The antioxidant activity of rosemary (Rosmarinus officinalis L.) extracts is mainly due to phenolic abietane diterpenes and phenolic acids such as rosmarinic acid. In this study a comprehensive characterization of non-water-soluble and water-soluble extracts from rosemary was achieved by liquid chromatography coupled to electrospray and mass spectrometry. The antioxidant activity of these extracts and their respective major compounds (carnosic acid, carnosol, rosmadial, genkwanin, and rosmarinic acid) was analyzed and compared by using different in vitro systems. Whereas rosmarinic acid, carnosic acid, and carnosol exhibited similar antioxidant activity in a phospholipid membrane-free assay, carnosol behaved as an extremely potent antioxidant in a membrane-based assay (4-6 times stronger than the rest of the compounds). This differential antioxidant behavior suggests that factors other than the radical scavenging capability may be involved. All of the diterpenes induced severe effects on lipid order and packing of phospholipid model membranes. Rosmadial and carnosol decreased the number and/or mobility of water molecules located at the polar head group region of the membrane phospholipids as seen by Laurdan fluorescence spectroscopy. Carnosol also strongly enhanced lipid order at the hydrophobic core of the membrane. These effects throughout the bilayer correlated to the stronger antioxidant capacity of carnosol to inhibit lipid peroxidation. On the contrary, carnosic acid decreased membrane fluidity at deeper regions of the bilayer as measured by bilayer-to-micelle transition assay and self-quenching measurements by using octadecylrhodamine B. These effects may contribute to membrane stabilization and hindrance of radical propagation, which may cooperate with the electron donor ability of rosemary diterpenes in protecting the membranes against oxidative damage.
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 b-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 b-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.
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