ENO 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 (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.

Keywords: AP2/ERF transcription factor; CLAVATA-WUSCHEL regulatory network; Solanum lycopersicum; floral meristem; fruit size.

Fig. 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].)

Fig. 2.

Characterization of CRISPR/Cas9-eno (CR-eno) lines. (A) Schematic illustrating single guide RNA targeting the ENO coding sequence (red arrow). Blue arrows indicate the PCR primers used to evaluate mutation type and efficiency. (B) CR-eno alleles identified by cloning and sequencing PCR products from the ENO targeted region from five T0 plants. Blue dashed lines indicate InDel mutations and black bold and underlined letters indicate protospacer-adjacent motif (PAM) sequences. (C) Quantification and statistical comparisons of floral organ number from wild-type (WT; cv. P73) and CR-eno flowers. Data were collected from five independent T₀ lines. Data are means ± SDs; n = 10 flowers per plant. A two-tailed, two-sample Student’s t test was performed, and significant differences are represented by asterisks: ns, no statistically significant differences; ***P < 0.0001. (D) Representative flower from CRISPR/Cas9-eno (CR-eno) lines compared with wild-type (WT) one. (Scale bar, 1 cm.)

Fig. 3.

Representative floral meristems, flowers, and fruits from the different allelic combinations of ENO, FAS, and LC loci. (A) ENO:FAS:LC. (B) ENO:FAS:lc. (C) eno:FAS:LC. (D) ENO:fas:LC. (E) eno:FAS:lc. (F) ENO:fas:lc. (G) eno:fas:LC. (H) eno:fas:lc. Se, sepals; Pe, petals; Sta, stamens; and Ca, carpels. Note: Sepals were removed in images of floral meristems. Number of petals and sepals are specified, and arrowheads indicate locules. (Scale bars, 200 μm [floral meristems] and 1 cm [flowers and fruits].) Number of sepals (I), petals (J), stamens (K), carpels (L), and fruit locules (M) in wild-type plants (gray) and single (blue), double (yellow), and triple (red) mutant lines for eno, fas, and lc alleles. For each genotype, 10 plants were phenotyped for 10 flowers and 10 fruits (100 measurements). Values are expressed as the mean ± SD. Significant differences were calculated by pairwise comparisons of means using the least significant difference test. Values followed by the same letter (a, b, c, d, e, or f) are not statistically different (P < 0.05).

Fig. 4.

Dynamic expression of ENO. (A) qRT-PCR for ENO transcripts in different developmental tissues and stages. Expression was compared to that of the control UBIQUTIN gene. SAM, shoot apical meristem; RM, reproductive meristem; FB0, floral bud of 3.0 to 5.9 mm in length; FB1, floral bud of 6.0 to 8.9 mm in length; FB2, floral bud of 9.0 to 12 mm in length; PA, flower at preanthesis stage; A, flower at anthesis stage; GF, green fruit; BF, breaker fruit; MF, mature fruit. (B) Reads per kilobase per million reads (RPKM) values for ENO across vegetative and reproductive meristem stages: EVM, early vegetative meristem; MVM, middle vegetative meristem; LVM, late vegetative meristem; TM, transition meristem; FM, floral meristem; SIM, sympodial inflorescence meristem; SYM, sympodial meristem. Data were obtained from the tomato meristem maturation atlas (17). (C–E) In situ mRNA hybridization of ENO in vegetative and reproductive meristems of wild-type plants. (Scale bars, 100 µm.)

Fig. 5.

ENO is involved in the transcriptional regulatory network that regulates floral meristem size. (A) GO terms enriched among significantly differentially expressed genes between wild-type and eno mutant reproductive meristems using agriGO v2.0 software. A FDR < 0.05 with the Fisher statistical test and the Bonferroni multitest adjustment was used to determined enriched GO terms. (B) RPKM values for SlWUS and SlCLV3 in wild-type (WT) and eno mutant. Genes with an FDR adjusted P value (Padj) < 0.05 were defined as significantly differentially expressed. (C–J) In situ mRNA hybridization of SlWUS (C–F) and SlCLV3 (G–J) in shoot apical and floral meristems of wild-type and eno plants. (Scale bars, 100 µm.) (K and L) EMSA of ENO protein revealing binding to the SlWUS promoter. Biotinylated probe containing the theoretical ERF binding site (GCCGTC, located at −9,326 bp relative to the translational start site) on the SlWUS promoter (K) incubated with purified ENO protein (L). Black triangle in L indicates the increasing amounts (100 and 1,000) of unlabeled probe used for competition. The specific complex formed is indicated by red arrow.

Fig. 6.

Natural allelic variation of ENO locus causes phenotypic variation in fruit locule number. (A) Multiple sequence alignment of ENO haplotypes identified in a set of 103 accessions producing fruits of different sizes, comprising of 92 S. lycopersicum, 7 S. lycopersicum var. cerasiforme, and 4 S. pimpinellifolium accessions. The ENO coding sequence is marked in pink. (B) Polymorphisms and deduced amino acid substitutions identified in the ENO coding sequence. (C) Functional effect of ENO promoter deletion allele on fruit locule number on the basis of genotypic information for LC and FAS loci. Fruits of S. pimpinellifolium accessions with the ENO wild allele (D) or the ENO promoter deletion allele (E). (Scale bars, 1 cm.) (F) Allele-specific ENO expression (copy number/μL) determined by TaqMan probe using the ddPCR assay. A two-tailed, two-sample Student’s t test was performed to determine significant differences between genotypes. (G) Frequencies of the ENO promoter, lc, and fas mutant alleles in phylogenetic groups representing sequential domestication steps as defined in Blanca et al. (30). Distant wild: wild tomato species; Spim: wild ancestor S. pimpinellifolium accessions; Slyc cer Andean: Andean accessions of S. lycopersicum var. cerasiforme; Slyc Vintage: S. lycopersicum Vintage varieties; Slyc Fresh: S. lycopersicum accessions for fresh market; Slyc Processing: S. lycopersicum accessions for industrial processing.