Direct isolation of a functional violaxanthin cycle domain from thylakoid membranes of higher plants

Abstract

A special domain of the thylakoid membrane of higher plants has been isolated which carries out the de-epoxidation of the xanthophyll cycle pigment violaxanthin to zeaxanthin. Recent models indicate that in the chloroplast of higher plants, the violaxanthin (V) cycle takes place within specialized domains in the thylakoid membrane. Here, we describe a new procedure to directly isolate such a domain in functional state. The procedure consists of a thylakoid membrane isolation at a pH value of 5.2 which realizes the binding of the enzyme V de-epoxidase (VDE) to the membrane throughout the preparation process. Isolated thylakoid membranes are then solubilized with the very mild detergent n-dodecyl α-D-maltoside and the pigment-protein complexes are separated by sucrose gradient ultracentrifugation. The upper main fraction of the sucrose gradient represents a V cycle domain which consists of the major light-harvesting complex of photosystem II (LHCII), a special lipid composition with an enrichment of the galactolipid monogalactosyldiacylglycerol (MGDG) and the VDE. The domain is isolated in functional state as evidenced by the ability to convert the LHCII-associated V to zeaxanthin. The direct isolation of a V cycle domain proves the most important hypotheses concerning the de-epoxidation reaction in intact thylakoid membranes. It shows that the VDE binds to the thylakoid membrane at low pH values of the thylakoid lumen, that it binds to membrane regions enriched in LHCII, and that the domain contains high amounts of MGDG. The last point is in line with the importance of the galactolipid for V solubilisation and, by providing inverted hexagonal lipid structures, for VDE activity.

Lipid Dependence of Xanthophyll Cycling in Higher Plants and Algae

“…It is thus possible that the local non-bilayer lipid phase enables the direct interaction between the VDE and the LHCII. A close association between the LHCII and the VDE is furthermore corroborated by the recent direct isolation of a functional Vx cycle membrane domain from thylakoid membranes of higher plants (Goss et al, 2017). With the use of the very mild detergent α-dodecylmaltoside and a preparation at pH5, which ensured the binding of VDE to the luminal side of the thylakoid membrane, the authors were able to isolate a membrane domain consisting of LHCII, VDE and MGDG.…”

“…Although recent measurements have begun to shed light on the existence and localization of inverted hexagonal phases in the thylakoid membrane of higher plants (Garab et al, 2017), and the isolation of a putative, functional Vx cycle membrane domain has been reported (Goss et al, 2017), there is still a wealth of open questions with respect to the lipid dependence of xanthophyll cycling. Future measurements have to show where the inverted hexagonal phase responsible for Vx de-epoxidation is located, i.e., whether it is located within the plane of the thylakoid membrane or whether it is attached to the luminal side of the membrane.…”

Do Galactolipid Synthases Play a Key Role in the Biogenesis of Chloroplast Membranes of Higher Plants?

“…This also raises the major but still unresolved question of the coexistence of HII+Lα phases. In particular, the location and regulation of MGDG-rich HII regions, required for the functional violaxanthin/asthaxanthin cycle ( Goss et al, 2017 ), and the relation with domains containing the photosystems, is clearly a major puzzling question for the future.…”

An optimized protocol for the preparation of oxygen-evolving thylakoid membranes from Cyclotella meneghiniana provides a tool for the investigation of diatom plastidic electron transport

“…However, to systematically address these topics the preparation needs further refinement, e.g. lower pH-values during thylakoid preparation to retain higher concentrations of DD de-epoxidase [ 17 ]. Nevertheless, the present data show for the first time Dt-dependent NPQ and DD cycle activity in isolated diatom thylakoids.…”