Abstract
Metabolism of molecular hydrogen (H2) 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 H2 in lateral root (LR) formation is still unclear. Here, our results showed that H2-induced lateral root formation is a universal event. Naphthalene-1-acetic acid (NAA; the auxin analog) was able to trigger endogenous H2 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 H2 production and thereafter lateral root development was rescued by exogenously applied H2. 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 H2-treated tomato seedlings. Furthermore, NO production and thereafter LR formation induced by auxin and H2 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 H2, but was impaired by the removal of endogenous NO. Genetic evidence suggested that in the presence of H2, 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 H2 production was associated with lateral root formation, at least partially via a NR-dependent NO synthesis.
Highlights
In higher plants, the formation of lateral root (LR) is influenced by phytohormones and a wide range of environmental cues, including water availability, nutrients, and abiotic stress [1,2,3]
To verify whether H2 had any effects on the lateral root formation in plants, tomato seedlings were incubated with solutions containing different concentrations of exogenous H2 (0.0078, 0.078, 0.39, and 0.78 mM)
Additive responses in lateral rooting were observed when naphthylacetic acid (NAA) was together with 0.39 mM H2 (Figure S1). These results suggested that endogenous H2 might be, at least partially, involved in lateral root formation promoted by auxin
Summary
The formation of lateral root (LR) is influenced by phytohormones and a wide range of environmental cues, including water availability, nutrients, and abiotic stress [1,2,3]. Ample evidence further revealed that nitric oxide (NO) is a ubiquitous and free radical gas that regulates a wide range of physiological processes in plants, and the important roles of NO in auxin-triggered lateral root formation [7], root hair development [8], and adventitious rooting [9], were discovered. Despite these discoveries, the understanding of the mechanisms of NO biosynthesis in plants is still incomplete. Two enzymes mainly involved in NO production were suggested, after the experiments with the inhibitor tests by the removal of endogenous NO, including the application of tungstate (a nitrate reductase (NR) inhibitor) and NG-nitro-L-arginine methyl ester (a nitric oxide synthetase (NOS)-like protein inhibitor) [10,11]
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