Pre-fertilization ovule development inCapsella: ultrastructure and ultracytochemical localization of acid phosphatase in the meiocyte

Summary Pre-meiotic and prophase I ovules ofCapsella bursa-pastoris (L.) Medic.(monosporic,Polygonum type of gametophyte development) were fixed routinely or incubated in a modified Gomori medium containing -glycerophosphate as a substrate. Prior to the beginning of meiosis the potential meiocyte is ultrastructurally similar to the other cells of the nucellus and is distinguished only by its size and position. At the initiation of prophase I dramatic ultrastructural and ultracytochemical changes take place in the female meiocyte. These include the sudden appearance of cytoplasmic structures composed of single and multiple concentric cisternae, distinctive changes in plastids and mitochondria, and the blebbing of 0.3 m double-membraned vesicles from the nuclear envelope. The concentric cisternae encapsulate portions of cytoplasm containing ribosomes, plastids, mitochondria, ER fragments and vesicles. Both single and multiple concentric cisternae localize high levels of acid phosphatase and function as autophagic vesicles (AVs) that sequester ribosomes and organelles for destruction during meiosis. Plastids stop dividing and become more spherical during prophase I. Some plastids localize acid phosphatase and many show continuities between the outer membrane and the plastid envelope and acid phosphatase-rich RER cisternae. Mitochondria appear as dense, contracted spheres or rods. Some mitochondria localize acid phosphatase but they do not show membrane confluencies with the ER. Some of the plastids and mitochondria that are segregated into the functional megaspore at meiosis II are destroyed but others apparantly survive meiosis and give rise to the plastid and mitochondrial populations of the young gametophyte (Schulz andJensen, unpublished). The lateral and end walls of the meiocyte show patches of intense aniline blue fluorescence and the chalazal end wall of the cell is perforated with large numbers of plasmodesmata.Research supported by NSF Grant PCM-79-11018. The authors gratefully acknowledge the valuable assistance of David Lee Ivans in this project.     

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.     

Ultrastructure of the vegetative cell ofBrassica napus pollen with particular reference to microbodies

Summary The ultrastructure of the vegetative cell ofBrassica napus tricellular pollen grains, just before anthesis with standard chemical fixation, is reported. The vegetative cell may be regarded as a highly differentiated and metabolically active fat-storage cell. It contains many mitochondria with a well developed internal membrane system, starchless plastids, microbodies, lipid bodies, dictyosomes and numerous vesicles thought to originate from the dictysomes. Rough endoplasmic reticulum organized in stacks of cisternae is also spatially associated with certain organelles, mainly lipid bodies, microbodies and plastids. There are also randomly distributed polyribosome areas. The microbodies are mainly polymorphic in shape and are often observed in contact with lipid bodies. The above spatial relationship implies that the microbodies may have a glyoxysomal function. In the late period of vegetative cell maturation, the microbodies are probably involved in the process of glyconeogenesis in which the conversion of lipid reserves to sugar takes place.Abbreviations VC vegetative cell - VN vegetative nucleus - SC sperm cell - M mitochondria - MB microbodies - L lipid body - P plastid - D dictyosomes     

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.     

Isolation and immobilization of various plastid subtypes by magnetic immunoabsorption

Antibodies have been prepared which immuno-localize to the outer membrane of the pea chloroplast envelope and cause agglutination of isolated chloroplasts. This antisera is immunoreactive with a variety of plastid forms from both monocotyledonous and dicotyledonous plants. Whether such antibodies might be effectively used for isolation and immobilization of plastids from whole cell lysates has been tested. A system has been developed for immunolabeling various forms of higher plant plastids with biotinylated antibody and streptavidin magnetic nano-particles followed by separation of the plastids in a 0.6 Tesla high gradient magnetic field. Using this magnetic immunoabsorption procedure it has been possible to achieve a high degree of positive enrichment for chromoplasts, amyloplasts, and chloroplasts from whole cell lysates of several plant species. The integrity of these plastids has been examined by in organellar protein synthesis, ¹⁴C-ADP-glucose uptake, flow cytometry, in vitro synthesized precursor import and FITC-cationized ferritin staining of the plastid envelope. Western blot analysis showed significant enrichment for amyloplasts from cytosolic sucrose synthase in maize endosperm. Magnetic immunoabsorption of subcellular structures from whole cell lysates is a new method that may be useful in the in vitro analysis of many different cellular compartments from a wide range of organisms.     

Plastids of sieve tube members in the stamen vascular bundle ofSorghum bicolor (Gramineae)

Summary The plastids in sieve tube members of the stamen vascular bundles ofSorghum bicolor, fixed in glutaraldehyde with postfixation in osmium tetroxide, are of the P-type containing cuneate crystalloids of a proteinaceous nature surrounded by a double envelope. Secondary inclusions are present in these P-type plastids. P-type plastids inSorghum often remain intact in the mature sieve tube members.This work was supported by a grant from the Iowa Agricultural and Home Economics Experiment Station, Ames, U.S.A. Project No. 1740 toHarry T.Horner, Jr., andNels R.Lersten and Project No. 1914 toHarry T.Horner, Jr. of the Department of Botany and Plant Pathology, Iowa State University, Ames, U.S.A. This work was completed by the author as part of the Ph. D. dissertation research.     

Preferential degradation of plastid DNA with preservation of mitochondrial DNA in the sperm cells ofPelargonium zonale during pollen development

Summary In the present study, we studied changes in organellar DNA in the sperm cells of maturing pollen ofPelargonium zonale, a plant typical to exhibit biparental inheritance, by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and by immunogold electron microscopy using anti-DNA antibody. Fluorescence intensities of DAPI-stained plastid nuclei in generative and sperm cells at various developmental stages were quantified with a video-intensified microscope photon counting system (VIMPCS). Results indicated that the amount of DNA per plastid in generative cells increased gradually during pollen development and reached a maximum value (about 70 T per plastid; 1 T represents the amount of DNA in a particle of T4 phage) in young sperm cells at 5 days before flowering. However, the DNA content of plastids was subsequently reduced to about 20% of the maximum value on the day of flowering. Moreover, the DNA content of the plastid further decreased to 4% of the maximum value when pollen grains were cultured for 6 h in germination medium. In contrast, the amount of DNA per mitochondrion did not decrease significantly around the flowering day. Similar results were also obtained by immunogold electron microscopy using anti-DNA antibody. The density of gold particles on plastids decreased during pollen maturation whereas labelling density on mitochondria remained relatively constant. The number of plastids and mitochondria per generative cell or per pair of sperm cells did not change significantly, indicating that the segregation of DNA by plastid division was not responsible for the decrease in the amount of DNA per plastid. These results indicate that the plastid DNA is preferentially degraded, but the mitochondrial DNA is preserved, in the sperm cells ofP. zonale. While the plastid DNA of the sperm cells decreased before fertilization, it was also suggested that the low DNA contents that remain in the plastids of the sperm cells are enough to account for the biparental inheritance of plastids inP. zonale.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon counting system     

Die Siebröhren-Plastiden der Monocotyledonen

Zusammenfassung In vergleichenden feinstrukturellen Beobachtungen an 24 Monocotyledonen aus 21 Familien wird ein für Monocotylen-Siebröhren charakteristischer Plastidentyp näher beschrieben. Neben gelegentlichen Ablagerungen von Siebröhrenstärke enthalten ausdifferenzierte Siebröhren-Plastiden zahlreiche keilförmige, kontrastreiche und proteinhaltige Kristalloide. Sie entstehen in der Matrix der noch amöboiden Formveränderungen unterworfenen Proplastiden; in reifem Zustand werden sie aus gekreuzten Reihen paralleler, gerader und kontrastreicher Filamente (50–60 Å) aufgebaut.Die Siebröhren-Plastiden von Nymphaea alba und Nuphar luteum bilden keine Kristalloide aus, dagegen läßt sich Siebröhrenstärke wie in den übrigen bisher untersuchten Dicotylen nachweisen.

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Sieve-tube plastids of monocotyledonsComparative investigations of the fine structure and distribution of specific plastids

Summary Fine-structural investigations of 24 monocotyledons from 21 families and all but one order succeeded in revealing a plastid with cuneate proteinaceous inclusion bodies as being typical of monocot sieve-tubes. Inclusion bodies originate in large numbers during plastid differentiation; they concentrate in the matrix and aggregate around an invisible centre, that mostly lies at one end of the elongated ameboid proplastid. The inclusion-free part of the young plastid contains countless vesicles and short membranes, presumably invaginations of the inner plastid envelope. Proteinaceous inclusion bodies show a crystal-like structure composed of 50–60 Å subunits in straight and parallel order. Besides these crystal-like inclusion bodies sieve-tube plastids of many monocotyledons also contain starch. — Sieve-tube plastids of Nuphar luteum and Nymphaea alba look like plastids in dicotyledon sieve-tubes, starch being their only inclusion.

     

The ontogeny of lipid bodies (spherosomes) in plant cells

Maturing embryos of 16 oil plants, anise suspension culture cells, and Neurospora crassa cells were prepared for electron microscopy at different stages during massive lipid accumulation. Lipid-rich structures of certain species were best preserved by dehydration of fixed tissues in ethanol without propylene oxide, embedding in Spurr's Medium, and polymerization at room temperature. In all cells examined, spherical lipid bodies (spherosomes) showed a moderately osmiophilic, amorphous matrix and displayed a delimiting half-unit membrane when sectioned medially. Associations with the endoplasmic reticulum (ER) were viewed at any stage during lipid body development but with different frequency in the different plant species. Plastids of fat-storing cells exhibited conspicuously undulate outer and inner envelope membranes that formed multiple contact sites with each other and protuberances into both cytoplasm and stroma. Some species, e.g., Linum, have plastids with tubular structures that connect the inner membrane to the thylakoid system; in addition, in the stroma vesicles fusing with or apparently passing through the envelope were observed. The outer envelope membrane may be associated with ER-like cytoplasmic membrane structures. In addition, lipid bodies of various sizes were found in contact with the plastid envelope. The ultrastructural observations are interpreted to match the published biochemical evidence, indicating that both plastids and ER may be involved in the synthesis of storage lipids and lipid body production.     

Ultrastructural studies on plastids of generative and vegetative cells inLiliaceae 3. Plastid distribution during the pollen development inGasteria verrucosa (Mill.) Duval

Summary This paper describes the development of pollen grains ofGasteria verrucosa from the late microspore to the mature two-cellular pollen grain. Ultrastructural changes and the distribution of plastids as a result of the first pollen mitosis have been investigated using light and electron microscopy. The microspores as well as the generative and the vegetative cell contain mitochondria and other cytoplasmic organelles during all of the observed developmental stages. In contrast, the generative cell and the vegetative cell show a different plastid content. Plastids are randomly distributed within the microspores before pollen mitosis. During the prophase of the first pollen mitosis the plastids become clustered at the proximal pole of the microspore. The dividing nucleus of the microspore is located at the distal pole of the microspore. Therefore, the plastids are not equally distributed into both the generative and the vegetative cell. The possible reasons for the polarization of plastids within the microspore are briefly discussed. The lack of plastids in the generative cell causes a maternal inheritance of plastids inGasteria verrucosa.     

Fine structure of a crystal-containing plastid in Colchicum autumnale

Summary A study has been made of plastids in the root meristem of Colchicum autumnale. These organelles contain simultaneously deposits of starch, lipid, and a crystalline material, probably protein. Two types of internal membrane are present within the plastid. One appears to be concerned with the transport of material across the plastid envelope. The other has a special relationship to the crystal. The crystal is seen as long columns when cut in longitudinal section, and the membrane there is dark staining material, which is sometimes seen in the form of a large more or less rectangular body. The possible significance of these internal structures is discussed in relation to recent ideas on the development and functions of plastids in general.     

Effects of nalidixic acid, chloramphenicol, cycloheximide, and anisomycin on structure and development of plastids and mitochondria in greening Euglena gracilis

The substructure of plastids and mitochondria and the alterations caused by the addition of antibiotics were investigated during light-induced proplastid-to-chloroplast transformation in Euglena gracilis. The organisms were grown in presence of the inhibitors up to 3 days (5 generations). Both 40 μg/ml nalidixic acid and 1–1.5 mg/ml chloramphenicol prevent the formation of chloroplasts of normal size and structure by blocking development during early stages. Under our conditions 2 to 5 straight thylakoids are formed beside 1 to 2 girdle-like thylakoids. The former rarely fuse into bands. Non-crystalline prolamellar bodies of considerable size are formed at the distended ends of the plastids in the presence of both drugs. Chloramphenicol also influences mitochondrial size, shape and internal structure. Giant mitochondria can be observed. Nalidixic acid does not change the size and shape of mitochondria, but the matrix frequently appears highly osmiophilic. Cycloheximide in sublethal doses (2–5 μg/ml) or 50 μg/ml anisomycin inhibits plastid development only in the early period after addition. In later culture periods chloroplasts are found enlarged in size with an increased number of thylakoids and bands per organelle. Insertions of new bands are noted at the inner membrane of the chloroplast envelope. The electronmicroscopic observations agree with the results of chloroplast-specific biochemical activities such as light-induced increase in chlorophyll synthesis and of two chloroplast-bound enzyme activities. The results are discussed with respect to metabolic and biogenetic correlations between the two types of organelles in E. gracilis cells.     

On the infiltration of Golgi bodies from the follicular epithelium to the egg

Summary 1. In a well advanced oocyte of the tortoise the egg membranes besides the theca and the single-layered epithelium consist of a zona pellucida often differentiated into zona striata and a homogeneous layer; underlying these two layers is a layer of cortical fibrillae or fibrillar layer, Next to this layer, is the limiting membrane of the egg which is not present in all stages and generally disappears in a well developed oocyte. In certain animals either the homogeneous layer or fibrillar layer is absent. 2. In certain animals,Golgi bodies seem to be extruded into the follicle cells from the theca cells. 3. At a particular stage of development the follicle cells become very active and produce a large number of smallGolgi bodies. TheseGolgi granules filter through canalicular passages of the zona radiata into either the homogeneous layer and from thence into the fibrillar layer or where a homogeneous layer is not present directly to the fibrillar layer. Where a fibrillar layer is not present they are transferred directly to the limiting membrane and from thence to the egg. 4. In certain cases e. g. in Fowl, Calotes and Uromastix, fairly large lumps ofGolgi bodies are extruded from the follicle cells through the zona pellucida into the egg. Here the fine canilicular passages do not seem to form a vehicle for the passage of these comparatively larger bodies. 5. The fine canalicular passages in the zona radiata ofTestudo graeca andKachuga smithii and the fibrillar prolongation of the cytoplasm which we have called the fibrillar layer are marked features of the egg membranes at certain stages of development of the egg. During the period when infiltration ofGolgi bodies through these passages takes place slides prepared by silver nitrate and osmic methods show black beaded chains ofGolgi granules in various stages of descent. 6. It is claimed that the extrusion and infiltration ofGolgi bodies from the follicular epithelium to the egg are established phenomena at least in the Vertebrates.     

Ultrastructural characterization of the reversible differentiation of chloroplasts in cucumber fruit

The changes in plastid ultrastructure in the pericarp of cucumber (Cucumis sativus L) fruit were studied during fruit yellowing (which accompanied maturation) and regreening. In the course of fruit maturation, the thylakoid system was progressively reduced, and only a small number of membranes remained in the plastids of mature fruit. At the same time, the plastoglobules increased in size, often remaining in close proximity to the degrading thylakoids. In pericarp tissue which turned green again, the thylakoid network in the plastids was gradually reconstituted. Morphological similarities between the plastids in mature and regreening fruit indicated that the chloroplasts in regreened tissue were redifferentiated from the plastids of mature fruit. Reconstitution of the thylakoid system appeared to start from two morphologically distinct types of membranes: from double membranes which resembled thylakoids and from membrane-bound bodies (MBBs). The latter appeared to form thylakoids by two mechanisms: by detachment of extensions from their surfaces and by fragmentation. The plastoglobules remained in the plastids during thylakoid system reconstitution and were often observed in close proximity to developing thylakoids. In the course of chloroplast redifferentiation, several types of membraneous structures were found to be associated with the plastid envelope: (i) vesicles which appeared to separate from the envelope and to fuse subsequently with the developing thylakoids, (ii) tubules, and (iii) double-membrane sheets which appeared asde novo forming thylakoids.     

IN VIVO CHARACTERIZATION OF THE DIATOM MULTIPARTATE PLASTID TARGETING SIGNAL

Diatoms and related algae have plastids that are surrounded by four membranes. The outer two membranes are continuous with the endoplasmic reticulum and the inner two membranes are analogous to the plastid envelope membranes of higher plants and green algae. Thus the plastids are completely compartmentalized within the ER membranes. The targeting presequences for nuclear‐encoded plastid proteins have two recognizable domains. The first domain is a classic signal sequence, which presumably targets the proteins to the endoplasmic reticulum. The second domain has characteristics of a transit peptide, which targets proteins to the plastids of higher plants. To characterize these targeting domains, the presequence from the nuclear‐encoded plastid protein AtpC was utilized. A series of deletions of this presequence were fused to Green Fluorescent Protein (GFP) and transformed into cells of the diatom, Phaeodactylum tricornutum. The intracelluar localization of GFP was visualized by fluorescence microscopy. This work demonstrates that the first domain of the presequence is responsible for targeting proteins to the ER lumen and is the essential first step in the plastid protein import process. The second domain is responsible to directing proteins from the ER and through the plastid envelope and only a short portion of the transit peptide‐like domain is necessary to complete this second processing step. In vivo data generated from this study in a fully homologous transformation system has confirmed Gibbs' hypothesis regarding a multistep import process for plastid proteins in chromophytic algae.     

Development of P-protein in sieve elements ofMimosa pudica

Summary The P-protein in sieve elements of leaves ofMimosa pudica L. is first discernible as fine fibrous material which forms homogeneous aggregates. Ribosomes, rough endoplasmic reticulum, and dictyosomes with associated vesicles occur in the cytoplasm surrounding the aggregates. The plastids and mitochondria are in a parietal position in the parts of the cell where the nascent P-protein accumulates. In a later stage, the fibrillar material is organized into a three-dimensional system of five- and six-sided elongated compartments. The corners of the compartments appear solid at first, then they become electron lucent in the center and assume tubular form. Aggregates of mature P-protein tubules usually occur near the compartmentalized system. Tubules in pentagonal or hexagonal arrangements may be present in the aggregates and may be partly interconnected. The conclusion was drawn that the P-protein tubules are assembled at the corners of compartments within a continuous orderly system. The fully formed tubules occur first in aggregates, the P-protein bodies. Later the aggregates become loose and partly dispersed. Many of the dispersed tubules assume a loose, extended, helical form characteristic of P-protein in older sieve elements.This work was supported in part by National Science Foundation grant GB-5506. I am also grateful to MissHatsume Kosakai and Mr.Robert H.Gill for technical assistance.     

Molecular evidence for the origin of plastids from a cyanobacterium-like ancestor

Summary The origin of plastids by either a single or multiple endosymbiotic event(s) and the nature of the progenitor(s) of plastids have been the subjects of much controversy. The sequence of the small subunit rRNA (Ssu rRNA) from the plastid of the chlorophyllc-containing algaCryptomonas is presented, allowing for the first time a comparison of this molecule from all of the major land plant and algal lineages. Using a distance matrix method, the phylogenetic relationships among representatives of these lineages have been inferred and the results indicate a common origin of plastids from a cyanobacterium-like ancestor. Within the plastid line of descent, there is a deep dichotomy between the chlorophyte/land plant lineage and the rhodophyte/chromophyte lineage, with the cyanelle ofCyanophora paradoxa forming the deepest branch in the latter group. Interestingly,Euglena gracilis and its colorless relativeAstasia longa are more related to the chromophytes than to the chlorophytes, raising once again the question of the origin of the euglenoid plastids.     

Some unusual features of the ultrastructure of the chloroplasts of the green algal orderCaulerpales and their development

Summary The ultrastructure of developing and mature chloroplasts of members of the green algal orderCaulerpales is described. The mature chloroplasts develop from small starch containing plastids. These small starch containing plastids may also develop into the large amyloplasts characteristic of this order. The thylakoid organizing body (TOB), a system of concentric lamellae found at one end of the plastid, appears to be involved in initial thylakoid membrane synthesis. During early plastid development the first formed thylakoids, the plastid DNA and lipid are closely associated with this body. Many developing plastids also have a number of microfilaments near the chloroplast envelope. These microfilaments extend from the TOB towards the opposite end of the plastid.The size and structure of the mature caulerpalean chloroplast varies greatly between species, as does the size and structure of the TOB. The simplest type of TOB occurs inAvrainvillea erecta and the most complex inCaulerpa cactoides. The membranes of the TOB are connected by crossbridges and they are also connected with the inner chloroplast envelope membrane. The structure of the TOB, its relation to the chloroplast envelope, its association with the thylakoids and its possible functions are described.