Auxin is a key hormone performing a wealth of functions throughout the plant life cycle. It acts largely by regulating genes at the transcriptional level through a family of transcription factors (TF) called auxin response factors (ARF). Even if all ARF monomers analysed so far bind a similar DNA sequence, there is evidence that ARFs differ in their target genomic regions and regulated genes. Here we use position weight matrices (PWM) to model ARF DNA binding specificity based on published DNA

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... ffinity purification sequencing (DAP-seq) data. We find that the genome binding of two ARFs (ARF2 and ARF5/Monopteros/MP) differ largely because these two factors have different preferred ARF binding site (ARFbs) arrangements (orientation and spacing). We illustrate why PWMs are more versatile to reliably identify ARFbs than the widely used consensus sequences and demonstrate their power with biochemical experiments on the regulatory regions of the IAA19 model gene. Finally, we combined gene regulation by auxin with ARF-bound regions and identified specific ARFbs configurations that are over-represented in auxin up-regulated genes, thus deciphering the ARFbs syntax functional for regulation. This provides a general method to exploit the potential of genome-wide DNA binding assays and decode gene regulation.