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?C100 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.