Chlorophyll intermediates and carotenoid content in chlorina and TM2016 mutants

Plants were cultivated under controlled greenhouse conditions at 200 μmol photons m−2 s−1 (PPFD) for 16 h light/8 h dark on acid soil (Vigor plant-growth medium based on Irish and Baltic peats; pH=6.0) supplemented with Osmocote fertiliser. Day/night temperatures were set to 22/16 ◦C with a relative humidity of 30%. Tetrapyrrole intermediates were quantified by HPLC coupled to fluorescence detection. Frozen leaf tissue was homogenised in extraction buffer containing 50% acetone, 45% methanol, and 5% NH4OH (0.1 N). Extracts were analysed using a Jasco Extrema LC-4000 HPLC system equipped with a fluorescence detector and a C18 reverse-phase column (Synergi Hydro-RP, Phenomenex), following a modified version of the method described by DOI 10.1007/s11103-009-9516. Separation was achieved using a gradient of solvent B (90:10 methanol:0.1 M ammonium acetate pH=5.2) in solvent A (10:90 methanol:0.1 M ammonium acetate pH=5.2): 0–100% solvent B over 8 min at 1.0 mL min−1, followed by 19 min at 100% solvent B and 1.5 mL min−1, and a return to 100% solvent A over 2 min at 1.0 mL min−1. Porphyrins were detected using the following excitation/emission wavelength pairs: 415/596 nm for Mg-protoporphyrin IX (Mg-proto IX) and Mg-protoporphyrin IX monomethyl ester (Mg-proto IX MME), 401/632 nm for protoporphyrin IX (proto IX), and 450/636 nm for both mono- and divinyl-protochlorophyllide (protochlorophyllide-a). Compounds were identified and quantified using authentic standards (Frontier Scientific) or, when standards were unavailable, by purification and spectroscopic characterization of chromatographic peaks followed by quantification using published extinction coefficients (https://doi.org/10.1046/j.1432-1327.1999.00627.x, https://doi.org/10.1016/j.ab.2004.03.008, https://doi.org/10.1093/mp/sss051).