Hydrogen sulfide - cysteine cycle system enhances cadmium tolerance through alleviating cadmium-induced oxidative stress and ion toxicity in Arabidopsis roots

Abstract

Cadmium (Cd²⁺) is a common toxic heavy metal ion. We investigated the roles of hydrogen sulfide (H₂S) and cysteine (Cys) in plant responses to Cd²⁺ stress. The expression of H₂S synthetic genes LCD and DES1 were induced by Cd²⁺ within 3 h, and endogenous H₂S was then rapidly released. H₂S promoted the expression of Cys synthesis-related genes SAT1 and OASA1, which led to endogenous Cys accumulation. The H₂S and Cys cycle system was stimulated by Cd²⁺ stress, and it maintained high levels in plant cells. H₂S inhibited the ROS burst by inducing alternative respiration capacity (AP) and antioxidase activity. H₂S weakened Cd²⁺ toxicity by inducing the metallothionein (MTs) genes expression. Cys promoted GSH accumulation and inhibited the ROS burst, and GSH induced the expression of phytochelatin (PCs) genes, counteracting Cd²⁺ toxicity. In summary, the H₂S and Cys cycle system played a key role in plant responses to Cd²⁺ stress. The Cd²⁺ tolerance was weakened when the cycle system was blocked in lcddes1-1 and oasa1 mutants. This paper is the first to describe the role of the H₂S and Cys cycle system in Cd²⁺ stress and to explore the relevant and specificity mechanisms of H₂S and Cys in mediating Cd²⁺ stress.

Figure 1. The effect of Cd²⁺ on root length, MDA and EL in Arabidopsis roots.

(a) Phenotype of root growth in WT seedlings. Bar = 1 cm. (b) The root lengths of WT seedlings (n > 25). (c) MDA contents in WT roots stressed by various concentrations of Cd²⁺. (d) EL in WT roots stressed by various concentrations of Cd²⁺. 7-d-old Arabidopsis seedlings were grown on 1/2 MS agar plates supplied with 0–150 μM Cd²⁺ for 5 d, and the lengths of the primary roots, MDA contents and EL were recorded. Mean values and SE were calculated from three replicates. Within each set of experiments, bars with different letters are significantly different (P < 0.05, Duncan’s multiple range tests).

Figure 2. Analysis of endogenous H₂S and Cys contents in WT roots of Arabidopsis.

(a) Time-course of H₂S content. (b) Time-course of Cys content. 7-d-old WT seedlings were treated with 50 μM Cd²⁺, 50 μM Cd²⁺ + 1 mM Cys and 50 μM Cd²⁺ + 50 μM NaHS for 0 to 48 h. (c) Changes of H₂S content in various Cd²⁺ concentrations. (d) Changes of Cys content in various Cd²⁺ concentrations. 7-d-old WT seedlings were supplied with 0–150 μM Cd²⁺ for 5 d. Mean values and SE are calculated from three replicates. Within each set of experiments, bars with different letters are significant different (P < 0.05, Duncan’s multiple range tests).

Figure 3. qRT-PCR analysis the synthesis genes of H₂S and Cys in Arabidopsis WT roots.

Relative expression levels were normalized with the internal standard EF1a. 7-d-old WT seedlings were grown on agar plates supplemented with 50 μM Cd²⁺, 50 μM NaHS, 1 mM Cys, 50 μM Cd²⁺ plus 50 μM NaHS, and 50 μM Cd²⁺ plus 1 mM Cys for 3 h, respectively. Mean values and SE were calculated from three replicates. Within each set of experiments, bars with different letters are significant different (P < 0.05, Duncan’s multiple range tests).

Figure 4. The effect of H₂S and Cys on root length, MDA and EL in lcddes1-1 and oasa1 mutant plants under Cd²⁺ stress.

(a) Phenotype of Arabidopsis root growth. Bar = 1 cm. (b) The root lengths of Arabidopsis seedlings (n > 25). (c) MDA contents in Arabidopsis roots. (d) EL in Arabidopsis roots. 7-d-old Arabidopsis seedlings were grown on 1/2 MS agar plates supplied with 50 μM Cd²⁺, 50 μM Cd²⁺ plus 50 μM NaHS and 50 μM Cd²⁺ plus 1 mM Cys for 5 d respectively, and the lengths of the primary roots, MDA contents and EL were recorded. Mean values and SE were calculated from three replicates. Within each set of experiments, bars with different letters are significantly different (P < 0.05, Duncan’s multiple range tests).

Figure 5. Effect of H₂S and Cys on the expression of AOX genes and the activity of TP, CP and AP in Cd²⁺ stress.

(a–c) The expression of AOX1A, AOX1C and AOX2. 7-d-old WT seedlings were grown on 1/2 MS agar plates supplied with 50 μM Cd²⁺, 50 μM NaHS, 1 mM Cys, 50 μM Cd²⁺ plus 50 μM NaHS, and 50 μM Cd²⁺ plus 1 mM Cys for 3 h, respectively. (d,e) Changes in TP, CP and AP activity in various Cd²⁺ concentrations for 5 d in WT. (f–l) AP activity in WT and mutants. 7-d-old Arabidopsis seedlings were grown on 1/2 MS agar plates treated with 50 μM Cd²⁺, 50 μM Cd²⁺ plus 50 μM NaHS and 50 μM Cd²⁺ plus 1 mM Cys for 5 d. Mean values and SE were calculated from three replicates. Within each set of experiments, bars with different letters are significantly different (P < 0.05, Duncan’s multiple range tests).

Figure 6. Effect of H₂S and Cys on antioxidant enzymes activity and GSH level in Cd²⁺ stress.

7-d-old Arabidopsis seedlings were grown on 1/2 MS agar plates supplemented with 50 μM Cd²⁺, 50 μM NaHS, 1 mM Cys, and 200 μM nPG for 6 h, and SOD activity (a,b), CAT activity (c,d) and GSH content (e,f) were recorded. Mean values and SE were calculated from three replicates. Within each set of experiments, bars with different letters are significantly different (P < 0.05, Duncan’s multiple range tests).

Figure 7. The changes of the endogenous H₂O₂ level in Arabidopsis root.

(a) H₂O₂ H₂DCF-DA fluorescence in WT roots (bar = 100 μm). (b) Quantification of H₂O₂ H₂DCF-DA fluorescence intensity. (c) Time-course of H₂O₂ level. 7-d-old Arabidopsis seedlings were grown on 1/2 MS agar plates supplemented with 50 μM Cd²⁺, 50 μM NaHS, 1 mM Cys, and 200 μM nPG for 6 h (a,b), 0–24 h (c). H₂DCF-DA fluorescence intensity data represents mean grey values. SE was calculated from measurements of at least five roots for each treatment, and the experiments were repeated three times. Within each set of experiments, bars with different letters are significantly different (P < 0.05, Duncan’s multiple range tests).

Figure 8. Effect of NaHS and Cys on Cd²⁺ percentage and qRT-PCR analysis the expression of heavy metal chelators genes in Arabidopsis roots.

(a) Cd²⁺ accumulation in Arabidopsis roots. (b) The expression of PCS1, PCS2, MT1A, MT1B and MT2B in WT. (c) Time-course of PCS1 gene expression. (d) Time-course of MT1A gene expression. 7-d-old Arabidopsis seedlings were grown on 1/2 MS agar plates supplied with 50 μM Cd²⁺, 50 μM NaHS and 1 mM Cys for 5 d (a), 3 h (b), 0–12 (c,d). Mean values and SE were calculated from three replicates. Within each set of experiments, bars with different letters are significantly different (P < 0.05, Duncan’s multiple range tests).

Figure 9. A diagram representing Cd²⁺-induced toxicity and protective mechanism of the H₂S and Cys cycle system in Arabidopsis roots.

Arrows indicate enhanced effects and hyphens indicate suppressed effects.