State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China.
Mol Plant. 2013 Mar;6(2):503-13. doi: 10.1093/mp/sss089. Epub 2012 Sep 14.
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.
拟南芥 Ib 亚家族 bHLH 基因家族包含四个成员（AtbHLH38、AtbHLH39、AtbHLH100 和 AtbHLH101）。AtbHLH38 和 AtbHLH39 先前被证实与 FER-样缺铁诱导转录因子（FIT）相互作用，直接作用于激活铁螯合还原酶 FRO2 和高亲和力亚铁转运蛋白 IRT1 的表达。在这项工作中，我们研究了 AtbHLH100 和 AtbHLH101 在缺铁反应和摄取中的功能。酵母双杂交分析和双分子荧光互补测定表明，AtbHLH100 和 AtbHLH101 都可以与 FIT 相互作用。在酵母细胞中，AtbHLH100 或 AtbHLH101 与 FIT 的双重表达激活了由 FRO2 和 IRT1 启动子驱动的 GUS 表达。过表达 FIT 与 AtbHLH101 的植物在根部表现出 FRO2 和 IRT1 的组成型表达，并在地上部分积累更多的铁。此外，还生成并表征了 AtbHLH38、AtbHLH39、AtbHLH100 和 AtbHLH101 的单、双和三缺失突变体。在 AtbHLH39、AtbHLH100 和 AtbHLH101 的三缺失突变体中，根部的 FRO2 和 IRT1 表达以及地上部分的铁含量比其他四个基因的双缺失和三缺失突变体降低得更明显。在缺铁条件下，对多个缺失突变体的生理反应以及 FRO2 和 IRT1 的表达进行比较表明，AtbHLH100、AtbHLH38、AtbHLH101 和 AtbHLH39 在缺铁反应和摄取中起着逐渐增加的重要作用。总的来说，我们得出结论，这四个 Ib 亚家族 bHLH 蛋白是必需的，并具有冗余功能，对拟南芥缺铁反应和摄取的激活具有不同的意义。
