Michael R. Gretz

Extracellular Matrix Assembly in Diatoms (Bacillariophyceae) (I. A Model of Adhesives Based on Chemical Characterization and Localization of Polysaccharides from the Marine Diatom Achnanthes longipes and Other Diatoms)

Extracellular adhesives from the diatoms Achnanthes longipes, Amphora coffeaeformis, Cymbella cistula, and Cymbella mexicana were characterized by monosaccharide and methylation analysis, lectin-fluorescein isothiocyanate localization, and cytochemical staining. Polysaccharide was the major component of adhesives formed during cell motility, synthesis of a basal pad, and/or production of a highly organized shaft. Hot water-insoluble/hot 0.5 M NaHCO3-soluble anionic polysaccharides from A. longipes and A. coffeaeformis adhesives were primarily composed of galactosyl (64–70%) and fucosyl (32–42%) residues. In A. longipes polymers, 2,3-, t-, 3-, and 4-linked/substituted galactosyl, t-, 3-, 4-, and 2-linked fucosyl, and t- and 2-linked glucuronic acid residues predominated. Adhesive polysaccharides from C. cistula were EDTA-soluble, sulfated, consisted of 83% galactosyl (4-, 4,6-, and 3,4-linked/substituted) and 13% xylosyl (t-, 4f/5p-, and 3p-linked/substituted) residues, and contained no uronosyl residues. Ulex europaeus agglutinin uniformly localized [alpha](1,2)-L-fucose units in C. cistula and Achnanthes adhesives formed during motility and in the pads of A. longipes. D-Galactose residues were localized throughout the shafts of C. cistula and capsules of A. coffeaeformis. D-Mannose and/or D-glucose, D-galactose, and [alpha](t)-L-fucose residues were uniformly localized in the outer layers of A. longipes shafts by Cancavalia ensiformis, Abrus precatorius, and Lotus tetragonolobus agglutinin, respectively. A model for diatom cell adhesive structure was developed from chemical characterization, localization, and microscopic observation of extracellular adhesive components formed during the diatom cell-attachment process.

Extracellular matrix assembly in diatoms (Bacillariophyceae). V. Environmental effects on polysaccharide synthesis in the model diatom, Phaeodactylum tricornutum

The effects of phosphate (P) limitation, varying salinity (5–65 psu), and solid media growth conditions on the polysaccharides produced by the model diatom, Phaeodactylum tricornutum Bohlin were determined. Sequential extraction was used to separate polymers into colloidal (CL), colloidal extracellular polymeric substances (cEPS), hot water soluble (HW), hot bicarbonate soluble (HB), and hot alkali (HA) soluble fractions. Media‐soluble polymers (CL and cEPS) were enriched in 4‐linked mannosyl, glucosyl, and galactosyl residues as well as terminal and 3‐linked xylosyl residues, whereas HW polymers consisted mainly of 3‐linked glucosyl as well as terminal and 2,4‐linked glucuronosyl residues. The HB fraction was enriched in terminal and 2‐linked rhamnosyl residues derived from the mucilage coating solubilized by this treatment. Hot alkali treatment resulted in the complete dissolution of the frustule releasing 2,3‐ and 3‐linked mannosyl residues. The fusiform morphotype predominated in standard and P‐limited cultures and cultures subjected to salinity variations, but growth on solid media resulted in an enrichment of the oval morphotype. The proportion and linkages of 15 residues, including neutral, uronic acid, and O‐methylated sugars, varied with environmental conditions. P limitation and salinity changes resulted in 1.5‐ to 2.5–fold increase in carbohydrate production, with enrichment of highly branched/substituted and terminal rhamnose, xylose, and fucose as well as O‐methylated sugars, uronic acids, and sulfate. The increased deoxy‐ and O‐methylated sugar content under unfavorable environments enhances the hydrophobicity of the polymers, whereas the anionic components may play important roles in ionic cross‐linking, suggesting that these changes could ameliorate the effects of salinity or P‐stress and that these altered polysaccharide characteristics may be useful as bioindicators for environmental stress.

The structure and biochemistry of charophycean cell walls: I. Pectins of Penium margaritaceum

Summary.Plant cell walls are essential for proper growth, development, and interaction with the environment. It is generally accepted that land plants arose from aquatic ancestors which are sister groups to the charophycean algae (i.e., Streptophyta), and study of wall evolution during this transition promises insight into structure–function relationships of wall components. In this paper, we explore wall evolutionary history by studying the incorporation of pectin polymers into cell walls of the model organism Penium margaritaceum, a simple single-cell desmid. This organism produces only a primary wall consisting of three fibrillar or fibrous layers, with the outermost stratum terminating in distinct, calcified projections. Extraction of isolated cell walls with trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid yielded a homogalacturonan (HGA) that was partially methyl esterified and equivalent to that found in land plants. Other pectins common to land plants were not detected, although selected components of some of these polymers were present. Labeling with specific monoclonal antibodies raised against higher-plant HGA epitopes (e.g., JIM5, JIM7, LM7, 2F4, and PAM1) demonstrated that the wall complex and outer layer projections were composed of the HGA which was significantly calcium complexed. JIM5 and JIM7 labeling suggested that highly methyl esterified HGA was secreted into the isthmus zone of dividing cells, the site of active wall secretion. As the HGA was displaced to more polar regions, de-esterification in a non-blockwise fashion occurred. This, in turn, allowed for calcium binding and the formation of the rigid outer wall layer. The patterning of HGA deposition provides interesting insights into the complex process of pectin involvement in the development of the plant cell wall.

Extracellular Matrix Assembly in Diatoms (Bacillariophyceae) (II. 2,6-Dichlorobenzonitrile Inhibition of Motility and Stalk Production in the Marine Diatom Achnanthes longipes).

The cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) and the DCB analogs 2-chloro-6-fluorobenzonitrile, 3-amino-2,6-dichlorobenzonitrile, and 5-dimethylamino-naphthalene-1-sulfonyl-(3-cyano-2, 4-dichloro)aniline (DCBF) inhibited extracellular adhesive production in the marine diatom Achnanthes longipes, resulting in a loss of motility and a lack of permanent adhesion. The effect was fully reversible upon removal of the inhibitor, and cell growth was not affected at concentrations of inhibitors adequate to effectively interrupt the adhesion sequence. Video microscopy revealed that the adhesion sequence was mediated by the export and assembly of polymers, and consisted of initial attachment followed by cell motility and eventual production of permanent adhesive structures in the form of stalks that elevated the diatom above the substratum. A. longipes adhesive polymers are primarily composed of noncellulosic polysaccharides (B.A. Wustman, M.R. Gretz, and K.D. Hoagland [1997] Plant Physiol 113: 1059–1069). These results, together with the discovery of DCB inhibition of extracellular matrix assembly in noncellulosic red algal unicells (S.M. Arad, O. Dubinsky, and B. Simon [1994] Phycologia 33: 158–162), indicate that DCB inhibits synthesis of noncellulosic extracellular polysaccharides. A fluorescent probe, DCBF, was synthesized and shown to inhibit adhesive polymer production in the same manner as DCB. DCBF specifically labeled an 18-kD polypeptide isolated from a membrane fraction. Inhibition of adhesion by DCB and its analogs provides evidence of a direct relationship between polysaccharide synthesis and motility and permanent adhesion.

MOVEMENT MODALITIES AND RESPONSES TO ENVIRONMENTAL CHANGES OF THE MUDFLAT DIATOM CYLINDROTHECA CLOSTERIUM (BACILLARIOPHYCEAE)1

Cylindrotheca closterium (Ehrenberg) Reiman et Lewin is a raphid diatom widely distributed in mudflat assemblages. Video microscopy showed various movement modalities defined as smooth and corkscrew gliding, pirouette, pivot, rock and roll, rollover, and simultaneous pirouette and gliding. Z‐axis projection analysis of images revealed a unique gliding motif with corkscrew motions, which may have important ecological implications for C. closterium movement in muds. The general response to salinity alteration was a decrease in gliding movements with a concomitant increase in other modalities listed above. Short‐term responses to salinity change include dramatic alteration in modalities in hypo‐saline conditions and cessation of motility in extreme hyper‐saline environments. Modality changes were rapid and occurred within 5 s in response to hyper‐saline conditions. Hypo‐ or hyper‐saline conditions resulted in decreased gliding speed in standard media. Five‐ and 15‐day acclimation to salinity changes resulted in a progressive reduction in gliding movement, increased non‐gliding modalities and increased cell aggregation. Aggregation in hypo‐saline conditions was accompanied by a large increase in the polymer extracted by hot bicarbonate‐ and ethylenediamine tetraaceticacid‐ fractions of extracellular polymeric substance (EPS), the polymers of which have been implicated in cell attachment/motility phenomena. The monosaccharide profiles of these fractions were altered in response to hypo‐saline conditions. In general, monosaccharide profiles showed increased diversity upon cessation of motility and aggregation of cultures. The movement responses of C. closterium in response to environmental changes, accompanied by modifications in EPS, may form part of an adaptive strategy to survive in mudflats and could be useful as bioindicators of environmental changes.

Extracellular matrix assembly in diatoms (Bacillariophyceae). iv. ultrastructure of Achnanthes longipes and Cymbella cistula as revealed by high-pressure freezing/freeze substituton and cryo-field emission scanning electron microscopy

Extracellular matrix (ECM) polymers secreted by the diatoms Achnanthes longipes Ag. and Cymbella cistula (Ehr.) Kirchn. completely encase the cell and are responsible for adhesion and other interactions with the external environment. To preserve details of the highly hydrophilic ECM in the native state and to preserve, with a high degree of fidelity, the intracellular structures involved in synthesis of extracellular polymers, we applied a suite of cryotechniques. The methods included high‐resolution visualization of surfaces using cryo‐field emission SEM (cryo‐FESEM) and preservation for TEM observation of thin sections by high‐pressure freezing (HPF) and freeze substitution (FS). The extracellular structures of diatoms plunge‐frozen in liquid ethane, etched at low temperature, and observed on a cryostage in the FESEM showed overall dimensions and shapes closely comparable to those observed with light microscopy. Cryo‐FESEM demonstrated the pervasive nature of the extracellular polymers and their importance in cell–substratum and cell–cell associations and revealed details of cell attachment processes not visible using other SEM techniques or light microscopy. The layer of ECM coating the frustule and entirely encapsulating cells of A. longipes and C. cistula was shown to have a significant role in initial cell adhesion and subsequent interaction with the environment. Trails of raphe‐associated ECM, generated during cell motility, were shown at high resolution and consist of anastomoses of coiled and linear strands. Cryo‐FESEM revealed a sheet‐like mucilage covering stalks. HPF/FS of A. longipes resulted in excellent preservation of intra‐ and extracellular structures comparable to previous reports for animals and higher plants and revealed several organelles not described previously. Three distinct vesicle types were identified, including a class closely associated with Golgi bodies and postulated to participate in formation of the extracellular adhesive structures. HPF/FS showed a number of continuous diatotepic layers positioned between the plasma membrane and the silicon frustule and revealed that extracellular adhesive extrusion through frustule pores during stalk production was closely related to the diatotepum. The stalks of A. longipes consist of highly organized, multilayered, fine fibrillar materials with an electron‐opaque layer organized as a sheath at the stalk periphery.

CELL-WALL DEVELOPMENT AND BIPOLAR GROWTH IN THE DESMID PENIUM MARGARITACEUM (ZYGNEMATOPHYCEAE, STREPTOPHYTA). ASYMMETRY IN A SYMMETRIC WORLD(1).

Cell‐wall (CW) development in the desmid Penium margaritaceum (Ehrenb.) Bréb. was studied using immunofluorescence labeling of living cells with the monoclonal antibodies (mAbs) JIM5 and JIM7, which recognize unesterified and methyl‐esterified homogalacturonan (HG), respectively. During cell expansion, HG was secreted in a high‐esterified form at a narrow band, called the HG secretion band or HGSB, at the isthmus or the polar tip of a daughter semicell. As newly secreted HG is displaced outward on the cell surface, deesterification and subsequent calcium (Ca2+)‐complexing occurred to yield a rigid covering. HG secretion and CW/cell expansion were reversibly inhibited by dark, brefeldin A (BFA), and incubation in 0.24–0.36 M sucrose but were not altered by treatment with actin/microfilament drugs. The HGSB was detected near the nucleus during most cell‐cycle events. Centrifugation displaced the nucleus away from the HGSB, but HG synthesis was not affected. HGSB activity was preceded by, and coordinated with, Calcofluor labeling, which suggests that cellulose production in CW/cell‐expansion sites was critical to expansion control. In many first‐cell‐division products, asymmetric patterning of HG was noted in the CW. These asymmetric patterns most likely were a result of timing mechanisms and displacement of the nucleus‐HGSB during the cell cycle.

Role of bacteria and bacterial exopolymer in the attachment of achnanthes longipes (Bacillariophyceae)

The attachment of diatoms to surfaces is an important and poorly understood step in the development of biofouling communities. Experiments were performed in vitro on a common fouling diatom (Achnanthes longipes) to determine the influence of the base material and bacterial conditioning on diatom attachment. The first series of experiments compared attachment of A. longipes to four different base materials, and the influence of a bacterial film on attachment to these materials. A. longipes preferentially attached to polystyrene, a hydrophobic surface, but was inhibited by the presence of a bacterial biofilm. On other surfaces, bacteria either facilitated or had no effect on algal attachment. The second series of experiments found no difference in the attachment of A. longipes to a surface covered with bacterial exopolymer compared to a surface with a film of living bacteria. Attachment of A. longipes was found to vary depending on the conditions under which the bacterial film developed and the species of b...

EFFECTS OF BROMIDE AND IODIDE ON STALK SECRETION IN THE BIOFOULING DIATOM ACHNANTHES LONGIPES (BACILLARIOPHYCEAE)1

Extracellular polymeric substance (EPS) secretion was examined in the stalked marine diatom Achnanthes longipes Ag. in defined medium. This common biofouling diatom exhibited an absolute requirement for bromide for stalk production and substratum attachment, whereas elevated iodide concentrations in the growth medium inhibited stalk formation and adhesion. Varying EPS morphologtes resulted from altering bromide and iodide levels: pads, stalk‐pads, stalks, and no EPS. Cells showed no differences in growth with bromide or iodide concentrations, indicating that they were not physiologically stressed under conditions that impaired EPS secretion. Cells grown in elevated iodide secreted significantly more soluble extracellular carbohydrate into the medium, suggesting that the EPS was soluble and unable to be polymerized into a morphologically distinct gel. By replacing sulfate with methionine, the diatom lost its ability to form stalks even in the presence of bromide, indicating that free sulphate may be required for proper cross‐linking of stalk polymers. Lotus‐FITC, a fluorescent‐tagged lectin, preferentially labeled the EPS and, thus, was used to visualize and quantify EPS secretion along a bromide gradient in conjunction with an image analysis system. This technique demonstrated a direct correlation between the amount of bromide present in the medium and the specific EPS morphology formed.

The extracellular polymeric substances of desmids (Conjugatophyceae, Streptophyta): chemistry, structural analyses and implications in wetland biofilms

S.N. Kiemle, D.S. Domozych and M.R. Gretz. 2007. The extracellular polymeric substances of desmids (Conjugatophyceae, Streptophyta): chemistry, structural analyses and implications in wetland biofilms. Phycologia 46: 617–627. DOI: 10.2216/06–97.1 Desmids represent a group of advanced green algae that are commonly found in biofilm communities of freshwater wetlands. Desmids secrete significant amounts of extracellular polymeric substances (EPS) that form an extensive mucilaginous sheath external to the cell wall and function in adhesion, gliding-based movements and ultimate ensheathment within the biofilm complex. We have initiated biochemical and structural analyses of the EPS of desmids isolated from biofilms from the southeastern Adirondack region of New York including Penium cylindris, Penium spirostriolatum, Cosmarium sp. 1, Cosmarium sp. 2, Pleurotaenium trabecula, Tetmemorus brebissonii, Netrium digitus, Netrium oblongum, Netrium interruptum and Netrium interruptum 2509. Cosmarium sp. 1 EPS appeared as a homogeneous sheath that encapsulated the cells, whereas P. trabecula EPS occurred as dispersed patches, and N. oblongum EPS was reticulated and striated. Polysaccharides were the major component of the EPS (52–76%), and lectin labeling revealed differences in polymer organization between saccoderm (labeled with Con-A, WGA and HPA) and placoderm (labeled with BS-I, PSA, UEA and HPA) desmids. Desmid EPS had significant uronic acid (3–29%) and protein (2–10%) content, and the polysaccharides were sulfated to varying degrees. Xylose and Fucose were the predominant monosaccharides with the major glycosyl linkages t-Xylp, 3,4-Fuc and t-Fuc. The unique EPS from N. oblongum was rich in galactose and uronic acid (29.3% w/w), extracellular polymers of P. spirostriolatum were composed predominantly of arabinose, and T. brebissonii EPS was enriched in glucose and galactose. EPS of P. trabecula and T. brebissonii was highly sulfated (10.2% and 14.7%, respectively). The EPS from New York and UTEX strains of N. interruptum were unique from each other. Overall, desmid EPS exhibits a conserved motif with the predominant component an anionic polysaccharide. The presence of deoxy-sugar subunits indicates the potential for hydrophobic interaction where anionic components may play important role in ionic cross-linking. The establishment of this baseline biochemistry provides the foundation for future dissection of core EPS molecules and associated functional groups and studies of EPS involvement in specific stages of biofilm development (e.g. adhesion).