Tomato (Solanum lycopersicum) is a globally important crop with an economic value in the tens of billions of dollars, and a significant supplier of essential vitamins, minerals, and phytochemicals in the human diet. Shelf life is a key quality trait related to alterations in cuticle properties and remodeling of the fruit cell walls. Studies with transgenic tomato plants undertaken over the last 20 years have indicated that a range of pectin-degrading enzymes are involved in cell wall remodeling. These studies usually involved silencing of only a single gene and it has proved difficult to compare the effects of silencing these genes across the different experimental systems. Here we report the generation of CRISPR-based mutants in the ripening-related genes encoding the pectin-degrading enzymes pectate lyase (PL), polygalacturonase 2a (PG2a), and β-galactanase (TBG4). Comparison of the physiochemical properties of the fruits from a range of PL, PG2a, and TBG4 CRISPR lines demonstrated that only mutations in PL resulted in firmer fruits, although mutations in PG2a and TBG4 influenced fruit color and weight. Pectin localization, distribution, and solubility in the pericarp cells of the CRISPR mutant fruits were investigated using the monoclonal antibody probes LM19 to deesterified homogalacturonan, INRA-RU1 to rhamnogalacturonan I, LM5 to β-1,4-galactan, and LM6 to arabinan epitopes, respectively. The data indicate that PL, PG2a, and TBG4 act on separate cell wall domains and the importance of cellulose microfibril-associated pectin is reflected in its increased occurrence in the different mutant lines.

Fruits are essential for a healthy diet, as they contribute to the prevention of cardiovascular diseases and some cancers, which is attributed to their high bioactive compound content contributing to their antioxidant capacity. Nevertheless, fruits have a short shelf life due to their high-water content, and freeze-drying is a well-known technique to preserve their nutritive quality. However, it is an expensive technology, both due to the use of low pressure and long processing time. Therefore, an optimisation of variables such as the freezing rate, working pressure and shelf temperature during freeze-drying may preserve fruit quality while reducing the time and costs. The impact of these variables on colour, porosity, mechanical properties, water content, vitamin C, total phenols, β-carotene, and antioxidant activity of a freeze-dried orange puree was evaluated. The results showed a great impact of pressure and shelf temperature on luminosity, chroma and water content. Vitamin C and β-carotene were more preserved with higher shelf temperatures (shorter times of processing) and lower pressure, respectively. The optimum freeze-drying conditions preserving the nutrients, and with an interesting structural property, perceived as a crunchy product by consumers, are low pressure (5 Pa) and high shelf temperature (50 °C).