Haruki Hashimoto

Double ring structure around the constricting neck of dividing plastids ofAvena sativa

SummaryUltrastructure of the constricting neck of dividing proplastids and young chloroplasts in the first leaves ofAvena sativa was examined by electron microscopy. An electron-dense, “double” ring structure (plastid-dividing ring doublet; PD ring doublet) with a width of 15–40 nm was revealed around the narrow neck of the constricted and dividing plastids by serial section technique. The inner and outer ring of the doublet coated the inside (stromal side) of the inner envelope membrane and the outside (cytoplasmic side) of the outer envelope membrane, respectively. However, electron-dense materials were not observed within the lumen between the outer and inner envelope membranes.Although the PD ring doublet was commonly observed in the constricted plastids with a 70–140 nm wide neck, they could be scarcely observed in the constricted plastids with a 160 or more nm wide neck. The components of the PD ring were assumed not to be concentrated enough to identify by electron microscopy in the early stage of constriction and the PD ring may be formed and recognized at the final stage.The significance of the formation of the PD ring and its role in plastokinesis (plastid kinesis) were discussed.

Role of galactolipid biosynthesis in coordinated development of photosynthetic complexes and thylakoid membranes during chloroplast biogenesis in Arabidopsis

The galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the predominant lipids in thylakoid membranes and indispensable for photosynthesis. Among the three isoforms that catalyze MGDG synthesis in Arabidopsis thaliana, MGD1 is responsible for most galactolipid synthesis in chloroplasts, whereas MGD2 and MGD3 are required for DGDG accumulation during phosphate (Pi) starvation. A null mutant of Arabidopsis MGD1 (mgd1-2), which lacks both galactolipids and shows a severe defect in chloroplast biogenesis under nutrient-sufficient conditions, accumulated large amounts of DGDG, with a strong induction of MGD2/3 expression, during Pi starvation. In plastids of Pi-starved mgd1-2 leaves, biogenesis of thylakoid-like internal membranes, occasionally associated with invagination of the inner envelope, was observed, together with chlorophyll accumulation. Moreover, the mutant accumulated photosynthetic membrane proteins upon Pi starvation, indicating a compensation for MGD1 deficiency by Pi stress-induced galactolipid biosynthesis. However, photosynthetic activity in the mutant was still abolished, and light-harvesting/photosystem core complexes were improperly formed, suggesting a requirement for MGDG for proper assembly of these complexes. During Pi starvation, distribution of plastid nucleoids changed concomitantly with internal membrane biogenesis in the mgd1-2 mutant. Moreover, the reduced expression of nuclear- and plastid-encoded photosynthetic genes observed in the mgd1-2 mutant under Pi-sufficient conditions was restored after Pi starvation. In contrast, Pi starvation had no such positive effects in mutants lacking chlorophyll biosynthesis. These observations demonstrate that galactolipid biosynthesis and subsequent membrane biogenesis inside the plastid strongly influence nucleoid distribution and the expression of both plastid- and nuclear-encoded photosynthetic genes, independently of photosynthesis.

The Assembly of the FtsZ Ring at the Mid-Chloroplast Division Site Depends on a Balance Between the Activities of AtMinE1 and ARC11/AtMinD1

Chloroplast division comprises a sequence of events that facilitate symmetric binary fission and that involve prokaryotic-like stromal division factors such as tubulin-like GTPase FtsZ and the division site regulator MinD. In Arabidopsis, a nuclear-encoded prokaryotic MinE homolog, AtMinE1, has been characterized in terms of its effects on a dividing or terminal chloroplast state in a limited series of leaf tissues. However, the relationship between AtMinE1 expression and chloroplast phenotype remains to be fully elucidated. Here, we demonstrate that a T-DNA insertion mutation in AtMinE1 results in a severe inhibition of chloroplast division, producing motile dots and short filaments of FtsZ. In AtMinE1 sense (overexpressor) plants, dividing chloroplasts possess either single or multiple FtsZ rings located at random intervals and showing constriction depth, mainly along the chloroplast polarity axis. The AtMinE1 sense plants displayed equivalent chloroplast phenotypes to arc11, a loss-of-function mutant of AtMinD1 which forms replicating mini-chloroplasts. Furthermore, a certain population of FtsZ rings formed within developing chloroplasts failed to initiate or progress the membrane constriction of chloroplasts and consequentially to complete chloroplast fission in both AtMinE1 sense and arc11/atminD1 plants. Our present data thus demonstrate that the chloroplast division site placement involves a balance between the opposing activities of AtMinE1 and AtMinD1, which acts to prevent FtsZ ring formation anywhere outside of the mid-chloroplast. In addition, the imbalance caused by an AtMinE1 dominance causes multiple, non-synchronous division events at the single chloroplast level, as well as division arrest, which becomes apparent as the chloroplasts mature, in spite of the presence of FtsZ rings.

INTERMEDIATE FEATURES OF CYANELLE DIVISION OF CYANOPHORA PARADOXA (GLAUCOCYSTOPHYTA) BETWEEN CYANOBACTERIAL AND PLASTID DIVISION1

Cyanelles of glaucocystophytes may be the most primitive of the known plastids based on their peptidoglycan content and the sequence phylogeny of cyanelle DNA. In this study, EM observations have been made to characterize the cyanelle division of Cyanophora paradoxa Korshikov and to gain insights into the evolution of plastid division. Constriction of cyanelles involves ingrowth of the septum at the cleavage site with the inner envelope membrane invaginating at the leading edge and the outer envelope membrane invaginating behind the septum. This means the inner and outer envelope membranes do not constrict simultaneously as they do in plastid division in other plants. The septum and the cyanelle envelope became stained after a silver‐methenamine staining was applied for in situ detection of polysaccharides. Septum formation was inhibited by β‐lactams and vancomycin, which are potent inhibitors of bacterial peptidoglycan biosynthesis. These results suggest the presence of peptidoglycan at the septum and the cyanelle envelope. In dividing cyanelles, a single electron‐dense ring (cyanelle ring) was observed on the stromal face of the inner envelope membrane at the isthmus, but no ring‐like structures were detected on the outer envelope membrane. Thus a single, stromal cyanelle ring such as this is quite unique and also distinct from FtsZ rings, which are not detectable by TEM. These features suggest that the cyanelle division of glaucocystophytes represents an intermediate stage between cyanobacterial and plastid division. If monophyly of all plastids is true, the cyanelle ring and the homologous inner plastid dividing ring might have evolved earlier than the outer plastid dividing ring.

Division and DNA distribution in ribosome-deficient plastids of the barley mutant "albostrians"

SummaryThe ribsome-deficient plastids of the albino leaves of the barley mutant ”albostrians“ divide at about the same rate as normal plastids and contain similar levels of plastids DNA to the normal plastids. Double-ring structures were observed around the neck of constricting dumbbell-shaped, ribosome-deficient plastids in the basal intercalary meristem of albino leaves. In the distal region of albino leaves the ribosome-deficient plastids contain a rudimentary thylakoid system often closely associated with DNA nucleoids. It is suggested that nuclear coded proteins synthesized within the cytoplasm are responsible for the formation of the double-ring structures and the rudimentary thylakoids of albino plastids.

Electron-opaque annular structure girdling the constricting isthmus of the dividing chloroplasts ofHeterosigma akashiwo (Raphidophyceae, Chromophyta)

SummaryThe plastokinesis (kinesis of chloroplasts) of a raphidophyte alga,Heterosigma akashiwo, was studied by electron microscopy using rapid freezing and freeze-substitution techniques. The chloroplasts are enveloped by two pairs of tightly appressed double membranes, the inner and the cytoplasmic outer pair. The inner pair constricts to divide in advance of the outer pair. By observation of serial sections an electron-opaque, annular structure (plastid-dividing ring) was observed at the isthmus of constricting chloroplasts, girdling the periplastidal outer surface of the inner pair of the four surrounding membranes. These observations suggest that the mechanisms underlying the constriction of the inner and outer pair may differ from each other. The localization of the annular structure (plastid-dividing ring) suggests that the inner pair of the surrounding membranes may be homologous to the double envelope membranes of the chloroplasts of Chlorophyta and Rhodophyta. In addition these findings provide a new evidence supporting the secondary endosymbiosis hypothesis for the origin of the chloroplasts in chromophyte algae.

Symbionin, an aphid endosymbiont-specific protein—II: Diminution of symbionin during post-embryonic development of aposymbiotic insects

Abstract Embryos from rifampicin-injected aphids lost their pre-existing symbionin, an endosymbiont-specific protein, exponentially over the course of their post-embryonic development. The amount of symbionin in the early offspring produced by rifampicin-injected insects was more than in those produced later by the same parent. The amount of nascent symbionin molecules in the embryos developing in the ovariole was much more than in the maternal tissues. These results suggest that symbionin is synthesized by the embryonic endosymbiont and is metabolized during the post-embryonic development of the host insect. Electron microscopic observations suggested that progeny of the rifampicin-injected insect do not have normal endosymbionts.

Involvement of actin filaments in chloroplast division of the algaClosterium ehrenbergii

SummaryStudies have been made of whether actin filaments and microtubules are involved in the chloroplast division ofClosterium ehrenbergii (Conjugatae). Fluorostaining with rhodamine-phalloidin showed 5 types of localization of F-actin: (1) cables of actin filaments running in the cortical cytoplasm along the cells long axis, (2) condensed actin filaments at the septum, (3) perinuclear distribution of actin filaments, (4) F-actins in a “marking pin-like” configuration adjacent to the nucleus of semicells just before completion of chloroplast kinesis, and (5) actin filaments girdling the isthmus of the constricted and dividing chloroplasts. Cytochalasin D (CD) at a concentration of 6 to 25 μM caused significant disruption of actin filaments and the arrest of chloroplast kinesis, nuclear division, septum formation and cytoplasmic streaming within 3 to 6h. Chloroplast kinesis and cytoplasmic streaming recovered when cells were transferred to the medium without CD after CD treatment, or were subjected to prolonged contact with CD for more than 9h. In these cells there was a coincidental reappearance of actin filaments. A tubulin inhibitor, amiprophos-methyl at 330 μM, did not inhibit chloroplast kinesis but did inhibit division and positioning of the nucleus. These results suggest that actin filaments do play a role in the mechanism of chloroplast kinesis but that microtubules do not appear to be involved in the process.

The ultrastructural features and division of secondary plastids.

Plastids in heterokonts, cryptophytes, haptophytes, dinoflagellates, chlorarachniophytes, euglenoids, and apicomplexan parasites derive from secondary symbiogenesis. These plastids are surrounded by one or two additional membranes covering the plastid-envelope double membranes. Consequently, nuclear-encoded plastid division proteins have to be targeted into the division site through the additional surrounding membranes. Electron microscopic observations suggest that the additional surrounding membranes are severed by mechanisms distinct from those for the division of the plastid envelope. In heterokonts, cryptophytes and haptophytes, the outermost surrounding membrane (epiplastid rough endoplasmic reticulum, EPrER) is studded with cytoplasmic ribosomes and connected to the rER and the outer nuclear envelope. In monoplastidic species belonging to these three groups, the EPrER and the outer nuclear envelope are directly connected to form a sac enclosing the plastid and the nucleus. This nuclear-plastid connection, referred to as the nucleus-plastid consortium (NPC), may be significant to ensure the transmission of the plastids during cell division. The plastid dividing-ring (PD-ring) is a conserved component of the division machinery for both primary and secondary plastids. Also, homologues of the bacterial cell division protein, FtsZ, may be involved in the division of secondary plastids as well as primary plastids, though in secondary plastids they have not yet been localized to the division site. It remains to be examined whether or not dynamin-like proteins and other protein components known to function in the division of primary plastids are used also in secondary plastids. The nearly completed sequencing of the nuclear genome of the diatom Thalassiosira pseudonana will give impetus to molecular and cell biological studies on the division of secondary plastids.

Unusual Nuclear Division in Nannochloropsis oculata (Eustigmatophyceae, Heterokonta) which May Ensure Faithful Transmission of Secondary Plastids

Nuclear and plastid division in the monoplastidic, unicellular eustigmatophyte alga Nannochloropsis oculata (Heterokonta) was investigated by electron microscopy. The outermost of four membranes of the secondary plastid is continuous with the outer nuclear envelope membrane to form a nucleus-plastid continuum (NPC). Such physical continuity between the nucleus and the plastid is maintained throughout the cell cycle. Mitosis takes place in a closed spindle. In prophase, a barrel-shaped nuclear pole body (BR-NPB), emanating microtubules towards the cytoplasm, was detected in the vicinity of the nuclear poles. In metaphase, instead of the BR-NPB, a boomerang-shaped nuclear pole body (BM-NPB) occupied the spindle poles, projecting microtubules towards the opposite pole. The BR- and BM-NPB may function as a microtubule organizing centre (MTOC) but are distinct in morphology from any known MTOCs. During anaphase/telophase, the nucleus undergoes constriction with the microtubules penetrating the nucleus along the pole-to-pole spindle axis. The final stage of the nuclear division takes place in an unusual fashion such that the compartment of the inner nuclear envelope divides in advance of that of the outer nuclear envelope. Such unusual nuclear division is discussed in relevance to transmission of the secondary plastid. The present study provides the first report for nuclear and plastid division in Eustigmatophyceae.