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.