MPK3-mediated phosphorylation inactivates MYB4 to promote FIT-dependent iron acquisition.

Iron (Fe) is an essential micronutrient for plant growth, and its homeostasis is critically regulated by the transcription factor FIT in response to deficiency. Here, we identify the R2R3-MYB transcription factor MYB4 as a previously unknown negative regulator of Fe acquisition. MYB4 directly interacts with FIT, competing with bHLH38/39 to prevent the formation of transcriptionally active heterodimers, thereby repressing the expression of the key Fe-uptake genes IRT1 and FRO2. We establish that the mitogen-activated protein kinase MPK3 acts upstream in this pathway. Under Fe-deficient conditions, MPK3 is activated and directly phosphorylates MYB4 at Serine 4, promoting its proteasome-dependent destabilization. This post-translational switch releases FIT to dimerize with bHLH partners, activating Fe uptake. Our work unveils the MPK3-MYB4-FIT signaling module as a previously unrecognized regulatory module that contributes to the dynamic control of Fe homeostasis in Arabidopsis.