The conserved function of bHLH121 and overexpression of N-terminal bHLH121 fragment suppresses iron deficiency response in Arabidopsis
- 1State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 211135, China
Abstract
Iron (Fe) homeostasis is fundamental to plant growth and development and is centrally regulated by a well-conserved network of basic helix-loop-helix (bHLH) transcription factors. In Arabidopsis thaliana, bHLH121 regulates the expression of FER-like iron deficiency-induced transcription factor (FIT) and clade Ib bHLH38/39/100/101 genes, among others. However, its functional conservation remains unexplored, and some contradictory results have been reported regarding the effects of bHLH121 overexpression. Here, we investigated the evolutionary conservation of bHLH121 through complementary tests in the Arabidopsis bhlh121 mutant created by CRISPR/Cas9 gene editing. We also investigated the effects of ectopically expressing full-length, N-terminal, and C-terminal fragments of bHLH121 on plant responses to Fe deficiency in Arabidopsis. Transgenic plants overexpressing the full-length bHLH121 driven by the cauliflower mosaic virus 35S promoter (bHLH121 ox) resulted in shorter roots and more severe leaf chlorosis compared to wild-type plants under Fe limiting conditions. Moreover, overexpression of the N-terminal fragment (N121 ox) rendered plants highly sensitive to Fe deficiency, whereas overexpression of the C terminal fragment (C121 ox) did not. Consistent with the phenotypic observations, N121 ox plants accumulated lower Fe content, and the ferric chelate reductase activity was significantly reduced. In N121 ox plants, the induction of key Fe regulatory genes, especially FIT, was significantly inhibited under Fe deficiency. Confocal imaging showed that both N121-GFP and C121-GFP fusion proteins could localize to the nucleus. Electrophoretic mobility shift assay showed that both full-length bHLH121 and N121 could effectively bind the probes derived from the promoter of FIT. The present work reveals a conserved function of bHLH121 in Fe homeostasis across several dicot species and demonstrates that the N121 may possess a dominant nature in Fe deficiency responses.
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BibTeX
@misc{zhou2026conserved,
title = {{The conserved function of bHLH121 and overexpression of N-terminal bHLH121 fragment suppresses iron deficiency response in Arabidopsis}},
author = {P. Zhou and C. Liu and Y. Pan and Y. Fei and R. Shen and R. Wang and P. Lan},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.27.754851},
url = {https://www.biorxiv.org/content/10.64898/2026.09.27.754851v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - The conserved function of bHLH121 and overexpression of N-terminal bHLH121 fragment suppresses iron deficiency response in Arabidopsis
AU - P. Zhou
AU - C. Liu
AU - Y. Pan
AU - Y. Fei
AU - R. Shen
AU - R. Wang
AU - P. Lan
PY - 2026
DA - 2026/09/27
PB - bioRxiv
DO - 10.64898/2026.09.27.754851
UR - https://www.biorxiv.org/content/10.64898/2026.09.27.754851v1
AB - Iron (Fe) homeostasis is fundamental to plant growth and development and is centrally regulated by a well-conserved network of basic helix-loop-helix (bHLH) transcription factors. In Arabidopsis thaliana, bHLH121 regulates the expression of FER-like iron deficiency-induced transcription factor (FIT) and clade Ib bHLH38/39/100/101 genes, among others. However, its functional conservation remains unexplored, and some contradictory results have been reported regarding the effects of bHLH121 overexpression. Here, we investigated the evolutionary conservation of bHLH121 through complementary tests in the Arabidopsis bhlh121 mutant created by CRISPR/Cas9 gene editing. We also investigated the effects of ectopically expressing full-length, N-terminal, and C-terminal fragments of bHLH121 on plant responses to Fe deficiency in Arabidopsis. Transgenic plants overexpressing the full-length bHLH121 driven by the cauliflower mosaic virus 35S promoter (bHLH121 ox) resulted in shorter roots and more severe leaf chlorosis compared to wild-type plants under Fe limiting conditions. Moreover, overexpression of the N-terminal fragment (N121 ox) rendered plants highly sensitive to Fe deficiency, whereas overexpression of the C terminal fragment (C121 ox) did not. Consistent with the phenotypic observations, N121 ox plants accumulated lower Fe content, and the ferric chelate reductase activity was significantly reduced. In N121 ox plants, the induction of key Fe regulatory genes, especially FIT, was significantly inhibited under Fe deficiency. Confocal imaging showed that both N121-GFP and C121-GFP fusion proteins could localize to the nucleus. Electrophoretic mobility shift assay showed that both full-length bHLH121 and N121 could effectively bind the probes derived from the promoter of FIT. The present work reveals a conserved function of bHLH121 in Fe homeostasis across several dicot species and demonstrates that the N121 may possess a dominant nature in Fe deficiency responses.
ER -