Maki Yamamoto

Complementation of sugary-1 phenotype in rice endosperm with the wheat isoamylase1 gene supports a direct role for isoamylase1 in amylopectin biosynthesis.

To examine the role of isoamylase1 (ISA1) in amylopectin biosynthesis in plants, a genomic DNA fragment from Aegilops tauschii was introduced into the ISA1-deficient rice (Oryza sativa) sugary-1 mutant line EM914, in which endosperm starch is completely replaced by phytoglycogen. A. tauschii is the D genome donor of wheat (Triticum aestivum), and the introduced fragment effectively included the gene for ISA1 for wheat (TaISA1) that was encoded on the D genome. In TaISA1-expressing rice endosperm, phytoglycogen synthesis was substantially replaced by starch synthesis, leaving only residual levels of phytoglycogen. The levels of residual phytoglycogen present were inversely proportional to the expression level of the TaISA1 protein, although the level of pullulanase that had been reduced in EM914 was restored to the same level as that in the wild type. Small but significant differences were found in the amylopectin chain-length distribution, gelatinization temperatures, and A-type x-ray diffraction patterns of the starches from lines expressing TaISA1 when compared with wild-type rice starch, although in the first two parameters, the effect was proportional to the expression level of TaISA. The impact of expression levels of ISA1 on starch structure and properties provides support for the view that ISA1 is directly involved in the synthesis of amylopectin.

Relationship between presence of a mother cell wall and speciation in the unicellular microalga Nannochloris (Chlorophyta)

The cell division mechanisms of seven strains from six species of Nannochloris Naumann were analyzed and compared with those of three species of Chlorella Beijerinck and Trebouxia erici Ahmadjian using differential interference microscopy and fluorescence microscopy. Nannochloris bacillaris Naumann divides by binary fission and N. coccoides Naumann divides by budding. Distinct triangular spaces or mother cell walls were found in the dividing autosporangia of the other five strains from four species of Nannochloris, three species of Chlorella, and T. erici. In an attempt to infer an evolutionary relationship between nonautosporic and autosporic species of Nannochloris, we constructed a phylogenetic tree of the actin genes using seven strains from six species of Nannochloris, three species of Chlorella, and T. erici. Nannochloris species were polyphyletic in the Trebouxiophyceae group. Two nonautosporic species of N. bacillaris and N. coccoides were monophyletic and positioned distally. Moreover, to determine their phylogenetic position within the Trebouxiophyceae, we constructed phylogenetic tree of 18S rRNA genes adding other species of Trebouxiophyceae. Nannochloris species were polyphyletic in the Trebouxiophyceae and appeared in two different lineages, a Chlorella–Nannochloris group and a Trebouxia–Choricystis group. The nonautosporic species, N. bacillaris and N. coccoides, and three autosporic species of Nannochloris belonged to the Chlorella–Nannochloris group. Nannochloris bacillaris and N. coccoides were also monophyletic and positioned distally in the phylogenetic tree of 18S rRNA genes. These results suggest that autosporulation is the ancestral mode of cell division in Nannochloris and that nonautosporulative mechanisms, such as binary fission and budding, evolved secondarily.

Magma supply path beneath Mt. Asama volcano, Japan

[1] Obtaining a sharp image of magma supply path through dense geophysical observations is important for forecasting time and magnitude of hazardous future eruptions. Here we reveal a clear magma plumbing system using dense seismic and geodetic networks around Mt. Asama, central Japan. Magma intrusions occurred several times beneath the western flank of Mt. Asama, forming a WNW-ESE directed zone with 1 km below sea level. The eastern end of this zone connects a narrow vertical pathway extending right under the summit crater, which erupted in 2004. Monitoring magmatic activity with a well-designed observational network is vital to mitigate future volcano hazards.

DNA content of Ulva compressa (Ulvales, Chlorophyta) nuclei determined with laser scanning cytometry

The green macroalgal genus Ulva (incl. Entemmorpha) contains economically valuable species, is of relevance for coastal management (green tides), and certain taxa serve as experimental organisms for fundamental research in green algae. The nuclear genome size of Ulva (Entemmorpha) compressa Linnaeus was measured in propidium iodide stained nuclei using laser scanning cytometry. Nuclei of fixed gametes yielded reproducible values, whereas nuclei extracted from multicellular gametophytes were unsuitable. With nuclei of Arabidopsis thaliana (L.) Heynh and Saccharomyces cerevisiae Hansen as references, the haploid nuclear genome size of U. compressa was calculated as 135 ± 7 Mbp. This is the smallest genome so far known from any species of Ulva.

Molecular divergence and characterization of two chloroplast division genes, FtsZ1 and FtsZ2, in the unicellular green alga Nannochloris bacillaris (Chlorophyta)

Two FtsZ paralogues (NbFtsZ1 and NbFtsZ2) were isolated from the unicellular green alga Nannochloris bacillaris Naumann. These sequences encoded proteins of 435 and 439 amino acids with tubulin signature motifs (GGGTG[T/S]G), which are important for GTP binding activity. NbFtsZ1 and NbFtsZ2 had four and three introns, respectively, and two different putative core promoters; a TATA box (TATAAAA) and an initiator element (CCCAGG) were located 40 bp and 80 bp upstream of the coding regions of NbFtsZ1 and NbFtsZ2, respectively. Southern blot hybridization and contour‐clamped homogeneous electric field electrophoresis showed that N. bacillaris contained at least one copy of each gene and that NbFtsZ1 was located on chromosome 5 and NbFtsZ2 on chromosome 3 or 4. Phylogenetically, NbFtsZ1 and NbFtsZ2 belong to the vascular plant protein families FtsZ1 and FtsZ2, respectively. The FtsZ1 proteins do not contain carboxy‐terminal consensus sequences, whereas all FtsZ2 proteins possess the consensus sequence (I/V)PxFL(R/K)(K/R)(K/R). Our study has shown that NbFtsZ2 possesses a similar consensus sequence (VPDFLRRK), whereas NbFtsZ1 does not, further supporting their classification as FtsZ2 and FtsZ1. Escherichia coli ftsZ mutants transformed with cloned NbFtsZ1, and NbFtsZ2 cDNAs were restored for the capacity to divide by binary fission, suggesting that the proteins retained the ability to function in the bacterium. An anti‐NbFtsZ2 antibody specifically recognized a single protein band of approximately 51 kDa on an immunoblot of N. bacillaris cellular proteins. Immunostaining of the algal cells with this antibody produced an intense fluorescent signal as a ring near the middle of the cell, which corresponded to the chloroplast division site.

Isolation and molecular characterization of rbcS in the unicellular green alga Nannochloris bacillaris (Chlorophyta, Trebouxiophyceae)

The small subunit of the chloroplast enzyme ribulose‐1,5‐bisphosphate carboxyase/oxygenase (Rubisco) is encoded by rbcS. We isolated and characterized three rbcS genes (NbrbcS1‐1, NbrbcS1‐2, and NbrbcS2) from the unicellular green alga Nannochloris bacillaris (Chlorophyta, Trebouxiophyceae). In the haploid N. bacillaris genome, each is a single‐copy gene located on different chromosomes. Each mature peptide contains 140 amino acid residues. NbrbcS1‐1 and NbrbcS1‐2 are identical and share 80% identity with NbrbcS2, while the respective transit sequences share only approximately 48% identity with that of NbrbcS2. NbrbcS1‐1 transcription was suppressed in the dark and recovered drastically after transfer to light. In contrast, NbrbcS1‐2 and NbrbcS2 expression were not reduced after transfer from light to dark. In chlorophyllic tobacco cells containing green fluorescent protein fusion proteins of the transit sequences of each peptide, green fluorescent protein signals were localized on particles matching chloroplasts. The first introns of NbrbcS1‐1 and NbrbcS1‐2 are identical to the corresponding introns of 37 rbcS genes in eight embryophyte species. While the second intron is conserved in the green algae, the NbrbcS2 intron appears to involve sliding by one base pair. The NbrbcS1‐1 and NbrbcS1‐2 intron conserved in green algae and embryophytes might be an ancestral intron from nuclear‐encoded rbcS.

DNA content of the cell nucleus in the macroalga Porphyra yezoensis (Rhodophyta)

Marine Biology and Ecology Research Program, Extremobiosphere Research Center, Japan Agency for Marine-Earth Science and Technology, Yokosuka, Kanagawa 236-0061, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba 277-8562, Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, School of Marine Sciences and Technology, Tokai University, Shimizu-Orido, Shizuoka 424-8610, and Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan