Diversity through repression

Researchers have uncovered how enhancer evolution contributed to differences in leaf shape in plants

To the Point

• Role of Repression: The study highlights that repressive elements in DNA play a significant role in creating evolutionary diversity, challenging the notion that repression merely silences gene activity.
• Gene Duplication Mechanism: The discovered repressive sequence emerged from a duplication within the RCO enhancer, demonstrating how nested duplications can drive regulatory and morphological innovations.
• Stability of Expression: RCO gene expression showed greater robustness to mutations compared to the ancestral LMI1 gene, suggesting that this new expression pattern could stabilize over time.
• Significance of Findings: These insights emphasize the creative potential of repressive regulatory elements and their impact on developmental outcomes in plant evolution.

For over two decades, scientists have known that evolutionary changes in DNA sequences controlling gene expression are a central driver of morphological diversity. Among these regulatory elements, enhancers play a crucial role — acting as genetic switches that determine when, where, and how strongly genes are expressed. While previous work has shown that modifications in enhancer activity can influence an organism’s form, the exact DNA changes and mechanisms that generate new patterns of gene expression are not well understood.

Using the crucifer Cardamine hirsuta, a close relative of Arabidopsis thaliana and a powerful model for studying the evolution of form, researchers at the Max Planck Institute for Plant Breeding Research uncovered how enhancer evolution contributed to differences in leaf shape within this plant family. By applying CRISPR/Cas9 genome editing to generate 17 different versions of enhancers controlling expression of the RCO (REDUCED COMPLEXITY) homeobox gene — and its related gene LMI1, from which RCO evolved through gene duplication — the team identified a repressive DNA sequence whose evolution helped generate a new domain of RCO gene expression relative to LMI1. This novel expression pattern drove the emergence of more complex leaf shapes in Cardamine species. 

Repressive elements can be just as important as activating ones

“While previous studies for example in fly wings have focused on how the gain of activating sequences can produce new traits, our work shows that repressive elements can be just as important in creating evolutionary diversity,” said Alessandro Popoli, first author of the study. “Repression is often viewed as simply silencing, but here we see it as a creative force — one that helped shape a new pattern of gene activity and, ultimately, a new form.”

The team further discovered that this repressive sequence arose through a duplication within the RCO enhancer, itself part of a larger gene duplication event. This “duplication within duplication” mechanism illustrates how nested sequence duplications can fuel regulatory and morphological innovation. In addition, the researchers found that RCO gene expression is more robust to mutations than its ancestral counterpart LMI1, potentially allowing this novel expression pattern to stabilize over evolutionary time.

“These results highlight the creative potential of repressive regulatory elements and provide an important example of how enhancer diversification can lead to new developmental outcomes,” said Miltos Tsiantis, senior author of the study and Director at MPIPZ. “It’s an exciting step toward understanding how small DNA changes can give rise to diversity in organismal form.”