The conserved function of bHLH121 and overexpression of N-terminal bHLH121 fragment suppresses iron deficiency response in Arabidopsis

Iron regulation is a critical aspect of plant growth and development, governed by a network of basic helix-loop-helix (bHLH) transcription factors. In Arabidopsis thaliana, bHLH121 plays a role in controlling the expression of various iron-related genes, such as the FER-like iron deficiency-induced transcription factor (FIT) and bHLH38/39/100/101 genes.

However, the exact effects of bHLH121 overexpression have been inconsistent in prior studies. To clarify, researchers examined the evolutionary conservation of bHLH121 in Arabidopsis by using CRISPR/Cas9 gene editing to create bhlh121 mutants. They also explored the effects of expressing various fragments of bHLH121, including the full-length, N-terminal, and C-terminal fragments, on plant responses to iron deficiency.

Plants overexpressing the full-length bHLH121 exhibited shorter roots and severe leaf chlorosis under iron-limited conditions. Additionally, overexpression of the N-terminal fragment (N121 ox) made plants highly susceptible to iron deficiency, while overexpression of the C-terminal fragment (C121 ox) did not impact their responses.

The reduced Fe content and diminished ferric chelate reductase activity in N121 ox plants supported these phenotypic changes. Furthermore, the induction of crucial Fe regulatory genes, particularly FIT, was significantly suppressed in N121 ox plants under iron deficiency. Both N121-GFP and C121-GFP fusion proteins were found to localize to the nucleus, and both full-length bHLH121 and N121 could effectively bind to FIT promoter probes.

This study confirms the conserved function of bHLH121 in iron homeostasis across multiple dicot species and highlights the potential dominant role of the N121 fragment in iron deficiency responses.