Copper-caused oxidative stress triggers the activation of antioxidant enzymes via ZmMPK3 in maize leaves

Abstract

Copper (Cu) is a necessary trace element participated in many physiological processes in plants. But excessive Cu2+ is toxic, which can activate intracellular signals that lead to cellular damage. The mitogen-activated protein kinase (MAPK) cascade is at the center of cell signal transduction and has been reported to be involved in stress-related signaling pathways. ZmMPK3, a kind of MAPKs in maize cells, can be activated by diverse abiotic stresses. In the present study, we investigated the effects of Cu2+ on hydrogen peroxide (H2O2) level, ZmMPK3 activity as well as the activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and ascorbic acid peroxidase (APX) using maize leaf as an experimental model. The results demonstrated that acute Cu2+ exposure for 24 hours led to rapid increases of H2O2 level and the increase in ZmMPK3 activity as well as the total activities of antioxidant enzymes SOD, CAT and APX. H2O2 scavenger, dimethylthiourea (DMTU), effectively inhibited the Cu2+-increased H2O2 level and the activity of ZmMPK3 as well as the activities of the antioxidant enzymes SOD, CAT and APX. Pre-treatment with the MAPK inhibitor, PD98059, significantly blocked the Cu2+-increased activities of ZmMPK3, CAT, APX and SOD, but didn't affect the accumulation of H2O2. Our results suggest that Cu2+ causes oxidative stress to the maize leaves which then activates defense antioxidant enzymes via MAPK pathway. Thus, the signaling pathway is Cu2+-H2O2-ZmMPK3-antioxidant enzymes.

Fig 1. H₂O₂ accumulation in the leaves of maize exposed to Cu²⁺.

(A) Histochemical detection of H₂O₂ production with DAB staining; (B) Determination of H₂O₂ content using spectrophotometric method. Results are presented as mean ± S.E. (n = 6) of three experiments. The mean value of the control is ascribed an arbitrary value of 1 and the mean value in each treated group is shown as a fold increase compared to the mean value in the control. The experiments were replicated three times. * denotes P<0.05, ** P<0.01.

Fig 2. Effects of excess Cu²⁺ exposure on ZmMPK3 activity in maize leaves.

(A) ZmMPK3 kinase activity. (B) Quantification of ZmMPK3 activity. In-gel images were analyzed by Image J image processing software. Data are shown as mean ± S.E. of three independent experiments. Plants were treated with various concentrations of Cu²⁺ (0, 10, 50 and 100 μM) for 0.5 h or 100 μM Cu²⁺ for different times (0, 0.5, 1, 2 and 4 h). All experiments were replicated three times. * denotes P<0.05, ** P<0.01.

Fig 3. Effects of Cu²⁺ stress on the activities of SOD, CAT and APX in leaves of maize.

Plants were treated with various concentrations of Cu²⁺ for 24 h. Results are expressed as mean ± S.E.(n = 6) of there different experiments. The mean value of the control is ascribed an arbitrary value of 1 and the mean value in each treated group is shown as a fold increase of that in the control. * denotes P<0.05, ** P<0.01.

Fig 4. Relationship between H₂O₂ production and ZmMPK3 activation induced by excess Cu²⁺ in maize leaves.

(A) Effects of pretreatment with PD98059 or DMTU on H₂O₂ production induced by excess Cu²⁺. Maize plants were pretreated with or without 100 μM PD98059/5 mM DMTU for 8 h, then exposed to 100 μM Cu²⁺ for 24 h. The letters on the lanes represent: H₂O = H₂O (8 h) + H₂O (24 h); Cu²⁺ = H₂O (8 h) + 100 μM Cu²⁺ (24 h); DMTU/ Cu²⁺ = 5 mM DMTU (8 h) + 100 μM Cu²⁺ (24 h) and PD98059/ Cu²⁺ = 100 μM PD98059 (8 h) + 100 μM Cu²⁺ (24 h). (B) Effects of pretreatment with PD98059 or DMTU on ZmMPK3 kinase activity. (C) Quantification of ZmMPK3 activity. In-gel images were analyzed by Image J image processing software. Data are shown as mean ± S.E. of three independent experiments. Maize plants were pretreated with or without 100 μM PD98059/5 mM DMTU for 8 h, then exposed to 100 μM Cu²⁺ for 0.5 h. The letters on the lanes represent: H₂O = H₂O (8 h) + H₂O (0.5 h); Cu²⁺ = H₂O (8 h) + 100 μM Cu²⁺ (0.5 h), PD98059/ Cu²⁺ = 100 μM PD98059 (8 h) + 100 μM Cu²⁺ (0.5 h) and DMTU/ Cu²⁺ = 5 mM DMTU (8 h) + 100 μM Cu²⁺ (0.5 h).The experiment was replicated three times.On comparing with the control, significance is shown by *P<0.05, **P<0.01; on comparing with Cu²⁺ treatment group, ^#P<0.05, ^##P<0.01.

Fig 5. Effects of pretreatment of DMTU or PD98059 on the activities of SOD, CAT and APX in maize leaves.

The maize plants were pretreated with or without 5 mM DMTU or 100 μM PD98059 for 8 h, then exposed to 100 μM Cu²⁺ or distilled water for 24 h. The letters on the lanes represent: Con = H₂O (8 h) + H₂O (24 h); Cu = H₂O (8 h) + 100 μM Cu²⁺ (24 h), DMTU+Cu = 5 mM DMTU (8 h) + 100 μM Cu²⁺ (24 h) and PD98059+Cu = 100 μM PD98059 (8 h) + 100 μM Cu²⁺ (24 h). Results are presented as mean ± S.E.(n = 6) of three experiments. The mean value of the control is ascribed an arbitrary value of 1 and the mean value in each treated group is shown as a fold increase compared to the mean value in the control. All experiments were replicated three times. On comparing with the control, significance is shown by *P<0.05, **P<0.01; on comparing with Cu²⁺ treatment group, ^#P<0.05, ^##P<0.01.