Brown algae, as multicellular organisms, possess plasmodesmata (PD) that connect adjacent cells and facilitate cell-to-cell interactions, similar to land plants. In brown algae, PD are tiny tubular cytoplasmic channels measuring 10-20 nm in diameter. The distribution of PD appears to depend on body structure: in uni- and multiseriate filaments, PD are scattered across cross walls, whereas in thalli undergoing cellular differentiation, PD are concentrated into localized regions known as pit fields (PF). However, it remains unclear how the developmental reorganization of PD contributes to the regulation of intercellular communication, as information on the size exclusion limit (SEL) in PF remains limited. In this study, we investigated early development from zygotes and protoplasts in sporophytes of Saccharina japonica, the brown alga with a highly differentiated thallus, to determine the timing of PD distribution and the properties of PF-mediated intercellular transport. We found that PF were already present at the two-celled stage in both the zygote- and protoplast-derived developmental pathways, in contrast to observations in Scytosiphon lomentaria, Mutimo cylindricus, Silvetia babingtonii, and Fucus distichus. Quantitative measurements revealed that PF width increased during development, whereas the spacing between individual PD remained relatively constant. Furthermore, analysis of intercellular transport using gold nanoparticles conjugated with 3 kDa and 10 kDa FITC-dextran, introduced into the epidermal cells of differentiated sporophytes by particle bombardment, demonstrated size-dependent movement through PF.

Ulva is a green seaweed that is widely distributed in coastal areas around the world, and genomic information has been reported for several species. However, research on reverse genetics methods is still in its early stages. Although our group has recently achieved efficient knockout of Ulva by introducing Cas9 ribonucleoprotein (RNP) through the PEG method, a precise knock-in transformation method is needed to understand gene and protein functions. Here, we report the development of a targeted insertional mutagenesis method by transfection of Cas9 RNP, donor dsDNA, and single-stranded DNAs homologous to the DSB site created by Cas9 and donor dsDNA. The target gene was RbcS, a well-known high-expression gene, and we attempted to knock in the EGFP or 2A peptide-EGFP sequence at the C-terminus region. This method was also applied to the adenine phosphoribosyltransferase (APT) gene, enabling selection by the toxic compound 2-fluoroadenine. A longer DNA fragment (2.6 kb) expressing EGFP under the RbcS promoter was inserted at the DSB site on the APT locus. As a result, EGFP knock-in transformants were obtained, and the transformant progeny exhibited high EGFP expression levels for several generations. Our method greatly advances the genetic engineering and functional analysis of target genes in Ulva.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated genome editing has been used for reverse genetics studies in many organisms. However, application to commercially important species of seaweeds is still limited. The genetics and breeding technologies for species of the Laminariales will be advanced by developing a genome editing tool. In this study, we attempted to edit a counter-selectable marker, the adenine phosphoribosyl transferase (APT) gene using CRISPR-Cas9 ribonucleoprotein (RNP) complexes delivered by microinjection into gametophytes of Saccharina japonica. After injection of CRISPR-Cas9 RNP, 2-fluoroadenine (2-FA) was added to the medium (10–40 μM) to select APT mutants. Twenty-three female and 12 male 2-FA resistant gametophytes had mutations in their APT genes. Genome editing efficiency for the injection trials was 8.64

Although the green seaweed Ulva is one of the most common seaweeds in the coastal regions with well-studied ecological characteristics, few reverse genetic technologies have been developed for it. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is a simple genome-editing technology based on a ribonucleoprotein (RNP) complex composed of an endonuclease and programmable RNA to target particular DNA sequences. Genome editing makes it possible to generate mutations on a target gene in non-model organisms without established transgenic technologies. In this study, we applied the CRISPR-Cas9 RNP genome-editing system to the green seaweed Ulva prolifera, using polyethylene glycol (PEG)-mediated transfection. Our experimental system disrupts a single gene (UpAPT) encoding adenine phosphoribosyl transferase (APT) and generates a resistant phenotype for gametophytes cultured in a medium with toxic compound 2-fluoroadenine. The PEG-mediated transfection used for gametes resulted in 2-fluoroadenine-resistant strains containing short indels or substitutions on UpAPT. Our results showed that the CRISPR-Cas9 system with PEG-mediated transfection was efficient for genome editing in Ulva.

The gametes of chlorophytes differ morphologically even in isogamy and are divided into two types (α and β) based on the mating type- or sex-specific asymmetric positioning of the mating structure (cell fusion apparatus) with respect to the flagellar beat plane and eyespot, irrespective of the difference in gamete size. However, the relationship between this morphological trait and the mating type or sex determination system is unclear. Using mating type-reversed strains of the isogamous alga Chlamydomonas reinhardtii , produced by deletion or introduction of the mating type-determining gene MID , we revealed that the positioning of the mating structure is associated with conversion of mating types (mt – and mt + ), implying that this trait is regulated by MID . Moreover, the dominant mating type is associated with the type β phenotype, as in the chlorophyte species Ulva prolifera . Our findings may provide a genetic basis for mating type- or sex-specific asymmetric positioning of the chlorophyte mating structure.

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.

Apomixis is an asexual reproduction system without fertilization, which is an important proliferation strategy for plants and algae. Here, we report on the apomeiosis in the green seaweed Ulva prolifera , which has sexual and obligate asexual populations. Genomic PCR of mating type (MT)-locus genes revealed asexual thalli carrying both MT genomes. Observation of the chromosomes during the formation of each type of reproductive cell revealed that cells in asexual thalli performed apomeiosis without chromosome reduction. Moreover, genotyping revealed that laboratory-cultured sporophytic thalli produced not only each type of gametophyte but also diploid thalli carrying the mt − and mt + genome (mt ± thallus strains). The mt ± thallus strain released diploid biflagellate zoids, with ultrastructure and behavior similar to mt + gametes. Additionally, a transcriptomic analysis revealed that some meiosis-related genes ( Mei2L and RAD1 ) were highly expressed in the quadriflagellate zoosporoids. Our results strongly suggest that asexual thalli originally evolved via apomeiosis in sporophytic thalli.

MAIN CONCLUSION:Among seaweed groups, brown algae had characteristically high concentrations of mannitol, and green algae were characterised by fructose. In red algae, metabolite profiles of individual species should be evaluated. Seaweeds are metabolically different from terrestrial plants. However, general metabolite profiles of the three major seaweed groups, the brown, red, and green algae, and the effect of various extraction methods on metabolite profiling results have not been comprehensively explored. In this study, we evaluated the water-soluble metabolites in four brown, five red, and two green algae species collected from two sites in northern Japan, located in the Sea of Japan and the Pacific Ocean. Freeze-dried seaweed samples were processed by methanol-water extraction with or without chloroform and analysed by capillary electrophoresis- and liquid chromatography-mass spectrometry for metabolite characterisation. The metabolite concentration profiles showed distinctive characteristic depends on species and taxonomic groups, whereas the extraction methods did not have a significant effect. Taxonomic differences between the various seaweed metabolite profiles were well defined using only sugar metabolites but no other major compound types. Mannitol was the main sugar metabolites in brown algae, whereas fructose, sucrose, and glucose were found at high concentrations in green algae. In red algae, individual species had some characteristic metabolites, such as sorbitol in Pyropia pseudolinearis and panose in Dasya sessilis. The metabolite profiles generated in this study will be a resource and provide guidance for nutraceutical research studies because the information about metabolites in seaweeds is still very limited compared to that of terrestrial plants.

Since 2008, the green seaweed Ulva prolifera has caused the world's largest green tide in the Yellow Sea, China. It has subsequently attracted considerable research interest. However, species identification is an essential step for advancing this research. Based on phylogenetic analyses using molecular sequences such as internal transcribed spacer (ITS) or ribulose bisphosphate carboxylase large chain (rbcL), specimens of U. prolifera collected worldwide were separated into a European Glade and the Ulva linza - procera - prolifera (LPP) complex Glade that included the Chinese bloom-forming strains and Japanese brackish strains. This has resulted in considerable controversy as to the identity of U. prolifera and the bloom-forming species in the Yellow Sea. To resolve this issue, we examined populations of U. prolifera from the type locality at Lolland Island, Denmark, and globally significant sites including sites from Japan and China using morphological, developmental, molecular and crossing studies. We found that almost all the Danish strains agree with the description of the type specimen and were included in the LPP Glade. They had a branched morphology in culture and an obligate asexual life history with quadriflagellate zoosporoids. We conclude that this taxon in the LPP Glade is true U. prolifera. Based on culture morphology, mating compatibility and the 5S rDNA spacer sequences, the Chinese bloom-forming strains were distinct, and the new subspecies U. prolifera subsp. qingdaoensis subsp. nov. is described. Strains of the European Glade showing gamete incompatibility with the sexual members of the LPP Glade were assigned to the species Ulva splitiana.

The evolution of sex chromosomes and mating loci in organisms with UV systems of sex/mating type determination in haploid phases via genes on UV chromosomes is not well understood. We report the structure of the mating type (MT) locus and its evolutionary history in the green seaweed Ulva partita, which is a multicellular organism with an isomorphic haploid-diploid life cycle and mating type determination in the haploid phase. Comprehensive comparison of a total of 12.0 and 16.6 Gb of genomic next-generation sequencing data for mt− and mt+ strains identified highly rearranged MT loci of 1.0 and 1.5 Mb in size and containing 46 and 67 genes, respectively, including 23 gametologs. Molecular evolutionary analyses suggested that the MT loci diverged over a prolonged period in the individual mating types after their establishment in an ancestor. A gene encoding an RWP-RK domain-containing protein was found in the mt− MT locus but was not an ortholog of the chlorophycean mating type determination gene MID. Taken together, our results suggest that the genomic structure and its evolutionary history in the U. partita MT locus are similar to those on other UV chromosomes and that the MT locus genes are quite different from those of Chlorophyceae.

Phylogenetic clades based on DNA sequence data are heavily used to delimit species in the current taxonomy of Ulva. However, because hybridisation within clades and among other clades for other species has been seldom tested, it remains unclear if molecular clades agree with species boundaries based on the biological species concept. An ITS-based Ulva clade including many specimens collected worldwide was provisionally named 'U. flexuosa'; its species boundary is ambiguous, and in the literature this group has been variously merged with the closely related U. californica clade. In the present study, we clarified the species boundary of this clade and its taxonomic status. Namely reproductive relationships among strains in this and closely related clades were examined by culturing and hybridisation. The culture experiment demonstrated that the ITS-based clade includes a sexual variant and an obligate asexual variant. The sexual strains having 0%-0.4% divergence in ITS2 sequence successfully crossed with each other. In combinations among the closely related clades including true U. flexuosa with. > 1.2% distance in the sequence, no hybridisation occurred. With U. californica strains having a 1.2% sequence divergence, the provisional 'U. flexuosa' strains had few hybrid sporophytes, and these produced zoids that failed to develop normally. Based on these observations of prezygotic and postzygotic isolation, we conclude that the ITS-based clade delimits a different species from U. flexuosa and U. californica. Taxonomically, both the sexual and the asexual variants are assigned to U. mediterranea.

Two Rhizoclonium strains thriving in contrasting spring types (slightly hot spring/ambient, shaded pool spring) and biogeographic areas (El-Farafra Oasis in the Western Desert of Egypt/River-Po Plain in Northern Italy) were studied in depth based on field and cultured material, bright-field and fluorescence microscopy, and phylogenetic molecular analyses (SSU and LSU rRNA gene sequences). This polyphasic approach revealed that the two Rh. strains clearly belonged to the Rhizoclonium clade but differed in some of their key diagnostic features. The Egyptian Rhizoclonium strain, isolated from the spring-fed (Ain El-Balad) agricultural ditch in El-Farafra Oasis, was described with the working name 'Rhizoclonium sp. 10.6 μm from a desert, slightly-hot spring' based on its smaller cell diameter, ecological and phylogenetic molecular traits compared to allied morphospecies. Moreover, it was highlighted that the L/D ratio can be more than 3.0 within this group. The Italian Rhizoclonium strain, collected from the Fontanile Valle Re-shaded, pool ambient spring (Emilia-Romagna Region), was virtually identical to other strains identified as Rh. hieroglyphicum from China and Japan, due to its highly supported congruence in morphological characteristics and phylogenetic position. This study provided the first partial LSU and SSU rRNA gene sequences for European Rh. hieroglyphicum based on available literature. Phenotypic plasticity of rhizoid formation was also observed in both Rhizoclonium strains studied using culturing approaching techniques. Our investigation also confirms that a lot of work with a variety of approaches is still needed to assess the ecological preferences, morphological plasticity, and phylogenetics of freshwater Rhizoclonium taxa worldwide.

Microfilamentous unbranched cladophoralean algae are distributed from high latitudes to the tropics in waters of varying salinities. Their simple morphology provides few diagnostic characteristics for distinguishing species, such that the taxonomic status of minute Cladophoraceae remains unclear. In a recent field survey in Japanese mangrove forests and brackish waters, we found Rhizoclonium-like cladophoralean algae (Cladophoraceae spp. 1-3). In molecular phylogenetic analyses based on partial small subunit ribosomal RNA (SSU) and large subunit ribosomal RNA (LSU) genes, these algae clustered with R. africanum Kutzing in the LSU tree and were separated from the true Rhizoclonium clade in both the LSU and SSU trees. An early divergence in the Cladophoraceae lineage was also strongly supported in both trees. Cladophoraceae spp. 1-3 and R. africanum shared a specific rhizoidal developmental structure, the "knee-like joint." Cladophoraceae spp. 1-3 also shared polypyramidal pyrenoids, while typical bilenticular pyrenoids are hardly found. Cladophoraceae sp. 1 is characterized by wider cells and more nuclei per cell than described taxa. Cladophoraceae sp. 3 had a smaller cell length/diameter ratio and fewer nuclei per cell than described taxa. Based on these distinct morphological features, we describe two new species: R. fractum Ichihara et Miyaji sp. nov. and R. umbraticum Ichihara et Miyaji sp. nov. We also revealed that Cladophoraceae sp. 2 should be assigned to the described species R. minutissimum (Zeller) P. C. Silva based on morphological characteristics.

In this report, Ulva partita Ichihara, sp. nov. (Ulvales, Ulvophyceae) was described from Japanese seashores. This species was characterized by (i) frequent branches in the basal region, but not in the middle to upper region, (ii) being up to 20 cm in height and 5mm in diameter, and (iii) containing chloroplasts covering the outer cell wall in the middle and upper regions of thalli, but with many chloroplasts leaning to one side of the cells in the basal region. Ulva partita is distinguished from morphologically similar species based on the branching pattern, early development pattern, and gamete size. Molecular phylogenetic analyses of nuclear-encoded ITS2 sequence and chloroplast-encoded rbcL sequences also strongly supported the independence of this species.