Violaxanthin De-Epoxidase (Purification of a 43-Kilodalton Lumenal Protein from Lettuce by Lipid-Affinity Precipitation with Monogalactosyldiacylglyceride)

Abstract

Violaxanthin de-epoxidase catalyzes the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin in the xanthophyll cycle. Its activity is optimal at approximately pH 5.2 and requires ascorbate. In conjunction with the transthylakoid pH gradient, the formation of antheraxanthin and zeaxanthin reduces the photochemical efficiency of photosystem II by increasing the nonradiative (heat) dissipation of energy i n the antennae. Previously, violaxanthin de-epoxidase had been partially purified. Here we report its purification from lettuce (Lactuca sativa var Romaine) to one major polypeptide fraction, detectable by two-dimensional isoelectic focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using anion-exchange chromatography on Mono Q and a nove1 lipid-affinity precipitation step with monogalactosyldiacylglyceride.l h e association of violaxanthin de-epoxidase and monogalactosyldiacyglyceride at pH 5.2 is apparently specific, since little enzyme was precipitated by eight other lipids tested. Violaxanthin de-epoxidase has an isoelectric point of 5.4 and an apparent molecular mass of 43 kD. Partia1 amino acid sequences of the N terminus and tryptic fragments are reported. The peptide sequences are unique i n the GenBank data base and suggest that violaxanthin de-epoxidase is nuclear encoded, similar to other chloroplast proteins localized in the lumen.VDE is an enzyme of the xanthophyll cycle that is localized in the chloroplast lumen and catalyzes the conversion of V to A and Z in the presence of an acidic lumen and ascorbate. In vivo, de-epoxidation is induced when a high transthylakoid pH gradient develops in plants exposed to high light intensities. The accumulation of Z and A, along with the transthylakoid pH gradient, mediates nonradiative dissipation of light energy in the antennae (reviewed by Bjorkman and Demmig-Adams, 1993). This nonradiative dissipation of light energy is an alternative energy path that diverts energy from PSII, effectively down-regulating PSII's efficiency. Although a large body of evidence has accumulated documenting the relationship between the presence of the de-epoxidized xanthophyll-cycle pigments (A and Z ) and a plant's ability to dissipate excess light '

Developmental Expression of Violaxanthin De-Epoxidase in Leaves of Tobacco Growing under High and Low Light

“…24 of the growth-chambergrown plants. It is well documented that the size of the xanthophyll cycle pool on a per-unit chlorophyll basis is lower in shade-adapted plants than in sun-adapted plants (Thayer and Bjö rkman, 1990;Demmig-Adams and Adams, 1992;Demmig-Adams et al, 1995, 1996. In our study, both types of plants also showed a gradient in the VAZ pool that decreased from the top to bottom leaves.…”

“…The blots were blocked for 1 h with 5% (w/v) nonfat dry milk in TBST (10 mm Tris-Cl, pH 8.0, 150 mm NaCl, and 0.05% [w/v] Tween 20), and probed for 1 h with a rabbit polyclonal antibody (0.5 g/mL of blocking solution) prepared against a synthetic peptide from the N terminus of lettuce VDE (VDALKTCACLLK) (Bugos and Yamamoto, 1996;Rockholm and Yamamoto, 1996). Blots were developed by im-munodetection using alkaline phosphatase-conjugated secondary antibodies.…”

Carotenoids in nature: insights from plants and beyond

“…The amount of zeaxanthin can be modulated by regulating the expression of the b-hydroxylase (b-OH) gene, which is photo-inducible and violaxanthin de-epoxidase (VDE), whose transcript levels are slightly downregulated by light (Rossel et al 2002). In addition, the luminal pH and ascorbate content appear to affect post-translation modulation of VDE activity and is critical for determining the levels of zeaxanthin upon exposure to excessive light (Rockholm and Yamamoto 1996). Further, the bLCY mutant (suppressor of zeaxanthin-less1, szl1) lacks zeaxanthin, yet accumulates higher levels of lutein and a-carotene, which partially restores quenching efficiency, revealing that lutein could substitute for zeaxanthin (Li et al 2009a).…”

A new regulatory role for the chloroplast ATP synthase

“…Two processes are known to be part of the mechanism of NPQ. One is the conversion of violaxanthin to zeaxanthin in the thylakoid membranes by activation of a membrane-associated violaxanthin deepoxidase (23)(24)(25). The other is protonation of the PsbS subunit of the photosystem II complex (26,27).…”