Shiwen Zhang, Shengqing Wu, Zhiqi Jia, Junhong Zhang, Ying Li, Xingyun Ma, Bingli Fan, Panqiao Wang, Yanna Gao, Zhibiao Ye, Wei Wang

Plant Biotechnology Journal; 16 April 2024; https://doi.org/10.1111/pbi.14352

Summary

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.

See https://onlinelibrary.wiley.com/doi/10.1111/pbi.14352

Fig. 3: Subcellular localization and expression of the SlEIN4 gene and characterization of overexpression and CRISPR/Cas9-engineered mutation lines. (a) The subcellular localization of SlEIN4 in tobacco protoplast. (a-1 to a-4) Fluorescence signals derived from 35 S::GFP and mCherry in tobacco protoplast. (a-5 to a-8) Fluorescence signals derived from 35 S::SlEIN4-GFP and mCherry in tobacco protoplast. Scale bars = 20 μm. (b) Relative expression of SlEIN4 in different Micro Tom (MT) tissues by qPCR. Root, roots; Stem, stems; YL, young leaves; ML, mature leaves; Flower, flower; IMG, immature green fruit; MG, mature green fruit; BR, breaker fruit; YR, yellow ripening fruit; RR, red ripening fruit. The quantitative fluorescence results are shown from three biological and three technical replicates. The Actin gene (Solyc11g005330) was used as the internal control. Data are presented as means ± SD (n = 3). Asterisks indicate the significant difference between root and other tissues revealed by t-test: *P < 0.05, **P < 0.01, ***P < 0.001. (c) The SlEIN4 promoter drives GUS gene expression in MT. Tissues of transgenic tomato plants were stained with X-Gluc. Root, roots; Stem, stems; YL, young leaves; ML, mature leaves; LB, leaf bud; Flower, flower; IMG, immature green fruit; MG, mature green fruit; BR, breaker fruit; YR, yellow ripening fruit; RR, red ripening fruit. Scale bars = 10 mm. (d) Schematic illustration of the two sgRNA target sites (red arrows) in SlEIN4. Black arrows represent the locations of PCR genotyping primers. (e) CR-SlEIN4 alleles were identified from two T0 mutant lines. Allele sequences, as determined by sequencing, are shown. Both CR-SlEIN4-11 and CR-SlEIN4-13 had base deletions near the sgRNA target site. Red text indicates the sgRNA target sequence, and black boxes indicate protospacer-adjacent motif (PAM) sequences. (f) The relative expression level of SlEIN4. Values are presented as means ± SD (n = 3). Asterisks indicate the significant difference between wild-type and transgenic plants revealed by t-test: *P < 0.05, **P < 0.01, ***P < 0.001.