Regulatory mutations at the LOCULE NUMBER and FASCIATED loci, the orthologues of the Arabidopsis WUSCHEL and CLAVATA3, have mainly contributed to enlarging fruit size by altering meristem activity. Here, we identify ENO as a tomato fruit regulator, which may function by regulating WUSCHEL gene expression to restrict stem-cell proliferation in a flower-specific manner. Our findings also show that a mutation in the ENO promoter was selected during domestication to establish the background for enhancing fruit size in cultivated tomatoes, denoting that transcriptional changes in key regulators have significant effects on agronomic traits.
A dramatic evolution of fruit size has accompanied the domestication and improvement of fruit-bearing crop species. In tomato (Solanum lycopersicum), 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 EXCESSIVE NUMBER OF FLORAL ORGANS (ENO), an AP2/ERF transcription factor which regulates floral meristem activity. Thus, the ENO gene mutation gives rise to plants that yield larger multilocular fruits due to an increased size of the floral meristem. Genetic analyses indicate that eno exhibits synergistic effects with mutations at the LOCULE NUMBER (encoding SlWUS) and FASCIATED (encoding SlCLV3) loci, two central players in the evolution of fruit size in the domestication of cultivated tomatoes. Our findings reveal that an eno mutation causes a substantial expansion of SlWUS 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 SlWUS promoter region, suggesting that ENO directly regulates SlWUS expression domains to maintain floral stem-cell homeostasis. Furthermore, the study of natural allelic variation of the ENO locus proved that a cis-regulatory mutation in the promoter of ENO 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.
Figure 1: Characterization and cloning of the eno mutant. Representative flower (A and B) and fruit (C and D) of wild-type (WT) and eno mutant plants. Images of the SAM from WT (E) and eno (F) plants at the transition meristem stage, before forming the first floral bud (L7, leaf 7). (G) Quantification of SAM size from WT and eno plants. (H) Yield performance of WT and eno plants. (I) Distribution of the average allele frequency of WT (blue line) and eno (red line) pools grouped by chromosomes. (J) Positional cloning of the ENO gene (coding and UTRs in dark and light gray, respectively). The SNP mutation in the start codon of the ENO gene is marked in red, and the SNP and the InDel localized in its 5′ UTR region are shown in blue. (K) Number of locules for each genotyped class identified in the interspecific eno × LA1589 (S. pimpinellifolium) F2 mapping population. (L) RNAi-mediated knockdown of ENO gene in S. pimpinellifolium (accession LA1589). Data are means ± SD; n = 20 (G, H, and K). A two-tailed, two-sample Student’s t test was performed, and significant differences are represented by asterisks: ***P < 0.0001; *P < 0.01. ns, no statistically significant differences. (Scale bars, 1 cm [A–D and L] and 200 μm [E and F].)