<i>ENO</i> regulates tomato fruit size through the floral meristem development network

Abstract

A dramatic evolution of fruit size has accompanied the domestication and improvement of fruit-bearing crop species. In tomato (<i>Solanum lycopersicum</i>), naturally occurring cis-regulatory mutations in the genes of the CLAVATA-WUSCHEL signaling pathway have led to a significant increase in fruit size generating enlarged meristems that lead to flowers with extra organs and bigger fruits. In this work, by combining mapping-by-sequencing and CRISPR/Cas9 genome editing methods, we isolated <i>EXCESSIVE NUMBER OF FLORAL ORGANS</i> (<i>ENO</i>), an AP2/ERF transcription factor which regulates floral meristem activity. Thus, the <i>ENO</i> gene mutation gives rise to plants that yield larger multilocular fruits due to an increased size of the floral meristem. Genetic analyses indicate that <i>eno</i> exhibits synergistic effects with mutations at the <i>LOCULE NUMBER</i> (encoding <i>SlWUS</i>) and <i>FASCIATED</i> (encoding <i>SlCLV3</i>) loci, two central players in the evolution of fruit size in the domestication of cultivated tomatoes. Our findings reveal that an <i>eno</i> mutation causes a substantial expansion of <i>SlWUS</i> expression domains in a flower-specific manner. In vitro binding results show that ENO is able to interact with the GGC-box cis-regulatory element within the <i>SlWUS</i> promoter region, suggesting that ENO directly regulates <i>SlWUS</i> expression domains to maintain floral stem-cell homeostasis. Furthermore, the study of natural allelic variation of the <i>ENO</i> locus proved that a cis-regulatory mutation in the promoter of <i>ENO</i> had been targeted by positive selection during the domestication process, setting up the background for significant increases in fruit locule number and fruit size in modern tomatoes.