MPK6-2-mediated phosphorylation of ERF4 releases the Fe uptake regulator FIT and enhances Fe uptake in apple

Iron (Fe) deficiency is a major limitation to apple (Malus domestica) growth in calcareous soils. Understanding the molecular mechanisms underlying Fe deficiency responses is crucial for improving Fe use efficiency in fruit trees. In this study, we identified the ETHYLENE RESPONSE FACTOR 4 (MxERF4) as a negative regulator of the Fe deficiency response in apple. Transgenic analysis revealed that overexpression of MxERF4 exacerbated leaf chlorosis and reduced root Fe content under Fe-deficient conditions, whereas RNA interference (RNAi) lines exhibited enhanced tolerance. We further identified the MITOGEN-ACTIVATED PROTEIN KINASE (MxMPK6-2) as an interactor of MxERF4. MxMPK6-2 phosphorylates MxERF4, reducing its protein stability and promoting its degradation. MxERF4 interacted with the key Fe uptake regulator FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (MxFIT) and colocalized with it in root tissues. Phosphorylation weakened the MxERF4-MxFIT interaction, thereby relieving the inhibition of MxFIT-basic Helix-Loop-Helix 38/39 (MxbHLH38/39) complex formation. This in turn restored the activation of Fe uptake genes IRON TRANSPORTER1 (MxIRT1) and FERRIC REDUCTASE OXIDASE 2 (MxFRO2), enhanced ferric chelate reductase (FCR) activity, and promoted active Fe accumulation. Together, these findings reveal a previously uncharacterized MPK6-2-ERF4 signaling module that regulates Fe deficiency responses in apple by modulating ERF4 stability and its interaction with FIT, providing insights into the molecular basis of Fe efficiency and offering potential strategies for breeding Fe-efficient rootstocks.