Leaf Pigment Extraction and Analysis

Pigment extraction and chromatographic analyses was performed as described in [57 (link)]. Briefly, liquid N₂-homogenized 200 mg fresh weight leaf tissue was spiked with beta-apo-8′-carotenal (Merck-Sigma, Darmstadt, Germany) as an internal standard at 2.5 or 5 µg 100 mg⁻¹. Samples were extracted twice with 1 mL of acetone:methanol 80:20 v/v% by vortexing for 10 s, followed by shaking in a MiniG 1600 instrument (SPEX Sam-plePrep.; Metuchen, NJ, USA). After centrifugation at 14,000× g (at 4 °C for 10 min), supernatants were collected, pooled, and filtered through 0.22 µm PTFE syringe filters and analyzed immediately. For LC-PDA-MS analysis, a Waters Acquity I-class UPLC coupled to a Xevo TQ-XS mass-spectrometry system and a Thermo Accucore C30 2.6 µm, 4.6 × 150 mm column was used. Eluent system A was methanol:water:tert-butyl methyl ether (TBME) 70:30:30 v/v%, while eluent B was methanol:TBME 50:50 v/v%. Solvents used were all at least HPLC grade and were purchased from VWR International (Radnor, Pennsylvania, United States). Absorbance was recorded at 250–700 nm with 1.2 nm resolution and 20 Hz with a PDA detector.