Susumu Takio

A 100%-complete sequence reveals unusually simple genomic features in the hot-spring red alga Cyanidioschyzon merolae

BackgroundAll previously reported eukaryotic nuclear genome sequences have been incomplete, especially in highly repeated units and chromosomal ends. Because repetitive DNA is important for many aspects of biology, complete chromosomal structures are fundamental for understanding eukaryotic cells. Our earlier, nearly complete genome sequence of the hot-spring red alga Cyanidioschyzon merolae revealed several unique features, including just three ribosomal DNA copies, very few introns, and a small total number of genes. However, because the exact structures of certain functionally important repeated elements remained ambiguous, that sequence was not complete. Obviously, those ambiguities needed to be resolved before the unique features of the C. merolae genome could be summarized, and the ambiguities could only be resolved by completing the sequence. Therefore, we aimed to complete all previous gaps and sequence all remaining chromosomal ends, and now report the first nuclear-genome sequence for any eukaryote that is 100% complete.ResultsOur present complete sequence consists of 16546747 nucleotides covering 100% of the 20 linear chromosomes from telomere to telomere, representing the simple and unique chromosomal structures of the eukaryotic cell. We have unambiguously established that the C. merolae genome contains the smallest known histone-gene cluster, a unique telomeric repeat for all chromosomal ends, and an extremely low number of transposons.ConclusionBy virtue of these attributes and others that we had discovered previously, C. merolae appears to have the simplest nuclear genome of the non-symbiotic eukaryotes. These unusually simple genomic features in the 100% complete genome sequence of C. merolae are extremely useful for further studies of eukaryotic cells.

Alkyl peroxyl radical-scavenging activity of catechins.

Alkyl peroxyl radical (ROO.) generated from the reaction between 20 mM t-butyl hydroperoxide (t-BuOOH) and 200 microM hematin could kill E. coli. The minimum concentrations of catechins sufficient to rescue the bacteria treated with ROO. were found to be 70 microM for (-)-epicatechingallate, 100 microM for (-)-epicatechin and 125 microM for (+)-catechin. These values were comparable with the value of alpha-tocopherol, a typical ROO. scavenger. On the other hand, L-ascorbate and beta-carotene revealed about one tenth the scavenging activity of catechins. No scavenging activity was found for superoxide dismutase even at 86 mM. These facts indicate that catechins have high ROO. scavenging activity.

Moss Chloroplasts Are Surrounded by a Peptidoglycan Wall Containing D-Amino Acids

A peptidoglycan wall surrounding each plastid in the moss Physcomitrella patens was visualized using metabolic labeling with a d-Ala-d-Ala dipeptide probe and click chemistry. It is believed that the plastids in green plants lost peptidoglycan (i.e., a bacterial cell wall-containing d-amino acids) during their evolution from an endosymbiotic cyanobacterium. Although wall-like structures could not be detected in the plastids of green plants, the moss Physcomitrella patens has the genes required to generate peptidoglycan (Mur genes), and knocking out these genes causes defects in chloroplast division. Here, we generated P. patens knockout lines (∆Pp-ddl) for a homolog of the bacterial peptidoglycan-synthetic gene encoding d-Ala:d-Ala ligase. ∆Pp-ddl had a macrochloroplast phenotype, similar to other Mur knockout lines. The addition of d-Ala-d-Ala (DA-DA) to the medium suppressed the appearance of giant chloroplasts in ∆Pp-ddl, but the addition of l-Ala-l-Ala (LA-LA), DA-LA, LA-DA, or d-Ala did not. Recently, a metabolic method for labeling bacterial peptidoglycan was established using ethynyl-DA-DA (EDA-DA) and click chemistry to attach an azide-modified fluorophore to the ethynyl group. The ∆Pp-ddl line complemented with EDA-DA showed that moss chloroplasts are completely surrounded by peptidoglycan. Our findings strongly suggest that the moss plastids have a peptidoglycan wall containing d-amino acids. By contrast, no plastid phenotypes were observed in the T-DNA tagged ddl mutant lines of Arabidopsis thaliana.

The effect of calcium on flavanol production in cell suspension cultures of Polygonum hydropiper

Abstract Cultured Polygonum hydropiper cells maintained in Murashige and Skoog (MS) medium supplemented with 10–6m 2,4-D, 10–6m kinetin, 0.1% casamino acids and 3% sucrose were transferred to medium containing a higher concentration of calcium chloride (15 mm). The content of flavanols in the cells on the 6th day was approximately twice that of the control culture (31.9–60.7 mg/g dry wt). However, the contents of other secondary metabolites such as chlorogenic acid and gallic acid were not changed. The levels of flavanols in the culture medium remained unchanged throughout the 21-day culture period. Of the the inorganic components supplemented to the culture medium , only elevated levels of calcium chloride induced an increase in flavanol contents of the cells. The results indicated that the elevated concentration of calcium in the culture medium played an important role in activating the accumulation of flavanols.

The peptidoglycan biosynthesis genes, MurA and MraY, are related to chloroplast division in the moss Physcomitrella patens.

In the moss Physcomitrella patens, 10 Mur genes involved in peptidoglycan biosynthesis were found, and the MurE and Pbp genes are related to plastid division. Although the MraY and MurG genes were missing in our previous expressed sequence tag screening, they were discovered in the P. patens genome in this study, indicating that P. patens has a full set of genes capable of synthesizing peptidoglycan. In addition, a second MurA gene (PpMurA2) was found. Whereas Northern analyses indicated that PpMurA1, PpMurG and PpMraY were expressed, transcripts of PpMurA2 were detected only when RT-PCR was employed. Whereas GFP fusion proteins with either PpMurA1 or PpMraY were detected in chloroplasts, the PpMurA2 fusion proteins were located in the cytoplasm. Protonema cells in the wild-type plants had an average of 46 chloroplasts. PpMurA1 gene-disrupted lines had <10 chloroplasts, whereas approximately 30 chloroplasts existed in the PpMurA2 knockout lines. The PpMurA1/A2 double-knockout lines had only a few macrochloroplasts, suggesting a redundant function for these two genes. Disruption of the PpMraY gene in P. patens resulted in the appearance of macrochloroplasts. Anabaena MraY fused to the N-terminal region of PpMraY and A. thaliana MraY could complement the macrochloroplast phenotype in the PpMraY knockout line. Electron microscopic observations showed no obvious differences in the shape or stacking of thylakoid membranes between all knockout transformants and wild-type plants, suggesting that these Mur genes are related only to plastid division in moss.

Ethylene regulation of sexual reproduction in the marine red alga Pyropia yezoensis (Rhodophyta)

Plant growth regulators (PGRs) play a pivotal role in vascular plants, regulating growth, development, and stress responses; however, the role of PGRs in algae remains largely unexplored. Here, the role of ethylene, a simple plant growth regulator, was demonstrated in sexual reproduction of the marine red alga Pyropia yezoensis. Application of the ethylene precursor 1-aminocylopropane-1-carboxylic acid (ACC) promoted the formation of spermatia and zygotospores in the gametophytes as well as ethylene production, whereas the growth rate was repressed in comparison to gametophytes not treated with ACC. In addition, gametophytes treated with ACC and mature gametophytes showed enhanced tolerance to oxidative stress. Gene expression profiles revealed upregulation of genes involved in cell division and stress response in gametophytes treated with ACC and in mature gametophytes. These results indicate that ethylene plays an important role in the regulation of gamete formation and protection against stress-induced damage during the sexual reproductive stage. Considered together, these findings demonstrate that ethylene is involved in regulating the switching from a vegetative to a sexual reproductive phase in P. yezoensis.

Chalcone synthase-like gene in the liverwort, Marchantia paleacea var. diptera

A chalcone synthase (CHS)-like gene, MpCHSLK1, was isolated from liverwort, Marchantia paleacea var. diptera. Phylogenetic analysis revealed that MpCHSLK1 is closely related to stilbene synthase of the whisk fern, Psilotum nudum. Southern blot analysis using an MpCHSLK1 probe revealed that the gene belongs to a small gene family. Northern blot analysis indicated that CHS-like genes were expressed in either the mother plants or photoautotrophic cells. In photoautotrophic cells, the CHS-like genes were expressed light-dependently, and this expression was completely inhibited by the photosynthetic electron transport inhibitor, DCMU.

Two types of plastid ftsZ genes in the liverwort Marchantia polymorpha

Summary. Two types of ftsZ genes (MpftsZ1 and MpftsZ2) were isolated from the liverwort Marchantia polymorpha by degenerate reverse transcription PCR. The MpFtsZ1 and MpFtsZ2 proteins are predicted to localize in chloroplasts. Genomic Southern analysis suggested that each ftsZ gene is a single-copy nuclear gene. Northern analysis confirmed that both genes are active. A phylogenetic tree constructed with the deduced MpFtsZ amino acid sequences suggests that MpFtsZ1 and MpFtsZ2 can be classified into the plant chloroplastic FtsZ1 and FtsZ2 families, respectively. This result suggests that two ftsZ families exist universally in land plants. The determination of the intron structures of both MpftsZ genes supported this hypothesis. The transformation of a sense MpftsZ2 overexpression construct into M. polymorpha produced a large chloroplast phenotype in a transgenic plant. The mean number of chloroplasts was 38.2 (standard deviation, 21.4; n = 200) in epidermal cells of wild-type plants, whereas the mean number of chloroplasts was 7.4 (standard deviation, 4.4; n = 200) in the transgenic plant. Southern analysis showed that the cauliflower mosaic virus 35S promoter-MpftsZ2 construct was inserted in at least three positions. Northern analysis suggested that the high accumulation of MpftsZ2 mRNA blocked plastid division. Determination of the chlorophyll content and the chlorophyll fluorescence parameters suggested that the macrochloroplasts function like chloroplasts in wild-type plants under normal light conditions. However, the transgenic plant grew more slowly than did wild-type plants.

Ammonium Induced Expression of the Red Algal Chloroplast Gene Ycf18, a Putative Homolog of the Cyanobacterial NblA Gene Involved in Nitrogen Deficiency-Induced Phycobilisome Degradation

In cyanobacteria, nutrient deficiency-induced phycobilisome degradation is controlled by the NblA gene. Red algae also have an NblA-related gene, Ycf18, in their chloroplast genomes. To elucidate the role of Ycf18, the expression pattern of Ycf18 in a red alga, Porphyra yezoensis, was investigated. Ycf18 expression was low in nitrate medium, but was greatly promoted in ammonium medium. Nitrogen starvation caused bleaching, but did not affect the expression of Ycf18. The responses of Ycf18 to nitrogen-starvation and the supply of ammonium were distinct from those of NblA, suggesting that Ycf18 has a role other than the regulation of phycobilisome degradation.

Isolation and characterization of the Larix gmelinii ANGUSTIFOLIA (LgAN) gene

ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.