Hydrogen Gas Is Involved in Auxin-Induced Lateral Root Formation by Modulating Nitric Oxide Synthesis

Metabolism of molecular hydrogen (H-2) in bacteria and algae has been widely studied, and it has attracted increasing attention in the context of animals and plants. However, the role of endogenous H-2 in lateral root (LR) formation is still unclear. Here, our results showed that H-2-induced lateral root formation is a universal event. Naphthalene-1-acetic acid (NAA; the auxin analog) was able to trigger endogenous H-2 production in tomato seedlings, and a contrasting response was observed in the presence of N-1-naphthyphthalamic acid (NPA), an auxin transport inhibitor. NPA-triggered the inhibition of H-2 production and thereafter lateral root development was rescued by exogenously applied H-2. Detection of endogenous nitric oxide (NO) by the specific probe 4-amino-5-methylamino-2 ',7 '-difluorofluorescein diacetate (DAF-FM DA) and electron paramagnetic resonance (EPR) analyses revealed that the NO level was increased in both NAA- and H-2-treated tomato seedlings. Furthermore, NO production and thereafter LR formation induced by auxin and H-2 were prevented by 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO; a specific scavenger of NO) and the inhibitor of nitrate reductase (NR; an important NO synthetic enzyme). Molecular evidence confirmed that some representative NO-targeted cell cycle regulatory genes were also induced by H-2, but was impaired by the removal of endogenous NO. Genetic evidence suggested that in the presence of H-2, Arabidopsis mutants nia2 (in particular) and nia1 (two nitrate reductases (NR)-defective mutants) exhibited defects in lateral root length. Together, these results demonstrated that auxin-induced H-2 production was associated with lateral root formation, at least partially via a NR-dependent NO synthesis.