Hydrogen Peroxide Pretreatment Mitigates Cadmium-Induced Oxidative Stress in Brassica napus L.: An Intrinsic Study on Antioxidant Defense and Glyoxalase Systems

Abstract

Cadmium (Cd) is considered as one of the most toxic metals for plant growth and development. In the present study, we investigated the role of externally applied hydrogen peroxide (H₂O₂) in regulating the antioxidant defense and glyoxalase systems in conferring Cd-induced oxidative stress tolerance in rapeseed (Brassica napus L.). Seedlings were pretreated with 50 μM H₂O₂ for 24 h. These pretreated seedlings as well as non-pretreated seedlings were grown for another 48 h at two concentrations of CdCl² (0.5 and 1.0 mM). Both the levels of Cd increased MDA and H₂O₂ levels and lipoxygenase activity while ascorbate (AsA) declined significantly. However, reduced glutathione (GSH) content showed an increase at 0.5 mM CdCl², but glutathione disulfide (GSSG) increased at any level of Cd with a decrease in GSH/GSSG ratio. The activities of ascorbate peroxidase (APX) and glutathione S-transferase (GST) upregulated due to Cd treatment in dose-dependent manners, while glutathione reductase (GR) and glutathione peroxidase (GPX) increased only at 0.5 mM CdCl² and decreased at higher dose. The activity of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT), glyoxalase I (Gly I), and glyoxalase II (Gly II) decreased under Cd stress. On the other hand, H₂O₂ pretreated seedlings, when exposed to Cd, AsA and GSH contents and GSH/GSSG ratio increased noticeably. H₂O₂ pretreatment increased the activities of APX, MDHAR, DHAR, GR, GST, GPX, and CAT of Cd affected seedlings. Thus enhancement of both the non-enzymatic and enzymatic antioxidants helped to decrease the oxidative damage as indicated by decreased levels of H₂O₂ and MDA. The seedlings which were pretreated with H₂O₂ also showed enhanced glyoxalase system. The activities of Gly I, and Gly II and the content of GSH increased significantly due to H₂O₂ pretreatment in Cd affected seedlings, compared to the Cd-stressed plants without H₂O₂ pretreatment which were vital for methylglyoxal detoxification. So, the major roles of H₂O₂ were improvement of antioxidant defense system and glyoxalase system which protected plants from the damage effects of ROS and MG. The mechanism of H₂O₂ to induce antioxidant defense and glyoxalase system and improving physiology under stress condition is not known clearly which should be elucidated. The signaling roles of H₂O₂ and its interaction with other signaling molecules, phytohormones or other biomolecules and their roles in stress protection should be explored.

Keywords: abiotic stress; antioxidant defense; cross tolerance; metal toxicity; methylglyoxal; oxidative stress; signaling molecule.

FIGURE 1

Malondialdehyde content (A), H₂O₂ content (B), and LOX activity (C) in rapeseed leaves induced by exogenous H₂O₂ under Cd stress. Mean (±SD) was calculated from three replicates for each treatment. Bars with different letters are significantly different at P < 0.05 applying Tukey’s HSD test.

FIGURE 2

3,3-Diaminobenzidine staining (A) of H₂O₂ and NBT staining (B) of superoxide ${\mathrm{O}}_{\mathrm{2}}^{\mathrm{•–}}$ in rapeseed leaves induced by exogenous H₂O₂ under Cd stress.

FIGURE 3

Ascorbate (AsA) content (A), DHA content (B), AsA/DHA ratio (C), GSH content (D), GSSG content (E), and GSH/GSSG ratio (F) in rapeseed leaves induced by exogenous H₂O₂ under Cd stress. Mean (±SD) was calculated from three replicates for each treatment. Bars with different letters are significantly different at P < 0.05 applying Tukey’s HSD test.

FIGURE 4

Activities of AsA-GSH cycle enzymes, APX (A), MDHAR (B), DHAR (C), and GR (D) in rapeseed leaves induced by exogenous H₂O₂ under Cd stress. Mean (±SD) was calculated from three replicates for each treatment. Bars with different letters are significantly different at P < 0.05 applying Tukey’s HSD test.

FIGURE 5

Activities of GPX (A), GST (B), and CAT (C) in rapeseed leaves induced by exogenous H₂O₂ under Cd stress. Mean (±SD) was calculated from three replicates for each treatment. Bars with different letters are significantly different at P < 0.05 applying Tukey’s HSD test.

FIGURE 6

Activities of Gly I (A) and Gly II (B) in rapeseed leaves induced by exogenous H₂O₂ under Cd stress. Mean (±SD) was calculated from three replicates for each treatment. Bars with different letters are significantly different at P < 0.05 applying Tukey’s HSD test.