Non-coding cis-regulatory variants in animal genomes are an important driving force in the evolution of transcription regulation and phenotype diversity. However, cistrome dynamics in plants remain largely underexplored. Here, we compare the binding of GOLDEN2-LIKE (GLK) transcription factors in tomato, tobacco, Arabidopsis, maize and rice. Although the function of GLKs is conserved, most of their binding sites are species-specific. Conserved binding sites are often found near photosynthetic genes dependent on GLK for expression, but sites near non-differentially expressed genes in the glk mutant are nevertheless under purifying selection. The binding sites’ regulatory potential can be predicted by machine learning model using quantitative genome features and TF co-binding information. Our study show that genome cis-variation caused wide-spread TF binding divergence, and most of the TF binding sites are genetically redundant. This poses a major challenge for interpreting the effect of individual sites and highlights the importance of quantitatively measuring TF occupancy.

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