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Determination of violaxanthin de-epoxidation by HPLC analysis

OD Ondřej Dlouhý
IK Irena Kurasová
VK Václav Karlický
UJ Uroš Javornik
PŠ Primož Šket
NP Nia Z. Petrova
SK Sashka B. Krumova
JP Janez Plavec
BU Bettina Ughy
VŠ Vladimír Špunda
GG Győző Garab
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The concentration of the xanthophyll cycle pigments was determined by HPLC pigment analysis. The pigments were extracted from thylakoid membranes by 80% acetone and analysed using an Agilent 1,200 HPLC–DAD system (Agilent, USA) equipped with a LiChroCART RP-18 (250 × 4 mm, 5 μm) chromatographic column (Merck, Germany) applying the eluent system and gradient programme described by72. Dark-adapted thylakoid membranes of appropriate pH were taken after 10 min of the dark period (control samples), and illuminated thylakoids, after 10 min of illumination (770 µmol photons m−2 s−1; after measurement of light-induced NPQ). The extent of Vx de-epoxidation was determined as de-epoxidation state of xanthophyll cycle pigments DEPS = (Zx + Ax)/(Vx + Ax + Zx) according to73.

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