Although auxin and ethylene play pivotal roles in leaf abscission, the subsequent signaling molecules are poorly understood. This is mainly because it is difficult to effectively treat the intact abscission zone (AZ) with pharmacological reagents. We developed an in vitro experimental system that reproduces stress-induced leaf abscission in planta. In this system, 1-mm-thick petiole strips, encompassing the AZ, were separated within 4 days of abscission at the AZ through cell wall degradation in an auxin depletion- and ethylene-dependent manner. The system allowed us to show that hydrogen peroxide (H2O2) is involved in abscission signaling. Microscopic analyses revealed continuous H2O2 production by AZ cells. H2O2 scavengers and diphenylene iodonium, an inhibitor of NADPH oxidase, suppressed in vitro abscission and cellulase expression. Conversely, the application of H2O2 promoted in vitro abscission and expression of cellulase. Ethephon-induced abscission was suppressed by inhibitors of H2O2 production, whereas the expression of ethylene-responsive genes was unaffected by both H2O2 and an H2O2 inhibitor. These results indicated that H2O2 acts downstream from ethylene in in vitro abscission signaling. In planta, salinity stress induced the expression of genes that respond to ethylene and reactive oxygen species, and also induced H2O2 production at the AZ, which preceded leaf abscission. These results indicate that H2O2 has roles in leaf abscission associated with ethylene both in vitro and in planta.
