CHLOROPLAST STRUCTURE AND FUNCTION IN ac-20, A MUTANT STRAIN OF CHLAMYDOMONAS REINHARDI : III. Chloroplast Ribosomes and Membrane Organization
TL;DR: These results, combined with earlier studies of the photosynthetic properties of the mutant strain, suggest that proper membrane organization, Photosystem II activity, and ribulose-1,5-diphosphate carboxylase formation are dependent on the presence of chloroplast ribosomes.
Abstract: The fine structure of the ac-20 strain of Chlamydomonas reinhardi is described. Cells grown mixotrophically in the presence of acetate have a highly disordered chloroplast membrane organization and usually lack pyrenoids. Chloroplast ribosome levels are only 5–10% of wild-type levels. Cells grown phototrophically without acetate possess more chloroplast ribosomes and have more normal membrane and pyrenoid organization. Chloroplast ribosome levels rise rapidly when cells are transferred from acetate to minimal medium, whereas membrane reorganization occurs only after a lag. These results, combined with earlier studies of the photosynthetic properties of the mutant strain, suggest that proper membrane organization, Photosystem II activity, and ribulose-1,5-diphosphate carboxylase formation are dependent on the presence of chloroplast ribosomes. Other chloroplast components tested are unaffected by a 10-fold reduction in levels of chloroplast ribosomes.
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TL;DR: It is concluded that metabolic alterations, the acquisition of mating ability, and the preparation for long-term survival are all elicited in this organism by nitrogen withdrawal, and how the various structural alterations observed in this study may relate to these three interrelated avenues of cellular differentiation is discussed.
Abstract: Gametogenesis in Chlamydomonas reinhardtii has been studied in mating-type plus cells utilizing several different culture conditions, all of which are shown to depend on the depletion of nitrogen from the medium, and the fine structure of gametes prepared under these conditions has been compared by using thin sections of fixed materials. We document alterations in ribosome levels, in chromatin morphology, in starch levels, in the organization of chloroplast membranes, and in the appearance of nuclear envelope and endoplasmic reticulum membranes during gametogenesis. We also noted the acquisition of two new organelles: a mating structure (Friedman, L., A. L. Colwin, and L. H. Colwin. 1968. j. cell Sci. 3:115-128; goodenough, U. W., and R. L. Weiss. 1975. J. Cell Biol. 67:623-637), and Golgi-derived vesicles containing a homogeneous material. We chart the time course of these morphological changes during synchronous gametogenesis. We note that many of these changes may represent adjustments to nitrogen starvation rather than direct features of gametic differentiation, and we also document that cells can differentiate so that they survive conditions of nitrogen starvation for many weeks after they become gametes. We conclude that metabolic alterations, the acquisition of mating ability, and the preparation for long-term survival are all elicited in this organism by nitrogen withdrawal, and we discuss how the various structural alterations observed in this study may relate to these three interrelated avenues of cellular differentiation.
192 citations
TL;DR: Chlamydomonas reinhardtii was shown to accumulate large amounts of starch following nutrient starvation under phototrophic, mixotrophic and heterotrophic conditions and this accumulation was clearly independent of plastome gene expression but depended on de novo cytoplasmic protein synthesis.
173 citations
TL;DR: It is concluded that certain particle distributions in the chloroplast membrane are created as a consequence of the stacking process, and that the ability of membranes to stack can be modified both by gene mutation and by the ionic environment in which the membranes are found.
Abstract: Wild-type chloroplast membranes from Chlamydomonas reinhardi exhibit four faces in freeze-etchreplicas: the complementary Bs and Cs faces are found where the membranes are stacked together; the complementary Bu and Cu faces are found in unstacked membranes The Bs face carries a dense population of regularly spaced particles containing the large, 160 ± 10 A particles that appear to be unique to chloroplast membranes Under certain growth conditions, membrane stacking does not occur in the ac-5 strain When isolated, these membranes remain unstacked, exhibit only Bu and Cu faces, and retain the ability to carry out normal photosynthesis Membrane stacking is also absent in the ac-31 strain, and, when isolated in a low-salt medium, these membranes remain unstacked and exhibit only Bu and Cu faces When isolated in a high-salt medium, however, they stack normally, and Bs and Cs faces are produced by this in vitro stacking process We conclude that certain particle distributions in the chloroplast membrane are created as a consequence of the stacking process, and that the ability of membranes to stack can be modified both by gene mutation and by the ionic environment in which the membranes are found
166 citations
TL;DR: Results showed that both chloroplast and cytoplasmic ribosomes were involved in synthesizing these proteins, and that most, and possibly all, of the disc membrane proteins were synthesized during this period of membrane formation.
139 citations
TL;DR: The mutant strain bald-2 is unique among "flagellaless" strains of Chlamydomonas reinhardtii isolated to date, in that it possesses a mutant basal body only capable of forming a ring of nine singlet microtubules, 180 nm in diameter, instead of the usual triplet basal body.
Abstract: The mutant strain bald-2 is unique among "flagellaless" strains of Chlamydomonas reinhardtii isolated to date, in that it possesses a mutant basal body: it is only capable of forming a ring of nine singlet microtubules, 180 nm in diameter, instead of the usual triplet basal body which is 225 nm in diameter. This singlet basal body lacks structural stability and the ability to associate with striated fiber material but retains two critical properties of basal bodies, namely, information specifying the length to which it should elongate and the ability to induce, albeit rarely, a flagellar transition region, a short, singlet-containing axoneme, and a specialized tunnel in the cell wall through which flagella normally emerge. The mutation seems to be specific for B- and C-microtubule synthesis or assembly since all other cytoplasmic sets of microtubules appear normal in numbers, orientation, and stability.
114 citations
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TL;DR: Electrophoresis in 2.2-2.6% gels gives a fractionation similar to density-gradient centrifugation, and the resolution is greater and more detailed than by centrifugations, and many samples can be analysed simultaneously and rapidly.
Abstract: 1. Gels were prepared with recrystallized acrylamide and bisacrylamide. Electrophoresis was in tris-sodium acetate-EDTA buffer for 0.5 to 3hr. Gels were scanned at 280 or 265mmu. Techniques are described for slicing and radioactive counting. 2. The mobility of RNA was inversely related to the sedimentation coefficient and varied with gel concentration. Electrophoresis in 2.2-2.6% gels gives a fractionation similar to density-gradient centrifugation. It shows the two ribosomal RNA components, the 45s precursor, transfer RNA and minor components. In 5% and 7.5% gels, 4s and 5s RNA separated and ribosomal RNA was excluded. 3. The resolution is greater and more detailed than by centrifugation, and many samples can be analysed simultaneously and rapidly.
1,956 citations
TL;DR: It is proposed that microtubules participate in the formation of the cleavage furrow in C. reinhardi and related to cell division in other organisms.
Abstract: Cell division in log-phase cultures of the unicellular, biflagellate alga, Chlamydomonas reinhardi, has been studied with the electron microscope. The two basal bodies of the cell replicate prior to cytokinesis; stages in basal body formation are presented. At the time of cell division, the original basal bodies detach from the flagella, and the four basal bodies appear to be involved in the orientation of the plane of the cleavage furrow. Four sets of microtubules participate in cell division. Spindle microtubules are involved in a mitosis that is marked by the presence of an intact nuclear envelope. A band of microtubules arcs over the mitotic nucleus, indicating the future cleavage plane. A third set of microtubules appears between the daughter nuclei at telophase, and microtubules comprising the "cleavage apparatus" radiate from the basal bodies and extend along both sides of the cleavage furrow during cytokinesis. Features of cell division in C. reinhardi are discussed and related to cell division in other organisms. It is proposed that microtubules participate in the formation of the cleavage furrow in C. reinhardi.
362 citations
TL;DR: It was observed that during growth in the dark the chloroplast lamellar system is gradually disorganized and drastically decreased in extent, while other subchloroplast components are either unaffected (pyrenoid and its tubular system, matrix) or much less affected (eyespot, ribosomes).
Abstract: This paper describes the morphology and photosynthetic activity of a mutant of Chlamydomonas reinhardi (y-1) which is unable to synthesize chlorophyll in the dark. When grown heterotrophically in the light, the mutant is indistinguishable from the wild type Chlamydomonas. When grown in the dark, chlorophyll is diluted through cell division and the photosynthetic activity (oxygen evolution, Hill reaction, and photoreduction of NADP) decays at a rate equal to or faster than that of chlorophyll dilution. However, soluble enzymes associated with the photosynthetic process (alkaline FDPase, NADP-linked G-3-P dehydrogenase, RuDP carboxylase), as well as cytochrome f and ferredoxin, continue to be present in relatively high concentrations. The enzymes involved in the synthesis of the characteristic lipids of the chloroplast (including mono- and digalactoside glycerides, phosphatidyl glycerol, and sulfolipid) are still detectable in dark-grown cells. Such cells accumulate large amounts of starch granules in their plastids. On onset of illumination, dark-grown cells synthesize chlorophyll rapidly, utilizing their starch reserve in the process. At the morphological level, it was observed that during growth in the dark the chloroplast lamellar system is gradually disorganized and drastically decreased in extent, while other subchloroplast components are either unaffected (pyrenoid and its tubular system, matrix) or much less affected (eyespot, ribosomes). It is concluded that the dark-grown mutant possesses a partially differentiated plastid and the enzymic apparatus necessary for the synthesis of the chloroplast membranes (discs). The advantage provided by such a system for the study of the biogenesis of the chloroplast photosynthetic membranes is discussed.
251 citations
TL;DR: The cytoplasmic organization of a normal green strain of the alga Chlamydomonas reinhardi has been investigated with the electron microscope using thin sections of OsO4 fixed material, identified on the basis of morphological comparability with structures seen in animal cells.
Abstract: The cytoplasmic organization of a normal green strain of the alga Chlamydomonas reinhardi has been investigated with the electron microscope using thin sections of OsO(4) fixed material. The detailed organization of the chloroplast has been of special interest. The chloroplast, a cup-shaped organelle, surrounded by a double membrane, consists of: (1) discs about 1 micron in diameter, considered to represent the basic structural unit of the chloroplast, and each composed of a pair of membranes joined at their ends to form a flat closed vesicle; the discs are grouped into stacks resembling the grana of higher plants; (2) matrix material of low density in which the discs are embedded; (3) starch grains; (4) the pyrenoid, a non-lamellar region associated with starch synthesis, and containing tubules which connect with the lamellae; (5) the eyespot, a differentiated region containing two or three plates of hexagonally packed, carotenoid-containing granules, located between discs, and associated with phototaxis. In addition to the chloroplast, the cytoplasm contains various membranous and granular components, including mitochondria, endoplasmic reticulum, and dictyosomes, identified on the basis of morphological comparability with structures seen in animal cells. The nucleus, not investigated in detail in this study, contains a large, granular nucleolus and is surrounded by a nuclear envelope which is provided with pores and exhibits instances of continuity with the endoplasmic reticulum of the cytoplasm.
233 citations
"CHLOROPLAST STRUCTURE AND FUNCTION ..." refers background in this paper
...The fine structure of the wild-type C. reinhardi chloroplast has been described in several other reports (6, 14, 18 ) . A representative field is shown at high magnification in Fig . 1 . Chloroplast discs (D) are organized into grana-like stacks (S) that exhibit a characteristic anastomosing pattern Chloroplast ribosomes (cr), smaller than the cytoplasmic ribosomes (cyr) (14), are densely packed within the chloroplast stroma . The structure ......
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TL;DR: The applicability to living or supravital tissues of the fluorochrome technique decreases errors due to fixation, dehydration, embedding and related procedures, and the low concentrations applied in fluorochroma staining further safeguard against histochemical artifacts.
Abstract: 1. The fluorochrome, acridin orange (AO), allows identification of basophilic cytoplasmic inclusions in the supravital state.2. The fluorescence picture of hepatic cells after treatment with AO shows the hyaloplasm faint gray-green; flake-like or granular structures in the cytoplasm, bright red; the nuclei, with chromatin structures, green; the central part of the nucleoli red, their periphery green.3. The red-fluorescent cytoplasmic inclusions correspond to those observed with the toluidine-blue technique, and shown, by the RNase test, to consist mainly of RNA. The same is demonstrated by the changes of AO fluorescence after application of RNase to fresh or fixed tissues.4. The fluorescence picture given by other cell elements and tissues in the rat liver is described.5. Changes in the AO fluorescence of liver tissue with varying pH, dye concentration, staining time, action of salts, prolonged U.V. illumination, fixation, as well as the fluorescence of some biologically important substances (RNA, DNA, he...
170 citations