Pyrenoid proteomics reveals independent evolution of the CO2-concentrating organelle in chlorarachniophytes.

Pyrenoids are microcompartments that are universally found in the photosynthetic plastids of various eukaryotic algae. They contain ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and play a pivotal role in facilitating CO2 assimilation via CO2-concentrating mechanisms (CCMs). Recent investigations involving model algae have revealed that pyrenoid-associated proteins participate in pyrenoid biogenesis and CCMs. However, these organisms represent only a small part of algal lineages, which limits our comprehensive understanding of the diversity and evolution of pyrenoid-based CCMs. Here we report a pyrenoid proteome of the chlorarachniophyte alga Amorphochlora amoebiformis, which possesses complex plastids acquired through secondary endosymbiosis with green algae. Proteomic analysis using mass spectrometry resulted in the identification of 154 potential pyrenoid components. Subsequent localization experiments demonstrated the specific targeting of eight proteins to pyrenoids. These included a putative Rubisco-binding linker, carbonic anhydrase, membrane transporter, and uncharacterized GTPase proteins. Notably, most of these proteins were unique to this algal lineage. We suggest a plausible scenario in which pyrenoids in chlorarachniophytes have evolved independently, as their components are not inherited from green algal pyrenoids.

The baseless mutant links protein phosphatase 2A with basal cell identity in the brown alga Ectocarpus.

The first mitotic division of the initial cell is a key event in all multicellular organisms and is associated with the establishment of major developmental axes and cell fates. The brown alga Ectocarpus has a haploid-diploid life cycle that involves the development of two multicellular generations: the sporophyte and the gametophyte. Each generation deploys a distinct developmental programme autonomously from an initial cell, the first cell division of which sets up the future body pattern. Here, we show that mutations in the BASELESS (BAS) gene result in multiple cellular defects during the first cell division and subsequent failure to produce basal structures during both generations. BAS encodes a type B″ regulatory subunit of protein phosphatase 2A (PP2A), and transcriptomic analysis identified potential effector genes that may be involved in determining basal cell fate. The bas mutant phenotype is very similar to that observed in distag (dis) mutants, which lack a functional Tubulin-binding co-factor Cd1 (TBCCd1) protein, indicating that TBCCd1 and PP2A are two essential components of the cellular machinery that regulates the first cell division and mediates basal cell fate determination.

Assembly and synthesis of the extracellular matrix in brown algae.

In brown algae, the extracellular matrix (ECM) and its constitutive polymers play crucial roles in specialized functions, including algal growth and development. In this review we offer an integrative view of ECM construction in brown algae. We briefly report the chemical composition of its main constituents, and how these are interlinked in a structural model. We examine the ECM assembly at the tissue and cell level, with consideration on its structure in vivo and on the putative subcellular sites for the synthesis of its main constituents. We further discuss the biosynthetic pathways of two major polysaccharides, alginates and sulfated fucans, and the progress made beyond the candidate genes with the biochemical validation of encoded proteins. Key enzymes involved in the elongation of the glycan chains are still unknown and predictions have been made at the gene level. Here, we offer a re-examination of some glycosyltransferases and sulfotransferases from published genomes. Overall, our analysis suggests novel investigations to be performed at both the cellular and biochemical levels. First, to depict the location of polysaccharide structures in tissues. Secondly, to identify putative actors in the ECM synthesis to be functionally studied in the future.

Low-CO2-inducible bestrophins outside the pyrenoid sustain high photosynthetic efficacy in diatoms.

Anion transporters sustain a variety of physiological states in cells. Bestrophins (BSTs) belong to a Cl- and/or HCO3- transporter family conserved in bacteria, animals, algae, and plants. Recently, putative BSTs were found in the green alga Chlamydomonas reinhardtii, where they are upregulated under low CO2 (LC) conditions and play an essential role in the CO2-concentrating mechanism (CCM). The putative BST orthologs are also conserved in diatoms, secondary endosymbiotic algae harboring red-type plastids, but their physiological functions are unknown. Here, we characterized the subcellular localization and expression profile of BSTs in the marine diatoms Phaeodactylum tricornutum (PtBST1 to 4) and Thalassiosira pseudonana (TpBST1 and 2). PtBST1, PtBST2, and PtBST4 were localized at the stroma thylakoid membrane outside of the pyrenoid, and PtBST3 was localized in the pyrenoid. Contrarily, TpBST1 and TpBST2 were both localized in the pyrenoid. These BST proteins accumulated in cells grown in LC but not in 1% CO2 (high CO2 [HC]). To assess the physiological functions, we generated knockout mutants for the PtBST1 gene by genome editing. The lack of PtBST1 decreased photosynthetic affinity for dissolved inorganic carbon to the level comparable with the HC-grown wild type. Furthermore, non-photochemical quenching in LC-grown cells was 1.5 to 2.0 times higher in the mutants than in the wild type. These data suggest that HCO3- transport at the stroma thylakoid membranes by PtBST1 is a critical part of the CO2-evolving machinery of the pyrenoid in the fully induced CCM and that PtBST1 may modulate photoprotection under CO2-limited environments in P. tricornutum.

Targeted CRISPR-Cas9-based gene knockouts in the model brown alga Ectocarpus.

Brown algae are an important group of multicellular eukaryotes, phylogenetically distinct from both the animal and land plant lineages. Ectocarpus has emerged as a model organism to study diverse aspects of brown algal biology, but this system currently lacks an effective reverse genetics methodology to analyse the functions of selected target genes. Here, we report that mutations at specific target sites are generated following the introduction of CRISPR-Cas9 ribonucleoproteins into Ectocarpus cells, using either biolistics or microinjection as the delivery method. Individuals with mutations affecting the ADENINE PHOSPHORIBOSYL TRANSFERASE (APT) gene were isolated following treatment with 2-fluoroadenine, and this selection system was used to isolate individuals in which mutations had been introduced simultaneously at APT and at a second gene. This double mutation approach could potentially be used to isolate mutants affecting any Ectocarpus gene, providing an effective reverse genetics tool for this model organism. The availability of this tool will significantly enhance the utility of Ectocarpus as a model organism for this ecologically and economically important group of marine organisms. Moreover, the methodology described here should be readily transferable to other brown algal species.

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.

Unusual Patterns of Mitochondrial Inheritance in the Brown Alga Ectocarpus.

Most eukaryotes inherit their mitochondria from only one of their parents. When there are different sexes, it is almost always the maternal mitochondria that are transmitted. Indeed, maternal uniparental inheritance has been reported for the brown alga Ectocarpus but we show in this study that different strains of Ectocarpus can exhibit different patterns of inheritance: Ectocarpus siliculosus strains showed maternal uniparental inheritance, as expected, but crosses using different Ectocarpus species 7 strains exhibited either paternal uniparental inheritance or an unusual pattern of transmission where progeny inherited either maternal or paternal mitochondria, but not both. A possible correlation between the pattern of mitochondrial inheritance and male gamete parthenogenesis was investigated. Moreover, in contrast to observations in the green lineage, we did not detect any change in the pattern of mitochondrial inheritance in mutant strains affected in life cycle progression. Finally, an analysis of field-isolated strains provided evidence of mitochondrial genome recombination in both Ectocarpus species.

DISTAG/TBCCd1 Is Required for Basal Cell Fate Determination in Ectocarpus.

Brown algae are one of the most developmentally complex groups within the eukaryotes. As in many land plants and animals, their main body axis is established early in development, when the initial cell gives rise to two daughter cells that have apical and basal identities, equivalent to shoot and root identities in land plants, respectively. We show here that mutations in the Ectocarpus DISTAG (DIS) gene lead to loss of basal structures during both the gametophyte and the sporophyte generations. Several abnormalities were observed in the germinating initial cell in dis mutants, including increased cell size, disorganization of the Golgi apparatus, disruption of the microtubule network, and aberrant positioning of the nucleus. DIS encodes a TBCCd1 protein, which has a role in internal cell organization in animals, Chlamydomonas reinhardtii, and trypanosomes. Our study highlights the key role of subcellular events within the germinating initial cell in the determination of apical/basal cell identities in a brown alga and emphasizes the remarkable functional conservation of TBCCd1 in regulating internal cell organization across extremely distant eukaryotic groups.

Thylakoid luminal θ-carbonic anhydrase critical for growth and photosynthesis in the marine diatom Phaeodactylum tricornutum.

The algal pyrenoid is a large plastid body, where the majority of the CO2-fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) resides, and it is proposed to be the hub of the algal CO2-concentrating mechanism (CCM) and CO2 fixation. The thylakoid membrane is often in close proximity to or penetrates the pyrenoid itself, implying there is a functional cooperation between the pyrenoid and thylakoid. Here, GFP tagging and immunolocalization analyses revealed that a previously unidentified protein, Pt43233, is targeted to the lumen of the pyrenoid-penetrating thylakoid in the marine diatom Phaeodactylum tricornutum The recombinant Pt43233 produced in Escherichia coli cells had both carbonic anhydrase (CA) and esterase activities. Furthermore, a Pt43233:GFP-fusion protein immunoprecipitated from P. tricornutum cells displayed a greater specific CA activity than detected for the purified recombinant protein. In an RNAi-generated Pt43233 knockdown mutant grown in atmospheric CO2 levels, photosynthetic dissolved inorganic carbon (DIC) affinity was decreased and growth was constantly retarded; in contrast, overexpression of Pt43233:GFP yielded a slightly greater photosynthetic DIC affinity. The discovery of a θ-type CA localized to the thylakoid lumen, with an essential role in photosynthetic efficiency and growth, strongly suggests the existence of a common role for the thylakoid-luminal CA with respect to the function of diverse algal pyrenoids.

Intercellular translocation of molecules via plasmodesmata in the multiseriate filamentous brown alga, Halopteris congesta (Sphacelariales, Phaeophyceae).

Despite the high number of studies on the fine structure of brown algal cells, only limited information is available on the intercelluar transportation of molecules via plasmodesmata in brown algae. In this study, plasmodesmatal permeability of Halopteris congesta was examined by observing the translocation of microinjected fluorescent tracers of different molecular sizes. The tip region of H. congesta consists of a cylindrical apical cell, while the basal region is multiseriate. Fluorescein isothiocyanate-dextran (FD; 3, 10, and 20 kDa) and recombinant green fluorescent protein (27 kDa) were injected into the apical cell and were observed to diffuse into the neighboring cells. FD of 40 kDa was detected only in the injected apical cell. The plasmodesmatal size exclusion limit was considered to be more than 20 kDa and less than 40 kDa. The extent of translocation of 3 and 10 kDa FD from the apical to neighboring cells 2 h postinjection was estimated based on the fluorescence intensity. It was suggested that the diffusing capacity of plasmodesmata varied according to molecular size. In order to examine acropetal and/or basipetal direction of molecular movement, 3 and 10 kDa FD were injected into the third cell from the apical cell. Successive observations indicated that the diffusion of fluorescence in the acropetal direction took longer than that in the basipetal direction. No ultrastructural difference in plasmodesmata was noted among the cross walls.

Phototaxis and chemotaxis of brown algal swarmers.

Brown algae exhibit three patterns of sexual reproduction: isogamy, anisogamy, and oogamy. Unicellular swarmers including gametes and zoospores bear two heterogenous flagella, an anterior flagellum with mastigonemes (fine tripartite hairs) and a posterior one. In seawater, these flagellates usually receive physico-chemical signals for finding partners and good habitats. It is well known that brown algal swarmers change their swimming direction depending on blue light (phototaxis), and male gametes do so, based on the sex pheromones from female gametes (chemotaxis). In recent years, the comparative analysis of chemotaxis in isogamy, anisogamy, and oogamy has been conducted. In this paper, we focused on the phototaxis and chemotaxis of brown algal gametes comparing the current knowledge with our recent studies.

Distribution of alginate and cellulose and regulatory role of calcium in the cell wall of the brown alga Ectocarpus siliculosus (Ectocarpales, Phaeophyceae).

MAIN CONCLUSION: This work investigated a correlation between the three-dimensional architecture and compound-components of the brown algal cell wall. Calcium greatly contributes to the cell wall integrity. Brown algae have a unique cell wall consisting of alginate, cellulose, and sulfated polysaccharides. However, the relationship between the architecture and the composition of the cell wall is poorly understood. Here, we investigated the architecture of the cell wall and the effect of extracellular calcium in the sporophyte and gametophyte of the model brown alga, Ectocarpus siliculosus (Dillwyn) Lyngbye, using transmission electron microscopy, histochemical, and immunohistochemical studies. The lateral cell wall of vegetative cells of the sporophyte thalli had multilayered architecture containing electron-dense and negatively stained fibrils. Electron tomographic analysis showed that the amount of the electron-dense fibrils and the junctions was different between inner and outer layers, and between the perpendicular and tangential directions of the cell wall. By immersing the gametophyte thalli in the low-calcium (one-eighth of the normal concentration) artificial seawater medium, the fibrous layers of the lateral cell wall of vegetative cells became swollen. Destruction of cell wall integrity was also induced by the addition of sorbitol. The results demonstrated that electron-dense fibrils were composed of alginate-calcium fibrous gels, and electron negatively stained fibrils were crystalline cellulose microfibrils. It was concluded that the spatial arrangement of electron-dense fibrils was different between the layers and between the directions of the cell wall, and calcium was necessary for maintaining the fibrous layers in the cell wall. This study provides insights into the design principle of the brown algal cell wall.

Phylogeographic data revealed shallow genetic structure in the kelp Saccharina japonica (Laminariales, Phaeophyta).

BACKGROUND: Population structure and genetic diversity of marine organisms in the Northwestern Pacific Ocean exhibited complex patterns. Saccharina japonica is a commercially and ecologically important kelp species widely distributed along the coast of Japan Sea. However, it is still poorly known about population genetics and phylogeographic patterns of wild S. japonica populations on a large geographic scale, which is an important contribution to breeding and conservation of this marine crop. RESULTS: We collected 612 mitochondrial COI and trnW-trnL sequences. Diversity indices suggested that S. japonica populations along the coast of Hokkaido exhibited the highest genetic diversity. Bayesian Analysis of Population Structure (BAPS) revealed four clusters in the kelp species (cluster 1: Hokkaido and South Korea; cluster 2: northwestern Hokkaido; cluster 3: Far Eastern Russia; cluster 4: China). The network inferred from concatenated data exhibited two shallow genealogies corresponding to two BAPS groups (cluster 2 and cluster 3). We did not detect gene flow between the two shallow genealogies, but populations within genealogy have asymmetric gene exchange. Bayesian skyline plots and neutrality tests suggested that S. japonica experienced postglacial expansion around 10.45 ka. CONCLUSIONS: The coast of Hokkaido might be the origin and diversification center of S. japonica. Gene exchange among S. japonica populations could be caused by anthropogenic interference and oceanographic regimes. Postglacial expansions and gene exchange apparently led to more shared haplotypes and less differentiation that in turn led to the present shallow phylogeographical patterns in S. japonica.

Spermatogenesis and auxospore structure in the multipolar centric diatom Hydrosera.

Spermatogenesis and auxospore development were studied in the freshwater centric diatom Hydrosera triquetra. Spermatogenesis was unusual, lacking depauperating cell divisions within the spermatogonangium. Instead, a series of mitoses occurred within an undivided cell to produce a multinucleate plasmodium with peripheral nuclei, which then underwent meiosis. 32 or 64 sperm budded off from the plasmodium leaving a large residual cell containing all the chloroplasts. Similar development apparently occurs in Pleurosira, Aulacodiscus, and Guinardia, these being so distantly related that independent evolution of plasmodial spermatogenesis seems likely. After presumed fertilization, the Hydrosera egg cell expanded distally to form a triangular end part. However, unlike in other triangular diatoms (Lithodesmium, Triceratium), the development of triradiate symmetry was not controlled by the "canonical" method of a perizonium that constrains expansion to small terminal areas of the auxospore wall. Instead, the auxospore wall lacked a perizonium and possessed only scales and a dense mat of thin, apparently entangled strips of imperforate silica. No such structures have been reported from any other centric diatoms, the closest analogs being instead the incunabular strips of some raphid diatoms (Nitzschia and Pinnularia). Whether these silica structures are formed by the normal method (intracellular deposition within a silica deposition vesicle) is unknown. As well as being more rounded than vegetative cells, the initial cell is aberrant in its structure, since it has a less polarized distribution of the "triptych" pores characteristic of the species.

Plasmodesmata of brown algae.

Plasmodesmata (PD) are intercellular connections in plants which play roles in various developmental processes. They are also found in brown algae, a group of eukaryotes possessing complex multicellularity, as well as green plants. Recently, we conducted an ultrastructural study of PD in several species of brown algae. PD in brown algae are commonly straight plasma membrane-lined channels with a diameter of 10-20 nm and they lack desmotubule in contrast to green plants. Moreover, branched PD could not be observed in brown algae. In the brown alga, Dictyota dichotoma, PD are produced during cytokinesis through the formation of their precursor structures (pre-plasmodesmata, PPD). Clustering of PD in a structure termed "pit field" was recognized in several species having a complex multicellular thallus structure but not in those having uniseriate filamentous or multiseriate one. The pit fields might control cell-to-cell communication and contribute to the establishment of the complex multicellular thallus. In this review, we discuss fundamental morphological aspects of brown algal PD and present questions that remain open.

Identification of three proteins involved in fertilization and parthenogenetic development of a brown alga, Scytosiphon lomentaria.

Metabolic pathways of cell organelles may influence the expression of nuclear genes involved in fertilization and subsequent zygote development through a retrograde regulation. In Scytosiphon lomentaria, inheritance of chloroplast is biparental but mitochondria are maternally inherited. Male and female gametes underwent different parthenogenetic outcomes. Most (>99%) male gametes did not differentiate rhizoid cells or survived beyond four-cell stage, while over 95% of female gametes grew into mature asexual plants. Proteomic analysis showed that the protein contents of male and female gametes differed by approximately 1.7%, 12 sex-specific proteins out of 700 detected proteins. Three sex-specific proteins were isolated and identified using CAF-MALDI mass spectrometry and RACE-PCR. Among them, a male gamete-specific homoaconitate hydratase (HACN) and a female gamete-specific succinate semialdehyde dehydrogenase (SSADH) were predicted to be the genes involved in mitochondrial metabolic pathways. The expression level of both mitochondrial genes was dramatically changed at the fertilization event. During parthenogenetic development the male-specific HACN and GTP-binding protein were gradually down-regulated but SSADH stayed up-regulated up to 48h. To observe the effect of chemicals on the expression of these genes, male and female gametes were treated with γ-aminobutyric acid (GABA), hydrogen peroxide and L-ascorbic acid. Among them GABA treatment significantly reduced SSADH gene expression in female gamete but the same treatment induced high upregulation of the gene in male gamete. GABA treatment affected the behavior of gametes and their parthenogenetic development. Both gametes showed prolonged motile stage, retarded settlement and subsequent parthenogenetic development. Our results suggest that male and female gametes regulate mitochondrial metabolic pathways differentially during fertilization, which may be the reason for their physiological and behavioral differences.

Sperm-specific histone H1 in highly condensed sperm nucleus of Sargassum horneri.

Spermatogenesis is one of the most dramatic changes in cell differentiation. Remarkable chromatin condensation of the nucleus is observed in animal, plant, and algal sperm. Sperm nuclear basic proteins (SNBPs), such as protamine and sperm-specific histone, are involved in chromatin condensation of the sperm nucleus. Among brown algae, sperm of the oogamous Fucales algae have a condensed nucleus. However, the existence of sperm-specific SNBPs in Fucales algae was unclear. Here, we identified linker histone (histone H1) proteins in the sperm and analyzed changes in their gene expression pattern during spermatogenesis in Sargassum horneri. A search of transcriptomic data for histone H1 genes in showed six histone H1 genes, which we named ShH1.1a, ShH1b, ShH1.2, ShH1.3, ShH1.4, and ShH1.5. Analysis of SNBPs using SDS-PAGE and LC-MS/MS showed that sperm nuclei contain histone ShH1.2, ShH1.3, and ShH1.4 in addition to core histones. Both ShH1.2 and ShH1.3 genes were expressed in the vegetative thallus and the male and female receptacles (the organs producing antheridium or oogonium). Meanwhile, the ShH1.4 gene was expressed in the male receptacle but not in the vegetative thallus and female receptacles. From these results, ShH1.4 may be a sperm-specific histone H1 of S. horneri.

Ultrastructural study of plasmodesmata in the brown alga Dictyota dichotoma (Dictyotales, Phaeophyceae).

Plasmodesmata are intercellular bridges that directly connect the cytoplasm of neighboring cells and play a crucial role in cell-to-cell communication and cell development in multicellular plants. Although brown algae (Phaeophyceae, Heterokontophyta) are phylogenetically distant to land plants, they nevertheless possess a complex multicellular organization that includes plasmodesmata. In this study, the ultrastructure and formation of plasmodesmata in the brown alga Dictyota dichotoma were studied using transmission electron microscopy and electron tomography with rapid freezing and freeze substitution. D. dichotoma possesses plasma membrane-lined, simple plasmodesmata without internal endoplasmic reticulum (desmotubule). This structure differs from those in land plants. Plasmodesmata were clustered in regions with thin cell walls and formed pit fields. Fine proteinaceous "internal bridges" were observed in the cavity. Ultrastructural observations of cytokinesis in D. dichotoma showed that plasmodesmata formation began at an early stage of cell division with the formation of tubular pre-plasmodesmata within membranous sacs of the cytokinetic diaphragm. Clusters of pre-plasmodesmata formed the future pit field. As cytokinesis proceeded, electron-dense material extended from the outer surface of the mid region of the pre-plasmodesmata and finally formed the nascent cell wall. From these results, we suggest that pre-plasmodesmata are associated with cell wall development during cytokinesis in D. dichotoma.

Ultrastructural observations of mitochondrial morphology through the life cycle of the brown alga, Mutiomo cylindricus (Cutleriaceae, Tilopteridales).

Mitochondrial morphology varies according to development and the physiological conditions of the cell. Here, we performed electron tomography using serial sections to analyze the number, individual volume, and morphological complexity of mitochondria in the cells across two generations in the life cycle of the brown alga Mutimo cylindricus. This species shows a heteromorphic alternation of generations between the macroscopic gametophyte and the crustose sporophyte during its life cycle and displays anisogamous sexual reproduction. We observed the mitochondria in the vegetative cells of gametophytes and sporophytes to mainly show tubular or discoidal shapes with high morphological complexity. The morphology of the mitochondria in the male and female gametes changed to a nearly spherical or oval shape from a tubular or discoidal shape before release. In this species, degradation of the paternal mitochondria was observed in the zygote 2 h after fertilization. Morphological changes in the mitochondria were not observed until 6 h after fertilization. Twenty-four-hour-old zygotes before and after cytokinesis showed a similar number of mitochondria as 6-h-old zygotes; however, the volume and morphological complexity increased. The results indicated that the maternal mitochondria did not undergo fission or fusion until this stage. Based on the analysis results of the number and total volume of mitochondria before and after the release of the gametes, it is possible that the mitochondria in the female gametes fuse immediately before release.

Ultrastructural Observation of Cytokinesis and Plasmodesmata Formation in Brown Algae.

Similar to land plant cells, brown algal cells possess plasmodesmata with minute cytoplasmic tunnels, which enable the direct connection between adjacent cells. Plasmodesmata are distributed depending on the association of their formation with cytokinesis. Primary plasmodesmata are formed during cytokinesis, while secondary plasmodesmata appear on the cell wall septum following cytokinesis. Typically, the plasmodesmata of brown algae are cylindrical without the penetration of desmotubules from the endoplasmic reticulum, and there are no morphological differences between primary and secondary plasmodesmata. This present chapter describes the observation of cytokinesis and primary plasmodesmata formation in brown algae using electron microscopy as well as the examination of polysaccharide distribution using antibodies and enzyme-gold probes.