Isolation of dividing chloroplasts with intact plastid-dividing rings from a synchronous culture of the unicellular red alga Cyanidioschyzon merolae

In order to obtain a three-dimensional view of the plastid-dividing ring (PD ring) and promote the biochemical study of plastid division, we developed a procedure to isolate structurally intact dividing chloroplasts (rhodoplasts) possessing PD rings from a highly synchronized culture of the unicellular red alga Cyanidioschyzon merolae. The procedure consists of five steps. (1) The chloroplast division cycle is synchronized by light/dark cycles and treatment with 5-fluorodeoxyuridine. (2) The synchronized cells are treated with hypotonic solution. (3) The swollen cells are lysed in a French Pressure Cell. (4) The lysate is treated with DNase I. (5) The intact chloroplasts are separated by density-gradient centrifugation. The PD ring was visualized by fluorescence microscopy, after labeling the surface proteins of isolated chloroplasts with N-hydroxy-sulfo-succinimidyl biotin and detecting them with fluorescein isothiocyanate avidin. Scanning electron microscopy (SEM) showed that the outer envelopes and PD rings were conserved on the isolated dividing chloroplasts. These are the first fluorescence microscopic and SEM images of the PD ring and they clearly show PD rings encircling isolated dividing chloroplasts in three dimensions. Received: 15 April 1999 / Accepted: 12 May 1999     

Synthesis of EF-Tu and distribution of its mRNA between stroma and thylakoids during the cell cycle of Chlamydomonas reinhardii

In Chlamydomonas reinhardii the elongation factor EF-Tu is encoded in the chloroplast DNA. We identified EF-Tu in the electrophoretic product pattern of chloroplast-made proteins and showed that this protein is only synthesized in the first half of the light period in synchronized cells. The newly synthesized EF-Tu contributed little to the almost invariable content of EF-Tu in chloroplasts during the light period of the cell cycle. However, increasing cell volume and the lack of EF-Tu synthesis in the second half of the light period led to a decrease in the concentration of EF-Tu in chloroplasts. At different times in the vegetative cell cycle, the RNA was extracted from whole chloroplasts and from free and thylakoid-bound chloroplast polysomes. The content of mRNA of EF-Tu in chloroplasts and the distribution between stroma and thylakoids were determined. During the light period, the content of the mRNA for EF-Tu varied in parallel to the rate of EF-Tu synthesis. However, in the dark, some mRNA was present even in the absence of EF-Tu synthesis. Most of the mRNA was bound to thylakoids during the whole cell cycle. This suggests that synthesis of EF-Tu is associated with thylakoid membranes.     

SYNCHRONIZATION OF CHLOROPLAST DIVISION IN THE ULTRAMICROALGA CYANIDIOSCHYZON MEROLAE (RHODOPHYTA) BY TREATMENT WITH LIGHT AND APHIDICOLIN

A system of highly synchronized chloroplast divisions was developed in the unicellular red alga Cyanidioschyzon merolae De Luca, Taddei, & Varano. Chloroplast divisions were examined by epifluorescence microscopy following treatments with light and inhibitors. When the cells during stationary phase were transferred into a new medium under a 12:12 h LD cycle, chloroplasts, mitochondria, and cell nuclei divided synchronously in that order soon after the initiation of dark periods. More than 40% of the cells contained dividing chloroplasts. To obtain a system of highly synchronized cell division and chloroplast division, the cells synchronized by a 12:12 h LD cycle were treated with various inhibitors. Nocodazole and propyzamide did not affect cell and organelle divisions, whereas aphidicolin markedly inhibited cell-nuclear divisions and cytokinesis and induced a delay in chloroplast division. More than 80% of the cells contained dividing chloroplasts when cells synchronized by light were treated with aphidicolin for 12 h. This synchronized system will be useful for studies of the molecular and cellular mechanisms of organelle divisions .     

Studies on the biochemistry and fine structure of silica shell formation in diatoms

Summary Cell division in Navicula pelliculosa (Bréb.) Hilse, strain 668 was synchronized with an alternating regime of 5 h light and 7 h dark. Cell volume and dry weight increased only during the light period. DNA synthesis, which began during the third h of light, was followed sequentially by mitosis, cytokinesis, silicic acid uptake, cell wall formation, and cell separation. Silicification and a small amount of net synthesis of DNA, RNA and protein occurred during the dark at the expense of carbohydrate reserves accumulated during the light period. Cells kept in continuous light, after synchronization with the light-dark regime, remained synchronized through a second division cycle; the sequence of morphological events was the same as that in the light-dark division cycle, but the biosynthesis of macromolecular components changed from a stepwise to a linear pattern. The silicon-starvation synchrony was improved by depriving light-dark synchronized cells of silicic acid at the beginning of their division cycle, then resupplying silicic acid to cells blocked at wall formation.Abbreviation L light - D dark Portions based on a thesis submitted by W.M.D. to the University of California, San Diego in partial fulfillment of the requirements for the PH.D degree     

Chloroplast replication in synchronously dividing Euglena gracilis

Summary 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.     

Interactions between the nucleus and cytoplasmic organelles during the cell cycle of Euglena gracilis in synchronized cultures. IV. An aggregate form of chloroplasts in association with the nucleus appearing prior to chloroplast division

Cells of Euglena gracilis were synchronized by applying a 14-h light:10-h dark regimen under photoautotrophic conditions and a 10-h light:14-h dark regimen under photoorganotrophic conditions. At a stage just prior to chloroplast division in the cell cycle of these synchronized cultures, chloroplasts temporarily gathered in the posterior part of the cell and were connected to each other by many bridges. Part of the chloroplast aggregate surrounded about half of the nuclear surface, making connections or close contacts at many sites. A chromosome was always attached to the inner membrane of the nuclear envelope at the site of association with the chloroplast. The nucleoids in these aggregate chloroplasts, examined by staining with 4',6-diamidino-2-phenylindole, a DNA fluorochrome, showed profiles of strings or strands with branchings, under photoorganotrophic conditions at least, and some parts of the branchings came close to the site of association with the nucleus. The association between the chloroplast aggregate and the nucleus was also observed in Euglena cells placed in continuous darkness after synchronization under photoorganotrophic conditions, suggesting that these organellar associations are related to the Euglena cell cycle but are not the result of light:dark alternations used for cell synchronization.     

A Diurnal Settling Rhythm in Platymonas subcordiformis Hazen*

SYNOPSIS. In cultures of Platymonas subcordiformis Hazen, grown in appropriate light-dark cycles, as many as 75% of the cells adhered to the surface of the glass culture vessel toward the end of the light period of each day. Cell division occurred primarily while the cells were attached. Subsequently, motile daughter cells were released into the growth medium by the rupture of the mother cell theca. The settling behavior appears to be an integral part of the life cycle being synchronized to the same extent as cell division.     

Translational regulation of protein synthesis, in response to light, at a critical stage of Volvox development

In Volvox cultures synchronized by a light-dark cycle, juveniles containing presumptive somatic and reproductive cells are produced during the dark, but their cells do not differentiate until after the lights come on. The pattern of protein synthesis changes rapidly after the lights come on. Action spectra and effects of photosynthesis inhibitors indicate that this protein synthetic change is not simply a consequence of renewed flow of energy from illuminated chloroplasts. Actinomycin, at a level adequate to block the response to heat shock, has virtually no effect on the response of the same cells to light; furthermore, RNAs isolated from unilluminated and illuminated juveniles yield indistinguishable in vitro translation products. We conclude, therefore, that this effect of light is exerted almost exclusively at the translational level, generating one of the most striking examples of translational regulation yet described.     

Sexual development in the homothallic green alga Chlamydomonas monoica Strehlow

In Chlamydomonas monoica, cell division and mating are interdependent processes, since under gametogenic conditions only newly born cells are mating competent. By refeeding nitrogen-starved cells with nitrate or ammonium ions, cell division and mating were synchronized. The mating competence of the progeny cells was dependent on the amount of the nitrogen source parent cells were refed, with an optimum around 0.1 mol·10⁵ cells. A second treatment with nitrate inhibited gametogenesis, but only when applied during the first part of the cell cycle, suggesting that an essential part of sexual development takes place during this period. During the latter part of the cell cycle, cells required light to acquire mating competence.     

Periodicity in cell division and physiological behavior of ditylum brightwellii,a marine planktonic diatom,during growth in light-dark cycles

Summary Cells of Ditylum brightwellii, a large marine centric diatom, were partially synchronized by employing an appropriate light-dark cycle. At 20°C this consisted of 8 hrs of illumination at an intensity of 0.05 cal/cm² min. A single 2.8 l culture was studied over a 20 day period by diluting the culture daily to a standard cell concentration. The sequence of events in cell development was as follows: daughter cells were formed late in the light period, in the dark they elongated and the numerous chromatophores began dividing. A minimum cell buoyancy was observed in the dark concurrent with cell elongation. Increase in cell phosphorus took place in the dark period. The photosynthetic rate of cells removed during the dark period decreased to a minimum. In the following light period photosynthetic rate increased to a maximum, photosynthetic pigments, cell carbon, nitrogen, and carbohydrate increased and cell division again took place. Cell silica content increased concomitant with cell division. Details of cell morphology during cell division, based upon light microscopy, are reported.Contribution of the Scripps Institution of Oceanography.     

Stage-dependent localization of LHCP II apoprotein in the Golgi of synchronized cells of Euglena gracilis by immunogold electron microscopy

We have localized LHCP II apoprotein in the Golgi and thylakoids of Euglena gracilis Klebs var. bacillaris Cori and strain Z Pringsheim by electron microscopy using a specific antibody and protein A-gold. Using synchronized cells (light, 14 h:dark, 10 h) we show that thylakoids are always immunoreactive. There is no reaction in the Golgi at 0 h (the beginning of the light period) but immunoreaction appears in the Golgi soon thereafter, rises to a peak at 8 h and declines to zero by 16 h (2 h into the dark period). The peak in immunoreaction in the Golgi immediately precedes the peak in cellular ¹⁴C-labeling of thylakoid LHCP II apoprotein seen by Brandt and von Kessel (Plant Physiol. (1983) 72, 616), supporting our suggestion that processing in the Golgi precedes deposition of LHCP apoprotein in the thylakoids. Substitution of preimmune serum for antiserum eliminates the immunoreaction in the Golgi, and thylakoids of synchronized cells of mutant Gr₁BSL which lacks LHCP II apoprotein show no immunoreaction in the Golgi or thylakoids at any stage. Random observations indicate that the compartmentalized osmiophilic structure (COS) shows an immunoreaction with anti-LHCP II apoprotein antibody at 1 h into the light period (when the Golgi is not immunoreactive) and at 10 h into the light period (when the Golgi is fully reactive), suggesting that the COS remains immunoreactive throughout the cell cycle.     

Varying competence for protein import into chloroplasts during the cell cycle in Chlamydomonas.

By studying the import of radioactively labelled small subunit of ribulose-1,5-bisphosphate carboxylase (pSS) into chloroplasts of the green alga C. reinhardtii cw-15 protein delivery to chloroplasts was found to vary during the cell cycle. Chloroplasts were isolated from highly synchronous cultures at different time points during the cell cycle. When pSS was imported into 'young' chloroplasts isolated early in the light period about three times less pSS was processed to small subunit SS than in 'mature' chloroplasts from the middle of the light period. In 'young' chloroplasts also, less pSS was bound to the envelope surface. During the second half of the light period the import competence of isolated chloroplasts decreased again when based on chlorophyll content or cell volume, but did not change significantly when related to chloroplast number. Measurements of pSS binding to the surface of chloroplasts of different age indicated that the adaptation of protein import competence during the cell cycle is due to a variation of the number of binding sites per chloroplast surface area, rather than to modulation of the binding constant.     

BEHAVIOR OF CHLOROPLAST NUCLEOIDS DURING THE CELL CYCLE OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA) IN SYNCHRONIZED CULTURE1

Cells of Chlamydomonas reinhardtii Dangeard were synchronized under a 12:12 h light: dark regimen. They increased in size during the light period, while nuclear division, chloroplast division and cytokinesis occurred during the dark period. Zoospores were liberated toward the end of the dark period. Changes in profile and distribution of chloroplast nucleoids were followed with a fluorescence Microscope after fixation with 0.1%(w/v) glutaraldehyde followed by staining with 4′.6-diamidino-2-phenylidole (DAPI), a DNA fluorochrome. About ten granular nucleoids were dispersed in the chloroplast at the beginning of the light period (0 h). Within 4 h the nucleoids aggregated around the pyrenoid giving a compact profile. The formation of the compact aggregate of cp-nucleoids around the pyrenoid occurred with maximal frequency twice during the light period. Toward the end of the light period the nucleoids were transformed into the form of threads interconnected with fine fibrils spreading throughout the chloroplast. Initially the thread-like nucleoids fluoresced only faintly. The fluorescence of some parts of the threadlike form became brighter over a period of 6 h; these nucleoids were divided into daughter chloroplasts during chloroplast division. Soon after chloroplast division, these thread-like nucleoids were transformed into about 20 granular forms, which were gradually combined to form about ten larger granular bodies in zoospores immediately prior to liberation from mother cells. Fixation of cells with glutaraldehyde at high concentrations or treatment of cells with protease significantly modified the profiles of DAPI-stained nucleoids. The different morphologies of chloroplast nucleoids are discussed in relation to changes in configuration of their protein components.     

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-³H]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.     

Subcellular localization and light-regulated expression of protoporphyrinogen IX oxidase and ferrochelatase in Chlamydomonas reinhardtii

Protoporphyrinogen IX oxidase (PPO) catalyzes the last common step in chlorophyll and heme synthesis, and ferrochelatase (FeC) catalyzes the last step of the heme synthesis pathway. In plants, each of these two enzymes is encoded by two or more genes, and the enzymes have been reported to be located in the chloroplasts or in the mitochondria. We report that in the green alga Chlamydomonas reinhardtii, PPO and FeC are each encoded by a single gene. Phylogenetic analysis indicates that C. reinhardtii PPO and FeC are most closely related to plant counterparts that are located only in chloroplasts. Immunoblotting results suggest that C. reinhardtii PPO and FeC are targeted exclusively to the chloroplast, where they are associated with membranes. These results indicate that cellular needs for heme in this photosynthetic eukaryote can be met by heme that is synthesized in the chloroplast. It is proposed that the multiplicity of genes for PPO and FeC in higher plants could be related to differential expression in differently developing tissues rather than to targeting of different gene products to different organelles. The FeC content is higher in C. reinhardtii cells growing in continuous light than in cells growing in the dark, whereas the content of PPO does not significantly differ in light- and dark-grown cells. In cells synchronized to a light/dark cycle, the level of neither enzyme varied significantly with the phase of the cycle. These results indicate that heme synthesis is not directly regulated by the levels of PPO and FeC in C. reinhardtii.     

AN ULTRASTRUCTURAL STUDY OF CHLOROPLAST STRUCTURE AND DEDIFFERENTIATION IN TISSUE CULTURES OF STREPTANTHUS TORTUOSUS (CRUCIFERAE)

Cells of Streptanthus tortuosus callus tissue contain chloroplasts when cultured in a liquid medium in the light. Similar cells grown in the dark contain proplastids that fail to develop prolamellar bodies but do contain a complex of loosely-associated membranes. When green, light-grown cultures are cut into small pieces and subcultured to a fresh culture medium, they become bleached even though maintained under the same illumination. The fine structure of the chloroplasts and the chlorophyll content of the cells indicate a dedifferentiation of the chloroplasts to a proplastid state during the early culture period. The changes in the ultrastructure of the plastids are paralleled by a dedifferentiation of the vacuolate cells to a less differentiated, meristematic state. Subsequent growth in the light results in a re-formation of chloroplasts and an increase in the chlorophyll content of the cells. The period of chloroplast redevelopment is associated with the re-formation of large central vacuoles in the cultured cells. Invaginations of the inner membrane of the plastid envelope occur at all stages of plastid development and are not lost during the period of grana degeneration. The proplastids formed from the dedifferentiation of the chloroplasts contain a large number of these invaginations and the redevelopment of grana is associated with a change in the electron density of the invaginating membranes. The degradation of the chlorophyll-containing membranes of the grana occurs during a period of rapid cytoplasmic synthesis induced by the fresh supply of nutrients in the culture medium. These results suggest that the high levels of nutrients may act directly on the chloroplasts and cause their dedifferentiation or that the rapid cell growth induced by the nutrients may cause a degradation of the membrane proteins in the grana of the chloroplasts and an incorporation of the released amino acids into non-plastid components of the cytoplasm.     

Studies on Halimeda IV. An endogenous rhythm of chloroplast migration in the siphonous green alga,H. distorta 1

Abstract

Halimeda has been found particularly suitable for studies of long‐distance chloroplast migration by virtue of its coenocytic structure and calcium carbonate skeleton. A circadian rhythm of chloroplast migration in Halimeda distorta was monitored by videography of segment surface pigmentation. In normal 12 h light/12 h dark treatments synchronised with dawn and dusk, the segments were green all day, began to become pale immediately the light was turned off, and then remained almost white for most of the night until beginning to re‐green a few hours before dawn. As a result of that, they were already quite green by the time the light was turned on. In continuous darkness a similar cycle, albeit with reducing amplitude and a period of about 23 hours, was maintained for at least 7 days. However, this cycle differed significantly from the normal one in that the segments did not remain green after the light was not switched on at dawn, but rather began to pale immediately thereafter. Conversely, in continuous light the segments did not become pale at any time. Thus, the rhythmical re‐emergence of the chloroplasts before dawn and their subsequent withdrawal appears to be controlled by an endogenous rhythm which is independent of light. However, light does completely, but reversibly, inhibit the chloroplast withdrawal component of the cycle. This behaviour of the chloroplasts in Halimeda is very similar to that in the related alga, Caulerpa, but it is quite different from that in another extensively Studied but unrelated siphonous green alga, Acetabularia, in which the circadian rhythm of chloroplast migration is maintained in continuous light.     

Light Dependence of Chloroplast Replication and Starch Metabolism in the Moss Polytrichum commune

Spores of Polytrichum conwtuine were grown on a mineral salt solution with or without sucrose and exposed to continuous white light, continuous darkness, red light and/or far-red light. With sucrose, germination and filament growth occurred in all conditions, Without sucrose, germination and filament growth occurred only in light. Two phytochrome mediated responses of the chloroplasts were demonstrated. Chloroplast replication occurred in continuous white light and red light of 15 min/6 hours. In continuous darkness and in far red light of 15 min/6 hours, the size of the chloroplasts increased; but no replication occurred. Both the chloroplast replication and chloroplast size were red, far-red light reversible. When changed from one continuous light environment to another, a lag period occurred before the chloroplasts responded to the new environment. Electron micrographs of sections and in vivo staining of the chloroplasts with iodine solution demonstrated that the change in size of the chloroplasts was at least partially due to the synthesis and degradation of starch.     

Change in Photosynthetic Capacity over the Cell Cycle in Light/Dark-Synchronized Amphidinium carteri Is Due Solely to the Photocycle

Cell cycle dependent photosynthesis in the marine dinoflagellate Amphidinium carteri was studied under constant illumination and light/dark (L/D) photocycles to distinguish intrinsic cell cycle control from environmental influences. Cells were grown in constant light and on a 14:10 L:D cycle at light intensities that would yield a population growth rate of 1 doubling per day. In the former case division was asynchronous, and cells were separated according to cell cycle stage using centrifugal elutriation. Cells grown on the L:D cycle were synchronized, with division restricted to the dark period. Cell cycle stage distributions were quantified by flow cytometry. Various cell age groups from the two populations were compared as to their photosynthetic response (photosynthetic rate versus irradiance) to determine whether or not the response was modulated primarily by cell cycle constraints or the periodic L/D cycle. Cell cycle variation in photosynthetic capacity was found to be determined solely by the L/D cycle; it was not present in cells grown in constant light.     

Growth retardation of plants transformed by overexpression of NtFtsZ1-2 in Tobacco

We transformed tobacco plants (Mcotiana tabacum L, Xanthi) by introducing a sense construct ofNtFtsZ1-2. This tobacco nuclear gene encodes a chloroplast-localized homologue of FtsZ, the bacterial cell-division protein. The overexpressing plants contained enlarged chloroplasts in their leaf mesophyll cells. In the T₁ progeny, we observed three different phenotypes: 1 ) plants with cells containing many small chloroplasts, which was the same as for wild-type plants; 2) plants in which the celts contained one to three enlarged chloroplasts (severe type); and 3) plants whose cells contained a combination of many small chloroplasts and one to three enlarged chloroplasts (intermediate type). The outward appearance of the severe and intermediate types of transgenic plants did not differ noticeably from the wild-types. However, the severe-type plants were most retarded in their growth under both high- and low-light conditions, followed by the intermediate-types. Under medium levels of light, the two types of transgenic plants exhibited growth rates comparable to that of the wild types. Based on the overall results, we suggest that many small chloroplasts, rather than a few large chloroplasts, are required for efficient use of light energy in the mesophyll cells.