A Three Amino Acid Sequence Gates Protein Stability Control of bHLH104 and Iron Uptake in Arabidopsis thaliana.

Iron (Fe) homeostasis is regulated to prevent iron imbalance, with the help of redundant basic helix-loop-helix (bHLH) IVc transcription factors that can be controlled by Fe-binding E3 ligases such as BRUTUS (BTS) in Arabidopsis thaliana. However, knowledge gaps remain to fully explain the mechanistic basis of this redundant protein interaction module. The C-terminus of bHLH104 is known to interact with BTS, and structural predictions implicate the three terminal amino acids, proline-alanine-alanine (PAA). However, the importance of the PAA sequence for post-translational regulation of bHLH104 and for the resulting plant phenotypes has not been tested experimentally. Here we show that transgenic plants expressing a bHLH104 variant lacking PAA (b104^dPAA) constitutively upregulate Fe acquisition and Fe transport genes and accumulate Fe, whereas plants expressing wild-type bHLH104 do not. The extent of Fe accumulation in independent b104^dPAA lines correlated positively with b104^dPAA protein abundance. Unlike the wild-type form, b104^dPAA showed no indication of interaction with BTS or of ubiquitination by it. Together, these findings support a model in which the PAA sequence serves as a recognition motif for binding to BTS and for subsequent post-translational regulation of bHLH104. Hence, targeted manipulation of the bHLH IVc protein PAA sequence may represent a strategy for crop biofortification.