Eiji Hase

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.

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.

Photocontrol and processing of LHCP II apoprotein in Euglena : possible role of Golgi and other cytoplasmic sites

Like other green photosynthetic eukaryotes, cells of Euglena gracilis var. bacillaris and strain Z contain a light-harvesting chlorophyll a/b complex associated with photosystem II. In Euglena, the formation of the 26.5 kDa principal light-harvesting chlorophyll a/b binding protein of photosystem II (LHCP II) has a number of unusual features. The precursors to LHCP II are large polyproteins containing multiple copies of LHCP II, and photocontrol of their formation is largely translational. Under conditions favoring LHCP II accumulation in the thylakoids, a reaction with anti-LHCP II antibody can be observed in the Golgi by immunogold electron microscopy. The timing of the immunoreaction in the Golgi in synchronous cells and in cells undergoing normal light-induced chloroplast development suggests that the nascent LHCP II passes through the Golgi on the way to the thylakoids. The compartmentalized osmiophilic structure (COS) also shows an immunoreaction. These observations, and other discussed in this paper, suggest that light permits translation of polyprotein LHCP II precursors on cytoplasmic ribosomes of the rough endoplasmic reticulum (ER) and that these pass through the ER to the Golgi where, presumably, further modifications take place. Since an LHCP II immunoreaction is found in Golgi vesicles, these may transport the nascent LHCP II to the plastid and facilitate its uptake.

Accumulation of LHCP II apoprotein in wax-rich cells of Euglena in low light or in the presence of streptomycin

Abstract Cells of Euglena gracilis var. bacillaris or Z strain grown under the usual conditions (“nonwax cells”) and exposed to low-intensity light at the developmental threshold (3–7 ft-c) fail to accumulate LHCP II apoprotein. However, cells grown in a medium rich in hexose without shaking accumulate wax; these wax-rich cells, after subsequent aeration in darkness on an inorganic medium for 6 days, accumulate LHCP II apoprotein on exposure to low-intensity light. Immunoelectron microscopy of these cells using anti-LHCP II antibody and protein A—gold shows LHCP II apoprotein first in the compartmentalized osmiophilic body (COS) and Golgi apparatus followed by the thylakoids of the plastid, as previously seen in nonwax cells at normal light intensities for chloroplast development. With time in the light the aerated wax-rich cells at low light intensity form curly thylakoids which react with the LHCP II antibody; exposure of these cells to high light intensity (500 ft-c) causes the curly thylakoids to assume the more normal straight configuration and these retain the reaction with LHCP II antibody. Aerated dark-grown wax-rich cells exposed to normal light intensities for chloroplast development (150 ft-c) in the presence of 0.1% streptomycin have plastids in which the disappearance of the prolamellar body (PLB) is inhibited; a paracrystalline body is formed in close proximity to the PLB which shows an immunoreaction with LHCP II antibody, but the immunoreaction is absent from the thylakoids. Thus, conditions in the aerated wax-rich cells allow an unusual accumulation of LHCP 11 apoprotein at low light intensities and streptomycin blocks the distribution of this apoprotein to the thylakoids in these cells at normal intensities.

Behaviour of proplastids and their nucleoids in dark-organotrophically grown cells of Euglena gracilis transferred to an inorganic medium

Euglena gracilis Z cells in the stationary phase of dark-organotrophic growth in a culture without agitation were rich in lipid, and contained six to eight proplastids dispersed in the cytoplasm (A-type proplastids). When these cells were transferred to an inorganic medium and aerated in darkness, they showed, with the disappearance of lipid, diphasic increase in number after an induction phase, concurrently with the development of prolamellar bodies and some primary thylakoids in their proplastids. The proplastids and their nucleoids, examined under a fluorescence microscope with and without staining with the DNA fluorochrome, 4′,6-diamidino-2-phenylindole, showed dynamic and sequential morphological changes. Before the first cell doubling, the A-type proplastids gathered to form three to four aggregates (B-type), that were themselves connected into a single elongated form with intricate windings and branchings (C-type). The nucleoid in the C-type proplastid was present as a branched string winding throu...

Number and Size of Developing Plastids in Greening Euglena Cells Depend on Nutritional Conditions

Euglena gracilis Klebs var. bacillaris Cori was grown on Hutner’s pH 3.5 medium to a cell density of 106 cells/ml. These cells were transferred to resting medium (cell density 4 x 105) and were allowed to shake for 72 hrs. in darkness. Cell division had ceased at a density of 1.1 x 106 cells/ml. At 72 hrs. the cells were placed under continuous illumination of 150 ft-c. Under these conditions, chloroplast development took place in non-dividing cells and the distribution of chloroplast number per cell and morphology followed by fluorescenece and electron microscopy. In these cells, plastid numbers are high (an average=14.5) and remain constant over the period of chloroplast development, although the plastids increase in size from proplastids to full-sized normal chloroplasts.

Development of Proplastids and Accumulation of LHCP II Apo-Protein in Golgi and Thylakoids of Dark-Grown Wax-Rich Cells of Euglena Gracilis at Low Light Intensities seen by Immuno-Electron Microscopy

When dark-grown resting cells of Euglena are exposed to light at the low intensity threshold of chloroplast development(7 ft-c) the antennas, including the LHCP II 26.5 kD apoprotein are very low or undetectable, but on exposure of these cells to more normal intensities of light for development (80–150 ft-c) the antennas, including the LHCP II apoprotein and chlorophyll b rapidly accumulate 1. Thin sections of similar cells at 3–7 ft-c do not show appreciable staining of either Golgi or thylakoids with rabbit immune serum against the 26.5 kD apoprotein plus protein A-gold, but when these cells are incubated at 80–150 ft-c specific staining with immunogold rapidly appears in both the Golgi and the thylakoids 2,3.

Low Intensity Light-Induced Development of Thylakoids in Proplastids of Dark-Grown Wax-Rich Cells of Euglena Gracilis

As shown previously 1,2), dark-grown, wax-rich cells of Euglena gracilis contain profoundly degenerate proplastids with no internal structure except for a single prothylakoid lying close to the envelope. When these cells are transferred to an inorganic medium containing ammonium salt as nitrogen source and aerated in darkness for 5 to 6 days, an early development of proplastids occurs using wax (and paramylum) as sources of carben and energy 3). The early development includes formation of rudimentary pyrenoid (propyrenoid) at the site adjacent to the prolamellar body, which appears earlier in the peripheral region of the proplastid. The peripheral single prothylakoid becomes paired along part of its length, and a portion of the paired prothylakoid becomes extended and enfolded in the propyrenoid. No further development of proplastids was observed during prolonged dark incubation of these Euglena cells. This paper describes the observations made when these dark-incubated Euglena cells were exposed to light at different intensities including the low intensity threshold (3–7 ft-c) for chloroplast development 4).

Immunogold Localization of Ribulose-1, 5-Bisphosphate Carboxylase in Synchronized Cells of Euglena gracilis Z

The pyrenoid is a unique protein complex in the chloroplast stroma of most eukaryotic algae. Schiff(1) has provided some evidence that Euglena pyrenoids contain RuBisCO. Cook et al.(2) reported that the pyrenoid is recognized in the chloroplasts of synchronized cells of E. gracilis only in the first half of the light period. Recently, Kiss et al.(3) showed by immunofluorescence method that anti-spinach RuBisCO binds to the pyrenoid region of the chloroplasts of E. gracilis.

Dynamic Morphological Changes of Proplastids and Mitochondria in Dark-Organotrophically Grown Cells of Euglena Gracilis Transferred to an Inorganic Medium

Previous studies1 have shown that Euglena gracilis Z cells in the stationary phase of dark-organotrophic growth in a culture without agitation were rich in lipid (mostly waxy ester) and contained proplastids showing no internal structures except for perces of thylakoid existing close to the envelope. When these cells were transferred to an inorganic medium and aerated in the dark, the proplastid stroma expanded, and prolamellar-like bodies and some primary thylakoids were newly produced, concurrently with the development of chlorophyll-synthesising potential. The lipid markedly decreased during the dark incubation of cells with aeration in the inorganic medium, in contrast to paramylum which showed no significant decrease. It was inferred that the dark development of proplastids, which had been repressed during the dark-organotrophic growth, could occur in the inorganic medium utilizing the lipid as the main carbon and energy source.