ENO regulates tomato fruit size through the floral meristem development network.

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.

A collection of enhancer trap insertional mutants for functional genomics in tomato.

With the completion of genome sequencing projects, the next challenge is to close the gap between gene annotation and gene functional assignment. Genomic tools to identify gene functions are based on the analysis of phenotypic variations between a wild type and its mutant; hence, mutant collections are a valuable resource. In this sense, T-DNA collections allow for an easy and straightforward identification of the tagged gene, serving as the basis of both forward and reverse genetic strategies. This study reports on the phenotypic and molecular characterization of an enhancer trap T-DNA collection in tomato (Solanum lycopersicum L.), which has been produced by Agrobacterium-mediated transformation using a binary vector bearing a minimal promoter fused to the uidA reporter gene. Two genes have been isolated from different T-DNA mutants, one of these genes codes for a UTP-glucose-1-phosphate uridylyltransferase involved in programmed cell death and leaf development, which means a novel gene function reported in tomato. Together, our results support that enhancer trapping is a powerful tool to identify novel genes and regulatory elements in tomato and that this T-DNA mutant collection represents a highly valuable resource for functional analyses in this fleshy-fruited model species.

Characterization of vegetative inflorescence (mc-vin) mutant provides new insight into the role of MACROCALYX in regulating inflorescence development of tomato.

Inflorescence development is a key factor of plant productivity, as it determines flower number. Therefore, understanding the mechanisms that regulate inflorescence architecture is critical for reproductive success and crop yield. In this study, a new mutant, vegetative inflorescence (mc-vin), was isolated from the screening of a tomato (Solanum lycopersicum L.) T-DNA mutant collection. The mc-vin mutant developed inflorescences that reverted to vegetative growth after forming two to three flowers, indicating that the mutated gene is essential for the maintenance of inflorescence meristem identity. The T-DNA was inserted into the promoter region of the MACROCALYX (MC) gene; this result together with complementation test and expression analyses proved that mc-vin is a new knock-out allele of MC. Double combinations between mc-vin and jointless (j) and single flower truss (sft) inflorescence mutants showed that MC has pleiotropic effects on the reproductive phase, and that it interacts with SFT and J to control floral transition and inflorescence fate in tomato. In addition, MC expression was mis-regulated in j and sft mutants whereas J and SFT were significantly up-regulated in the mc-vin mutant. Together, these results provide new evidences about MC function as part of the genetic network regulating the development of tomato inflorescence meristem.

Mutation at the tomato excessive number of floral organs (ENO) locus impairs floral meristem development, thus promoting an increased number of floral organs and fruit size.

A novel tomato (Solanum lycopersicum L.) mutant affected in reproductive development, excessive number of floral organs (eno), is described in this study. The eno plants yielded flowers with a higher number of floral organs in the three innermost floral whorls and larger fruits than those found in wild-type plants. Scanning-electron microscopy study indicated that the rise in floral organ number and fruit size correlates with an increased size of floral meristem at early developmental stages. It has been reported that mutation at the FASCIATED (FAS) gene causes the development of flowers with supernumerary organs; however, complementation test and genetic mapping analyses proved that ENO is not an allele of the FAS locus. Furthermore, expression of WUSCHEL (SlWUS) and INHIBITOR OF MERISTEM ACTIVITY (IMA), the two main regulators of floral meristem activity in tomato, is altered in eno but not in fas flowers indicating that ENO could exert its function in the floral meristem independently of FAS. Interestingly, the eno mutation delayed the expression of IMA leading to a prolonged expression of SlWUS, which would explain the greater size of floral meristem. Taken together, results showed that ENO plays a significant role in the genetic pathway regulating tomato floral meristem development.