Membrane curvature stress controls the maximal conversion of violaxanthin to zeaxanthin in the violaxanthin cycle—influence of α-tocopherol, cetylethers, linolenic acid, and temperature

Zeaxanthin, an important component in protection against overexcitation in higher plants, is formed from violaxanthin by the enzyme violaxanthin de-epoxidase. We have investigated factors that may control the maximal degree of conversion in the violaxanthin cycle. The conversion of violaxanthin to zeaxanthin in isolated spinach thylakoids was followed at different temperatures and in the presence of lipid packing modifiers. The maximum degree of conversion was found to be 35%, 70% and 80% at 4 °C, 25 °C and 37 °C respectively. In the presence of membrane modifying agents, known to promote non-lamellar structures (H_II), such as linolenic acid the conversion increased, and the maximal level of violaxanthin de-epoxidation obtained was close to 100%. In contrast, substances promoting lamellar phases (L_α), such as α-tocopherol and 8-cetylether (C₁₆EO₈), only 55% and 35% of the violaxanthin was converted at 25 °C, respectively. The results are interpreted in light of the lipid composition of the thylakoid membrane, and we propose a model where a negative curvature elastic stress in the thylakoid lipid bilayer is required for violaxanthin de-epoxidase activity. In this model zeaxanthin with its longer hydrophobic stretch is proposed to promote lamellar arrangements of the membrane. As a result, zeaxanthin relieves the curvature elastic stress, which in turn leads to inactivation of violaxanthin de-epoxidase.     

Violaxanthin accessibility and temperature dependency for de-epoxidation in spinach thylakoid membranes

Using DTT and iodoacetamide as a novel irreversible method to inhibit endogenous violaxanthin de-epoxidase, we found that violaxanthin could be converted into zeaxanthin from both sides of the thylakoid membrane provided that purified violaxanthin de-epoxidase was added. The maximum conversion was the same from both sides of the membrane. Temperature was found to have a strong influence both on the rate and degree of maximal violaxanthin to zeaxanthin conversion. Thus only 50% conversion of violaxanthin was detected at 4 °C, whereas at 25 °C and 37 °C the degree of conversion was 70% and 80%, respectively. These results were obtained with isolated thylakoids from non-cold acclimated leafs. Pigment analysis of sub-thylakoid membrane domains showed that violaxanthin was evenly distributed between stroma lamellae and grana partitions. This was in contrast to chlorophyll a and -carotene which were enriched in stroma lamellae fractions while chlorophyll b, lutein and neoxanthin were enriched in the grana membranes. In combination with added violaxanthin de-epoxidase we found almost the same degree of conversion of violaxanthin to zeaxanthin (73–78%) for different domains of the thylakoid membrane. We conclude that violaxanthin de-epoxidase converts violaxanthin in the lipid matrix and not at the proteins, that violaxanthin does not prefer one particular membrane region or one particular chlorophyll protein complex, and that the xanthophyll cycle pigments are oriented in a vertical manner in order to be accessible from both sides of the membrane when located in the lipid matrix.     

Kinetics of violaxanthin de-epoxidation by violaxanthin de-epoxidase, a xanthophyll cycle enzyme, is regulated by membrane fluidity in model lipid bilayers.

This paper describes violaxanthin de-epoxidation in model lipid bilayers. Unilamellar egg yolk phosphatidylcholine (PtdCho) vesicles supplemented with monogalactosyldiacylglycerol were found to be a suitable system for studying this reaction. Such a system resembles more the native thylakoid membrane and offers better possibilities for studying kinetics and factors controlling de-epoxidation of violaxanthin than a system composed only ofmonogalactosyldiacylglycerol and is commonly used in xanthophyll cycle studies. The activity of violaxanthin de-epoxidase (VDE) strongly depended on the ratio of monogalactosyldiacylglycerol to PtdCho in liposomes. The mathematical model of violaxanthin de-epoxidation was applied to calculate the probability of violaxanthin to zeaxanthin conversion at different phases of de-epoxidation reactions. Measurements of deepoxidation rate and EPR-spin label study at different temperatures revealed that dynamic properties of the membrane are important factors that might control conversion of violaxanthin to antheraxanthin. A model of the molecular mechanism of violaxanthin de-epoxidation where the reversed hexagonal structures (mainly created by monogalactosyldiacylglycerol) are assumed to be required for violaxanthin conversion to zeaxanthin is proposed. The presence of monogalactosyldiacylglycerol reversed hexagonal phase was detected in the PtdCho/monogalactosyldiacylglycerol liposomes membrane by 31P-NMR studies. The availability of violaxanthin for de-epoxidation is a diffusion-dependent process controlled by membrane fluidity. The significance of the presented results for understanding themechanism of violaxanthin de-epoxidation in native thylakoid membranes is discussed.     

Ascorbate deficiency can limit violaxanthin de-epoxidase activity in vivo

As a response to high light, plants have evolved non-photochemical quenching (NPQ), mechanisms that lead to the dissipation of excess absorbed light energy as heat, thereby minimizing the formation of dangerous oxygen radicals. One component of NPQ is pH dependent and involves the formation of zeaxanthin from violaxanthin. The enzyme responsible for the conversion of violaxanthin to zeaxanthin is violaxanthin de-epoxidase, which is located in the thylakoid lumen, is activated by low pH, and has been shown to use ascorbate (vitamin C) as its reductant in vitro. To investigate the effect of low ascorbate levels on NPQ in vivo, we measured the induction of NPQ in a vitamin C-deficient mutant of Arabidopsis, vtc2-2. During exposure to high light (1,500 micromol photons m(-2) s(-1)), vtc2-2 plants initially grown in low light (150 micromol photons m(-2) s(-1)) showed lower NPQ than the wild type, but the same quantum efficiency of photosystem II. Crosses between vtc2-2 and Arabidopsis ecotype Columbia established that the ascorbate deficiency cosegregated with the NPQ phenotype. The conversion of violaxanthin to zeaxanthin induced by high light was slower in vtc2-2, and this conversion showed saturation below the wild-type level. Both the NPQ and the pigment phenotype of the mutant could be rescued by feeding ascorbate to leaves, establishing a direct link between ascorbate, zeaxanthin, and NPQ. These experiments suggest that ascorbate availability can limit violaxanthin de-epoxidase activity in vivo, leading to a lower NPQ. The results also demonstrate the interconnectedness of NPQ and antioxidants, both important protection mechanisms in plants.     

Laurdan fluorescence spectroscopy in the thylakoid bilayer: the effect of violaxanthin to zeaxanthin conversion on the galactolipid dominated lipid environment

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.     

The xanthophyll cycle, its regulation and components

During the last few years much interest has been focused on the photoprotective role of zeaxanthin. In excessive light zeaxanthin is rapidly formed in the xanthophyll cycle from violaxanthin, via the intermediate antheraxanthin, a reaction reversed in the dark. The role of zeaxanthin and the xanthophyll cycle in photoprotection, is based on fluorescence quenching measurements, and in many studies a good correlation to the amount of zeaxanthin (and antheraxanthin) has been found. Other suggested roles for the xanthophylls involve, protection against oxidative stress of lipids, participation in the blue light response, modulation of the membrane fluidity and regulation of abscisic acid synthesis. The enzyme violaxanthin de-epoxidase has recently been purified from spinach and lettuce as a 43-kDa protein. It was found as 1 molecule per 20–100 electron-transport chains. The gene has been cloned and sequenced from Lactuca sativa, Nicotiana tabacum and Arabidopsis thaliana. The transit peptide was characteristic of nuclear-encoded and lumen-localized proteins. The activity of violaxanthin de-epoxidase is controlled by the lumen pH. Thus, below pH 6.6 the enzyme binds to the thylakoid membrane. In addition ascorbate becomes protonated to ascorbic acid (pK_a= 4.2) the true substrate (K_m= 0.1 m M ) for the violaxanthin de-epoxidase. We present arguments for an ascorbate transporter in the thylakoid membrane. The enzyme zeaxanthin epoxidase requires FAD as a cofactor and appears to use ferredoxin rather than NADPH as a reductant. The zeaxanthin epoxidase has not been isolated but the gene has been sequenced and a functional protein of 72.5 kDa has been expressed. The xanthophyll cycle pigments are almost evenly distributed in the thylakoid membrane and at least part of the pigments appears to be free in the lipid matrix where we conclude that the conversion by violaxanthin de-epoxidase occurs.     

Functional roles of the major chloroplast lipids in the violaxanthin cycle

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 μM) 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 μM) 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.     

Violaxanthin de-epoxidase, the xanthophyll cycle enzyme, requires lipid inverted hexagonal structures for its activity

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.     

Comparison of violaxanthin de-epoxidation from the stroma and lumen sides of isolated thylakoid membranes from Arabidopsis: implications for the mechanism of de-epoxidation

The enzyme violaxanthin de-epoxidase (VxDE) is localized in the thylakoid lumen and catalyzes the de-epoxidation of membrane-bound violaxanthin (Vx) to zeaxanthin. De-epoxidation from the opposite, stroma side of the membrane has been investigated in the npq1 mutant from Arabidopsis thaliana (L.) Heynh. - which lacks VxDE - by adding partially purified VxDE from spinach thylakoids. The accessibility of Vx to the exogenously added enzyme (exoVxDE) and the kinetics of Vx conversion by the exoVxDE in thylakoids from npq1 plants were very similar to the characteristics of Vx conversion by the endogenous enzyme (endoVxDE) in thylakoids from wild-type plants. However, the conversion of Vx by exoVxDE was clearly retarded at lower temperatures when compared with the reaction catalyzed by endoVxDE. Since the exoVxDE - in contrast to the endoVxDE - has no access to the stacked regions of the membrane, where the xanthophylls bound to photosystem II are located, these results support the assumption of pronounced diffusion of xanthophylls within the thylakoid membrane.     

Laurdan fluorescence spectroscopy in the thylakoid bilayer: The effect of violaxanthin to zeaxanthin conversion on the galactolipid dominated lipid environment

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 °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.     

Mechanism and regulation of the violaxanthin cycle: The role of antenna proteins and membrane lipids

The violaxanthin cycle describes the reversible conversion of violaxanthin to zeaxanthin via the intermediate antheraxanthin. This light-dependent xanthophyll conversion is essential for the adaptation of plants and algae to different light conditions and allows a reversible switch of photosynthetic light-harvesting complexes between a light-harvesting state under low light and a dissipative state under high light. The photoprotective functions of zeaxanthin have been intensively studied during the last decade, but much less attention has been directed to the mechanism and regulation of xanthophyll conversion. In this review, an overview is given on recent progress in the understanding of the role of (i) xanthophyll binding by antenna proteins and of (ii) the lipid properties of the thylakoid membrane in the regulation of xanthophyll conversion. The consequences of these findings for the mechanism and regulation of xanthophyll conversion in the thylakoid membrane will be discussed.     

Dicyclohexylcarbodiimide alters the pH dependence of violaxanthin de-epoxidation

The de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin in the xanthophyll cycle of higher plants is controlled by the pH of the thylakoid lumen. The influence of N,N′-dicyclohexylcarbodiimide (DCCD) on the pH dependence of the de-epoxidation reactions has been investigated in isolated pea thylakoids. In the presence of DCCD, the decrease in de-epoxidase activity at increasing pH was found to be shifted by about 0.3 pH units to more-alkaline pH values. This was paralleled by a less-pronounced cooperativity for the pH dependence of de-epoxidation. Comparative studies with antenna-depleted thylakoids from plants grown in intermittent light and with unstacked thylakoids indicated that binding of DCCD to antenna proteins is most probably not responsible for the altered pH dependence. Analyses of the zeaxanthin content of different antenna subcomplexes showed that the DCCD-induced de-epoxidation at high pH leads to zeaxanthin formation in all antenna proteins from both photosystems. Our data support the view that DCCD binding to the violaxanthin de-epoxidase may be responsible for the altered pH dependence. Received: 4 July 1998 / Accepted: 9 September 1998     

Phase behavior of phosphatidylglycerol in spinach thylakoid membranes as revealed by 31P-NMR

Non-bilayer lipids account for about half of the total lipid content in chloroplast thylakoid membranes. This lends high propensity of the thylakoid lipid mixture to participate in different phases which might be functionally required. It is for instance known that the chloroplast enzyme violaxanthin de-epoxidase (VDE) requires a non-bilayer phase for proper functioning in vitro but direct evidence for the presence of non-bilayer lipid structures in thylakoid membranes under physiological conditions is still missing. In this work, we used phosphatidylglycerol (PG) as an intrinsic bulk lipid label for 31P-NMR studies to monitor lipid phases of thylakoid membranes. We show that in intact thylakoid membranes the characteristic lamellar signal is observed only below 20 degrees C. But at the same time an isotropic phase is present, which becomes even dominant between 14 and 28 degrees C despite the presence of fully functional large membrane sheets that are capable of generating and maintaining a transmembrane electric field. Tris-washed membranes show a similar behavior but the lamellar phase is present up to higher temperatures. Thus, our data show that the location of the phospholipids is not restricted to the bilayer phase and that the lamellar phase co-exists with a non-bilayer isotropic phase.     

Localization of the xanthophyll-cycle enzyme violaxanthin de-epoxidase within the thylakoid lumen and abolition of its mobility by a (light-dependent) pH decrease

The formation of zeaxanthin (Zea) from violaxanthin (Vio) in chloroplasts of leaves and algae upon strong illumination is currently suggested to play a role in the photoprotection of plants. Properties and location of the enzyme Vio de-epoxidase, which is responsible for the transformation of Vio to Zea, were studied using thylakoid membrane vesicles isolated from leaves of Spinacia oleracea L. Without using detergents a repeated freeze-thaw treatment of thylakoid vesicles was sufficient to release the enzyme into the medium. With the same procedure the mobile electron carrier plastocyanin, known to occur in the thylakoid lumen, was also released. The enzyme was demonstrated by its activity in the supernatant of the pelleted thylakoid vesicles in the presence of the added substrates Vio and ascorbic acid, as well as by staining of the released proteins after polyacrylamide gel electrophoresis. The release of the deepoxidase from the vesicles was pH-dependent, declined below pH 6.5 and ceased in the pH range around 5, which corresponds to the pH optimum of the enzyme activity. By using thylakoid vesicles isolated from pre-illuminated and therefore Zea-containing leaves the release by freeze-thaw cycles of both the de-epoxidase and plastocyanin was diminished compared with the dark control. However, the reason for this effect was not the Zea content but an unknown effect of the illumination on the thylakoid membrane properties. The de-epoxidase collected at pH 7 was able to re-bind to thylakoid membranes at pH 5.5 and to transform intrinsic Vio to Zea in the presence of ascorbate. The isolated de-epoxidase, as well as the endogenous membrane-bound de-epoxidase, was inhibited by dithiothreitol. From these results it is concluded that Vio de-epoxidase, like plastocyanin, is mobile within the thylakoid lumen at neutral pH values which occur under in-vivo conditions in the dark. However, upon strong illumination, when the lumen pH drops (pH < 6.5) due to the formation of a proton gradient, the properties of the de-epoxidase are altered and the enzyme becomes tightly bound to the membrane (in contrast to plastocyanin) thus gaining access to its substrate Vio. These findings corroborate the assumption of a transmembrane opposite location of the two enzymes of the xanthophyll cycle, the ascorbate-dependent Vio deepoxidase at the lumenal side and the NADPH-dependent Zea epoxidase at the stromal side. Indications in favour of a location of Vio within the lipid bilayer of the thylakoid membrane and of a binding of the active deepoxidase to these areas are discussed.     

Amino sugars: new inhibitors of zeaxanthin epoxidase, a violaxanthin cycle enzyme

The effect of three sugars and their amino derivatives on violaxanthin cycle enzymes activity was investigated in duckweed (Lemna trisulca), a model water-plant. No effect of sugars and amino sugars on violaxanthin de-epoxidase was observed independent of incubation time; however, epoxidation of zeaxanthin to violaxanthin was inhibited. The minimum amino sugar concentrations causing maximum inhibition of zeaxanthin epoxidation have been estimated. Amino sugars but not sugars caused more than a 50% inhibition of zeaxanthin epoxidation in duckweed after a 24h incubation when applied at a concentration of 0.5%. Incubation with amino sugars under a 6d photoperiod enhanced the inhibitory effect. Zeaxanthin epoxidation was completely inhibited under such conditions, whereas only a minor inhibitory effect was observed in sugar treated plants. The strong amino sugar inhibition of zeaxanthin epoxidase activity represents additional evidence for the creation of an unstable carotenoid carbocation in the molecular mechanism of epoxidation.     

Membrane barriers and Mehler-peroxidase reaction limit the ascorbate available for violaxanthin de-epoxidase activity in intact chloroplasts

The presence of an acidic lumen and the xanthophylls, zeaxanthin and antheraxanthin, are minimal requirements for induction of non-radiative dissipation of energy in the pigment bed of Photosystem II. We recently reported that ascorbate, which is required for formation for these xanthophylls, also can mediate the needed lumen acidity through the Mehler-peroxidase reaction [Neubauer and Yamamoto (1992) Plant Physiol 99: 1354–1361]. It is demonstrated that in non-CO₂-fixing intact chloroplasts and thylakoids of Lactuca sativa, L. c.v. Romaine, the ascorbate available to support de-epoxidase activity is influenced by membrane barriers and the ascorbate-consuming Mehler-peroxidase reaction. In intact chloroplasts, this results in biphasic kinetic behavior for light-induced de-epoxidation. The initial relatively high activity is due to ascorbate preloaded into the thylakoid before light-induction and the terminal low activity due to limiting ascorbate from the effects of chloroplast membranes barriers and a light-dependent process. A five-fold difference between the initial and final activities was observed for light-induced de-epoxidation in chloroplasts pre-incubated with 120 mM ascorbate for 40 min. The light-dependent activity is ascribed to the competitive use of ascorbic acid by ascorbate peroxidase in the Mehler-peroxidase reaction. Thus, stimulating ascorbic peroxidase with H₂O₂ transiently inhibited de-epoxidase activity and concomitantly increased photochemical quenching. Also, the effects inhibiting ascorbate peroxidase with KCN, and the K_M values for ascorbate peroxidase and violaxanthin de-epoxidase of 0.36 and 3.1 mM, respectively, support this conclusion. These results indicate that regulation of xanthophyll-dependent non-radiative energy dissipation in the pigment bed of Photosystem II is modulated not only by lumen acidification but also by ascorbate availability.Abbreviations APO ascorbate peroxidase - MP Mehler ascorbate-peroxidase - NIG nigericin - NPQ non-photochemical quenching - F_o dark fluorescence - F fluorescence at any time - F_M maximal fluorescence of the (dark) non-energized state - F_M maximal fluorescence of the energized state - q_P coefficient for photochemical fluorescence quenching - VDE violaxanthin de-epoxidase - k first-order rate constant for violaxanthin de-epoxidase activity     

The effects of moderately high temperature on zeaxanthin accumulation and decay

Moderately high temperature reduces photosynthetic capacities of leaves with large effects on thylakoid reactions of photosynthesis, including xanthophyll conversion in the lipid phase of the thylakoid membrane. In previous studies, we have found that leaf temperature of 40°C increased zeaxanthin accumulation in dark-adapted, intact tobacco leaves following a brief illumination, but did not change the amount of zeaxanthin in light-adatped leaves. To investigate heat effects on zeaxanthin accumulation and decay, zeaxanthin level was monitored optically in dark-adapted, intact tobacco and Arabidopsis thaliana leaves at either 23 or 40°C under 45-min illumination. Heated leaves had more zeaxanthin following 3-min light but had less or comparable amounts of zeaxanthin by the end of 45?min of illumination. Zeaxanthin accumulated faster at light initiation and decayed faster upon darkening in leaves at 40°C than leaves at 23°C, indicating that heat increased the activities of both violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE). In addition, our optical measurement demonstrated in vivo that weak light enhances zeaxanthin decay relative to darkness in intact leaves of tobacco and Arabidopsis, confirming previous observations in isolated spinach chloroplasts. However, the maximum rate of decay is similar for weak light and darkness, and we used the maximum rate of decay following darkness as a measure of the rate of ZE during steady-state light. A simulation indicated that high temperature should cause a large shift in the pH dependence of the amount of zeaxanthin in leaves because of differential effects on VDE and ZE. This allows for the reduction in ΔpH caused by heat to be offset by increased VDE activity relative to ZE.     

Effect of monogalactosyldiacylglycerol and other thylakoid lipids on violaxanthin de-epoxidation in liposomes

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.     

Phase behavior of phosphatidylglycerol in spinach thylakoid membranes as revealed by P-NMR

Non-bilayer lipids account for about half of the total lipid content in chloroplast thylakoid membranes. This lends high propensity of the thylakoid lipid mixture to participate in different phases which might be functionally required. It is for instance known that the chloroplast enzyme violaxanthin de-epoxidase (VDE) requires a non-bilayer phase for proper functioning in vitro but direct evidence for the presence of non-bilayer lipid structures in thylakoid membranes under physiological conditions is still missing.In this work, we used phosphatidylglycerol (PG) as an intrinsic bulk lipid label for ³¹P-NMR studies to monitor lipid phases of thylakoid membranes. We show that in intact thylakoid membranes the characteristic lamellar signal is observed only below 20 °C. But at the same time an isotropic phase is present, which becomes even dominant between 14 and 28 °C despite the presence of fully functional large membrane sheets that are capable of generating and maintaining a transmembrane electric field. Tris-washed membranes show a similar behavior but the lamellar phase is present up to higher temperatures. Thus, our data show that the location of the phospholipids is not restricted to the bilayer phase and that the lamellar phase co-exists with a non-bilayer isotropic phase.     

Plant lipocalins: violaxanthin de-epoxidase and zeaxanthin epoxidase

Violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the interconversions between the carotenoids violaxanthin, antheraxanthin and zeaxanthin in plants. These interconversions form the violaxanthin or xanthophyll cycle that protects the photosynthetic system of plants against damage by excess light. These enzymes are the first reported lipocalin proteins identified from plants and are only the second examples of lipocalin proteins with enzymatic activity. This review summarizes the discovery and characterization of these two unique lipocalin enzymes and examines the possibility of other potential plant lipocalin proteins.