Petal samples from GB_Pe, WF_Pe and YF_Pe were used for carotenoid extraction. Three biological replicates were performed for each developmental stage. Each replicate (~ 1 g of fresh weight) was freeze-dried and ground into fine powder, and then about 100 mg of powder was dissolved in 1 mL of a solution of n-hexane: acetone: ethanol (2:1:1 (V:V:V)). The solution sample was vortexed (30 s), followed by ultrasound-assisted extraction at room temperature for 20 min, and centrifuged at 12,000 g for 5 min to collect the supernatant. The extraction steps above were repeated, and the supernatants from the two centrifugations were combined. Subsequently, the combined supernatant was evaporated to dryness using a nitrogen gas stream and then reconstituted in 200 μL of (acetonitrile:methanol = 3:1 (V:V)): methyl tert-butyl ether = 85:15 (V:V). Finally, the solution was centrifuged at 12,000 g for 2 min to collect the supernatant for LC-MS/MS analysis. To monitor the stability of the LC-MS/MS analytical conditions, a quality control (QC) sample was used with equal mixing of all measured samples and was run at intervals during the assay. Stock solutions of standards purchased from Sigma-Aldrich (St. Louis, MO, USA) or Olchemim Ltd. (Olomouc, Czech Republic) were prepared at a concentration of 10 mg/mL in HPLC-grade acetonitrile (ACN) and then diluted with ACN to working solutions. For each carotenoid, five successive concentration gradients were used to plot the standard curve.

The prepared sample solutions were analysed by an LC-APCI-MS/MS system (UPLC, Shim-pack UFLC SHIMADZU CBM30A system,; MS, Applied Biosystems 6500 Triple Quadrupole, equipped with an APCI Turbo Ion-Spray interface and operated in positive ion mode and controlled by Analyst 1.6.3 software (Applied Biosystems Company, Framingham, MA, USA) at Wuhan Metware Biotechnology Co., Ltd. (Wuhan, China). The APCI source operation parameter settings and multiple reaction monitoring (MRM) transitions were performed according to a previous study [25]. Finally, the carotenoid levels were calculated according to the following formula: carotenoid content (μg/g) = A*(B/1000)/C (A: the carotenoid concentration calculated by the standard curve using chromatographic peak area integrals, μg/mL; B: the volume of solution for redissolution, μL; C: the dry weight of plant tissue powder, g). SPSS 16.0 was used for the analysis of variance (ANOVA). The differences in means were compared using Duncan’s multiple range test.

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