OsbHLH064, an IVb bHLH Transcription Factor, Regulates Iron Homeostasis and Enhances Grain Fe Accumulation in Rice

Iron (Fe) is an essential micronutrient for plant growth and development. The maintenance of Fe homeostasis relies on sophisticated regulatory networks where bHLH transcription factors play a key role. However, how these factors coordinate to regulate this vital process is not fully understood. Here, we characterise OsbHLH064, a previously unstudied IVb bHLH transcription factor, and reveal its critical role in iron homeostasis in rice. Loss of OsbHLH064 results in constitutive activation of Fe homeostasis-related genes under Fe-sufficient conditions, whereas its overexpression strongly suppresses their expression. Remarkably, OsbHLH064 overexpression leads to excessive Fe accumulation in roots, shoots and brown rice. Under Fe deficiency, it also triggers ROS overproduction, highlighting its essential role in balancing Fe homeostasis and oxidative stress. Mechanistically, OsbHLH064 forms heterodimers with IVc bHLH transcription factors, and these interactions facilitate its nuclear localisation. OsbHLH064 represses OsIRO2 and OsIRO3 by competing with or sequestering IVc activators at shared promoters, thereby limiting downstream transcriptional activation. Furthermore, OsbHLH064 directly binds not only canonical IVb/IVc targets but also a broader set of genes involved in Fe uptake, translocation and signalling. Collectively, these findings establish OsbHLH064 as a central upstream regulator integrating multiple pathways to maintain Fe homeostasis and suggest its potential as a target for biofortification strategies aimed at enhancing iron content in rice grains.