Kyoko Hatano

Transport of storage proteins to protein storage vacuoles is mediated by large precursor-accumulating vesicles

Novel vesicles that accumulate large amounts of proprotein precursors of storage proteins were purified from maturing pumpkin seeds. These vesicles were designated precursor-accumulating (PAC) vesicles and had diameters of 200 to 400 nm. They contained an electron-dense core of storage proteins surrounded by an electron-translucent layer, and some vesicles also contained small vesicle-like structures. Immunocytochemical analysis revealed numerous electrondense aggregates of storage proteins within the endoplasmic reticulum. It is likely that these aggregates develop into the electron-dense cores of the PAC vesicles and then leave the endoplasmic reticulum. Immunocytochemical analysis also showed that complex glycans are associated with the peripheral region of PAC vesicles but not the electron-dense cores, indicating that Golgi-derived glycoproteins are incorporated into the PAC vesicles. These results suggest that the unique PAC vesicles might mediate a transport pathway for insoluble aggregates of storage proteins directly to protein storage vacuoles.

Light and low-CO2 dependent LCIB/LCIC complex localization in the chloroplast supports the carbon-concentrating mechanism in Chlamydomonas reinhardtii

The carbon-concentrating mechanism (CCM) is essential to support photosynthesis under CO2-limiting conditions in aquatic photosynthetic organisms, including the green alga Chlamydomonas reinhardtii. The CCM is assumed to be comprised of inorganic carbon transport systems that, in conjunction with carbonic anhydrases, maintain high levels of CO2 around ribulose-1, 5-bisphosphate carboxylase/oxygenase in a specific compartment called the pyrenoid. A set of transcripts up-regulated during the induction of the CCM was identified previously and designated as low-CO2 (LC)-inducible genes. Although the functional importance of one of these LC-inducible genes, LciB, has been shown recently, the biochemical properties and detailed subcellular localization of its product LCIB remain to be elucidated. Here, using yeast two-hybrid, immunoprecipitation and mass spectrometry analyses we provide evidence to demonstrate that LCIB interacts with the LCIB homologous protein LCIC in yeast and in vivo. We also show that LCIB and LCIC are co-localized in the vicinity of the pyrenoid under LC conditions in the light, forming a hexamer complex of approximately 350 kDa, as estimated by gel filtration chromatography. LCIB localization around the pyrenoid was dependent on light illumination and LC conditions during active operation of the CCM. In contrast, in the dark or under high-CO2 conditions when the CCM was inactive, LCIB immediately diffused away from the pyrenoid. Based on these observations, we discuss possible functions of the LCIB-LCIC complex in the CCM.

A morphological study of the mycorrhiza of Entoloma clypeatum f. hybridum on Rosa multiflora

Mycorrhizas ofEntoloma clypeatum f.hybridum onRosa multiflora in the field in Japan were studied by stereo, light and electron microscopy. In most mycorrhizas, the root cap, meristem, and apical region of the cortex disappeared, but in a few mycorrhizas, these tissues remained. Fungal hyphae of the mycorrhizas invaded root tissues and branched palmately. Hyphae in contact with cortical cells were larger than those far from the root cells and contained many mitochondria, cisternae of endoplasmic reticulum and transitional vesicles. Invading hyphae were undulate in the apical part of the mycorrhiza, and some of them lacked distinct organelles. Electron-dense granules accumulated in the root cells adjacent to the fungal hyphae. Both the remnants of the plant cells and the fungal hyphae were included in the amorphous materials on the tip of the stele. These observations suggest the destructive infection by fungal hyphae of the root cells and their collapse near the tip of the stele.

Cytosolic subunits of ATP synthase are localized to the cortical endoplasmic reticulum-rich domain of the ascidian egg myoplasm

Previously, we revealed that p58, one of the ascidian maternal factors, is identical to the alpha‐subunit of F1‐ATP synthase (ATPα), a protein complex of the inner mitochondrial membrane. In the current study, we used immunological probes for ascidian mitochondria components to show that the ascidian ATPα is ectopically localized to the cytosol. Virtually all mitochondrial components were localized to the mitochondria‐rich myoplasm. However, in detail, ATP synthase subunits and the matrix proteins showed different localization patterns. At least at the crescent stage, transmission electron microscopy (TEM) distinguished the mitochondria‐less, endoplasmic reticulum (ER)‐rich cortical region and the mitochondria‐rich internal region. ATPα was enriched in the cortical region and MnSOD was limited to the internal region. Using subcellular fractionation, although all of the mitochondria components were highly enriched in the mitochondria‐enriched fraction, a considerable amount of ATPα and F1‐ATP synthase beta‐subunit (ATPβ) were recovered in the insoluble cytoplasmic fraction. Even under these conditions, F1‐ATP synthase gamma‐subunit (ATPγ) and F0‐ATP synthase subunit b (ATPb) were not recovered in the insoluble cytoplasmic fraction. This result strongly supports the exomitochondrial localization of both ATPα and ATPβ. In addition, the detergent extraction of eggs supports the idea that these cytosolic ATP synthase subunits are associated with the egg cytoskeleton. These results suggest that the subunits of ATP synthase might play dual roles at different subcellular compartments during early development.

Growth pattern of isolated zoospores in Hydrodictyon reticulatum (Chlorococcales, Chlorophyceae)

Zoospores at various developmental stages in Hydrodictyon reticulatum were isolated from parent cells and cultured in Waris medium. Isolated zoospores grew to mature vegetative cells, and were able to reproduce zoo‐spores that formed daughter hexagonal nets. Three types of shape appeared in cells 24 h after isolation: cylindrical, Y‐shaped and 4‐armed type. Protrusions of Y‐shaped or 4‐armed cells were formed at an angle of about 120° to the long axis of the cell. When cells were isolated at later stages, more cells became cylindrical in shape and fewer ceils became Y‐shaped or 4‐armed, Direction of cell growth also seemed to depend largely on the developmental stages of the zoospores. The later the isolated stages were, the more the cells elongated along the long axis of the zoospores.

The AP-1 Complex is Required for Proper Mucilage Formation in Arabidopsis Seeds

The adaptor protein (AP) complexes play crucial roles in vesicle formation in post-Golgi trafficking. Land plants have five types of AP complexes (AP-1 to AP-5), each of which consists of two large subunits, one medium subunit and one small subunit. Here, we show that the Arabidopsis AP-1 complex mediates the polarized secretion and accumulation of a pectic polysaccharide called mucilage in seed coat cells. Previously, a loss-of-function mutant of AP1M2, the medium subunit of AP-1, has been shown to display deleterious growth defects because of defective cytokinesis. To investigate the function of AP-1 in interphase, we generated transgenic Arabidopsis plants expressing AP1M2-GFP (green fluorescent protein) under the control of the cytokinesis-specific KNOLLE (KN) promoter in the ap1m2 background. These transgenic plants, designated pKN lines, successfully rescued the cytokinesis defect and dwarf phenotype of ap1m2. pKN lines showed reduced mucilage extrusion from the seed coat. Furthermore, abnormal accumulation of mucilage was found in the vacuoles of the outermost integument cells of pKN lines. During seed development, the accumulation of AP1M2-GFP was greatly reduced in the integument cells of pKN lines. These results suggest that trans-Golgi network (TGN)-localized AP-1 is involved in the trafficking of mucilage components to the outer surface of seed coat cells. Our study highlights an evolutionarily conserved function of AP-1 in polarized sorting in eukaryotic cells.

Isolation and characterization of Chlamydomonas autophagy-related mutants in nutrient-deficient conditions

Autophagy is a recycling system for amino acids and carbon- and nitrogen (N)-containing compounds. To date, the functional importance of autophagy in microalgae in nutrient-deficient conditions has not been evaluated by using autophagy-defective mutants. Here, we provide evidence which supports the following notions by characterizing an insertional mutant of the autophagy-related gene ATG8, encoding a ubiquitin-like protein necessary for the formation of the autophagosome in the green alga, Chlamydomonas reinhardtii. First, ATG8 is required for maintenance of cell survival and Chl content in N-, sulfur- and phosphate-deficient conditions. Secondly, ATG8 supports the degradation of triacylglycerol and lipid droplets after the resupply of N to cells cultured in N-limiting conditions. Thirdly, ATG8 is also necessary for accumulation of starch in phosphate-deficient conditions. Additionally, autophagy is not essential for maternal inheritance of the organelle genomes in gametogenesis.

Zoospore‐specific antigen as a useful marker for molecular analysis of net formation in Hydrodictyon reticulatum (Chlorococcales, Chlorophyceae)

In the green alga Hydrodictyon reticulatum zoospores are arranged in a regular fashion to form an intricate hexagonal network during the asexual reproductive cycle. A monoclonal antibody which was raised against a homogenate of zoospores recognized a single poly‐peptide in zoospores with a molecular mass of 31 kDa. The antigenic polypeptide, which was designated Amy1, was localized within the cytoplasm of zoospores. The accumulation of Amy1 occurred concomitantly with the transition from multinuclear vegetative cells to mononuclear zoospores, and the degradation of Amy1 occurred concomitantly with the further development of zoospores. Amy1 was constantly expressed during the period of mononuclear zoospores. Thus, we conclude that Amy1 is a zoospore‐specific polypeptide. Using the anti‐Amy1 monoclonal antibody, we could easily distinguish between mononuclear zoospores and multinuclear vegetative net‐cells. This provides an important tool for analysing the molecular mechanisms involved in the hexagonal net formation by zoospores.

Effects of concanavalin A on the net formation of zoospores in Hydrodictyon reticulatum (Chlorococcales, Chlorophyceae)

In the green alga Hydrodictyon reticulatum (L.) Lager‐heim (Chlorococcales, Chlorophyceae), zoospores are arranged in a regular fashion to form an intricate hexagonal network during the asexual reproductive cycle. Polypeptides that bind concanavalin A (Con A) in zoospores increased in amount during net formation and decreased after the completion of the adhesion between zoospores. Fluorescein isothiocyanate‐Con A‐binding sites corresponded to the contact sites of zoospores immediately after cessation of the movement. Treatment with 25 μg mL−1 Con A inhibited adhesion of isolated immotile zoospores obtained from parental cells, and Con A‐treated zoospores could not form hexagonal nets. Moreover, when isolated immotile zoospores were treated with Con A, the cessation of the zoospore movement was retarded in dose‐dependent manner. These results suggest that the Con A‐binding sites may participate in the adhesion of zoospores during hexagonal net formation.