A three amino acid sequence gates protein stability control of bHLH104 and iron uptake in Arabidopsis thaliana
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Iron (Fe) homeostasis is precisely regulated to prevent iron imbalance. The basic helix-loop-helix (bHLH) transcription factor bHLH104, a member of bHLH subgroup IVc, activates Fe uptake. bHLH104 is negatively controlled by the Fe-binding E3 ligase BRUTUS (BTS) that interacts with the C-terminus of bHLH104, according to predictions with involvement of the last three amino acids proline-alanine-alanine (PAA). Yet, experimental evidence for the PAA interaction site and importance for post-translational regulation are lacking. Here, we demonstrate the role of PAA for plant growth and bHLH104 regulation. Transgenic plants expressing a bHLH104 variant lacking the terminal PAA ( b104^dPAA ) have a constitutive Fe deficiency response. Strong accumulation of Fe causes b104^dPAA plants to have abnormal shoot and root development and delayed flowering, accompanied by mis-regulated Fe response and upregulated Fe acquisition genes. The severity of Fe accumulation phenotypes coincides with bHLH104 protein amounts. In plants, bHLH104 levels are controlled by proteasomal degradation in a PAA-dependent manner. Cell assays show that the PAA sequence is required for bHLH104 interaction with BTS and subsequent ubiquitination. Taken together, bHLH104 requires the terminal PAA to be post-translationally controlled by BTS. Manipulation of bHLHIVc protein PAA sequence represents a biofortification strategy for crop plants.
Plain language summary
Plants need iron but excess is harmful. Iron uptake transcription factor bHLH104 is negatively controlled by Fe-binding E3 ligase BTS. We show that three–amino acid terminal sequence of bHLH104 controls its breakdown by the BTS pathway. Removing PAA keeps bHLH104 active, driving constant iron uptake and iron accumulation phenotypes. Targeting this motif can be a strategy for crop biofortification.
Highlights of this study
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A conserved C-terminal PAA sequence in bHLH104 is required for its post-translational control in Arabidopsis thaliana .
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Expression of bHLH104 lacking the PAA sequence ( b104^dPAA ) perturbs Fe regulation, resulting in constitutive iron accumulation in plants depending on b104^dPAA protein levels.
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The PAA sequence mediates interaction with E3 ligases BTS and is essential for bHLH104 ubiquitination.
