Hydrogen sulfide toxicity inhibits primary root growth through the ROS-NO pathway

Abstract

High concentrations of hydrogen sulfide (H₂S) are toxic to plants and inhibit their growth. Previous research indicated that high concentrations of H₂S modulate the root system architecture (RSA) by affecting auxin transport; however, the signaling pathway underlying this process remains unclear. Here, we investigated the effects of exogenous sodium hydrosulfide (NaHS), an H₂S donor, on primary root (PR) growth in Arabidopsis using pharmacological, physiological, and genetic approaches. H₂S toxicity repressed PR growth by triggering a signal transduction pathway involving reactive oxygen species (ROS) accumulation, MITOGEN-ACTIVATED PROTEIN KINASE 6 (MPK6) activation, and nitric oxide (NO) production. Respiratory burst oxidase homolog mutants and an NO synthase mutant were less sensitive to NaHS, suggesting that both ROS and NO mediate the inhibitory effects of H₂S on PR growth. We found that exogenous H₂S-activated ROS production was required for NO generation and that MPK6 mediated H₂S-induced NO production. MPK6 was shown to function downstream of ROS and upstream of NO. Finally, we demonstrated that exogenous H₂S repressed the distribution of auxin and reduced the meristematic cell division potential in root tips, and NO was involved in this process.

Figure 1

NaHS treatment inhibited PR growth. (a,b) Five-day-old wild-type seedlings grown in 1/2 MS medium were treated with 100–800 μM NaHS for 2 d, and (a) PR growth and (b) the length of the meristem zone were measured after treatment. ck, untreated control. n = 60. Error bars represent ± SD. Different letters indicate significantly different values (P < 0.05 by Tukey’s test).

Figure 2

NaHS induces the accumulation of ROS. (a,b) Detection of ROS production in the roots of 5-d-old Col-0 seedlings exposed to 500 μM NaHS for periods of up to 2 d using the ROS-specific fluorescent probe DCFH-DA (a) and quantification of ROS-specific fluorescence intensities (b) in plants treated as described in (a). The fluorescence intensity of the untreated roots was set to 100. Bars, 100 μm. n = 30. (c) Relative root growth of Col-0 seedlings treated with 500 μM NaHS in the presence or absence of 1 μM DPI, 1 mM KI, and 1 mM H₂O₂ for 2 d compared with untreated seedlings. (d) Relative root growth of col-0, rbohF, and rbohD/F seedlings treated with 500 μM NaHS for 2 d compared with untreated seedlings. n = 45. Error bars represent ± SD. Different letters indicate significantly different values (P < 0.05 by Tukey’s test).

Figure 3

NO is involved in the NaHS-mediated inhibition of PR growth. (a,b) Detection of NO production in the roots of 5-d-old wild-type seedlings exposed to 500 μM NaHS for periods of up to 2 d using the NO-specific fluorescence probe DAF-2 DA (a) and quantification of NO-specific fluorescence intensities (b) in plants treated as described in (a). The fluorescence intensity of the untreated roots was set to 100. Bars, 100 μm. n = 30. (c) Relative root growth of Col-0 seedlings treated with 500 μM NaHS in the presence or absence of 500 μM L-NAME and 200 μM cPTIO for 2 d compared with untreated seedlings. (d) Relative root growth of Col-0, nia1/2, and noa1 seedlings treated with 500 μM NaHS for 2 d compared with untreated seedlings. n = 45. Error bars represent ± SD. Different letters indicate significantly different values (P < 0.05 by Tukey’s test).

Figure 4

(a,b) Detection of NO production in the roots of 5-d-old wild-type Col-0 and rbohD/F seedlings exposed to 500 μM NaHS with or without 1 μM DPI, 1 mM KI, and 1 mM H₂O₂ for 24 h using the NO-specific fluorescent probe DAF-2 DA (a) and quantification of NO-specific fluorescence intensities (b) in plants treated as described in (a). (c,d) Detection of H₂O₂ production in the roots of 5-d-old wild-type Col-0, nia1/2, and noa1 seedlings exposed to 500 μM NaHS with or without 500 μM L-NAME for 24 h using the ROS-specific fluorescent probe DCFH-DA (c) and quantification of H₂O₂-specific fluorescence intensities (d) in plants treated as described in (c). The fluorescence intensity of the untreated roots was set to 100. Bars, 100 μm. n = 30. ck, untreated control. Error bars represent ± SD.

Figure 5

(a) Relative root growth of Col-0, rbohF, and rbohD/F seedlings exposed to 500 μM NaHS with or without 1 mM H₂O₂, 100 μM SNAP, and 500 μM L-NAME for 2 d compared with untreated seedlings. (b) Relative root growth of Col-0 and noa1 seedlings exposed to 500 μM NaHS with or without 1 mM H₂O₂ and 100 μM SNAP for 2 d compared with untreated seedlings. n = 45. Error bars represent ± SD. Different letters indicate significantly different values (P < 0.05 by Tukey’s test).

Figure 6

MPK6 mediates PR growth inhibition by NaHS. (a) Relative root growth of Col-0 seedlings treated with 500 μM NaHS in the presence or absence of 150 μM PD98059 for 2 d compared with untreated seedlings. (b) Relative root growth of Col-0 and mpk6 seedlings exposed to 500 μM NaHS with or without 1 mM H₂O₂, 100 μM SNAP, 1 mM KI, and 500 μM L-NAME for 2 d compared with untreated seedlings. n = 45. (c,d) Detection of ROS production in the roots of 5-d-old wild-type col-0 and mpk6 seedlings exposed to 500 μM NaHS with or without 150 μM PD98059 for 24 h by the ROS-specific fluorescent probe DCFH-DA (c) and the quantification of the ROS-specific fluorescence intensities (d) in plants treated as described in (c). (e,f) Detection of NO production in the roots of 5-d-old wild-type col-0 and mpk6 seedlings exposed to 500 μM NaHS with or without 1 mM H₂O₂ or 150 μM PD98059 for 24 h using the NO-specific fluorescence probe DAF-2 DA (e) and quantification of NO-specific fluorescence intensities (f) in plants treated as described in (e). n = 30. Bars, 100 μm. ck, untreated control. Error bars represent ± SD. Different letters indicate significantly different values (P < 0.05 by Tukey’s test).

Figure 7

NO is involved in the NaHS-mediated reduction of the distribution of auxin in root tips. YFP fluorescence in the roots of 5-d-old DII-VENUS seedlings exposed to 500 μM NaHS with or without 500 μM L-NAME for 24 h. Bars, 50 μm. n = 30. ck, untreated control.

Figure 8

H₂S toxicity inhibits PR growth via the ROS-MPK6-NO signaling pathway. High-concentration H₂S induced ROS production via the NADPH oxidase pathway, which directly inhibited PR growth and activated MPK6. MPK6 then promoted NO production through both L-Arg-dependent and NR-dependent routes. Elevated NO repressed auxin distribution, ultimately inhibiting PR growth.