The hormone auxin controls many aspects of the plant life cycle by regulating the expression of thousands of genes. The transcriptional output of the nuclear auxin signaling pathway is determined by the activity of AUXIN RESPONSE transcription FACTORs (ARFs), through their binding to cis-regulatory elements in auxin-responsive genes. Crystal structures, in vitro, and heterologous studies have fueled a model in which ARF dimers bind with high affinity to distinctly spaced repeats of canonical AuxRE motifs. However, the relevance of this "caliper" model, and the mechanisms underlying the binding affinities in vivo, have remained elusive. Here we biochemically and functionally interrogate modes of ARF–DNA interaction. We show that a single additional hydrogen bond in Arabidopsis ARF1 confers high-affinity binding to individual DNA sites. We demonstrate the importance of AuxRE cooperativity within repeats in the Arabidopsis TMO5 and IAA11 promoters in vivo. Meta-analysis of transcriptomes further reveals strong genome-wide association of auxin response with both inverted (IR) and direct (DR) AuxRE repeats, which we experimentally validated. The association of these elements with auxin-induced up-regulation (DR and IR) or down-regulation (IR) was correlated with differential binding affinities of A-class and B-class ARFs, respectively, suggesting a mechanistic basis for the distinct activity of these repeats. Our results support the relevance of high-affinity binding of ARF transcription factors to uniquely spaced DNA elements in vivo, and suggest that differential binding affinities of ARF subfamilies underlie diversity in cis-element function.

生长素激素通过调节数千种基因的表达来控制植物生命周期的许多方面。生长素信号转导途径的转录输出是由AUXIN RESPONSE转录因子（ARF）的活性决定的，通过其与生长素响应基因中的顺式调节元件的结合来实现。晶体结构，体外和异源研究促进了一个模型，在该模型中，ARF二聚体以高亲和力结合到规范的AuxRE图案的不同间隔重复序列上。但是，这种“卡尺”模型的相关性以及体内结合亲和力的潜在机制仍然难以捉摸。在这里，我们对ARF-DNA相互作用进行生化和功能研究。我们表明拟南芥中有一个额外的氢键ARF1赋予与单个DNA位点的高亲和力结合。我们证明了拟南芥TMO5和IAA11重复中AuxRE合作的重要性。体内启动子。转录组的荟萃分析进一步揭示了生长素应答与反向（IR）和直接（DR）AuxRE重复序列在全基因组范围内的强烈关联，我们通过实验进行了验证。这些元素与生长素诱导的上调（DR和IR）或下调（IR）的关联分别与A类和B类ARF的差异结合亲和力相关，这为不同活性的机制基础提供了依据这些重复。我们的结果支持体内ARF转录因子与独特间隔的DNA元件高亲和力结合的相关性，并表明ARF亚家族的不同结合亲和力是顺式元件功能多样性的基础。