Cadmium-Induced Hydrogen Sulfide Synthesis Is Involved in Cadmium Tolerance in <i>Medicago sativa</i> by Reestablishment of Reduced (Homo)glutathione and Reactive Oxygen Species Homeostases

<div><p>Until now, physiological mechanisms and downstream targets responsible for the cadmium (Cd) tolerance mediated by endogenous hydrogen sulfide (H₂S) have been elusive. To address this gap, a combination of pharmacological, histochemical, biochemical and molecular approaches was applied. The perturbation of reduced (homo)glutathione homeostasis and increased H₂S production as well as the activation of two H₂S-synthetic enzymes activities, including _L-cysteine desulfhydrase (LCD) and _D-cysteine desulfhydrase (DCD), in alfalfa seedling roots were early responses to the exposure of Cd. The application of H₂S donor sodium hydrosulfide (NaHS), not only mimicked intracellular H₂S production triggered by Cd, but also alleviated Cd toxicity in a H₂S-dependent fashion. By contrast, the inhibition of H₂S production caused by the application of its synthetic inhibitor blocked NaHS-induced Cd tolerance, and destroyed reduced (homo)glutathione and reactive oxygen species (ROS) homeostases. Above mentioned inhibitory responses were further rescued by exogenously applied glutathione (GSH). Meanwhile, NaHS responses were sensitive to a (homo)glutathione synthetic inhibitor, but reversed by the cotreatment with GSH. The possible involvement of cyclic AMP (cAMP) signaling in NaHS responses was also suggested. In summary, LCD/DCD-mediated H₂S might be an important signaling molecule in the enhancement of Cd toxicity in alfalfa seedlings mainly by governing reduced (homo)glutathione and ROS homeostases.</p></div

Concentrations of low molecular weight thiols and their disulfides, and hGSH/hGSSGh ratio in root tissues.

<p>Seedlings were pretreated with or without 100 µM NaHS, 2 mM PAG, 1 mM GSH, individual or combination for 6 h, and then exposed to 200 µM CdCl₂ for another 12 h. Values are means ± SD of three independent experiments with three replicates for each. Different letters within columns indicate significant differences (<i>P</i><0.05) according to Duncan's multiple range test.</p><p>Concentrations of low molecular weight thiols and their disulfides, and hGSH/hGSSGh ratio in root tissues.</p

NaHS, GSH and BSO pretreatments differentially regulated seedling growth, TBARS accumulation, (h)GSH content, and (h)GSH/(h)GSSG(h).

<p>Fresh weight of 10 roots (A), TBARS accumulation (B), (h)GSH contents (C), and (h)GSH/(h)GSSG(h) ratio (D) in root tissues were determined after seedlings were pretreated with or without 100 µM NaHS, 1 mM GSH, 1 mM BSO, individual or combination for 6 h, and then exposed to 200 µM CdCl₂ for 72 h (A), 24 h (B) and 12 h (C and D). Values are means ± SD of three independent experiments with three replicates for each. Bars denoted by the same letter did not differ significantly at <i>P</i><0.05 according to Duncan's multiple range test.</p

Time course changes of GSH pool and H₂S synthesis upon Cd stress.

<p>Upon 200 µM CdCl₂ treatment for 12 h, contents of (h)GSH (A), (h)GSSG(h) (B) and H₂S (D), the ratio of (h)GSH/(h)GSSG(h) (C), and the activities of LCD (E) and DCD (F) in root tissues were analyzed. Values are means ± SD of three independent experiments with three replicates for each. Bars denoted by the same letter did not differ significantly at <i>P</i><0.05 according to Duncan's multiple range test.</p

NaHS increased endogenous H₂S and (h)GSH contents, and the ratio of (h)GSH/(h)GSSG(h) upon Cd stress.

<p>Endogenous H₂S concentration in root tissues (A) was detected at 3 h after the beginning of 100 µM NaHS pretreatment (−3 h), and 200 µM CdCl₂ or chemical-free control treatments for 12 h (12 h). Meanwhile, contents of (h)GSH (B) and the ratio of (h)GSH/(h)GSSG(h) (C) in root tissues were detected at the indicated time points of treatments. Values are means ± SD of three independent experiments with three replicates for each. Bars denoted by the same letter did not differ significantly at <i>P</i><0.05 according to Duncan's multiple range test.</p

NaHS and GSH pretreatments alleviated Cd-induced ROS production, but blocked by PAG.

<p>LSCM results (A). Seedlings were pretreated with or without 100 µM NaHS, 2 mM PAG, 1 mM GSH, individual or combination for 6 h, and then exposed to 200 µM CdCl₂ for another 6 h. After various treatments, the roots were respectively stained with H₂DCFDA, then washed thoroughly to removal extra dye and immediately photographed by LSCM. Scale bar, 0.5 mm. The relative DCF fluorescence intensity in the corresponding roots (B).</p

NaHS, PAG and GSH pretreatments differentially regulated seedling growth, (h)GSH content, and (h)GSH/(h)GSSG(h) ratio.

<p>Corresponding phenotypes were photographed after 200 µM CdCl₂ treatment for 72 h, with or without 100 µM NaHS, 2 mM PAG, 1 mM GSH, individual or combination pretreatments for 6 h (A). Scale bar, 2 cm. Contents of (h)GSH (B), and the ratio of (h)GSH/(h)GSSG(h) (C) in root tissues were also analyzed after 200 µM CdCl₂ treatment for 12 h, with or without 100 µM NaHS, 2 mM PAG, 1 mM GSH, individual or combination pretreatment for 6 h. Values are means ± SD of three independent experiments with three replicates for each. Bars denoted by the same letter did not differ significantly at <i>P</i><0.05 according to Duncan's multiple range test.</p

Time course of transcripts responsible for (h)GSH metabolism regulated by NaHS and Cd.

<p>Seedlings were pretreated with or without 100 µM NaHS for 6 h and then exposed to 200 µM CdCl₂ for another 24 h. The expression levels of <i>ECS</i> (A), <i>GS</i> (B) and <i>GR1</i> (C) in root tissues analyzed by real-time RT-PCR are presented as values relative to the control at the beginning of pretreatment, normalized against expression of two internal reference genes in each sample. Values are means ± SD of three independent experiments with three replicates for each.</p

cAMP pathway might be involved in H₂S-alleviated Cd toxicity.

<p>Fresh weight of 10 roots (A), TBARS accumulation (B), <i>GR1</i> (C), <i>Cu,Zn-SOD</i> (D), <i>APX1</i> (E), and <i>GPX</i> (F) gene expression in alfalfa seedling roots upon Cd stress. Seedlings were pretreated with or without 100 µM NaHS, 50 µM 8-Br-cAMP (8Br), 2 mM PAG, 200 µM alloxan (All), 500 µM IBMX, 1 mM DDA alone, or the combination of treatments for 6 h, and then exposed to 200 µM CdCl₂ for 72 h (A), 24 h (B) and 12 h (C–F). The expression levels of corresponding genes analyzed by real-time RT-PCR are presented as values relative to the control, normalized against expression of two internal reference genes in each sample. Values are means ± SD of three independent experiments with three replicates for each. Bars denoted by the same letter did not differ significantly at <i>P</i><0.05 according to Duncan's multiple range test.</p