Enzyme Violaxanthin De-epoxidase Research Articles

Influence of the compatible solute sucrose on thylakoid membrane organization and violaxanthin de-epoxidation

Main conclusionThe compatible solute sucrose reduces the efficiency of the enzymatic de-epoxidation of violaxanthin, probably by a direct effect on the protein parts of violaxanthin de-epoxidase which protrude from the lipid phase of the thylakoid membrane.The present study investigates the influence of the compatible solute sucrose on the violaxanthin cycle of higher plants in intact thylakoids and in in vitro enzyme assays with the isolated enzyme violaxanthin de-epoxidase at temperatures of 30 and 10 °C, respectively. In addition, the influence of sucrose on the lipid organization of thylakoid membranes and the MGDG phase in the in vitro assays is determined. The results show that sucrose leads to a pronounced inhibition of violaxanthin de-epoxidation both in intact thylakoid membranes and the enzyme assays. In general, the inhibition is similar at 30 and 10 °C. With respect to the lipid organization only minor changes can be seen in thylakoid membranes at 30 °C in the presence of sucrose. However, sucrose seems to stabilize the thylakoid membranes at lower temperatures and at 10 °C a comparable membrane organization to that at 30 °C can be observed, whereas control thylakoids show a significantly different membrane organization at the lower temperature. The MGDG phase in the in vitro assays is not substantially affected by the presence of sucrose or by changes of the temperature. We conclude that the presence of sucrose and the increased viscosity of the reaction buffers stabilize the protein part of the enzyme violaxanthin de-epoxidase, thereby decreasing the dynamic interactions between the catalytic site and the substrate violaxanthin. This indicates that sucrose interacts with those parts of the enzyme which are accessible at the membrane surface of the lipid phase of the thylakoid membrane or the MGDG phase of the in vitro enzyme assays.

Modulation of non-bilayer lipid phases and the structure and functions of thylakoid membranes: effects on the water-soluble enzyme violaxanthin de-epoxidase

The role of non-bilayer lipids and non-lamellar lipid phases in biological membranes is an enigmatic problem of membrane biology. Non-bilayer lipids are present in large amounts in all membranes; in energy-converting membranes they constitute about half of their total lipid content—yet their functional state is a bilayer. In vitro experiments revealed that the functioning of the water-soluble violaxanthin de-epoxidase (VDE) enzyme of plant thylakoids requires the presence of a non-bilayer lipid phase. 31P-NMR spectroscopy has provided evidence on lipid polymorphism in functional thylakoid membranes. Here we reveal reversible pH- and temperature-dependent changes of the lipid-phase behaviour, particularly the flexibility of isotropic non-lamellar phases, of isolated spinach thylakoids. These reorganizations are accompanied by changes in the permeability and thermodynamic parameters of the membranes and appear to control the activity of VDE and the photoprotective mechanism of non-photochemical quenching of chlorophyll-a fluorescence. The data demonstrate, for the first time in native membranes, the modulation of the activity of a water-soluble enzyme by a non-bilayer lipid phase.

Protein redox regulation in the thylakoid lumen: The importance of disulfide bonds for violaxanthin de-epoxidase

When exposed to saturating light conditions photosynthetic eukaryotes activate the xanthophyll cycle where the carotenoid violaxanthin is converted into zeaxanthin by the enzyme violaxanthin de-epoxidase (VDE). VDE protein sequence includes 13 cysteine residues, 12 of which are strongly conserved in both land plants and algae. Site directed mutagenesis of Arabidopsis thaliana VDE showed that all these 12 conserved cysteines have a major role in protein function and their mutation leads to a strong reduction of activity. VDE is also shown to be active in its completely oxidized form presenting six disulfide bonds. Redox titration showed that VDE activity is sensitive to variation in redox potential, suggesting the possibility that dithiol/disulfide exchange reactions may represent a mechanism for VDE regulation.

Enhancement of Non-Photochemical Quenching in the Bryophyte Physcomitrella patens During Acclimation to Salt and Osmotic Stress

Drought and salt stress are major abiotic constraints affecting plant growth worldwide. Under these conditions, the production of reactive oxygen species (ROS) is a common phenomenon taking place mainly in chloroplasts, peroxisomes, mitochondria and apoplasts, especially when associated with high light stress. ROS are harmful because of their high reactivity to cell components, thereby leading to cytotoxicity and cell death. During the Ordovician and early Devonian period, photosynthetic organisms colonized terrestrial habitats, and the acquisition of desiccation tolerance has been a major component of their evolution. We have studied the capacity for acclimation to drought and salt stress of the moss Physcomitrella patens, a representative of the early land colonization stage. Exposure to high concentrations of NaCl and sorbitol strongly affects chloroplast development, the Chl content and the thylakoid protein composition in this moss. Under sublethal conditions (0.2 M NaCl and 0.4 M sorbitol), the photosynthetic apparatus of P. patens responds to oxidative stress by increasing non-photochemical quenching (NPQ). Surprisingly, the accumulation of PSBS and LHCSR, the two polypeptides essential for NPQ in P. patens, was not up-regulated in these conditions. Rather, an increased NPQ amplitude correlated with the overaccumulation of zeaxanthin and the presence of the enzyme violaxanthin de-epoxidase. These results suggest that the regulation of excess energy dissipation through control of PSBS and LHCSR is mainly driven by light conditions, while osmotic and salt stress act through acclimative regulation of the xanthophyll cycle. We conclude that regulation of the xanthophyll cycle is an important anticipatory strategy against photoinhibition by high light.

Photoprotection in the brown alga Macrocystis pyrifera: Evolutionary implications

The dissipation of energy as heat is essential for photosynthetic organisms to protect themselves against excess light. We compared Photosystem II florescence changes (non-photochemical quenching, NPQ) in the brown alga Macrocystis pyrifera with that of Ficus sp., a higher plant to examine if the mechanism of heat dissipation (energy-dependent quenching, qE) differs between these evolutionary distant groups of phototrophs. We discovered that M. pyrifera had a slower rise of NPQ upon illumination than the Ficus sp. Further, the NPQ relaxation phase that takes place in the first minutes after light to dark transition is absent in this brown alga. We found that the NPQ induction rate in this alga was 1.5 times faster in preilluminated samples than in dark-adapted samples; this was associated with an increase in the rate of accumulation of the carotenoid zeaxanthin. Therefore, we conclude that NPQ in M. pyrifera is associated only with the formation of zeaxanthin. These results indicate that M. pyrifera lacks the fast component of qE that is related to allosteric changes in the light harvesting complexes of Ficus sp., a representative of higher plants. Although the xanthophyll cycle of this brown alga is similar to that of Ficus sp., yet, the transthylakoid proton gradient (ΔpH) does not influence NPQ beyond the activation of the violaxanthin de-epoxidase enzyme. These findings suggest that NPQ control mechanisms are not universal and we suggest that it may have diverged early in the evolution of different groups of eukaryotic phototrophs.

PsbS-specific zeaxanthin-independent changes in fluorescence emission spectrum as a signature of energy-dependent non-photochemical quenching in higher plants

The PsbS protein of photosystem II is necessary for the development of energy-dependent quenching of chlorophyll (Chl) fluorescence (qE), and PsbS-deficient Arabidopsis plant leaves failed to show qE-specific changes in the steady-state 77 K fluorescence emission spectra observed in wild-type leaves. The difference spectrum between the quenched and un-quenched states showed a negative peak at 682 nm. Although the level of qE development in the zeaxanthin-less npq1-2 mutant plants, which lacked violaxanthin de-epoxidase enzyme, was only half that of wild type, there were no noticeable changes in this qE-dependent difference spectrum. This zeaxanthin-independent DeltaF682 signal was not dependent on state transition, and the signal was not due to photobleaching of pigments either. These results suggest that DeltaF682 signal is formed due to PsbS-specific conformational changes in the quenching site of qE and is a new signature of qE generation in higher plants.

Thioredoxin targets of the plant chloroplast lumen and their implications for plastid function

The light-dependent regulation of stromal enzymes by thioredoxin (Trx)-catalysed disulphide/dithiol exchange is known as a classical mechanism for control of chloroplast metabolism. Recent proteome studies show that Trx targets are present not only in the stroma but in all chloroplast compartments, from the envelope to the thylakoid lumen. Trx-mediated redox control appears to be a common feature of important pathways, such as the Calvin cycle, starch synthesis and tetrapyrrole biosynthesis. However, the extent of thiol-dependent redox regulation in the thylakoid lumen has not been previously systematically explored. In this study, we addressed Trx-linked redox control in the chloroplast lumen of Arabidopsis thaliana. Using complementary proteomics approaches, we identified 19 Trx target proteins, thus covering more than 40% of the currently known lumenal chloroplast proteome. We show that the redox state of thiols is decisive for degradation of the extrinsic PsbO1 and PsbO2 subunits of photosystem II. Moreover, disulphide reduction inhibits activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase, which participates in thermal dissipation of excess absorbed light. Our results indicate that redox-controlled reactions in the chloroplast lumen play essential roles in the function of photosystem II and the regulation of adaptation to light intensity.

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 °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.

The influence of phase transitions in phosphatidylethanolamine models on the activity of violaxanthin de-epoxidase

In the present study, the influence of the phospholipid phase state on the activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase (VDE) was analyzed using different phosphatidylethanolamine species as model lipids. By using 31P NMR spectroscopy, differential scanning calorimetry and temperature dependent enzyme assays, VDE activity could directly be related to the lipid structures the protein is associated with. Our results show that the gel (L β) to liquid-crystalline (L α) phase transition in these single lipid component systems strongly enhances both the solubilization of the xanthophyll cycle pigment violaxanthin in the membrane and the activity of the VDE. This phase transition has a significantly stronger impact on VDE activity than the transition from the L α to the inverted hexagonal (H II) phase. Especially at higher temperatures we found increased VDE reaction rates in the presence of the L α phase compared to those in the presence of H II phase forming lipids. Our data furthermore imply that the H II phase is better suited to maintain high VDE activities at lower temperatures.

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 (C 16EO 8), 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.

Dependence of reaction center-type energy-dependent quenching on photosystem II antenna size

The effects of photosystem II antenna size on reaction center-type energy-dependent quenching (qE) were examined in rice plants grown under two different light intensities using both wild type and qE-less (OsPsbS knockout) mutant plants. Reaction center-type qE was detected by measuring non-photochemical quenching at 50 μmol photons m − 2 s − 1 white light intensity. We observed that in low light-grown rice plants, reaction center-type qE was higher than in high light-grown plants, and the amount of reaction center-type qE did not depend on zeaxanthin accumulation. This was confirmed in Arabidopsis npq1–2 mutant plants that lack zeaxanthin due to a mutation in the violaxanthin de-epoxidase enzyme. Although the electron transport rate measured at a light intensity of 50 μmol photons m − 2 s − 1 was the same in high light- and low light-grown wild type and mutant plants lacking PsbS protein, the generation of energy-dependent quenching was completely impaired only in mutant plants. Analyses of the pigment content, Lhcb proteins and D1 protein of PSII showed that the antenna size was larger in low light-grown plants, and this correlated with the amount of reaction center-type qE. Our results mark the first time that the reaction center-type qE has been shown to depend on photosystem II antenna size and, although it depends on the existence of PsbS protein, the extent of reaction center-type qE does not correlate with the transcript levels of PsbS protein. The presence of reaction center-type energy-dependent quenching, in addition to antenna-type quenching, in higher plants for dissipation of excess light energy demonstrates the complexity and flexibility of the photosynthetic apparatus of higher plants to respond to different environmental conditions.

The giant kelp Macrocystis pyrifera presents a different nonphotochemical quenching control than higher plants.

Here the mechanisms involved in excitation energy dissipation of Macrocystis pyrifera were characterized to explain the high nonphotochemical quenching of chlorophyll a (Chla) fluorescence (NPQ) capacity of this alga. We performed a comparative analysis of NPQ and xanthophyll cycle (XC) activity in blades collected at different depths. The responses of the blades to dithiothreitol (DTT) and to the uncoupler NH4Cl were also assayed. The degree of NPQ induction was related to the amount of zeaxanthin synthesized in high light. The inhibition of zeaxanthin synthesis with DTT blocked NPQ induction. A slow NPQ relaxation upon the addition of NH4Cl, which disrupts the transthylakoid proton gradient, was detected. The slow NPQ relaxation took place only in the presence of de-epoxidated XC pigments and was related to the epoxidation of zeaxanthin. These results indicate that in M. pyrifera, in contrast to higher plants, the transthylakoid proton gradient alone does not induce NPQ. The role of this gradient seems to be related only to the activation of the violaxanthin de-epoxidase enzyme.

On Why Thylakoids Energize ATP Formation Using Either Delocalized or Localized Proton Gradients – A Ca2+Mediated Role in Thylakoid Stress Responses

By the early 1970s, the chemiosmotic hypothesis of Peter Mitchell was widely accepted by bioenergetics researchers as the best conceptual scheme to explain how ATP is formed in oxidative and photosynthetic phosphorylation. At about the same time, however, work from a few laboratories suggested that some aspects of that elegant, relatively simple hypothesis required revision - not abandonment, but refinement to accommodate more complex movements of protons in the ATP formation mechanism than originally envisioned by Peter Mitchell. In some situations it appeared that protons were constrained to localized domains rather than always delocalized within an enclosed vesicle as envisioned by chemiosmosis. This minireview tells that story from my perspective, as one of the researchers involved in the experimental approaches that revealed more complex energy coupling proton flux patterns. Ionic conditions during isolated thylakoid storage were found to reversibly switch the [Formula: see text] gradient driving ATP formation between delocalized and localized energy coupling modes. Thylakoid accessible Ca(2+) ions proved to be the switching factor that was responding to the ionic conditions in the storage treatment. The mechanism of Ca(2+) was at least partially demystified when it was shown that the reversible switching between [Formula: see text] energy coupling modes involved Ca(2+) interactions with the 8 kDa CF(0) (the H(+) channel) subunit in a type of H(+) flux gating action. Other experiments showed that the Ca(2+) gating of H(+) flux into the lumen may be a critical regulatory factor in controlling the lumen pH and thereby help regulate the activity of the violaxanthin de-epoxidase enzyme, a key part of the chloroplast photoprotective response to over-energization (excess light) stress.

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.

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.

PH-dependent reversible inhibition of violaxanthin de-epoxidase by pepstatin related to protonation-induced structural change of the enzyme

The redox enzyme violaxanthin de-epoxidase (VDE) was found to be sensitive to pepstatin, a specific inhibitor of aspartic protease. The inhibition was similar to that of aspartic protease in that it was reversible and accompanied by the protonation of the enzyme. Of the two peaks of VDE appearing on anion exchange chromatography, VDE-I predominated at pH 7.2. On lowering the pH of the chromatography, VDE-I decreased and VDE-II increased. Furthermore, re-chromatography of either peak yielded both peaks. These results suggest that VDE-I and VDE-II are interconvertible depending on pH, and thus, they represent the de-protonated and protonated forms of the enzyme, respectively. Presumably the protonation-induced structural change of the enzyme is responsible for the interaction with pepstatin, and also with substrate.

Photodamage of the Photosynthetic Apparatus and Its Dependence on the Leaf Developmental Stage in the npq1 Arabidopsis Mutant Deficient in the Xanthophyll Cycle Enzyme Violaxanthin De-epoxidase

The npq1 Arabidopsis mutant is deficient in the violaxanthin de-epoxidase enzyme that converts violaxanthin to zeaxanthin in excess light (xanthophyll cycle). We have compared the behavior of mature leaves (ML) and developing leaves of the mutant and the wild type in various light environments. Thermoluminescence measurements indicated that high photon flux densities (>500 micromol m(-2) s(-1)) promoted oxidative stress in the chloroplasts of npq1 ML, which was associated with a loss of chlorophyll and an inhibition of the photochemical activity. Illuminating leaf discs in the presence of eosin, a generator of singlet oxygen, brought about pronounced lipid peroxidation in npq1 ML but not in wild-type leaves. No such effects were seen in young leaves (YL) of npq1, which were quite tolerant to strong light and eosin-induced singlet oxygen. Non-photochemical energy quenching was strongly inhibited in npq1 YL and ML and was not improved with high-light acclimation. Our results confirm that the xanthophyll cycle protects chloroplasts from photooxidation by a mechanism distinct from non-photochemical energy quenching and they reveal that the absence of xanthophyll cycle can be compensated by other protective mechanisms. npq1 YL were observed to accumulate considerable amounts of vitamin E during photoacclimation, suggesting that this lipophilic antioxidant could be involved in the high phototolerance of those leaves.

Influence of Ca(2+) on the thylakoid lumen violaxanthin de-epoxidase activity through Ca(2+) gating of H(+) flux at the CF(o) H(+) channel.

Part of the chloroplast photoprotection response to excess light absorption involves formation of zeaxanthin (and antheraxanthin) via the violaxanthin deepoxidase enzyme, the activity of which requires lumen acidity near or below pH 6.0. Clearly, the violaxanthin de-epoxidase activity is strongly regulated because at equivalent energization levels (including the parameters of H(+) accumulation and ATP formation rates), there can be either low or high violaxanthin de-epoxidase enzyme activity. This work shows that the factor or factors responsible for regulating the violaxanthin deepoxidase correlate directly with those which regulate the expression of membrane-localized or delocalized proton gradient (Delta[Formula: see text] (H+)) energy coupling. The most clearly identified factor regulating switching between localized and delocalized energy coupling modes is Ca(2+) binding to the lumen side of the thylakoid membrane; in particular, Ca(2+) binding to the 8 kDA subunit III of the CF(o) H(+) channel. The activity of violaxanthin deepoxidase in pea (Pisum sativa) and spinach (Spinacea oleracea) thylakoids is shown here to be strongly correlated with conditions known from previous work to displace Ca(2+) from the CF(o) H(+) channel and thus to modulate the extent of lumenal acidification while maintaining a fairly constant rate of ATP formation.

Developmental expression of violaxanthin de-epoxidase in leaves of tobacco growing under high and low light.

Violaxanthin de-epoxidase (VDE) is a lumen-localized enzyme that catalyzes the de-epoxidation of violaxanthin in the thylakoid membrane upon formation of a transthylakoid pH gradient. We investigated the developmental expression of VDE in leaves of mature tobacco (Nicotiana tabacum) plants grown under high-light conditions (in the field) and low-light conditions (in a growth chamber). The difference in light conditions was evident by the increased pool size (violaxanthin + antheraxanthin + zeaxanthin, VAZ) throughout leaf development in field-grown plants. VDE activity based on chlorophyll or leaf area was low in the youngest leaves, with the levels increasing with increasing leaf age in both high- and low-light-grown plants. However, in high-light-grown plants, the younger leaves in early leaf expansion showed a more rapid increase in VDE activity and maintained higher levels of VDE transcript in more leaves, indicating that high light may induce greater levels of VDE. VDE transcript levels decreased substantially in leaves of mid-leaf expansion, while the levels of enzyme continued to increase, suggesting that the VDE enzyme does not turn over rapidly. The level of VDE changed in an inverse, nonlinear relationship with respect to the VAZ pool, suggesting that enzyme levels could be indirectly regulated by the VAZ pool.

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 (pKa= 4.2) the true substrate (Km= 0.1 mM) 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.