IRON MAN interacts with Cu-DEFICIENCY INDUCED TRANSCRIPTION FACTOR 1 to maintain copper homeostasis.

Copper (Cu) is essential for plant growth and development. IRON MAN (IMA) is a family of small peptides that can bind both iron (Fe) and Cu ions. It was reported that IMAs mediate Fe homeostasis in Arabidopsis thaliana. However, it remains unclear whether IMAs are involved in Cu homeostasis. The transcript abundance of IMA genes decreased in response to Cu deficiency. The combined disruption of all IMA genes caused enhanced tolerance to Cu deficiency and resulted in an increase in the transcript abundance of Cu uptake genes, whereas the overexpression of IMA1 or IMA3 led to the opposite results. Protein interaction assays indicated that IMAs interact with Cu-DEFICIENCY INDUCED TRANSCRIPTION FACTOR1 (CITF1), which is a positive regulator of the Cu uptake genes. Further studies showed that IMAs not only interfere with the DNA binding of CITF1 but also repress the transcriptional activation activity of CITF1, hence resulting in downregulation of the Cu uptake genes. Genetic analyses indicated that IMAs modulate Cu homeostasis in a CITF1-dependent manner. Our findings indicate that IMAs inhibit the functions of CITF1 in regulating Cu deficiency responses, thereby providing a conceptual framework for comprehending the regulation of Cu homeostasis.

CITF1 Functions Downstream of SPL7 to Specifically Regulate Cu Uptake in Arabidopsis.

Copper (Cu) is one of the most indispensable micronutrients, and proper Cu homeostasis is required for plants to maintain essential cellular functions. Plants activate the Cu uptake system during Cu limitation. Although SPL7 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 7) and CITF1 (Cu-DEFICIENCY INDUCED TRANSCRIPTION FACTOR 1) are two transcription factors in Cu homeostasis, it remains unclear how SPL7 and CITF1 control the Cu uptake system. Here, we reveal that overexpression of CITF1 causes the enhanced tolerance to Cu deficiency and the elevated expression of Cu uptake genes COPT2, FRO4 and FRO5. Electrophoretic mobility shift assays (EMSA) and transient expression assays indicate that SPL7 directly binds to and activates the promoter of CITF1. The overexpression of CITF1 partially rescues the sensitivity of spl7-1 to Cu deficiency. Transcriptome data suggest that SPL7 and CITF1 coregulate the Cu-homeostasis-signaling network, and CITF1 has its own independent functions. Moreover, both SPL7 and CITF1 can directly bind to and activate the promoters of three Cu uptake genes COPT2, FRO4 and FRO5. This work shows the functions of CITF1 in the Cu-homeostasis-signaling network, providing insights into the complicated molecular mechanism underlying Cu homeostasis.

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.

FIT and bHLH Ib transcription factors modulate iron and copper crosstalk in Arabidopsis.

Although the crosstalk between iron (Fe) and copper (Cu) homeostasis signalling networks exists in plants, the underlined molecular mechanism remains unclear. FIT (FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR) and four bHLH Ib members (bHLH38, bHLH39, bHLH100 and bHLH101) are the key regulators of Fe homeostasis. Here, we reveal that FIT and bHLH Ib control the up-regulation of Cu-uptake genes (COPT2, FRO4 and FRO5) by Fe deficiency, and Cu is required for improving plant growth under Fe-deficiency conditions. The induction of Cu-uptake gene expression and the elevation of Cu concentration are inhibited in the fit-2 or bhlh4x (the quadruple mutant of four bHLH Ib genes) under Fe-deficiency conditions. The dual overexpression of both bHLH38 (or bHLH39) and FIT activates the expression of COPT2, FRO4 and FRO5 and increases Cu accumulation. Furthermore, bHLH Ib proteins directly bind to the promoters of COPT2, FRO4 and FRO5. Either Cu supplement or overexpression of COPT2 or FRO4 improves the growth of fit-2 under Fe-deficiency conditions. Moreover, the induction of COPT2, FRO4 and FRO5 by Fe deficiency is independent of SPL7, a central regulator of Cu-deficiency responses. This work through the link between bHLH Ib/FIT and COPT2/FRO4/FRO5 under Fe-deficiency conditions establishes a new relationship between Cu and 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.

bHLH104 confers tolerance to cadmium stress in Arabidopsis thaliana.

Cd is a non-essential heavy metal that is toxic to both plants and animals. Here, we reveal that the transcription factor bHLH104 positively regulates Cd tolerance in Arabidopsis thaliana. We show that Fe deficiency-responsive genes were induced by Cd treatment, and that their upregulation was suppressed in bhlh104 loss-of-function mutants, but enhanced upon overexpression of bHLH104. Correspondingly, the bhlh104 mutants displayed sensitivity to Cd stress, whereas plants overexpressing bHLH104 exhibited enhanced Cd tolerance. Further analysis suggested that bHLH104 positively regulates four heavy metal detoxification-associated genes, IREG2, MTP3, HMA3 and NAS4, which play roles in Cd sequestration and tolerance. The bHLH104 overexpression plants accumulated high levels of Cd in the root but low levels of Cd in the shoot, which might contribute to the Cd tolerance in those lines. The present study thus points to bHLH104 as a potentially useful tool for genetic engineering of plants with enhanced Cd tolerance.