Rhizoid Tip Research Articles

Regulation of ROP GTPase cycling between active and inactive states is essential for vegetative organogenesis in Marchantia polymorpha.

Rho/Rac of plant (ROP) GTPases are plant-specific proteins that function as molecular switches, activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). The bryophyte Marchantia polymorpha contains single copies of ROP (MpROP), GEFs [ROPGEF and SPIKE (SPK)] and GAPs [ROPGAP and ROP ENHANCER (REN)]. MpROP regulates the development of various tissues and organs, such as rhizoids, gemmae and air chambers. The ROPGEF KARAPPO (MpKAR) is essential for gemma initiation, but the functions of other ROP regulatory factors are less understood. This study focused on two GAPs: MpROPGAP and MpREN. Mpren single mutants showed defects in thallus growth, rhizoid tip growth, gemma development, and air-chamber formation, whereas Mpropgap mutants showed no visible abnormalities. However, Mpropgap Mpren double mutants had more severe phenotypes than the Mpren single mutants, suggesting backup roles of MpROPGAP in processes involving MpREN. Overexpression of MpROPGAP and MpREN resulted in similar gametophyte defects, highlighting the importance of MpROP activation/inactivation cycling (or balancing). Thus, MpREN predominantly, and MpROPGAP as a backup, regulate gametophyte development, likely by controlling MpROP activation in M. polymorpha.

Establishment and application of novel culture methods in Marchantia polymorpha: persistent tip growth is required for substrate penetration by rhizoids

ABSTRACT A NIMA-related protein kinase, MpNEK1, directs tip growth of rhizoids through microtubule depolymerization in a liverwort Marchantia polymorpha. The Mpnek1 knockouts were shown to develop curly and spiral rhizoids due to the fluctuated direction of growth. Still, physiological roles and mechanisms of MpNEK1-dependent rhizoid tip growth remain to be clarified. Here, we developed novel culture methods to further study rhizoid growth of M. polymorpha, in which plants were grown on vertical plates. We applied the established methods to investigate MpNEK1 function in rhizoid growth. Rhizoids of the wild-type and Mpnek1 plants grew toward the gravity. The aerial rhizoids were longer in Mpnek1 than in the wild type. When the rhizoids were grown on the surface of a cellophane sheet, rhizoid length was comparable between the wild type and Mpnek1, whereas Mpnek1 developed more rhizoids compared to the wild type. We also applied gellan gum, which is more transparent than agar, to analyze rhizoids grown in the medium. Rhizoids of Mpnek1 displayed defect on entering into the solid medium. These results suggest that Mpnek1 rhizoids have the deficiency in invasive tip growth. Thus, stable directional growth is important for rhizoids to get into the soil to anchor plant body and to adsorb water and nutrients. Collectively, our newly designed growth systems are valuable for analyzing rhizoid growth.

Changes in Cell Wall Structure During Rhizoid Formation of Silvetia babingtonii (Fucales, Phaeophyceae) Zygotes.

We examined the ultrastructure of the cell wall and immunolocalization of alginates using specific antibodies against M-rich alginates and MG blocks during rhizoid formation in fucoid zygotes, Silvetia babingtonii. The thallus region of 24-h-old zygotes had a cell wall made of three layers with different fiber distribution. In the 12-h-old zygotes, three layers in the thallus were observed before rhizoid formation, namely the inner, middle, and outer layers. During rhizoid elongation, only the inner layer was apparent close to the rhizoid tip area. Immunoelectron microscopy detected M-rich blocks of alginate on the inner half of the cell wall, irrespective of the number of layers in the thallus and rhizoid regions. The MG blocks were seen to cover a slightly wider area than M-rich alginate blocks. It was suggested that parts of M in mannuronan would be rapidly converted to G, and MG-blocks are generated. Transcriptome analysis was performed using 3 -, 10 -, and 24-h-old zygotes after fertilization to examine the relationship between gene expression and alginate synthesis over time. The expression of two mannuronan C5-epimerase homologs that convert mannuronic acid into guluronic acid in alginates was upregulated or downregulated over the course of the examination.

The Ca2+ signaling, Glu, and GABA responds to Cd stress in duckweed

Cadmium (Cd) affects plants and animal health seriously. Ca2+ signals in plant cells are important for adaptive responses to environmental stresses. Here we showed that 50 μM Cd shock stimulated the Ca2+ signal via modifying the instantaneous Ca2+ flux from influx of 17 pmol·cm-2·s-1 to the efflux of 240 pmol·cm-2·s-1 at 100 μm from rhizoid tip. And the Ca2+ signal transferred to the vein and mesophyll cell. The Ca addition decreased the accumulation of Cd. The gene expression of glutamate receptor–like (GLR) proteins, which is activated by Glu and triggers Ca2+ flux, was increased significantly by 24 h Cd stress. Glu content was increased under Cd stress and exogenous Glu triggered the Ca2+ signal in duckweed, while Ca2+ addition caused no influence to Glu content. GABA, which is synthesized from Glu and acts as an inhibitory neurotransmitter, has been decreased with 24 h Cd treatment. GABA addition increased the abscission rate and Glu addition decreased the abscission rate during Cd stress, suggesting that the Glu/GABA ratio is important for responding to Cd. This research shows the sight of the Glu, Ca2+, GABA signaling networks during Cd stress.

Shining new lights on chytrid cell biology: quantitative live cell imaging of rhizoid development in an early-diverging fungus

Chytridiomycota (Chytrids) are the most basal lineage within the true fungi, however they have largely remained in the dark in terms of their fundamental cell biology. In aquatic ecosystems, chytrids can dominate ‘dark matter’ surveys and are important saprotrophs of recalcitrant organic carbon. They therefore play an integral biogeochemical role in carbon cycling. Unlike ‘higher’ dikaryan fungi that feed via multicellular hyphae, chytrids are unicellular and develop an anucleate rhizoid system that acts as the trophic interface of the cell. Understanding the functions of the rhizoid has the potential to shed light on the trophic biology of ‘dark matter’ chytrids. We applied 3D and 4D live-cell confocal microscopy to morphometrically quantify rhizoid development in the model saprotrophic chytrid Rhizoclosmatium globosum under different nutrient treatments. Rhizoid branching was highest under carbon-rich conditions, whereas under carbon-starved treatments, rhizoids grew significantly longer and were less branched, in what we interpret to be a ‘search strategy’ for nutrient sources. F-actin and the cell wall were identified throughout the rhizoid system. Chemical inhibition of actin and cell wall glucan synthesis induced the development of hyperbranched paramorphs, suggesting that these components underpin rhizoid branching and organising cell polarity at the rhizoid tip. Previous studies have shown that inhibition of these components induces an identical phenotype in dikaryan fungi. These findings represent an important step in understanding the trophic biology of a biogeochemically important microbe and unveil striking similarities in cell development between early-diverging and ‘higher’ dikaryan fungi.

More than meets the eye: regional specialisation and microbial cover of the blade of Porphyra umbilicalis (Bangiophyceae, Rhodophyta)

Abstract Completion of the Porphyra umbilicalis genome and ongoing research on this species by many investigators suggest the need for wider appreciation of regional specialisation of the P. umbilicalis blade. Here we use light and electron microscopy to describe four distinct regions of the blade: rhizoid cells with abundant floridean starch, vegetative cells, differentiating neutral sporangia, and mature neutral spores. The holdfast, densely covered by microorganisms, presents an intriguing biomechanical structure: thousands of very thin, long rhizoid tips course through the thick, secreted polysaccharide to the substratum. Wild blades in culture have more microorganisms than when collected, including filamentous cyanobacteria.

An evolutionarily conserved NIMA-related kinase directs rhizoid tip growth in the basal land plant Marchantia polymorpha.

Tip growth is driven by turgor pressure and mediated by the polarized accumulation of cellular materials. How a single polarized growth site is established and maintained is unclear. Here, we analyzed the function of NIMA-related protein kinase 1 (MpNEK1) in the liverwort Marchantia polymorpha In the wild type, rhizoid cells differentiate from the ventral epidermis and elongate through tip growth to form hair-like protrusions. In Mpnek1 knockout mutants, rhizoids underwent frequent changes in growth direction, resulting in a twisted and/or spiral morphology. The functional MpNEK1-Citrine protein fusion localized to microtubule foci in the apical growing region of rhizoids. Mpnek1 knockouts exhibited increases in both microtubule density and bundling in the apical dome of rhizoids. Treatment with the microtubule-stabilizing drug taxol phenocopied the Mpnek1 knockout. These results suggest that MpNEK1 directs tip growth in rhizoids through microtubule organization. Furthermore, MpNEK1 expression rescued ectopic outgrowth of epidermal cells in the Arabidopsis thaliana nek6 mutant, strongly supporting an evolutionarily conserved NEK-dependent mechanism of directional growth. It is possible that such a mechanism contributed to the evolution of the early rooting system in land plants.

Wilsonosiphonia gen. nov. (Rhodomelaceae, Rhodophyta) based on molecular and morpho-anatomical characters.

Morphological, anatomical, and molecular sequence data were used to assess the establishment and phylogenetic position of the genus Wilsonosiphonia gen. nov. Phylogenies based on rbcL and concatenated rbcL and cox1 loci support recognition of Wilsonosiphonia gen. nov., sister to Herposiphonia. Diagnostic features for Wilsonosiphonia are rhizoids located at distal ends of pericentral cells and taproot-shaped multicellular tips of rhizoids. Wilsonosiphonia includes three species with diagnostic rbcL and cox1 sequences, Wilsonosiphonia fujiae sp. nov. (the generitype), W.howei comb. nov., and W.indica sp. nov. These three species resemble each other in external morphology, but W.fujiae is distinguished by having two tetrasporangia per segment rather than one, W.indica by having abundant and persistent trichoblasts, and W.howei by having few and deciduous trichoblasts.

Thidiazuron Triggers Morphogenesis in Rosa canina L. Protocorm-Like Bodies by Changing Incipient Cell Fate.

Thidiazuron (N-phenyl-N′-1,2,3-thiadiazol-5-ylurea; TDZ) is an artificial plant growth regulator that is widely used in plant tissue culture. Protocorm-like bodies (PLBs) induced by TDZ serve as an efficient and rapid in vitro regeneration system in Rosa species. Despite this, the mechanism of PLB induction remains relatively unclear. TDZ, which can affect the level of endogenous auxins and cytokinins, converts the cell fate of rhizoid tips and triggers PLB formation and plantlet regeneration in Rosa canina L. In callus-rhizoids, which are rhizoids that co-develop from callus, auxin and a Z-type cytokinin accumulated after applying TDZ, and transcription of the auxin transporter gene RcPIN1 was repressed. The expression of RcARF4, RcRR1, RcCKX2, RcCKX3, and RcLOG1 increased in callus-rhizoids and rhizoid tips while the transcription of an auxin response factor (RcARF1) and auxin transport proteins (RcPIN2, RcPIN3) decreased in callus-rhizoids but increased in rhizoid tips. In situ hybridization of rhizoids showed that RcWUS and RcSERK1 were highly expressed in columella cells and root stem cells resulting in the conversion of cell fate into shoot apical meristems or embryogenic callus. In addition, transgenic XVE::RcWUS lines showed repressed RcWUS overexpression while RcWUS had no effect on PLB morphogenesis. Furthermore, higher expression of the root stem cell marker RcWOX5 and root stem cell maintenance regulator genes RcPLT1 and RcPLT2 indicated the presence of a dedifferentiation developmental pathway in the stem cell niche of rhizoids. Viewed together, our results indicate that different cells in rhizoid tips acquired regeneration competence after induction by TDZ. A novel developmental pathway containing different cell types during PLB formation was identified by analyzing the endogenous auxin and cytokinin content. This study also provides a deeper understanding of the mechanisms underlying in vitro regeneration in Rosa.

Cytological and Proteomic Analyses of Osmunda cinnamomea Germinating Spores Reveal Characteristics of Fern Spore Germination and Rhizoid Tip Growth*

Fern spore is a good single-cell model for studying the sophisticated molecular networks in asymmetric cell division, differentiation, and polar growth. Osmunda cinnamomea L. var. asiatica is one of the oldest fern species with typical separate-growing trophophyll and sporophyll. The chlorophyllous spores generated from sporophyll can germinate without dormancy. In this study, the spore ultrastructure, antioxidant enzyme activities, as well as protein and gene expression patterns were analyzed in the course of spore germination at five typical stages (i.e. mature spores, rehydrated spores, double-celled spores, germinated spores, and spores with protonemal cells). Proteomic analysis revealed 113 differentially expressed proteins, which were mainly involved in photosynthesis, reserve mobilization, energy supplying, protein synthesis and turnover, reactive oxygen species scavenging, signaling, and cell structure modulation. The presence of multiple proteoforms of 25 differentially expressed proteins implies that post-translational modification may play important roles in spore germination. The dynamic patterns of proteins and their encoding genes exhibited specific characteristics in the processes of cell division and rhizoid tip growth, which include heterotrophic and autotrophic metabolisms, de novo protein synthesis and active protein turnover, reactive oxygen species and hormone (brassinosteroid and ethylene) signaling, and vesicle trafficking and cytoskeleton dynamic. In addition, the function skew of proteins in fern spores highlights the unique and common mechanisms when compared with evolutionarily divergent spermatophyte pollen. These findings provide an improved understanding of the typical single-celled asymmetric division and polar growth during fern spore germination.

Morphohistological analysis of the origin and development of Rosa canina protocorm-like bodies

A protocorm-like bodies (PLBs) regeneration system has been established for several Rosa spp. Greater understanding of the PLB formation process, in particular how PLBs are triggered from the rhizoid meristem, would contribute to the establishment of PLB protocols for cut roses that could be applied for rapid propagation and molecular breeding. The present study investigated the morphogenesis of Rosa canina PLBs. Callus rhizoids of R. canina were cultured on thidiazuron medium to induce PLBs, and the developmental process was examined by stereoscope, scanning electron microscopy, and transmission electron microscopy via paraffin sections and ultrathin sections. The results showed that 5 days after callus rhizoids were transferred to thidiazuron medium (DAT), vascular tissue comprising phloem and xylem was formed from the leaf callus to the rhizoid meristem, where a group of cells dividing periclinally emerged at 9 DAT. In the rhizoid tip, the division and enlargement of those dividing cells induced the formation of starch-rich parenchyma cells, and the totipotent cells adjacent to the epidermis differentiated into meristematic centers at about 20 DAT. PLBs were formed from meristematic centers through globular embryos at approximately 35 DAT, and then germinated precociously without going through the heart, torpedo, and cotyledon stages. This research suggests that R. canina PLBs were derived from parenchyma cells adjacent to the epidermis of the rhizoid tip. Vascular tissue of callus rhizoid was essential for the formation of parenchyma cells in the rhizoid meristem.

Inducible growth mode switches influence Valonia rhizoid differentiation

Cell differentiation and cell type commitment are an integral part of plant growth and development. Investigations on how environmental conditions affect the formation of shoots, roots, and rhizoids can help illustrate how plants determine cell fate and overall morphology. In this study, we evaluated the role of substratum and light on rhizoid differentiation in the coenocytic green alga, Valonia aegagropila. Elongating rhizoids displayed varying growth modes and cell shape upon exposure to different substrata and light conditions. It was found that soft substrata and dark incubation promoted rhizoid elongation via tip growth while subsequent exposure to light prevented tip growth and instead induced swelling in the apical region of rhizoids. Swelling was accompanied by the accumulation of protoplasm in the rhizoid tip through expansion of the cell wall and uninhibited cytoplasmic streaming. Subsequent diffuse growth led to the transformation from slender, rod-shaped rhizoids into spherical thallus-like structures that required photosynthesis. Further manipulation of light regimes caused vacillating cell growth redirections. An elongating V. aegagropila rhizoid cell thus appears capable of growth mode switching that is regulated by immediate environmental conditions thereby influencing ultimate cell shape and function. This is the first description of inducible, multiple growth mode shifts in a single intact plant cell that directly impact its differentiation.

Plant regeneration through protocorm-like bodies induced from rhizoids using leaf explants of Rosa spp.

A new protocol for plant regeneration via protocorm-like bodies (PLBs) induced from rhizoids that developed from leaf explants of Rosa spp. (R. canina L., R. multiflora var. cathayensis Rehd. et Wils., and R. multiflora f. carnea Thory.) has been established. Rhizoids were induced from calli of leaf explants incubated under dark conditions on Murashige and Skoog (MS) medium containing 1.5 mg/l 2, 4-D. PLBs developed from the tip of rhizoids cultured under light conditions on (1/2) MS medium containing 20 mg/l TDZ. About 90, 17 and 93% of rhizoid formation were achieved for the above-mentioned Rosa spp., respectively using this protocol. The frequency of PLB clusters formation and the number of PLB clusters per explant reached 50% and 5.1 for R. canina, 46.7% and 0.8 for R. multifolra var. cathayensis, 46.7% and 4.2 for R. multiflora f. carnea, respectively. PLB clusters regenerated on MS medium supplemented with 2 mg/l 6-BA, 0.1 mg/l IBA, and 0.1 mg/l GA(3). The best result of regenerated plantlets per leaf explant achieved via PLBs for the three Rosa spp. mentioned above was 3.6, 0.1, and 1.2, respectively. Environmental scanning electron microscope and histological studies revealed that rhizoids were structurally different from roots grown in vitro, and PLBs developed from proembryos.

The Rac1 inhibitor, NSC23766, depolarizes adhesive secretion, endomembrane cycling, and tip growth in the fucoid alga, Silvetia compressa.

Proper cell morphogenesis is dependent on the establishment and expression of cellular polarity. In the fucoid zygote, cell shape is critical for establishing the developmental pattern of the adult, and is achieved by guiding insertion of new membrane and wall to the rhizoid tip. Selection and growth of the appropriate tip site are accompanied by formation of dynamic actin arrays associated with the actin-nucleating Arp2/3 complex. In eukaryotes, a major pathway for activation of the Arp2/3 complex is via the Rho family GTPase, Rac1, which stimulates the Scar/WAVE complex. To determine whether Rac1 controls actin nucleation in Silvetia compressa (J. Agardh) E. Serrao, T. O. Cho, S. M. Boo et Brawley, we tested the effects of the Rac1-specific inhibitory compound, NSC23766, on actin dependent processes and on actin arrays. We found that NSC23766 disrupted polar secretion of adhesive, polarization of endomembranes, and tip-focused growth in the rhizoid. Similarly, NSC23766 altered actin and Arp2 localization in the growing rhizoid. In contrast, NSC23766 had no effect on selection of the growth site or on cytokinesis. These data suggest that Rac1 participates in nucleation of specific actin arrays in the developing zygote.

A tip-high, Ca(2+) -interdependent, reactive oxygen species gradient is associated with polarized growth in Fucus serratus zygotes.

We report the existence of a tip-high reactive oxygen species (ROS) gradient in growing Fucus serratus zygotes, using both 5-(and 6-) chloromethyl-2',7'-dichlorodihydrofluorescein and nitroblue tetrazolium staining to report ROS generation. Suppression of the ROS gradient inhibits polarized zygotic growth; conversely, exogenous ROS generation can redirect zygotic polarization following inhibition of endogenous ROS. Confocal imaging of fluo-4 dextran distributions suggests that the ROS gradient is interdependent on the tip-high [Ca(2+)](cyt) gradient which is known to be associated with polarized growth. Our data support a model in which localized production of ROS at the rhizoid tip stimulates formation of a localized tip-high [Ca(2+)](cyt) gradient. Such modulation of intracellular [Ca(2+)](cyt) signals by ROS is a common motif in many plant and algal systems and this study extends this mechanism to embryogenesis.

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.

Interactions between parasitic fungi and mosses: pegged and swollen-tipped rhizoids in Funaria and Bryum

Two kinds of parasitic fungal infections are described in the rhizoidal systems of both wild and cultured materials of Funaria hygrometrica, Bryum capillare and B. pseudotriquetrum. Protonemal and rhizoidal cells of Funaria produce pegs of host wall materialaroundsites of hyphal penetration. Similar structures appear to be much more widespread in hepatics and in Monoclea have been misinterpreted as rudimentary pegs, the characteristic feature ofpeggedrhizoids.Swollen side branch initials and the tip cells of major rhizoidal axescontaina single rozellopsidalean (Oomycota formerly Chytridiomycota) zoosporangium in Bryum pseudotriquetrum and up to three similar structures in B. capillare. Each zoosporangium produces a single exit tube. Whereas the infected cells die the subapical cells remain alive and,afterzoospore discharge, produce side branches whose tip cells also become infected. Restriction of the parasites to sidebranchinitials and rhizoid tip cells is perhaps because these are the only cells that the zoospores are able to penetrate. Nutrients translocated along rhizoids to sustaintipgrowth are diverted to nurture the fungi in infected plants. Differences between the number of zoosporangia per host cell and the number of exit tubes between different mosses suggest that more than one species of rozellopsidalean fungus may form rhizoid galls in mosses and not just Pleotracheluswildemanidescribedpreviously.Rhizoid galls appear to be genuinely uncommon in mosses but, following clarification of the differences between these and rhizoidal tubers, further discoveries may be anticipated.

The Arp2/3 complex nucleates actin arrays during zygote polarity establishment and growth.

Previous work has demonstrated that dynamic actin arrays are important for axis establishment and polar growth in the fucoid zygote, Silvetia compressa. Transitions between these arrays are mediated by depolymerization of an existing array and polymerization of a new array. To begin to understand how polymerization of new arrays might be regulated, we investigated the role of the highly conserved, actin-nucleating, Actin-related protein 2/3 (Arp2/3) complex. Arp2, a subunit of the complex, was cloned and peptide antibodies were raised to the C-terminal domain. In immunolocalization studies of polarizing zygotes, actin and Arp2 colocalized around the nucleus and in a patch at the rhizoid pole. In germinated zygotes, a cone of Arp2 and actin extended from the nucleus to the subapex. Within the rhizoid tip, three structural zones were observed in the majority of zygotes: the extreme apex was devoid of label, the subapex was enriched for Arp2, and further back both actin and Arp2 were present. This zonation suggests that actin nucleation occurs at the leading edge of the cone, in the Arp2-enriched region. In two sets of experiments, we showed that tip zonation is important for growth. First, pharmacological treatments that disrupted Arp2/actin zonation arrested tip growth. Second, changes in the direction of tip growth during negative phototropism were preceded by a reorientation of the zonation in accordance with the new growth direction. This work represents the first investigation of Arp2/3 complex localization in tip-growing algal cells.

Localization to the rhizoid tip implicates a Fucus distichus Rho family GTPase in a conserved cell polarity pathway.

Generation and expression of cell polarity in brown algal zygotes of the Fucales involve regulation of the actin cytoskeleton and localized secretion. We used degenerate PCR to isolate cDNAs that encode two small GTPases, FdRac1 and FdRab8, from zygotes of Fucus distichus (L.) Powell. Sequence analysis placed FdRac1 in the Rho family, which regulates actin, and FdRab8 in the Rab family, which regulates vesicle transport. As expected, bacterially expressed forms of both proteins bound GTP in vitro. When expressed in budding yeast, FdRac1 showed some functional overlap with CDC42, the Saccharomyces cerevisiae Rho family gene required for yeast cell polarity. Immunolocalization revealed an asymmetric distribution of FdRac1 in polarized zygotes and embryos, with FdRac1 concentrated at or near the growing tip of the algal rhizoid. Our data support the hypothesis that FdRac1 regulates algal cell polarity, possibly via the actin cytoskeleton. Because brown algae belong to the heterokont group, which diverged from other groups early in eukaryotic evolution, we argue that the Rho family function of regulating cell polarity is ancient and may extend throughout the eukaryotes.

Cortical Actin Filaments Form Rapidly during Photopolarization and Are Required for the Development of Calcium Gradients in Pelvetia compressa Zygotes

Previous research has shown that cortical gradients of cytosolic Ca2+ are formed during the photopolarization of Pelvetia compressa zygotes, with elevated Ca2+ on the shaded hemisphere that will become the site of rhizoid germination. We report here that the marine sponge toxin, latrunculin B, which blocks photopolarization at nanomolar concentrations, inhibited the formation of the light-driven Ca2+ gradients. Using low concentrations of microinjected fluorescent phalloidin as a tracer for actin filaments, we found that exposure to light induced a striking increase in actin filaments in the cells as indicated by an increase in fluorescence. The increase was quantified in the cortex, where it was most apparent, and the fluorescence there was found to increase by about a factor of 3. This increase in cortical phalloidin fluorescence was inhibited by latrunculin B at the same concentration required to inhibit Ca2+ gradient formation and photopolarization. The distribution of the increasing phalloidin fluorescence was uniform with respect to the developing rhizoid–thallus axis during the formation of the axis, and no intense patches of fluorescence were observed. After germination, fluorescence suggestive of an apical ring of actin filaments was seen near the rhizoid tip. Finally, inhibitor studies indicated that myosin may be involved in the photopolarization process.