MhMAPK4 from Malus hupehensis Rehd. decreases cell death in tobacco roots by controlling Cd2+ uptake

Cadmium (Cd) induces cell death in plant roots. Mitogen-activated protein kinase (MAPK) plays a role in the regulation of cell death induced by Cd in plant roots. In this study, MhMAPK4 was isolated from the roots of Malus hupehensis. Subcellular localization showed that the MhMAPK4 protein was located in the cell membrane and cytoplasm and is a transmembrane protein that is characterized by hydrophily. The expression of MhMAPK4 in the roots of M. hupehensis was up-regulated by Cd sulfate and Cd chloride. Phenotypic comparison under Cd stress showed that the growth of wild-type (WT) tobacco was lower than the transgenic lines overexpressing MhMAPK4. The fresh weight and the root length of WT also was lower than that of the transgenic tobacco. The net Cd2+ influx in the tobacco roots was decreased by the overexpression of MhMAPK4, as was root Cd accumulation. The recovery time of the Cd2+ influx to stable state in the transgenic tobacco was also shorter than the WT. The expression of iron-regulated transporter 1 (NtIRT1) and natural resistance associated macrophage protein 5 (NtNRAMP5) was relatively low in the transgenic lines under Cd stress. Cell death and apoptosis in the tobacco roots was reduced following the overexpression of MhMAPK4. The activity of vacuolar processing enzyme (VPE) and the transcript level of VPE in the transgenic tobacco was lower than that of WT under Cd stress. In addition, the electrolyte leakage and malondialdehyde and hydrogen peroxide contents in the transgenic tobacco were lower than those of WT, whereas the antioxidant enzyme activity and expression were higher. These results suggest that MhMAPK4 regulates Cd accumulation by mediating Cd2+ uptake by the roots, and controls Cd-caused cell death by adjusting VPE activity.