Teresa P. Dibbayawan

Filamentous cyanophytes containing phycourobilin and in symbiosis with sponges and an ascidian of coral reefs

A study was made of the ultrastructure and pigment composition of filamentous cyanophytes living in symbiosis with several sponges and a colonial didemnid ascidian collected from the southern end of the Great Barrier Reef, Australia, between 1983 and 1986. The sponges were Dysidea herbacea Keller and several other encrusting sponges which have not been identified; the ascidian was Trididemnum miniatum Kott (1977). The cyanophyte Oscillatoria spongeliae (Shultz) Hauck was identified as the symbiont of several of the sponges, including D. herbacea. Two other unidentified Oscillatoria species were found in a bristly papillate sponge and in T. miniatum. Chlorophyll a, alone, was present in all the symbionts with the exception of T. miniatum, which contained the cosymbiont Prochloron and where chlorophyll b was also present. Two phycoerythrins were isolated by chromatography and chromatofocusing. Both resembled C-phycoerythrin, but one of the two carried the chromophore phycourobilin as well as phycoerythrobilin possibly on both the α and β subunits, which had apparent molecular masses of 18 and 20 kdaltons. No γ subunit was present. Ultrastructurally, the three Oscillatoria species were distinguished by an unusual type of parallel, longitudinal, thylakoid organisation; the arrangement was different in detail in each species.

A γ-tubulin that associates specifically with centrioles in HeLa cells and the basal body complex in Chlamydomonas

γ‐Tubulin is a putative component of microtubule initiating material. To further explore its subcellular distribution in plant and animal cells, we have raised a polyclonal antibody, Rb27, directed towards a conserved region (EEFATEGTDRKDVFFY) of the γ‐tubulin molecule. Immunoblotting of cell protein extracts with Rb27 reveals a polypeptide band of Mr 49 kD in HeLa and a 58 kD band in Chlamydomonas. Although these polypeptides are comparable in size to forms of γ‐tubulin detected previously in mammalian and plant protein extracts by other antibodies to γ‐tubulin, by immunofluorescence microscopy Rb27 gives localization patterns not previously seen. It localizes specifically with the centrioles in HeLa cells and with the basal body complex in Chlamydomonas. Other γ‐tubulin antibodies label pericentriolar material. Because of the similarities in the size of the polypeptides recognized by our and other γ‐tubulin antibodies, and a restricted co‐localization with known microtubule‐organizing centres in evolutionarily distant organisms, we propose that Rb27 recognizes a novel conserved γ‐tubulin isotype.

High resolution scanning electron microscopy of plasmodesmata.

Symplastic transport occurs between neighbouring plant cells through functionally and structurally dynamic channels called plasmodesmata (PD). Relatively little is known about the composition of PD or the mechanisms that facilitate molecular transport into neighbouring cells. While transmission electron microscopy (TEM) provides 2-dimensional information about the structural components of PD, 3-dimensional information is difficult to extract from ultrathin sections. This study has exploited high-resolution scanning electron microscopy (HRSEM) to reveal the 3-dimensional morphology of PD in the cell walls of algae, ferns and higher plants. Varied patterns of PD were observed in the walls, ranging from uniformly distributed individual PD to discrete clusters. Occasionally the thick walls of the giant alga Chara were fractured, revealing the surface morphology of PD within. External structures such as spokes, spirals and mesh were observed surrounding the PD. Enzymatic digestions of cell wall components indicate that cellulose or pectin either compose or stabilise the extracellular spokes. Occasionally, the PD were fractured open and desmotubule-like structures and other particles were observed in their central regions. Our observations add weight to the argument that Chara PD contain desmotubules and are morphologically similar to higher plant PD.

Field emission scanning electron microscopy of plant cells

SummaryTwo basic specimen preparation protocols that allow field emission scanning electron microscope imaging of intracellular structures in a wide range of plants are described. Both protocols depend on freeze fracturing to reveal areas of interest and selective removal of cytosol. Removal of cytosol was achieved either by macerating fixed tissues in a dilute solution of osmium tetroxide after freeze fracturing or by permeabilizing the membranes in saponin before fixation and subsequent freeze fracturing. Images of a variety of intracellular structures including all the main organelles as well as cytoskeletal components are presented. The permeabilization protocol can be combined with immunogold labelling to identify specific components such as microtubules. High-resolution three-dimensional imaging was combined with immunogold labelling of microtubules and actin cables in cell-free systems. This approach should be especially valuable for the study of dynamic cellular processes (such as cytoplasmic streaming) in live cells when used in conjunction with modern fluorescence microscopical techniques.

Intercellular Alignments of the Plant Cytoskeleton

The cytoskeleton orchestrates many processes in plant development, including division and control of the direction of cell expansion, and is therefore central in the coordination of plant growth. There are a number of situations in which there is a precise alignment of the cytoskeleton, in particular microtubules, between neighboring cells. However, it is not known how these intercellular alignments are brought about. We discuss the possibility that the intercellular alignments are due to individual cells each responding in turn to an external orienting vector, without a need for direct communication between cells. Alternatively, there may be information exchange between the cells about the orientation of the cytoskeleton to allow for coordination. This exchange could take place directly via the plasmodesmata or more indirectly through the intervening cell wall. The final possibility, discussed here, is that orientation of the cytoskeleton in neighboring cells is coordinated via direct continuity of the cytoskeleton between neighboring cells, presumably via the plasmodesmata.

High-resolution microscopy of cell wall formation in regenerating Mougeotia (Chlorophyceae) protoplasts

Field emission scanning electron microscopy (FESEM) preparation techniques have been successfully adapted for visualization of the internal and external ultrastructure of Mougeotia filaments and protoplasts. FESEM of the innermost layer of cell wall in Mougeotia filaments revealed that microfibrils are deposited parallel to each other in an interconnected mesh and are oriented perpendicular to the direction of elongation. For the first time, the surface of protoplasts at different stages of regeneration has been observed using FESEM. Nascent microfibril deposition occurs between 1 and 2 h after isolation and arrangement of these microfibrils is random for at least 8 h. Observation of the inner surface of the plasma membrane in burst protoplasts showed that microtubules are not strongly attached for at least 3 h after protoplast isolation.

COMBINED IMMUNOFLUORESCENCE AND FIELD EMISSION SCANNING ELECTRON MICROSCOPE STUDY OF PLASMA MEMBRANE-ASSOCIATED ORGANELLES IN HIGHLY VACUOLATED SUSPENSOR CELLS OF WHITE SPRUCE SOMATIC EMBRYOS

Highly vacuolated suspensor cells of spruce somatic embryos were examined by immunofluorescence light microscopy using butyl‐methyl‐methacrylate (BMM) and polyethylene glycol (PEG) embedded sections, transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM). The use of PEG embedded embryos provided a rapid method for light microscope detection of antigens before committing to FESEM analysis. BMM embedded specimens provided well preserved suspensor cells for immunofluorescence. FESEM permitted high resolution observation of large areas of the inner surface of the plasma membrane and associated cell organelles. Suspensor cells contained mostly transversely oriented cortical microtubules linked to the plasma membrane and adjacent microtubules by cross‐ bridges. Light and electron microscopy revealed numerous clathrin coated structures on the plasma membrane. These included flat patches of clathrin, coated pits and coated vesicles. Many coated vesicles were associated with microtubules. Both tubular and lamellar endoplasmic reticulum were observed on the plasma membrane by FESEM.

Cell wall and plasma membrane architecture of Butyrivibrio spp.

The cell wall and plasma membrane of four strains of Butyrivibrio (anaerobic rumen bacteria) have been studied by freeze-etching and thin sectioning. Some strains have a wall of gram-positive ultrastructure, which is vulnerable to lysozyme, while others have a wall which resembles the outer envelope of a gram-negative bacterium and is less susceptible to lysozyme. The plasma membrane of all strains is rather rigid, and its lipids have a very high phase-transition temperature.

Multiphoton microscopy of cell division in plant cells

Using confocal and multiphoton microscopy we have mapped the three-dimensional arrangements of chromosomes, microtubules and gamma tubulin during cell division in plants. We have also for the first time imaged diivision in living, intact plant tissue. These results are preliminary, but exciting and we anticipate considerable further progress will be possible with advances in hardware which are now becoming available.

Cord-Like Phycobilisomes of Rhodosorus marinus Geitler: Fine Structure and its Functional Implications

Rhodosorus marinus is a unicellular, marine, red alga common in tropical and subtropical waters. We recently isolated it for the first time in Australia, and in the course of a routine ultrastructural investigation found that it had phycobilisomes of most unusual structure and arrangement.