Suppression of Pectinase Genes Confers Stable Enhancement of Fruit Firmness through Modulation of Pectin Structure in Strawberry

Fruit softening is primarily determined by modifications in the cell wall architecture, which are mediated by the coordinated activity of cell wall-degrading enzymes. In strawberry (Fragaria x ananassa Duch.), transgenic suppression of the genes encoding the pectinases polygalacturonase (PG), {beta}-galactosidase ({beta}Gal), and rhamnogalacturonan lyase (RGLyase), reduced fruit softening. In this study, we evaluated the fruit firmness phenotype of selected transgenic lines across several harvest years, from 3 to 9 years depending on the line, and analyzed the cell wall composition of ripe fruits using a carbohydrate microarray. Multiple linear regression analysis revealed that fruit firmness was significantly affected by both genotype and harvest year, while fruit size and soluble solids content showed no significant contribution. All pectinase-silenced lines exhibited increased firmness relative to the wild type, with those lines with PG down-regulated showing the most significant effects, followed by B-Gal and RGLyase fruits. The firmer phenotype was maintained stably in all the transgenic lines during the different years analyzed. Carbohydrate microarray analyses of sequentially extracted cell wall fractions demonstrated that transgenic ripe fruits retained higher levels of low methyl-esterified homogalacturonan (HG) and rhamnogalacturonan I (RG-I) epitopes compared to wild-type ripe fruits, resembling the composition of white-stage control fruits. Principal component analysis of microarray data revealed a clear separation between wild-type ripe fruits and transgenic lines, with the latter clustering near the earlier developmental stages of wild-type fruits. Correlation analysis further revealed positive associations between increased firmness and the abundance of high-methylated HG pectic epitopes in the water fraction, recognized with JIM7, and low-methylated HG abundance in the rest of the fractions (JIM5, LM18, and LM19). Overall, these results suggest that suppressing pectinase genes alters pectin remodeling during ripening, resulting in the retention of structurally intact pectin domains and increased fruit firmness. These genes are therefore excellent candidates for the improvement of this key quality trait in strawberry.