Hydrogen peroxide acts downstream of melatonin to induce lateral root formation.

Background and Aims: Although several studies have confirmed the beneficial roles of exogenous melatonin in lateral root (LR) formation, the molecular mechanism is still elusive. Here, the role of hydrogen peroxide (H2O2) in the induction of LR formation triggered by melatonin was investigated.

Methods: Alfalfa (Medicago sativa 'Biaogan') and transgenic Arabidopsis seedlings were treated with or without melatonin, diphenyleneiodonium (DPI, NADPH oxidase inhibitor), N,N'-dimethylthiourea (DMTU, H2O2 scavenger), alone or combined. Then, H2O2 content was determined with 2',7'-dichlorofluorescein diacetate (H2DCFDA)-dependent fluorescence and spectrophotography. Transcript levels of cell cycle regulatory genes were analysed by real-time reverse transcription-PCR.

Key Results: Application of exogenous melatonin not only increased endogenous H2O2 content but also induced LR formation in alfalfa seedlings. Consistently, melatonin-induced LR primordia exhibited an accelerated response. These inducible responses were significantly blocked when DPI or DMTU was applied. Compared with the wild-type, transgenic Arabidopsis plants overexpressing alfalfa MsSNAT (a melatonin synthesis gene) increased H2O2 accumulation and thereafter LR formation, both of which were blocked by DPI or DMTU. Similarly, melatonin-modulated expression of marker genes responsible for LR formation, including MsCDKB1;1, MsCDKB2;1, AtCDKB1;1 and AtCDKB2;1, was obviously impaired by the removal of H2O2 in both alfalfa and transgenic Arabidopsis plants.

Conclusions: Pharmacological and genetic evidence revealed that endogenous melatonin-triggered LR formation was H2O2-dependent.