Conserved pleiotropy of an ancient plant homeobox gene uncovered by cis-regulatory dissection

Divergence of gene function is a hallmark of evolution, but assessing functional divergence over deep time is not trivial. The few alleles available for cross-species studies often fail to expose the entire functional spectrum of genes, potentially obscuring deeply conserved pleiotropic roles. Here, we explore the functional divergence of WUSCHEL HOMEOBOX9 (WOX9), suggested to have species-specific roles in embryo and inflorescence development. Using a cis-regulatory editing drive system, we generated a comprehensive allelic series in tomato, which revealed hidden pleiotropic roles for WOX9. Analysis of accessible chromatin and conserved cis-regulatory sequences identified the regions responsible for this pleiotropic activity, the functions of which were conserved in groundcherry, a tomato relative. Mimicking these alleles in arabidopsis, distantly related to tomato and groundcherry, revealed new inflorescence phenotypes, exposing a deeply conserved pleiotropy. We suggest that targeted cis-regulatory mutations can uncover conserved gene functions and reduce undesirable effects in crop improvement. Six replicate of ATAC-seq data from anantha inflorescences (meristematic enriched tissue) were generated using Illumina MiSeq