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.
SUMMARYIron (Fe) deficiency significantly affects the growth and development, fruit yield and quality of apples. Apple roots respond to Fe deficiency stress by promoting H+ secretion, which acidifies the soil. In this study, the plasma membrane (PM) H+‐ATPase MxHA2 promoted H+ secretion and root acidification of apple rootstocks under Fe deficiency stress. H+‐ATPase MxHA2 is upregulated in Fe‐efficient apple rootstock of Malus xiaojinensis at the transcription level. Fe deficiency also induced kinase MxMPK6‐2, a positive regulator in Fe absorption that can interact with MxHA2. However, the mechanism involving these two factors under Fe deficiency stress is unclear. MxMPK6‐2 overexpression in apple roots positively regulated PM H+‐ATPase activity, thus enhancing root acidification under Fe deficiency stress. Moreover, co‐expression of MxMPK6‐2 and MxHA2 in apple rootstocks further enhanced PM H+‐ATPase activity under Fe deficiency. MxMPK6‐2 phosphorylated MxHA2 at the Ser909 site of C terminus, Thr320 and Thr412 sites of the Central loop region. Phosphorylation at the Ser909 and Thr320 promoted PM H+‐ATPase activity, while phosphorylation at Thr412 inhibited PM H+‐ATPase activity. MxMPK6‐2 also phosphorylated the Fe deficiency‐induced transcription factor MxbHLH104 at the Ser169 site, which then could bind to the promoter of MxHA2, thus enhancing MxHA2 upregulation. In conclusion, the MAP kinase MxMPK6‐2‐mediated phosphorylation directly and indirectly regulates PM H+‐ATPase MxHA2 activity at the protein post‐translation and transcription levels, thus synergistically enhancing root acidification under Fe deficiency stress.
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