2.7 Analysis of anthocyanins
Bilberry (S4) samples collected after 6 days of light treatment (equal time-point with transcriptomics samples) were ground and freeze dried in a lyophilizer (Virtis benchtop-K; SP Scientific, Gardiner, NY, USA). Approximately 43 mg dry weight (DW) of lyophilized bilberry powder from each sample was used in extraction. The samples were extracted twice with 500µl MeOH:IPA:acetic acid (20:79:1) for anthocyanin analysis. The extracts were evaporated to dryness and resuspended in 100 µl MeOH. The extracts were analyzed with UPLC-PDA-Synapt G2 QTOF/HDMS (Waters, Milford, MA, USA) in positive (ESI+) resolution ion mode. Samples were analyzed with capillary voltage at 3.0 kV. The source temperature was 120°C and desolvation temperature was set to 360°C, cone gas flow rate was 20 L h-1 and desolvation gas flow rate 800 L h-1. The compounds were separated on an Acquity UPLC-BEH C18 column (1.7 µm, 50 x 2.1 mm, Waters) in 40°C. The mobile phase consisted of (A) H2O and (B) acetonitrile (Chromasolv grade, Sigma-Aldrich, Steinheim, Germany) both containing 0.1% HCOOH (Sigma-Aldrich). A gradient of eluents was used as follows: linear gradient of 95 % of A to 5 % in 10 min, then back to 95 % at 10.1 min and left to equilibrate for 1 min. The injection volume was 2 µl and flow-rate of the mobile phase was 0.6 ml min-1. Tray temperature was set to 10°C. Mass range was set from 100 to 1500. Peak picking and integration of the peaks was executed with MassLynx V4.2 (Waters) and identification was performed by comparing the exact mass/chemical formula, retention time, UV-spectra and/or previously published data of bilberry secondary metabolites. The relative content of the anthocyanins in arbitrary units (AU) was calculated by normalizing the analyte peaks area with the dry weight of the samples (AU mg-1 DW). Total anthocyanins from S4 berries after 12 days from light treatment were measured according to Karppinen et al. (2018).
To determine the anthocyanins from S5 stage fully ripe berries, 100 mg of samples were ground to a fine powder and were freeze-dried overnight to remove water content. The samples were further extracted with methanol acidified with 0.1% HCl (v/v) for two hours at room temperature before being centrifuged and the supernatant vacuum spin dried. The pellet was resuspended in 500 µl 20% methanol and filtered using 0.45 µM PVDF syringe filter (Phenomenex, Torrance, CA, USA). The samples were diluted to 1:10 with 20% methanol before a 5 μL aliquot was injected into a C18 Acclaim Polar Advantage II column (150 × 2.1 mm i.d., 3 μm particle size; Dionex, Sunnyvale, CA, USA) in a HPLC system (Ultimate 3000; Thermo Fisher Dionex) coupled with a diode array detector (DAD). The column oven temperature was set to 35°C and the flow rate was adjusted to 0.350 ml min-1. The mobile phases consisted of 10% formic acid (A) and a mixture of 45% methanol, 45% acetonitrile and 10% formic acid (B). The gradient was as follows: 100% A followed by 9% B in A for 0-12 min, 35% B in A for 12-25 min, 50% B in A for 25 min, and 9% B in A for 30-35 min. The identified anthocyanin peaks were compared with that of known authentic standards and monitored at 254 nm, 280 nm, 320 nm, and 520 nm. The samples were quantified using a calibration curve and expressed as cyanidin 3-O -galactoside equivalents. All analyses were performed with three biological replicates.