Kenji Katoh

Transcriptional activation of the alternative oxidase gene of the fungus Magnaporthe grisea by a respiratory-inhibiting fungicide and hydrogen peroxide

Alternative oxidase (AOX) is dramatically induced when the fungus Magnaporthe grisea is incubated with the fungicide SSF-126, which interacts with the cytochrome bc1 complex in the electron transport system of mitochondria. A full-length cDNA for the alternative oxidase gene (AOX) was obtained, and the deduced amino acid sequence revealed marked similarity to other AOXs, but lacks two cysteine residues at corresponding sites which are conserved in plant AOXs and play essential roles in the post-translational regulation. Northern blot experiments showed that treatment of M. grisea cells with SSF-126 induces accumulation of AOX mRNA in a dose-dependent manner, and the level was correlated with the activity of alternative respiration. H2O2 also induced the accumulation of the transcript with a short half-life (<15 min). Nuclear run-on experiments showed that the AOX gene was transcribed constitutively in unstimulated cells. Cycloheximide did not change the basal level of transcription, but induced the accumulation of the transcript, indicating that active degradation of the transcript occurs by factor(s) sensitive to cycloheximide. On the other hand, SSF-126 enhanced the transcriptional activity of AOX gene threefold compared to that of control cells, and H2O2 was also potent for enhancement of the transcription. From these results, it is concluded that the respiratory inhibitor-dependent activation of the transcription is a primary determinant for the induction of alternative respiration in M. grisea. Because we have previously shown that SSF-126 treatment of M. grisea mitochondria induced the generation of superoxide, active oxygen species are thought to be signal mediators to activate AOX gene transcription in M. grisea.

Growth and sesquiterpenoid production by Calypogeia granulata inoue cells in suspension culture.

Growth and the production of volatile sesquiterpenoids by a chlorophyllous cell suspension culture from gametophytes of C. granulata, a leafy liverwort, were examined. Glucose was more effective than 2,4-dichlorophenoxyacetic acid for callus induction, and elimination of glucose from the medium resulted in prompt redifferentiation of plantlets. The cells grew photoheterotrophically, but not in the dark. 1,4-Dimethylazulene, a trinorsesquiterpenoid, was produced as the major volatile sesquiterpenoid in the cultured cells; bicyclogermacrene, compound II, an indene-type aldehyde (a trinorsesquiterpenoid aldehyde), compound I and tetrahydro-1,4-dimethylazulene (a trinorsesquiterpenoid) followed in decreasing order. The azulene was produced both in light and the dark, and its yield was proportional to the growth in light. The yield in light was four times higher than that in the dark. The content of 1,4-dimethylazulene was 0.9–10.% and that of total essential oils was 2.0–3.3% of the dry werght of the cultured cells. The quantity, quality, and proportions of the volatile sesquiterpenoids of the cell culture were almost equal to those of intact (original) plants and redifferentiated plantlets.

Sterol analysis of DMI-resistant and -sensitive strains of Venturia inaequalis

The sterol composition of Venturia inaequalis strains sensitive and resistant to sterol demethylation-inhibiting fungicides (DMI) were analysed. Without DMI treatment, both strains contained ergosta-5,7-dien-3β-ol, ergosterol, ergosta-7,24(241)-dien-3β-ol and ergost-7-en-3β-ol. Ergosta-5,7-dien-3β-ol was the main sterol in both strains. Treatment with fenarimol decreased the 4-desmethyl sterols and accumulated eburicol in both strains, indicating that this fungicide inhibits C14-demethylase of not only the sensitive strain but also the resistant strain. The accumulation of eburicol in the resistant strain occurred at higher concentration of fenarimol than in the sensitive strain.

Metal-tolerant moss Scopelophila cataractae accumulates copper in the cell wall pectin of the protonema.

The growth kinetics in the presence of copper (Cu) of the protonema of the moss Scopelophila cataractae and the matrix polysaccharides of its cell walls have been analyzed in this study. Protonemal cells cultured in a medium containing 0.2mM CuSO(4) showed a rapid accumulation of Cu, reaching a maximum between 30 and 60d at approximately 65 micromolg(-1) DW. Uronic acids were found in similar amounts in cell walls of both control and Cu-treated cells, whereas arabinose and galactose decreased to 61-67% in the presence of Cu. Cell wall polysaccharides were determined after successive extraction with 50mM CDTA, 50mM Na(2)CO(3), 1M KOH, and 4M KOH. The pectic fractions (CDTA- and Na(2)CO(3)-soluble) decreased to 47% and the hemicellulosic fractions (1M KOH- and 4M KOH-soluble) to 86% under Cu application. Approximately 43% of the Cu taken into cell walls was released following endo-pectate lyase treatment, suggesting that two-fifths of the total Cu accumulation was tightly bound to the homogalacturonan of the cell wall pectin.

Ring-A cleavage of 3-oxo-olean-12-en-28-oic acid by the fungus Chaetomium longirostre

Abstract 3-Oxo-olean-12-en-28-oic acid was transformed by the filamentous fungus Chaetomium longirostre into 3,4- seco -olean-12-en-4-ol-3,28-dioic acid and the 21β-hydroxylated compound. A cell-free preparation derived from the fungus converted 3-oxo-olean-12-en-28-oic acid into 3,4- seco -olean-12-en-4-ol-3,28-dioic acid. the ring-A cleavage activity was detected in the soluble fraction of the cell-free preparation and showed a requirement for NADPH.

Establishment and growth characteristics of a cell suspension culture of Marchantia polymorpha L. with high chlorophyll content

A green-pigmented cell suspension culture of Marchantia polymorpha was established using the medium of Murashige and Skoog with addition of organic acids of the tricarboxylic-acid cycle, vitamins and sugars plus sugar alcohols, exclusion of kinetin, and replacement of sucrose with glucose. In continuous light, the cells grew exponentially for ca. 10 days; in the dark, they grew only to a slight extent. The light-grown cells contained well-developed chloroplasts, and chlorophyll content reached almost twice that of the intact gametophyte.

Photoautotrophic growth of Marchantia polymorpha L. cells in suspension culture

A cell line of M. polymorpha was grown photoautotrophically in liquid suspension culture using 1% CO2 in air as sole carbon source. The growth rate in terms of cell dry-weight during the exponential phase was 0.171 and the doubling time was 1.76 d. The rate of increase in chlorophyll was 1.6 times higher than the growth rate. The highest content of chlorophyll was 24 mg g-1 dry weight, and the photosynthetic activity of the cells in the exponential phase, as calculated from the growth rate, was at least 60 μmol mg-1 chlorophyll h-1.

A β-glucosidase associated with cell walls from cell suspension cultures of carrot

Abstract The activity of β-glucosidase (EC 3.2.1.21) in the protein fraction solubilized with 3 M LiCl from cell walls of carrot cell cultures was found to be much higher than those of the other glycan-hydrolases. The cell wall-associated β-glucosidase was purified to electrophoretic homogeneity. The M r of the purified enzyme was estimated to be 46 000 by Sephacryl S-200HR gel-permeation, and 48 000–52 000 by SDS-PAGE under denaturing conditions. The enzyme contained carbohydrate and protein in a ratio of 1 : 15 (w/w) and was rich in Ser, Gly, Glx and Ala. The isoelectric point was pH 8.2, the pH optimum 4.6–5.2 and the temperature optimum 50°. The activity was inhibited by Cu 2+ , Ag + , Hg 2+ , p -chloromercuribenzoate, and d -glucono-1,5-lactone. The K m and V max values for p -nitrophenyl (PNP)-β-glucopyranoside were 0.12 mM and 0.13 mmol (mg protein) −1 hr −1 , respectively. The enzyme also acted on PNP-β-cellobioside, lichenan and laminarin, but was not capable of hydrolysing the glucose-containing polymers isolated from cell walls of carrot cell cultures.

Subunit structure and amino acid analyses of β-galactosidase purified from carrot cell cultures

Abstract β-Galactosidase has been purified to electrophoretic homogeneity from cell suspension cultures of carrot. It has an apparent Mr of 104 000 and consists of two identical subunits with an apparent Mr of 50 000. The amino acid composition of the enzyme is characterized by a relatively large content of Asx and Leu, and a small content of His and Met. The amino-terminal amino acid of the enzyme is Glu.

Characteristics, hydrolysis of cell wall polymers, and response to calcium deficiency of a cell wall-associated β-galactosidase from carrot cells

Summary The activity of β-galactosidase (EC 3.2.1.23) in the protein fraction solubilized with 3 mol/L LiCl from cell walls of suspension-cultured carrot ( Daucus carota ) cells has been found to be higher than those of the other glycosyl-hydrolytic enzymes. The cell wall-associated β-galactosidase/exo-galactanase was purified to electrophoretic homogeneity. Analysis by denaturing PAGE revealed a single polypeptide chain with a molecular mass of 50 kDa, similar to 52 kDa estimated for the native protein by size-exclusion chromatography. The enzyme contained carbohydrate and protein in a ratio of 1 : 10 (w/w), and was rich in Gly and Phe, followed by Ser, Ala and Thr. The isoelectric point was pH 6.5, and pH and temperature optima 4.4 and 45-50 °C, respectively. The β-galactosidase activity was inhibited by Mg 2+ , Ag + , Hg 2+ , p -chloromercuribenzoate and D-galactono-1,4-lactone. The K m and V max values for p -nitrophenyl-β-D-galactopyranoside were 0.77 mmol/L and 0.32 mmol (mg protein) −1 h −1 , respectively. The enzyme hydrolyzed citrus galactan in an exo-fashion and was capable of hydrolysing an acidic pectic polymer containing galactosyl and arabinosyl residues from carrot cell walls and, therefore, is an exo-galactanase. However, even after an exhaustive reaction, the enzyme had no effect on a galactose-rich hemicellulosic polymer from carrot cell walls. Under calcium deficiency the activities of a cell wall-associated α-galactosidase (EC 3.2.1.22) and β-galactosidase increased until 15 days of culture and then decreased gradually, whereas β-glucosidase (EC 3.2.1.21) activity increased markedly with time despite poor cell growth.