Toshihide Ohya

Light-enhanced induction of ascorbate peroxidase in Japanese radish roots during postgerminative growth

Abstract During postgerminative growth of Japanese radish ( Raphanus sativus L. var. hortensis ) in continuous light, the specific activity of APX (EC 1.11.1.11) in roots markedly increased 2 days after imbibition. This increase in APX activity in roots was accompanied by increases in activities of the enzymes in the ascorbate-dependent H 2 O 2 -scavenging system, superoxide dismutase (EC 1.15.1.1), monodehydroascorbate reductase (EC 1.6.5.4), dehydroascorbate reductase (EC 1.8.5.1) and glutathione reductase (EC 1.6.4.2) which may participate in the detoxification of H 2 O 2 generated via the β-oxidation of fatty acid in seeds and consequent active cell division in the meristem/determination zone of the root. Catalase (EC 1.11.1.6) activity was not detected and guaiacol peroxidase (EC 1.11.1.7) activity was extremely low in roots at this stage of postgermination. A prominent increase in APX activity in roots under light conditions reflected the increases in protein level and mRNA accumulation of cytosolic APX. These results indicate that at the early stages of postgermination of Japanese radish, cytosolic APX is rapidly induced in growing roots and its induction may be regulated by light. The role of cytosolic APX in roots is discussed in relation to detoxification of H 2 O 2 generated in the process of postgerminative growth.

Purification and characterization of ascorbate peroxidase in roots of Japanese radish

Abstract Ascorbate peroxidase (APX) was purified to homogeneity from roots of Japanese radish (Raphanus sativus L.). The root APX was monomeric with a molecular mass of 28 kD and was stabilized by ascorbate. The enzyme utilized mainly ascorbate as a substrate, within a narrow optimum around pH 6.0, but could not use guaiacol, 3,3′-diaminobenzidine, pyrocatechol or d -iso-ascorbate. The purified APX was labile in the absence of ascorbate. Spectral analysis and inhibitor studies revealed the presence of a heme moiety and the participation of an SH group for enzymic activity. Antibodies raised against root APX reacted to extracts of spinach leaf, maize seedling and Brassica root, but not to guaiacol peroxidase from Japanese radish roots. The amino acid sequence of the N-terminal region of the root APX exhibited homology to the cytosolic forms of APX from pea, maize, and a deduced sequence from the Arabidopsis genome, but not to tea chloroplastic APX. These results suggest that the Japanese radish root APX is a novel cytosolic enzyme.

Presence of ascorbate-peroxidizing enzymes in roots of Brassica campestris L. cv. Komatsuna

Abstract Three peroxidases which could utilize ascorbate as a reductant were found in roots of Brassica campestris , and designated P I, P II and P III according to their order of elution from a DEAE-Cellulofine chromatography column. P I was not adsorbed to the column and oxidized ascorbate at only 4% of the rate for pyrogallol. Both P II and P III had a high preference for ascorbate as an electron donor and no activity with guaiacol. P II was detected in all tissues of B. campestris , whereas P III was detected characteristically in roots and accounted for 70% of the total ascorbate-peroxidizing activity in 9-day-old roots. Partially purified P III was inactivated in the absence of ascorbate and markedly inhibited by cyanide, 5,5′-dithiobis-(2-nitrobenzoic acid) and p -chloromercuribenzoate. The molecular weight of P III was estimated to be 28 000 by both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both P II and P III strongly reacted with a monoclonal antibody raised against spinach leaf ascorbate peroxidase whereas P I did not react with it. P III is left to be defined ascorbate peroxidase but these observations suggest that it can be considered as an ascorbate peroxidase peculiar to roots. The content of ascorbate in roots increased rapidly in the early stages of germination and this was followed by an increase in ascorbate-peroxidizing activity accompanied by increased superoxide dismutase activity. Oxidation of ascorbate by peroxidase activity and the absence of ascorbate oxidase activity suggest that P II and P III function to remove H 2 O 2 at an early stage in roots, whose catalase activity is less than 2% that of the described peroxidase activity.

Cytokinin-promoted polyribosome formation in excised cucumber cotyledons

Summary Polyribosome (polysome) levels based on total ribosomal materials in excised etiolated cotyledons of Cucumis sativus L. cv. Ohio were examined during incubation with or without N 6 -benzyladenine (BA) in the dark. Within the first 2 h after excision, polysome levels in both cotyledons with and without BA increased about 5-fold. The polysome level in control cotyledons subsequently declined, approximating the initial level after 12-18h. In BA-treated cotyledons, however, it remained high during the entire period of incubation. BA application to the water-pretreated (18h) cotyledons also increased the polysome level and did not affect RNase activity or RNA content. BA-promoted polysome formation was inhibited by α-amanitin and cordycepin but not by 5-fluorouracil. These results are consistent with the hypothesis that cytokininpromoted polysome formation depends on de novo synthesis of mRNA but not of rRNA.

Effect of Benzyladenine on Change in Plastid Membrane Proteins of Etiolated Cucumber Cotyledons During Greening

Summary Developmental changes of plastid membrane proteins in Cucumis sativus cotyledons and the effect of benzyladenine (BA) thereon were examined by SDS-polyacrylamide gel electrophoresis. Quantitative and qualitative changes of plastid membrane proteins were observed during greening. A protein which became undetectable in the light disappeared following BA treatment in the dark. On the other hand, after illumination BA treatment eliminated the time lag of synthesis of light-harvesting chlorophyll complex proteins observed in the control. In additional illumination, BA conspicuously increased light-harvesting chlorophyll complex proteins in comparison to other plastid membrane proteins.

Combined Effect of Benzyladenine and Potassium on the Level of Light-harvesting Chlorophyll a/b Protein in Detached Cucumber Cotyledons

Summary Benzyladenine (BA) and KCl were applied to detached cucumber ( Cucumis sativus L. cv. Ohio) cotyledons under dark followed by light, or under continuous light conditions. Pretreatment with BA in the dark resulted in loss of the lag in the increase of the apoprotein of the light-harvesting chlorophyll a/b protein (LHCP-apoprotein) normally occuring upon illumination. KCl pretreatment did not shorten this lag phase, but did enhance the rate of increase during greening. BA + KCl pretreatment markedly increased the effect of BA on the LHCP-apoprotein level during the early phase of greening. In contrast, neither BA, KCl or BA + KCl exhibited any significant effect on the LHCP-apoprotein level in continuous light.

Differential Effects of Benzyladenine and Potassium on Ribulose-l,5-Bisphosphate Carboxylase Synthesis in Detached Cucumber Cotyledons

Summary Excised cucumber (Cucumis sativus L. cv. Ohio) cotyledons were incubated with benzyladenine (BA) and/or KCl in the dark, the dark followed by light or continuous light, and ribulose-1,5-bisphosphate carboxylase (RuBPCase) content was determined immunologically. BA, not KCl, significantly increased RuBPCase content per cotyledon in the dark. On the other hand, KCl, not BA, significantly promoted RuBPCase synthesis in light. BA+KCl enhanced BA or KCl effect on RuBPCase synthesis in both the dark and light. Dark pretreatment with water or KCl produced a lag phase before RuBPCase synthesis occurred on exposure tolight, while pretreatment with BA+KCl eliminated this lag phase. These facts indicate that BA in the dark can act instead of light in RuBPCase synthesis, while KCl increases the light effect on RuBPCase synthesis, and that potassium, cytokinin and light affect RuBPCase gene expression differentially.