bHLH11 inhibits bHLH IVc proteins by recruiting the TOPLESS/TOPLESS-RELATED corepressors.

Iron (Fe) homeostasis is essential for plant growth and development. Many transcription factors (TFs) play pivotal roles in the maintenance of Fe homeostasis. bHLH11 is a negative TF that regulates Fe homeostasis. However, the underlying molecular mechanism remains elusive. Here, we generated two loss-of-function bhlh11 mutants in Arabidopsis (Arabidopsis thaliana), which display enhanced sensitivity to excess Fe, increased Fe accumulation, and elevated expression of Fe deficiency responsive genes. Levels of bHLH11 protein, localized in both the cytoplasm and nucleus, decreased in response to Fe deficiency. Co-expression assays indicated that bHLH IVc TFs (bHLH34, bHLH104, bHLH105, and bHLH115) facilitate the nuclear accumulation of bHLH11. Further analysis indicated that bHLH11 represses the transactivity of bHLH IVc TFs toward bHLH Ib genes (bHLH38, bHLH39, bHLH100, and bHLH101). The two ethylene response factor-associated amphiphilic repression motifs of bHLH11 provided the repression function by recruiting the TOPLESS/TOPLESS-RELATED (TPL/TPRs) corepressors. Correspondingly, the expression of Fe uptake genes increased in the tpr1 tpr4 tpl mutant. Moreover, genetic analysis revealed that bHLH11 has functions independent of FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR. This study provides insights into the complicated Fe homeostasis signaling network.

Oryza sativa POSITIVE REGULATOR OF IRON DEFICIENCY RESPONSE 2 (OsPRI2) and OsPRI3 are involved in the maintenance of Fe homeostasis.

Iron (Fe) is an essential micronutrient for plant growth development and plays a key role in regulating numerous cellular processes. In rice, OsHRZ1, an Fe-binding ubiquitin ligase, is a putative sensor of Fe homeostasis that negatively regulates iron acquisition. Despite its apparent importance, only a single basic-Helix-Loop-Helix (bHLH) transcription factor, OsPRI1, has been identified as a direct target of OsHRZ1. In this study, we identified and functionally characterized OsPRI2 and OsPRI3, two paralogs of OsPRI1, observing that they directly interact with OsHRZ1. Additional analyses suggested that OsHRZ1 promotes the degradation of OsPRI2 and OsPRI3. The translocation of Fe from roots to shoots was impaired in plants with loss-of-function mutations in OsPRI2 or OsPRI3, causing the downregulation of Fe-deficiency-responsive genes. In contrast, overexpression of OsPRI2 and OsPRI3 promotes Fe accumulation and activates the expression of Fe-deficiency-responsive genes. We also provide evidence that OsPRI2 and OsPRI3 bind to the promoters of OsIRO2 and OsIRO3, two key regulators of Fe homeostasis. Moreover, OsPRI2 and OsPRI3 directly induce expression of the metal-nicotianamine transporter, OsYSL2, by associating with the promoter in response to Fe deficiency. Our results provide insights into the complex network regulating Fe homeostasis in rice.

Oryza sativa FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (OsFIT/OsbHLH156) interacts with OsIRO2 to regulate iron homeostasis.

Iron (Fe) is indispensable for the growth and development of plants. It is well known that FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) is a key regulator of Fe uptake in Arabidopsis. Here, we identify the Oryza sativa FIT (also known as OsbHLH156) as the interacting partner of IRON-RELATED BHLH TRANSCRIPTION FACTOR 2 (OsIRO2) that is critical for regulating Fe uptake. The OsIRO2 protein is localized in the cytoplasm and nucleus, but OsFIT facilitates the accumulation of OsIRO2 in the nucleus. Loss-of-function mutations of OsFIT result in decreased Fe accumulation, severe Fe-deficiency symptoms, and disrupted expression of Fe-uptake genes. In contrast, OsFIT overexpression promotes Fe accumulation and the expression of Fe-uptake genes. Genetic analyses indicate that OsFIT and OsIRO2 function in the same genetic node. Further analyses suggest that OsFIT and OsIRO2 form a functional transcription activation complex to initiate the expression of Fe-uptake genes. Our findings provide a mechanism understanding of how rice maintains Fe homeostasis.

bHLH121 Functions as a Direct Link that Facilitates the Activation of FIT by bHLH IVc Transcription Factors for Maintaining Fe Homeostasis in Arabidopsis.

Iron (Fe) deficiency is prevalent in plants grown in neutral or alkaline soil. Plants have evolved sophisticated mechanisms that regulate Fe homeostasis, ensuring survival. In Arabidopsis, FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) is a crucial regulator of Fe-deficiency response. FIT is activated indirectly by basic helix-loop-helix (bHLH) IVc transcription factors (TFs) under Fe deficiency; however, it remains unclear which protein(s) act as the linker to mediate the activation of FIT by bHLH IVc TFs. In this study, we characterize the functions of bHLH121 and demonstrate that it directly associates with the FIT promoter. We found that loss-of-function mutations of bHLH121 cause severe Fe-deficiency symptoms, reduced Fe accumulation, and disrupted expression of genes associated with Fe homeostasis. Genetic analysis showed that FIT is epistatic to bHLH121 and FIT overexpression partially rescues the bhlh121 mutant. Further investigations revealed that bHLH IVc TFs interact with and promote nuclear accumulation of bHLH121. We demonstrated that bHLH121 has DNA-binding activity and can bind the promoters of the FIT and bHLH Ib genes, but we did not find that it has either direct transcriptional activation or repression activity toward these genes. Meanwhile, we found that bHLH121 functions downstream of and is a direct target of bHLH IVc TFs, and its expression is induced by Fe deficiency in a bHLH IVc-dependent manner. Taken together, these results establish that bHLH121 functions together with bHLH IVc TFs to positively regulate the expression of FIT and thus plays a pivotal role in maintaining Fe homeostasis in Arabidopsis.