Fucus Zygotes Research Articles

Vanadium-dependent haloperoxidase activity and phlorotannin incorporation into the cell wall during early embryogenesis of Fucus vesiculosus (Phaeophyceae)

ABSTRACT Vanadium-dependent haloperoxidases (V-HPO) are specific enzymes involved in the metabolism of reactive oxygen species in marine algae. These enzymes contribute to early embryogenesis of algae in the order Fucales, catalyzing the reactions which result in phlorotannin incorporation into the newly forming zygote cell wall and adhesive material. In this study we showed that early embryogenesis in Fucus vesiculosus is accompanied by synchronous changes of V-HPO activity, hydrogen peroxide content and content of cell-wall-associated phlorotannins. Phlorotannin incorporation into the cell wall of Fucus zygotes had already begun 1 h after fertilization, and this process was accompanied by rapid increase of H2O2 content and V-HPO activity. We suggest that altogether this initiates polymerization of phenolics and their cross-linking with the major cell wall constituent, alginic acid. Presumably, de novo phlorotannin biosynthesis is involved in this process, as GC-MS analysis showed dramatic changes in content of intracellular phloroglucinol and low-molecular-weight phlorotannins during the first 9 days of F. vesiculosus embryogenesis.

Dynamics of cell wall assembly during early embryogenesis in the brown alga Fucus.

Zygotes from Fucus species have been used extensively to study cell polarization and rhizoid outgrowth, and in this model system cell wall deposition aligns with the establishment of polarity. Monoclonal antibodies are essential tools for the in situ analysis of cell wall glycans, and here we report the characteristics of six monoclonal antibodies to alginates (BAM6-BAM11). The use of these, in conjunction with monoclonal antibodies to brown algal sulfated fucans, has enabled the study of the developmental dynamics of the Fucus zygote cell walls. Young zygotes are spherical and all alginate epitopes are deposited uniformly following cellulose deposition. At germination, sulfated fucans are secreted in the growing rhizoid wall. The redistribution of cell wall epitopes was investigated during treatments that cause reorientation of the growth axis (change in light direction) or disrupt rhizoid development (arabinogalactan-protein-reactive Yariv reagent). Alginate modeling was drastically impaired in the latter, and both treatments cause a redistribution of highly sulfated fucan epitopes. The dynamics of cell wall glycans in this system have been visualized in situ for the first time, leading to an enhanced understanding of the early developmental mechanisms of Fucus species. These sets of monoclonal antibodies significantly extend the available molecular tools for brown algal cell wall studies.

Photopolarization of Fucus zygotes is determined by time sensitive vectorial addition of environmental cues during axis amplification.

Fucoid zygotes have been extensively used to study cell polarization and asymmetrical cell division. Fertilized eggs are responsive to different environmental cues (e.g., light, gravity) for a long period before the polarity is fixed and the cells germinate accordingly. First, it is commonly believed that the direction and sense of the polarization vector are established simultaneously as indicated by the formation of an F-actin patch. Secondly, upon reorientation of the zygote, a new polar gradient is formed and it is assumed that the position of the future rhizoid pole is only influenced by the latter. Here we tested these two hypotheses investigating photopolarization in Fucus zygotes by reorienting zygotes 90° relative to a unilateral light source at different time points during the first cell cycle. We conclude that fixation of direction and sense of the polarization vector is indeed established simultaneously. However, the experiments yielded a distribution of polarization axes that cannot be explained if only the last environmental cue is supposed to determine the polarization axis. We conclude that our observations, together with published findings, can only be explained by assuming imprinting of the different polarization vectors and their integration as a vectorial sum at the moment of axis fixation. This way cells will average different serially perceived cues resulting in a polarization vector representative of the dynamic intertidal environment, instead of betting exclusively on the perceived vector at the moment of axis fixation.

Spatial re-organisation of cortical microtubules in vivo during polarisation and asymmetric division of Fucus zygotes.

Fucus zygotes polarise and germinate a rhizoid before their first asymmetrical division. The role of microtubules (MTs) in orienting the first division plane has been extensively studied by immunofluorescence approaches. In the present study, the re-organisation of MT arrays during the development of Fucus zygotes and embryos was followed in vivo after microinjection of fluorescent tubulin. A dynamic cortical MT array that shows dramatic reorganization during zygote polarization was detected for the first time. Randomly distributed cortical MTs were redistributed to the presumptive rhizoid site by the time of polarisation and well before rhizoid germination. The cortical MT re-organisation occurs independently of centrosome separation and nucleation. By the time of mitosis the cortical array depolymerised to cortical foci in regions from which it also reformed following mitosis, suggesting that it is nucleated from cortical sites. We confirm previous indications from immunodetection studies that centrosomal alignment and nuclear rotation occur via MT connexions to stabilised cortical sites and that definitive alignment is post-metaphasic. Finally, we show that cortical MTs align parallel to the growth axis during rhizoid tip growth and our results suggest that they may be involved in regulating rhizoid growth by shaping the rhizoid and containing turgor pressure.

Marine plants may polarize remoteFucus eggs via luminescence

Fucus zygotes can be polarized by many environmental vectors. These include those created by pieces of all the intertidal marine plants tested. At distances of up to 5-10 mm away from such pieces, Fucus zygotes form their intitial outgrowths or germinate towards them. Earlier papers had inferred that this so-called 'thallus effect' is mediated by diffusing molecules. The present reinvestigation indicates that the thallus effect is exerted by influences that can go right through glass barriers. This indicates action via luminescence. This luminescence may come from bacteria growing in biofilms on the similar surfaces of these otherwise unrelated source plants. Moreover, this directive luminescence is inferred to lie at wavelengths in the red or longer and may take the form of more or less coherent biophotons.

Marine Plants May Polarize Remote Fucus Eggs via Luminescence.

Fucus zygotes can be polarized by many environmental vectors. These include those created by pieces of all the intertidal marine plants tested. At distances of up to 5-10 mm away from such pieces, Fucus zygotes form their intitial outgrowths or germinate towards them. Earlier papers had inferred that this so-called 'thallus effect' is mediated by diffusing molecules. The present reinvestigation indicates that the thallus effect is exerted by influences that can go right through glass barriers. This indicates action via luminescence. This luminescence may come from bacteria growing in biofilms on the similar surfaces of these otherwise unrelated source plants. Moreover, this directive luminescence is inferred to lie at wavelengths in the red or longer and may take the form of more or less coherent biophotons.

Pattern formation of stationary transcellular ionic currents in Fucus.

Stationary and nonstationary spatiotemporal pattern formations emerging from the cellular electric activity are a common feature of biological cells and tissues. The nonstationary ones are well explained in the framework of the cable model. Inversely, the formation of the widespread self-organized stationary patterns of transcellular ionic currents remains elusive, despite their importance in cell polarization, apical growth, and morphogenesis. For example, the nature of the breaking symmetry in the Fucus zygote, a model organism for the experimental investigation of embryonic pattern formation, is still an open question. Using an electrodiffusive model, we report here an unexpected property of the cellular electric activity: a phase-space domain that gives rise to stationary patterns of transcellular ionic currents at finite wavelength. The cable model cannot predict this instability. In agreement with experiments, the characteristic time is an ionic diffusive one (<2 min). The critical radius is of the same order of magnitude as the cell radius (30 microm). The generic salient features are a global positive differential conductance, a negative differential conductance for one ion, and a difference between the diffusive coefficients. Although different, this mechanism is reminiscent of Turing instability.

ARE “GREEN TIDES” HARMFUL ALGAL BLOOMS? TOXIC PROPERTIES OF WATER‐SOLUBLE EXTRACTS FROM TWO BLOOM‐FORMING MACROALGAE, ULVA FENESTRATA AND ULVARIA OBSCURA (ULVOPHYCEAE)

Toxic properties are more often associated with microalgal blooms than with macroalgal blooms, although herbivore deterrents are well known in macroalgae, and inhibition of invertebrate larval development by extracts from Ulva spp. has been suggested. This study was prompted from our observation that substantial discoloration of seawater occurred in small bays after mass desiccation‐induced mortality of Ulvaria obscura (Kützing) Gayral. We examined the effects of extracts from Ulva fenestrata Postels et Ruprecht and Ulvaria obscura on Fucus gardneri Silva zygote development, growth of Ulva and Ulvaria, epiphytic algal accumulation, and oyster larval development. Fucus zygote development was inhibited by extracts from both species, although the effects of Ulvaria extracts were significantly greater. Epiphytic algal accumulation and the growth of Ulva and Ulvaria were inhibited by extracts from both species. Oyster larval development was arrested by the presence of extracts from each species. We conclude that extracts from both Ulva fenestrata and Ulvaria obscura have allelopathic properties. The effects are more widespread and occur at lower concentrations for extracts from Ulvaria than Ulva. These properties could alter competitive interactions by inhibiting germination or development of algae and invertebrates.

Are ‘Green TIDES’ Harmful Algal Blooms? Allelopathic Properties of Extracts from Ulva Fenestrata and Ulvaria Obscura

AbstractToxic properties are rarely associated with macroalgal blooms. A few studies have demonstrated inhibition of invertebrate larval development by extracts from Ulva spp. and we have observed substantial discoloration of the seawater in small bays following mass desiccation‐induced mortality of Ulvaria obscura. We examined the effects of extracts from Ulva fenestrata and U. obscura on enzyme systems, larval oyster development, Fucus gardneri zygote development, epiphytic diatom growth, and growth of Ulva and Ulvaria. Extracts from Ulvaria inhibited trypsin activity, but not pepsin or amyloglucosidase activity when tested at pH optimized for each enzyme system (pH 8, 2, and 5.5, respectively). When tested at neutral to slightly alkaline pH (7 or 8), Ulvaria extracts also inhibited amyloglucosidase activity. ‘Aged’ extract caused greater enzyme inhibition than fresh extract. Maximum inhibition occurred in 6‐h‐old‐extract. Oyster larval development was inhibited by extracts from both algae, although the visible effects were much greater when the larvae were exposed to extracts of Ulvaria. Fucus zygote development was inhibited by extracts of both species, although the effect was measurably greater in extracts from Ulvaria. Epiphytic diatom growth and growth of Ulva and Ulvaria were inhibited by extracts from both species. Thus, extracts from both Ulva fenestrata and U. obscura have allelopathic properties, although the impacts are more widespread for Ulvaria extracts. These properties could alter competitive interactions by inhibiting germination or development of algal and animal competitors. Multiple toxins are probably involved, since the two species tested cause visibly different responses.

Cell cycle-dependent control of polarised development by a cyclin-dependent kinase-like protein in the Fucus zygote.

Although iterative development can be uncoupled from morphogenesis in plant organs, the relationship between the cell cycle and developmental events is not well established in embryos. Zygotes of fucoid algae, including Fucus and Pelvetia are particularly well suited for studying the interaction(s) between cell cycle progression and the early morphogenetic events, as the establishment of polarity and its morphogenetic expression, i.e. germination, and the first cell cycle are concomitant. We have previously demonstrated that, in Fucus zygotes, various aspects of cell cycle progression are tightly controlled by cyclin-dependent kinase (CDK)-like proteins, including two PSTAIRE CDK-like proteins, p34 and p32, which are synthesised after fertilisation. We show that specific inhibition of CDK-like proteins, either with purine derivatives such as olomoucine and amino-purvalanol or by microinjection of the CDK inhibitor p21(cip1), prevents germination and cell division. Whereas direct inhibition of DNA replication by aphidicolin did not affect polarised development, olomoucine, which has previously been shown to prevent entry in S phase, and other purine derivatives also inhibited photopolarisation. Early microinjection of a monoclonal anti-PSTAIRE antibody also prevented germination and cell division. Only p34 had affinity for amino-purvalanol, suggesting that among PSTAIRE CDKs, this protein is the main target of purine derivatives. Models to account for the simultaneous control of early cell cycle progression and polarisation are proposed.

Cell Cycle in the Fucus Zygote Parallels a Somatic Cell Cycle but Displays a Unique Translational Regulation of Cyclin-Dependent Kinases

Cell Cycle in the Fucus Zygote Parallels a Somatic Cell Cycle but Displays a Unique Translational Regulation of Cyclin-Dependent Kinases

FUCUS VANADIUM PEROXIDASE: MINIMUM CATALYTIC DOMAIN SIZE RETAINING PEROXIDASE ACTIVITY

Vanadium peroxidase catalyzes the extracellular assembly of Fucus zygote cell surface adhesive (Vreeland &amp; Epstein 1996, Modern Meth. Plant Anal. 17, 95–116). Our goal is to identify the catalytic, self‐associating and wall targeting functional domains of algal vanadium peroxidase to understand its role in algal propagule adhesion. As a first step, we truncated our recombinant Fucus vanadium peroxidase (GenBank AF053411) for catalytic domain identification. Recombinant constructs were prepared which reduced the C‐terminal catalytic domain at either or both N‐ and C‐terminal ends. Recombinant proteins were expressed in E. coli, refolded from cytoplasm and inclusion bodies and tested for vanadium‐specific o‐dianisidine peroxidase activity. Preliminary results demonstrated peroxidase activity when the 40 kDa catalytic domain was truncated on both ends to 24 kDa. Further terminal and internal truncation is needed to fully define the minimal catalytic unit, which could be as small as 15–20 kDa within the 73 kDa monomer. The very small catalytic unit in Fucus vanadium peroxidase is not unexpected considering the rigid bundled helical vanadate frame in the Curvularia fungal vanadium peroxidase (Macedo‐Ribeiro et al. 1999, J. Biol. Inorg. Chem. 4, 209–219). We conclude that interactions between the N‐terminal noncatalytic domain and the C‐terminal catalytic domain, found in the crystalline Ascophyllum enzyme (Weyland et al. 1999, J. Mol. Biol. 293, 595–611), are unnecessary for peroxidase activity. Other conserved amino acids in the C‐terminal half of Fucus vanadium peroxidase, peripheral to the helical core, could participate in protein surface functions such self‐association and wall targeting.

Membrane recycling occurs during asymmetric tip growth and cell plate formation inFucus distichus zygotes

Zygotes of the brown algaFucus distichus undergo a series of intracellular changes resulting in the establishment of a polar growth axis prior to the first embryonic cell division. In order to examine the dynamics of membrane recycling which occur in the zygote during polar growth of the rhizoid, we probed living Fucus zygotes with the vital stain FM4-64, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylammo)phenyl)hexatrienyl)pyridinium dibromide. In newly fertilized, spherical zygotes, FM4-64 staining is symmetric and predominantly in the perinuclear region which is rich in endoplasmic reticulum, Golgi, and vacuolar membranes. As rhizoid or tip growth is initiated, this population of stained membranes becomes asymmetrically redistributed, concentrating at the rhizoid tip and extending centrally to the perinuclear region. This asymmetric localization is maintained in the zygote throughout polar growth of the rhizoid and during karyokinesis. Subsequently, FM4-64 staining also begins to accumulate in a central location between the daughter nuclei. As cytokinesis proceeds, this region of stain expands laterally from this central location, perpendicular to the plane of polar rhizoid outgrowth. The staining pattern thus delineates the formation of a cell plate, similar spatially to the accumulation of nascent plate membranes of higher plants. Treatment of Fucus zygotes with brefeldin-A inhibits both asymmetric growth of the rhizoid and formation of a new cell plate. These data suggest that inF. distichus FM4-64 is labeling a Golgi-derived membrane fraction that appears to be recycling between the site of tip growth, perinuclear region, and new cell plate.

Soft bottom versus hard rock:: Community ecology of macroalgae on intertidal mussel beds in the Wadden Sea

On the extensive mud and sand flats of the Wadden Sea, intertidal mussel beds ( Mytilus edulis L.) represent one of few secondary hard substrata and thus potentially serve as attachment surfaces for macroalgae in an environment which is largely dominated by sediments. However, the only abundant colonizing seaweed on mussel beds is a special form of rockweed, Fucus vesiculosus forma mytili (Nienburg), which lacks a holdfast and reproduces only vegetatively. Ephemeral macroalgae are absent from mussel beds, and large mussel patches remain completely uncolonized by seaweeds. I tested the effect of grazing by periwinkles, Littorina littorea L., on algal cover through experimental exclusion of snails. Under reduced grazing pressure, ephemeral green and red algae ( Enteromorpha, Ulva and Porphyra) developed and either partially or completely covered the surface on mussel bed plots, but only in the absence of Fucus. Additional experiments showed that Fucus dispersal from fertile populations to mussel beds is possible within a range of at least 25 m. Between 5 and 25 m from a fertile population on rocky substratum F. vesiculosus recruited successfully on artificial settlement surfaces, in the absence and to a lesser extent also in the presence, of littorinid grazing. If F. vesiculosus zygotes settle on mussel surfaces, they are subject to very high post-settlement mortality. Field experiments showed that survival of Fucus zygotes inoculated onto live mussels was close to zero, under high and low grazing intensities. Survival was elevated on mortar-filled mussel valves when snail grazing pressure was reduced, and especially if barnacles were present. As demonstrated in laboratory experiments, the recruitment failure on live mussels was due to the accumulation of faeces and pseudofaeces which shade, bury and eventually kill Fucus zygotes underneath. Adult thalli are either not susceptible to these adverse effects of mussel biodeposition or can counterbalance them by growth. Since propagation can occur through fragmentation of adults, sexual reproduction is unnecessary for the survival of the Fucus population. Just as on rocky shores, the abundance of ephemeral seaweeds on mussel beds of the Wadden Sea tidal flats is controlled by grazing. With opportunistic life strategies ephemerals are characterized by high growth rates and reproductive capabilities which allow them to respond rapidly to spatial and temporal reductions in grazing pressure. In contrast, adults of the long-lived perennial seaweed, Fucus, are largely unaffected by grazing, and juvenile stages, which would be subject to herbivory as well as strong biodeposition on Wadden Sea mussel beds, are omitted from the life cycle of Fucus in this environment.

Settlement and survival of epiphytes on two intertidal crustose coralline alga

The effects of the microtopography of the surface and the antifouling ability of crustose coralline alga on the recruitment of epiphytes were tested under different shading conditions. In experiments set up on the south coast of Isle of Man, British Isles, during autumn and winter, epiphyte settlement and survival on Phymatolithon lenormandii (Aresch) Adey and P. purpureum (P. & H. Crouan) Woelkerling and Irvine were compared on epoxy replicas as well as dead and living crusts. Differences in the density of epiphyte germlings between replicas of the surface of two crust species were due to their surface textures. The rough-surfaced P. lenormandii had a higher density of epiphytes, probably because it provides a better refuge for Ulva and Enteromorpha spp. zoospores than for Fucus zygotes, which are larger and thus less protected by crust protuberances on wave washed shores. On the shore, shading with neutral roofs was less effective than canopy algae in cutting out light and suppressing Fucus germlings. Living crusts strongly retarded epiphyte recruitment, but this was dependent on light levels on the shore. The factors influencing this process need to be identified, because mechanical and chemical mechanisms cannot yet be distinguished.

Cortical asymmetries direct the establishment of cell polarity and the plane of cell division in the Fucus embryo.

External gradients, such as unilateral light, applied to apolar zygotes of Fucus result in a cortical asymmetry expressed as the actin-dependent translocation of existing plasma membrane molecules (e.g., DHP receptors) to the shaded side (Fig. 1a). This process corresponds to the alignment of the polar axis. The localized cortical domain identified by the accumulation of DHP receptors, F-actin, and free calcium forms a target site for Golgi vesicle (F granule) secretion. Localized secretion of F granules is essential to stabilize the polar axis (Fig. 1b), and to complete a structural complex at the site for polar growth, postulated to span the plasma membrane, from the actin cytoskeleton to the cell wall (Fig. 2). Furthermore, targeted secretion of the contents of F granules into the plasma membrane and/or cell wall appears to provide localized positional information required to orient the first cell division plane and to differentiate the rhizoid and thallus cells of the two-celled embryo (Fig. 1c). Our cytological approaches using Fucus zygotes point to the importance of directed vesicle movement and secretion in creating asymmetries in the plasma membrane/cell wall during embryogenesis, which appear to have a critical role in cell morphogenesis. Conclusions drawn from these results may provide a useful paradigm for the study of cell morphogenesis and pattern formation in higher plant embryos and vegetative tissues.

Spatial Organization of Calcium Signaling Involved in Cell Volume Control in the Fucus Rhizoid.

Subprotoplasts prepared from different regions of rhizoid and thallus cells of Fucus zygotes displayed mechanosensitive plasma membrane channels in cell-attached patch-clamp experiments by using laser microsurgery. In excised patches, this channel was found to be voltage gated, carrying K+ outward and Ca2+ inward, with a relative permeability of Ca2+/K+ of 0.35 to 0.5, and an increased open probability at membrane potentials more positive than -80 mV. No significant difference was found in the density of this channel type from different regions of rhizoid or thallus cells. Hypoosmotic treatment of intact zygotes induced dramatic transient elevations of cytoplasmic Ca2+, initiating at the rhizoid apex and propagating in a wavelike manner to subapical regions. Localized initiation of the Ca2+ transient correlated with greater osmotic swelling at the rhizoid apex compared with other regions of the zygote. Ca2+ transients exhibited a refractory period between successive hypoosmotic shocks, during which additional transients could not be elicited and the ability to osmoregulate was impaired. Buffering the Ca2+ transients with microinjected Br2BAPTA similarly reduced the ability of rhizoid cells to osmoregulate. Ca2+ influx was associated with the initiation of the Ca2+ transient in apical regions, whereas intracellular sources contributed to its propagation. Thus, localized signal transduction is patterned by interactions of the cell wall, plasma membrane, and intracellular Ca2+ stores.

The role of targeted secretion in the establishment of cell polarity and the orientation of the division plane in Fucus zygotes.

In this study, we investigate the role of polar secretion and the resulting asymmetry in the cell wall in establishing polarity in Fucus zygotes. We have utilized brefeldin-A to selectively interrupt secretion of Golgi-derived material into the cell wall as assayed by toluidine blue O staining of sulfated fucoidin. We show that the polar secretion of Golgi-derived material is targeted to a cortical site of the zygote identified by the localization of actin filaments and dihydropyridine receptors. The deposition of Golgi-derived material into the cell wall at this target site is temporally coincident with and required for polar axis fixation. We propose that local secretion of Golgi-derived material into the cell wall transforms the target site into the fixed site of polar growth. We also found that polar secretion of Golgi-derived material at the fixed site is essential for growth and differentiation of the rhizoid, as well as for the proper positioning of the first plane of cell division. We propose that the resulting asymmetry in the cell wall serves as positional information for the underlying cortex to initiate these polar events. Our data supports the hypothesis that cell wall factors in embryos, previously shown to be responsible for induction of rhizoid cell differentiation, are deposited simultaneously with and are responsible for polar axis fixation. Furthermore, the pattern of polar growth is attributable to a positional signal at the fixed site and appears to be independent of the orientation of the first cell division plane. Thus, the establishment of zygotic cell polarity and not the position of the first division plane, is critical for the formation of the initial embryonic pattern in Fucus.

Polar localization of a dihydropyridine receptor on living Fucus zygotes.

We have used a fluorescently-labeled dihydropyridine (FL-DHP) to vitally stain living Fucus zygotes during the establishment of cell polarity. Localization of FL-DHP is primarily at the plasma membrane and FL-DHP binding is competitively blocked by an unlabeled dihydropyridine. Distribution of FL-DHP is initially symmetrical before fixation of the polar axis, but becomes asymmetrical in response to a unilateral light gradient. The distribution of FL-DHP receptors can be relocalized when the direction of the photopolarizing stimulus is changed. Treatment of cells with cytochalasin B prior to axis fixation reversibly prevents localization of FL-DHP receptors. Observation of FL-DHP labeling by time-lapse fluorescence microscopy indicates that the existing receptors are redistributed during polar axis formation. The asymmetric distribution of FL-DHP receptors coincides temporally and spatially with increased local intracellular calcium ion concentrations, as measured by calcium green dextran. Based on the site, timing, photo-reversibility, and actin dependence of the asymmetric localization of FL-DHP receptors, we conclude that FL-DHP is a vital probe for the later stage of polar axis formation in Fucus zygotes. Furthermore, we propose that FL-DHP receptors correspond to ion channels that are transported to the future site of polar growth to create the changes in local calcium concentration required for polarity establishment.

The role of targeted secretion in the establishment of cell polarity and the orientation of the division plane in Fucus zygotes

The role of targeted secretion in the establishment of cell polarity and the orientation of the division plane in Fucus zygotes