Kinase MxMPK4-1 and calmodulin-binding protein MxIQM3 enhance apple root acidification during Fe deficiency.

Iron (Fe) deficiency is a long-standing issue in plant mineral nutrition. Ca2+ signals and the mitogen-activated protein kinase (MAPK) cascade are frequently activated in parallel to perceive external cues, but their interplay under Fe deficiency stress remains largely unclear. Here, the kinase MxMPK4-1, which is induced during the response to Fe deficiency stress in apple rootstock Malus xiaojinensis, cooperates with IQ-motif containing protein3 (MxIQM3). MxIQM3 gene expression, protein abundance, and phosphorylation level increased under Fe deficiency stress. The overexpression of MxIQM3 in apple calli and rootstocks mitigated the Fe deficiency phenotype and improved stress tolerance, whereas RNA interference or silencing of MxIQM3 in apple calli and rootstocks, respectively, worsened the phenotype and reduced tolerance to Fe deficiency. MxMPK4-1 interacted with MxIQM3 and subsequently phosphorylated MxIQM3 at Ser393, and co-expression of MxMPK4-1 and MxIQM3 in apple calli and rootstocks enhanced Fe deficiency responses. Furthermore, MxIQM3 interacted with the central-loop region of the plasma membrane (PM) H+-ATPase MxHA2. Phospho-mimicking mutation of MxIQM3 at Ser393 inhibited binding to MxHA2, but phospho-abolishing mutation promoted interaction with both the central-loop and C terminus of MxHA2, demonstrating phosphorylation of MxIQM3 caused dissociation from MxHA2 and therefore increased H+ secretion. Moreover, Ca2+/MxCAM7 (Calmodulin7) regulated the MxMPK4-1-MxIQM3 module in response to Fe deficiency stress. Overall, our results demonstrate that MxMPK4-1-MxIQM3 forms a functional complex and positively regulates PM H+-ATPase activity in Fe deficiency responses, revealing a versatile mechanism of Ca2+/MxCAM7 signaling and MAPK cascade under Fe deficiency stress.