Gamete dimorphism of the isogamous green alga (Chlamydomonas reinhardtii), is regulated by the mating type-determining gene, MID.

The gametes of chlorophytes differ morphologically even in isogamy and are divided into two types (α and ß) 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 ß 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.

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.

Asexual thalli originated from sporophytic thalli via apomeiosis in the green seaweed Ulva.

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.

Metabolome profiling of various seaweed species discriminates between brown, red, and green algae.

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.

Genomic structure and evolution of the mating type locus in the green seaweed Ulva partita.

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.

Culture and hybridization experiments on an ulva clade including the Qingdao strain blooming in the yellow sea.

In the summer of 2008, immediately prior to the Beijing Olympics, a massive green tide of the genus Ulva covered the Qingdao coast of the Yellow Sea in China. Based on molecular analyses using the nuclear encoded rDNA internal transcribed spacer (ITS) region, the Qingdao strains dominating the green tide were reported to be included in a single phylogenetic clade, currently regarded as a single species. On the other hand, our detailed phylogenetic analyses of the clade, using a higher resolution DNA marker, suggested that two genetically separate entities could be included within the clade. However, speciation within the Ulva clade has not yet been examined. We examined the occurrence of an intricate speciation within the clade, including the Qingdao strains, via combined studies of culture, hybridization and phylogenetic analysis. The two entities separated by our phylogenetic analyses of the clade were simply distinguished as U. linza and U. prolifera morphologically by the absence or presence of branches in cultured thalli. The inclusion of sexual strains and several asexual strains were found in each taxon. Hybridizations among the sexual strains also supported the separation by a partial gamete incompatibility. The sexually reproducing Qingdao strains crossed with U. prolifera without any reproductive boundary, but a complete reproductive isolation to U. linza occurred by gamete incompatibility. The results demonstrate that the U. prolifera group includes two types of sexual strains distinguishable by crossing affinity to U. linza. Species identification within the Ulva clade requires high resolution DNA markers and/or hybridization experiments and is not possible by reliance on the ITS markers alone.

ISOLATION AND TEMPORAL EXPRESSION ANALYSIS OF FRESHWATER-INDUCED GENES IN ULVA LIMNETICA (ULVALES, CHLOROPHYTA)1.

The macroalga Ulva limnetica K. Ichihara et S. Shimada is the only known Ulva species to be distributed exclusively in freshwater and is restricted to freshwater bodies in the Ryuku archipelago. Molecular phylogenetic analysis suggests that U. limnetica originally evolved from marine forms of Ulva. The mechanisms of adaptation to freshwater in Ulva spp. are poorly understood. In this study, we isolated genes potentially involved in adaptation or tolerance to freshwater conditions in U. limnetica, using suppression subtractive hybridization between mRNAs of samples cultured in freshwater and seawater conditions. A total of 219 genes, up-regulated by the exposure of the macroalga to freshwater, were isolated. Reverse transcription-PCR (RT-PCR) revealed 39 clones, including malate dehydrogenase, soluble starch synthase, triosephosphate isomerase, plastid ribosomal protein, DnaJ-like protein, and chloroplast ascorbate peroxidase (APX), which were specifically or preferentially expressed in freshwater conditions. These 39 clones were also analyzed for their temporal transcriptional response to freshwater conditions. A large majority of these up-regulated genes showed a transient peak of expression after 1-4 h, followed in the next 24 h by a decrease to a stable level (over the 7 d of the experiment). After the initial response peak, the level of expression either remained higher than in the control (long-term response) or returned to a level similar to pretreatment level. A few genes showed a more delayed response (i.e., after several days) to freshwater exposure. Finally, we discussed the possible contributions of the freshwater-induced genes in the acquisition of freshwater adaptation or tolerance of U. limnetica.