A Structural Basis for the pH-Dependent Xanthophyll Cycle in Arabidopsis thaliana

Plants adjust their photosynthetic activity to changing light conditions. A central regulation of photosynthesis depends on the xanthophyll cycle, in which the carotenoid violaxanthin is converted into zeaxanthin in strong light, thus activating the dissipation of the excess absorbed energy as heat and the scavenging of reactive oxygen species. Violaxanthin deepoxidase (VDE), the enzyme responsible for zeaxanthin synthesis, is activated by the acidification of the thylakoid lumen when photosynthetic electron transport exceeds the capacity of assimilatory reactions: at neutral pH, VDE is a soluble and inactive enzyme, whereas at acidic pH, it attaches to the thylakoid membrane where it binds its violaxanthin substrate. VDE also uses ascorbate as a cosubstrate with a pH-dependent K_m that may reflect a preference for ascorbic acid. We determined the structures of the central lipocalin domain of VDE (VDE_cd) at acidic and neutral pH. At neutral pH, VDE_cd is monomeric with its active site occluded within a lipocalin barrel. Upon acidification, the barrel opens up and the enzyme appears as a dimer. A channel linking the two active sites of the dimer can harbor the entire carotenoid substrate and thus may permit the parallel deepoxidation of the two violaxanthin β-ionone rings, making VDE an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate.     

The xanthophyll cycle - molecular mechanism and physiological significance

The light-dependent, cyclic changes of xanthophyll pigments: violaxanthin, antheraxanthin and zeaxanthin, called the xanthophyll cycle, have been known for about fifty years. This process was characterised for higher plants, several fern and moss species and in some algal groups. Two enzymes, violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE), belonging to the lipocalin protein family, are engaged in the xanthophyll cycle. VDE requires for its activity ascorbic acid and reversed hexagonal structure formed by monogalactosyldiacylglycerol. ZE, postulated to be a flavoprotein, has not been purified yet and it is known from its gene sequence only. Zeaxanthin epoxidation is dependent on the reducing power of NADPH and presence of additional proteins. The xanthophyll cycle is postulated to play a role in many important physiological processes. Zeaxanthin, formed from violaxanthin under high light conditions, is thought to be a main photoprotector in autotrophic cells due to its ability to dissipate excess of absorbed light energy that can be measured as a non-photochemical quenching. In addition the zeaxanthin formation is important in protection of the thylakoid membranes against lipid peroxidation. Other postulated functions of the xanthophyll cycle, which include regulation of membrane physical properties, blue light reception and regulation of abscisic acid synthesis, are also discussed.     

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.     

Alternative Electron Transports Participate in the Maintenance of Violaxanthin De-Epoxidase Activity of Ulva sp. under Low Irradiance

The xanthophyll cycle (Xc), which involves violaxanthin de-epoxidase (VDE) and the zeaxanthin epoxidase (ZEP), is one of the most rapid and efficient responses of plant and algae to high irradiance. High light intensity can activate VDE to convert violaxanthin (Vx) to zeaxanthin (Zx) via antheraxanthin (Ax). However, it remains unclear whether VDE remains active under low light or dark conditions when there is no significant accumulation of Ax and Zx, and if so, how the ΔpH required for activation of VDE is built. In this study, we used salicylaldoxime (SA) to inhibit ZEP activity in the intertidal macro-algae Ulva sp. (Ulvales, Chlorophyta) and then characterized VDE under low light and dark conditions with various metabolic inhibitors. With inhibition of ZEP by SA, VDE remained active under low light and dark conditions, as indicated by large accumulations of Ax and Zx at the expense of Vx. When PSII-mediated linear electron transport systems were completely inhibited by SA and DCMU, alternative electron transport systems (i.e., cyclic electron transport and chlororespiration) could maintain VDE activity. Furthermore, accumulations of Ax and Zx decreased significantly when SA, DCMU, or DBMIB together with an inhibitor of chlororespiration (i.e., propyl gallate (PG)) were applied to Ulva sp. This result suggests that chlororespiration not only participates in the build-up of the necessary ΔpH, but that it also possibly influences VDE activity indirectly by diminishing the oxygen level in the chloroplast.     

Dynamics of chromophore binding to Lhc proteins in vivo and in vitro during operation of the xanthophyll cycle

Three plant xanthophylls are components of the xanthophyll cycle in which, upon exposure of leaves to high light, the enzyme violaxanthin de-epoxidase (VDE) transforms violaxanthin into zeaxanthin via the intermediate antheraxanthin. Previous work () showed that xanthophylls are bound to Lhc proteins and that substitution of violaxanthin with zeaxanthin induces conformational changes and fluorescence quenching by thermal dissipation. We have analyzed the efficiency of different Lhc proteins to exchange violaxanthin with zeaxanthin both in vivo and in vitro. Light stress of Zea mays leaves activates VDE, and the newly formed zeaxanthin is found primarily in CP26 and CP24, whereas other Lhc proteins show a lower exchange capacity. The de-epoxidation system has been reconstituted in vitro by using recombinant Lhc proteins, recombinant VDE, and monogalactosyl diacylglycerol (MGDG) to determine the intrinsic capacity for violaxanthin-to-zeaxanthin exchange of individual Lhc gene products. Again, CP26 was the most efficient in xanthophyll exchange. Biochemical and spectroscopic analysis of individual Lhc proteins after de-epoxidation in vitro showed that xanthophyll exchange occurs at the L2-binding site. Xanthophyll exchange depends on low pH, implying that access to the binding site is controlled by a conformational change via lumenal pH. These findings suggest that the xanthophyll cycle participates in a signal transduction system acting in the modulation of light harvesting versus thermal dissipation in the antenna system of higher plants.     

Antisense suppression of violaxanthin de-epoxidase in tobacco does not affect plant performance in controlled growth conditions

Violaxanthin de-epoxidase (VDE) catalyzes the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin in the xanthophyll cycle. Tobacco was transformed with an antisense VDE construct under control of the cauliflower mosaic virus 35S promoter to determine the effect of reduced levels of VDE on plant growth. Screening of 40 independent transformants revealed 18 antisense lines with reduced levels of VDE activity with two in particular (TAS32 and TAS39) having greater than 95% reduction in VDE activity. Northern analysis demonstrated that these transformants had greatly suppressed levels of VDE mRNA. De-epoxidation of violaxanthin was inhibited to such an extent that no zeaxanthin and only very low levels of antheraxanthin could be detected after exposure of leaves to high light (2000 μmol m⁻² s⁻¹ for 20 min) with no observable effect on levels of other carotenoids and chlorophyll. Non-photochemical quenching was greatly reduced in the antisense VDE tobacco, demonstrating that a significant level of the non-photochemical quenching in tobacco requires de-epoxidation of violaxanthin. Although the antisense plants demonstrated a greatly impaired de-epoxidation of violaxanthin, no effect on plant growth or photosynthetic rate was found when plants were grown at a photon flux density of 500 or 1000 μmol m⁻² s⁻¹ under controlled growth conditions as compared to wild-type tobacco. This revised version was published online in June 2006 with corrections to the Cover Date.     

Overexpression of violaxanthin de-epoxidase: properties of C-terminal deletions on activity and pH-dependent lipid binding

Violaxanthin de-epoxidase (VDE) is localized in the thylakoid lumen and catalyzes the de-epoxidation of violaxanthin to form antheraxanthin and zeaxanthin. VDE is predicted to be a lipocalin protein with a central barrel structure flanked by a cysteine-rich N-terminal domain and a glutamate-rich C-terminal domain. A full-length Arabidopsis thaliana (L.) Heynh. VDE and deletion mutants of the N- and C-terminal regions were expressed in Escherichia coli and tobacco (Nicotiana tabacum L. cv. Xanthi) plants. High expression of VDE in E. coli was achieved after adding the argU gene that encodes the E. coli arginine AGA tRNA. However, the specific activity of VDE expressed in E. coli was low, possibly due to incorrect folding. Removal of just 4 amino acids from the N-terminal region abolished all VDE activity whereas 71 C-terminal amino acids could be removed without affecting activity. The difficulties with expression in E. coli were overcome by expressing the Arabidopsis VDE in tobacco. The transformed tobacco exhibited a 13- to 19-fold increase in VDE specific activity, indicating correct protein folding. These plants also demonstrated an increase in the initial rate of nonphotochemical quenching consistent with an increased initial rate of de-epoxidation. Deletion mutations of the C-terminal region suggest that this region is important for binding of VDE to the thylakoid membrane. Accordingly, in vitro lipid-micelle binding experiments identified a region of 12 amino acids that is potentially part of a membrane-binding domain. The transformed tobacco plants are the first reported example of plants with an increased level of VDE activity.     

Chlamydomonas Xanthophyll Cycle Mutants Identified by Video Imaging of Chlorophyll Fluorescence Quenching

The photosynthetic apparatus in plants is protected against oxidative damage by processes that dissipate excess absorbed light energy as heat within the light-harvesting complexes. This dissipation of excitation energy is measured as nonphotochemical quenching of chlorophyll fluorescence. Nonphotochemical quenching depends primarily on the [delta]pH that is generated by photosynthetic electron transport, and it is also correlated with the amounts of zeaxanthin and antheraxanthin that are formed from violaxanthin by the operation of the xanthophyll cycle. To perform a genetic dissection of nonphotochemical quenching, we have isolated npq mutants of Chlamydomonas by using a digital video-imaging system. In excessive light, the npq1 mutant is unable to convert violaxanthin to antheraxanthin and zeaxanthin; this reaction is catalyzed by violaxanthin de-epoxidase. The npq2 mutant appears to be defective in zeaxanthin epoxidase activity, because it accumulates zeaxanthin and completely lacks antheraxanthin and violaxanthin under all light conditions. Characterization of these mutants demonstrates that a component of nonphotochemical quenching that develops in vivo in Chlamydomonas depends on the accumulation of zeaxanthin and antheraxanthin via the xanthophyll cycle. However, observation of substantial, rapid, [delta]pH-dependent nonphotochemical quenching in the npq1 mutant demonstrates that the formation of zeaxanthin and antheraxanthin via violaxanthin de-epoxidase activity is not required for all [delta]pH-dependent nonphotochemical quenching in this alga. Furthermore, the xanthophyll cycle is not required for survival of Chlamydomonas in excessive light.     

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.     

Reaction System for Violaxanthin De-Epoxidase with PSII Membranes

PSII membranes were used as a substrate for violaxanthin de-epoxidase(VDE) that had been solubilized from spinach thylakoids by sonication.Inclusion of Tween 20 in the assay mixture was essential, althoughthe detergent apparently inhibited the activity in the conventionalassay with purified violaxanthin and lipid as substrate. Themaximum enhancing effect of the detergent was observed nearits critical micellar concentration. It is likely that the monomerof the detergent helped VDE react with the substrate in themembranes. Dependence of the activity on the substrate concentrationsuggested that VDE functions at least at two sites in the membranes,probably on both their lumenal and stromal surfaces. The abilityof the enzyme to function on the stromal surface in in vitroassays was demonstrated by using intact thylakoids as the substrate.Under such conditions where the endogenous VDE was functioningin the lumen, the exogenously added VDE converted an-theraxanthinto zeaxanthin in the absence of Tween 20. This result suggeststhat, in the reaction with PSII membranes, the detergent wasrequired for VDE to react with violaxanthin but not with antheraxanthin.Otherwise, the detergent was necessary for the reaction on thelumenal surface. (Received September 5, 1997; Accepted October 19, 1997)     

Physiology and xanthophyll cycle activity of Nannochloropsis gaditana

The physiology of the violaxanthin-producing microalga Nannochloropsis gaditana is examined and the effect of environmental factors on the growth and cellular pigment content investigated in batch and continuous cultures. N. gaditana is slow-growing, with a maximum specific growth rate of 0.56 day(-1) at 23 degrees C. The xanthophyll cycle is present in this strain, but has a much lower activity than in higher plants and other species of Nannochloropsis. At 30 degrees C, under high light (1500 micromol photons m(-2) s(-1)), 33% of the violaxanthin pool was deepoxidated to antheraxanthin (76%) and zeaxanthin (24%) over 60 min. Addition of iodoacetamide dramatically affected the xanthophyll cycle activity: 50% of the violaxanthin was replaced by zeaxanthin (90%) within 30 min. This was attributed to an increase in membrane fluidity following iodoacetamide addition, resulting in a larger pool of violaxanthin available for conversion. Batch culture studies showed that a decrease in irradiance (from 880 to 70 micromol photons m(-2) s(-1)) can increase chlorophyll a and violaxanthin content by as much as 80% and 60%, respectively. Continuous cultures indicated that violaxanthin is a growth-rate-dependent product, but the violaxanthin content is less affected by dilution rate (in the range 0.12 to 0.72 day(-1)) and pH (6.8 to 7.8) than chlorophyll a. The optimum conditions for growth and violaxanthin production in continuous culture were found to occur at a dilution rate of 0.48 day(-1), a temperature of between 24 degrees C and 26 degrees C, and pH in the range 7.1 to 7.3.     

The de-epoxidase and epoxidase reactions of Mantoniella squamata (Prasinophyceae) exhibit different substrate-specific reaction kinetics compared to spinach

In vivo the prasinophyceaen alga Mantoniella squamata Manton et Parke uses an incomplete violaxanthin (Vx) cycle, leading to a strong accumulation of antheraxanthin (Ax) under conditions of high light. Here, we show that this zeaxanthin (Zx)-depleted Vx/Ax cycle is caused by an extremely slow second de-epoxidation step from Ax to Zx, and a fast epoxidation from Ax back to Vx in the light. The rate constant of Ax epoxidation is 5 to 6 times higher than the rate constant of Zx formation, implying that Ax is efficiently converted back to Vx before it can be de-epoxidated to Zx. It is, however, only half the rate constant of the first de-epoxidation step from Vx to Ax, thus explaining the observed net accumulation of Ax during periods of strong illumination. When comparing the rate constant of the second de-epoxidation step in M. squamata with Zx formation in spinach (Spinacia oleracea L.) thylakoids, we find a 20-fold reduction in the reaction kinetics of the former. This extremely slow Ax de-epoxidation, which is also exhibited by the isolated Mantoniella violaxanthin de-epoxidase (VDE), is due to a reduced substrate affinity of M. squamata VDE for Ax compared with the VDE of higher plants. Mantoniella VDE, which has a similar Km value for Vx, shows a substantially increased Km for the substrate Ax in comparison with spinach VDE. Our results furthermore explain why Zx formation in Mantoniella cells can only be found at low pH values that represent the pH optimum of VDE. A pH of 5 blocks the epoxidation reaction and, consequently, leads to a slow but appreciable accumulation of Zx.     

Changes in Violaxanthin Deepoxidase Activity and Unsaturation of Thylakoid Membrane Lipids in Indica and Japonica RiceUnder Chill and Strong Light

To explore the differences of sesitivities to chill and strong light in indica and japonica rice (Oryza sativa), the changes in unsaturation of thylakoid membrane lipids and xanthophyll cycle were studied under Chill condition and strong light. The contents of unsaturated fatty acids of thylakoid membrane lipids decreased and that of the saturated ones increased with the time of Chill- and strong lighttreatment, resulting in the reduction of the index of unsaturation of fatty acids (IUFA). The activities of violaxanthin deepoxidase (VDE), a key enzyme of xanthophyll cycle, also reduced. The content of violaxanthin (V) increased, and the contents of antheraxanthin (A) and zeaxanthin (Z) decreased, the ratio of (A+Z)/(A+Z+V) decreased correspondingly. Arrhenius analysis showed that VDE was sensitive to both chill and unsaturation level of thylakoid membrane lipids. Correlation analysis showed that there was distinctly positive relationships between IUFA of thylakoid membrane lipids and the activity of VDE, Fv/Fm, and D1 protein content. Lower IUFA values, less fluidity and stability of thylakoid membrane lipids, lower VDE activity and (A+Z)/(A+Z+V) ratio were found in indica rice cv. Shanyou 63 than in japonica rice cv. 9516 under chill and strong light.     

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.

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 in 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 novel lipid-affinity precipitation step with monogalactosyldiacylglyceride. The 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. Partial amino acid sequences of the N terminus and tryptic fragments are reported. The peptide sequences are unique in the GenBank data base and suggest that violaxanthin de-epoxidase is nuclear encoded, similar to other chloroplast proteins localized in the lumen.     

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     

A functional zeaxanthin epoxidase from red algae shedding light on the evolution of light‐harvesting carotenoids and the xanthophyll cycle in photosynthetic eukaryotes

The epoxy‐xanthophylls antheraxanthin and violaxanthin are key precursors of light‐harvesting carotenoids and participate in the photoprotective xanthophyll cycle. Thus, the invention of zeaxanthin epoxidase (ZEP) catalyzing their formation from zeaxanthin has been a fundamental step in the evolution of photosynthetic eukaryotes. ZEP genes have only been found in Viridiplantae and chromalveolate algae with secondary plastids of red algal ancestry, suggesting that ZEP evolved in the Viridiplantae and spread to chromalveolates by lateral gene transfer. By searching publicly available sequence data from 11 red algae covering all currently recognized red algal classes we identified ZEP candidates in three species. Phylogenetic analyses showed that the red algal ZEP is most closely related to ZEP proteins from photosynthetic chromalveolates possessing secondary plastids of red algal origin. Its enzymatic activity was assessed by high performance liquid chromatography (HPLC) analyses of red algal pigment extracts and by cloning and functional expression of the ZEP gene from Madagascaria erythrocladioides in leaves of the ZEP‐deficient aba2 mutant of Nicotiana plumbaginifolia. Unlike other ZEP enzymes examined so far, the red algal ZEP introduces only a single epoxy group into zeaxanthin, yielding antheraxanthin instead of violaxanthin. The results indicate that ZEP evolved before the split of Rhodophyta and Viridiplantae and that chromalveolates acquired ZEP from the red algal endosymbiont and not by lateral gene transfer. Moreover, the red algal ZEP enables engineering of transgenic plants incorporating antheraxanthin instead of violaxanthin in their photosynthetic machinery.     

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.     

A mathematical model describing kinetics of conversion of violaxanthin to zeaxanthin via intermediate antheraxanthin by the xanthophyll cycle enzyme violaxanthin de-epoxidase

The xanthophyll cycle is one of the mechanisms protecting the photosynthetic apparatus against the light energy excess. Its action is still not well understood on the molecular level.Our model makes it possible to follow independently the kinetics of the two de-epoxidation steps occurring in the xanthophyll cycle: the conversion of violaxanthin into antheraxanthin and the conversion of antheraxanthin into zeaxanthin. Using a simple form of the transition rates of these two conversions, we model the time evolution of the concentration pattern of violaxanthin, antheraxanthin and zeaxanthin during the de-epoxidation process. The model has been applied to describe the reactions of de-epoxidation in a system of liposome membranes composed of phosphatidylcholine and monogalactosyldiacylglycerol. Results obtained within the model fit very well with the experimental data. Values of the transition probabilities of the violaxanthin conversion into antheraxanthin and the antheraxanthin conversion into zeaxanthin calculated by means of the model indicate that the first stage of the de-epoxidation process is much slower than the second one.