BHLH34 And BHLH104 Transcription Factors Regulate Iron Homeostasis In Arabidopsis Thaliana

Iron (Fe) is of particular importance as a cofactor for a wide variety of functional proteins for plants growth and development. In humans, Fe deficiency is one of the major reasons for anemia. A plant diet is a major resource of iron for human; thus it is important to clarify the mechanisms of Fe uptake, translocation and regulation in plants. bHLH38/39/100/101 are four genes involved in Fe-deficient response in Arabidopsis thaliana; however, it is still unknown how they are activated by Fe-deficiency. Here, we performed the yeast one-hybrid (Y1H) screening using the promoter of bHLH101 as bait. bHLH34 was identified as a poteintial candidate. Further experiments confirmed that both bHLH34 and its homolog, bHLH104 can acitivate the promoter of bHLH101. These data suggest that bHLH34 and bHLH104 may be involved in regulating Fe-deficient response in Arabidopsis thaliana.To investigate their functioons, we obtained two T-DNA insertion alleles for the bHLH34 and bHLH104 genes from the Arabidopsis Biological Resource Center. The loss of full-length transcripts in the T-DNA lines was determined by RT-PCR, indicating that both T-DNA lines are knockout mutants. Considering their potential functional redundancy, we constructed bhlh34bhlh104 double mutants by crossing two single knock-out mutants. Phenotype analysis revealed that bhlh34, bhlh104 and bhlh34bhlh104 double mutants showed interveinal chlorosis in leaves and the inhibition of the root growth on Fe-deficient media, which is a typical Fe deficiency symptom, and the Fe deficiency symptoms of the bhlh34bhlh104 double mutants were particularly severe. To further confirm the functions of bHLH34 and bHLH104, we designed an artificial miRNA, amiR-bhlh34/104, which specifically targets both bHLH34 and bHLH104, and generated amiR-bhlh34/104 transgenic plants. As expected, the amiR-bhlh34/104 plants displayed Fe-deficient symptoms similar to those of bhlh34bhlh104 mutants. These data demonstrate that the amiR-bhlh34/104 plants phenocopy the bhlh34bhlh104 mutants.Fe reductase activity is a typical indicator of Fe deficiency. In response to Fe deficiency, both wild-type and mutant plants increased their Fe reductase activity. However, the Fe reductase activity of the mutant plants was significantly lower than that of wild type, and the bhlh34bhlh104 double mutants had the lowest Fe reductase activity. The measurement of Fe content demonstrates that the roots and shoots of bhlh34bhlh104 plants contained less Fe than wild-type plants under both Fe-sufficient and -deficient conditions. To further analyze the molecular functions of bHLH34 and bHLH104, we determined the expression levels of Fe deficiency responsive genes. Compared with wild-type plants, the expression levels of two Fe-uptake associated genes IRT1 and FRO2 were lower in the mutants. Moreover, MYB10/72, bHLH38/39/100/101 and FIT which positively regulate Fe-deficient response, were also down-regulated in the mutants. These data suggest that bHLH34 and bHLH104 positively regulate Fe-deficient response in Arabidopsis thaliana. Considering that their positive roles in Fe deficiency reposne, we are interested in whether overexpression of bHLH34 and bHLH104 will increase Fe content in plants. we constructed the overexpression transgenic plants of bHLH34 and bHLH104. When grown in normal soils, about 10% of bHLH34 overexpression transgenic plants and 33% of bHLH104 overexpression transgenic plants displayed visible phenotypes (small rosettes and leaf necrosis) and no visible phenotype was observed for the other transgenic plants. All overexpression plants have higher Fe content than wild type and constitutively activate FIT, bHLH38/39/100/101, MYB10/72, IRT1 and FRO2. These data also suggest that bHLH34 and bHLH104 positively regulate Fe deficiency response.Yeast one-hybrid assay indicated that bHLH34 and bHLH104 can activate the promoter of bHLH101, which suggest that bHLHl 01 is one direct target gene of bHLH34 and bHLH104. Considering the fact that bHLH38/39/100 are homologous genes of bHLH101, we proposed that bHLH38/39/100 are also the target genes of bHLH34 and bHLH104. To confirm this hypothesis, we designed a reporter-effector transient expression assay system. The promoter of bHLH38/39/100/101 was fused with the GFP reporter gene containing a nuclear localization sequence (NLS) as a reporter expression cassette. For the construction of effectors, bHLH34 and bHLHl 04 were fused with the CaMV 35S promoter. Co-expression assays indicated that bHLH34 and bHLH104 specifically activate the expression of bHLH101. Similarly, we also found that bHLH34 and bHLH104 specifically activate the expression of bHLH38/39/100, but not FIT. These data suggest that bHLH38/39/100/101 are the target genes of bHLH34 and bHLH104. When the Pro35S:bHLH101 was introduced into the bhlh34bhlh104 mutants, the Fe-deficient sympotoms of bhlh34bhlh104 mutants were dramatically relieved. Gene expression analysis confirmed that IRT1 and FRO2 were elevated in the transgenic plants compared with the bhlh34bhlh104 mutants. These data reveal that the overexpression of bHLH101 partially rescues the bhlh34bhlh104 mutants.Yeast-two-hybrid assays, bimolecular fluorescence complementation assays and co-immunoprecipitation assays confirmed that bHLH34 can physically interact with bHLH104. Further yeast-two-hybrid assays confirmed that each of bHLH34, bHLH104, bHLH105 can interact with itself and the other two proteins, implying that they function as homodimers or heterodimers. To further investigate their genetic interactions, we further produced two double mutants, bhlh34bhlh105 and bhlh104bhlh105. We found that under Fe deficiency conditions bhlh34bhlh104, bhlh34bhlh105 and bhlh104bhlhl105 mutants displayed the enhanced Fe-deficiency symptoms compared with the three single mutants.These data suggest that bHLH34, bHLH104 and bHLH105 play non-redundant but additive roles in modulating Fe homeostasis. We also observed differential expression patterns in roots and hypocotyls among ProbHLH34:GUS, ProbHLH104:GUS and ProbHLH105:GUS, which might explain their non-redundant roles.This work reveals that both bHLH34 and bHLH104 positively regulate Fe homeostasis by affecting the expression of bHLH38/39/100/101, and the elevated expression of bHLH34 and bHLH104 can increase the Fe content of plants, which provide a novel strategy directed at increasing Fe content in the edible portions of crops.