IRON MAN interacts with BRUTUS to maintain iron homeostasis in Arabidopsis

Significance

bHLH105 and bHLH115, key transcription factors that positively regulate Fe homeostasis, are both ubiquitinated and degraded by BRUTUS (BTS). How plants activate bHLH105 and bHLH115 under Fe-deficient conditions remains unclear. IRON MAN peptides (IMAs) are a class of small peptides that are induced by Fe deficiency and are required for Fe homeostasis, but how they regulate Fe homeostasis is also not known. Here, we established that IMA peptides sequester BTS and promote the accumulation of bHLH105 and bHLH115, thereby activating the Fe deficiency response in Arabidopsis.

Abstract

IRON MAN (IMA) peptides, a family of small peptides, control iron (Fe) transport in plants, but their roles in Fe signaling remain unclear. BRUTUS (BTS) is a potential Fe sensor that negatively regulates Fe homeostasis by promoting the ubiquitin-mediated degradation of bHLH105 and bHLH115, two positive regulators of the Fe deficiency response. Here, we show that IMA peptides interact with BTS. The C-terminal parts of IMA peptides contain a conserved BTS interaction domain (BID) that is responsible for their interaction with the C terminus of BTS. Arabidopsis thaliana plants constitutively expressing IMA genes phenocopy the bts-2 mutant. Moreover, IMA peptides are ubiquitinated and degraded by BTS. bHLH105 and bHLH115 also share a BID, which accounts for their interaction with BTS. IMA peptides compete with bHLH105/bHLH115 for interaction with BTS, thereby inhibiting the degradation of these transcription factors by BTS. Genetic analyses suggest that bHLH105/bHLH115 and IMA3 have additive roles and function downstream of BTS. Moreover, the transcription of both BTS and IMA3 is activated directly by bHLH105 and bHLH115 under Fe-deficient conditions. Our findings provide a conceptual framework for understanding the regulation of Fe homeostasis: IMA peptides protect bHLH105/bHLH115 from degradation by sequestering BTS, thereby activating the Fe deficiency response.

Iron (Fe) is a crucial mineral nutrient that is essential for plant growth and development and for various biochemical processes, such as photosynthesis, respiration, and chlorophyll biosynthesis (1). When exposed to Fe deficiency, plants usually develop interveinal chlorosis in the leaves. However, Fe is redox active and hence prone to generating reactive oxygen species when it is present at excess levels in a free state (2). Thus, plants must maintain Fe homeostasis. To do so, plants tightly control Fe uptake, transport, and distribution in response to environmental Fe availability. Plants have developed two types of strategies to acquire Fe (3). Dicots and nongraminaceous monocot plants use strategy I, which involves three steps: the solubilization of Fe in the rhizosphere, reduction of Fe(III) to Fe(II), and transport of Fe into root epidermal cells (4). Graminaceous plants employ strategy II, which involves phytosiderophore secretion and Fe(III) chelation (5).

To withstand a fluctuating environmental Fe status, plants must modulate the expression of a number of genes, including those involved in Fe uptake and transport. In Arabidopsis thaliana, a group of transcription factors participate in signal transduction in response to Fe deficiency (6, 7). Of these, FIT (FER-LIKE IRON DEFICIENCY–INDUCED TRANSCRIPTION FACTOR) is a key component that interacts with four basic helix–loop–helix subgroup Ib (bHLH Ib) transcription factors (bHLH38, bHLH39, bHLH100, and bHLH101) to form heterodimers that modulate the expression of Fe uptake genes, including IRT1 (IRON REGULATED TRANSPORTER 1) and FRO2 (FERRIC REDUCTASE OXIDASE 2) (8, 9). In plants subjected to Fe deficiency, the expression of both the FIT and bHLH group Ib transcription factor genes is induced in a process that depends on four bHLH IVc proteins (bHLH34, bHLH104, bHLH105/ILR3–IAA-LEUCINE RESISTANT3, and bHLH115) (10–12). These proteins interact with bHLH121 (a bHLH IVb subgroup member) to up-regulate the expression of FIT and bHLH Ib genes as well as other Fe deficiency–responsive genes (13–15). Like bHLH121, bHLH11 and PYE (POPEYE) belong to the bHLH IVb subgroup. However, unlike bHLH121, both bHLH11 and PYE are negative regulators of Fe deficiency responses and also interact with bHLH IVc proteins (16–18). Tanabe et al. (18) determined that bHLH11 negatively regulates the Fe deficiency response in an FIT-dependent manner. In contrast to bHLH11, which is repressed by Fe deficiency, PYE is markedly induced. PYE directly targets FRO3 and NAS4 (NICOTIANAMINE SYNTHASE 4), both of which are up-regulated under Fe-deficient conditions (16). In addition, PYE can dimerize with bHLH105 to negatively regulate the expression of ferritin genes (19).

Plants must adjust their transcriptomes in response to fluctuating Fe concentrations. Plants sense the internal and external Fe status and modulate the abundance and activity of transcription factors, which control the transcription of downstream structural genes. Potential plant Fe sensors include OsHRZ1 (HEMERYTHRIN MOTIF-CONTAINING REALLY INTERESTING NEW GENE [RING] AND ZINC-FINGER PROTEIN 1) and OsHRZ2 in rice (Oryza sativa) and BRUTUS (BTS) in A. thaliana, which negatively regulate Fe homeostasis (17, 20). These proteins contain several hemerythrin domains and a RING domain; the former is responsible for Fe binding and the latter for E3 ligase activity. bHLH105 and bHLH115 stimulate the Fe deficiency response, and their levels increase under Fe-deficient conditions (17, 19). BTS interacts with and destabilizes bHLH105 and bHLH115, thereby negatively regulating the Fe deficiency response (17, 21). However, BTS is induced and BTS protein is stable under Fe-deficient conditions. Therefore, the finding that both BTS and bHLH105/bHLH115 proteins are stable under Fe-deficient conditions appears to be paradoxical.

IRON MAN (IMA)/FE-UPTAKE-INDUCING PEPTIDEs (FEPs), a recently identified class of small peptides, control Fe transport in plants (22, 23). The overexpression of IMA genes enhances IRT1 and FRO2 expression and promotes Fe uptake in A. thaliana, whereas the loss of function of IMA genes has the opposite effects. IMA peptides also promote the transcription of bHLH Ib genes, implying that IMA peptides function upstream of these genes. However, the molecular mechanism by which IMA peptides regulate the Fe deficiency response remains to be elucidated.

Here, we show that both IMA peptides and bHLH105/bHLH115 proteins contain a BID (BTS interaction domain), which accounts for their interaction with BTS. Similar to bHLH105/bHLH115 proteins, IMA peptides are degraded by BTS. Under Fe-deficient conditions, IMA genes are up-regulated, and IMAs interfere with the interaction between bHLH105/bHLH115 and BTS. These findings indicate that IMA peptides act as inhibitors of BTS and buffer the degradation of bHLH105/bHLH115 proteins by BTS, thereby activating the Fe deficiency response.

Results

IMAs Physically Interact with BTS.

To identify the partners of IMAs, we chose IMA3 as a representative IMA for which to identify interacting proteins in a yeast two-hybrid assay. We used the full-length IMA3 as a bait to identify targets in a complementary DNA library generated from A. thaliana under Fe-deficiency treatment. Sequencing analysis suggested that two positive clones contained the same insert sequence, encoding the C-terminal region of BTS (BTSc; SI Appendix, Fig. S1A). To confirm the interaction between IMA3 and BTS, we performed coimmunoprecipitation assays (Fig. 1A). MYC-tagged BTSc (or MYC-tagged GUS as a negative control) and GFP-tagged IMA3 were transiently coexpressed in Nicotiana benthamiana leaves, and total proteins were extracted and incubated in GFP-trap agarose. Immunoblot analysis indicated that MYC-BTSc, but not MYC-GUS, coimmunoprecipitated with GFP-IMA3, supporting the interaction between BTS and IMA3.

Fig. 1.

IMAs interact with BTS. (A) Immunoprecipitation showing IMA3 interacts with BTSc. Immunoprecipitation was performed using GFP-Trap agarose beads, and immunoblot analyses were probed with anti-MYC antibody and anti-GFP antibody. (B) Interaction of IMA3 and BTS in plant cells. Tripartite split-sfGFP complementation assays were performed. IMA3 was fused with GFP10, and BTS was fused with GFP11. The combinations indicated were introduced into Agrobacterium and coexpressed in N. benthamiana leaves. (C) IMAs interact with BTS. Yeast cotransformed with different BD and AD plasmid combinations was spotted. Growth on selective plates lacking leucine, tryptophan, adenine, and histidine (−4) or lacking leucine and tryptophan (−2) is shown. (D) Identification of BID. Yeast cotransformed with different BD and AD plasmid combinations was spotted. Growth on selective plates lacking leucine, tryptophan, adenine, and histidine (−4) or lacking leucine and tryptophan (−2) is shown.

IMA3 is expressed both in the nucleus and cytosol (22), and BTS is localized to the nucleus (17). To verify the cellular compartments in which the protein–protein interaction occurs, we performed tripartite split-GFP complementation assays. The GFP10 fragment was fused with the N-terminal end of IMA3 (GFP10-IMA3) and the GFP11 fragment with the C-terminal end of BTS (BTS-GFP11). When GFP10-IMA3 and BTS-GFP11 were transiently coexpressed with GFP1-9 in N. benthamiana cells, GFP signals were visible in the nuclei of transformed cells (Fig. 1B). By contrast, the negative controls did not generate GFP signals. These data suggest that IMA3 and BTS interact with each other in the nucleus.

IMA3 belongs to the IMA family, which has eight members in A. thaliana (22). To investigate whether other IMA family members also interact with BTS, we tested their interactions in yeast cells (Fig. 1C). Six of the eight family members, IMA1 to IMA4, IMA6, and IMA7, interacted with BTS. The IMA family members share a conserved 17-amino-acid motif in their C-terminal regions (SI Appendix, Fig. S1B). We therefore investigated whether the conserved motif was required for their interaction with BTS. We divided IMA3 into two parts, the N-terminal part (IMA3-N) without the conserved motif and the C-terminal 17 amino acid residues comprising the conserved motif, and examined their interactions with BTSc in yeast cells. The results indicated that the conserved motif was sufficient for the interaction with BTSc (Fig. 1D). Thus, we designated this motif as the BID.

The BID contains a conserved four-residue region APAA (SI Appendix, Fig. S1B), which corresponds to amino acid residues 44 to 47 of IMA3. To investigate whether these residues are responsible for the interaction with BTS, we performed site-specific mutagenesis of each residue separately, resulting in IMA3^A44P, IMA3^P45A, IMA3^A46V, and IMA3^A47V. Interaction tests showed that only IMA3^A47V failed to interact with BTSc. These data suggest that the last residue (A) is crucial for the interaction of BID with BTS. We noted that in IMA5, which does not interact with BTSc, the BID sequence contains a seven-amino-acid deletion resulting from a premature stop codon caused by a single nucleotide mutation (SI Appendix, Fig. S1B). We speculated that this partial deletion of the BID accounts for the failure of IMA5 to interact with BTSc. Thus, we generated a mutated version of IMA5 (IMA5^*50Y) containing a perfect BID by converting the stop codon to a tyrosine (Y) codon (SI Appendix, Fig. S1C). Interaction tests in yeast indicated that IMA5^*50Y physically interacted with BTSc (SI Appendix, Fig. S1D). Unlike other IMAs, IMA8 contains a seven-amino-acid appendix at the end of BID (SI Appendix, Fig. S1B). Interaction tests indicated that a truncated IMA8 (IMA8del) without the seven-amino-acid appendix could interact with BTSc (SI Appendix, Fig. S1D).

Constitutive Expression of BTS-Interacting IMAs Mimics the bts-2 Mutation.

Given that six of the eight IMAs interact with BTS, we next examined whether this interaction is relevant to their biological functions. We generated overexpression lines of all eight IMAs and investigated their phenotypes (SI Appendix, Fig. S2A). Overexpression of BTS-interacting IMAs (IMA1oe, IMA2oe, IMA3oe, IMA4oe, IMA6oe, and IMA7oe) led to reduced fertility, as did the bts-2 mutation, whereas plants overexpressing IMA5 or IMA8 (IMA5oe and IMA8oe) developed as well as the wild type (Fig. 2A). We then examined plant growth on medium with or without sufficient Fe and established that the reduced fertility corresponded with the enhanced tolerance to Fe deficiency (Fig. 2B). We also measured the Fe concentration (Fig. 2C), Fe chelate reductase activity (Fig. 2D), and expression of Fe deficiency–responsive genes IRT1, FRO2, bHLH38, and bHLH39 (SI Appendix, Fig. S2B). The results further support the notion that overexpressing BTS-interacting IMAs resulted in plants with phenotypes similar to those of bts-2.

Fig. 2.

Phenotypes of IMA overexpression plants. (A) Siliques of IMA overexpression plants. Plants were grown in soil. (B) The 7-d-old seedlings. Plants were grown on Fe-sufficient (+Fe; 100 μM Fe²⁺) or Fe-deficient (−Fe + Frz; Fe free plus 50 μM Frz) medium for 7 d. (C) Fe concentration. Leaves from plants grown in soil for 4 wk were used for Fe measurement. (D) Ferric-chelate reductase activity. Plants were grown on +Fe medium for 4 d and then transferred to +Fe or −Fe medium for 3 d. (C and D) The data represent means ± SD. The different letters above each bar indicate statistically significant differences as determined by one-way ANOVA followed by Tukey’s multiple comparison test (P < 0.05).

To investigate whether the BID of the IMAs is required for their biological functions, we generated A. thaliana plants overexpressing IMA5^*50Y (SI Appendix, Fig. S3A). Indeed, the IMA5^*50Y overexpression plants had phenotypes similar to those of bts-2 (SI Appendix, Fig. S3 B and C), indicating that IMA5^*50Y restored the ability to activate the Fe deficiency response. We also generated plants overexpressing IMA3^A47V (SI Appendix, Fig. S3A) and noted that they showed no visible difference from the wild type (SI Appendix, Fig. S3 B and C), indicating that IMA3^A47V lacked the ability to activate the Fe deficiency response. qRT-PCR analysis showed that the overexpression of IMA5^*50Y, but not IMA3^A47V, activated the expression of IRT1, FRO2, bHLH38, and bHLH39 (SI Appendix, Fig. S3D). Considering that the BID is responsible for the interaction with BTS, we generated overexpression lines in which only the BID of IMA3 (IMA3BID) was driven by the cauliflower mosaic virus (CaMV) 35S promoter (SI Appendix, Fig. S3A). Similar to IMA3oe, IMA3BIDoe also constitutively activated the Fe deficiency response (SI Appendix, Fig. S3 B, C, and E), suggesting that the BID is sufficient for activating this response. These data suggest that the BTS-interacting BID is necessary and sufficient for the roles of IMAs in the Fe deficiency response.

IMAs Are Ubiquitinated and Degraded by BTS.

Considering the finding that BTS interacts with IMAs and the status of BTS as an E3 ligase involved in the ubiquitination process (17), we next explored whether BTS could ubiquitinate and degrade IMAs. We performed ubiquitination assays in plant cells using IMA3 as a representative IMA. When IMA3 was coexpressed with UBQ11, we detected ubiquitinated forms of IMA3 (Fig. 3A). When IMA3 was coexpressed with both UBQ11 and BTSc, the levels of ubiquitinated forms of IMA3 increased significantly, suggesting that IMA3 can be ubiquitinated by BTS.

Fig. 3.

Ubiquitination and degradation of IMAs by BTS. (A) Ubiquitination of IMA3 by BTS. Pro35S:MYC-UBQ11 and Pro35S:GFP-IMA3 were coexpressed with Pro35S:MYC or Pro35S:MYC-BTSc. Crude lysates (input) were immunoprecipitated with GFP-trap agarose, and then the immunoprecipitates were detected with anti-MYC and anti-GFP antibody. (B) Degradation of IMA3 by BTS. Pro35S:GFP-IMA3 was coexpressed with Pro35S:MYC-GUS, Pro35S:MYC-BTS, or Pro35S:MYC-BTSc. Total protein was extracted and immunoblotted with anti-MYC, anti-GFP antibody, and anti–β-tubulin. β-tubulin was used as an internal control. (C) IMA3^A47V is resistant to BTS. Pro35S:GFP-IMA3^A47V was coexpressed with Pro35S:MYC-GUS or Pro35S:MYC-BTSc. Total protein was extracted and immunoblotted with anti-MYC, anti-GFP antibody, and anti–β-tubulin, respectively. β-tubulin was used as an internal control. (D) IMA1 is stable in bts-2. The 7-d-old seedlings grown on +Fe medium were shifted to +Fe or −Fe medium for 3 d. Total protein of roots was immunoblotted with anti-GFP antibody and anti–β-tubulin, respectively. β-tubulin was used as an internal control. The numbers indicate the relative protein abundance of EYFP-IMA1.

Subsequently, we examined whether BTS promotes the degradation of IMA3. To visibly monitor their protein levels, we fused IMA3 and BTS with mCherry and GFP, respectively, and performed transient expression assays by coexpressing mCherry-IMA3 with BTS-GFP or GFP. Compared with the strong mCherry signals in leaves expressing the combination of GFP and mCherry-IMA3, we observed significantly weaker mCherry signals in leaves expressing the combination of BTS-GFP and mCherry-IMA3, which is in agreement with the lower mCherry-IMA3 protein levels in the presence of BTS-GFP (SI Appendix, Fig. S4). Similarly, mCherry-IMA1 levels also decreased in the presence of BTS-GFP (SI Appendix, Fig. S4). Since we could not detect the full-length BTS protein in our immunoblot assays, we performed immunoblot analysis with BTSc. GFP-IMA3 was coexpressed with MYC-GUS or MYC-BTSc. GFP-IMA3 protein levels declined dramatically in the presence of BTSc (Fig. 3B). Similarly, IMA1 protein was degraded by BTSc (SI Appendix, Fig. S5). Given that the IMA3^A47V mutant did not interact with BTSc, we hypothesized that it would be insensitive to BTS. To investigate this hypothesis, we constructed GFP-IMA3^A47V and coexpressed it with BTSc. Compared with the negative control (GUS), the presence of BTSc did not alter the protein abundance of GFP-IMA3^A47V (Fig. 3C). To further investigate whether IMAs are stable in bts-2, the ProIMA1:EYFP-IMA1 construct (23) was introduced to wild-type and bts-2 plants, respectively. We found that the EYFP-IMA1 was only detected under Fe-deficient conditions in the ProIMA1:EYFP-IMA1/WT plants. In contrast, the EYFP-IMA1 protein was abundant in the ProIMA1:EYFP-IMA1/bts-2 plants regardless of Fe status (Fig. 3D). Taken together, these results suggest that BTS mediates the ubiquitination and degradation of IMAs.

bHLH105 and bHLH115 Also Contain a BID.

bHLH105 and bHLH115 physically interact with BTS, and these proteins are stable in the bts mutant (17). However, the regions of bHLH105 and bHLH115 that interact with BTS were unclear. To investigate this issue, we divided bHLH105 protein into two parts: the N-terminal part containing the bHLH domain and the C-terminal part. Interaction tests suggested that the C-terminal part of bHLH105 is required for its interaction with BTS (Fig. 4).

Fig. 4.

Identification of BID in the bHLH105 protein. The schematic diagram (Left) shows the various truncated bHLH105 protein fragments and the positions of mutated residues. Yeast cotransformed with different BD and AD plasmid combinations was spotted. Growth on selective plates lacking leucine, tryptophan, adenine, and histidine (−4) or lacking leucine and tryptophan (−2) is shown.

To narrow down the region that accounts for the interaction between bHLH105 and BTS, we constructed two shorter peptides containing 34 and 23 amino acids from the C-terminal part of bHLH105, respectively (Fig. 4). Interaction analysis indicated that the 34-amino-acid peptide, but not the 23-amino-acid peptide, interacted with BTS. Interestingly, the last three residues of bHLH105 and bHLH115 are PVA, which is similar to the PAA sequence of IMAs. To investigate whether the interaction of bHLH105 with BTS depends on these three residues, we performed residue substitution analysis. Indeed, substitution of the last A (to create the bHLH105^A234V variant) affected the protein’s interaction with BTS (Fig. 4). bHLH IVc subgroup members share a PPxA motif in their C-terminal ends (SI Appendix, Fig. S6A). To establish whether the two-residue sequence PP is required for the interaction of bHLH105 with BTS, we constructed another mutant version of bHLH105 (bHLH105^P231AP232A). Interaction tests showed that mutation of PP did not block the interaction of this protein with BTS (Fig. 4). We also confirmed that the C-terminal region of bHLH115 and its last residue (A) are required for the interaction with BTS (SI Appendix, Fig. S6B). These data suggest that the C-terminal regions of bHLH105 and bHLH115 comprise BIDs.

IMA Peptides Stabilize bHLH105/bHLH115 Proteins by Competitively Interacting with BTS.

The overexpression of IMA genes leads to the up-regulation of Fe deficiency–responsive genes that function downstream of bHLH105/bHLH115, such as bHLH38, bHLH39, bHLH100, bHLH101, IRT1, and FRO2 (22, 23). However, it is unclear how IMAs activate the expression of these genes. Since both IMAs and bHLH105/bHLH115 interacted with BTS via their BIDs, we hypothesized that IMAs influence Fe signaling by shielding bHLH105/bHLH115 from targeting by BTS. We therefore investigated whether IMAs affect the interaction between bHLH105/bHLH115 and BTS. We conducted LUC-based complementation assays. The N-terminal part of LUC was fused with the C-terminal end of BTS and the C-terminal part of LUC with the N-terminal end of bHLH105 (or bHLH115). When BTS-nLUC was coexpressed with cLUC-bHLH105 (or bHLH115), we observed LUC fluorescence. The addition of GFP, as a negative control, did not affect the LUC fluorescence intensity. By contrast, the addition of GFP-IMA1 or GFP-IMA3 significantly reduced the LUC fluorescence density resulting from the BTS/bHLH105 (or BTS/bHLH115) interaction (Fig. 5A and SI Appendix, Fig. S7A). To investigate whether there is a dose-dependent inhibition effect, we compared the LUC fluorescence density with different concentrations of GFP-IMA1/3. We found that the LUC fluorescence density decreased as the GFP-IMA1/3 increased (Fig. 5B and SI Appendix, Fig. S7B). These results suggest the repressive effects of IMA1 and IMA3 on the interactions of bHLH105/bHLH115 with BTS.

Fig. 5.

IMAs stabilize bHLH105 protein by inhibiting its interaction with BTS. (A) IMA1 and IMA3 interfere with the interaction of bHLH105 and BTS. Compared with the two negative controls (BTS-LUCn/LUCc and LUCn/LUCc-bHLH105), the combination of BTS-LUCn/LUCc-bHLH105 generated LUC activity. Compared with GFP, both GFP-IMA1 and GFP-IMA3 alleviated the LUC activity resulting from BTS-LUCn/LUCc-bHLH105. The OD600 value of LUCn/BTS-LUCn, LUCc/LUCc-bHLH115, and GFP/GFP-IMA1/GFP-IMA3 is 0.5, 0.5, and 1.0, respectively. The numbers indicate the relative LUC fluorescence density. (B) IMA1 and IMA3 competitively inhibit the interaction of bHLH105 and BTS. BTS-LUCn and LUCc-bHLH105 were coexpressed with the third party (GFP, GFP-IMA1, or GFP-IMA3) in tobacco leaves. The y-axis shows the relative LUC fluorescence density resulting from the BTS-LUCn/LUCc-bHLH105 complex. The x-axis shows the concentration of the third party. The OD600 value of BTS-LUCn and LUCc-bHLH105 is 0.5. (C) IMA1 and IMA3 stabilize bHLH105 protein. Pro35S:GFP-bHLH105 and Pro35S:MYC-BTSc were coexpressed with Pro35S:mCherry, Pro35S:mCherry-IMA1, or Pro35S:mCherry-IMA3. Total protein was extracted and immunoblotted with anti-MYC, anti-GFP, and anti-mCherry antibody. (D) bHLH105^A234V is resistant to BTSc. Pro35S:GFP-bHLH105 or Pro35S:GFP-bHLH105^A234V was coexpressed with Pro35S:MYC-GUS or Pro35S:MYC-BTSc. Total protein was extracted and immunoblotted with anti-MYC, anti-GFP, and anti–β-tubulin antibody. The asterisk labels a nonspecific signal that is used as a loading control. (E) bHLH105 protein abundance. Roots of 10-d-old seedlings grown on −Fe medium were used for total protein extraction. Immunoblot was conducted with anti-bHLH105 antibody. The numbers indicate the relative protein abundance of bHLH105. Coomassie blue staining indicates equal amount of protein extract loaded.

Subsequently, we speculated that IMA peptides could stabilize bHLH105/bHLH115 proteins. To test this possibility, we carried out transient expression assays. When GFP-bHLH105 (or GFP-bHLH115) was coexpressed with MYC-BTSc, the addition of mCherry-IMA1 (or mCherry-IMA3), but not mCherry, significantly increased GFP-bHLH105 (or GFP-bHLH115) protein abundance (Fig. 5C and SI Appendix, Fig. S7C).

Because IMAs stabilize bHLH105/bHLH115 proteins by interfering with their interactions with BTS, we reasoned that the mutated versions of bHLH105^A234V/bHLH115^A226V were not degraded by BTS because they were unable to physically interact with BTS. To verify this hypothesis, we constructed GFP-tagged bHLH105^A234V and bHLH115^A226V and coexpressed them with BTSc. Whereas GFP-bHLH105 and GFP-bHLH115 levels decreased in the presence of BTSc, GFP-bHLH105^A234V and GFP-bHLH115^A226V were unaffected (Fig. 5D and SI Appendix, Fig. S7D). Taken together, these results suggest that IMAs stabilize bHLH105/bHLH115 by interfering with their interactions with BTS.

We then measured the level of native bHLH105 in A. thaliana plants. We transferred 7-d-old seedlings grown on Fe-sufficient medium to Fe-deficient medium, grew them for 3 d, extracted proteins from the roots of these plants, and subjected the proteins to immunoblot analysis. bHLH105 protein levels were higher in IMA1oe and IMA3oe plants than in wild-type plants but comparable to those in bts-2 plants (Fig. 5E). These data suggest that IMA peptides promote bHLH105 protein accumulation.

Genetic Interactions Exist among IMA3, bHLH105/bHLH115, and BTS.

To identify the genetic loci of IMAs that function in the regulatory network of the Fe deficiency response, we generated two ima3 mutants by CRISPR/Cas9-mediated gene editing (SI Appendix, Fig. S8A). In agreement with a previous report (22), both mutants were sensitive to Fe deficiency. When grown on a Fe-deficient medium, similar to bhlh105-1 and bhlh115-1 plants, the ima3 plants showed increased sensitivity to Fe deficiency (SI Appendix, Fig. S8B). We then generated bhlh105 ima3 and bhlh115 ima3 double mutants by crossing the corresponding single mutants. When grown on Fe-deficient medium, both double mutants were more sensitive to Fe deficiency than the single mutants, with significantly shorter roots (Fig. 6A and SI Appendix, Fig. S9A). qRT-PCR analysis indicated that IRT1, FRO2, bHLH38, and bHLH39 expression was lower in the double mutants than in the single mutants (Fig. 6B). These results suggest that bHLH105/bHLH115 and IMA3 play additive roles in regulating the Fe deficiency response.

Fig. 6.

Genetic interactions between IMA3, bHLH105/bHLH115, and BTS. (A) Phenotypes of various combinations of ima3-1, bhlh105-1, and bhlh115-1. (B) Expression of Fe deficiency responsive genes in various combinations of ima3-1, bhlh105-1, and bhlh115-1. (C) Phenotypes of various combinations of bts-2, IMAoe-2, and bhlh105/bhlh115. (D) Expression of Fe deficiency responsive genes in various combinations of bts-2, IMAoe-2, and bhlh105/bhlh115. (A and C) Plants were grown on Fe-sufficient (+Fe; 100 μM Fe²⁺) or Fe-deficient (−Fe+Frz; Fe free plus 50 μM Frz) medium for 7 d. (B and D) Seedlings grown on +Fe medium for 4 d were transferred to +Fe or –Fe medium for 3 d. Roots were used for RNA extraction. Data represent means ± SD (n = 3). Different letters above each bar indicate statistically significant differences (ANOVA, P < 0.01).

Subsequently, we generated bts bhlh105, bts bhlh115, and bts ima3 double mutants. bts-2 displayed enhanced tolerance to Fe deficiency, and the introduction of bhlh105-1 or bhlh115-1 into the bts-2 background significantly alleviated this enhanced tolerance (SI Appendix, Fig. S10). When ima3-1 was introduced into the bts-2 background, the tolerance of bts-2 for Fe deficiency was also dramatically reduced (SI Appendix, Fig. S10). Furthermore, IMA3oe-2 or bts-2 was crossed into the bhlh105 bhlh115 double mutant background, the tolerance of IMA3oe-2 and bts-2 for Fe deficiency was almost completely compromised (Fig. 6C and SI Appendix, Fig. S9B), and the up-regulation of bHLH38, bHLH39, IRT1, and FRO2 was considerably suppressed (Fig. 6D). These data suggest that bHLH105/bHLH115 function downstream of IMAs and BTS.

bHLH105 and bHLH115 Directly Regulate the Transcription of IMA Genes and BTS.

Both BTS and IMA genes are up-regulated by Fe deficiency. bHLH105 and bHLH115, two key positive regulators of the Fe deficiency response, control the expression of numerous Fe deficiency–responsive genes. To investigate whether the up-regulation of BTS and IMA genes by Fe deficiency requires bHLH105 and bHLH115, we examined their expression levels in the bhlh105-1, bhlh115-1, and bhlh105 bhlh115 mutants. The loss of both bHLH105 and bHLH115 function caused a reduction in BTS and IMA transcript levels (Fig. 7A). Thus, bHLH105 and bHLH115 positively regulate BTS and IMA transcription. To further investigate whether their promoter activity is affected in the bhlh105-1 mutant background, we generated pBTS:GUS and pIMA3:IMA3-GUS transgenic plants and crossed them with the bhlh105-1 mutant. Both promoters were less active in the bhlh105-1 mutant than in the wild type, as revealed by GUS staining (SI Appendix, Fig. S11).

Fig. 7.

bHLH105 directly targets the promoters of IMA3 and BTS. (A) Expression of BTS, IMA1, IMA2, and IMA3. Seedlings grown on +Fe medium for 4 d were transferred to +Fe or –Fe medium for 3 d. Roots were used for RNA extraction. Data represent means ± SD (n = 3). Different letters above each bar indicate statistically significant differences (ANOVA, P < 0.01). (B) ChIP-qPCR analysis. The 10-d-old seedlings grown on +Fe medium were harvested for ChIP assays using anti-GFP antibody, and the immunoprecipitated DNA was quantified by qPCR and normalized to its counterpart in the input. The Pro35S:MYC-GFP plants were used as the negative control. Data represent means ± SD (n = 3). The value that is significantly different from the corresponding control (Pro35S:MYC-GFP) value was indicated by * (P < 0.01), as determined by Student’s t test. (C) EMSA analysis. The recombinant His-bHLH105 protein was used. Biotin probe, biotin-labeled probe; Cold-Probe, unlabeled probe; Cold-mProbe, unlabeled mutated probe with a mutated E-box.

To further investigate whether bHLH105 directly binds to the promoters of IMAs and BTS, we conducted chromatin immunoprecipitation (ChIP) assays with Pro35S:MYC-GFP and ProbHLH105:bHLH105-GFP plants, and the GFP-tagged proteins were immunoprecipitated using anti-GFP antibody. The promoter of bHLH38, a direct target gene of bHLH105 (10), was used as a positive control in the ChIP assays, and a fragment of the TUB2 promoter containing an E-box was used as a negative control. We observed bHLH105-specific enrichment at the bHLH38, BTS, and IMA3 promoters but not the IMA1, IMA2, or TUB2 promoters (Fig. 7B).

To investigate whether bHLH105 and bHLH115 recognize the promoters of BTS and IMA3 by direct protein–DNA binding, we performed the electrophoretic mobility shift assays (EMSA). Specifically, we used His-bHLH105 and His-bHLH115 recombinant proteins purified from Escherichia coli to verify the direct interactions with the BTS and IMA3 promoters. When the BTS promoter fragment was used as a probe, a DNA–protein complex was detected. The abundance of this complex decreased following the addition of unlabeled wild-type competitors. By contrast, the unlabeled mutant probe carrying a mutated E-box had no effect on DNA–protein binding (Fig. 7C and SI Appendix, Fig. S12). A similar result was obtained when the IMA3 promoter was used as the probe (Fig. 7C and SI Appendix, Fig. S12). Taken together, these results suggest that bHLH105 and bHLH115 bind to the promoters of BTS and IMA3.

Discussion

To meet the demands for Fe in different tissues and organs, the uptake and transport of Fe must be precisely controlled. Plants have evolved a sophisticated regulatory network involving the sensing of Fe concentrations and signal transduction. BTS, a potential Fe sensor, is known to interact with bHLH105 and bHLH115 to coordinate the Fe deficiency response in A. thaliana. Here, we reveal that IMAs interact with BTS and modulate the abundance of bHLH105 and bHLH115.

The IMA family consists of eight members in A. thaliana. Although their roles as positive regulators of the Fe deficiency response are well established, the underlying molecular mechanism remains elusive. Here, we clarified the molecular mechanism underlying the roles of IMAs in regulating the response of A. thaliana to Fe deficiency. We discovered that six of the eight IMA family members (all except IMA5 and IMA8) interact with BTS and activate the Fe deficiency response. IMA5 contains a premature stop codon in its BID, and repairing this BID rescued not only its interaction with BTS but also its biological function in activating the Fe deficiency response (SI Appendix, Fig. S3 B–D). Thus, the disruption of BID is the reason that IMA5 fails to interact with BTS and is nonfunctional. By contrast, IMA3^A47V, a mutated version of IMA3, neither interacted with BTS nor activated the Fe deficiency response (Fig. 1D and SI Appendix, Fig. S3 B–D). Thus, there is a direct relationship between the biological functions of IMAs and their interactions with BTS. It is noteworthy that the removal of the appendix from the C-terminal end of IMA8 BID restores its interaction with BTS (SI Appendix, Fig. S1D), implying that an intact C-terminal end without an appendix is required for BID’s interaction with BTS.

Similar to IMAs, bHLH105 and bHLH115 also contain a BID in their C termini. When the last residue (A) of their BIDs was mutated, both IMAs and bHLH105/bHLH115 failed to interact with BTS (Figs. 1D and 4 and SI Appendix, Fig. S6B) and were therefore stable and resistant to degradation by BTS (Figs. 3C and 5D). However, disruption of the BID made the IMAs lose their function. The loss of IMA function occurred when they were no longer able to interact with BTS (Fig. 2 and SI Appendix, Fig. S3). IMAs interact with BTS and competitively inhibit the interactions between bHLH105/bHLH115 and BTS, thus stabilizing bHLH105/bHLH115, which also explains why IMA overexpression plants can phenocopy the bts-2 mutant (Fig. 2 A and B). Xing et al. (21) recently reported a similar mechanism in which a bacterial effector protects bHLH105/bHLH115 from degradation by BTS. The observation that the overexpression of IMA genes activated the target genes of bHLH105/bHLH115 and promoted bHLH105 protein accumulation further supports the positive roles of IMAs in stabilizing bHLH105/bHLH115.

We established that the BID of bHLH105 is required for its degradation by BTS (Fig. 5D). Another line of evidence supporting the role of the bHLH105 BID comes from the ilr3-1 mutant, which is a gain-of-function mutant due to the lack of the 64 C-terminal amino acids of bHLH105 protein (19, 24). We noted that the two other members of the bHLH IVc subgroup, bHLH34 and bHLH104, also contain a potential BID. However, it was reported that bHLH34 cannot interact with BTS (16) and bHLH104 is insensitive to BTS (17). Interestingly, the mutation of the last residue (A) in bHLH34 resulted in the constitutive activation of the Fe deficiency regulatory pathway (25). Perhaps these two bHLH IVc subgroup members are degraded by unknown proteins.

We found that IMA1, IMA2, and IMA3 are down-regulated in the bhlh105 bhlh115 mutants (Fig. 7A); however, bHLH105 only binds to the promoter of IMA3 (Fig. 7B). It is plausible that bHLH105 regulates other IMAs by interacting with other bHLH IVc members or bHLH121, which directly binds to the promoters of these three IMAs (14). Constitutive expression of IMA genes causes reduced fertility, which might be related to Fe toxicity resulting from the increased expression of Fe uptake genes (SI Appendix, Fig. S2B). By contrast, their loss of function hinders Fe uptake and results in Fe deficiency symptoms (22, 23). Therefore, appropriate levels of IMA peptides are required for the maintenance of Fe homeostasis. We provided evidence that IMA peptides undergo ubiquitination and degradation mediated by BTS. Notably, the BID of IMAs, but not of bHLH105 or bHLH115, is rich in aspartic acid (D), which can bind ferrous Fe. Grillet et al. (23) reported that IMA peptides are unstable when saturated with metal ions. Taken together, these findings indicate that the peptide levels of IMAs are under the control of Fe status via multiple layers of regulation: 1) peptide translation from IMA transcripts induced by Fe deficiency, 2) protein ubiquitination and degradation mediated by BTS, and 3) protein precipitation after saturation with ferrous Fe. Further investigation is required to clarify whether and how the D repeat region of IMAs responds to Fe status in vivo.

The ability of a plant to turn on the transcription of Fe uptake genes upon Fe starvation and turn off their transcription under Fe excess is an important factor in determining its growth and development. BTS is a potential Fe sensor, which is unstable in the presence of Fe and possesses a RING domain with E3 ligase activity (17). As its substrates, both IMAs and bHLH105/bHLH115 are degraded by BTS. Whereas bHLH105 and bHLH115 are barely responsive to Fe deficiency (10, 12), IMA genes are considerably induced by Fe deficiency (23). It is likely that the rapid accumulation of IMAs results in the lock-in of BTS and the release of bHLH105/bHLH115, thus activating the expression of Fe deficiency–responsive genes (Fig. 8). bHLH105 and bHLH115 are the key activators of this process, and thus their abundance is tightly regulated. bHLH105 and bHLH115 directly activate the expression of BTS and thus their own degradation, thereby forming a negative feedback loop between bHLH105/bHLH115 and BTS (Fig. 8). On the other hand, bHLH105 and bHLH115 directly activate IMA3 (or indirectly other IMAs) and thus their own accumulation, forming a positive feedback loop between bHLH105/bHLH115 and IMAs (Fig. 8). These two feedback loops balance bHLH105/bHLH115 levels, and hence Fe homeostasis, in plants. When the negative feedback loop is blocked in the bts-2 mutant or the positive feedback loop is enhanced in the IMA overexpression plants, bHLH105/bHLH115 proteins accumulate, thereby activating the expression of Fe deficiency–responsive genes. When the interaction between bHLH105/bHLH115 and BTS was uncoupled by mutation, both bHLH105^A234V and bHLH115^A226V were not affected by BTS any more (Fig. 5D and SI Appendix, Fig. S7D). Therefore, the maintenance of bHLH105/bHLH115 levels by these two feedback loops is crucial for maintaining Fe homeostasis.

Fig. 8.

Working model of IMAs in Fe homeostasis. BTS, a potential Fe sensor, is unstable in the presence of Fe. bHLH105 and bHLH115 are two major regulators of Fe homeostasis, which directly activate the transcription of BTS, IMA3, and other Fe deficiency–responsive genes including PYE and bHLH Ib. BTS can ubiquitinate and degrade IMAs and bHLH105/115. Under Fe-sufficient conditions, most of bHLH105/115 are degraded by BTS and the transcription of Fe deficiency–responsive genes is limited. Under Fe-deficient conditions, although BTS is induced, a large number of IMAs are synthesized and then competitively inhibit the interactions of bHLH105/115 with BTS, thus alleviating their degradation by BTS. Finally, bHLH105 and bHLH115 activate the expression of Fe deficiency–responsive genes. The width of orange arrows indicates relative protein amount. The question mark indicates an unknown transcription complex which might consist of bHLH121, bHLH IVc members, or other transcription factors. The 26 S indicates 26 S proteasome complex. Dotted lines indicate the results that have not been validated in vivo.

FBXL5, which functions as an Fe sensor in mammals, negatively regulates Fe homeostasis and is stable under Fe-sufficient conditions (26, 27). By contrast, BTS, as a potential Fe sensor, also negatively regulates Fe homeostasis but is unstable in vitro under Fe-sufficient conditions (17). If BTS is unstable in vivo under Fe-sufficient conditions, it seems unlikely for plants to switch off the Fe deficiency response because bHLH115 and bHLH115 are stable without BTS. Thus, elucidating the stability and activity of BTS in vivo will be helpful to address this issue. IMA peptides exist across different plant species but not in animals (23), implying that plants have evolved the specific peptides that antagonize the functions of their Fe sensors. Further investigation is required to analyze whether Fe ions affect the interactions between BTS and IMAs and whether IMAs affect the stability of BTS. Finally, the identification of an IMA–BTS interaction module revealed an intervention point for the directed modulation of Fe acquisition. Given their sequence conservation across different plant species (23), perhaps IMA peptides could be engineered and used as universal tools to manipulate plant uptake of Fe from the soil.

Materials and Methods

Plant Material, Cultivation, and Generation of Mutants and Transgenic Plants.

A. thaliana wild type, mutant lines, transgenic plants, growth conditions, Fe treatments, histochemical staining, ferric-chelate reduction activity, and Fe content measurement are described in detail in SI Appendix, Materials and Methods.

Protein Production and Protein Interaction Studies.

Details of the constructs for protein expression in E. coli, protein purification, protein interactions, ubiquitination and degradation assays, firefly luciferase assays, and immunoblot analysis are provided in SI Appendix, Materials and Methods. The primer sequences for generation of constructs are listed in SI Appendix, Table S1.

Gene Expression Analysis and ChIP-qPCR.

Details of RNA extraction, qRT-PCR, ChIP, and EMSA are described in SI Appendix, Materials and Methods. The primer sequences for qRT-PCR, ChIP, and EMSA are listed in SI Appendix, Table S1.

Data Availability

All study data are included in the article and/or SI Appendix.