Suppression of pectinase genes confers stable enhancement of fruit firmness through modulation of pectin structure in strawberry

Strawberry ( Fragaria × ananassa Duch.) fruit softening is primarily determined by modifications in the cell wall architecture, and previous studies demonstrated that silencing individual genes encoding pectinases reduced strawberry softening. In this study, we performed an integrative study of the fruit firmness phenotype and cell wall composition of selected transgenic lines with polygalacturonase (PG), β-galactosidase (BGAL) and rhamnogalacturonan lyase (RG) genes down-regulated. Fruit firmness was evaluated across several harvest years, from 3 to 9 years depending on the line. 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 BGAL and RG 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 homogalacturonan (HG) and rhamnogalacturonan I (RG-I) epitopes tightly associated to xyloglucan-cellulose network 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 HG pectins, high-methylated HG pectic epitopes in the water fraction (recognized with JIM7), and low-methylated HG epitopes in the fractions of polysaccharides tightly bound to the cell wall (recognized with JIM5 and LM18). 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.