Quantifying Leaf Isoprenoid Metabolites

Leaf carotenoids, chlorophylls, and tocopherols were extracted in 2 mL Eppendorf tubes from 4 mg of freeze-dried leaf tissue, using 375 µL of methanol as the extraction solvent and a 25 µL of 10% (w/v) solution of canthaxanthin in chloroform (Sigma) as the internal standard. Tissue was lysed by adding 4 mm glass beads and grinding for 1 min at 30 Hz in a TissueLyser II (QIAGEN, Venlo, Netherlands) and the extraction was carried out by adding 400 µL of Tris-NaCl pH 7.5 and 800 µL of chloroform. Thoroughly mixed samples were centrifuged for 5 min at 13,000 rpm at 4 °C and the organic phase was transferred into a new tube and evaporated using a SpeedVac system (Eppendorf Concentrator plus, Hamburg, Germany). The extracted metabolites were then completely redissolved in 200 µL of acetone, filtered with 0.2 µm filters into amber-colored 2 mL glass vials and a 10-µL aliquot of each sample was then injected onto an Agilent Technologies 1200 series HPLC system (Agilent Technologies, Santa Clara, CA, USA). A C30 reverse-phase column (YMC Carotenoid, 250 × 4.6 mm × 3 µm, YMC CO., Kyoto, Japan) was used, with three mobile phases consisting of methanol (solvent A), water/methanol (20/80 v/v) containing 0.2% ammonium acetate (w/v) (solvent B), and tert-methyl butyl ether (solvent C). Metabolites were separated following the following gradient: 95% A, 5% B isocratically for 12 min, a step-up to 80% A, 5% B, 15% C at 12 min, followed by a linear gradient up to 30% A, 5% B, and 65% C by 30 min. The flow rate was maintained at 1 mL/min. The HPLC equipment was coupled to a Photometric Diode Array (PDA) detector (Santa Clara, CA, USA) allowing the detection of the full UV-visible absorption spectra of the different metabolites. Peak areas of chlorophylls at 650 nm and carotenoids at 472 nm were determined using Agilent ChemStation software. A fluorescence detector at 330 nm was used for tocopherol identification. The quantification of the compounds of interest was done by using a concentration curve built with a commercial standard (Sigma-Aldrich, Steinheim, Germany) [51 (link)]. For the rest of isoprenoid metabolites, approximately 7 mg of freeze-dried tissue were mixed with 500 µL of THF:MeOH (Analytical grade, Normapur) 1:1 buffered with 10% of water (v/v), thoroughly mixed, centrifuged, transferred to an amber vial, and injected into a Waters Acquity UPLC™ (Milford, MA, USA) coupled to a Waters Synapt G2 MS QTOF equipped with an atmospheric pressure chemical ionization (APCI) source. Prenyllipids were separated on an Acquity BEH C18 column (50 × 2.1 mm, 1.7 µm) under the following conditions: Solvent A = water; Solvent B = MeOH; 80–100% B in 4.0 min, 100% B for 2.5 min, re-equilibration at 80% B for 2.0 min. The flow rate was 500 µL/min, and the injection volume was 2.5 µL. Standards of HPLC grade (≥99.5%) were purchased from Sigma-Aldrich. PQ-9 and PC-8 standards were purified in house [52 ].