ENO Regulates Tomato Fruit Size Through the Floral Meristem Development Network
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ENO regulates tomato fruit size through the floral meristem development network Fernando J. Yuste-Lisbonaa,b,1, Antonia Fernández-Lozanoa,1, Benito Pinedac, Sandra Bretonesa, Ana Ortíz-Atienzaa, Begoña García-Sogoc, Niels A. Müllerb,d, Trinidad Angostoa, Juan Capela, Vicente Morenoc, José M. Jiménez-Gómezb,e, and Rafael Lozanoa,2 aCentro de Investigación en Biotecnología Agroalimentaria,Universidad de Almería, 04120 Almería, Spain; bDepartment of Plant Breeding and Genetics, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany; cInstituto de Biología Molecular y Celular de Plantas, Universitat Politècnica de València–Consejo Superior de Investigaciones Científicas, 46022 Valencia, Spain; dGenome Research Group, Thünen Institute of Forest Genetics, 22927 Grosshansdorf, Germany; and eInstitut Jean-Pierre Bourgin, Institut National de la Recherche Agronomique (INRA), AgroParisTech, CNRS, UniversitéParis- Saclay, 78026 Versailles, France Edited by James J. Giovannoni, US Department of Agriculture, Ithaca, NY, and approved February 6, 2020 (received for review August 8, 2019) A dramatic evolution of fruit size has accompanied the domesti- Extreme fruit size in tomato (Solanum lycopersicum), which cation and improvement of fruit-bearing crop species. In tomato evolved from the small fruited wild ancestor S. pimpinellifolium, (Solanum lycopersicum), naturally occurring cis-regulatory muta- is determined mainly by the number of carpels in a flower and, tions in the genes of the CLAVATA-WUSCHEL signaling pathway hence, by the final number of locules (seed compartments) have led to a significant increase in fruit size generating enlarged forming the mature fruit (9, 10). During tomato breeding, the joint meristems that lead to flowers with extra organs and bigger fruits. action of fasciated (fas)andlocule number (lc) mutations allowed In this work, by combining mapping-by-sequencing and CRISPR/ for the development of large-fruited cultivars bearing more than EXCESSIVE NUMBER Cas9 genome editing methods, we isolated eight locules, in contrast with the bilocular fruits of tomato wild OF FLORAL ORGANS ENO ( ), an AP2/ERF transcription factor which species and most small-fruited varieties (10, 11). The fas mutation ENO regulates floral meristem activity. Thus, the gene mutation is caused by a 294-kb inversion disrupting the tomato CLV3 gives rise to plants that yield larger multilocular fruits due to an (SlCLV3) promoter (2), whereas lc is associated with two single- increased size of the floral meristem. Genetic analyses indicate nucleotide polymorphisms (SNPs) in a putative CArG box regu- that eno exhibits synergistic effects with mutations at the LOCULE PLANT BIOLOGY latory element downstream of the tomato WUS (SlWUS) (12, 13). NUMBER (encoding SlWUS) and FASCIATED (encoding SlCLV3) loci, two central players in the evolution of fruit size in the domestica- The fas and lc mutations are partial loss-of-function and gain-of- tion of cultivated tomatoes. Our findings reveal that an eno mu- function alleles, respectively, and both mutations positively affect tation causes a substantial expansion of SlWUS expression domains the FM size (14). A tomato mutant, excessive number of floral in a flower-specific manner. In vitro binding results show that ENO is organs (eno), was recently reported to show alterations in FM size able to interact with the GGC-box cis-regulatory element within the leading to the development of flowers with supernumerary organs SlWUS promoter region, suggesting that ENO directly regulates SlWUS expression domains to maintain floral stem-cell homeostasis. Significance Furthermore, the study of natural allelic variation of the ENO locus proved that a cis-regulatory mutation in the promoter of ENO had Fruit-size increase is one of the major changes associated with been targeted by positive selection during the domestication pro- tomato domestication, and it currently represents an important cess, setting up the background for significant increases in fruit objective for breeding. Regulatory mutations at the LOCULE locule number and fruit size in modern tomatoes. NUMBER and FASCIATED loci, the orthologues of the Arabi- dopsis WUSCHEL and CLAVATA3, have mainly contributed to Solanum lycopersicum | fruit size | floral meristem | CLAVATA-WUSCHEL enlarging fruit size by altering meristem activity. Here, we regulatory network | AP2/ERF transcription factor identify ENO as a tomato fruit regulator, which may function by regulating WUSCHEL gene expression to restrict stem-cell uring the domestication process, fruit-bearing crop species proliferation in a flower-specific manner. Our findings also Dhave largely increased their fruit size compared with those show that a mutation in the ENO promoter was selected during normally found in progenitor wild species. Accordingly, a large domestication to establish the background for enhancing fruit rise in fruit size has been achieved through breeding to increase size in cultivated tomatoes, denoting that transcriptional changes the final size of floral meristems (FM) in crops such as tomato or in key regulators have significant effects on agronomic traits. maize (1–3). Modification of the CLAVATA (CLV)-WUSCHEL Author contributions: F.J.Y.-L. and R.L. designed research; F.J.Y.-L., A.F.-L., B.P., S.B., (WUS) negative feedback loop has led to this increase in meristem A.O.-A., and B.G.-S. performed research; F.J.Y.-L., A.F.-L., N.A.M., T.A., J.C., V.M., size. The homeodomain transcription factor WUS specifies stem- J.M.J.-G., and R.L. contributed new reagents/analytic tools; F.J.Y.-L., A.F.-L., B.P., S.B., cell fate and promotes CLV3 expression, which is a peptide ligand A.O.-A., N.A.M., T.A., J.C., V.M., J.M.J.-G., and R.L. analyzed data; and F.J.Y.-L., A.F.-L., that binds to different plasma membrane-localized receptor and R.L. wrote the paper. complexes to initiate a signaling cascade that subsequently re- The authors declare no competing interest. presses WUS activity (4, 5). The core signaling module of the This article is a PNAS Direct Submission. CLV-WUS feedback loop is deeply conserved in diverse plants This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY). such as Arabidopsis, tomato, and maize, while dosage compensa- Data deposition: The sequencing datasets for this study have been deposited in the tion mechanisms that operate to buffer stem-cell homeostasis in National Center for Biotechnology Information Short Read Archive under the BioProject diverse lineages have diversified (6). In this way, mutations in the accession codes PRJNA503558 and PRJNA495568. CLV-WUS circuit have played a relevant role in crop yield im- 1F.J.Y.-L. and A.F.-L. contributed equally to this work. provement of both dicots and monocots (5, 7). Thus, in tomato, 2To whom correspondence may be addressed. Email: [email protected]. mutations in the CLV3 signal peptide promote stem-cell over- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ proliferation resulting in the development of extra organs in doi:10.1073/pnas.1913688117/-/DCSupplemental. flowers and bigger fruits (8). First published March 16, 2020. www.pnas.org/cgi/doi/10.1073/pnas.1913688117 PNAS | April 7, 2020 | vol. 117 | no. 14 | 8187–8195 Downloaded by guest on September 23, 2021 and the formation of larger multilocular fruits (15). In this study, knockout mutations by using the CRISPR/Cas9 system with a ENO was identified as a member of the APETALA2/Ethylene single guide RNA (Fig. 2A) in the cultivar P73 genetic back- Responsive Factor (AP2/ERF) superfamily of transcription fac- ground. We evaluated five independent first-generation (T0) tors. Our findings suggest that ENO regulates SlWUS expression diploid lines (CR-eno) that were homozygous or biallelic for to restrict stem-cell proliferation in a flower-specific manner. edited mutant alleles (Fig. 2B). In all cases, CR-eno lines yield Moreover, the analysis of genetic variation in tomato germplasm fasciated flowers and fruits resembling the phenotype observed has shown that ENO played an important role in the increase of in eno mutants (Fig. 2 C and D and SI Appendix, Table S3). fruit size during tomato domestication. Hence, our results revealed that mutations in Solyc03g117230 (hereafter referred to as ENO) in combination with lc are re- Results sponsible for the fasciation observed in flowers and fruits de- Eno Mutation Affects FM Size, Giving Rise to Plants with Higher Yield. veloped by eno mutant plants. Previously, we reported that eno mutant plants developed an increased number of floral organs and multilocular fruits (Fig. 1 eno, lc, and fas Loci Exhibit Synergistic Effects. To determine the A–D) (15), a phenotype reminiscent of the CLV gene mutants, phenotypic effect of eno locus in a wild-type LC background, the shoot apical meristems (SAMs) of which are enlarged (2). allele-specific markers for the ENO and LC loci were evaluated Based on this evidence, we examined SAM size at the transition in the interspecific eno × LA1589 F2 mapping population. Thus, from vegetative to reproductive growth. eno plants showed plants bearing single lc or eno mutations showed an increased slightly wider and shorter SAM than the wild type (Fig. 1 E–G), number of locules with respect to wild-type ones, whereas a in contrast to the 1.8-fold increase in the size of FM previously significant nonadditive increase in the number of locules (de- detected in the mutant from petal initiation and stamen pri- termined by a two-way ANOVA; P = 0.004) was observed in mordia onward (15). Consistently with this, the increased floral plants carrying both the eno and lc mutations (Fig. 1K). The organ number of eno is more evident in the three innermost effect of eno on locule number was additionally confirmed by an whorls than in the outermost one (SI Appendix, Table S1). As a RNA interference (RNAi)-mediated knockdown of ENO in S.