Taizo Motomura

Distribution and phylogeny of the blue light receptors aureochromes in eukaryotes

The new type blue light (BL) receptor aureochrome (AUREO) was recently discovered in a stramenopile alga, Vaucheria (Takahashi et al. Proc Natl Acad Sci USA 104(49):19625–19630, 2007). AUREO has a bZIP (basic region/leucine zipper) and BL-sensing light-oxygen-voltage (LOV) domain and functions as a BL-activated transcription factor. It mediates BL-induced branching and regulates the development of the sex organ in V. frigida. Although AUREO sequences have previously been found in Fucus and some diatoms, here we report that AUREO orthologs are commonly conserved in photosynthetic stramenopiles. Five AUREO orthologs were isolated from three stramenopile genera (Fucus, Ochromonas, and Chattonella). By BLAST search, several AUREO sequences were also detected in genomes in Aureococcus anophagefferens (Pelagophyceae). However, AUREO was not found in heterotrophic stramenopiles or in closely related phyla, such as haptophytes and cryptophytes, or in green plants. Stramenopiles do not possess phototropin, the well-known BL receptor for phototropism of green plants. From comparative analysis of LOV domains, together with kinship analysis of AUREO bZIP domains, AUREO can be regarded as the BL receptor specific to phototrophic stramenopiles. The evolution of AUREO and the phylogeny of LOV domains in stramenopiles and green plants are discussed.

Overexpression of the NAC transcription factor family gene ANAC036 results in a dwarf phenotype in Arabidopsis thaliana

NAC proteins comprise one of the largest families of transcription factors in the plant genome. They are known to be involved in various aspects of plant development, but the functions of most of them have not yet been determined. ANAC036, a member of the Arabidopsis NAC transcription factor family, contains unique sequences that are conserved among various NAC proteins found in other plant species. Expression analysis of the ANAC036 gene indicated that this gene was strongly expressed in leaves. Transgenic plants overexpressing the ANAC036 gene showed a semidwarf phenotype. The lengths of leaf blades, petioles and stems of these plants were smaller than those in wild-type plants. Microscopy revealed that cell sizes in leaves and stems of these plants were smaller than those in wild-type plants. These findings suggested that ANAC036 and its orthologues are involved in the growth of leaf cells.

Inheritance of mitochondrial and chloroplast genomes in the isogamous brown alga Scytosiphon lomentaria (Phaeophyceae)

Patterns of inheritance of chloroplasts and mitochondria were examined by fluorescence microscopy and haplotype genome markers in the isogamous brown alga Scytosiphon lomentaria (Lyngbye) Link. Germination of the zygote in this species was unilateral, the growing thallus developed entirely from the germ tube, and the original zygote cell did not develop except for the formation of a hair. Inheritance of chloroplasts was biparental, and partitioning of the two parental chloroplasts into the first sporophytic cells was accidental: either the maternal or the paternal chloroplast was migrated from the zygote into the germ tube cell, whereas the other chloroplast remained in the original cell. In contrast, the mitochondrial genome in all cells of the sporophyte came only from the female gamete (maternal inheritance). These inheritance patterns are similar to those of the isogamous brown alga Ectocarpus siliculosus (Dillwyn) Lyngbye. Maternal inheritance of mitochondria might be universal in brown algae.

Proteomics analysis of heterogeneous flagella in brown algae (stramenopiles).

Flagella are conserved organelles among eukaryotes and they are composed of many proteins, which are necessary for flagellar assembly, maintenance and function. Stramenopiles, which include brown algae, diatoms and oomycetes, possess two laterally inserted flagella. The anterior flagellum (AF) extends forward and bears tripartite mastigonemes, whilst the smooth posterior flagellum (PF) often has a paraflagellar body structure. These heterogeneous flagella have served as crucial structures in algal studies especially from a viewpoint of phylogeny. However, the protein compositions of the flagella are still largely unknown. Here we report a LC-MS/MS based proteomics analysis of brown algal flagella. In total, 495 flagellar proteins were identified. Functional annotation of the proteome data revealed that brown algal flagellar proteins were associated with cell motility, signal transduction and various metabolic activities. We separately isolated AF and PF and analyzed their protein compositions. This analysis led to the identification of several AF- and PF-specific proteins. Among the PF-specific proteins, we found a candidate novel blue light receptor protein involved in phototaxis, and named it HELMCHROME because of the steering function of PF. Immunological analysis revealed that this protein was localized along the whole length of the PF and concentrated in the paraflagellar body.

Identification and characterization of a fluorescent flagellar protein from the brown alga Scytosiphon lomentaria (Scytosiphonales, Phaeophyceae): A flavoprotein homologous to Old Yellow Enzyme

The posterior flagellum of the zoospore of the brown alga Scytosiphon lomentaria exhibits bright green autofluorescence. To identify the fluorescent flagellar substance(s), we isolated flagella from zoospores and partially purified a flavoprotein by anion-exchange and gel-filtration chromatography. Spectrofluorometric and chromatographic analyses showed that the flavoprotein had an apparent molecular mass of 41 kDa and a non-covalently bound flavin mononucleotide as a chromophore. Based on partial amino acid sequences of the protein, a cDNA of the 41-kDa flavoprotein was cloned and sequenced. The deduced amino acid sequence of the cDNA was homologous to that of the Old Yellow Enzyme family distributed in proteobacteria, yeasts and vascular plants.

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.

Membrane fusion process and assembly of cell wall during cytokinesis in the brown alga, Silvetia babingtonii (Fucales, Phaeophyceae)

During cytokinesis in brown algal cells, Golgi-derived vesicles (GVs) and flat cisternae (FCs) are involved in building the new cell partition membrane. In this study, we followed the membrane fusion process in Silvetia babingtonii zygotes using electron microscopy together with rapid freezing and freeze substitution. After mitosis, many FCs were formed around endoplasmic reticulum clusters and these then spread toward the future cytokinetic plane. Actin depolymerization using latrunculin B prevented the appearance of the FCs. Fusion of GVs to FCs resulted in structures that were thicker and more elongated (EFCs; expanded flat cisternae). Some complicated membranous structures (MN; membranous network) were formed by interconnection of EFCs and following the arrival of additional GVs. The MN grew into membranous sacs (MSs) as gaps between the MNs disappeared. The MSs were observed in patches along the cytokinetic plane. Neighboring MSs were united to form the new cell partition membrane. An immunocytochemical analysis indicated that fucoidan was synthesized in Golgi bodies and transported by vesicles to the future cytokinetic plane, where the vesicles fused with the FCs. Alginate was not detected until the MS phase. Incubation of sections with cellulase-gold showed that the cellulose content of the new cross wall was not comparable to that of the parent cell wall.

Reexamination of the Pit Plugs and the Characteristic Membranous Structures in Porphyra Yezoensis (Bangiales, Rhodophyta)

C. Ueki, C. Nagasato, T. Motomura and N. Saga. 2008. Reexamination of the pit plugs and the characteristic membranous structures in Porphyra yezoensis (Bangiales, Rhodophyta). Phycologia 47: 5–11. DOI: 10.2216/07–12.1 Pit plug formation during conchosporogenesis in Porphyra yezoensis Ueda was studied with electron microscopy using freeze substitution. Just after nuclear division, cytokinesis started with furrowing of the septum. During furrowing, small vesicles were produced by projections of the plasma membrane in the space between the plasma membrane and the cell wall material and occasionally formed long chains. Afterwards, the plug core was gradually formed by endoplasmic reticulum, becoming vertical to the septum. The plasma membrane expanded like a loop along the pit plug from both sides of the furrowing septum. Lomasomes were located near the plasma membrane, beside the pit plug. With maturation of conchosporangia, the number of lomasomes increased near the pit plug. P. yezoensis has a characteristic expanding process of the plasma membrane along the pit plug, which is unique in the red algae.

Comparative immunofluorescence and ultrastructural analysis of microtubule organization in Uronema sp., Klebsormidium flaccidum, K. subtilissimum, Stichococcus bacillaris and S. chloranthus (Chlorophyta).

A detailed comparative examination of microtubule (MT) organization in interphase and dividing cells of Uronema sp., Klebsormidium flaccidum, K. subtilissimum, Stichococcus bacillaris and S. chloranthus was made using tubulin immunofluorescence and transmission electron microscopy (TEM). During interphase all the species bear a well-organized cortical MT system, consisting of parallel bundles with different orientations. In Uronema sp. the cortical MT bundles are longitudinally oriented, whereas in the other species they are in transverse orientation to the axis of the cells. Considerable differences in MT organization were also observed during stages of mitosis, mainly preprophase, as well as cytokinesis. In Uronema sp., a particular radial MT assembly is organized during preprophase-early prophase, which was not observed in the other species. In Stichococcus a fine MT ring surrounded the nucleus during preprophase and prophase. An MT ring, together with single cytoplasmic MTs, was also found associated with the developing diaphragm during cytokinesis in Stichococcus. A phycoplast participates in cytokinesis in Uronema sp., but not in the other species. In Uronema sp. the centrosome functions as a microtubule organizing center (MTOC) during mitosis, but not during interphase and cytokinesis. The phylogenetic significance of these differences is discussed in combination with SSU/ITS sequencing and other, existing molecular data.

Ultrastructural analysis of flagellar development in plurilocular sporangia of Ectocarpus siliculosus (Phaeophyceae)

Flagellar development in the plurilocular zoidangia of sporophytes of the brown alga Ectocarpus siliculosus was analyzed in detail using transmission electron microscopy and electron tomography. A series of cell divisions in the plurilocular zoidangia produced the spore-mother cells. In these cells, the centrioles differentiated into flagellar basal bodies with basal plates at their distal ends and attached to the plasma membrane. The plasma membrane formed a depression (flagellar pocket) into where the flagella elongated and in which variously sized vesicles and cytoplasmic fragments accumulated. The anterior and posterior flagella started elongating simultaneously, and the vesicles and cytoplasmic fragments in the flagellar pocket fused to the flagellar membranes. The two flagella (anterior and posterior) could be clearly distinguished from each other at the initial stage of their development by differences in length, diameter and the appendage flagellar rootlets. Flagella continued to elongate in the flagellar pocket and maintained their mutually parallel arrangement as the flagellar pocket gradually changed position. In mature zoids, the basal part of the posterior flagellum (paraflagellar body) characteristically became swollen and faced the eyespot region. Electron dense materials accumulated between the axoneme and the flagellar membrane, and crystallized materials could also be observed in the swollen region. Before liberation of the zoospores from the plurilocular zoidangia, mastigoneme attachment was restricted to the distal region of the anterior flagellum. Structures just below the flagellar membrane that connected to the mastigonemes were clearly visible by electron tomography.