Exploring the influence of a single-nucleotide mutation in EIN4 on tomato fruit firmness diversity through fruit pericarp microstructure

Tomato (Solanum lycopersicum) stands as one of the most valuable vegetable crops globally, and fruit firmness significantly impacts storage and transportation. To identify genes governing tomato firmness, we scrutinized the firmness of 266 accessions from core collections. Our study pinpointed an ethylene receptor gene, SlEIN4, located on chromosome 4 through a genome-wide association study (GWAS) of fruit firmness in the 266 tomato core accessions. A single-nucleotide polymorphism (SNP) (A -> G) of SlEIN4 distinguished lower (AA) and higher (GG) fruit firmness genotypes. Through experiments, we observed that overexpression of SlEIN4(AA) significantly delayed tomato fruit ripening and dramatically reduced fruit firmness at the red ripe stage compared with the control. Conversely, gene editing of SlEIN4(AA) with CRISPR/Cas9 notably accelerated fruit ripening and significantly increased fruit firmness at the red ripe stage compared with the control. Further investigations revealed that fruit firmness is associated with alterations in the microstructure of the fruit pericarp. Additionally, SlEIN4(AA) positively regulates pectinase activity. The transient transformation assay verified that the SNP (A -> G) on SlEIN4 caused different genetic effects, as overexpression of SlEIN4(GG) increased fruit firmness. Moreover, SlEIN4 exerts a negative regulatory role in tomato ripening by impacting ethylene evolution through the abundant expression of ethylene pathway regulatory genes. This study presents the first evidence of the role of ethylene receptor genes in regulating fruit firmness. These significant findings will facilitate the effective utilization of firmness and ripening traits in tomato improvement, offering promising opportunities for enhancing tomato storage and transportation capabilities.