagellate alga which contains many small discoidal chloroplasts. This naturally wall-less organism can be axenically maintained on a defined nonprecipitating artificial seawater medium. Sufficient light, the presence of bicarbonate, minimum mechanical turbulence, and the addition of vitamin B(12) to the culture medium are important factors in the maintenance of a good growth response. Cells can be induced to divide synchronously when subject to a 12-hour light/12-hour dark cycle. The chronology of cell division, DNA synthesis, and plastid replication has been studied during this synchronous growth cycle. Cell division begins at hour 4 in the dark and terminates at hour 3 in the light, whereas DNA synthesis initiates 3 hours prior to cell division and terminates at hour 10 in the dark. Synchronous replication of the cell's numerous chloroplasts begins at hour 10 in the light and terminates almost 8 hours before cell division is completed. The average number of chloroplasts found in an exponentially growing synchronous culture is rather stringently maintained at 20 to 21 plastids per cell, although a large variability in plastid complement (4-50) is observed within individual cells of the population. A change in the physiological condition of an Olisthodiscus cell may cause an alteration of this chloroplast complement. For example, during the linear growth period, chloroplast number is reduced to 14 plastids per cell. In addition, when Olisthodiscus cells are grown in medium lacking vitamin B(12), plastid replication continues in the absence of cell division thereby increasing the cell's plastid complement significantly.

The effects of spectinomycin and ethidium bromide on the synthesis of organelle rRNA and on ultrastructure in Ochromonas danica.

The effects of 24-h exposure to spectinomycin (100 microgram/ml) and ethidium bromide (1 microgram/ml) on the accumulation of chloroplast and mitochondrial rRNAs and on organelle ultrastructure were studied in greening cells of Ochromonas danica. Cells treated with ethidium bromide for 24 h divide at the same rate as controls but contain less than one third the normal amount of mitochondrial rRNA. Ultrastructural observations showed that these cells contain only 10% the number of mitochondrial ribosomes found in controls as well as fewer mitochondrial cristae. Ethidium bromide has no effect on chloroplast ultrastructure in Ochromonas. Greening cells treated with spectinomycin grow at close to control rates but contain 30-40% less chloroplast rRNA than do controls. Electron microscopy showed that spectinomycin disrupts the organization of chloroplast membranes and reduces the number of chloroplast ribosomes by 30%. Under these conditions, spectinomycin has no effect on mitochondrial rRNA or ultrastructure. Since spectinomycin is a specific inhibitor of translation on 70S ribosomes, these results are consistent with the possibility that at least some chloroplast ribosomal proteins are synthesized in the chloroplast of Ochromonas.

Immunocytochemical Localization of Phosphoribulose Kinase in the Cyanelles of Cyanophora paradoxa and Glaucocystis nostochinearum.

The distribution of phosphoribulose kinase (PRK) in the cyanelles of Cyanophora paradoxa Korschikoff and Glaucocystis nostochinearum Itzigsohn was studied by protein A-gold immunoelectron microscopy. In both endocyanomes, antiserum against PRK heavily labeled the thylakoid region of the cyanelles, whereas little or no label was present over the carboxysomes. Antiserum against ribulose 1,5-bisphosphate carboxylase/oxygenase by contrast heavily labeled the carboxysomes of each endocyanome. In vitro studies of PRK distribution in cell-free extracts of C. paradoxa showed that 93% of the enzyme was in the soluble fraction. Quantitative immunoelectron microscopy showed that more than 99% of the PRK in the cyanelle of C. paradoxa was localized in the thylakoid region. We conclude that the carboxysomes of cyanelles like the carboxysomes of autotrophic prokaryotes and the pyrenoids of green algal chloroplasts do not contain phosphoribulose kinase.