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     

Inhibition of zeaxanthin formation and of rapid changes in radiationless energy dissipation by dithiothreitol in spinach leaves and chloroplasts

Dithiothreitol, which completely inhibits the de-epoxidation of violaxanthin to zeaxanthin, was used to obtain evidence for a causal relationship between zeaxanthin and the dissipation of excess excitation energy in the photochemical apparatus in Spinicia oleracea L. In both leaves and chloroplasts, inhibition of zeaxanthin formation by dithiothreitol was accompanied by inhibition of a component of nonphotochemical fluorescence quenching. This component was characterized by a quenching of instantaneous fluorescence (F_o) and a linear relationship between the calculated rate constant for radiationless energy dissipation in the antenna chlorophyll and the zeaxanthin content. In leaves, this zeaxanthin-associated quenching, which relaxed within a few minutes upon darkening, was the major component of nonphotochemical fluorescence quenching determined in the light, i.e. it represented the `high-energy-state' quenching. In isolated chloroplasts, the zeaxanthin-associated quenching was a smaller component of total nonphotochemical quenching and there was a second, rapidly reversible high-energy-state component of fluorescence quenching which occurred in the absence of zeaxanthin and was not accompanied by F_o quenching. Leaves, but not chloroplasts, were capable of maintaining the electron acceptor, Q, of photosystem II in a low reduction state up to high degrees of excessive light and thus high degrees of nonphotochemical fluorescence quenching. When ascorbate, which serves as the reductant for violaxanthin de-epoxidation, was added to chloroplast suspensions, zeaxanthin formation at low photon flux densities was stimulated and the relationship between nonphotochemical fluorescence quenching and the reduction state in chloroplasts then became more similar to that found in leaves. We conclude that the inhibition of zeaxanthin-associated fluorescence quenching by dithiothreitol provides further evidence that there exists a close relationship between zeaxanthin and potentially photoprotective dissipation of excess excitation energy in the antenna chlorophyll.     

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.     

Linear models relating xanthophylls and lumen acidity to non-photochemical fluorescence quenching. Evidence that antheraxanthin explains zeaxanthin-independent quenching

Zeaxanthin has been correlated with high-energy non-photochemical fluorescence quenching but whether antheraxanthin, the intermediate in the pathway from violaxanthin to zeaxanthin, also relates to quenching is unknown. The relationships of zeaxanthin, antheraxanthin and pH to fluorescence quenching were examined in chloroplasts ofPisum sativum L. cv. Oregon andLactuca sativa L. cv. Romaine. Data matrices as five levels of violaxanthin de-epoxidation against five levels of light-induced lumen-proton concentrations were obtained for both species. The matrices included high levels of antheraxanthin as well as lumen-proton concentrations induced by subsaturating to saturation light levels. Analyses of the matrices by simple linear and multiple regression showed that quenching is predicted by models where the major independent variable is the product of lumen acidity and de-epoxidized xanthophylls, the latter as the sum of zeaxanthin and antheraxanthin. The interactions of lumen acidity and xanthophyll concentration are shown in three-dimensional plots of the best-fit multiple regression models. Antheraxanthin apparently contributes to quenching as effectively as zeaxanthin and explains quenching previously not accounted for by zeaxanthin. Hence, we propose that all high-energy dependent quenching is xanthophyll dependent. Quenching requires a threshold lumen pH that varies with xanthophyll composition. After the threshold, quenching is linear with lumen acidity or xanthophyll composition.     

Mehler-peroxidase reaction mediates zeaxanthin formation and zeaxanthin-related fluorescence quenching in intact chloroplasts

Induction of zeaxanthin formation and the associated nonphotochemical quenching in iodoacetamide-treated, non-CO₂-fixing intact chloroplasts of Lactuca sativa L. cv Romaine is reported. The electron transport needed to generate the required ΔpH for zeaxanthin formation and nonphotochemical quenching are ascribed to the Mehler-ascorbate peroxidase reaction. KCN, an inhibitor of ascorbate peroxidase, significantly affected these activities without affecting linear electron transport to methyl viologen or violaxanthin deepoxidase activity. At 1 millimolar KCN, zeaxanthin formation and ΔpH were inhibited 60 and 55%, respectively, whereas ascorbate peroxidase activity was inhibited almost totally. The KCN-resistant activity, which apparently was due to electron transport mediated by the Mehler reaction alone, however, was insufficient to support a high level of nonphotochemical quenching. We suggest that in vivo, as CO₂ fixation becomes limiting, the Mehler-peroxidase reaction protects photosystem II against the excess light by supporting the electron transport needed for zeaxanthin-dependent nonphotochemical quenching and concomitantly scavenging H₂O₂. Ascorbate is essential for this process to occur.     

The Kinetics of Zeaxanthin Formation Is Retarded by Dicyclohexylcarbodiimide

The de-epoxidation of violaxanthin to antheraxanthin (Anth) and zeaxanthin (Zeax) in the xanthophyll cycle of higher plants and the generation of nonphotochemical fluorescence quenching in the antenna of photosystem II (PSII) are induced by acidification of the thylakoid lumen. Dicyclohexylcarbodiimide (DCCD) has been shown (a) to bind to lumen-exposed carboxy groups of antenna proteins and (b) to inhibit the pH-dependent fluorescence quenching. The possible influence of DCCD on the de-epoxidation reactions has been investigated in isolated pea (Pisum sativum L.) thylakoids. The Zeax formation was found to be slowed down in the presence of DCCD. The second step (Anth → Zeax) of the reaction sequence seemed to be more affected than the violaxanthin → Anth conversion. Comparative studies with antenna-depleted thylakoids from plants grown under intermittent light and with unstacked thylakoids were in agreement with the assumption that binding of DCCD to antenna proteins is probably responsible for the retarded kinetics. Analyses of the DCCD-induced alterations in different antenna subcomplexes showed that Zeax formation in the PSII antenna proteins was predominantly influenced by DCCD, whereas Zeax formation in photosystem I was nearly unaffected. Our data support the suggestion that DCCD binding to PSII antenna proteins is responsible for the observed alterations in xanthophyll conversion.     

Dynamics of Xanthophyll-Cycle Activity in Different Antenna Subcomplexes in the Photosynthetic Membranes of Higher Plants (The Relationship between Zeaxanthin Conversion and Nonphotochemical Fluorescence Quenching)

The generation of nonphotochemical quenching of chlorophyll fluorescence (qN) in the antenna of photosystem II (PSII) is accompanied by the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin. The function of zeaxanthin in two mechanisms of qN, energy-dependent quenching (qE) and photoinhibitory quenching (qI), was investigated by measuring the de-epoxidation state in the antenna subcomplexes of PSII during the generation and relaxation of qN under varying conditions. Three different antenna subcomplexes were separated by isoelectric focusing: Lhcb1/2/3, Lhcb5/6, and the Lhcb4/PSII core. Under all conditions, the highest de-epoxidation state was detected in Lhcb1/2/3 and Lhcb5/6. The kinetics of de-epoxidation in these complexes were found to be similar to the formation of qE. The Lhcb4/PSII core showed the most pronounced differences in the de-epoxidation state when illumination with low and high light intensities was compared, correlating roughly with the differences in qI. Furthermore, the epoxidation kinetics in the Lhcb4/PSII core showed the most pronounced differences of all subcomplexes when comparing the epoxidation after either moderate or very strong photoinhibitory preillumination. Our data support the suggestion that zeaxanthin formation/epoxidation in Lhcb1-3 and Lhcb5/6 may be related to qE, and in Lhcb4 (and/or PSII core) to qI.     

Cold acclimation and photoinhibition of photosynthesis in Scots pine

Cold acclimation of Scots pine did not affect the susceptibility of photosynthesis to photoinhibition. Cold acclimation did however cause a suppression of the rate of CO₂ uptake, and at given light and temperature conditions a larger fraction of the photosystem II reaction centres were closed in cold-acclimated than in nonacclimated pine. Therefore, when assayed at the level of photosystem II reaction centres, i.e. in relation to the degree of photosystem closure, cold acclimation caused a significant increase in resistance to photoinhibition; at given levels of photosystem II closure the resistance to photoinhibition was higher after cold acclimation. This was particularly evident in measurements at 20° C. The amounts and activities of the majority of analyzed active oxygen scavengers were higher after cold acclimation. We suggest that this increase in protective enzymes and compounds, particularly Superoxide dismutase, ascorbate peroxidase, glutathione reductase and ascorbate of the chloroplasts, enables Scots pine to avoid excessive photoinhibition of photosynthesis despite partial suppression of photosynthesis upon cold acclimation. An increased capacity for light-induced de-epoxidation of violaxanthin to zeaxanthin upon cold acclimation may also be of significance.Abbreviations APX ascorbate peroxidase - DHA dehydroascorbate - DHAR dehydroascorbate reductase - Fm maximal fluorescence when all reaction centres are closed - Fv/Fm maximum photochemical yield of PSII - GR glutathione reductase - GSH reduced glutathione - Je rate of photosynthetic electron transport - MDAR monodehydroascorbate reductase - q_N nonphotochemical quenching of fluorescence - q_P photochemical quenching of fluorescence - SOD superoxide dismutase This work was supported by the Swedish Natural Science Research Council and the National Natural Science Foundation of China.     

Expression of vde Gene Integrated Into Tobacco Genome in Antisense and Overexpressed Ways

Violaxanthin de-epoxidase (VDE) is localised in the thylakoid lumen of chloroplasts and catalyses de-epoxidation of violaxanthin into antheraxanthin and zeaxanthin. Tobacco vde gene was inserted into a binary vector pCAMBIA1301 with the hygromycin resistant gene for selection in antisense and overexpressed ways. Two constructs with antisense and overexpressed vde gene were introduced in tobacco (Nicotiana tabacum L.) using Agrobacterium tumefaciens strain LBA4404, PCR and Southern blot analyses demonstrated that the exogenous gene was integrated into genome of tobacco plants. VDE activity assay and HPLC analysis of pigments showed that the vde gene was expressed in the overexpressed transformants, whereas suppressed in the antisense ones. The chlorophyll fluorescence measurements proved that the contents of VDE in transgenic plants have a significant function in non-photochemical quenching.     

A pronounced light-induced zeaxanthin formation accompanied by an unusually slight increase in non-photochemical quenching: a study with barley leaves treated with methyl viologen at moderate light

Light-induced deepoxidation of violaxanthin to antheraxanthin and zeaxanthin in plants is associated with the induction of pronounced xanthophyll-dependent non-photochemical quenching (NPQ). To date, a misbalance between a high amount of zeaxanthin in thylakoid membranes and low NPQ has been explained by an absence of lumen acidification (e.g. when NPQ is measured in the dark after high light stress). In this study, we report that this misbalance can also be observed under moderate light. We found this result (deepoxidation state, DEPS, above 55% and NPQ0.9) in barley leaves treated with 10 μM methyl viologen (MV) under white light (100 μmol photons m⁻² s⁻¹, photosynthetically active radiation (PAR), growth irradiance). The addition of MV at this moderate light did not accelerate electron transport in thylakoid membranes, and induced only slight oxidative stress (no lipid peroxidation, almost unchanged maximum yield of photosystem II photochemistry, a decrease in activity of ascorbate peroxidase, and an increase in that of glutathion reductase). We suggest that, in leaves treated under the conditions used here, the lumen acidification induced by light-limited electron transport in thylakoid membranes was high enough to activate violaxanthin deepoxidase, but not sufficiently high to form the expected number of zeaxanthin-dependent quenching centers in photosystem II antennae.     

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.     

Relationship between xanthophyll cycle and non-photochemical quenching in rice (Oryza sativa L.) plants in response to light stress

Thirty days old rice plants grown under low and moderate light conditions were transferred to full sunlight to observe the extent of photoinhibitory damage and protective mechanism, and the relationship between xanthophyll cycle and nonphotochemical quenching (qN) under changing light environment. Control plants (low, moderate and sun grown) exhibited similar Fv/Fm ratio, indicating similar photosynthetic efficiency prior to light stress. On exposure to the high light treatment, low light grown plants exhibited faster and higher degree of photoinhibition compared to moderate and high light grown plants. Moderate and high light grown plants showed relatively less photoinhibition and also showed higher qN, indicating better capacity of energy dissipation. Increase in qN in moderate light and sun grown plants was accompanied by conversion of violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z) indicating operation of Z-dependent thermal dissipation. Rice plants fed with ascorbate (AsA), a stimulator of the de-epoxidation state of V to Z, showed higher Fv/Fm ratio and qN than the plants fed with dithiothreitol (DTT) an inhibitor of xanthophyll cycle. This indicated that an increased amount of energy reached PS II reaction centre, due to absence of A and Z formation, thereby causing greater damage to photosynthesis in DTT fed rice plants. The present data confirmed the relationship between qN and Z in dissipating the excess light energy, thereby protecting plants against photodamage.     

Characterization of a nonphotochemical quenching-deficient Arabidopsis mutant possessing an intact PsbS protein, xanthophyll cycle and lumen acidification

Arabidopsis thaliana plants grown from ethyl methane sulfonate-treated seeds were screened for so-called que mutants, which are affected in non-photochemical energy quenching. Based on video imaging of chlorophyll fluorescence an energy dissipation mutant, que1, was identified, isolated and characterized. Similar to the npq mutants, the que1 mutant showed a drastically reduced capacity for pH-dependent energy dissipation, qE, but without affecting the Δ pH-dependent conformational changes at 535 nm (ΔA ₅₃₅), which have been supposed to be obligatorily correlated with qE and to reflect pH-regulated binding of zeaxanthin to the PsbS protein. Western blot and DNA sequence analysis revealed that neither a reduced expression of the PsbS protein nor a mutation in the PsbS gene was responsible for the missing qE in que1. Measurements of 9-aminoacridine fluorescence quenching showed that the acidification of the thylakoid lumen was also not affected in the mutant. Furthermore, que1 was able to convert violaxanthin to zeaxanthin. However, unusual characteristics of zeaxanthin formation in the mutant pointed at an altered availability of violaxanthin for de-epoxidation. This was further accompanied by a decrease of the photochemical quenching of chlorophyll fluorescence (qP), an increase of the portion of oxidized P700 and a reduction of the electron transport rate. These characteristics indicate changes in the organization of the thylakoid membrane that affect linear electron transport (but not lumen acidification) and the formation of energy dissipation in photosystem II. Preliminary genetic analysis revealed that the phenotype of que1 is related to two different mutations, mapped to the lower arms of chromosomes 1 and 4.     

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.     

The xanthophyll cycle of Mantoniella squamata converts violaxanthin into antheraxanthin but not to zeaxanthin: consequences for the mechanism of enhanced non-photochemical energy dissipation

The prasinophycean alga Mantoniella squamata uses in vivo an incomplete violaxanthin cycle. Although the violaxanthin cycle in Mantoniella is capable of converting violaxanthin to zeaxanthin, in intact cells only antheraxanthin accumulates during periods of strong illumination. Antheraxanthin enhances non-photochemical quenching of chlorophyll fluorescence. Inhibition of antheraxanthin synthesis by the de-epoxidase inhibitor dithiothreitol abolishes increased thermal energy dissipation. Antheraxanthin-dependent non-photochemical quenching, like zeaxanthin-mediated non-photochemical quenching in higher plants, is uncoupler-sensitive. Mantoniella squamata cells cultivated at high light intensities contain higher amounts of violaxanthin than cells grown at low light. The increased violaxanthin-cycle pool size in high-light-grown Mantoniella cells is accompanied by higher de-epoxidation rates in the light and by a greater capacity to quench chlorophyll fluorescence non-photochemically. Antheraxanthin-dependent amplification of non-photochemical quenching is discussed in the light of recent models developed for zeaxanthin- and diatoxanthin-mediated enhanced heat dissipation. Received: 4 September 1997 / Accepted: 22 December 1997     

Seasonal changes of violaxanthin cycle pigment de-epoxidation in wintergreen and evergreen plants

We studied carotenoids composition and the activities of the xanthophylls pigments in evergreen conifers (Abies sibirica, Juniperus communis, Picea obovata) and dwarf-shrub (Vaccinium vitis-idaea), and in wintergreen herbaceous plants (Ajuga reptans, Pyrola rotundifolia) growing near Syktyvkar (61°67(/) N 50°77(/) E). The carotenoid pool consisted mainly of following xanthophylls: lutein (70%), neoxanthin (7-10%) and a xanthophylls cycle component - violaxanthin (3-15%). Zeaxanthin and antheraxanthin were found in conifer samples collected in December-March while in other species - during all year. A direct connection between xanthophyll pigment de-epoxidation level and light energy thermal dissipation was shown only for boreal conifer species. It is proposed that zeaxanthin plays a central role in the dissipation of excess excitation energy (nonphotochemical quenching) in the antenna of photosystem II (PSII). We conclude that the increase in the extent of de-epoxidation is beneficial for the retention of PSII activity for conifers in early spring and for herbs in summer.     

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.     

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.     

Inhibition of violaxanthin deepoxidation by ultraviolet-B radiation in isolated chloroplasts and intact leaves

The effect of pretreatment with ultraviolet-B (UV-B) light (280-320 nanometers) on the enzymatic conversion of the diepoxyxanthophyll violaxanthin to the epoxy-free zeaxanthin occurring in thylakoid membranes was investigated. When isolated chloroplasts of pea (Pisum sativum) were exposed to UV-B, a biologically effective fluence of 7000 joules per square meter caused about 50% inhibition of the activity of the violaxanthin deepoxidase, measured as the first order rate constant of the absorbance change at 505 nanometers. The dose requirement for the inhibition of the deepoxidase in intact leaves, however, was about 2 orders of magnitude higher. The inhibition of the rate constant was observed for both the dark deepoxidation at pH 5, and for the light-driven deepoxidation induced by the lumen acidification due to electron transport from H₂O to methylviologen or due to a photosystem I partial reaction with duroquinol as the electron donor. The availability of violaxanthin was not directly affected by UV-B radiation, as shown for UV-B-treated chloroplasts by the final extent of the 505 nanometer change measured in the dark at pH 5 or by the partial photosystem I reaction. A significant decrease in the violaxanthin availability was observed when lumen acidification was caused by electron transport from H₂O to methylviologen. That effect was probably caused by the wellknown UV-B inhibition of photosystem II with a subsequent decreased ability to reduce the plastoquinone pool, the redox state of which is believed to regulate the final amount of converted violaxanthin.