In vitro chloroplast protein synthesis by the chromophytic alga Olisthodiscus luteus

The chloroplasts of chlorophytic and chromophytic plants exhibit significant morphological and biochemical differences. Presently, it is impossible to compare the influence of ctDNA on the structure and function of organelles within these two phylogenetic groups for no data exist in the literature on the profile of protein products synthesized by a chromophytic plastid. In this paper, the chloroplast DNA coded proteins of the chromophytic plant Olisthodiscus luteus are investigated by labeling isolated chloroplasts in vitro. Isolated plastids of excellent morphological condition are pulse labeled with [35S]methionine. Approximately 100 proteins are detected by two-dimensional gel electrophoresis and fluorography. However, these isolated plastids have a number of unusual characteristics: (1) they are photosynthetically inactive; (2) in vitro protein synthesis is light independent; (3) all proteins synthesized in vitro are membrane associated.     

Heat shock Hsp70 protein is chloroplast-encoded in the chromophytic alga Pavlova lutherii

Heat shock proteins are ubiquitous and highly conserved. Recently they have become implicated in the import of proteins into organelles. All the heat shock genes characterized to date, however, are known or assumed to be encoded in the nuclear genome even if the corresponding protein can be localised in the mitochondrion or chloroplast. In contrast, we identify here an hsp70 gene in the unicellular chromophytic alga Pavlova lutherii which is located on the chloroplast genome. Localisation of this gene to the chloroplast chromosome is confirmed by Southern blot analysis and pulse-field gel electrophoresis which also reveals that the length of the P. lutherii chloroplast chromosome is 115 kb. We compare the predicted protein of this hsp70 gene with that of maize and of the analogous proteins in the prokaryotic organisms Escherichia coli and Synechocystis PCC6803. The greatest identity is found with the cyanobacterium Synechocystis PCC6803.     

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 B₁₂ 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 B₁₂, plastid replication continues in the absence of cell division thereby increasing the cell's plastid complement significantly.     

Chloroplast Protein Synthesis in the Chromophytic Alga Olisthodiscus luteus: Cell Cycle Analysis

This study represents the first report on chloroplast protein synthesis during the synchronous cell growth of a chromophytic (chlorophyll a,c) plant. When the unicellular alga Olisthodiscus luteus is maintained on a 12-hour light:12-hour dark cycle, cell and chloroplast number double every 24 hours. A temporal separation between these two events occurs. Measurements of chloroplast and total cellular protein values suggest that polypeptide synthesis occurs mainly in the light portion of the cell cycle, and pulse chase studies demonstrate that chloroplast proteins made in the light are not degraded in the dark. Data support the following conclusions: (a) a similar complement of chloroplast DNA coded proteins is made at all phases of the light portion of the cell cycle, and (b) chloroplast protein synthesis is a light rather than a cell cycle mediated response.     

Characterisation of a chloroplast-encoded secY homologue and atpH from a chromophytic alga. Evidence for a novel chloroplast genome organisation.

secY is a prokaryotic gene that encodes the SecY protein, an integral membrane component of the prokaryotic protein translocation apparatus. A chloroplast-encoded secY homologue has been identified in the unicellular, chromophytic alga, Pavlova lutherii. The gene predicts a protein composed of ten membrane-spanning regions, that is approximately 25% homologous and 50% similar to bacterial and plastid SecY proteins. The secY gene from P. lutherii is independent of the ribosomal protein (rp) gene cluster to which it is closely linked in other organisms. In P. lutherii secY is located 5' to atpI and atpH. Since, in higher plants the atpIHFA gene cluster and the rp gene cluster are separated by approximately 50 kb, we conclude, this indicates a novel chloroplast gene arrangement in P. lutherii.     

Effects of Olisthodiscus luteus on the feeding and reproduction of the marine rotifer Synchaeta cecilia

The chrysophyte Olisthodiscus luteus is not ingested by Synchaetacecilia. It inhibits the feeding on other, acceptable food atO. luteus densities as low as 50 cells ml^–1 and reducessurvival and reproduction at O. luteus densities > 10³ cellsml^–1. The possible mechanisms and implications of thisphenomenon for the distribution and abundance of S. ceciliaare discussed.     

Chromatin structure in the unicellular algae Olisthodiscus luteus,Crypthecodinium cohnii and Peridinium balticum

Isolated nuclei of the unicellular alga Olisthodiscus luteus, the uninucleate dinoflagellate Crypthecodinium cohnii and the binucleate dinoflagellate Peridinium balticum were lysed and deposited on grids by the microcentrifugation technique. The ultrastructure of the released chromatin fibers was compared to that of mouse liver nuclei. Chromatin from nuclei of Olisthodiscus luteus and the eukaryotic¹ nuclei of Peridinium balticum, appeared as linear arrays of regularly repeating subunits which were identical in size and morphology to mouse nucleosomes. In contrast, the chromatin fibers from Crypthecodinium cohnii nuclei appeared as smoothe threads with a diameter of about 6.5 nm. Nuclear preparations containing mixtures of dinokaryotic and eukaryotic nuclei of Peridinium balticum also contained smooth fibers which most likely originated from the dinokaryotic nuclei. These and other results demonstrating the presence of nucleosomes in lower eukaryotes suggest that the subunit structure of chromatin arose very early in the evolution of the eukaryotic cell.     

In vivo study of chloroplast volume regulation.

This paper describes a new technique that can be used to study chloroplast volume regulation in vivo. Nuclear magnetic resonance spectroscopy was used to measure relative amounts of chloroplast water in Acer platanoides leaves as they dried in air, and also in leaf disks exposed to aqueous polyethylene glycol, sucrose, or glycerol. The chloroplasts retained a constant quantity of water as leaf water potentials varied between -0.05 and -1.90 MPa, indicating that volume regulation was effective throughout this range. The chloroplasts lost water when the water potential fell below -1.90 MPa, except when leaf disks were exposed to glycerol, suggesting that the lower limit of effective volume regulation is determined by physiological levels of osmotic solutes and that glycerol can be used for chloroplast osmoregulation.     

In vivo localization of centrin in the green alga Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii has been used as a model system to study flagellar assembly, centriole assembly, and cell cycle events. These processes are dynamic. Therefore, protein targeting and protein-protein interactions should be evaluated in vivo. To be able to study dynamic processes in C. reinhardtii in vivo, we have explored the use of the green fluorescent protein (GFP). A construct containing a fusion of centrin and GFP was incorporated into the genome as a single copy. The selected clone shows expression in 25-50% of the cells. Centrin-GFP was targeted in vivo to the nuclear basal body connectors and the distal connecting fibers. At the electron microscopic level, it was also localized to the flagellar transitional regions. EM data of transformants indicate that there are some abnormalities in the centrin-containing structures. The transitional region consists of only the transverse septum or has lesions in the H-piece. The distal connecting fibers are thinner and their characteristic crossbands seem to be incomplete. Deflagellation is not affected since more than 95% of the cells deflagellate. Also basal body segregation is not affected since cells with an abnormal flagellar number were not detected. Functional studies of the centrin-GFP fusion show the characteristic calcium-induced mobility shift in SDS-PAGE. Immunofluorescence revealed that during cell division, centrin-GFP remains associated with the basal bodies. In vivo localization of the fusion protein during cell division shows that in metaphase centrin-GFP appears as two opposing spots located close to the spindle poles. The distance between the spots increases as the cells progress through anaphase and then decreases during telophase. GFP is a useful tool to study dynamic processes in the cytoskeleton of C. reinhardtii.     

In vivo synthesis of glycogen by phosphorylase system

Summary Application of conventional histochemical techniques to the living chick retina demonstrates that phosphorylase can synthesize glycogen (polyglucose) in vivo, in the paraboloid of the accessory cone. Natural in vivo glycogen synthesis may therefore be due to glycogen synthetase and phosphorylase systems, although phosphorylase is normally regarded as a glycolytic enzyme.     

Kinetic Complexity, Homogeneity, and Copy Number of Chloroplast DNA from the Marine Alga Olisthodiscus luteus

The kinetic complexity of chloroplast DNA isolated from the chromophytic alga Olisthodiscus luteus has been determined. Using optical reassociation techniques, it was shown that the plastid DNA of this alga reacted as a single component with a second order rate constant of 4.1 molar⁻¹ and second⁻¹ (Cot_½ 0.24 molar second) under conditions equivalent to 180 millimolar Na⁺ and 60°C. Given the 92 × 10⁵ dalton complexity calculated for this chloroplast genome, an Olisthodiscus cell contains 650 plastome copies. Although this complement remains constant throughout the growth cycle of the organism, the ploidy level of an individual chloroplast shows significant plasticity and is dependent upon the number of chloroplasts present per cell. Experiments with the DNA fluorochrome Hoechst dye 33258 (bisbenzimide) demonstrate that plastids isolated from all phases of cell growth each possess a ring-shaped nucleoid containing detectable DNA. Olisthodiscus chloroplast DNA showed no sequence mismatch when thermal denaturation profiles of reassociated chloroplast DNA were examined, thus all plastome copies are essentially identical. Finally, reassociation studies demonstrated that no foldback (short inverted repeat) sequences were present in the plastid genome although significant hairpin loop structures were observed in control nuclear DNA samples.     

In vivo assessment of the significance of phosphorylation of the Arabidopsis chloroplast protein import receptor, atToc33

atToc33 is a transit peptide receptor of the chloroplast outer envelope membrane, and possesses GTPase activity. In vitro, its transit peptide- and GTP-binding properties are abrogated by its phosphorylation at serine 181, which was proposed to represent an important regulatory mechanism. We mutated S181 to alanine (to prevent phosphorylation), and to aspartate and glutamate (to mimic the effects of phosphoserine), and expressed all three proteins in ppi1 (atToc33 knockout) plants using the native promoter. The mutants complemented ppi1 with equal efficiency in respect of all criteria tested, including protein import efficiency and light stress tolerance. The data suggest that atToc33 phosphorylation may not play an important role in vivo.