Malformed fruits depreciate a plant's market value. In tomato (Solanum lycopersicum), fruit malformation is associated with the multi‐locule trait, which involves genes regulating shoot apical meristem (SAM) development. The expression pattern of TOPLESS3 (SlTPL3) throughout SAM development prompted us to investigate its functional significance via RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats/CRISPR‐associated nuclease 9 (Cas9)‐mediated gene editing. Lower SlTPL3 transcript levels resulted in larger fruits with more locules and larger SAMs at the 5 d after germination (DAG5) stage. Differentially expressed genes in the SAM of wild‐type (WT) and SlTPL3‐RNAi plants, identified by transcriptome deep sequencing (RNA‐seq), were enriched in the gibberellin (GA) biosynthesis and plant hormone signaling pathways. Moreover, exogenous auxin and paclobutrazol treatments rescued the multi‐locule phenotype, indicating that SlTPL3 affects SAM size by mediating auxin and GA levels in the SAM. Furthermore, SlTPL3 interacted with WUSCHEL (SlWUS), which plays an important role in SAM size maintenance. We conducted RNA‐seq and DNA affinity purification followed by sequencing (DAP‐seq) analyses to identify the genes regulated by SlTPL3 and SlWUS in the SAM and to determine how they regulate SAM size. We detected 24 overlapping genes regulated by SlTPL3 and SlWUS and harboring an SlWUS‐binding motif in their promoters. Furthermore, functional annotation revealed a notable enrichment for functions in auxin transport, auxin signal transduction, and GA biosynthesis. Dual‐luciferase assays also revealed that SlTPL3 enhances SlWUS‐mediated regulation (repression and activation) of SlPIN3 and SlGA2ox4 transcription, indicating that the SlTPL3–SlWUS module regulates SAM size by mediating auxin distribution and GA levels, and perturbations of this module result in enlarged SAM. These results provide novel insights into the molecular mechanism of SAM maintenance and locule formation in tomato and highlight the SlTPL3–SlWUS module as a key regulator.

Glycoside hydrolases (GHs), enzymes that break down glycosidic bonds in carbohydrates and between carbohydrates and non-carbohydrates, are prevalent in plants, animals, microorganisms, and other organisms. The tomato is a significant crop that contains the GH17 gene family. However, its role in tomatoes has yet to be fully investigated. In this study, we identified 43 GH17 genes from the tomato genome, distributed unevenly across 12 chromosomes. We further analyzed their gene structure, phylogenetic relationships, promoter elements, and expression patterns. The promoter element analysis indicated their potential roles in response to biotic and abiotic stresses as well as phytohormone effects on growth and development. The expression studies across different tomato tissues revealed that 10 genes were specifically expressed in floral organs, with SlA6 prominently expressed early during bud formation. By using CRISPR/Cas9 gene-editing technology, SlA6 knockout plants were generated. Phenotypic characterization showed that pollen viability, pollen tube germination, fruit weight, and seed number were significantly reduced in the Sla6 mutant, but the soluble solids content (TSS) was significantly higher in the Sla6 mutant, suggesting that SlA6 affects pollen development and fruit quality.