Requirement and Functional Redundancy of Ib Subgroup bHLH Proteins for Iron Deficiency Responses and Uptake in Arabidopsis thaliana

Abstract

The Ib subgroup of the bHLH gene family in Arabidopsis contains four members (AtbHLH38, AtbHLH39, AtbHLH100, and AtbHLH101). AtbHLH38 and AtbHLH39 were previously confirmed to interact with FER-like iron deficiency induced transcription factor (FIT), directly functioning in activation of the expression of ferric-chelate reductase FRO2 and high-affinity ferrous iron transporter IRT1. In this work, we characterized the functions of AtbHLH100 and AtbHLH101 in the regulation of the iron-deficiency responses and uptake. Yeast two-hybrid analysis and bimolecular fluorescence complementation assay demonstrated that both AtbHLH100 and AtbHLH101 could interact with FIT. Dual expression of either AtbHLH100 or AtbHLH101 with FIT in yeast cells activated the GUS expression driven by promoters of FRO2 and IRT1. The plants overexpressing FIT together with AtbHLH101 showed constitutive expression of FRO2 and IRT1 in roots, and accumulated more iron in shoots. Further, the single, double, and triple knockout mutants of AtbHLH38, AtbHLH39, AtbHLH100, and AtbHLH101 were generated and characterized. The FRO2 and IRT1 expression in roots and the iron content in shoots were more drastically decreased in the triple knockout mutant of AtbHLH39, AtbHLH100, and AtbHLH101 than that of the other available double and triple mutants of the four genes. Comparison of the physiological responses as well as the expression of FRO2 and IRT1 in the multiple knockout mutants under iron deficiency revealed that AtbHLH100, AtbHLH38, AtbHLH101, and AtbHLH39 played the gradually increased important role in the iron-deficiency responses and uptake. Taken all together, we conclude that the four Ib subgroup bHLH proteins are required and possess redundant functions with differential significance for activation of iron-deficiency responses and uptake in Arabidopsis.

The Arabidopsis Mediator subunit MED16 regulates iron homeostasis by associating with EIN3/EIL1 through subunit MED25

“…During iron uptake in Arabidopsis, the bHLH transcription factor FIT, the master regulator, forms heterodimers with other bHLH proteins to regulate the ferrous iron transporter IRT1 and the ferric-chelate reductase FRO2 in response to iron deficiency in plants (Robinson et al, 1999;Varotto et al, 2002;Vert et al, 2002;Connolly et al, 2003;Colangelo and Guerinot, 2004;Yuan et al, 2008;Wang et al, 2012). Our data support that FIT, rather than the interacting proteins bHLH38 or bHLLH39, plays a significant role in regulation of iron homeostasis in the yid1/med16 or med25 mutants ( Figure S6), since the induction of the two genes encoding FIT-interacting-protein, bHLH38 and bHLH39, was unaffected under iron-deficient conditions in the yid1/med16 or med25 mutants.…”

“…The role of FIT is in iron homeostasis, regulating the expression of IRT1 and FRO2 in species of Arabidopsis and tomato (Ling et al, 2002;Colangelo and Guerinot, 2004;Jakoby et al, 2004;Yuan et al, 2005). Yuan et al (2008) and Wang et al (2012) proposed that FIT forms a complex with other iron-deficiencyinduced bHLH transcription factors such as bHLH38, bHLH39, bHLH100 and bHLH101. Recently, two transcription factors ethylene-insensitive 3 (EIN3) and ethyleneinsensitive 3-like 1 (EIL1) in the ethylene signaling pathway were shown to be involved in the regulation of iron homeostasis.…”

Similarities and Differences in the Acquisition of Fe and P by Dicot Plants

“…Although ethylene's mode of action is not fully understood, a signalling pathway has been proposed in Arabidopsis which includes the transcription factors EIN3 and EIL1 [71]. Both transcription factors are upregulated under Fe deficiency [29] and are involved in FIT stabilization and activity [72,73].…”

Polarization of IRON-REGULATED TRANSPORTER 1 (IRT1) to the plant-soil interface plays crucial role in metal homeostasis

“…Intricate regulatory networks control plant responses to low iron conditions and, more specifically, IRT1 gene expression. Several transcription factors directly binding to the IRT1 promoter in root epidermal cells have been identified and control its inducibility by low iron conditions (9)(10)(11)(12). Other pathways including the cytokinin-mediated root growth control and the stress hormone ethylene impinge on iron uptake by converging at the level of the IRT1 promoter (13,14).…”