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.

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.

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.

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.

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.

Cytoplasmic inheritance of mitochondria and chloroplasts in the anisogamous brown alga Mutimo cylindricus (Phaeophyceae).

Based on the morphology of gametes, sexual reproduction in brown algae is usually classified into three types: isogamy, anisogamy, and oogamy. In isogamy, chloroplasts and chloroplast DNA (chlDNA) in the sporophyte cells are inherited biparentally, while mitochondria (or mitochondrial DNA, mtDNA) is inherited maternally. In oogamy, chloroplasts and mitochondria are inherited maternally. However, the patterns of mitochondrial and chloroplast inheritance in anisogamy have not been clarified. Here, we examined derivation of mtDNA and chlDNA in the zygotes through strain-specific PCR analysis using primers based on single nucleotide polymorphism in the anisogamous brown alga Mutimo cylindricus. In 20-day-old sporophytes after fertilization, mtDNA and chlDNA derived from female gametes were detected, thus confirming the maternal inheritance of both organelles. Additionally, the behavior of mitochondria and chloroplasts in the zygotes was analyzed by examining the consecutive serial sections using transmission electron microscopy. Male mitochondria were isolated or compartmentalized by a double-membrane and then completely digested into a multivesicular structure 2 h after fertilization. Meanwhile, male chloroplasts with eyespots were observed even in 4-day-old, seven-celled sporophytes. The final fate of male chloroplasts could not be traced. Organelle DNA copy number was also examined in female and male gametes. The DNA copy number per chloroplast and mitochondria in male gametes was lower compared with female organelles. The degree of difference is bigger in mtDNA. Thus, changes in different morphology and DNA amount indicate that maternal inheritance of mitochondria and chloroplasts in this species may be based on different processes and timing after fertilization.

Organelle inheritance and genome architecture variation in isogamous brown algae.

Among the brown algal lineages, Ectocarpales species have isogamous fertilization in which male and female gametes are morphologically similar. In contrast, female gametes are much larger than male gametes in the oogamous species found in many other brown algal lineages. It has been reported that the plastids of isogamous species are biparentally inherited whereas the plastids of oogamous species are maternally inherited. In contrast, in both isogamous and oogamous species, the mitochondria are usually inherited maternally. To investigate whether there is any relationship between the modes of inheritance and organellar genome architecture, we sequenced six plastid genomes (ptDNA) and two mitochondrial genomes (mtDNA) of isogamous species from the Ectocarpales and compared them with previously sequenced organellar genomes. We found that the biparentally inherited ptDNAs of isogamous species presented distinctive structural rearrangements whereas maternally inherited ptDNAs of oogamous species showed no rearrangements. Our analysis permits the hypothesis that structural rearrangements in ptDNAs may be a consequence of the mode of inheritance.

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.

Phylogeographic diversification and postglacial range dynamics shed light on the conservation of the kelp Saccharina japonica.

Studies of postglacial range shifts could enhance our understanding of seaweed species' responses to climate change and hence facilitate the conservation of natural resources. However, the distribution dynamics and phylogeographic diversification of the commercially and ecologically important kelp Saccharina japonica in the Northwest Pacific (NWP) are still poorly surveyed. In this study, we analyzed the evolutionary history of S. japonica using two mitochondrial markers and 24 nuclear microsatellites. A STRUCTURE analysis revealed two partially isolated lineages: lineage H, which is scattered along the coast of Japan; and lineage P, which occurs along the west coast of the Japan Sea. Ecological niche modeling projections to the Last Glacial Maximum (LGM) revealed that the southern coasts of the Japan Sea and the Pacific side of the Oshima and Honshu Peninsulas provided the most suitable habitats for S. japonica, implying that these regions served as ancient refugia during the LGM. Ancient isolation in different refugia may explain the observed divergence between lineages P and H. An approximate Bayesian computation analysis indicated that the two lineages experienced post-LGM range expansion and that postglacial secondary contact occurred in Sakhalin. Model projections into the year 2,100 predicted that S. japonica will shift northwards and lose its genetic diversity center on the Oshima Peninsula in Hokkaido and Shimokita Peninsula in Honshu. The range shifts and evolutionary history of S. japonica improve our understanding of how climate change impacted the distribution range and diversity of this species and provide useful information for the conservation of natural resources under ongoing environmental change in the NWP.

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.

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.

Calcium Control of the Sign of Phototaxis in Brown Algal Gametes of Mutimo cylindricus.

Brown algal swarmers usually exhibit positive or negative phototaxis. Such behaviors influence the increasing or decreasing dispersal distance or colonization on the new substratum. We confirmed that the sign of phototaxis (negative or positive) in male gametes of Mutimo cylindricus was affected by extracellular Ca2+ influx through Ca2+ channels. Under the control condition (10-2 m [Ca2+ ]), male gametes swimming with a helical rotation of their cell body mostly showed positive phototaxis. At 10-3 m [Ca2+ ], more than half of the male gametes showed positive phototaxis, whereas the others showed negative phototaxis. From 10-4 -10-5 m [Ca2+ ], the phototactic sign changed to negative. When these negative phototactic gametes were transferred back to the control condition, the phototactic sign reverted to positive. At 10-6 m [Ca2+ ], some of male gametes showed negative phototaxis, but most showed no phototaxis or flagellar beating. Lanthanum, a Ca2+ channel blocker, affected the sign of phototaxis at 10-4 m [La3+ ] under 10-2 m [Ca2+ ], and male gametes mostly showed negative phototaxis. A further increase in [La3+ ] inhibited phototaxis and flagellar beating. These results pointed out the involvement of Ca2+ channels that were blocked by La3+ in phototaxis and flagellar beating.

Mitotic spindle formation in Triparma laevis NIES-2565(Parmales, Heterokontophyta).

The parmalean algae possess a siliceous wall and represent the sister lineage of diatoms; they are thought to be a key group for understanding the evolution of diatoms. Diatoms possess well-characterized and unique mitotic structures, but the mitotic apparatus of Parmales is still unknown. We observed the microtubule (MT) array during interphase and mitosis in Triparma laevis using TEM. The interphase cells had four or five centrioles (∼80 nm in length), from which MTs emanated toward the cytoplasm. In prophase, the bundle of MTs arose at an extranuclear site. The position of centrioles with respect to an MT bundle changed during its elongation. Centrioles were observed on the lateral side of a shorter MT bundle (∼590 nm) and on either side of an extended MT bundle (∼700 nm). In metaphase, the spindle consisted of two types of MTs-MT bundle that passed through a cytoplasmic tunnel in the center of the nucleus and single MTs (possibly kinetochore MTs) that extended from the poles into the nucleus. The nuclear envelope disappeared only at the regions where the kinetochore MTs penetrated. In telophase, daughter chromosomes migrated toward opposite poles, and the MT bundle was observed between segregating chromosomes. These observations showed that MT nucleation does not always occur at the periphery of centrioles through cell cycle and that the spindle of T. laevis has a similar configuration to that of diatoms.

Branch regeneration induced by sever damage in the brown alga Dictyota dichotoma (dictyotales, phaeophyceae).

Tissue wounds are mainly caused by herbivory, which is a serious threat for macro-algae, and brown algae are known to regenerate branches or buds in response to wounding. In the present paper, we describe a branch regeneration system, induced by sever damage, in the brown alga Dictyota dichotoma. Segmentations of juvenile thalli induced branch regenerations unless explants possessed apical cells. Apical excisions in distinct positions elucidated that disruption of an apical cell or disconnection of tissue with an apical cell triggered the branch regeneration. Furthermore, spatial positions of regenerated branches seemed to be regulated by the apical region, which was assumed to generate inhibitory effects for lateral branch regeneration. Mechanical incision, which disrupted tissue continuity with the apical region, induced branch regeneration preferentially below the incision. Although we were unable to identify the candidate inhibitory substance, our results suggested that the apical region may have an inhibitory effect on lateral branch regeneration. Additionally, observations of branch regeneration showed that all epidermal cells in D. dichotoma possess the ability to differentiate into apical cells, directly. This may be the first report of algal transdifferentiation during the wound-stress response.

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.

Chemotactic movement in sperm of the oogamous brown algae, Saccharina japonica and Fucus distichus.

In oogamous species of brown algae such as Saccharina japonica and Fucus distichus, the sperm possess an unusual long posterior flagellum, which oscillates actively and produces a propulsive force during swimming. In this study, we quantitatively analyzed the effect of chemotactic responses on sperm swimming and flagellar waveforms by high-speed video recordings. We found that the thigmotactic response to the chemo-attractant was not enhanced during chemotactic swimming and that the swimming velocity of sperm did not decrease. As concentration of the chemo-attractant decreased, the sperm performed drastic U-turn movements, which was caused by a rapid and large bend of the posterior flagellum. Unilateral bending of the posterior flagellum when sensing a decrease in the concentration of the chemo-attractant may be a common response in male gametes during fertilization of brown algae both oogamous and isogamous species.