Heavy metal stress. Activation of distinct mitogen-activated protein kinase pathways by copper and cadmium

Abstract

Excessive amounts of heavy metals adversely affect plant growth and development. Whereas some regions naturally contain high levels of heavy metals, anthropogenic release of heavy metals into the environment continuously increases soil contamination. The presence of elevated levels of heavy metal ions triggers a wide range of cellular responses including changes in gene expression and synthesis of metal-detoxifying peptides. To elucidate signal transduction events leading to the cellular response to heavy metal stress we analyzed protein phosphorylation induced by elevated levels of copper and cadmium ions as examples for heavy metals with different physiochemical properties and functions. Exposure of alfalfa (Medicago sativa) seedlings to excess copper or cadmium ions activated four distinct mitogen-activated protein kinases (MAPKs): SIMK, MMK2, MMK3, and SAMK. Comparison of the kinetics of MAPK activation revealed that SIMK, MMK2, MMK3, and SAMK are very rapidly activated by copper ions, while cadmium ions induced delayed MAPK activation. In protoplasts, the MAPK kinase SIMKK specifically mediated activation of SIMK and SAMK but not of MMK2 and MMK3. Moreover, SIMKK only conveyed MAPK activation by CuCl(2) but not by CdCl(2). These results suggest that plants respond to heavy metal stress by induction of several distinct MAPK pathways and that excess amounts of copper and cadmium ions induce different cellular signaling mechanisms in roots.

Figure 1.

CuCl₂ and CdCl₂ induce the activation of 44- and 46-kD protein kinases. Alfalfa roots of hydroponically grown plants were exposed for 30 min to 0, 1, 10, 50, 100, 500, and 1,000 μm CuCl₂ (A) or CdCl₂ (B). In-gel kinase assays were performed with 20 μg of protein extracts using MBP as substrate.

Figure 2.

Concentration-dependent activation of MAPKs by CuCl₂ and CdCl₂. Seedlings were exposed to increasing concentrations of CuCl₂ or CdCl₂ for 30 min. Protein extracts from roots were immunoprecipitated with M23, M11, M14, or M24 antibodies that are specific for SIMK, MMK2, MMK3, and SAMK, respectively. Subsequently, kinase activities were determined by in vitro kinase assays with γ-[³²P]ATP and MBP as substrate. The experiment was performed independently three times showing similar results.

Figure 3.

Differential activation of MAPKs by CuCl₂ and CdCl₂. Seedlings were treated with 100 μm CuCl₂ or CdCl₂. Roots were harvested at the given time points. A, Immunokinase assays were performed with M23, M11, M14, or M24 antibodies that specifically recognize SIMK, MMK2, MMK3, or SAMK, respectively. B, The same protein extracts were used for protein gel-blot analysis with M23, M11, M14, or M24 antibodies. The experiment was repeated twice revealing similar kinetics in all cases.

Figure 4.

SIMKK enhances the activation of SIMK and SAMK by CuCl₂. SIMK-HA (A) and SAMK-HA (B) were either expressed alone (1, 5, and 9) or coexpressed with myc-tagged MEK1 (2, 6, and 10), PRKK (3, 7, and 11), or SIMKK (4, 8, and 12) in protoplasts. The protoplasts were treated either for 30 min with 100 μm CuCl₂ (lanes 5–8), 100 μm CdCl₂ (lanes 9–12), or as control with equal amount of water (lanes 1–4). Subsequently, SIMK and SAMK activities were determined by immunokinase assays using anti-HA antibodies for immunoprecipitation and MBP as substrate. The same protein extracts were analyzed for the presence of the coexpressed proteins by protein gel-blot analysis with anti-HA or anti-Myc antibodies. Three independent experiments yielded comparable results.

Figure 5.

Ion-specific MAPK activation. Seedlings were exposed to (1) water or 100 μm (2) Al₂(SO₄)₃, (3) CdCl₂, (4) CoCl₂, (5) CuCl₂, (6) FeCl₂, (7) Pb(NO₃)₂, or (8) ZnCl₂ for 30 min. Root protein extracts were used to immunoprecipitate SIMK, MMK2, MMK3, and SAMK with M23, M11, M14, or M24 antibodies, respectively. Subsequently, kinase activities were determined using MBP as substrate. Independent repetitions of the experiment showed the same activity profile.

Figure 6.

Excess levels of CuCl₂ induce hydrogen peroxide accumulation. DAB staining of alfalfa roots exposed to water, 100 μm and 500 μm CuCl₂ (A), or 100 μm and 500 μm CdCl₂ (B). The experiment was repeated five times showing comparable stainings.

Figure 7.

Copper and cadmium-induced MAPK signaling pathways. Excess copper and cadmium ions induce distinct MAPK pathways with different kinetics. A, As a redox-active metal ion, copper leads to the production of ROS that might trigger SIMK and SAMK activation via SIMKK. B, Cadmium activates SIMK, MMK2, MMK3, and SAMK. The upstream components mediating MAPK activation by cadmium remain to be identified.