Nitric oxide is involved in hydrogen gas-induced cell cycle activation during adventitious root formation in cucumber

BACKGROUND: Adventitious root development is a complex process regulated through a variety of signaling molecules. Hydrogen gas (H₂) and nitric oxide (NO), two new signaling molecules are both involved in plant development and stress tolerance. RESULTS: To investigate the mechanism of adventitious root development induced by hydrogen-rich water (HRW), a combination of fluorescence microscopy and molecular approaches was used to study cell cycle activation and cell cycle-related gene expression in cucumber (Cucumis sativus ‘Xinchun 4’) explants. The results revealed that the effect of HRW on adventitious root development was dose-dependent, with maximal biological responses at 50 % HRW. HRW treatment increased NO content in a time-dependent fashion. The results also indicated that HRW and NO promoted the G1-to-S transition and up-regulated cell cycle-related genes: CycA (A-type cyclin), CycB (B-type cyclin), CDKA (cyclin-dependent kinase A) and CDKB (cyclin-dependent kinase B) expression. Additionally, target genes related to adventitious rooting were up-regulated by HRW and NO in cucumber explants. While, the responses of HRW-induced adventitious root development and increase of NO content were partially blocked by a specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt, NO synthase (NOS)-like enzyme inhibitor Nᴳ –nitro-L-arginine methylester hydrochloride, or nitrate reductase inhibitors tungstate and NaN₃. These chemicals also partially reversed the effect of HRW on cell cycle activation and the transcripts of cell cycle regulatory genes and target genes related adventitious root formation. CONCLUSIONS: Together, NO may emerge as a downstream signaling molecule in H₂-induced adventitious root organogenesis. Additionally, H₂ mediated cell cycle activation via NO pathway during adventitious root formation.