VALVE MORPHOGENESIS IN THE PENNATE DIATOM ACHNANTHES COARCTATA

Mitosis and valve morphogenesis in the pennate diatom Achnanthes coarctata (Bréb. in W. Sm.) Grun. are described. After cytokinesis, both daughter nuclei and their microtubule centers (MCs) are found near one side of the cell. Each new tubular silica deposition vesicle (SDV) arises centrally, forming a single rib running the length of the cell. Each MC then migrates around its nucleus and positions itself directly adjacent to the new SDV. The enlarging silicalemmas with their associated MCs, nuclei, microtubules (MTs) and microfilaments (MFs) appear in mirror image in the daughter cells. Both SDVs soon generate a second longitudinal rib alongside the first; the gap between the ribs ultimately becomes the future raphe fissure. The MC, MTs and nucleus are associated with each fissure. However, the subsequent behavior of the valve secreting machinery now becomes quite different in the daughter cells. In the cell that will form a raphid valve, the silicalemma, flanked by MFs, expands laterally in both directions over the cleavage furrow. Within the expanding SDV, silica secretion continues, eventually generating the structure of the mature valve, and during this phase the raphe fissure becomes delineated as in other raphid diatoms. In the other daughter cell, however, the MC and its MTs withdraw from the silicalemma, and the SDV moves laterally across the cleavage furrow until the double rib is at the corner of the cell. As silica is secreted into this expanding SDV, the raphe fissure completely fills in. This valve, therefore, lacks a raphe when mature and has a symmetry quite different from that of the valve formed in the other daughter cell. These events are compared with the course of morphogenesis described for other raphid diatoms.     

Cell division and morphogenesis of the labiate process in the centric diatomDitylum brightwellii

Summary Cells of the centric diatomDitylum brightwellii were filmed undergoing cell division and valve secretion, and were fixed for transmission electron microscopy. Attention was directed particularly at the origin of the Labiate Process Apparatus (LPA).As reported previously (li andVolcani 1985 a), the nucleus, centrally situated during interphase, moves laterally to undergo mitosis against the girdle bands. We describe the spindle which splits up into numerous fibres of overlapped polar microtubules (MTs) by metaphase. The chromosomes are diffuse and the spindle elongates rapidly during anaphase. A complex of organelles is found at the poles and ill-defined, dense material extends to the nearby plasmalemma from prophase on. The two Silica Deposition Vesicles (SDVs) are initiated during anaphase close to the poles and by midcleavage, the dense LPA arises on each SDV close to dense polar material. After cleavage, the daughter protoplasts round up and the SDV, already containing a nascent valve, expands over the cleavage furrow. The labiate process, a long straight hollow tube of silica, is rapidly (ca. 25 minutes) secreted from directly under the LPA; a fibrous plug (polysaccharide?) always appears in the SDV immediately adjacent to the LPA during the initiation of this secretion. The ill-defined Microtubule-Organizing Center (MC) from the spindle pole remains close to the LPA and in it can be seen the tiny presumptive primordial spindle on the nuclear envelope.The raphe and the labiate process (LP), both highly differentiated apertures in the valve, probably function in a specialized form of the mucilage secretion involved in generation of movement in raphid diatoms, and in a simple form of movement in some centrics. Morphogenesis of the LP is associated with the LPA while differentiation of the raphe is almost associated with the MC; both MC and LPA have an intimate ontological relationship with the spindle pole and the postmitotic cytoskeletal system of MTs. This association also is seen in the formation of the LP in an araphid pennate,Diatoma (work in progress). Therefore, from functional, morphogenetic and ontogenetic observations, we support the proposal that the raphe of pennate diatoms arose from the LP of centric diatoms.     

VALVE MORPHOGENESIS IN THE PENNATE DIATOM NAVICULA CUSPIDATA1

The deposition of siliceous valves during asexual reproduction of the pennate diatom, Navicula cuspidata Kütz., is described with emphasis on the cytoplasmic components involved. The events accompanying valve secretion are similar to those already known from other pennate species. After mitosis, the microtubule centre (MC) moves to the center of the cleavage furrow where silica deposition is initiated inside a tubular silicalemma, and it remains associated with the prospective central nodule during valve growth. Microtubules (MTs), emanating from the MC, run parallel to the prospective raphe and together with the raphe fibres, appear to be involved with raphe development. Multiple raphe fibres occupy the maturing raphe fissure, in contrast to the single fibre of Pinnularia viridis, P. maior and Hantzschia amphioxys. The fibers exhibit a periodic substructure and are often opposed to the silicalemma where they may inhibit silica deposition and control the shaping of the raphe fissure. In contrast with the above species, in N. cuspidata MTs are clustered strictly opposite the raphe and lose their association with the MC which degenerates before the valves are mature. The primary role of MTs may be the stabilization of the cytoplasmic region where initial silicification occurs. Mitochondria and endoplasmic reticulum are not involved in molding valve growth in this species. Evidence for vesicle involvement in silica transport and deposition was limited. The possible contributions provided by comparative studies on the ultrastructure of valve morphogenesis towards elucidating the control of valve formation and the taxonomy of diatoms are discussed briefly.     

VALVE MORPHOGENESIS IN SURIRELLA (BACILLARIOPHYCEAE)1

Valve morphogenesis in two Surirellae (S. ovalis Brebisson and S. robusta Ehrenberg) is described. Mitosis takes place at the broad end of the cell. After cleavage, a new Microtubule Center (MC) arises near each spindle pole and moves to the adjacent plasmalemma. Soon, a specific group of microtubules (MTs) extends from very near the MC around the periphery of the cell. Concurrently, the new tubular Silica Deposition Vesicle (SDV) grows around the periphery of the cell close to these MTs. A double rib of silica is rapidly formed inside the SDV; the space between the ribs becomes the raphe. Mitochondria line up along the MTs, and the SDV may be molded around these to create the canal raphe. Soon, the SDV expands in two directions to create the face and the mantle of the new valve. Meanwhile, each daughter nucleus, accompanied by the MC, moves to its interphase position at the center of the cell; this movement is colchicine-sensitive. As in several other pennate diatoms, an interruption in the raphe of the mature valve coincides with the initial position of the MC. The canal raphe thickens rapidly around the mitochondria; a rudimentary raphe fiber may be associated with the creation of a tiny curvature at the inner raphe fissure. As the SDV expands in the large S. robusta, the daughter cell protoplasts slowly shrink by plasmolysis, thereby creating the complex curved surface of the new valve surmounted by the arching canal raphes which are now quite rigid. In S. ovalis, the daughter cell protoplasts remain appressed and therefore the new valve surface is basically flat. The symmetry of Surirella is quite different from that of other pennate diatoms. However, the cytoplasmic events accompanying valve morphogenesis are similar in all important respects to those described in other raphid pennate diatoms, and clearly supports a naviculoid origin for this genus.     

Fine-structural and chemical analyses on inner and outer sheath of the cyanobacteriumGloeothece sp. PCC 6909

Cells of the unicellular cyanobacteriumGloeothece sp. PCC 6909 are surrounded by an inner (enclosing 1–2 cells) and an outer (enclosing cell groups) sheath. Using conventional Epon-embedding in combination with ruthenium-red staining, the inner and outer sheaths appeared similar and displayed multiple bands of electron-dense subunits. However, embedding in Nanoplast resin to avoid shrinkage led to the detection of two distinct zones (inner and outer zone) each with several distinct layers. The zone delimited by the electron-dense thick inner sheath layer, and the zone enclosed by the thin electron-dense outer sheath layer, are composed of a homogeneous material of little electron-contrast. Whereas the outer zone appears to be of even contrast, the inner zone is characterized by a distinct electron-transparent layer. Element distribution analysis revealed that the electron-transparent layer contained relatively large amounts of sulfur, carbon, and oxygen but only little nitrogen.Inner and outer sheath fractions were isolated by differential mechanical cell breakage and centrifugation. The outer sheath fraction was less hydrated than the inner one. The two fractions differed little in their contents of uronic acids, carbohydrate and protein, although the outer sheath fraction contained less sulfate. A soluble polysaccharide with a chemical composition similar to that of inner and outer sheath fractions was also obtained from the culture supernatant.     

Substratum adhesion and gliding in a diatom are mediated by extracellular proteoglycans

Diatoms are unicellular microalgae encased in a siliceous cell wall, or frustule. Pennate diatoms, which possess bilateral symmetry, attach to the substratum at a slit in the frustule called the raphe. These diatoms not only adhere, but glide across surfaces whilst maintaining their attachment, secreting a sticky mucilage that forms a trail behind the gliding cells. We have raised monoclonal antibodies to the major cell surface proteoglycans of the marine raphid diatom Stauroneis decipiens Hustedt. The antibody StF.H4 binds to the cell surface, in the raphe and to adhesive trails and inhibits the ability of living diatoms to adhere to the substratum and to glide. Moreover, StF.H4 binds to a periodate-insensitive epitope on four frustule-associated proteoglycans (relative molecular masses 87, 112, and >200 kDa). Another monoclonal antibody, StF.D5, binds to a carbohydrate epitope on the same set of proteoglycans, although the antibody binds only to the outer surface of the frustule and does not inhibit cell motility and adhesion. Received: 2 December 1996 / Accepted: 6 March 1997     

SILICON METABOLISM IN DIATOMS: IMPLICATIONS FOR GROWTH

Diatoms are the world's largest contributors to biosilicification and are one of the predominant contributors to global carbon fixation. Silicon is a major limiting nutrient for diatom growth and hence is a controlling factor in primary productivity. Because our understanding of the cellular metabolism of silicon is limited, we are not fully knowledgeable about intracellular factors that may affect diatom productivity in the oceans. The goal of this review is to present an overview of silicon metabolism in diatoms and to identify areas for future research. Numerous studies have characterized parameters of silicic acid uptake by diatoms, and molecular characterization of transport has begun with the isolation of genes encoding the transporter proteins. Multiple types of silicic acid transporter gene have been identified in a single diatom species, and multiple types appear to be present in all diatom species. The controlled expression and perhaps localization of the transporters in the cell may be factors in the overall regulation of silicic acid uptake. Transport can also be regulated by the rate of silica incorporation into the cell wall, suggesting that an intracellular sensing and control mechanism couples transport with incorporation. Sizable intracellular pools of soluble silicon have been identified in diatoms, at levels well above saturation for silica solubility, yet the mechanism for maintenance of supersaturated levels has not been determined. The mechanism of intracellular transport of silicon is also unknown, but this must be an important part of the silicification process because of the close coupling between silica incorporation and uptake. Although detailed ultrastructural analyses of silica deposition have been reported, we know little about the molecular details of this process. However, proteins occluded within silica that promote silicification in vitro have recently been characterized, and the application of molecular techniques holds the promise of great advances in this area. Cellular energy for silicification and transport comes from aerobic respiration without any direct involvement of photosynthetic energy. As such, diatom silicon metabolism differs from that of other major limiting nutrients such as nitrogen and phosphorous, which are closely linked to photosynthetic metabolism. Cell wall silicification and silicic acid transport are tightly coupled to the cell cycle, which results in a dependency in the extent of silicification on growth rate. Silica dissolution is an important part of diatom cellular silicon metabolism, because dissolution must be prevented in the living cell, and because much of the raw material for mineralization in natural assemblages is supplied by dissolution of dead cells. Perhaps part of the reason for the ecological success of diatoms is due to their use of a silicified cell wall, which has been calculated to impart a substantial energy savings to organisms that have them. However, the growth of diatoms and other siliceous organisms has depleted the oceans of silicon, such that silicon availability is now a major factor in the control of primary productivity. Much new progress in understanding silicon metabolism in diatoms is expected because of the application of molecular approaches and sophisticated analytical techniques. Such insight is likely to lead to a greater understanding of the role of silicon in controlling diatom growth, and hence primary productivity, and of the mechanisms involved in the formation of the intricate silicified structures of the diatom cell wall.     

NANOSTRUCTURE OF THE DIATOM FRUSTULE AS REVEALED BY ATOMIC FORCE AND SCANNING ELECTRON MICROSCOPY

The cell wall (frustule) of the freshwater diatom Pinnularia viridis (Nitzsch) Ehrenberg is composed of an assembly of highly silicified components and associated organic layers. We used atomic force microscopy (AFM) to investigate the nanostructure and relationship between the outermost surface organics and the siliceous frustule components of live diatoms under natural hydrated conditions. Contact mode AFM imaging revealed that the walls were coated in a thick mucilaginous material that was interrupted only in the vicinity of the raphe fissure. Analysis of this mucilage by force mode AFM demonstrated it to be a nonadhesive, soft, and compressible material. Application of greater force to the sample during repeated scanning enabled the mucilage to be swept from the hard underlying siliceous components and piled into columns on either side of the scan area by the scanning action of the tip. The mucilage columns remained intact for several hours without dissolving or settling back onto the cleaned valve surface, thereby revealing a cohesiveness that suggested a degree of cross-linking. The hard silicified surfaces of the diatom frustule appeared to be relatively smooth when living cells were imaged by AFM or when field-emission SEM was used to image chemically cleaned walls. AFM analysis of P. viridis frustules cleaved in cross-section revealed the nanostructure of the valve silica to be composed of a conglomerate of packed silica spheres that were 44.8 ± 0.7 nm in diameter. The silica spheres that comprised the girdle band biosilica were 40.3 ± 0.8 nm in diameter. Analysis of another heavily silicified diatom, Hantzschia amphioxys (Ehrenberg) Grunow, showed that the valve biosilica was composed of packed silica spheres that were 37.1 ± 1.4 nm and that silica particles from the girdle bands were 38.1 ± 0.5 nm. These results showed little variation in the size range of the silica particles within a particular frustule component (valve or girdle band), but there may be differences in particle size between these components within a diatom frustule and significant differences are found between species.     

Lorica Production in Choanoflagellates–A Model System for Investigating the Mechanics, Controls and Dynamics of Secretion

BIOMINERALIZATION is the process by which living organisms assemble structures from naturally occurring inorganic compounds. Mineral deposition is common and widespread amongst Protozoa and in most instances the mineralized structures provide skeletal support and protection for softer organic parts [10]. The 2 most common minerals to be deposited by Protozoa are silica and calcium carbonate. Groups of Protozoa that deposit silica, which we are concerned with here, include the diatoms, chrysophytes, choanoflagellates, Radiolar-ia, Heliozoa and testate amoebae [10]. In the majority of silica-depositing protista, silica is taken up from the medium in the form of monomelic orthosilicic acid Si(OH)₄ (soluble reactive silicate) and deposited as amorphous, polymerised biogenic silica or opal within membrane-bounded vesicles known as silica deposition vesicles (SDV). Often biogenic silica is characteristically patterned and ornamented and for most protozoan groups the morphology of silicified parts is of prime taxonomic importance. By far the most extensively studied group of silica-depositing organisms are the diatoms [1, 12, 13]. To date most of our knowledge of silica metabolism in protists has been based on investigations into this group. Diatoms require silica for the production of their frustules. Uptake and deposition of silica occurs within a closely denned portion of the cell cycle, between nuclear division and cell separation. It occupies about ± of the cell cycle and without an adequate supply of silica diatoms are unable to produce new frustule valves with the result that cell division cannot be completed. Diatoms, therefore, have an obligate requirement for silica and without this nutrient they cease to grow [11]. In contrast to diatoms a number of other silica-depositing protistan groups, such as loricate choanoflagellates and certain chrysophytes, have a facultative requirement for silica. In the past decade the ultras true ture, physiology and ecology of loricate choanoflagellates have been extensively studied by a number of different workers [7] and the significance of these studies to our understanding of the mechanisms, controls and dynamics of silica secretion is summarised and discussed here.     

VARIATION IN PATTERNS OF VALVE MORPHOGENESIS BETWEEN REPRESENTATIVES OF SIX BIRAPHID DIATOM GENERA (BACILLARIOPHYCEAE)

Scanning electron microscopic studies of silica valve formation in naviculoid diatoms representing six different genera revealed that the precise sequence of depositional events varied among genera. Valve deposition begins with the formation of the raphe sternum, from which virgae (lateral outgrowths) extend. Areolae (pores) are formed between the virgae by the fusion of cross-extensions (vimines). In most of the species studied ( Craticula ambigua (Kützing) D. G. Mann, Frustulia vulgaris (Thwaites) De Toni, Craspedostauros australis E. J. Cox, and Gomphonema truncatum Ehrenberg), areola (pore) formation began near the raphe sternum before completion of the valve margin, but in Pinnularia gibba Ehrenberg the valve margin fused before the areolae were formed. Silica deposition in all these taxa was mainly distal to proximal (with respect to the cytoplasm), but in Haslea sp. it was mainly proximal to distal. Haslea also differed in that areolae were defined as the valve margin was completed. These data have also contributed to the interpretation of taxonomically important features, such as raphe endings. In P. gibba the internal central raphe fissures were laterally deflected but subsequently obscured by additional silicification of the valve, whereas in G. truncatum they were initially straight, becoming laterally deflected as valves mature. External raphe fissures in Frustulia became Y-shaped only just before maturity; in immature valves they were dotlike, as in Amphipleura Kützing. The comparison of developmental pathways in diatoms is a useful adjunct to morphological and other approaches in diatom systematics and warrants renewed attention.     

THE ROLE OF DIATOMS IN STROMATOLITE GROWTH: TWO EXAMPLES FROM MODERN FRESHWATER SETTINGS1

Some modern laminated and calcified stromatolitic structures are partially or completely formed by eukaryotes. Diatom populations in freshwater environments with elevated ionic concentrations contribute to calcite precipitation, and the formation of distinctive mineral-rich stromatolitic laminae. Two types of stromatolite-forming diatom populations were observed. In the first example, in stromatolies growing on a quarry ledge near Laegerdorf, North Germany, calcite crystals with biogenic imprints form around polysaccharide stalks of the diatom Gomphonema olivaceum var. calcarea (Cleve) Cleve-Euler. These individually precipitated crystals eventunally become cemented together in layers, forming rigid, laminated stromatolitic deposits which drape over the quarry ledge. In the second example, in stromatolites forming in a shallow stream near Cuatro Ciénegas, Coahuila, Mexico, diatomaceous laminae also form by the accumulation of carbonate particles in a matrix of diatoms and their extracellular polysaccharide products. These laminae become thick enough to drape over individual stromatolite heads. The diatoms responsible for these deposits are Amphora aff. A. Katii Selva, Nitzschia denticula Grun., and six other species. At Cuatro Ciénegas, in addition to the diatomaceous laminae, carbonate-rich cyanobacterial layers, dominated by two cyanobacterial species with different fabrics and porosities, are also present and contribute substantially to the growth of the stromatolites. In both the Laegerdorf and Cuatro Ciénegas examples, entire stromatolites or thick laminations on stromatolites are built by a small number of diatom species which produce copious amounts of extracellular stalk, gel, and sheath material, a property they share with cyanobacterial stromatolite builders.     

Origin and evolution of the canal raphe system in diatoms

One lineage of pennate diatoms has a slit through the siliceous cell wall, called a "raphe," that functions in motility. Raphid pennate diatoms number in the perhaps tens of thousands of species, with the diversity of raphe forms potentially matching this number. Three lineages-the Bacillariales, Rhopalodiales, and Surirellales-possess a complex and presumably highly derived raphe that is physically separated from the cell interior, most often by a set of siliceous braces. Because the relationship among these three lineages is unclear, the number of origins of the canal raphe system and the homology of it and its constitutive parts among these lineages, is equally unclear. We reconstructed the phylogeny of raphid pennate diatoms and included, for the first time, members of all three canal raphid diatom lineages, and used the phylogeny to test specific hypotheses about the origin of the canal raphe. The canal raphe appears to have evolved twice, once in the common ancestor of Bacillariales and once in the common ancestor of Rhopalodiales and Surirellales, which form a monophyletic group in our analyses. These results recommend careful follow-up morphogenesis studies of the canal raphe in these two lineages to determine the underlying developmental basis for this remarkable case of parallel evolution.     

DIATOM SILICA BIOMINERALIZATION: AT NANOSCALE LEVEL A CHEMICALLY UNIFORM PROCESS

Using a high-brilliance synchrotron X-ray source, combined small- and wide-angle X-ray scattering (SAXS and WAXS) was applied to study nanoscale characteristics, in particular pore size in the range of 3 to 65 nm, of a variety of unialgal cultures of centric and pennate diatoms, and of mixed diatom populations sampled in the field. Results of scattering analysis were compared with details of pore size, structure and orientation visible at the electron microscopic level. WAXS patterns did not reveal any crystalline phase or features of microcrystallinity (resolution 0.07 to 0.51 nm), which implies a totally amorphous character of the SiO₂ matrix of the frustule material. SAXS data (resolution 3 to 65 nm) provided information on geometry, size, and distribution of pores in the silica. Overall, two pore regions were recognized that were common to the silica of all samples: the smallest (d less than 10 nm) regularly spaced and shaped spherically, the larger (up to 65 nm) being cylinders or slits. Apparently, at a nanoscale level diatomaceous silica is quite homologous among species, in agreement with the chemical principles of silica polymerization under the conditions of pH and precursor concentrations inside the silicon deposition vesicle. The final frustule "macro"-morphology is of course species-specific, being determined genetically. Synthetically-derived MCM-type silicas have a similarly organized pore distribution in an amorphous silica matrix as we found in all diatom species studied. We therefore suggest that organic molecules of a kind used as structure-directing agents to produce these artificial silicas play a role in the nucleation of the silica polymerization reaction and the shaping of pore morphology inside the silicon deposition vesicle of diatoms. Structure-directing molecules now await isolation from the SDV, followed by identification and characterisation by molecular techniques.     

THE ROLE OF DIATOMS IN STROMATOLITE GROWTH: TWO EXAMPLES FORM MODERN FRESHWATER SETTINGS1

Some modern laminated find calcified stromatolitic structures are partially or completely formed by eukaryotes. Diatom populations in freshwater environments with elevated ionic concentrations contribute to calcite precipitation, and the formation of distinctive mineral-rich stromatolitic laminae. Two types of stromatolite-forming diatom populations were observed. In the first example, in stromatolites growing on a quarry ledge near Laegerdorf, North Germany, calcite crystals with biogenic imprints form around polysaccharide stalks of the diatom Gomphonema olivaceum var. calcarea (Cleve) Cleve-Euler. These individually precipitated crystals eventually become cemented together in layers, forming rigid, laminated stromatolitic deposits which drape over the quarry ledge. In the second example, in stromatolites forming in a shallow stream near Cuatro Ciénegas, Coahuila, Mexico, diatomaceous laminae also form by the accumulation of carbonate particles in a matrix of diatoms and their extracellular polysaccharide products. These laminae become thick enough to drape over individual stromatolite heads. The diatoms responsible for these deposits are Amphora aff. A. katii Selva, Nitzschia denticula Grun., and six other species. At Cuatro Ciénegas, in addition to the diatomaceous laminae, carbonate-rich cyanobacterial layers, dominated by two cyanobacterial species with different fabrics and porosities, are also present and contribute substantially to the growth of the stromatolites. In both the Laegerdorf and Cuatro Ciénegas examples, entire stromatolites or thick laminations on stromatolites are built by a small number of diatom species which produce copious amounts of extracellular stalk, gel, and sheath material, a propertuy they share with cyanobacterial stromatolite builders.     

Conservative motif CMLD in silicic acid transport proteins of diatom algae

Sequencing of fragments of genes coding for silicic acid transport (SIT) proteins of diatoms of evolutionary distant classes (centric Chaetoceros muelleri Lemmermann, pennate araphid Synedra acus Kützing, pennate raphid Phaeodactylum tricornutum Bohlin, and pennate with keeled raphe system Cylindrotheca fusiformis Reimann et Lewin), revealed the presence in these proteins of a conservative amino acid motif CMLD. Hydropathy profiles suggest that CMLD occupies a position between two transmembrane strands which do not contain lysine and arginine residues. The two strands are good candidates for the role of the channel along which transport of silicic acid occurs. CMLD is a rare motif. Diatoms are known to need Zn2+ for the incorporation of silica. Presumably, CMLD is the site of Zn2+ binding of SITs. We found that the growth of diatoms is inhibited by a negatively charged alkylating reagent 5-(2-iodoacetamidoethyl)aminonaphtalene-1-sulfonic acid which cannot penetrate through the cell membrane. Cysteine of CMLD can be a target of this reagent. Synthetic peptide NCMLDY forms a complex with Zn2+, as revealed by the fact that the ion considerably reduces the rate of alkylation of the peptide.     

SILICEOUS SCALE PRODUCTION IN CHRYSOPHYTE AND SYNUROPHYTE ALGAE. I. EFFECTS OF SILICA-LIMITED GROWTH ON CELL SILICA CONTENT,SCALE MORPHOLOGY,AND THE CONSTRUCTION OF THE SCALE LAYER OF SYNURA PETERSENII1

The cells of synurophyte flagellates (algal class Synurophyceae, formerly included in the Chrysophyceae) are enclosed within a regularly imbricate layer of ornamented siliceous scales. Scale morphology is of critical taxonomic importance within this group of algae, and the scales are valuable indicator microfossils in paleolimnological studies. The data presented here demonstrate that scale morphology and the integrity of the scale layer can exhibit extreme variability in culture as a function of the cellular quota of silica under silica-limited growth. Silica-limited, steady-state populations of the colonial flagellate Synura petersenii Korsh. were maintained over a range of specific growth rates (μ= 0.11–0.69 days^?1) and silica cell quotas (Q_si= 0.13–2.40 pmoles Si · cell¹). Scale morphology and the organization of the scale layer became increasingly aberrant as silica stress increased. Under severe stress, scale deposition was completely suppressed so that cells appeared scale-free. This depression of scale deposition was reversible; populations of silica-starved, scale-free cells rapidly regenerated new scale layers when placed in batch culture and spiked with dissolved silica. During recovery from silica stress, cell division was repressed for 24 h while mean cell silica quota increased 25-fold. The first new scales appeared within 2 h after the silica addition, and development of the new scale layer proceeded in an approximately synchronous manner, residting in normal scale layers on virtually all cells after 48 h of recovery in Sirich medium. Silica content of silica-replete Synura cells is comparable to freshwater diatoms of siynilar size, but Synura has much greater potential quota variability than diatoms and no apparent threshold silica requirement. Silica-limited growth kinetics and competition between diatoms and Synura for silica are discussed. The results suggest that morphological variability of siliceous scales in natural populations of synurophyte flagellates may result from silica stress and that the experimental approach developed here has great potential value as a means for circumscribing ecotypic variation in scale morphology. Results also demonstrate that scale production can be uncoupled from cell division, suggesting that cell cycle regulation of silica biomineralization in the Synurophyceae may be fundamentally different from that of diatoms (algal class Bacillariophyceae). This experimental system has application in the future study of the intracellular membrane systems and the regulatory processes involved in silica biomineralization.     

Diversity of mineral cell coverings and their formation processes: a review focused on the siliceous cell coverings

Mineral cell coverings are found in various protists. Some macroalgae accumulate calcium carbonate in the intercellular space, and some unicellular organisms use calcium carbonate or silica for the construction of loricas, scales, and frustules. Diatoms are representatives of those utilizing silica for the material of the cell covering called a frustule. The development of the frustule is initiated in a silica-deposition vesicle (SDV), which occurs just beneath the plasma membrane and, subsequently, the silicified cell covering expands its area, following the expansion of the SDV from valve face to valve mantle. Sequential valve development with whole valves is reviewed in several diatoms placed in different phylogenetic positions. Every diatom commences its valve formation from its pattern center and then develops by means of individual procedures. The results indicate that the valve development reflects the phylogeny of diatoms. In addition, recent progress in silica biomineralization is briefly reviewed, and the phylogeny of ability concerning siliceous cell covering formation is inferred. Electronic Publication     

Ultrastructure of the Interaction of Cells of Pseudomonas phaseolicola with Cell Walls of a Resistant and Susceptible Bean Cultivar

Cells of Pseudomonas phaseolicola were observed entrapped against plant cell walls in both susceptible (Red Kidney) and resistant (Red Mexican) cultivars of French bean (Phaseolus vulgaris). After staining of samples with ruthenium red for electron microscopy pectic polysaccharide within plant cell walls became particularly well contrasted as did fibrillar material connecting bacteria to the plant cell walls. In places this fibrillar material appeared to emanate from the pectic polysaccharide in the plant cell wall, and the plant cell wall surface was eroded at such points. Ruthenium red also stains acidic, bacterial extracellular polysaccharide (EPS) and some of the fibrillar material in intercellular spaces is probably from this source. It is possible that bacteria become attached through an interaction between EPS and Pectic polysaccharide in plant cell walls.     

Heterogeneity of silica and glycan-epitope distribution in epidermal idioblast cell walls in Adiantum raddianum laminae

Laminae of Adiantum raddianum Presl., a fern belonging to the family Pteridaceae, are characterised by the presence of epidermal fibre-like cells under the vascular bundles. These cells were thought to contain silica bodies, but their thickened walls leave no space for intracellular silica suggesting it may actually be deposited within their walls. Using advanced electron microscopy in conjunction with energy dispersive X-ray microanalysis we showed the presence of silica in the cell walls of the fibre-like idioblasts. However, it was specifically localised to the outer layers of the periclinal wall facing the leaf surface, with the thick secondary wall being devoid of silica. Immunocytochemical experiments were performed to ascertain the respective localisation of silica deposition and glycan polymers. Epitopes characteristic for pectic homogalacturonan and the hemicelluloses xyloglucan and mannan were detected in most epidermal walls, including the silica-rich cell wall layers. The monoclonal antibody, LM6, raised against pectic arabinan, labelled the silica-rich primary wall of the epidermal fibre-like cells and the guard cell walls, which were also shown to contain silica. We hypothesise that the silicified outer wall layers of the epidermal fibre-like cells support the lamina during cell expansion prior to secondary wall formation. This implies that silicification does not impede cell elongation. Although our results suggest that pectic arabinan may be implicated in silica deposition, further detailed analyses are needed to confirm this. The combinatorial approach presented here, which allows correlative screening and in situ localisation of silicon and cell wall polysaccharide distribution, shows great potential for future studies.     

Spermatogenesis and auxospore structure in the multipolar centric diatom Hydrosera

Spermatogenesis and auxospore development were studied in the freshwater centric diatom Hydrosera triquetra. Spermatogenesis was unusual, lacking depauperating cell divisions within the spermatogonangium. Instead, a series of mitoses occurred within an undivided cell to produce a multinucleate plasmodium with peripheral nuclei, which then underwent meiosis. 32 or 64 sperm budded off from the plasmodium leaving a large residual cell containing all the chloroplasts. Similar development apparently occurs in Pleurosira, Aulacodiscus, and Guinardia, these being so distantly related that independent evolution of plasmodial spermatogenesis seems likely. After presumed fertilization, the Hydrosera egg cell expanded distally to form a triangular end part. However, unlike in other triangular diatoms (Lithodesmium, Triceratium), the development of triradiate symmetry was not controlled by the “canonical” method of a perizonium that constrains expansion to small terminal areas of the auxospore wall. Instead, the auxospore wall lacked a perizonium and possessed only scales and a dense mat of thin, apparently entangled strips of imperforate silica. No such structures have been reported from any other centric diatoms, the closest analogs being instead the incunabular strips of some raphid diatoms (Nitzschia and Pinnularia). Whether these silica structures are formed by the normal method (intracellular deposition within a silica deposition vesicle) is unknown. As well as being more rounded than vegetative cells, the initial cell is aberrant in its structure, since it has a less polarized distribution of the “triptych” pores characteristic of the species.