Isolation of mutant lines with decreased numbers of chloroplasts per cell from a tagged mutant library of the moss Physcomitrella patens.

Eleven mutant lines exhibiting decreased numbers of chloroplasts per cell were isolated from 8 800 tagged mutant lines of Physcomitrella patens by microscopic observations. Chloronema subapical cells in wild-type plants had a mean of 48 chloroplasts, whereas chloroplast numbers in subapical cells in mutant lines 215 and 222 decreased to 75 % of that in the wild type. Seven mutant lines - 473, 122, 221, 129, 492, 207, and 138 - had about half as many chloroplasts as the wild type. Mutant line 11 had a few remarkably enlarged chloroplasts, and mutant line 347 had chloroplasts of various sizes. Whereas the cell volume was the same as in the wild type in mutant lines 222, 473, 221, 129, 492, and 207, the cell volume of the other mutants increased. The chloroplast number of leaf cells was the same as that of chloronema cells in each mutant line when gametophores could be formed. Treatment with ampicillin decreased the number of chloroplasts in all mutant lines. Southern hybridization using DNA in tags as probes showed that only one insertion occurred in mutant lines 473 and 221. To determine whether the tagged DNA inserted into the known genes for plastid division, we isolated the PpMinD1, PpMinD2, and PpMinE1 genes. Genomic polymerase chain reaction analysis showed that the PpFtsZ and PpMinD/E genes were not disrupted by the insertion of the tags in mutant lines 11 and 347, respectively.

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.

Effect of cytokinin on morphological changes of suspension cultured cells of the moss, Barbula unguiculata.

Suspension cultured cells of the moss, Barbula unguiculata, grow actively in both light and dark culture. Light-grown cells contain chlorophyll and exhibit an undifferentiated callus form. When cells are transferred to a dark condition, they develop into protonemata. Protonemata formation in the dark can be inhibited by the addition of 5 µM benzyladenine or 6-furfurylaminopurine but is not affected by the addition of 5 µM 2,4-dichlorophenoxyacetic acid or naphthalene acetic acid.

Photosynthetic ability in dark-grown Reboulia hemisphaerica and Barbula unguiculata cells in suspension culture.

Suspension cultured cells of the liverwort, Reboulia hemisphaerica and of the moss, Barbula unguiculata were independently subcultured in the medium containing 2% glucose in the dark or in the light for more than one year, and the photosynthetic activities of the final cultures were determined. Throughout the culture period light-grown cells of both species contained high amount of chlorophyll (4 to 34 µg mg(-1) dry weight) and showed a high photosynthetic activity (10 to 84 µmol O2 mg(-1) chlorophyll h(-1)). Dark-grown cells of R. hemisphaerica showed the same level of chlorophyll content and photosynthetic O2 evolving activity as light-grown cells. Although chlorophyll content in dark-grown B. unguiculata cells was ten-fold lower than that in light-grown cells, the photosynthetic activity of these dark-grown cells was higher than that of light-grown cells based on chlorophyll content.

Purification of the cytosolic CuZn-superoxide dismutase (CuZn-SOD) of Marchantia paleacea var. diptera and its resemblance to CuZn-SOD from chloroplasts.

Suspension-cultured cells of Marchantia paleacea var. diptera contain a single form of CuZn-superoxide dismutase (SOD; EC 1.15.1.1) which is localized in the cytosol. SOD activity was found in cells cultured under heterotrophic, photoheterotrophic and photoautotrophic conditions. The CuZn-SOD was purified to homogeneity from liverwort cells that had been cultured heterotrophically. Its molecular mass was 32.6 kDa, and it contained 17.5 dDa subunits, an indication that the enzyme is a homodimer. The enzyme had peaks of absorption at 252, 258 and 264 nm in the ultraviolet region, due to the presence of phenylalanine, and a peak at 680 nm in the visible region, which is characteristic of CuZn-SODs from chloroplasts. The amino acid sequence of the amino-terminal region of the enzyme exhibited a very high degree of homology to those of chloroplast CuZn-SODs. An antiserum raised against the CuZn-SOD from liverwort cross-reacted more strongly with the enzyme from spinach chloroplasts, than with the enzyme from spinach cytosol. These results indicate that the CuZn-SOD of liverwort resembles CuZn-SOD in chloroplasts even though the former is located in the cytosol.

Involvement in denitrification of the napKEFDABC genes encoding the periplasmic nitrate reductase system in the denitrifying phototrophic bacterium Rhodobacter sphaeroides f. sp. denitrificans.

Seven genes, napKEFDABC, encoding the periplasmic nitrate reductase system were cloned from the denitrifying phototrophic bacterium Rhodobacter sphaeroides f. sp. denitrificans IL106. Two transmembrane proteins, NapK and NapE, an iron-sulfur protein NapF, a soluble protein NapD, a catalytic subunit of nitrate reductase precursor NapA, a soluble c-type diheme cytochrome precursor NapB, and a membrane-anchored c-type tetraheme cytochrome NapC were deduced as the gene products. Every mutant in which each nap gene was disrupted by omega-cassette insertion lost nitrate reductase activity as well as the ability of cells to grow with nitrate under anaerobic-dark conditions. A transconjugant of the napD-disrupted mutant with a plasmid bearing the napKEFDABC genes recovered both nitrate reductase activity and nitrate-dependent anaerobic-dark growth of cells. Denitrification activity, which was not observed in the napD mutant, was also restored by the conjugation. These results indicate that the periplasmic nitrate reductase encoded by the napKEFDABC genes is the enzyme responsible for denitrification in this phototroph, although the presence of a membrane-bound nitrate reductase has been reported in the same strain.

Characterization of a cDNA encoding CuZn-superoxide dismutase from the liverwort Marchantia paleacea var. diptera.

Suspension-cultured cells of the liverwort Marchantia paleacea var. diptera contain a cytosolic CuZn-superoxide dismutase (SOD) whose N-terminal amino acid sequence is similar to those of the isozymes found in chloroplasts of higher plants [Tanaka et al. (1996) Plant Cell Physiol. 37: 523]. A cDNA (MSODCc) encoding the cytosolic CuZn-SOD was isolated from cDNA library constructed from a liverwort cell suspension culture. The deduced amino acid sequence showed a higher degree of homology with the sequences of CuZn-SODs in chloroplasts than those in the cytosol of higher plants and an unique additional peptide in the C-terminal region, but no plastid transit sequence. Northern blotting using MSODCc as a probe and immunoblot analysis with antiserum against the enzyme revealed that the steady state level of transcript was not affected by copper, but both CuZn-SOD protein and its activity increased.

Two types of plastid ftsZ genes in the liverwort Marchantia polymorpha.

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.

Characterization of cDNA of the liverwort phytochrome gene, and phytochrome involvement in the light-dependent and light-independent protochlorophyllide oxidoreductase gene expression in Marchantia paleacea var. diptera.

The cDNA of the phytochrome gene in the liverwort Marchantia paleacea var. diptera (MpdPHY1) was isolated. MpdPHY1 encoded a conventional phytochrome apoprotein. The MpdPHY1 transcript was accumulated in the dark and suppressed in the light. The degradation of the MpdPHY1 transcript by red light irradiation had red/far-red reversibility, suggesting that the liverwort phytochrome gene expression was regulated by a phytochrome. Northern blot analysis of the transcripts in cells irradiated by red/far-red light revealed that the liverwort phytochrome was involved in the expressions of chlB, chlL, chlN, or por, which encode subunits of light-independent and light-dependent protochlorophyllide oxidoreductase, respectively.

Differential light regulation of the rbcS gene expression in two cell lines of the liverwort Marchantia paleacea var. diptera.

A cDNA of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) gene (rbcS) was isolated from cells of the liverwort Marchantia paleacea var. diptera. The rbcS was expressed light-independently in a green line, but light-dependently in a yellow line. The accumulation of the large subunit of RuBisCO was regulated cooperatively with that of the small subunit.

Light-dependent expression of protochlorophyllide oxidoreductase gene in the liverwort, Marchantia paleacea var. diptera.

A cDNA encoding the NADPH:protochlorophyllide oxidoreductase (EC 1.6.99.1) was isolated from suspension-cultured cells of the liverwort, Marchantia paleacea var. diptera. In contrast to the situation in most higher plants, the liverwort gene was expressed in a light-dependent manner.

Hypermethylation of retrotransposons in the liverwort Marchantia paleacea var. diptera.

Three DNA fragments encoding part of a reverse transcriptase similar to that of Ty1- copia retrotransposons were isolated from suspension-cultured cells of the liverwort Marchantia paleacea var. diptera. Digestion of total DNA with methylation-sensitive and -insensitive isoschizomers revealed that, like those of the subtelomeric region, CpG sequences in retrotransposon genes were hypermethylated. The methylation status and expression of these retrotransposons was less affected by 5-azadeoxycytidine treatment compared to the subtelomeric region, which was considerably demethylated.

Isolation of a germin-like protein with manganese superoxide dismutase activity from cells of a moss, Barbula unguiculata.

A novel extracellular Mn-superoxide dismutase (SOD) was isolated from a moss, Barbula unguiculata. The SOD was a glycoprotein; the apparent molecular mass of its native form was 120 kDa, as estimated by gel filtration chromatography, and that of its monomer was 22,072 Da, as estimated by time of flight mass spectroscopy. The protein had manganese with a stoichiometry of 0.80 Mn/monomer. The cDNA clone for a gene encoding the extracellular Mn-SOD was isolated. Sequence analysis showed that it has a strong similarity to germin (oxalate oxidase) and germin-like proteins (GLPs) of several plant species and possesses all the characteristic features of members of the germin family. The clone encoding this extracellular Mn-SOD was therefore designated B. unguiculata GLP (BuGLP). BuGLP had no oxalate oxidase activity. In addition, the cDNA for a gene encoding the moss mitochondrial Mn-SOD was isolated. Its amino acid sequence had little similarity to that of BuGLP, even though a close similarity was observed among the mitochondrial Mn-SODs of various organisms. BuGLP was the first germin-like protein that was really demonstrated to be a metalloprotein with Mn-SOD activity but no oxalate oxidase activity.