Total anthocyanin quantifications by HPLC

The contents of anthocyanin were measured in both Arabidopsis seedlings and tobacco flowers according to previous method reported by predecessors (Li et al., 2017). Briefly, 0.1 g samples of seedlings as well as tobacco flowers were mashed into a fine powder and placed into extraction solution (water: methanol: hydrochloric acid = 75:24:1) at 4° C for 12 h. After centrifugation at 12,000 rpm for 5 min at 4° C, the supernatant was collected and filtered by a 0.22 μm nylon membrane filter for the following analysis. Chromatographic detection was performed on a Shimadzu HPLC system with the absorbance at 520 nm based on the procedure described by Li et al. (2017). Total anthocyanin content was quantified by using the external standard curve calibration of cyanidin 3-O-glucoside with three biological replicates (Fanali et al., 2001).