Maret Vesk

Actin associated with plasmodesmata

SummaryWe have used several methods to localise actin associated with plasmodesmata. In meristematic plant material fixed in 0.1% glutaraldehyde/1% paraformaldehyde and embedded in LR White resin, actin was localised (in TEM using 5 nm gold-labelled secondary antibody to C4 anti-actin primary antibody) in the neck region by the plasma membrane and endoplasmic reticulum, and also down the length of the plasmodesma, deep in the cell wall. When the chemical fixation was replaced by rapid freezing in liquid propane (without cryoprotectants) and substitution in acetone, the plasmodesmata were labelled in similar positions, but with less background label on sections. While only 8–20% of plasmodesmata were labelled, the label was 10 to 100 fold denser over plasmodesmata than over the surrounding wall indicating specific association with plasmodesmata. We presume the apparent extracellular location of some label was due to the size of the antibodies between the site of attachment and the observed position of the gold particle. Gold label was found in similar locations in material fixed in 3% paraformaldehyde, infiltrated with sucrose, frozen, sectioned (10–12 μm thick), then labelled with antibodies before resin embedding. Furthermore, cell walls in epidermal peels stained with rhodamine-phalloidin showed localised patches of fluorescence, presumably at the site of plasmodesmata (or primary pit-fields), which were connected on either side to fluorescent strands of actin in the cytoplasm. Suspension cultured cells ofNicotiana plumbaginifolia similarly stained showed very faint, narrow fluorescent strands crossing the walls of sister cells, which may indicate actin associated with individual plasmodesmata, shown in TEM to be sparsely distributed in these walls. In addition, the neck regions of cytochalasin-treated plasmodesmata were greatly enlarged and lacked the normal extracellular ring of particles. We propose that actin associated with plasmodesmata stabilizes the neck region and possibly also the cytoplasmic sleeve, and may be actively involved in regulating cell-to-cell transport.

CALLOSE DEPOSITION AT PLASMODESMATA

SummaryThe transport of ions and metabolites through plasmodesmata has been thought to be controlled at the neck region where the cytoplasmic annulus is constricted and where callose has also been localised. In order to determine the possible structural and functional effects of callose, its deposition was inhibited through incubation of the plant tissue with 2-deoxy-D-glucose (DDG) for 1 h prior to fixation in 2.5% glutaraldehyde. The inhibition of callose formation was monitored through aniline blue-induced fluorescence of callose. The neck region of the plasmodesmata fromAllium cepa L. roots treated with DDG exhibited a funnel-shaped configuration. This is in contrast to the plasmodesmata from tissue not incubated with DDG, which exhibited constricted necks similar to those previously reported. Both initial dissection and glutaraldehyde fixation induced neck constriction in plasmodesmata, however, dissection of tissue increased the frequency of constrictions. The inhibition of callose formation by chemical means showed that the neck constrictions and raised collars in this area are artefacts due to physical wounding and glutaraldehyde fixation. The external electron-dense material observed when tannic acid is included in the primary fixative appears to be unrelated to the deposition of callose at the neck region.

EFFECT OF BLUE-GREEN LIGHT ON PHOTOSYNTHETIC PIGMENTS AND CHLOROPLAST STRUCTURE IN UNICELLULAR MARINE ALGAE FROM SIX CLASSES1

The photosynthetic pigments of 17 species of unicellular marine algae grown in white and blue‐green light were examined. Blue‐green light (400 μW·cm−2; 12:12 LD cycle) caused major chlorophyll increases (55–146%) in five diatoms, one dinoflagellate and one cryptomonad; minor chlorophyll increases (17–39%) in two diatoms, two dinoflagellates, one prymnesiophyte (haptophyte), one chrysophyte and one chlorophyte; and no chlorophyll increase in two diatoms and one pyrmnesiophyte (haptophyte). The relative proportions of major chlorophylls and carotenoids did not change, but in six of eight species tested small increases in the concentration of chlorophyll c occurred. Blue‐green light caused a small increase in the concentration of phycoerythrin relative to chlorophyll a in the cryptomonad. A larger number of thylakoids per chloroplast were observed in six species grown in blue‐green light compared to white light controls. The ultrastructure changes observed depended not only on the magnitude of the chlorophyll increase but also on the architecture of the chloroplast.

FURTHER EVIDENCE FOR A MEMBRANE‐BOUND ENDOSYMBIONT WITHIN THE DINOFLAGELLATE PERIDINIUM FOLIACEUM1

The fine structure of the binucleate, fucoxanthin‐containing dinoflagellate Peridinium foliaceum (Stein) Biechler was re‐examined for evidence of an endosymbiout. The eucaryotic nucleus, chloroplasts and associated ribosome‐dense cytoplasm were separated by a single invaginating membrane from the rest of the dinoflagellate cell. The triple membrane‐enclosed eyespot, mesocaryotic nucleus, trichocysts and accumulation bodies resided in the dinoflagellate cytoplasm. These observations suggest that P. foliaceum contains a membrane‐bound endosymbiont, similar to that already described for the closely related species. P. balticum (Levander) Lemmermann.

ULTRASTRUCTURE AND PIGMENTS OF TWO STRAINS OF THE PICOPLANKTONIC ALGA PELAGOCOCCUS SUBVIRIDIS (CHRYSOPHYCEAE)

The organelle ultrastructure and photosynthetic pigments of a new isolate of the picoplanktonic alga Pelagococcus subviridis Norris from the East Australian Current was compared with the North Pacific Ocean type species. No differences in the ultrastructure of the two isolates were observed. Mitosis was studied in detail in the Australian strain, and showed two unusual features: the de novo appearance of centrioles prior to mitosis, and the formation of a small, extra‐nuclear spindle.

The fine structure of leaf cells of manganese-deficient spinach

Observations have been made by phase contrast, fluorescence, and electron microscopy on the leaf cells of normal (full nutrient) and manganese-deficient spinach. In manganese-deficient cells the structure of the chloroplasts is markedly changed. The structure of other cell organelles such as mitochondria, spherosomes, and the nucleus is not altered. Initially, in manganese-deficient chloroplasts there is a proliferation of the stroma or “jacket” material of the plastid together with a reduction in the number of intergrana connections. As the deficiency progresses the grana increase in size and take on an almost circular shape. There are fewer but larger grana, giving a greatly altered distribution of chlorophyll within the plastids. At this stage there are virtually no intergrana connections. Within the stroma numerous electron transparent areas are found and, as well, vacuole-like structures at the margins of the plastids. It is suggested that the “vacuoles” are formed by the folding of the chloroplast about itself. In a number of sections objects similar in size to mitochondria and containing crista-like tubules occur at the margins of the plastids.

THE FINE STRUCTURE OF TWO PHOTOSYNTHETIC SPECIES OF DINOPHYSIS (DINOPHYSIALES, DINOPHYCEAE)1

Two closely related, photosynthetic species belonging to the genus Dinophysis were examined, D. acuminata Claparède et Lachmann and D. fortii Pavillard. Typical dinoflagellate features include the amphiesmal covering enclosing the cells and the structure of the nucleus and mitochondria. Many other characteristics seem to be specific to the order Dinophysiales. Many rhabdosomes are present, and complex mucocysts are found beneath the amphiesma. The thecal pores are unusual with the base of the pore occluded by a thin disc that is continuous with the main amphiesmal plate. The structure of the apical pore is also distinctive. Chloroplasts are grouped together in chromatospheres, enclosed by a double membrane, and contain paired thylakoids with electron dense contents in the lumen. The two pusules are extensive, each branching off the flagellar canal, and consisting of a large antechamber and a number of convoluted sacs. The entrance of each antechamber, and site of an emerging flagellum, is surrounded by a striated fibrous collar. Near the flagellar pore is a prominent microtubular/microbody complex which penetrates deep into the cell cytoplasm. Consideration is given to taxonomic position of the Dinophysiales and also to the nature and origins of the chloroplasts.

Structure and function in the euglenoid eyespot apparatus: The fine structure, and response to environmental changes.

SummaryThe microanatomy of the eyespot apparatus of Euglena gracilis Z was examined with the electron microscope. The stigma was found to be a membrane-bounded organelle showing no close homology with the chloroplast or any other organelle. The structure and pigment content of the stigma both diminish with extended hetrotrophic growth, and quickly regain normal dimensions upon exposure to light. Synthesis of the red pigment is particularly sensitive to inhibition by chloramphenicol, whereas construction of the structure itself is specifically inhibited by cycloheximide.The paraflagellar body appears to consist of two sets of parallel 80 Å striations intersecting at 60°. It is within the flagellar membrane, but separated from the axoneme by another structure, the paraflagellar rod. This elongated structure has an ordered substructure which appears as intersecting sets of parallel striations; part of its basal portion projects as a circular flange which makes contact with the paraflagellar body.

EFFECT OF BLUE‐GREEN LIGHT ON PHOTOSYNTHETIC PIGMENTS AND CHLOROPLAST STRUCTURE IN THE MARINE DIATOM STEPHANOPYXIS TURRIS1

Blue‐green light increased the chlorophyll concentration and chloroplast number of cells of Stephanopyxis turris (Grev.) Ralfs, compared to white light controls. Light fields for growth were 400 μW·cm−2 (12:12 LD cycles). Chlorophyll increased up to 100%/cell, but no change in the ratio of chlorophylls to major carotenoids occurred. The effect was, therefore, not that of complementary chromatic adaptation. At the same time, blue‐green light enhanced the photosynthetic fixation of CO2. At the ultrastructure level, an increase in, and rearrangement of, the thylakoid system occurred.

Ultrastructure of the corallinaceae. I. The vegetative cells of Corallina officinalis and C. cuvierii

A technique utilizing combined fixation and gentle decalcification has been employed to study the ultrastructure of the vegetative cells of the articulated calcareous coralline algae Corallina officinalis Linnaeus and C. cuvierii Lamouroux (Rhodophyta: Cryptonemiales). The epidermal cells are distinctive, with many cell wall inggrowths which pass between the chloroplasts. It is suggested that these cells function as “transfer cells”. The epidermal cells contain no starch, although the chloroplasts have well developed photosynthetic lamellae. Damage to these epidermal cells leads to formation of new cells by renewed division of sub-epidermal meristematic cells. The outer cortical cells have few small vacuoles and many plastids, with an extensive photosynthetic lamellar system. Deeper into the thallus, the vacuoles increase in size and free cytoplasmic starch grains occur. The medullary cells have a very large vacuole and in older tissue often appear dead. The long genicular cells have calcareous walls at either end while the wall in the middle of these cells is non-calcareous and has an inner fibrillar layer and a thin outer “cuticle”. In partially decalcified material, the orientation of the CaCO3 (calcite) crystals next to the cells can clearly be seen. Immediately next to the cell the crystals are fairly small and arranged at right angles to the plasmalemma. Further away from the cell the crystal size is larger and their orientation is more random. The crystals are surrounded by organic material, and the possible rôle of this material in calcification in coralline algae is discussed.