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bioRxiv · 10.1101/2025.11.18.689088

A three amino acid sequence gates protein stability control of bHLH104 and iron uptake in Arabidopsis thaliana

Baby, D.·Weldi, S.·Knopf, M.·Venkadasamy, V. L.·Erickson, J. L.·Bauer, P.
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Abstract

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. Plain language summaryPlants tightly control iron levels. In Arabidopsis thaliana, transcription factor bHLH104 steers iron uptake. E3 ligases like BTS regulate bHLH104. We show that a three-amino acid terminal sequence of bHLH104 is needed for its targeting by the BTS pathway. bHLH104 lacking PAA leads to iron accumulation. Targeting this short sequence can be a strategy for crop biofortification.

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Baby, D., Weldi, S., Knopf, M., Venkadasamy, V. L., Erickson, J. L., Bauer, P.. 2025-11-18. A three amino acid sequence gates protein stability control of bHLH104 and iron uptake in Arabidopsis thaliana. https://doi.org/10.1101/2025.11.18.689088

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