Hydrogen peroxide generated by copper amine oxidase involved in adventitious root formation in mung bean hypocotyl cuttings

Abstract

The role of hydrogen peroxide (H2O2) generated by copper-containing diamine oxidases (CuAO) in adventitious rooting in mung bean hypocotyl was studied. The physiological data show that aminoguanidine (AG), an irreversible inhibitor of CuAO, inhibited adventitious rooting significantly. Among major catalysed and metabolised products or substrates of CuAO, only H2O2 obviously induced adventitious rooting and notably reversed the inhibitory effect of AG on adventitious rooting. The spatio-temporal change of CuAO activity in the rooting region of hypocotyls was histochemically measured using a peroxidase-coupled assay with the artificial substrate 3,3-diaminobenzidine tetrahydrochloride (DAB) as chromogen. DAB staining was detected in the parenchyma tissue between the vascular bundles, where roots originated before the emergence of root primordium, and then in the forming root primordium. With the origination of adventitious roots, the density and area of DAB staining increased rapidly. Most cells in root primodium had an evidently DAB staining, and strong staining mostly distributed in root meristem. H2O2, which was detected with the H2O2-sensitive probe 2,7-dichlorofluorescein diacetate, had a similar change to CuAO activity in this process. AG largely suppressed DAB staining and H2O2 fluorescence, and prevented root primordium formation. Taken together, these results clearly prove that, CuAO is responsible for H2O2 production during adventitious rooting, and H2O2 generated by CuAO is required for adventitious rooting in mung bean hypocotyls.