Two bHLH Transcription Factors, bHLH34 and bHLH104, Regulate Iron Homeostasis in Arabidopsis thaliana

Abstract

The regulation of iron (Fe) homeostasis is critical for plant survival. Although the systems responsible for the reduction, uptake, and translocation of Fe have been described, the molecular mechanism by which plants sense Fe status and coordinate the expression of Fe deficiency-responsive genes is largely unknown. Here, we report that two basic helix-loop-helix-type transcription factors, bHLH34 and bHLH104, positively regulate Fe homeostasis in Arabidopsis (Arabidopsis thaliana). Loss of function of bHLH34 and bHLH104 causes disruption of the Fe deficiency response and the reduction of Fe content, whereas overexpression plants constitutively promote the expression of Fe deficiency-responsive genes and Fe accumulation. Further analysis indicates that bHLH34 and bHLH104 directly activate the transcription of the Ib subgroup bHLH genes, bHLH38/39/100/101 Moreover, overexpression of bHLH101 partially rescues the Fe deficiency phenotypes of bhlh34bhlh104 double mutants. Further investigation suggests that bHLH34, bHLH104, and bHLH105 (IAA-LEUCINE RESISTANT3) function as homodimers or heterodimers to nonredundantly regulate Fe homeostasis. This work reveals that plants have evolved complex molecular mechanisms to regulate Fe deficiency response genes to adapt to Fe deficiency conditions.

Figure 1.

Identification of bHLH34 and bHLH104 by yeast one-hybrid assay. The promoter of bHLH101 was used as bait and bHLH34/104 as prey. The representative growth status of yeast cells is shown on synthetic dextrose medium agar plates without Leu (SD/-Leu) with or without aureobasidin A (AbA) from triplicate independent trails.

Figure 2.

Characterization of various mutant plants. A, T-DNA insertion position in the corresponding gene. Black boxes indicate the coding sequence. White boxes indicate untranslated regions. Triangles indicate the position of T-DNA. B, The corresponding full-length complementary DNAs (cDNAs) were amplified by reverse transcription-PCR to confirm the mutants. C, Ten-day-old seedlings germinated directly on Fe-deficient (–Fe) medium. D, Phenotypes of amiR-bhlh34/104 and bhlh34bhlh104 plants in soils. Four-week-old plants grown in normal soil are shown. WT, Wild type.

Figure 3.

Fe deficiency response of various mutants. A, Three-week-old plants germinated in soil with various concentrations of calcium oxide. B, Iron reductase activity of plants germinated and grown on Fe-sufficient (+Fe) medium for 10 d and then shifted to Fe-deficient (–Fe) medium for 3 d. The ferrozine assay was performed, in triplicate, on 10 pooled plant roots. Significant differences from the wild type (WT) are indicated by asterisks (P < 0.05). C and D, Fe content of shoots and roots. Seedlings were germinated and grown on +Fe medium for 11 d and then shifted to +Fe or –Fe medium for 3 d. Significant differences from the wild type are indicated by asterisks (P < 0.05). DW, Dry weight.

Figure 4.

Expression of Fe deficiency-responsive genes in various mutants. Wild-type (WT) and mutant plants were grown on Fe-sufficient (+Fe) medium for 10 d and then transferred to +Fe or Fe-deficient (–Fe) medium for 3 d. RNA was prepared from root tissues. The data represent means ± sd of three technical repeats from one representative experiment. Significant differences from the corresponding wild type are indicated by asterisks (P < 0.05). DW, Dry weight.

Figure 5.

Phenotypes of bHLH34 and bHLH104 overexpression plants. A, Four-week-old plants grown in normal soil. B, Ten-day-old seedlings grown on Fe-deficient (–Fe) medium. Numbers indicate the frequency of transgenic plants with the corresponding phenotypes. C, Relative transcript levels of bHLH34 (left) and bHLH104 (right) in overexpression plants. Significant differences from the wild type (WT) are indicated by asterisks (P < 0.05). D, Fe content of leaves in 4-week-old plants grown in normal soil. Significant differences from the wild type are indicated by asterisks (P < 0.05). DW, Dry weight.

Figure 6.

Expression of Fe deficiency-responsive genes in various transgenic plants. Wild-type (WT) and transgenic plants were grown on Fe-sufficient (+Fe) medium for 10 d and then transferred to +Fe or Fe-deficient (–Fe) medium for 3 d. RNA was prepared from root tissues. The data represent means ± sd of three technical repeats from one representative experiment. Significant differences from the corresponding wild type are indicated by asterisks (P < 0.05).

Figure 7.

bHLH34 and bHLH104 activate the promoter of bHLH101. A, Schematic representation of the constructs used for transient expression assays. The reporter construct consists of a bHLH101 promoter, a nuclear localization sequence (NLS) fused with the GFP coding sequence, and a poly(A) terminator. Effector constructs express Myc-bHLH34, Myc-bHLH104, and Myc-MYC2 under the control of the cauliflower mosaic virus (CaMV) 35S promoter. B, bHLH34 and bHLH104 activate the promoter of bHLH101 in transient expression assays. The results are one representative of three biological repeats. C, GFP transcript abundance. In the transient assays, Pro_35S:Myc-GUS was expressed as a control. GFP transcript abundance was normalized to GUS transcript. The value with the empty vector as an effector was set to 1. The results are means ± sd of three technical repeats from one of three biological repeats. Significant differences from the empty vector are indicated by asterisks (P < 0.05).

Figure 8.

Overexpression of bHLH101 partially rescues bhlh34bhlh104. A, Ten-day-old seedlings grown on Fe-sufficient (+Fe) or Fe-deficient (–Fe) medium. B, Root length of seedlings on +Fe or –Fe medium. Values are means ± sd of 10 plants for each genotype. C, Chlorophyll content of seedlings on +Fe or –Fe medium. Significant differences from the wild type (WT) are indicated by asterisks (P < 0.05). D, Expression of IRT1 and FRO2. Plants were grown on +Fe medium for 10 d and then transferred to +Fe or –Fe medium for 3 d. RNA was prepared from root tissues. Significant differences from the corresponding wild type are indicated by asterisks (P < 0.05).

Figure 9.

Interaction between bHLH34 and bHLH104. A, Yeast two-hybrid assays. Representative growth status of yeast cells is shown on synthetic dextrose medium agar plates without Leu/Trp/His/adenine. B, BiFC assays. N. benthamiana leaves were infiltrated with different combinations of the constructs. C, Coimmunoprecipitation (IP) assays. Total protein was immunoprecipitated using Flag antibody, and coimmunoprecipitated protein was then detected using Myc antibody.

Figure 10.

Interactions between bHLH34, bHLH104, and bHLH105. A, Yeast two-hybrid assays. Interaction was indicated by the ability of cells to grow on synthetic dropout medium lacking Leu/Trp/His/adenine. C-terminally truncated bHLH34 and bHLH104 and full-length bHLH105 were cloned into pGBKT7, and full-length cDNAs of bHLH34, bHLH104, and bHLH105 were cloned into pGADT7. B, Fe deficiency symptoms of various single and double mutants. Ten-day-old seedlings were germinated directly on Fe-deficient (–Fe) or Fe-sufficient (+Fe) medium. WT, Wild type.

Figure 11.

GUS staining of Pro_bHLH34:GUS, Pro_bHLH104:GUS, and Pro_bHLH105:GUS seedlings. Two-week-old seedlings were used for GUS staining. GUS staining of the root maturation regions, early lateral roots, root tips, leaves, hypocotyls, and whole seedlings is shown. c, Cortex; e, endodermis; el, elongation region of the root tip; ep, epidermis; m, meristem zone of the root tip; p, pericycle.

Figure 12.

Fe deficiency-responsive signaling pathway. Fe deficiency stabilizes the BTS protein that interacts with bHLH104 and bHLH105. bHLH34, bHLH104, and bHLH105 can form homodimers or heterodimers to activate the transcription of bHLH38/39/100/101 and PYE. FIT interacts with bHLH38/39/100/101 to activate the transcription of FRO2 and IRT1. PYE regulates the expression of ZIF1 and NAS4 negatively. MYB10 and MYB72 regulate the expression of NAS4 positively. The induction of MYB10/72 by Fe deficiency is partially dependent on FIT; bHLH34/104/105 are required for the induction of FIT and MYB10/72.