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

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.

Keywords: H2; NO; auxin; lateral root formation; nitrate reductase.

Figure 1

Effects of 1-naphthylacetic acid (NAA), N-1-naphthylphthalamic acid (NPA), and H₂ on lateral root formation and H₂ release. 3-day-old tomato seedlings were incubated with solutions containing various concentrations of NAA, NPA, and H₂. (a) The number of emerged lateral roots (LRs) (>1 mm) per seedling and LR length were calculated after 3-day of treatments (n = 60); (b) The H₂ production in roots was detected after 6-h treatment (n = 5); (c) Time-course of H₂ production in response to 200 nM NAA, 500 nM NPA, and 0.39 mM H₂ (n = 5). Distilled water was used for the control (Con) treatment. Data are the means ± SE of three independent experiments with at least three replicates for each. Within each set of experiments, bars denoted by the same letter did not differ significantly at the p < 0.05 level according to Duncan’s multiple range test.

Figure 2

NAA- and NPA-regulated lateral root formation and H₂ production were regulated by exogenous H₂. 3-day-old tomato seedlings were incubated with solutions containing 200 nM NAA, 500 nM NPA, and 0.39 mM H₂, alone or the combination treatments. The number of emerged LRs (>1 mm) per seedling and LR length were calculated after 3-day of treatment (n = 60). The H₂ production in roots was determined after 6-h treatment (n = 5). Distilled water was used for the control (Con) treatment. Data are the means ± SE of three independent experiments with at least three replicates for each. Within each set of experiments, bars denoted by the same letter did not differ significantly at the p < 0.05 level according to Duncan’s multiple range test.

Figure 3

H₂-induced nitric oxide (NO) production and lateral rooting were sensitive to the removal of NO by 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a NO scavenger. 3-day-old tomato seedlings were incubated with solutions containing 200 nM NAA, 0.39 mM H₂, and 200 µM cPTIO, alone or the combination treatments. (a) The number of emerged LRs (>1 mm) per seedling and LR length were calculated after 3-day of treatments. Meanwhile, the NO fluorescence in tomato roots was analyzed by fluorescence probe DAF-FM DA at 36 h (b) and over 48 h; (c) of treatments, respectively, using laser scanning confocal microscopy (LSCM) (TCS-SP2 system; Leica Lasertechnik GmbH). The DAF-FM DA fluorescence density was analyzed using Leica software. (d) The NO signal was also detected by electron paramagnetic resonance (EPR) after being treated for 36 h. Distilled water was used for the control (Con) treatment. Data are the means ± SE of three independent experiments with at least three replicates for each (n = 60 for lateral root formation analysis; n = 5 for NO detection). Within each set of experiments, bars denoted by the same letter did not differ significantly at p < 0.05 level according to Duncan’s multiple range test.

Figure 4

NR might be the enzymatic source of H₂-triggered NO generation. 3-day-old tomato seedlings were incubated with solutions containing 200 nM NAA, 0.39 mM H₂, and 20 µM tungstate (Tg), alone or the combination treatments. (a) The number of emerged LRs (>1 mm) per seedling and LR length were calculated after 3-day of treatments. Meanwhile, the activity of nitrate reductase (NR) was determined at 36 h (b) and over 48 h (d) of treatments, respectively; (c) The NO fluorescence in tomato roots was analyzed by fluorescence probe DAF-FM DA at 36 h of treatments, using LSCM (TCS-SP2 system; Leica Lasertechnik GmbH). The DAF-FM DA fluorescence density was analyzed using Leica software; (e) The NO signal was also detected by EPR after being treated for 36 h. Distilled water was used for the control (Con) treatment. Data are the means ± SE of three independent experiments with at least three replicates for each (n = 60 for lateral root formation analysis; n = 5 for NR activity and NO detection). Within each set of experiments, bars denoted by the same letter did not differ significantly at P < 0.05 level according to Duncan’s multiple range test.

Figure 5

H₂-induced cell cycle regulatory gene transcripts were sensitive to the removal of NO with its scavenger and NR inhibitor. Three-day-old tomato seedlings were incubated with solutions containing 200 nM NAA, 0.39 mM H₂, 200 µM cPTIO, and 20 µM tungstate (Tg), alone or the combination treatments for 36 h. The transcripts of tomato CDKA1, CYCD3;1, and CYCA2;1 were analyzed using qPCR. Distilled water was used for the control (Con) treatment. Data are the means ± SE of three independent experiments with at least three replicates for each. Within each set of experiments, bars denoted by the same letter did not differ significantly at p < 0.05 level according to Duncan’s multiple range test.

Figure 6

Genetic evidence supporting the involvement of NR in H₂-induced lateral root formation. 7-day-old Arabidopsis seedlings of wild-type (WT), nia1, and nia2 mutants were treated with semi-strength Murashige and Skoog (MS) medium in the presence or absence of 0.078 mM H₂. (a,b) The number of emerged LRs (>1 mm) per seedling and LR length were calculated after 3-day treatment; (c) Representative photographs were then taken. Bar = 1 cm. Treatment without H₂ was regarded as control (Con). Data are the means ± SE of three independent experiments with at least three replicates for each (n = 20). Within each set of experiments, bars denoted by the same letter or labeled with an asterisk did not differ significantly at p < 0.05 level according to Duncan’s multiple range test.