A STRESS‐INDUCED PROTEIN ASSOCIATED WITH THE GIRDLE BAND REGION OF THE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYTA)1

We report the characterization of a cell‐surface protein isolated from the centric diatom Thalassiosira pseudonana Hasle and Heimdal. This protein has an apparent molecular weight of 150 kDa, is highly acidic, and is intimately associated with the cell wall. Although originally identified in cells experiencing copper toxicity, it is also induced by silicon and iron limitation but not by phosphate or nitrate limitation. Using immunofluorescence techniques, the 150‐kDa protein was localized to the girdle band region and covered the elongated girdle band region of morphologically aberrant cells suffering from copper toxicity. Although having biochemical similarities to girdle band associated proteins identified in pennate diatoms known as pleuralins, the 150‐kDa protein is not a sequence homolog and is predicted to have a number of unique features, such as chitin binding domains and a possible RGD cell attachment motif. Results presented here suggest that this protein is normally cell cycle regulated and may be involved in stabilizing cells during the division process.     

THE POLYMORPHIC DIATOM PHAEODACTYLUM TRICORNUTUM: ULTRASTRUCTURE OF ITS MORPHOTYPES1,2

The ultrastructure of the oval, fusiform and triradiate morphotypes of Phaeodactylum tricornutum Bohlin is described. The organization and structure of the cytoplasmic organelles is similar in all three morphotypes, except that the vacuoles occupy the extra volume created by the arms of the fusiform and triradiate cells. The frustule in fusiform and triradiate cells is organic; in the oval type it may be organic or one of the valves may have a silica frustule surrounded by an organic wall. In all cells, the organic cell wall has up to 10 silica bands (13 nm wide) embedded in its surface in the girdle region, lacks girdle bands, and has an outer corrugated cell wall layer, except in the girdle region. Cell division, organic wall formation and silica deposition are described in detail. Four types of oval cells are also described. The relation to other diatoms is discussed.     

Silicon uptake and metabolism of the marine diatom Thalassiosira pseudonana: Solid-state 29Si NMR and fluorescence microscopic studies

Uptake and metabolism of silicon by diatoms are studied by the combined use of solid-state 29Si NMR spectroscopy and confocal laser fluorescence microscopy especially with respect to the presence and nature of an intracellular silicon-storage pool. Cells of the marine diatom Thalassiosira pseudonana were synchronized by silicon starvation and frozen without any freeze-drying or chemical treatment in order to analyze integer and unmodified diatoms. The frozen samples were investigated by solid-state 29Si NMR spectroscopy to identify potential silica precursors. The developmental state of the cell culture and the formation of new siliceous girdle bands and valves were monitored by laser fluorescence microscopic studies. A comparison of fluorescence microscopic and NMR data allows the assignment of NMR spectra to the various developmental stages of the dividing diatom cells. A detailed analysis of solid-state 29Si NMR spectra suggests that the silicon-storage pool-if present-consists of four-coordinated, condensed silicon; possibly a silica sol.     

Ontogenetic analysis of siliceous cell wall formation in Triparma laevis f. inornata (Parmales,Stramenopiles)

Triparma laevis f. inornata is a unicellular alga belonging to the Bolidophyceae, which is most closely related to diatoms. Like diatoms, T. laevis f. inornata has a siliceous cell wall. The cell wall of T. laevis f. inornata consists of four round plates (three shields and one ventral plate) and one dorsal and three girdle plates. But, unlike diatoms, T. laevis f. inornata cells can grow when concentrations of silica are depleted. We took advantage of this ability, using TEM to study the ontogeny of the siliceous plate, pattern center formation, and development. Two types of pattern centers (annulus and sternum) were observed in the early and middle stage of plate formation. During their formation, the annuli were initially crescent‐shaped but eventually their ends fused to make a ring. Only outward silica deposition of the branching ribs occurred on the growing annulus until it became a ring, resulting in an unfilled circle inside the annulus. The pattern center of the shield plate was always an annulus, but in ventral plates both annulus and sternum were observed. The annuli and sterna in T. laevis f. inornata round plates were very similar to the annuli and sterna in diatom valves. These results suggested that the round plates of Parmales are homologous to diatom valves. This information on the plate ontogeny of T. laevis f. inornata provides new insights into the evolution of the siliceous cell wall in the Parmales and diatoms.     

Thalassiosira pseudonana (Cyclotella nana) (Hustedt) Hasle et Heimdal (Bacillariophyceae): A genetically tractable model organism for studying diatom biology,including biological silica formation

In 2004, Thalassiosira pseudonana was the first eukaryotic marine alga to have its genome sequenced. Since then, this species has quickly emerged as a valuable model species for investigating the molecular underpinnings of essentially all aspects of diatom life, particularly bio-morphogenesis of the cell wall. An important prerequisite for the model status of T. pseudonana is the ongoing development of increasingly precise tools to study the function of gene networks and their encoded proteins in vivo. Here, we briefly review the current toolbox for genetic manipulation, highlight specific examples of its application in studying diatom metabolism, and provide a peek into the role of diatoms in the emerging field of silica biotechnology.     

MICROMORPHOGENESIS DURING DIATOM WALL FORMATION PRODUCES SILICEOUS NANOSTRUCTURES WITH DIFFERENT PROPERTIES1

Micromorphogenesis within the silica deposition vesicle (SDV) of the diatom Pinnularia viridis (Nitzsh) Ehrenb. resulted in distinct silica nanostructures and layers within forming valves and girdle bands. These siliceous components were similarly disclosed following alkaline etching of mature valves/girdle bands, where their different susceptibilities to dissolution over time resulted from apparent differences in silica density and/or chemistry. The bulk of silica appeared to be deposited at the interface of the forming valve or girdle band with the silicalemma and occurred by the outward expansion of microfibrils of silica that aligned perpendicularly to the silicalemma. Microfibrils originated from both sides of the “silica lamella,” the first nanostructure formed within the SDV, and several silica species of distinct nanostructure and density resulted, including distinctive inner and outermost silica “coverings” of mature valves/girdle bands and the central and terminal nodules. Not all silica deposition and micromorphogenesis occurred in contact with the expanding silicalemma, but was somehow directed within the SDV cavity, and resulted in the distinct silica layers that lined the raphe fissures and poroids. Following alkaline etching, the inner surfaces of valves/girdle bands, as well as the silica layers lining the raphes, poroids, and slits, were determined to be significantly more resistant to alkaline etching than the exterior surfaces, while the outer silica coating and the nodules were quickly dissolved. The processes of micromorphogenesis must have exerted precise control over the chemical nature of the silica formed at different positions within the SDV and affected the overall structure and function of the diatom wall.     

Dynamics of silica cell wall morphogenesis in the diatom Cyclotella cryptica: Substructure formation and the role of microfilaments

Diatoms are unicellular algae that make cell walls out of silica with highly ornate features on the nano- to microscale. The complexity and variety of diatom cell wall structures exceeds those possible with synthetic materials chemistry approaches. Understanding the design and assembly processes involved in diatom silicification should provide insight into patterning on the unicellular level, and information for biomimetic approaches for materials synthesis. In this report we examine the formation of distinct cell wall structures (valves and girdle bands) in the diatom Cyclotella cryptica by high resolution imaging using SEM, AFM, and fluorescence microscopy. Special attention was paid to imaging structural intermediates, which provided insight into the underlying design and assembly principles involved. Distinct stages in valve formation were identified, indicating a transition from a fractally organized structure to a dynamic pathway-dependent process. Substructures in the valves appeared to be pre-positioned prior to complete silicification, suggesting that organics responsible for these structures were pre-assembled and put in place. Microtubules and microfilamentous actin play significant roles in the positioning process, and actin is also important in the pathway-dependent expansion of the front of silicification. Our results indicate that even though all silica structures in C. cryptica are made of assemblies of nanoparticulate silica, control of meso- and microscale structure occurs on a higher order. It is apparent that diatoms integrate bottom up and top down control and synthesis mechanisms to form the diversity of structures possible.     

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.     

Dissection of the frustules of the diatom Synedra acus under the action of picosecond impulses of submillimeter laser irradiation

Diatom algae realize highly intriguing processes of biosynthesis of siliceous structures in living cells under moderate conditions. Investigation of diatom physiology is complicated by frustule (siliceous exoskeleton). Frustules consist of valves and girdle bands which are adhered to each other by means of organic substances. Removal of the frustule from the lipid membrane of diatom cells would open new possibilities for study of silicon metabolism in diatoms. We found that submillimeter laser irradiation produced by a free-electron laser causes splitting of diatom frustules without destruction of cell content. This finding opens the way to direct study of diatom cell membrane and to isolation of cell organelles, including silica deposition vesicles. We suppose that the dissection action of the submillimeter irradiation results from unusual ultrasonic waves produced by the short (30–100 ps) but high-power (1 MW) terahertz laser impulses at 5.6 MHz frequency.     

Triacylglycerol accumulation and profiling in the model diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum (Baccilariophyceae) during starvation

Although substantial economic barriers exist, marine diatoms such as Thalassiosira pseudonana and Phaeodactylum tricornutum hold promise as feedstock for biodiesel because of their ability to manufacture and store triacylglycerols (TAGs). The recent sequencing of these two marine diatom genomes by the United States Department of Energy Joint Genome Institute and the development of improved systems for genetic manipulation should allow a more systematic approach to understanding and maximizing TAG production. However, in order to best utilize these genomes and genetic tools, we must first gain a deeper understanding of the nutrient-mediated regulation of TAG anabolism. By determining both the yield and molecular species distribution of TAGs we will, in the future, be able to fully characterize the effects of genetic manipulation. Here, we lay the groundwork for understanding TAG production in T. pseudonana and P. tricornutum, as a function of nitrate and silicate depletion. Diatoms were starved of either nitrate or silicate, and TAGs were extracted with hexane from lyophilized samples taken at various time intervals following starvation. The timing of TAG production and the relative abundance of TAGs were estimated by fluorescence spectroscopy using Nile red and the total yield per biomass determined by gravimetric assay. TAGs were analyzed using thin layer chromatography, gas chromatography–mass spectrometry, and electrospray ionization mass spectrometry to identify the major TAG species produced during the growth curve. Under our conditions, the TAG yield from T. pseudonana is about 14–18% of total dry weight. The TAG yield from P. tricornutum is about 14% of total dry weight. Silicate-starved T. pseudonana accumulated an average of 24% more TAGs than those starved for nitrate; however, the chemotypes of the TAGs produced were generally similar regardless of the starvation condition employed.     

Frustule morphogenesis of raphid pennate diatom <Emphasis Type="Italic">Encyonema ventricosum</Emphasis> (Agardh) Grunow

Diatoms stand out among other microalgae due to the high diversity of species-specific silica frustules whose components (valves and girdle bands) are formed within the cell in special organelles called silica deposition vesicles (SDVs). Research on cell structure and morphogenesis of frustule elements in diatoms of different taxonomic groups has been carried out since the 1950s but is still relevant today. Here, cytological features and valve morphogenesis in the freshwater raphid pennate diatom Encyonema ventricosum (Agardh) Grunow have been studied using light and transmission electron microscopy of cleaned frustules and ultrathin sections of cells, and scanning electron and atomic force microscopy of the frustule surface. Data have been obtained on chloroplast structure: the pyrenoid is spherical, penetrated by a lamella (a stack of two thylakoids); the girdle lamella consists of several short lamellae. The basic stages of frustule morphogenesis characteristic of raphid pennate diatoms have been traced, with the presence of cytoskeletal elements near SDVs being observed throughout this process. Degradation of the plasmalemma and silicalemma is shown to take place when the newly formed valve is released into the space between sister cells. The role of vesicular transport and exocytosis in the gliding of pennate diatoms is discussed.     

INTER‐ AND INTRASPECIFIC RELATIONSHIPS BETWEEN NUCLEAR DNA CONTENT AND CELL SIZE IN SELECTED MEMBERS OF THE CENTRIC DIATOM GENUS THALASSIOSIRA (BACILLARIOPHYCEAE)1

The enormous species diversity of diatoms correlates with the remarkable range of cell sizes in this group. Nuclear DNA content relates fundamentally to cell volume in other eukaryotic cells. The relationship of cell volume to G1 DNA content was determined among selected members of the genus Thalassiosira, one of the most species‐rich and well‐studied centric diatom genera. Both minimum and maximum species‐specific cell volume correlated positively with G1 DNA content. Phylogeny based on 5.8 S and ITS rDNA sequences indicated that multiple changes in G1 DNA content and cell volume occurred in Thalassiosira evolution, leading to a 1,000‐fold range in both parameters in the group. Within the Thalassiosira weissflogii (Grunow) G. A. Fryxell et Grunow species complex, G1 DNA content varied 3‐fold: differences related to geographic origin and time since isolation; doubling and tripling of G1 DNA content occurred since isolation in certain T. weissflogii isolates; and subcultures of T. weissflogii CCMP 1336 diverged in DNA content by 50% within 7 years of separation. Actin, β‐tubulin, and Spo11/TopVIA genes were selected for quantitative PCR estimation of haploid genome size in subclones of selected T. weissflogii isolates because they occur only once in the T. pseudonana Hasle et Heimdal genome. Comparison of haploid genome size estimates with G1 DNA content suggested that the most recent T. weissflogii isolate was diploid, whereas other T. weissflogii isolates appeared to be polyploid and/or aneuploid. Aberrant meiotic and mitotic cell divisions were observed, which might relate to polyploidization. The structural flexibility of diatom genomes has important implications for their evolutionary diversification and stability during laboratory maintenance.     

Ultrastructure of diatom <Emphasis Type="Italic">Synedra acus</Emphasis> subsp. <Emphasis Type="Italic">radians</Emphasis> as revealed by transmission electron microscopy after mild silica dissolution

A diatom Synedra acus subsp. radians (Kotz.) Skabitsch. has been studied by transmission electron microscopy. Examination of ultrathin sections demonstrated that silica dissolution in ammonium fluoride pH 5 under mild conditions leaves the key ultrastructural elements intact. The ultrastructure and arrangement of the cell organelles was studied during ontogeny. Silicalemma-surrounded silica deposition vesicles (SDVs) with maturating daughter valves and forming girdle bands have been identified. This method of SDV visualization offers considerable advantages over the standard approach without silica dissolution.     

MOLECULAR GENETIC MANIPULATION OF THE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1

Here, we describe the first system for genetic transformation of Thalassiosira pseudonana (Hustedt) Hasle et Heimdal, the only diatom for which a complete genome sequence is presently available. This method is based on microparticle bombardment followed by selection of transformants using the antibiotic nourseothricin. It exhibits the highest transformation efficiency compared with transformation systems for other diatom species. To achieve the high transformation efficiency, it is important to allow recovery of the bombarded T. pseudonana cells in non‐selective suspension culture before spreading on nourseothricin containing agar plates. It is demonstrated that T. pseudonana is readily susceptible to co‐transformation allowing for the simultaneous introduction of a non‐selective gene together with the selection marker gene. Both introduced genes are stably inherited even in the absence of the antibiotic selection pressure. We have developed two T. pseudonana‐specific expression vectors that can drive constitutive expression (vector pTpfcp) and inducible expression (vector pTpNR) of introduced genes. In combination with the available genome data the T. pseudonana transformation system is expected to provide a powerful tool for functional genomics in diatoms.     

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.     

Genome-wide analysis of the diatom cell cycle unveils a novel type of cyclins involved in environmental signaling

Background

Despite the enormous importance of diatoms in aquatic ecosystems and their broad industrial potential, little is known about their life cycle control. Diatoms typically inhabit rapidly changing and unstable environments, suggesting that cell cycle regulation in diatoms must have evolved to adequately integrate various environmental signals. The recent genome sequencing of Thalassiosira pseudonana and Phaeodactylum tricornutum allows us to explore the molecular conservation of cell cycle regulation in diatoms.

Results

By profile-based annotation of cell cycle genes, counterparts of conserved as well as new regulators were identified in T. pseudonana and P. tricornutum. In particular, the cyclin gene family was found to be expanded extensively compared to that of other eukaryotes and a novel type of cyclins was discovered, the diatom-specific cyclins. We established a synchronization method for P. tricornutum that enabled assignment of the different annotated genes to specific cell cycle phase transitions. The diatom-specific cyclins are predominantly expressed at the G1-to-S transition and some respond to phosphate availability, hinting at a role in connecting cell division to environmental stimuli.

Conclusion

The discovery of highly conserved and new cell cycle regulators suggests the evolution of unique control mechanisms for diatom cell division, probably contributing to their ability to adapt and survive under highly fluctuating environmental conditions.     

Characterizing the silica deposition vesicle of diatoms

Summary The present study on a centric diatom,Ditylum brightwellii, includes two parts: detection of sugars in the silica deposition vesicle (SDV) with lectins and labeling the developing siliceous cell wall in the SDV with rhodamine 123. Cells with developing valves are treated with SDS to remove all the cytoplasmic contents, then either stained with fluorescein labeled lectins or thin-sectioned and stained with colloidal gold labeled lectins. The results show that mannose is part of the organic matrix in the SDV. Rhodamine 123, a non-toxic fluorescent laser dye, enters the cell immediately and is trapped in the SDV probably by the high reducing potential of the SDV. Silica is co-deposited with rhodamine 123 in the SDV, and the resulting valves and girdle bands become fluorescent. Implications of this study for the mechanism of silicification are discussed.Abbreviation SDV Silica deposition vesicle