MxMPK6-2-mediated phosphorylation enhances the response of apple rootstocks to Fe deficiency by activating PM H -ATPase MxHA2.

Iron (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.