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Kenny A Bogaert, Tom Beeckman, Olivier De Clerck, Egg activation-triggered shape change in the Dictyota dichotoma (Phaeophyceae) zygote is actin–myosin and secretion dependent, Annals of Botany, Volume 120, Issue 4, October 2017, Pages 529–538, https://doi.org/10.1093/aob/mcx085
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Abstract
Background and Aims Cellular morphogenesis in land plants and brown algae is typically a slow process involving growth established by an interplay of turgor pressure and cell wall rigidity. However, a recent study showed that zygotes of the brown alga Dictyota dichotoma undergo a rapid shape change from a sphere to an elongated spheroid in about 90 s, establishing the first body axis.
Methods Using a combination of pharmacology, staining techniques, membrane depolarization and microscopy techniques (brightfield, transmission electron microscopy and confocal laser scanning microscopy), egg activation and the shape change of the egg cell of D. dichotoma was studied.
Key Results It was established that elongation of the zygote does not involve growth, i.e. a positive change in size. The elongation is dependent on F-actin and myosin but independent of microtubules. Secretion was also found to be necessary for elongation after addition of brefeldin A. Moreover, a temporal correlation between extracellular matrix secretion and elongation was observed. Ionomycin and high potassium seawater are capable of triggering the onset of elongation, suggesting a role for membrane depolarization and calcium influx in the signalling mechanism. The elongated cells are shorter in the presence of ionomycin, suggesting a role for calcium in elongation.
Conclusions A model is proposed in which the fast elongation of the fertilized egg in Dictyota is accomplished by a force generated by F-actin and myosin, regulated by cytoplasmic calcium concentrations and by secretion during elongation lowering the antagonistic force. The finding of early extracellular matrix secretion, membrane depolarization and ionophore-triggered egg activation suggest significant differences in the mechanism of egg activation signalling between D. dichotoma and the oogamous brown algal model system Fucus.
INTRODUCTION
During growth and development, cells of land plants and brown algae change their shape to create a functional adult body. Due to the rigidity of the extracellular matrix (ECM), however, this is a slow process involving directional growth (Hamant et al., 2010; for a review, see Harold, 2002). Typically, osmotic water uptake leads to a turgor pressure which causes the cells to swell (Kropf et al., 1995; Zonia and Munnik, 2007). Due to the presence of rigid cellulose microfibrils, cross-linked via a polysaccharide and protein matrix, an antagonistic force is provided, avoiding bursting of the cells. An increase of turgor pressure or a decrease in strength of the ECM may cause cells to grow in size. The direction of cell expansion is determined by the orientation of cellulose microfibrils, which are deposited preferentially parallel to cortical microtubules in land plants (Brown and Montezinos, 1976) or F-actin filaments in brown algae (Katsaros et al., 2002). Additionally, localized cell wall loosening may cause cell expansion or tip growth (Hable and Kropf, 1998). Other schemes for directional growth have evolved in filamentous algae such as telescoping cell walls in Microspora (Pickett-Heaps, 1973) and Tribonema (Chi et al., 1999) or rings of pliant cell walls in Oedogonium (Ohashi, 1930). Importantly, all these mechanisms for shape change rely on the presence of a cell wall and are relatively slow processes, accompanied by cell growth.
Natural protoplasts (i.e. freshly released spores, gametes, plasmodial tapetum, etc.) present an exception to the mechanism described above. Protoplasts may change their shape more quickly due to the absence of the rigid ECM. However, the general perception is that protoplasts are only capable of passive shape changes. Despite this assumption, zygotes of D. dichotoma undergo a fast elongation immediately following fertilization (Bogaert et al., 2017) which cannot be explained in the framework of passive forces acting on the membrane. In about 90 s the spherical cell elongates into a prolate spheroid with a major axis twice as long as the minor axis. The mechanistic basis of this fast and pronounced shape change, however, is unknown.
Dictyota dichotoma eggs can develop parthenogenetically for up to a maximum of 2–3 divisions. They are able to polarize and divide, but lack centrosomes and centrioles, and show irregular spindle formation and scattered chromosomes during mitosis. After a maximum of three divisions, they invariably die (Williams, 1904). This situation is in contrast to the egg activation in fucoids, which has been extensively studied (Brownlee, 1994; Dumas and Gaude, 2006). In Fucus, the calcium ionophore ionomycin is not sufficient to induce complete egg activation (Brawley and Bell, 1987). Similarly, depolarization of the plasma membrane using 450 mm K+ artificial sewater (ASW) is not able to activate the eggs, suggesting the involvement of an additional signal triggered by sperm entry (Roberts et al., 1994). This second signal would be necessary to propagate around the egg beneath the plasma membrane and activate cell wall secretion in concert with the first signal which is better characterized.
Here we provide data suggesting a model in which the fast elongation in D. dichotoma eggs is mediated by actin, myosin and increased exocytosis. Additionally, a role for intracellular calcium signals is suggested for cell elongation. Application of pharmacological agents and ASW suggests a role for membrane depolarization and an increase of cytoplasmic calcium concentrations in the activation of the elongation mechanism; however, unlike Fucus, D. dichotoma eggs do not require an additional signal for egg activation.
MATERIALS AND METHODS
Biological material
Experiments were carried out using unialgal laboratory cultures of gametophytes of D. dichotoma (KB07 and ODC1387) collected near Roscoff Biological Station in Brittany (France) and near l’Ancient Fort Croix in Wimereux (France). Thalli were grown in aerated 1 L culture flasks at approx. 19 °C or in 250 mL crystallizing dishes in white fluorescent light with a photon flux rate of 60 μmol m–2 s–1 (Lumilux, Cool White, Osram GmbH, Augsburg, Germany) using modified Provasoli enriched seawater (mPES) (West and Mcbride, 1999). Gametes were released by placing male and female gametophytes during release periods (Bogaert et al. 2016) in natural daylight in fresh medium after incubation in the dark overnight.
Membrane depolarization using high potassium ASW
Eggs were released in ASW (450 mm NaCl, 2·5 mm NaHCO3, 30 mm MgCl2, 16 mm MgSO4, 10 mm KCl, 10 mm CaCl2 buffered with 5 mm Tris at pH 7·9), sieved with a 10 μm Nitex nylon mesh 10 min after release, rinsed three times and transferred to high potassium ASW (140 mm NaCl, 2·5 mm NaHCO3, 30 mm MgCl2, 16 mm MgSO4, 320 mm KCl, 10 mm CaCl2 buffered with 5 mm Tris at pH 7·9) or normal ASW. The percentage of activated eggs, as scored by their elongated shape, was counted approx. 15 min after transfer. To stain the cell wall, suspended eggs were diluted ten times in high K+ ASW, or fresh ASW in the case of the control treatment, and stained with 0·0001 % (w/v) Calcofluor white (CFW). Eggs were photographed with a Zeiss Axiophot 2 epifluorescence microscope (Zeiss).
Size measurements
Eggs and zygotes were photographed 5 and 15 min after release, and 5 and 30 min after fertilization at × 200 using an Axiovert 40C inverted microscope (Zeiss) equipped with a Canon Powershot G11 camera for volume and area calculations. Diameters of spherical eggs and axes for prolate spheroidal zygotes were measured using ImageJ (https://imagej.nih.gov/ij/). Volume and area were calculated based on these results.
Pharmacological treatments
Pharmacological agents known to affect F-actin (latrunculin B, cytochalasin D), myosin [2,3-butanedione monoxime (BDM), 1-(5-iodonaphtalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine (ML-7)], microtubules (colchicine and oryzalin), Golgi-mediated secretion [brefeldin A (BFA)], cellulose synthesis (isoxaben) and calcium influx (ionomycin) were applied to zygotes to observe their effects on the fast shape change of egg cells. Stock solutions of inhibitors were prepared as follows: latunculin B (Sigma, Belgium), 50 μm in dimethylsulphoxide (DMSO); ML-7 (Sigma), 100 mm in DMSO at –20 °C; colchicine (Sigma, Belgium) at 4 °C, 100 mg mL in DMSO:filtered seawater (FSW) (50:50); BFA, 5 mg mL in ethanol at 4 °C; isoxaben (Sigma Belgium), 0·1 m isoxaben in DMSO at room temperature. A stock solution of ionomycin (Calbiochem, UK) was prepared at 5 μM in DMSO and stored at –80 °C. Ionomycin was added at 1 and 10 μm between 5 and 15 min after egg release.
Stock solutions were diluted to appropriate concentrations in autoclaved FSW. In all experiments, the final DMSO concentrations did not exceed 0·001 %. This concentration of DMSO did not induce mortality in D. dichotoma. The DMSO concentrations seemed to have no apparent effects on elongation and the final length of the zygotes. BDM (Sigma, Belgium) and cytochalasin D (Sigma, Belgium) were dissolved in ASW or high potassium ASW and immediately used. Eggs were pre-treated for 15 min before fertilization or parthenogenetic egg activation. When eggs were activated parthenogenetically, 9 vols of 450 μm K+ ASW were added to 1 vol. of ASW containing the freshly released eggs, bringing the final concentration to 406 mm K+.
Epifluorescence, calcoflour staining and confocal laser scanning microscopy (CLSM) brightfield imaging
Cell wall secretion was assayed using 0·0001 % CFW with a confocal laser imaging system (model TCS SP5, Leica, France) equipped with a helium–neon laser at an excitation wavelength of 405 nm. An AOBS (Leica, France) was used to monitor CFW fluorescence between 555 and 625 nm using a HCX PL APO ×40, 0·85 dry objective (Leica, France). CFW, brightfield and autofluorescence were captured simultaneously every 15 s.
Transmission electron microscopy (TEM)
For TEM analyses, an identical protocol was used to that outlined in Bogaert et al. (2017).
RESULTS
Freshly discharged eggs are free from ECM as assayed by TEM (Fig. 1A). In contrast, the zygotes show a thin cell wall at 30 min after fertilization (Fig. 1B, C). Confocal laser scanning microscopy on CFW-stained elongating cells revealed that cell wall is secreted almost immediately after addition of male gametes and simultaneously with the onset of elongation (Fig. 1D, E). Confocal images show that secretion of cell wall material is homogeneously spread over the cell surface (Fig. 1D).
The diameter of freshly released eggs and both major and minor axes of the prolate spheroidal cells after addition of sperm (n = 200, five pooled replicate populations) were measured. Freshly released eggs had a mean volume of 201 491 μm3 (± 2202 μm3 ), while the volume of zygotes was significantly smaller: 185 892 μm3 (± 2371 μm3) (Welch two-sample t-test, t = 4·82, d.f. = 396, P < 0·05) [mean (± s.e.)] (Fig. 2A). Surface area, in contrast, increased slightly, but significantly, from 16 575 (± 120 μm3) to 16 965 (± 150 μm3) (Welch two-sample t-test, t = –2·02, d.f. = 379, P < 0·05) (Fig. 2B).
To test the role of intracellular calcium, ionomycin was applied in different concentrations. A concentration of 1 or 10 μm Ca2+ ionomycin applied to freshly released eggs triggered a 4- to 8-fold increase in the percentage of elongating cells, respectively (Fig. 3B). A significant dose-dependent effect on the percentage of elongation can be observed [Type III test, generalized linear mixed model (GLMM), F = 39·76, d.f.numerator, denominator = 2, 4, P < 0·05]. However, while ionomycin induces elongation, the cells are significantly shorter than the control (Fig. 3C) (Type III test, GLMM, F = 39·76, d.f.numerator, denominator = 2, 4, P < 0·05). At 10 μm ionomycin, the length increase is about half that of cells in control experiments, indicating that ionomycin also interferes with the cell shape. High potassium seawater similarly triggers egg activation (assayed by elongation and CFW staining). The percentage of eggs that elongates parthenogenetically can be increased by artificially activating the eggs using high K+ ASW (Fig. 3A) (Cochran–Mantel–Haenszel test, F = 447·43, d.f. = 1, P < 0·05).
The role of the cytoskeleton in the shape change was examined with pharmacological agents. Latrunculin B, shown to disrupt F-actin arrays in brown algae at concentrations of 10–100 nm (Alessa and Kropf, 1999; Varvarigos et al., 2005), was used at a concentration of 15 nm during fertilization. Only a minute fraction of zygotes showed elongation, suggesting the dependence of elongation on the F-actin cytoskeleton (Type III test, GLMM, F = 184·74, d.f.n.umerator, denominator = 1, 2, P < 0·001) (Fig. 4A). Cytochalasin D also disrupts the actin cytoskeleton in brown algae (Brawley and Quatrano, 1979; Brawley and Robinson, 1985). Addition of a freshly prepared stock solution of cytochalasin D at 10 μm (with 15 min pre-incubation at 12·5 μm) showed less severe effects. Fertilized eggs were able to elongate, but were significantly shorter than the control (parametric Wilcoxon rank sum test, W = 2738·50, P < 0·001) (Fig. 4B, C). Latrunculin B did not inhibit fertilization or egg activation because cells divided and developed, in contrast to parthenogenetic eggs (Fig. 5). Zygotes developed into spherical, instead of spheroidal, embryos and rarely showed rhizoids at 15 nm unless the inhibitor was removed. While normal embryos typically show one rhizoidal and one thalloid pole (Bogaert et al., 2017), often multiple apical cells emerged following some seemingly random divisions when treated with latrunculin B (Fig. 5B, C). This results in multiple thalloid growth axes that do not necessarily emerge on diametrically opposed positions of the spheroidal embryo, and therefore cannot be pre-determined along the elongation axis (Fig. 5C).
Myosin functioning was inhibited using BDM, which is a general inhibitor of myosin ATPases in a wide range of eukaryotes including brown and red algae (Herrmann et al., 1992; May et al., 1998; Pu et al., 2000; Samaj et al., 2000; Ackland et al., 2007). BDM at 50 μm immediately inhibited motility of the male gametes and thereby precluded fertilization. BDM at 50 μm, without pre-incubation of the eggs, resulted in almost complete inhibition of elongation triggered by parthenogenetic egg activation using high 50 μm K+ ASW (Type III test, GLMM, F = 247·60, d.f.numerator, denominator = 1, 2, P < 0·005). The cells were rinsed after 30 min and managed to divide parthenogenetically, highlighting their survival (Fig. 6A). After staining with CFW, the round cells appeared to be activated, indicating that BDM selectively inhibits the elongation but not the activation of the egg (Fig. 6B–D). BDM at 20 μm had less severe effects and allowed elongation in most of the cells; however, the length of the prolate spheroidal cells was significantly smaller than those of untreated controls (Type III test, GLMM, F = 64·11, d.f.numerator, denominator = 1, 296, P < 0·0001) (Fig. 4D, E). ML-7 is a specific inhibitor of myosin light-chain kinase (MLCK) (Saitoh et al., 1987). Eggs were pre-treated for 15 min in 62·5 μm ML-7 seawater and fertilized by diluting the medium with seawater containing freshly released male gametes to 50 μm. The inhibitor did not prevent fertilization. Elongation was assayed categorically and shown to be inhibited by 50 μm ML-7 (χ2-test, χ = 29·85, d.f. = 1, P < 0·0001) (Fig. 4F). The fraction of zygotes that was still able to elongate was significantly shorter than control cells (parametric Wilcoxon rank sum test, W = 1820, P < 0·001) (Fig. 4G).
Colchicine and oryzalin, two drugs used to disrupt the microtubules, were previously applied to brown algae (Peters and Kropf, 2010). To confirm whether the dosage of colchicine is high enough and the effect is conserved in this species, the effect on rhizoid development in the zygote was assessed at 10 and 200 μg mL–1. At 200 μg mL–1, rhizoid development was significantly inhibited, while rhizoid morphology was significantly altered at low concentrations of 10 μg mL–1 colchicine (Supplementary Data Fig. S1). At a concentration of 200 μg mL–1 colchicine, with a pre-treatment of 15 min at 222 μg mL–1, no significant inhibition of elongation could be observed both when assayed categorically (Type III test, GLMM, F = 18·11, d.f.numerator, denominator = 1, 2, P > 0·05) and when assayed by measuring the length of the spheroids (Type III test, GLMM, F = 0·15, d.f.numerator, denominator = 1, 296, P > 0·05) (Fig. 4H, I). Similar results were obtained with 2 μM oryzalin, with pre-treatment of the eggs for 15 min at 2·22 μm. No significant inhibition of the elongation could be detected when analysed categorically (Fisher’s exact test, F = 0·25, d.f. = 1, P > 0·05) and by analysing the lengths of the elongated cells (Welch two-sample t-test, t = 1·0694, d.f. = 97, P > 0·05) (Fig. 4J, K).
The onset of elongation coincides with the first indication of cell wall secretion, as visualized by a faint CFW staining. Additionally a halo that stains with Toluidine blue O is observed after fertilization (Fig. 7B, D). The halo encases superfluous D. dichotoma gametes at a distance from the plasma membrane (Fig. 7C). When Ectocarpus siliculosus male gametes are added to freshly released D. dichotoma eggs, the male gametes gather around eggs and accumulate at the adhesive layer of parthenogenetically activated eggs. The layer is completely impermeable to distantly related E. siliculosus male gametes (Fig. 7A).
To interrupt Golgi-mediated secretory processes selectively (Nagasato and Motomura, 2009), we utilized the vesicular transport inhibitor BFA. Eggs were pre-treated for 15 min in FSW containing 35 μg mL–1 BFA and fertilized in 25 μg mL–1 BFA in FSW. This partially inhibited the elongation of the zygotes (Fig. 8A) (Type III test, GLMM, F = 575·59, d.f.numerator, denominator = 1, 296, P < 0·0001).
In order to test whether secretion of cellulose synthase complexes and/or deposition of cellulose microfibrils in the cell wall were essential for the elongation process, cellulose synthesis was inhibited using the drug isoxaben, which was earlier reported to be effective in brown algae (Heim et al., 1990; Bisgrove and Kropf, 2001). When isoxaben (25 μm) was added at 5 h after fertilization, we observed a significant increase in lysed cells at 24 h after fertilization (Type III test, GLMM, F = 486·48, d.f.numerator, denominator = 1, 2, P < 0·005) (Fig. 8C). The application of isoxaben (25 μm) before fertilization did not have any significant effect on the length of the zygotes (Type III test, GLMM, F = 0·01, d.f.numerator, denominator = 1, 397, P > 0·05) (Fig. 8B), ruling out a potential role for cellulose synthesis in the emergence of the elongation axis.
DISCUSSION
Actin–myosin-dependent shape change
It is widely assumed that size increase is the driving factor for cell morphogenesis of plant systems (Szymanski, 2009). Typical examples of morphogenetic programmes that accomplish polar growth are tip growth and diffuse growth (Kropf et al., 1998).
Bogaert et al. (2017), however, described a fast shape change during development of the zygote of D. dichotoma. The speed of the process suggests a mechanism other than elongation by tip growth or polar diffuse growth by oriented deposition of cellulose microfibrils (Kropf et al. 1998). Based on our calculations of the volumes of the egg and zygote, no growth could be established during the shape change of zygotes, which rules out the possibility that the fast shape change is established by bidirectional polar growth along the elongation axis. While the cell diameter of eggs was about 70 μm, the short axis of the prolate spheroid has decreased to 55 μm, illustrating that the cells change shape by deforming their dimensions.
This shape change is probably achieved by F-actin and myosin function as suggested by the pharmacological results. The microtubular cytoskeleton did not seem to play a crucial role in the generation of the force that accomplishes the elongation, while the pharmacological inhibitors were effective in inhibiting and affecting rhizoid growth of the zygote as reported for Fucus (Corellou et al., 2005). Even though elongation of zygotes is commonly observed, it is typically a slow process, involving growth (West and Harada, 1993; He et al., 2007).
The effect of ionomycin on the elongation process, as evidenced by the shorter length of elongated cells, may indicate a role for [Ca2+]cyt signalling in the pathway controlling the elongating force. Myosin is shown to be directly under control of [Ca2+]cyt–calmodulin in unikonts (Mockrin and Spudich, 1976; Nakamura and Kohama, 1999), and a similar control is suggested in land plants (Yokota et al., 1999; Tominaga et al., 2012). Therefore, we speculate that a similar regulation of myosin–actin interaction occurs in brown algae.
Although the exact function of the axial chloroplast distribution (Bogaert et al., 2017) is unclear, the notion that chloroplast in the eggs are clustered along the elongation axis and the cargo-binding tail domain of myosins may interact with organelles (Madison and Nebenführ, 2013) could suggest that the chloroplast distribution has a role as a structural component interacting with the actin–myosin cytoskeleton. If, for example, the chloroplasts would be pushed outwards along the F-actin cytoskeleton, an elongation in the direction determined by the chloroplast distribution can be expected. Alternatively, actin polymerization of the cortical actin cytoskeleton of brown algae (Katsaros et al., 2002) via the Arp2/3 complex and myosin pulling activity may induce tension that can drive a shape change (Carvalho et al., 2013), while the internal distribution of chloroplasts may determine the preferential elongation direction. This can be expected in the framework of the poroelastic model (Moeendarbary et al., 2013) which states that the density of the porous elastic meshwork has a negative influence on the contractibility. The local density of organelles, macromolecules and filaments determines the hydraulic pore size through which water and solutes can flow. This would cause the cell to contract its less dense hyaline zone faster than the polar regions and consequently elongate according to the cytoplasmic heterogeneity axis.
Role of secretion in the shape change
A shape change can be accomplished by changing both the forces actively generated by the cell (e.g. actin–myosin dependent) and the antagonistic forces that need to be overcome by this active force. A sphere is the most economic shape in terms of volume per unit of surface. A cell membrane behaves as an elastic two-dimensional fluid which can withstand forces of up to 4 mN m–2 and can expand by only about 2·6 % in area before lysing (Wolfe and Steponkus, 1983; Kell and Glaser, 1993; Nichol and Hutter, 1996). Therefore, the force generated by the cell membrane contributes to the total antagonistic force that needs to be overcome by the actin–myosin system. Increasing the available cell membrane material would enable the elongation by lowering the antagonistic force and would lower the chance of cell lysis.
Secondly, the need for increased cell surface may be (partially) compensated by lowering the volume of the cell content while elongating. Indeed, instead of growing, the cells shrink by 7–8 % in volume. This may be explained by the loss of volume caused by secretion of cell wall and adhesive halo material. The ECM secretion is temporally correlated with the shape change, because zygotes become sticky immediately after fertilization, while still elongating, and cell wall (assayed by CFW staining) is observed homogeneously all over the cell surface during elongation. Therefore, the cells are expected to lose volume during the fast elongation phase and they do not have to rely on osmoregulation only. However, the decrease in volume does not compensate completely because the surface area still increases by approx. 2·35 %, which is dangerously close to the lysis point of a biological membrane. We speculate that activation-induced secretion of the ECM provides at least a fraction of the additional membrane and thereby enables the shape change. Indeed, the role of secretion in cell elongation is also suggested by the effect of BFA, which partially inhibits elongation, resulting in shorter cells. This may be explained by the increased antagonistic force counteracting the elongation because of the increased tension on the membrane.
While actin–myosin interaction accompanied by secretion-induced membrane addition is suggested to be the motive force for the shape change, the rigidity of the cell wall will help in fixing the acquired cell shape. Already during elongation the cell wall is detectable using CFW staining and will have reached a considerable thickness. It therefore is tempting to speculate that the fast uniform cell wall secretion has a role in fixing the cell shape early after completion of the elongation. The most important structural components of brown algal cell wall are cellulose and alginate (Michel et al., 2010). While it may be questionable whether cellulose is synthesized during the first minutes after fertilization, alginate is expected to be among the early synthesized cell wall components (Evans et al., 1982). As alginate immediately starts gelating with the calcium ions of the medium (Fang et al., 2007), the incipient cell wall immediately gains rigidity to withstand the isodiametric hydrostatic force rounding up the cell.
The importance of secretion in female gametes is reflected in the gamete transcriptome by the upregulation of endomembrane-related and cell wall synthesizing enzymes (Bogaert et al. 2017). Ontology terms ‘SNARE interactions in vesicular transport’ (ko04130), ‘endocytosis’ (ko04144), ‘protein export’ (ko03060) and ‘exocytosis’ (GO:0006887) are all transcriptionally upregulated in eggs (contrast eggs vs. embryos). Three clathrin coat genes, involved in vesicle secretion, show significant upregulation relative to embryos of at least 1·5-fold change (FC), suggesting that the secretory event at egg activation requires more gene products than secretion at rhizoid development. Cell walls in brown algae consist of cellulose, alginate, sulphated fucans and phenolic compounds (Michel et al., 2010), and their individual expression patterns are less conclusive than that of gene set enrichment analysis (GSEA) of ontology terms. Relative to embryos, five (out of 15 found) mannuronan C-5-epimerase genes responsible for alginate synthesis and one cellulose synthase (out of four found) were differentially expressed [FC > 1·5, false discovery rate (FDR) <0·05] in eggs. Eight mannuronan C-5-epimerase genes, however, were significantly upregulated in embryos relative to eggs; and out of the 15 found sulphotransferases, required for fucan biosynthesis, the four differentially expressed genes (FC ≥ 1·5, FDR <0·05) were significantly biased to eggs. Out of the 19 identified putative orthologues annotated as mannuronan C-5-epimerase genes (Michel et al., 2010), with roles in cell wall biogenesis and modification (Nyvall et al., 2003), eight are upregulated by the time of asymmetrical cell division, possibly related to tip growth, while five are upregulated in the eggs (FDR <0·05). Out of four identified orthologues of cellulose synthase, one (orthologous to Esi0097_0016) shows significant upregulation in eggs (FDR < 0·05) (contrast eggs vs. embryos).
Egg activation
Besides the mechanisms behind the elongation, our data also provide some insights on how eggs are activated and elongation is triggered. In fucoids, after the localized onset of CFW staining (presumably at the sperm entry point), cell wall secretion traverses through the cell in a wave lasting 3–10 min in fucoid algae depending on the species (Brawley and Bell, 1987; Brawley, 1991; Bothwell et al., 2008). It is assumed that this wave starts at the sperm entry point (Brawley and Bell, 1987). The fact that application of calcium ionophores (Brawley and Bell, 1987) or mere depolarization of the plasma membrane using high K+ ASW (Roberts et al., 1994) is not sufficient for complete parthenogenetic activation, suggests that an additional signal is necessary, which propagates around the egg beneath the plasma membrane (Brownlee, 1994). The dependency on two signals for egg activation may explain why parthenogenesis of fucoids is only rarely reported (Overton, 1913; Nagasato et al., 2000). Moreover, the capacity of parthenogenesis is thought to differ seasonally (Brawley and Bell, 1987). In D. dichotoma, the cell wall secretion signal is released uniformly and simultaneously all over the cell surface at the onset of the elongation. No wave traversing through the cell could be observed, reflecting the fast propagation of cell wall secretion throughout the cell surface. Increased calcium influx triggered by the addition of ionomycin triggers egg activation and the onset of elongation. Together these findings suggest that no such additional factor supplied by the sperm is involved in the signal transduction leading to egg activation as mere membrane depolarization and Ca2+ influx (triggered by voltage-gated Ca2+ channels) is sufficient for full egg activation.
Sperm blocks are categorically sub-divided into a fast and slow category depending on whether they act on the scale of seconds or minutes after fertilization, respectively. In fucoids, land plants, sea urchin and Xenopus, the secretion of an ECM barrier (a cell wall, an activation calyx or a fertilization envelope, respectively) is a slow polyspermy block (Grey et al., 1976; Dumas and Gaude, 2006). In these organisms, membrane depolarization is the only prevention for polyspermy during the first minutes after fertilization (Jaffe, 1976; Grey et al., 1982; Brawley, 1991).
This situation is in contrast to the fast release of cell wall material here in D. dichotoma, where the ECM barrier qualifies as a fast efficient sperm block. The onset of elongation coincides with the first indication of cell wall secretion, as visualized by a faint CFW staining. Additionally, an adhesive halo that stains by TBO is observed after fertilization. Besides an obvious adhesive role, an additional polyspermy-blocking function may be speculated, because the halo seems to encase arriving superfluous D. dichotoma gametes at a distance and is completely impermeable to distantly related E. siliculosus male gametes. In our experiments, male gametes need only 1 min to find the egg, fuse, induce homogenous cell wall secretion and start showing adhesive properties, underlying the role of the ECM as a fast sperm block in D. dichotoma.
SUPPLEMENTARY DATA
Supplementary data are available online at https://academic.oup.com/aob and consist of Figure S1: colchicine affects rhizoid growth.
ACKNOWLEDGEMENTS
The authors are indebted to the Research Foundation Flanders (FWO) (PhD fellowship to K.B.) and ASSEMBLE (grant agreement no. 227799). We would like to thank Myriam Claeys for practical assistance, and Taizo Motomura, Christos Katsaros, Susana Coelho, John Bothwell, Agnieszka Lipinska and Wouter Houthoofd for helpful comments.
LITERATURE CITED