Plant growth is severely affected by toxic concentrations of heavy metals. On characterizing the heavy metal-induced signalling pathways, the effects of cadmium (CdCl₂) and copper (CuCl₂) on MBP (myelin basic protein) kinase activities in Oryza sativa L. cv. TNG67 were analysed and it was found that Cd²⁺-induced 42 kDa MBP kinase has the characteristics of a mitogen-activated protein (MAP) kinase. This study confirmed that the 42 kDa kinase-active band contains, at least, the activities of OsMPK3 and OsMPK6. Then, the heavy metal signal transduction pathways leading to MAP kinase activation in rice roots were examined. Pretreatment with sodium benzoate, a hydroxyl radical scavenger, attenuated Cd²⁺- or Cu²⁺-induced MAP kinase activation. The Cd²⁺-, but not Cu²⁺-, induced MAP kinase activities were suppressed by diphenylene iodonium (DPI), an NADPH oxidase inhibitor, and Cd²⁺ induced NADPH oxidase-like activities, suggesting that NADPH oxidases may be involved in Cd²⁺-induced MAP kinase activation. Using a Ca²⁺ indicator, it was demonstrated that Cd²⁺ and Cu²⁺ induce Ca²⁺ accumulation in rice roots. The Cd²⁺- and Cu²⁺-induced MAP kinase activation required the involvement of Ca²⁺-dependent protein kinase (CDPK) and phosphatidylinositol 3-kinase (PI3 kinase) as shown by the inhibitory effect of a CDPK antagonist, W7, and a PI3 kinase inhibitor, wortmannin, respectively. Furthermore, bongkrekic acid (BK), a mitochondrial permeability transition pore opening blocker, suppressed Cd²⁺-, but not Cu²⁺-, induced MAP kinase activation, indicating that Cd²⁺-induced MAP kinase activities are dependent on the functional state of mitochondria. Collectively, these findings imply that Cd²⁺ and Cu²⁺ may induce MAP kinase activation through distinct signalling pathways. Moreover, it was found that the 42 kDa MAP kinase activities are higher in Cd-tolerant cultivars than in Cd-sensitive cultivars. Therefore, the Cd-induced 42 kDa MAP kinase activation may confer Cd tolerance in rice plants.

Mitogen-activated protein kinase (MAPK) pathways are modules involved in the transduction of extracellular signals to intracellular targets in all eukaryotes. In plants, it has been evidenced that MAPKs play a role in the signaling of biotic and abiotic stresses, plant hormones, and cell cycle cues. However, the effect of heavy metals on plant MAPKs has not been well examined. The Northern blot analysis of OsMAPK mRNA levels has shown that only OsMAPK2, but not OsMAPK3 and OsMAPK4, expressed in suspension-cultured cells in response to 100–400 µM Cd treatments. The OsMAPK2 transcripts increased within 12 h upon 400 µM Cd treatment. In addition, we found that 42- and 50-kDa MBP kinases were significantly activated by Cd treatment in rice suspension-cultured cells. And 40-, 42-, 50- and 64-kDa MBP kinases were activated in rice roots. Furthermore, GSH inhibits Cd-induced 40-kDa MBP kinase activation. By immunoblot analysis and immunoprecipitation followed by in-gel kinase assay, we confirmed that Cd-activated 42-kDa MBP kinase is a MAP kinase. Our results suggest that a MAP kinase cascade may function in the Cd-signalling pathway in rice.