Noriaki Kondo

Expression of arabidopsis cytosolic ascorbate peroxidase gene in response to ozone or sulfur dioxide

The effects of ozone or sulfur dioxide on antioxidant enzymes were investigated in Arabidopsis thaliana. Plants were fumigated with 0.1–0.15 ppm ozone or sulfur dioxide up to about 1 week in an environment-controlled chamber. Both pollutants increased the activities of ascorbate peroxidase and guaiacol per-oxidase in leaves, but had little effect on the activities of superoxide dismutase, catalase, monodehydroascorbate reductase, dehydroascorbate reductase or glutathione reductase. Ozone was more effective than sulfur dioxide in increasing the activities of the peroxidases. Ascorbate peroxidase activity increased 1.8-fold without a lag period during fumigation with 0.1 ppm ozone, while guaiacol peroxidase activity increased 4.4-fold with a 1-day lag. Expression of the APX1 gene encoding cytosolic ascorbate peroxidase was further investigated. Its protein levels in leaves exposed to 0.1 ppm ozone for 4 or 8 days were 1.5-fold higher than in controls. Both ozone and sulfur dioxide elevated APX1 mRNA levels in leaves at 4 and 7 days, whereas at 1 day only ozone was effective. The induction of APX1 mRNA levels by ozone (3.4- to 4.1-fold) was more prominent than that by sulfur dioxide (1.6-to 2.6-fold). The APX1 mRNA level increased by day and decreased by night. Exposure of plants to 0.1 ppm ozone enhanced the APX1 mRNA level within 3 h, which showed a diurnal rhythm similar to that of the control. These results demonstrate that near-ambient concentrations of ozone as well as similar concentrations of sulfur dioxide can induce APX1 gene expression in A. thaliana.

Cloning and sequencing of a cDNA encoding ascorbate peroxidase from Arabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage λgt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)+ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3′ untranslated region of the cDNA.

Distribution of growth regulators in relation to the light-induced geotropic responsiveness in Zea roots

Growth regulators were measured in extracts from the upper and lower halves of 7-mm apical segments of horizontally oriented, red-light-irradiated and non-irradiated roots of Zea mays L. cv. Golden Cross Bantam 70 which exhibit a georesponse only after an exposure to light. Abscisic acid (ABA) was measured by gas-liquid chromatography, auxin (indole-3-acetic acid, IAA) by the Avena straight-growth assay, and an unidentified growth inhibitor by a Zea root-growth assay. The ratio of ABA in the upper and lower halves was 1.6 in the irradiated roots and 1.0 in the non-irradiated ones. The total amount of ABA after irradiation was increased by a factor of ca. 1.8. The ratio of IAA in the upper and lower halves of irradiated and non-irradiated roots was 1:3.4 and 1:2.9, respectively. The content (or activity) of an unidentified growth inhibitor was highest in the lower halves of horizontally oriented roots which had been irradiated with red light. The unidentified growth inhibitor, rather than IAA or ABA, may be the major factor in the light-induced geotropic responsiveness in Zea roots.

Monoclonal antibodies to spinach ascorbate peroxidase and immunochemical detection of the enzyme in eight different plant species

Abstract Eight monoclonal antibodies were raised against ascorbate peroxidase purified from spinach ( Spinach oleracea L.) leaves. All of these antibodies were specifically bound to a protein (antigen) having a molecular mass of about 30,000 and an isoelectric point of about 6.5 when examined against electrophoretically-separated soluble proteins of spinach leaves. Binding of these monoclonal antibodies to spinach ascorbate peroxidase resulted in various degrees of inhibition of the enzyme activity. Seven other plant species examined ( Arabidopsis thaliana Heynh., Beta vulgaris , L., Chenopodium album L., Nicotiana tabacum L., Pharbitis nil Choisy, Pisum sativum L., Oryza sativa L.) were shown to have immunochemically related proteins, most likely ascorbate peroxidase.

A model system to study the effect of SO2 on plant cells. II: Effect of sulfite on fern spore germination and rhizoid development

Fumigation with 0.05 μl/l SO2 decreased the germination rate of fern spores ofAdiantum capillus-veneris L. and influenced greatly the rhizoid development for abnormities such as apical swelling. Even 10 μM sulfite derived from SO2 fumigation was influential. These SO2 effects on germination and following rhizoid development were proved to be independent of each other, and could be induced separately by pulse treatment with K2SO3 solution given at different timings. According to the analysis using a time-lapse video recorder, growth retardation and apical swelling of rhizoid occurred just after the application of sulfite. It became clear that the fern gametophytes are very sensitive to SO2, (that is sulfite) as compared with other plants used for air pollutant experiments so far.

Differences in the probability of cloning specific DNA between primary and amplified libraries: Theoretical considerations

We present a theoretical study of the probability of isolating a particular clone from a DNA library. There are differences in this probability between primary and amplified libraries even if the desired clone represents the same fraction of both libraries. As the result, we must screen severalfold more phage or bacteria in an amplified library than in a primary library.

The change in leaf protease and protease inhibitor activities after supplying various chemicals

Protease activity inSpinacia oleracea leaves, but not roots, increased when sodium sulfite, hydrogen peroxide and sodium azide, but not sulfuric acid, were injected through the petiole under light conditions. On the other hand, protease inhibitory activity in both the leaves and roots decreased by the injection.Protease activity inRicinus communis leaves increased when hydrogen peroxide and sodium sulfite were injected through the petiole and kept for 4 h under light conditions. No visible injuries were caused to the leaf. On the other hand, protease inhibitory activity in leaves decreased by the injection of hydrogen peroxide.Changes in the activity of protease caused the senescence of leaves such as chlorosis and necrosis which were observed with leaves injected with hydrogen peroxide after one week. These results suggested that in the healthy leaf, the protease inhibitor protects the cellular components from the protease.

A model system to study the effect of SO2 on plant cells. III. Effects of sulfite on the ultrastructure of fern protonemal cells

The mechanism of the toxic effects on plant cells of sulfite, a product of the air pollutant sulfur dioxide, is not well understood. Therefore, changes in the fine structure and organization of microtubules and microfibrils induced by sulfite were studied by electron and light microscopy in the protonemata of the fernAdiantum capillusveneris L.Under red-light conditions, growing protonemata fumigated with 0.05 or 0.1 μ1/1 SO2 for 1 to 4 days showed abnormalities, such as apical swelling, and they sometimes burst at the apex. The incidence of abnormalities seemed to be correlated with the concentration of the sulfite dissolved in the culture medium.At an appropriate concentration (3.3–6.6. mM) of sulfite (applied as K2SO3), cell swelling at the apical region of protonema was also induced. When the concentration of sulfite was as high as 6.6 mM, more than 60% of protonemata burst at the tip. During the apical swelling, no distinct changes were observed in the fine structure of organelles, such as the chloroplasts, mitochondria, microbodies, Golgi bodies and nucleus. However, the arrangement of cortical microtubules and that of the innermost layer of microfibrils around the subapical region of protonemata were changed from transverse to the cell axis (i.e., circular) to random and the cell wall was thickened. These observations suggest that sulfite may influence the mechanisms that maintain the transverse orientation of microtubules in the subapical region of a protonema and that the resultant random arrangement of microtubules induces the random arrangement of microfibrils and leads to apical swelling.

Detection and evaluation of serine proteinase by affinity chromatography on immobilized-aprotinin inRicinus communia

Neutral proteinase was found in the leaves ofRicinus communie as assayed with α-casein and H-D-Val-Leu-Lys-pNA as substrates. The enzyme is maximally active at pH around 7.4. A selective adsorbent for serine proteinase was prepared by attaching aprotinin to aminoalkyl-porous glass.When partially purified leaf proteinase was passed through a column containing this adsorbent, the proteinase activity present was bound to the porous glass. The proteinase eluted at IM NaCl was inhibited by aprotinin, leupeptin, DFP, phenylmethylsulfonyl fluoride (PMSF) and serine proteinase inhibitor fromR, communis leaves, whereas pepstatin, EDTA, EGTA, and DTT had no effect on the enzyme. This inhibition profile suggests the leaf proteinase is a neutral proteinase, such as a serine proteinase.