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 interacts with BTS. Structural predictions suggest involvement of the three terminal amino acids, proline-alanine-alanine (PAA). However, the importance of the PAA short sequence for post-translational regulation of bHLH104 and resulting plant phenotypes has not been experimentally tested. Here, we demonstrate that transgenic plants expressing a bHLH104 variant lacking PAA (b104^dPAA) constitutively upregulated Fe acquisition and Fe transport genes and consequently accumulated Fe in contrast to wild-type bHLH104 protein-expressing plants. The extent of Fe accumulation in independent b104^dPAA lines correlated positively with b104^dPAA protein abundance. In contrast to the wild-type bHLH104 form, there was no indication that b104^dPAA interacted with BTS or was ubiquitinated by it. Together, these findings corroborated that the PAA sequence is required for post-translational control of bHLH104 by BTS. Hence, targeted manipulation of bHLHIVc protein PAA sequence may represent a strategy for crop biofortification.