Figure 10.

The superoxide-scavenging ability of flavonoid compounds is associated with the number of –OH groups on the B-ring and determines the roles of flavonoids in the susceptibility of tomato fruit to B. cinerea. A, Structures of different flavonoid compounds in tomato. B, Effects of flavonoid supplements to B. cinerea inocula on lesion size in wild-type tomato fruit. Purified flavonoids at different concentrations, as well as a control treatment (0 mm compound and 1% methanol [MeOH]), were supplied to different B. cinerea inoculation sites on the same fruit. Data are presented as the ratio of lesion diameter to the average lesion diameter of normal treatments (10 μL of B05.10 at 1.25 × 10⁵ mL⁻¹ in one-quarter-strength PDB). Error bars show se (n = 6). Asterisks indicate significant differences between the tested concentration and the PDB control (**, P < 0.01) in Student’s t test. For comparison, the maximum concentrations of Pet-Cou-Rut-Glc are about 0.8 and 3.4 mm in Del/Ros1 and Indigo tomatoes, respectively. Maximum concentrations of Del-Cou-Rut-Glc are about 0.6 and 2.8 mm in Del/Ros1 and Indigo tomatoes, respectively. For myricetin derivatives, the maximum concentration found in Indigo tomatoes is about 0.6 mm. The maximum concentrations of Kae-Rut in AtMYB12 tomatoes is about 4.8 mm, and the maximum rutin content in AtMYB12 tomatoes is about 2.4 mm. Calculations were made based on data shown in Supplemental Table S1 (assuming that 1 g fresh weight of tomato is equivalent to 1 mL). C, Data for superoxide scavenging by tomato flavonoids. Suppression of the increase in fluorescence when different flavonoids were added to the xanthine oxidase reaction was assayed. Different flavonoids purified from tomatoes were added at 0.025 mm to the xanthine oxidase assay. For each compound, the fluorescence increase compared with initial fluorescence over a 15-min reaction time was calculated. Error bars show se (n = 3). Different letters indicate significantly different values at P < 0.05 (one-way ANOVA, Tukey’s posthoc test). D, Superoxide scavenging by different tomato flavonoids. The superoxide-scavenging assay was set up using the Amplex Red Xanthine/Xanthine Oxidase Assay Kit (Life Technologies). Xanthine oxidase catalyzes the oxidation of purine bases of xanthine to uric acid and superoxide. Superoxide spontaneously degrades to H₂O₂, which, in the presence of horseradish peroxidase, reacts with the Amplex Red reagent to generate resorufin. The presence of resorufin can be detected by fluorescence. Fluorescence was measured every 1 min for 15 min by a Varioskan Flash Multimode Reader (Thermo Scientific) using excitation at 560 nm and emission detection at 590 nm. For each sample, a blank control (Ctr) without xanthine was also measured at the same time. For each time point, background fluorescence was corrected by subtracting the value derived from the no-xanthine control. Error bars show se (n = 6).