ELECTRON MICROSCOPY OF MEMBRANOUS BODIES AND GENOPHORE IN CHLOROPLASTS OF BOTRYOCLADIA BOTRYOIDES (RHODYMENIALES, RHODOPHYTA)1

An ultrastructural study on the chloroplasts of the red alga Botryocladia botryoides( Wulfen) J. Feldmann has been carried out to investigate the morphology of globular membranaous bodies (MBs) located between thylakoids. These membranous bodies show an electron dense lumen, in which a pair of denser cores occasionally occurs. The lumen is electron transparent in the chloroplasts of vacuolized, non-dividing cells. The surface of MBs is in contact with the fibrils representing the plastidial genophore; the fibrils sometimes bridge the surface of MBs with the nearest thylakoid. In some planes of section a membranous stalk connecting a MB with thylakoids has been made evident .
Because the presence of similar bodies in chloroplasts and mitochondria of other red algae, as well as in mitochondria of yeast in which an intense synthesis of DNA occurs, the authors propose that MBs in red algae have a role in the replication of the genophore .     

Unusual cell structures in tumor-like formations of Gracilaria (Rhodophyta)

This paper deals with electron microscopic observations on cultivated plants of the marine red alga Gracilaria verrucosa which developed simple galls; also sea collected material, without galls, had been studied. The galls showed unusual but characteristic cell structures, caterpillar-like bodies, containing rows of fusiform bodies. These were found mostly in the cytoplasm near the plastids, in one case connected with the endoplasmic reticulum, occasionally even inside the nucleus, and are described here, as far as we know, for the first time. It does not seem probable that the caterpillar-like bodies represent mitochondria or bacteria, but the hypothesis that fusiform bodies are related to virus-like structures is discussed. The normal tissues as well as the gall tissue of the laboratory plants contained, besides plastids typical for the red algae, another type of plastids characterized by tubular thylakoids.This work was supported by grants from Consiglio Nazionale delle Ricerche (Rome) and Ministero dell'Agricoltura e delle Foreste of Italy.     

Plastid development in seedlings of Echinochloa crus-galli var. oryzicola under anoxic germination conditions

Plastid development in the primary leaf of Echinochloa crus-galli (L.) Beauv. var. oryzicola (Vasing.) Ohwi was followed during 5 d of anoxic germination and growth. Plastids develop slowly from simple spheroidal proplastids into larger pleomorphic plastids with several stromal membranes and many peripheral membrane vesicles. A small prolamellar body is present at 96 h with perforated (pro)thylakoids extending into the stroma. Changes in starch grains and plastoglobuli are evidence of carbohydrate and lipid metabolism. Plastid division is indicated by dumbbell plastid profiles after 4 d of anoxia. These results demonstrate that plastids not only maintain their integrity during anaerobic germination but also show developmental changes involving an increase in internal membrane complexity, although to a lesser extent than in etiolated shoots.Abbreviation PLB prolamellar body Scientific paper No. 6167. College of Agriculture, Washington State University, Pullman     

The capacity of plastids from developing pea cotyledons to synthesise acetyl CoA

In order to determine whether the enzymes required to convert triose phosphate to acetyl CoA were present in pea (Pisum sativum L.) seed plastids, a rapid, mechanical technique was used to isolate plastids from developing cotyledons. The plastids were intact and the extraplastidial contamination was low. The following glycolytic enzymes, though predominantly cytosolic, were found to be present in plastids: glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12), phosphoglycerate kinase (EC 2.7.2.3), and pyruvate kinase(EC 2.7.1.40). Evidence is presented which indicates that plastids also contained low activities of enolase (EC 4.2.1.11) and phosphoglycerate mutase (EC 2.7.5.3). Pyruvate dehydrogenase, although predominantly mitochondrial, was also present in plastids. The plastidial activities of the above enzymes were high enough to account for the rate of lipid synthesis observed in vivo.Abbreviations FPLC fast protein liquid chromatography - PPi pyrophosphate     

Insertion of the precursor of the light-harvesting chlorophylla/b-protein into the thylakoids requires the presence of a developmentally regulated stromal factor

The precursor of the major light-harvesting chlorophylla/b-proteins of photosystem II was synthesizedin vitro from a gene fromLemna gibba. When the labelled precursor was incubated with developing barley plastids, the precursor and the processed polypeptide were incorporated in the thylakoids in proportions that varied depending on the developmental stage of plastids. At early stages of development most of the precursor associated with the thylakoids could be removed by washing with 0.1 M NaOH, while in more mature plastids most of its was resistant to a NaOH wash. Insertion of the precursor into thylakoids required the presence of a stromal factor and Mg-ATP. The stromal factor is probably a protein. The insertion reaction has an optimal temperature of 25°C and a pH of 8. The appearance of the stromal factor and the thylakoid membrane's receptivity for the insertion of the precursor depended on the stage of plastid development. These observations are consistent with the hypothesis that the insertion of the precursor into the thylakoid prior to its proteolytic processing, is one of the steps involved in the assembly of the light-harvesting complex of photosystem II.     

Immunogold localization of light-harvesting and photosystem I complexes in the thylakoids ofFucus serratus (Phaeophyceae)

Summary The repartition of light-harvesting complex (LHC) and photosystem I (PS I) complex has been examined in isolated plastids ofFucus serratus by immunocytochemical labelling. LHC is distributed equally all along the length of thylakoid membranes, without any special repartition in the appressed membranes of the three associated thylakoids ofFucus. PS I is present on all the thylakoid membranes, but the external membranes of the three associated thylakoids are largely enriched relatively to the inner ones. This specific repartition of PSI on non-appressed membranes can be compared to the localization of PSI on stroma thylakoid membranes of higher plants and green algae. Consequently, although they share some common features with those of higher plants and green algae, the appressions of thylakoids in brown algae has neither the same structure nor the same functional role as typical grana stacked membranes in the repartition of the harvested energy.Abbreviations BSA bovine serum albumin - GAR goat anti-rabbit immunoglobulin G - LHC light-harvesting complex - PBS phosphatebuffered saline - PS I photosystem I - PS II photosystem II     

Morphology and phylogeny of the pseudocolonial dinoflagellates Polykrikos lebourae and Polykrikos herdmanae n. sp

Both the photosynthetic and heterotrophic forms of the only known marine benthic (sand-dwelling) species of Polykrikos, namely P. lebourae, were investigated using light and electron microscopy and molecular phylogenetic analyses. The pseudocolonies usually contained eight integrated zooids and two nuclei. Pseudocolonies consisting of four or five zooids and one nucleus were observed for the first time for this species; some of these reduced pseudocolonies contained plastids, while others were heterotrophic and contained taeniocyst-nematocyst complexes. The ultrastructure of the plastids in P. lebourae did not conform to the organization of thylakoids and enveloping membranes present in the peridinin-containing plastids of other photosynthetic dinoflagellates (i.e. stacks of 3 thylakoids and 3 outer membranes). Instead, the plastids in P. lebourae had thylakoids arranged in pairs and appeared to be enveloped by only two membranes. Molecular phylogenetic data using small subunit rDNA demonstrated that the photosynthetic and heterotrophic forms of P. lebourae represent two distinct clades. The more inclusive clade containing both forms of P. lebourae was most closely related to heterotrophic polykrikoids, namely P. kofoidii. These results led us to conclude that the photosynthetic and heterotrophic forms of P. lebourae are in fact two distinct lineages, and the heterotrophic form is described here as Polykrikos herdmanae n. sp.     

Ultrastructure of carpospores inGastroclonium clavatum (Roth)Ardissone (Rhodymeniales)

Summary Carpospores ofG. clavatum have been studied under the light and electron microscopes. They are wedge-shaped cells of 80–100 m at their longest diameters. The nucleus is an uncondensed structure provided with a regular outline and a large nucleolus. The plastids constitute heterogeneous populations of organelles differing in size and shape as well as in number and arrangement of the thylakoids. Multiplicating plastids are also present. The mitochondria are small but have well developed cristae. The Golgi apparatus consists of very numerous active dictyosomes. Starch is the main storage substance but some large lipid bodies are also present. Labyrinthine polysaccharide aggregations are present in the carposporial cytoplasm. Multilayered bodies constitute a sui generis very conspicuous cell component.     

Assembly of the barley light-harvesting chlorophyll a/b proteins in barley etiochloroplasts involves processing of the precursor on thylakoids

A barley gene encoding the major light-harvesting chlorophyll a/b-binding protein (LHCP) has been sequenced and then expressed in vitro to produce a labelled LHCP precursor (pLHCP). When barley etiochloroplasts are incubated with this pLHCP, both labelled pLHCP and LHCP are found as integral thylakoid membrane proteins, incorporated into the major pigment-protein complex of the thylakoids. The presence of pLHCP in thylakoids and its proportion with respect to labelled LHCP depends on the developmental stage of the plastids used to study the import of pLHCP. The reduced amounts of chlorophyll in a chlorophyll b-less mutant of barley does not affect the proportion of pLHCP to LHCP found in the thylakoids when import of pLHCP into plastids isolated from the mutant plants is examined. Therefore, insufficient chlorophyll during early stages of plastid development does not seem to be responsible for their relative inefficiency in assembling pLHCP. A chase of labelled pLHCP that has been incorporated into the thylakoids of intact plastids, by further incubation of the plastids with unlabelled pLHCP, reveals that the pLHCP incorporated into the thylakoids can be processed to its mature size. Our observations strongly support the hypothesis that after import into plastids, pLHCP is inserted into thylakoids and then processed to its mature size under in vivo conditions.     

Ultrastructural studies on the secretory cavities ofCitrus deliciosa ten. I. Early stages of the gland cells differentiation

Summary Secretory cavities ofCitrus deliciosa seem to originate from a pair of meristematic cells (an epidermal cell and a second one placed under it). These cells undergo successive divisions resulting in the formation of a globular/oval gland situated in the parenchyma, and a conical stalk, which joins the gland with the epidermis. The young gland consists of a central group of polyhedral cells ensheathed by layers of radially flattened cells.During the early differentiation stages of the gland cells a close association of cytoplasmic microtubules with various organelles is observed. Plastids increase progressively in number and size and their matrix locally contains tubular networks accompanied by small oil droplets. In peripheral cytoplasm numerous myelin-like lomasomes have been observed. Peripheral cells of the gland are gradually modified from the inner cells following a different developmental pattern. Their walls become thicker and plastids do not contain tubular complexes, but only a few thylakoids partly surrounding the newly formed starch grains.     

Photosystem II characteristics of a constructedSynechocystis 6803 mutant lacking synthesis of the D1 polypeptide

Photosystem II (PSII) composition was studied in a mutant of the cyanobacteriumSynechosystis 6803 in which synthesis of the reaction center polypeptide D1 has been inactivated. The mutant thylakoids had lost also the other reaction center polypeptide D2 and the chlorophylla-binding protein CP47. Cytochromeb559 and the chlorophylla-binding protein CP43 accumulated to almost wild-type amounts in mutant thylakoids. Also the 33 kDa polypeptide involved in water oxidation was present and membrane-bound in mutant thylakoids. The intrinsic 22 kDa polypeptide, so far known only from plants, was detected both in wild-type and mutant thylakoids.     

Green tissue within the haustorium of the dwarf mistletoe Korthalsella (Viscaceae). An ultrastructural comparison between chloroplasts of sucker and aerial stem tissues

Summary Korthalsella (Viscaceae) is a dwarf mistletoe attached to its host branch by a single haustorium. Plants are leafless with flattened or cylindrical stems that function in photosynthesis. When a fresh haustorium is cut the sucker within the host appears bright green. Transmission electron microscopy reveals that this greening is due to chloroplasts, but that their organization differs from those of the aerial stem. The three representatives of Korthalsella endemic to New Zealand were the main species investigated. In the stem, chloroplasts have short stacks of cylindrical grana interconnected by stroma thylakoids typical of normal chloroplasts. Sucker chloroplasts have a more variable organization, with most containing extensive granal stacks and poorly differentiated stroma thylakoids. These granal thylakoids exhibit extensive partitions formed by appression of adjacent membranes. Some sucker plastids also approach etioplasts in having a prominent prolamellar body from which radiate thylakoids with short partitions. Sucker chloroplasts usually contain a few large starch grains, plastoglobuli, and sometimes also a stroma centre. The extensive granal thylakoids in sucker chloroplasts of Korthalsella resemble that found in certain shade plants and tissue grown under low light conditions. Sucker chloroplasts probably have a low level of photosynthesis. This activity might provide a local source of osmotically active material used to assist transport between host and parasite.     

Overexpression of the triose phosphate translocator (TPT) complements the abnormal metabolism and development of plastidial glycolytic glyceraldehyde‐3‐phosphate dehydrogenase mutants

The presence of two glycolytic pathways working in parallel in plastids and cytosol has complicated the understanding of this essential process in plant cells, especially the integration of the plastidial pathway into the metabolism of heterotrophic and autotrophic organs. It is assumed that this integration is achieved by transport systems, which exchange glycolytic intermediates across plastidial membranes. However, it is unknown whether plastidial and cytosolic pools of 3‐phosphoglycerate (3‐PGA) can equilibrate in non‐photosynthetic tissues. To resolve this question, we employed Arabidopsis mutants of the plastidial glycolytic isoforms of glyceraldehyde‐3‐phosphate dehydrogenase (GAPCp) that express the triose phosphate translocator (TPT) under the control of the 35S (35S:TPT) or the native GAPCp1 (GAPCp1:TPT) promoters. TPT expression under the control of both promoters complemented the vegetative developmental defects and metabolic disorders of the GAPCp double mutants (gapcp1gapcp2). However, as the 35S is poorly expressed in the tapetum, full vegetative and reproductive complementation of gapcp1gapcp2 was achieved only by transforming this mutant with the GAPCp1:TPT construct. Our results indicate that the main function of GAPCp is to supply 3‐PGA for anabolic pathways in plastids of heterotrophic cells and suggest that the plastidial glycolysis may contribute to fatty acid biosynthesis in seeds. They also suggest a 3‐PGA deficiency in the plastids of gapcp1gapcp2, and that 3‐PGA pools between cytosol and plastid do not equilibrate in heterotrophic cells.     

Electron microscopical studies ofThorea ramosissima (Thoreaceae,Rhodophyta): Taxonomic implications ofThorea pit plug ultrastructure

The thallus ofThorea ramosissima was studied electron microscopically. The cells of the medulla, the cortex and the assimilatory hairs differ not only in size and number of plastids and their equipment with thylakoids but also in cell wall structure, the number of mitochondria and the activity of the Golgi apparatus, with dictyosomes transforming complete cisternae into Golgi vesicles with mucilaginous contents in the outer region of the cortex. The pit connections have plugs with a distinct plate—like (not dome-like) outer cap layer. BecauseT. riekei was reported to have dome-like outer cap layers and because this character was the main reason to place theThoreaceae into theBatrachospermales (Pueschel & Cole 1982),T. riekei was reinvestigated, too. A distinct outer cap could not be detected. The reliability of pit plug structure as a taxonomic character and the taxonomic position ofThorea is discussed.     

Further studies of the sieve-element plastids of theCaryophyllales includingBarbeuia,Corrigiola, Lyallia,Microtea, Sarcobatus,andTelephium

The sieve-element plastids of 69 species of theCaryophyllales were investigated by transmission electron microscopy. All contained the specific subtype-P3 plastids characterized by a peripheral ring of protein filaments. The presence or absence of an additional central protein crystal and their shape being either polygonal or globular as well as the average sizes of the sieve-element plastids are useful features in the characterization of some families.—Barbeuia contains sieve-element plastids that confirm its placement within thePhytolaccaceae. Lyallia differs fromHectorella by including small starch grains in their sieve-element plastids, which otherwise by their globular crystals negate a closer connection to theCaryophyllaceae. The lack of a central protein crystal in its form-P3fs plastids placesMicrotea best within theChenopodiaceae. Sarcobatus, a so far uncontested member of theChenopodiaceae, contains form-P3cf plastids, i.e., including a central crystal not found elsewhere in this family.Telephium andCorrigiola, shifted back and forth betweenMolluginaceae andCaryophyllaceae, have form-P3cf(s) plastids with a polygonal crystal which favor their placement within theCaryophyllaceae.     

The biology ofChlamydomyxa montana: Ultrastructure of the cyst

Summary Cells ofChlamydomyxa montana Lankester photosynthesize within a cyst under bright light and ingest diatoms in an excysted amoeboid form during periods of low light intensity. Cyst cell walls are partially comprised of cellulose and vary in thickness. The cells are multinucleate and intracellular bacteria are closely associated with the nuclei. A pair of centrioles is also present adjacent to individual nuclei. Chloroplasts are numerous and thylakoids are generally organized into bands of three. No endoplasmic reticulum was found surrounding the plastids, nor was a complete girdling lamella ever observed within.C. montana chloroplasts appear to be its own but this protist does not precisely fit into an algal class.     

Comparative ultrastructure of pyrenoids inTrebouxia (Microthamniales,Chlorophyta)

Pyrenoid ultrastructure has been investigated from cultures of all 26 species ofTrebouxia with the aim of establishing pyrenoids as a taxonomic character. Different arrangements and forms of thylakoid lamellae within the pyrenoid matrix allow eight pyrenoid types to be distinguished. Each type is characteristic of a group of species. Thegigantea- andimpressa-type are similar, differing only in the form of the tubules: short, branched tubules mark thegigantea-type; ± long and straight invaginations theimpressa-type. Thearboricola-type is characterized by meandering pyrenoid membranes developing from lamellae parallel with each other in young autospores. Pyrenoids of thegelatinosa-type are traversed by thin parallel-arranged tubules. Few thylakoids with a curved profile are typical of theirregularis-type. Thecorticola-type is different from all others in having a distinct starch sheath closely connected with the pyrenoid matrix and no pyrenoglobuli being associated with the pyrenoid membranes. No true pyrenoids have been found inT. magna andT. erici. Within the chloroplast, they have indistinct areas with pyrenoglobuli, but without differentiated thylakoids. Pyrenoid morphology is stable in culture on different media as well as in phycobionts within lichen thalli. Comparing the pyrenoid of a lichenizedTrebouxia with that from cultured species, the identification of the phycobiont within the lichen thallus is possible, without the need of culturing the algae. This has been shown in species ofParmelia andHypogymnia. New aspects for the taxonomy and systematics ofTrebouxia are discussed.Dedicated to Prof. DrLothar Geitler on the occasion of the 90th anniversary of his birthday.     

P-type sieve-element plastids present in members of the tribesTriplareae andCoccolobeae (Polygonaceae) renew the links between thePolygonales and theCaryophyllales

Form-Pfs sieve-element plastids were found inTriplaris, Ruprechtia, andCoccoloba (Polygonaceae) while other genera of the family and those studied from the often associatedPlumbaginaceae contain S-type sieve-element plastids. The rareness of form-Pfs plastids among the angiosperms, their similarity to the peculiar form-P3fs plastids of theChenopodiineae, and the comparatively small plastid diameters measured for all forms present in theCaryophyllales, Polygonales, andPlumbaginales suggest close relationships between these taxa. The restriction inPolygonaceae of form-Pfs plastids to the closely allied tribesTriplareae andCoccolobeae is discussed with regard to both the intrafamilial and ordinal phylogeny, and also considering possible connections to the only magnoliidaean Pfs-taxonCanella. Dedicated to Univ.-Prof. DrF. Ehrendorfer on the occasion of his 70th birthday.     

Plastids with densely staining thylakoid contents inNymphoides indica

Summary Most chloroplasts of the lower epidermal and sub-epidermal cells in mature floating leaves ofNymphoides indica have densely staining thylakoid loculi which contrast markedly with electron translucent loculi of chloroplasts elsewhere in the leaves. Examination of leaf primordia (< 1 mm long) showed that granal membranes arise within the plastid stroma and already at that stage the stainable substance can be seen associated with the lamellae. In slightly enlarged primordia (5 mm long), nearly all plastids of the young leaf contain thylakoids with stained loculi, but by the time the leaf reaches 25 mm in length, the staining is restricted to the lower epidermal layers.     

The role of chloroplast membranes in the location of chloroplast DNA during the greening ofPhaseolus vulgaris etioplasts

Summary The location of DNA containing nucleoids has been studied in greening bean (Phaseolus vulgaris L.) etioplasts using electron microscopy of thin sections and the staining of whole leaf cells with the fluorochrome DAPI. At 0 hours illumination a diffuse sphere of cpDNA surrounds most of the prolamellar body. It appears to be made up of a number of smaller nucleoids and can be asymmetric in location. The DNA appears to be attached to the outside of the prolamellar body and to prothylakoids on its periphery. With illumination the nucleoid takes on a clear ring-like shape around the prolamellar body. The maximum development of the ring-like nucleoid at 5 hours illumination is associated with the outward expansion of the prolamellar body and the outward growth of the prothylakoids. At 5 hours the electron transparent areas lie in between the prothylakoids radiating out from the prolamellar body. Between 5 hours and 15 hours observations are consistent with the growing thylakoids separating the nucleoids as the prolamellar body disappears and the chloroplast becomes more elongate. At 15 hours the fully differentiated chloroplast has discrete nucleoids distributed throughout the chloroplast with evidence of thylakoid attachment. This is the SN (scattered nucleoid) distribution ofKuroiwa et al. (1981) and is also evident in 24 hours and 48 hours chloroplasts which have more thylakoids per granum. The changes in nucleoid location occur without significant changes in DNA levels per plastid, and there is no evidence of DNA or plastid replication.The observations indicate that cpDNA partitioning in dividing SN-type chloroplasts could be achieved by thylakoid growth and effectively accomplish DNA segregation, contrasting with envelope growth segregating nucleoids in PS-type (peripheral scattered nucleoids) chloroplasts. The influence of plastid development on nucleoid location is discussed.