β carotene

Carotenoids were extracted as described above and analyzed using high-performance liquid chromatography (1260 Infinity II, Agilent, CA, USA) equipped with a C30 column (YMC Carotenoid column, 250 mm, 5 μm pore size). Mobile phase A consisted of 15:81:4 Methyl tert-Butyl Ether (MTBE):methanol:water by volume, and mobile phase B consisted of 81:15:4MTBE: methanol:water by volume. Using a flow rate of 1.0 mL/min at 20 °C, a linear elution gradient from 100% A to 100% B over 15 min was followed by 12 min of 100% B before returning to mobile phase A over 3 min. HPLC standards (astaxanthin, lycopene, β-carotene, zeaxanthin, and canthaxanthin) were purchased from Santa Cruz Biotechnology for identification of carotenoid retention times. zeaxanthin was used to identify isozeaxanthin as this compound cannot be purchased, and these isomers are known to co-elute using C18 chromatography [20 (link)].

Chlorophylls and carotenoids were extracted with 2 mL of acetone by shaking for 6 h at 4 °C in the dark. After centrifugation for 5 min at 5000 rpm at 4 °C, 800 µL of the extract was transferred into a new light-protected tube and 200 µL of water was added. After centrifugation, the supernatant was transferred to brown glass vials for analysis on an HPLC Agilent 1100 Series with UV diode-array-detector. UV detection was set at 445 nm for the detection of carotenoids and at 650 nm for the chlorophylls. A Supelcosil column LC-18 (7.5 cm × 4.6 mm × 3 µm; Sigma Aldrich) was used to separate the pigments using an acetone (solvent A): 1 mM NaHCO₃ (in water, solvent B) gradient with a flow rate of 1.5 mL min⁻¹. The gradient started with an initial mobile phase of 65% (v/v) solvent A. Then, solvent A was increased to 90% in 12 min and to 100% in 8 min. Solvent A was then held for 2 min and then decreased again to 65% after 3 min. Quantification was done by using external standard curves prepared with authentic standards of chlorophylls and β-carotene (Santa Cruz Biotechnology) analyzed in a range from 0.1 mg mL⁻¹ to 0.00625 mg mL⁻¹. The quantities of lutein, neoxanthin and violaxanthin were determined by reference to the β-carotene standard curve assuming a similar response factor.

Leaves sampled from the isoprene experiment at 10 dpi were ground in liquid nitrogen and 50 mg of fresh tissue was extracted in light-protected tubes with 1 ml of acetone by shaking for 6 h at 4°C in the dark. After centrifugation for 5 min at 2350 g at 4°C, 800 μl of the extract was transferred into a new light-protected tube and 200 μl of water was added. After spinning the samples for 1 min at 5000 rpm at 4°C, they were transferred to brown glass vials for analysis on an Agilent 1100 Series HPLC with UV/VIS diode array detector. The detector was set at 445 nm for the detection of carotenoids and at 650 nm for the chlorophylls. The pigments were separated on a Supelcosil column LC-18 (7.5 cm × 4.6 mm × 3 μm; Sigma-Aldrich) using an acetone (solvent A)/1 mM NaHCO₃ (in water, solvent B) gradient with a flow rate of 1.5 ml min^-1. The initial mobile phase consisted of 65/35% (v/v) solvent A/solvent B. Then, solvent A was linearly increased to 90% within 12 min and to 100% over 8 min. 100% solvent A was kept for 2 min and then decreased to 65% again within 3 min. Quantification was done using external standard curves. Authentic standards of the chlorophylls and β-carotene (Santa Cruz Biotechnology) were analyzed in a range from 0.1 to 0.00625 mg ml^-1. Lutein, neoxanthin, and violaxanthin were assumed to have the same response factor as β-carotene.