Hydrogen sulfide negatively regulates cd-induced cell death in cucumber (Cucumis sativus L) root tip cells

Abstract

Background: Hydrogen sulfide (H₂S) is a gas signal molecule involved in regulating plants tolerance to heavy metals stress. In this study, we investigated the role of H₂S in cadmium-(Cd-) induced cell death of root tips of cucumber seedlings.

Results: The results showed that the application of 200 μM Cd caused cell death, increased the content of reactive oxygen species (ROS), chromatin condensation, the release of Cytochrome c (Cyt c) from mitochondria and activated caspase-3-like protease. Pretreatment of seedlings with 100 μM sodium hydrogen sulfide (NaHS, a H₂S donor) effectively alleviated the growth inhibition and reduced cell death of root tips caused by Cd stress. Additionally, NaHS + Cd treatment could decrease the ROS level and enhanced antioxidant enzyme activity. Pretreatment with NaHS also inhibited the release of Cyt c from the mitochondria, the opening of the mitochondrial permeability transition pore (MPTP), and the activity of caspase-3-like protease in the root tips of cucumber seedling under Cd stress.

Conclusion: H₂S inhibited Cd-induced cell death in cucumber root tips by reducing ROS accumulation, activating the antioxidant system, inhibiting mitochondrial Cyt c release and reducing the opening of the MPTP. The results suggest that H₂S is a negative regulator of Cd-induced cell death in the root tips of cucumber seedling.

Keywords: Caspase-3-like protease; Cell death; Cyt c; Mitochondria; Oxidative damage.

Fig. 1

Effects of Cd stress on root length and fresh weight of cucumber seedlings. a Root length under Cd stress. b Fresh weight of seedlings under Cd stress. Seeds germinated for 2 d were exposed to different concentrations of CdCl₂ (50, 100, 200, and 300 μM) for 48 h. The data are means ± SE of three independent experiments (n = 15). Different letters indicate significant differences (P < 0.05; Duncan’s multiple range test)

Fig. 2

Effects of 200 μM Cd treatment on cell death in root tips of cucumber seedlings. a Roots stained with Evans blue at different times (0, 12, 24, 36, and 48 h). b Quantitative analysis of root tip cell death in cucumber seedlings. Scale bar indicates 500 μm. The data are means ± SE of three independent experiments. Different letters indicate significant differences (P < 0.05; Duncan’s multiple range test). FW, fresh weight

Fig. 3

Effects of NaHS on root length, fresh weight and cell death of cucumber seedlings under Cd stress. a Effects of different concentrations (1, 10, 100 and 200 μM) of NaHS (a H₂S donor) on root length (b) and fresh weight of cucumber seedlings under Cd stress. c Effects of different concentrations (1, 10, 100 and 200 μM) of NaHS on root length of cucumber seedlings under without Cd stress. d Roots stained with Evans blue were observed. e Quantitative analysis of root tip cell death in cucumber seedlings. Scale bar indicates 500 μm. The results are means ± SE of three independent experiments (n = 10). Different letters indicate significant differences (P < 0.05; Duncan’s multiple range test). FW, fresh weight

Fig. 4

Effects of different treatments on endogenous H₂S content in cucumber seedling roots. Cucumber seedlings treated with distilled water (Con), 200 μM CdCl₂ for 48 h, 100 μM NaHS pretreatment for 24 h or 100 μM NaHS pretreatment + Cd for 48 h. The content of endogenous H₂S after 24 h and 48 h were measured. The data are means ± SE of three independent experiments. Different letters indicate significant differences (P < 0.05; Duncan’s multiple range test). FW, fresh weight

Fig. 5

Effects of H₂S on H₂O₂, O2·−, MDA and ELP under Cd stress in cucumber seedling roots. Cucumber seedlings pretreated with 100 μM NaHS were exposed to Cd stress for 48 h and analyzed for the content of H₂O₂ (a), O₂^·− (b), MDA (c), and ELP (d). The data are means ± SE of three independent experiments. Different letters indicate a statistically significant difference (P < 0.05; Duncan’s multiple range test). FW, fresh weight

Fig. 6

Effects of H₂S on antioxidant enzyme activity under Cd stress in cucumber seedling roots. Cucumber seedlings pretreated with 100 μM NaHS were exposed to Cd stress for 48 h and analyzed the activity of SOD (a), CAT (b), POD (c), and APX (d). The data are means ± SE of three independent experiments. Different letters indicate a statistically significant difference (P < 0.05; Duncan’s multiple range test). FW, fresh weight

Fig. 7

DAPI staining (a) and fluorescence quantitative analysis (b). Con: distilled water; Cd: 200 μM CdCl₂; NaHS: 100 μM NaHS pretreatment for 24 h; NaHS + Cd: 100 μM NaHS pretreatment for 24 h + Cd treatment for 48 h. The data are means ± SE of three independent experiments. Different letters indicate significant differences (P < 0.05; Duncan’s multiple range test)

Fig. 8

Effects of NaHS on mitochondrial Cyt c/a, MPTP and caspase-3-like activity of cucumber seedlings root tips under Cd stress. Con: distilled water; Cd: 200 μM Cd stress for 48 h; NaHS: pretreated with 100 μM NaHS for 24 h; NaHS + Cd; seedlings were pretreated with 100 μM NaHS for 24 h and then treated with 200 μM Cd for 48 h. The ratio of Cyt c/a (a), mitochondrial membrane absorbance (b) and caspase-3-like (c) were measured after 48 h in different treatments. The data are means ± SE of three independent experiments. Different letters indicate significant differences (P < 0.05; Duncan’s multiple range test)