In Vitro Oxidation of Indoleacetic Acid by Soluble Auxin-Oxidases and Peroxidases from Maize Roots

Abstract

Soluble auxin-oxidases were extracted from Zea mays L. cv LGlI apical root segments and partially separated from peroxidases (EC 1.11.1.7) by size-exclusion chromatography. Auxinoxidases were resolved into one main peak corresponding to a molecular mass of 32.5 kilodaltons and a minor peak at 54.5 kilodaltons. Peroxidases were separated into at least four peaks, with molecular masses from 32.5 to 78 kilodaltons. In vitro activity of indoleacetic acid-oxidases was dependent on the presence of MnCI2 and p-coumaric acid. Compound(s) present in the crude extract and several synthetic auxin transport inhibitors (including 2,3,5-triiodobenzoic acid and N-1-naphthylphthalamic acid) inhibited auxin-oxidase activity, but had no effect on peroxidases. The products resulting from the in vitro enzymatic oxidation of [3H] indoleacetic acid were separated by HPLC and the major metabolite was found to cochromatograph with indol-3yl-methanol.Two pathways for the oxidative degradation of IAA have been established. The first involves oxidation of the indole nucleus (32) by a specific enzyme which catalyzes the formation of oxindole-3-acetic acid (OxIAA2; 23), whereas the second pathway consists of the oxidative decarboxylation of the side chain. Peroxidases (EC 1.11.1.7) from numerous plant species have been shown to catalyze this oxidative decarboxylation (27), leading to the formation of either indol3yl-methanol (17) or 3-methyleneoxindole (6). The ratio of the production of these last two compounds is influenced by several factors including the nature of the cofactors added to study the reaction in vitro, the pH, and the enzyme/substrate ratio (4, 26).The breakdown of IAA catalyzed by peroxidases has been studied in a number of systems, and the requirement for added cofactors and for H202 differs between species. The degradation of auxin by HRP may occur without added cofactors, and in the presence or absence of H202 (6). However, tomato peroxidases are dependent on the presence of H202 for the oxidation of IAA (9) and fail to catalyze the reaction in the presence of compounds such as 2,4-dichloro-'Present address:

Effect of exogenous abscisic acid on the level of antioxidants in Atractylodes macrocephala Koidz under lead stress

“…POD activity was assayed according to Beffa et al (1990). Briefly, the assay mixture contained 2.95 mL 100 mM NaH 2 PO 4 -Na 2 HPO 4 buffer (pH 6.0), 2 mM hydrogen peroxide (H 2 O 2 ), 9 mM guaiacol, and 0.05 mL enzyme extract in a total volume of 3.0 mL.…”

Role of salicylic acid in alleviating oxidative damage in rice roots (Oryza sativa) subjected to cadmium stress

“…The POD activity was assayed following the method of Beffa et al (1990). Briefly, the assay mixture contained 2.95 mL 100 mM NaH 2 PO 4 eNa 2 HPO 4 buffer (pH 6.0), 2 mM H 2 O 2 , 9 mM guaiacol and 0.05 mL enzyme extract in a total volume of 3.0 mL.…”

Does salicylic acid regulate antioxidant defense system, cell death, cadmium uptake and partitioning to acquire cadmium tolerance in rice?

“…One unit SOD activity was defined as the amount of enzyme required to result in a 50% inhibition of the rate of NBT (r-nitro blue tetrazolium chloride) reduction measured at 560 nm. The POD activity was assayed following the method of Beffa et al (1990). Changes in the absorbance of the brown guaiacol at 460 nm in the presence of H 2 O 2 were recorded for calculating POD activity.…”