CRISPR mutants targeting genes involved in tomato softening: Effects on fruit ripening and localisation of pectic polysaccharides in pericarp cell walls

The challenge is to breed tomato varieties that have a reasonable postharvest shelf life while maintaining excellent eating quality. Shelf life is greatly influenced by texture changes that occur due to cell wall remodelling during softening and is a consequence of the combined action of multiple gene products. More than 50 cell wall modifying genes are expressed during fruit development and ripening (Tomato Genome Consortium, 2012). In this project, we focused on several of the most highly expressed ripening-related cell wall genes including POLYGALACTURONASE (PG2a), PECTATE LYASE (PL), BETA-GALACTANASE (TBG4), and XYLOGLUCAN ENDOTRANSGLUCOSYLASE-HYDROLASE (XTH5) to test if knocking them out by DNA editing using the CRISPR/Cas9 system could influence the progress of tomato fruit softening and extend shelf life. Gene expression and enzyme activity were significantly reduced in CRISPR transgenic lines compared with the control. Only the PL CRISPR lines showed substantial changes in fruit firmness. CRISPR edited PG2a, TBG4 and XTH5 produced fruits that showed a similar degree of softening to wild type controls. Experiments to investigate the effect of the CRISPR mutations on processing quality indicated that genes modifying pectin metabolism and especially polyuronide chain length, such as PG2a and PL had a greater impact on paste viscosity than those that acting on pectin side chains or hemicelluloses. Immunocytochemistry indicated that PL was necessary for pectin solubilisation, perhaps through degradation of branched homogalacturonan, thereby allowing disaggregation of polymers for subsequent sidechains cleavage by TBG4 and depolymerization by PG2a. The results of this study provide new insights into the role of cell wall enzymes in tomato fruit softening and allow fruit pectin metabolism to be placed in the context of recent advances in our understanding of primary cell wall structure. This information will be useful for crop improvement through molecular breeding