digalactosyldiacylglycerol and violaxanthin

Laurdan (6-lauroyl-2-dimethylaminonaphthalene) fluorescence spectroscopy has been applied to probe the physical status of the thylakoid membrane upon conversion of violaxanthin to zeaxanthin. So far, only phospholipid-dominated membranes have been studied by this method and hereby we report the first use of laurdan in mono- and digalactosyldiacylglycerol-dominated membrane systems. The generalised polarisation (GP) of laurdan was used as a measure of the structural effect of xanthophyll cycle pigments in isolated spinach (Spinacia oleracea) thylakoids and in model membrane vesicles composed of chloroplast galactolipids. Higher GP values indicate a membrane in a more ordered structure, whereas lower GP values point to a membrane in a less ordered fluid phase. The method was used to probe the effect of violaxanthin and zeaxanthin in thylakoid membranes at different temperatures. At 4, 25 and 37 degrees C the GP values for dark-adapted thylakoids in the violaxanthin-form were 0.55, 0.28 and 0.26. After conversion of violaxanthin to zeaxanthin, at the same temperatures, the GP values were 0.62, 0.36 and 0.34, respectively. GP values increased gradually upon conversion of violaxanthin to zeaxanthin. Similar results were obtained in the liposomal systems in the presence of these xanthophyll cycle pigments. We conclude from these results that the conversion of violaxanthin to zeaxanthin makes the thylakoid membrane more ordered.

Topics: 2-Naphthylamine; Fatty Acids; Galactolipids; Laurates; Lipid Bilayers; Liposomes; Spectrometry, Fluorescence; Spinacia oleracea; Temperature; Thylakoids; Xanthophylls; Zeaxanthins

Monogalactosyldiacylglyceride (MGDG) and digalactosyldiacylglyceride (DGDG) are the major membrane lipids of chloroplasts. The question of the specialized functions of these unique lipids has received limited attention. One function is to support violaxanthin de-epoxidase (VDE) activity, an enzyme of the violaxanthin cycle. To understand better the properties of this system, the effects of galactolipids and phosphatidylcholines on VDE activity were examined by two independent methods. The results show that the micelle-forming lipid (MGDG) and bilayer forming lipids (DGDG and phosphatidylcholines) support VDE activity differently. MGDG supported rapid and complete de-epoxidation starting at a threshold lipid concentration (10 microM) coincident with complete solubilization of violaxanthin. In contrast, DGDG supported slow but nevertheless complete to nearly complete de-epoxidation at a lower lipid concentration (6.7 microM) that did not completely solubilize violaxanthin. Phosphotidylcholines showed similar effects as DGDG except that de-epoxidation was incomplete. Since VDE requires solubilized violaxanthin, aggregated violaxanthin in DGDG at low concentration must become solubilized as de-epoxidation proceeds. High lipid concentrations had lower activity possibly due to formation of multilayered structures (liposomes) that restrict accessibility of violaxanthin to VDE. MGDG micelles do not present such restrictions. The results indicate VDE operates throughout the lipid phase of the single bilayer thylakoid membrane and is not limited to putative MGDG micelle domains. Additionally, the results also explain the differential partitioning of violaxanthin between the envelope and thylakoid as due to the relative solubilities of violaxanthin and zeaxanthin in MGDG, DGDG and phospholipids. The violaxanthin cycle is hypothesized to be a linked system of the thylakoid and envelope for signal transduction of light stress.

Topics: Chloroplasts; Chromatography, High Pressure Liquid; Galactolipids; Oxidoreductases; Plant Leaves; Signal Transduction; Spectrophotometry; Spinacia oleracea; Thylakoids; Xanthophylls

Bilayer-forming lipids were shown to be ineffective in sustaining the enzymatic activity of violaxanthin de-epoxidase. On the other hand, non-bilayer-forming lipids, regardless of their different chemical character, ensured high activity of violaxanthin de-epoxidase, resulting in conversion of violaxanthin to zeaxanthin. Our data indicates that the presence of lipids forming reversed hexagonal structures is necessary for violaxanthin de-epoxidase activity and this activity is dependent on the degree of unsaturation of the fatty acids. The significance of the reversed hexagonal phase domains in the conversion of violaxanthin into zeaxanthin in model systems and in the native thylakoid membranes is discussed.

Topics: beta Carotene; Chromatography, Thin Layer; Enzyme Activation; Galactolipids; Kinetics; Lipid Bilayers; Liposomes; Narcissus; Oxidoreductases; Plant Proteins; Structure-Activity Relationship; Xanthophylls

In this study we present evidence that one of two reactions of the xanthophyll cycle, violaxanthin de-epoxidation, may occur in unilamellar egg phosphatidylcholine vesicles supplemented with monogalactosyldiacylglycerol (MGDG). Activity of violaxanthin de-epoxidase (VDE) in this system was found to be strongly dependent on the content of MGDG in the membrane; however, only to a level of 30 mol%. Above this concentration the rate of violaxanthin de-epoxidation decreased. The effect of individual thylakoid lipids on VDE-independent violaxanthin transformation was also investigated and unspecific effects of phosphatidylglycerol and sulphoquinovosyldiacyglycerol, probably related to the acidic character of these lipids, were found. The presented results suggest that violaxanthin de-epoxidation most probably takes place inside MGDG-rich domains of the thylakoid membrane. The described activity of the violaxanthin de-epoxidation reaction in liposomes opens new possibilities in the investigation of the xanthophyll cycle and may contribute to a better understanding of this process.

Topics: beta Carotene; Diglycerides; Enzyme Inhibitors; Galactolipids; Glycolipids; Kinetics; Liposomes; Lutein; Medicago sativa; Oxidoreductases; Phosphatidylcholines; Plant Leaves; Thylakoids; Triticum; Xanthophylls