Mitogen‐activated protein kinase MxMPK3‐2 mediated phosphorylation of MxZR3.1 participates in regulating iron homoeostasis in apple rootstocks

The micronutrient iron plays a crucial role in the growth and development of plants, necessitating meticulous regulation for its absorption by plants. Prior research has demonstrated that the transcription factor MxZR3.1 restricts iron absorption in apple rootstocks; however, the precise mechanism by which MxZR3.1 contributes to the regulation of iron homoeostasis in apple rootstocks remains unexplored. Here, MxMPK3-2, a protein kinase, was discovered to interact with MxZR3.1. Y2H, bimolecular fluorescence complementation and pull down experiments were used to confirm the interaction. Phosphorylation and cell semi-degradation tests have shown that MxZR3.1 can be used as a substrate of MxMPK3-2, which leads to the MxZR3.1 protein being more stable. In addition, through tobacco transient transformation (LUC and GUS) experiments, it was confirmed that MxZR3.1 significantly inhibited the activity of the MxHA2 promoter, while MxMPK3-2 mediated phosphorylation at the Ser94 site of MxZR3.1 further inhibited the activity of the MxHA2 promoter. It is tightly controlled to absorb iron during normal growth and development of apple rootstocks due to the regulatory effect of the MxMPK3-2-MxZR3.1 module on MxHA2 transcription level. Consequently, this research has revealed the molecular basis of how the MxMPK3-2-MxZR3.1 module in apple rootstocks controls iron homoeostasis by regulating the MxHA2 promoter's activity.