Evolution of repressive sequences within an enhancer contributed to morphological diversity in crucifer plants.

Abstract

<i>Cis</i>-regulatory sequences, such as enhancers, play a crucial role in morphological evolution, but how their diversification leads to evolution of novel expression patterns that underpin diversity is still poorly understood. Recent work suggests that the function of enhancers is influenced by their genomic sequence context more than previously thought, further complicating our understanding of their contribution to diversity. To address these issues, we investigated the regulation of the <i>RCO</i> homeobox gene, which contributed to the evolution of complex leaves following its tandem duplication from <i>LMI1</i>. By characterizing seventeen regulatory alleles at the <i>LMI1</i>/<i>RCO</i> locus that we generated by genome editing, we found that the evolved <i>RCO</i> enhancer is subject to pronounced negative regulation that helps delimit the <i>RCO</i> expression domain. While the <i>LMI1</i> enhancer was strictly required for <i>LMI1</i> gene expression, the same was not true for the <i>RCO</i> enhancer, which caused only partial loss of function when deleted. We mapped both positively and negatively acting sequences within the <i>RCO</i> enhancer, validated them in reporter gene assays and showed that a repressive sequence arose in association with a nested duplication within the evolved <i>RCO</i> enhancer. This repressive sequence played a key role in shaping the specific <i>RCO</i> expression domain that underlies its role in leaf complexity, and we provide evidence that it helped to prevent potential pleiotropic effects arising from evolutionary diversification or <i>RCO</i> expression. Our findings highlight the regulatory features of a diversity-linked enhancer and show that the evolution of repressive sequences is a powerful force in regulatory evolution.