Quantitative Carotenoid and Chlorophyll Analysis

Carotenoid (Car) contents were assessed using frozen leaf discs (each 0.5 cm²) cut under the growth chamber conditions. All procedures were carried out exactly as previously described in detail for leaf coffee samples (Vinci et al., 2022 (link)), with minor adjustments. Briefly, after extraction in aqueous 90% acetone (v/v), and sample processing and filtration (13 mm, nylon), the separation of the pigments was achieved through a reverse phase HPLC analysis using an end-capped C18, 5 µm Spherisorb ODS 2 column (250 mm × 4.6 mm). Detection was performed at 440 nm using an HPLC system (Beckman, System Gold, Tulsa, OK, USA) coupled to a diode detector (Model 168, Beckman). Identification and quantification of each compound was achieved using individual pigment standards. The de-epoxidation state (DEPS), involving the components of the xanthophyll cycle, was calculated as DEPS = (zeaxanthin (Z)+0.5 antheraxanthin (A))/(violaxanthin (V)+A+Z).
Chlorophyll (Chl) content was obtained from spectrophotometric measurements of the same homogenates, diluted to 80% acetone and using the formulae of Lichtenthaler (1987) (link).