Distribution of hydrogen peroxide during adventitious roots initiation and development in mung bean hypocotyls cuttings

Previous studies have shown that hydrogen peroxide (H2O2) may mediate the auxin response during the formation of adventitious roots (AR). However, the mechanism and distribution of H2O2 during AR formation remains unclear. In this study, we investigate the spatiotemporal changes and role of H2O2 in AR initiation and development. Application of 5-100 mM H2O2 to Mung bean (Phaseolus radiatus L.) hypocotyl cuttings induced AR formation in a dose-dependent manner. The effect was blocked by ascorbic acid (AA), an important reducing substrate for H2O2 reduction. Depletion of endogenous H2O2 by AA resulted in the significant reduction of AR emergence, suggesting a physiological role for H2O2 in the regulation of AR formation. Determination of H2O2 content showed that the level of H2O2 increased gradually and reached the highest value 60 h after induction of AR. Further detection of endogenous H2O2 by the specific fluorescent probe dichlorofluorescein diacetate (H2DCF-DA) and 3,3'-diaminobenzidine (DAB) staining in transverse sections of the basal region of cuttings revealed that obvious H2O2 signals were observed in the pericycle cells between the vascular bundles 24 h after the primary roots were removed. With the development of root primordia, H2O2 signals increased gradually and were mainly distributed in the root meristem. AA significant inhibited the H2O2-dependent fluorescence and the formation of AR, suggesting an essential role of H2O2 generation during AR initiation and development. Furthermore, the involvement of Ca2+ during H2O2-mediated AR formation was evaluated. Ca2+ channel inhibitors LaCl3 and ruthenium red (RR) and Ca2+ chelator ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) prevent H2O2-induced AR formation, which indicate that the hypocotyl cuttings response to H2O2 depends on the availability of both intracellular and extracellular Ca2+ pools, and Ca2+ is a downstream messenger in the signaling pathway triggered by H2O2 to promote adventitious root formation.