BIOGENESIS OF CHLOROPLAST MEMBRANES : II. Plastid Differentiation during Greening of a Dark-Grown Algal Mutant (Chlamydomonas reinhardi)

Dark-grown cells of the y-1 mutant of Chlamydomonas reinhardi contain a partially differentiated plastid lacking the photosynthetic lamellar system. When exposed to the light, a rapid synthesis of photosynthetic membranes occurs accompanied by synthesis of chlorophyll, lipids, and protein and extensive degradation of the starch reserve. The process is continuously dependent on illumination and is completed within 6–8 hr in the absence of cell division. Photosynthetic activity (O₂ evolution, Hill reaction, NADP photo-reduction, and cytochrome f photooxidation) parallels the synthesis of pigment and membrane formation. During the greening process, only slight changes occur in the levels of soluble enzymes associated with the photosynthetic process (RuDP-carboxylase, NADP-linked G-3-P dehydrogenase, alkaline FDPase (pH 8)) as compared with the dark control. Also cytochrome f concentration remains almost constant during the greening process. The kinetics of the synthesis of chlorophyll, formation of photosynthetic membranes, and the restoration of photosynthetic activity suggest that the membranes are assembled from their constituents in a single-step process.     

Photosynthetic oxygen production and the size of the photosynthetic unit in a cell-free preparation from Cyanidium caldarium

A cell-free preparation has been isolated from a mutant of Cyanidium caldarium, grown under conditions such that there is 15 times less chlorophyll per photosynthetic unit than in normal green algae. The preparation is sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea and shows the well-characterized oscillation of O₂ yield, from saturating flashes, following a period of dark adaptation. Greening experiments with dark-grown, wild-type Cyanidium show that the synthesis of photosynthetic units precedes that of bulk chlorophyll and that the O₂-producing system is assembled before the total system coupled to CO₂. No large-scale cooperation of chlorophyll molecules is required for O₂ production.     

Chloroplast-cytoplasmic interrelations involved in chloroplast development in Chlamydomonas reinhardi y-1: effect of selective depletion of chloroplast translates

Chlamydomonas reinhardi y-1 cells grown in the dark in the presence of chloramphenicol (CD cells) are depleted of photosynthetic membranes and 70S translates. These cells were found to be unable to synthesize chlorophyll in the light until chloroplast protein synthesis was resumed. On the other hand, CD cells acquired the capacity to partially green in the presence of cycloheximide. This greening was characterized by the development of photosynthetic activity, as demonstrated by light- dependent oxygen evolution of whole cells and by measurements of ribulose-1,5-bisphosphate carboxylase and fluorescence kinetics. The chlorophyll synthesized de novo during greening in the absence of 80S ribosomal activity was organized in chlorophyll-protein complexes, as ascertained by low-temperature fluorescence-emission spectra. The morphology of these cells appeared to be normal. A model has been proposed as a working hypothesis, which could account for the phenomena described above and previously reported data pertaining to chloroplast development.     

A chloroplast-associated fatty acid synthetase system in Euglena

Fatty acid synthetase activity in etiolated Euglena gracilis strain Z is independent of added ACP and associated with a high-molecular-weight complex of the type found in yeast. Cells grown in the dark and then greened by illumination in a resting medium develop a second enzyme system which is dependent on added ACP and generally resembles the corresponding E. coli and plant enzymes. Cycloheximide has no effect on the appearance of the ACP-dependent fatty acid synthetase in greening cells whereas chloramphenicol causes complete inhibition at concentrations which decrease chlorophyll synthesis by 66%. An induction of the ACP-dependent fatty acid synthetase in the absence of chloroplast development occurs on exposure of dark-grown cells to doses of ultraviolet light which selectively affect proplastid nucleoprotein. This enzyme induction by ultraviolet light is inhibited by chloramphenicol. The protein synthesis machinery of the chloroplast appears to be responsible, either directly or indirectly, for the appearance of the ACP-dependent fatty acid synthetase of Euglena.     

BIOGENESIS OF CHLOROPLAST MEMBRANES : V. A Radioautographic Study of Membrane Growth in a Mutant of Chlamydomonas reinhardi y-1

The development of photosynthetic lamellae during greening of dark-grown Chlamydomonas y-1 cells was investigated by radioautography. Acetate-³H was used as a marker for membrane lipids. In short pulse-labeling experiments, about 50–60% of the radioactivity incorporated was found in the lipid fraction and about 25–50% in starch granules present in the chloroplast of these algae. The relative specificity of acetate-³H used as a marker for membranes was artificially increased through quantitative removal of the starch granules from fixed cells by amylase treatment. Analysis of turnover coefficients of different membrane constituents and of the contribution of turnover and net synthesis to the total label incorporated in pulse experiments indicated that the incorporation of acetate into specific lipids was mainly due to net synthesis. The distribution of radioactivity in the different lipid constituents at the end of a short pulse and after 30- and 60-min chases indicated that transacylation is minimal and may be disregarded as a possible cause of randomization of the label. Statistical analysis of radioautographic grain distribution and measurements of different structural parameters indicate that (a) the chloroplast volume and surface remain constant during the process, whereas the growth of the photosynthetic lamellae parallels the increase in chlorophyll; (b) the lamellae do not develop from the chloroplast envelope or from the tubular system of the pyrenoid; (c) all the lamellae grow by incorporation of new material within preexisting structures; (d) different types of lamellae grow at different rates. The pyrenoid tubular system develops faster than the thylakoids, and single thylakoids develop about twice as fast as those which are paired or fused to grana. It is concluded that growth of the membranes occurs by a mechanism of random intussusception of molecular complexes within different types of preexisting membranes.     

THE FINE STRUCTURE OF RHODOSPIRILLUM RUBRUM

The fine structure of Rhodospirillum rubrum grown under a series of defined conditions has been examined in thin sections prepared by the methods of Ryter and Kellenberger. In cells grown anaerobically at different light intensities, the abundance of 500 A membrane-bounded vesicles in the cytoplasm is inversely related to light intensity, and directly related to cellular chlorophyll content. When the chlorophyll content of the cell is low, the vesicles are exclusively peripheral in location; they extend more deeply into the cytoplasm when the chlorophyll content is high. Typical vesicles also occur, though rarely, in cells grown aerobically in the dark, which have a negligible chlorophyll content. When synthesis of the photosynthetic pigment system is induced in a population of aerobically grown cells by incubation under semianaerobic conditions in the dark, the vesicles become increasingly abundant with increasing cellular chlorophyll content, and the cells eventually acquire the cytoplasmic structure that is characteristic of cells growing anaerobically at a high light intensity. Poststaining with lead hydroxide reveals that the membranes surrounding the 500 A vesicles are indistinguishable in structure from the cytoplasmic membrane, and continuous with it in some areas of the sections. The bearing of these observations on current notions concerning the organization of the bacterial photosynthetic apparatus is discussed.     

Light-Induced Chloroplast Differentiation in Soybean Cells in Suspension Culture : Ultrastructural Changes during the Bleaching and Greening Cycles

Suspension cultures of SB-P cells of soybean (Glycine max) provide a novel, reproducible, and readily manipulable greening system useful for inducing chloroplast differentiation. The cells are subcultured and grown heterotrophically (3% sucrose) in the dark for at least three successive 14-day periods, subcultured and grown in the dark for 7 days more, and finally placed under white light and grown photoautotrophically. Chlorophyll begins to accumulate by 1 hour of light and continues up to 12 days. The chlorophyll a:chlorophyll b ratio is 3:1. Dark-grown cells contain a small amount of total carotenoids which increase 10-fold during greening. Chloroplast differentiation is strictly light dependent, with photosynthetic pigments accumulating in the light and being lost from cells returned to the dark. In the dark, the chloroplasts dedifferentiate to amyloplasts as the organized thylakoid network is lost and starch accumulates. Under continuous light, the amyloplasts differentiate into mature chloroplasts as the organelle elongates, becomes spanned by several bands of thylakoids, and undergoes grana formation. Chloroplast differentiation in SB-P cells is similar to that in intact angiosperms developing under normal light-dark cycles.     

Structural-functional organization of the cells of Brc-1 mutant of Chlamydomonas reinhardtii accumulating protoporphyrin IX in the dark

The structural-functional characteristics of the cells of wild type CC-124 and Brc-1 mutant of the unicellular green algae Chlamydomonas reinhardtii grown in the dark and in the light were studied. The cells of the wild type in heterotrophic and mixotrophic conditions had a well developed structure and high functional activity due to the ability of the cells to synthesize chlorophyll both in the light and in the dark. The cells of Brc-1 mutant lost their ability to synthesize chlorophyll in the dark and the cell color was orange due to brc-1 mutation in the nuclear gene LTS3 that regulated the activity of Mg-chelatase enzyme. In the dark the mutant cells accumulated protoporphyrin IX and had weakly developed structure with low functional activity. Because of the high content of protoporphyrin IX, even a short-term exposure of the Brc-1 mutant cells to the light was accompanied by very strong destructive changes in all the membranes in a cell: plasmalemma, chloroplast, mitochondrion, envelopes of the nucleus and vacuoles. The causes of significant impairment of the membrane components and O₂-gas exchange in the Brc-1 mutant cells are discussed.     

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.     

Light-induced Enzyme Formation in a Chlorophyll-less Mutant of Euglena gracilis

A mutant strain, Y₉, of Euglena gracilis strain Z that is unable to produce protochlorophyll or chlorophyll has been isolated following treatment of wild type cells with nalidixic acid. Dark-grown cells of the mutant contain proplastids that show only limited ultrastructural development when placed in the light. Treatment of Y₉ cells with ultraviolet light brings about permanent cell bleaching with a target number similar to wild type Euglena, and with a slightly greater sensitivity to ultraviolet. Three enzymes of the reductive pentose phosphate cycle, fructose-1,6-diphosphate aldolase (class I), NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and 3-phosphoglycerate kinase, are detectable in dark-grown Y₉ cells at the low concentrations characteristic of dark-grown wild type cells, and increase substantially when these cells are exposed to light. The activity of ribulose-1,5-diphosphate carboxylase increases in the light to a lesser extent. Cytochrome 552, a carrier in the photosynthetic electron transport chain, is not present in light-grown cells of Y₉. The significance of this mutant for an understanding of the role of light in Euglena chloroplast development is discussed.     

Functional and structural organization of chlorophyll in the developing photosynthetic membranes of Euglena gracilis Z. I. Formation of System II photosynthetic units during greening under optimal light intensity

The relationships between light-harvesting chlorophyll and reaction centers in Photosystem II were analyzed during the chloroplast development of dark-grown, non-dividing Euglena gracilis Z. Comparative measurements included light saturation of photosynthesis, oxygen evolution under flashing-light and fluorescence induction. The results obtained can be summarized as follows: (1) Photosystem II photocenters are formed in parallel with chlorophyll synthesis, but after a longer lag phase. (2) As a consequence, the chlorophyll: reaction center ratio (Emerson's type photosynthetic unit) decreases during greening. (3) This decrease is accompanied by considerable changes in the energy transfer and trapping properties of Photosystem II. Most of the initially synthesized chlorophylls are inactive in the transfer of excitations to active photochemical centers and are shared among newly formed Photosystem II photocenters; as a consequence, the number of chlorophylls functionally connected to each Photosystem II photocenter decreases and cooperativity between these centers appears. Results are discussed in terms of chlorophyll organization in developing photosynthetic membranes with reference to the lake or puddle models of photosynthetic unit organization.     

CHLOROPLAST DEVELOPMENT IN OCHROMONAS DANICA

When dark-grown cells of Ochromonas danica are placed in the light, the amount of chlorophyll a per cell increases 82-fold; the content of carotenoid pigment, 24-fold. Concomitantly with this increase in chlorophyll and carotenoid pigment, the small proplastid of dark-grown cells develops into a large lamellate chloroplast. During the first 12 hours in the light, vesicles appear within the loose clusters of dense chloroplast granules, enlarge, align themselves into rows (plates in three dimensions), and fuse into discs. Double discs may form from the more or less simultaneous fusion of two adjacent plates of vesicles or by the addition of vesicles to an already formed single disc. Three-disc bands arise by the addition of a disc to an already formed two-disc band through the approach and fusion of more vesicles. After 24 hours in the light, most of the chloroplast bands contain three discs, but the chloroplasts are still small. After 48 hours in the light, almost all the cells contain full-sized chloroplasts with a full complement of three-disc bands. However, at this time the amount of chlorophyll a and carotenoid pigment is only one-half of maximum. During the next 3 days in the light, as the number of chlorophyll and carotenoid molecules per chloroplast approximately doubles, there is a compression of the discs in each band (from 180 to 130 A) and a precise alignment of their membranes. Changes also occur in the nucleus when dark-grown cells are placed in the light. There is an increase in the number of small nucleolar bodies, many of which lie directly against the nuclear envelope, and in a few cells a dense mass of granules is seen between the two membranes of the nuclear envelope.     

CHANGES IN CHEMICAL COMPOSITION OF THYLAKOID MEMBRANES DURING GREENING OF THE y-1 MUTANT OF CHLAMYDOMONAS REINHARDI

Two fractions of thylakoid membranes (TMF) have been isolated from disrupted (French press) algal cells by using a discontinuous sucrose gradient. TMF-II consists mostly of thylakoid membranes still partially organized in grana; it contains also fragments of chloroplast envelope, pyrenoid tubules, and starch granules; thus it amounts to a fraction of chloroplast fragments which have lost practically all matrix components. TMF-I consists of smaller chloroplast fragments and is contaminated to a larger extent than TMF-II by other subcellular components, primarily mitochondria. TMF-II accounts for about 12% of the protein and 30% of the chlorophyll of the whole cell; it contains cytochrome 554 and carotenoids in the same ratio to chlorophyll as the latter, and shows photosystems I and II activities but lacks enzymatic activities characteristic of the dark reactions. During the greening of the y-1 mutant of Chlamydomonas, TMF's have been isolated over a range of chlorophyll concentrations from 5 to 25 µg/10⁷ cells. The results showed that during this period the ratios of chlorophyll to cytochrome 554 and of chlorophyll to carotenoids, and the relative concentrations of individual carotenoids were continuously changing. The findings support the view that during greening, thylakoid membranes are produced by multistep assembly.     

Formation of protochlorophyll(ide) in wild type and mutant C-2A' cells of Scenedesmus obliquus

Protochlorophyll(ide) was isolated from dark-grown wild typeand mutant C-2A' cells of Scenedesmus obliquus after dark incubationwith 5-aminolevulinate. Proto-chlorophyll(ide) was detectedin mutant cells grown heterotrophically at 29°C or at 21°C.At the latter temperature chlorophyll synthesis was significant.Regulation of chlorophyll synthesis in algae is discussed. ¹Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, Otsuka, Hachioji, Tokyo 192-03, Japan. (Received July 14, 1980; )     

Formation of protochlorophyll(ide) in wild type and mutant C-2A' cells of Scenedesmus obliquus

Protochlorophyll(ide) was isolated from dark-grown wild typeand mutant C-2A' cells of Scenedesmus obliquus after dark incubationwith 5-aminolevulinate. Proto-chlorophyll(ide) was detectedin mutant cells grown heterotrophically at 29°C or at 21°C.At the latter temperature chlorophyll synthesis was significant.Regulation of chlorophyll synthesis in algae is discussed. ¹Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, Otsuka, Hachioji, Tokyo 192-03, Japan. (Received July 14, 1980; )     

THE EFFECTS OF INHIBITORS OF RNA AND PROTEIN SYNTHESIS ON CHLOROPLAST STRUCTURE AND FUNCTION IN WILD-TYPE CHLAMYDOMONAS REINHARDI

Wild-type cells of the unicellular green alga Chlamydomonas reinhardi have been grown for several generations in the presence of rifampicin, an inhibitor of chloroplast DNA-dependent RNA polymerase, spectinomycin and chloramphenicol, two inhibitors of protein synthesis on chloroplast ribosomes, and cycloheximide, an inhibitor of protein synthesis on cytoplasmic ribosomes. The effects of cycloheximide are complex, and it is concluded that this inhibitor cannot give meaningful information about the cytoplasmic control over the synthesis of chloroplast components in long-term experiments with C. reinhardi. In the presence of acetate and at the appropriate concentrations, the three inhibitors of chloroplast protein synthesis retard growth rates only slightly and do not affect the synthesis of chlorophyll; however, photosynthetic rates are reduced fourfold after several generations of growth. Each inhibitor produces a similar pattern of lesions in the organization of chloroplast membranes. Only rifampicin prevents the production of chloroplast ribosomes.