Ascorbate-glutathione pathways mediated by cytokinin regulate H2O2 levels in light-controlled rose bud burst

Rosebush (Rosa "Radrazz") plants are an excellent model to study light control of bud outgrowth since bud outgrowth only arises in the presence of light and never occurs in darkness. Recently, we demonstrated high levels of hydrogen peroxide (H2O2) present in the quiescent axillary buds strongly repress the outgrowth process. In light, the outgrowing process occurred after H2O2 scavenging through the promotion of Ascorbic acid-Glutathione (AsA-GSH)-dependent pathways and the continuous decrease in H2O2 production. Here we showed Respiratory Burst Oxidase Homologs expression decreased in buds during the outgrowth process in light. In continuous darkness, the same decrease was observed although H2O2 remained at high levels in axillary buds, as a consequence of the strong inhibition of AsA-GSH cycle and GSH synthesis preventing the outgrowth process. Cytokinin (CK) application can evoke bud outgrowth in light as well as in continuous darkness. Furthermore, CKs are the initial targets of light in the photocontrol process. We showed CK application to cultured buds in darkness decreases bud H2O2 to a level that is similar to that observed in light. Furthermore, this treatment restores GSH levels and engages bud burst. We treated plants with buthionine sulfoximine, an inhibitor of GSH synthesis, to solve the sequence of events involving H2O2/GSH metabolisms in the photocontrol process. This treatment prevented bud burst, even in the presence of CK, suggesting the sequence of actions starts with the positive CK effect on GSH that in turn stimulates H2O2 scavenging, resulting in initiation of bud outgrowth.