Activation of Diadinoxanthin De-Epoxidase Due to a Chiororespiratory Proton Gradient in the Dark in the Diatom Phaeodactylum tricornutum

Abstract: An exponential dynamic light regime with prolonged dark periods (light/dark cycle 8/40 h) was used to simulate deep mixing of algae in natural waters and to investigate the adaptation of the diatom Phaeodactylum tricornutum to these extreme light conditions. After prolonged dark periods Phaeo dactylum cells showed surprisingly high contents of diatoxan-thin, low photosynthetic efficiency and high non-photochemical quenching (NPQ) of chlorophyll fluorescence. Diatoxanthin con centrations and NPQ were low at the beginning of the dark peri od and increased with the duration of the dark incubation. Addi tion of the diadinoxanthin de-epoxidase inhibitor, DTT, prevent ed the formation of diatoxanthin, thereby excluding de novo synthesis of diatoxanthin during the prolonged dark period. Evi dence of chlororespiratory electron flow and the establishment of a diadinoxanthin de-epoxidase activating proton gradient in the dark was derived from two observations. First, uncoupling of electron transport with NH₄CI at the beginning of the dark period prevented the development of non-photochemical quenching of chlorophyll fluorescence and the formation of diatoxanthin during the dark period. Second, inhibition of the electron and proton consuming terminal redox component of chlororespiratory electron transport, cytochrome oxidase, by addition of KCN induced stronger NPQ and a higher de-epoxidation state of the xanthophyll cycle. These results strongly indi cate that the activation of diadinoxanthin de-epoxidase in the dark is the consequence of a chlororespiratory proton gradient. We furthermore present evidence that diatoxanthin formed by the chlororespiratory proton gradient has the same efficiency in the mechanism of enhanced heat dissipation as diatoxanthin induced by a light-driven ApH.     

The regulation of xanthophyll cycle activity and of non-photochemical fluorescence quenching by two alternative electron flows in the diatoms Phaeodactylum tricornutum and Cyclotella meneghiniana

Intact cells of diatoms are characterized by a rapid diatoxanthin epoxidation during low light periods following high light illumination while epoxidation is severely restricted in phases of complete darkness. The present study shows that rapid diatoxanthin epoxidation is dependent on the availability of the cofactor of diatoxanthin epoxidase, NADPH, which cannot be generated in darkness due to the inactivity of PSI. In the diatom Phaeodactylum tricornutum, NADPH production during low light is dependent on PSII activity, and addition of DCMU consequently abolishes diatoxanthin epoxidation. In contrast to P. tricornutum, DCMU does not affect diatoxanthin epoxidation in Cyclotella meneghiniana, which shows the same rapid epoxidation in low light both in the absence or presence of DCMU. Measurements of the reduction state of the PQ pool and PSI activity indicate that, in the presence of DCMU, NADPH production in C. meneghiniana occurs via alternative electron transport, which includes electron donation from the chloroplast stroma to the PQ pool and, in a second step, from PQ to PSI. Similar electron flow to PQ is also observed during high light illumination of DCMU-treated P. tricornutum cells. In contrast to C. meneghiniana, the electrons are not directed to PSI, but most likely to a plastoquinone oxidase. This chlororespiratory electron transport leads to the establishment of an uncoupler-sensitive proton gradient in the presence of DCMU, which induces diadinoxanthin de-epoxidation and NPQ. In C. meneghiniana, electron flow to the plastoquinone oxidase is restricted, and consequently, diadinoxanthin de-epoxidation and NPQ is not observed after addition of DCMU.     

The lipid dependence of diadinoxanthin de-epoxidation presents new evidence for a macrodomain organization of the diatom thylakoid membrane

The present study shows that thylakoid membranes of the diatom Cyclotella meneghiniana contain much higher amounts of negatively charged lipids than higher plant or green algal thylakoids. Based on these findings, we examined the influence of SQDG on the de-epoxidation reaction of the diadinoxanthin cycle and compared it with results from the second negatively charged thylakoid lipid PG. SQDG and PG exhibited a lower capacity for the solubilization of the hydrophobic xanthophyll cycle pigment diadinoxanthin than the main membrane lipid MGDG. Although complete pigment solubilization took place at higher concentrations of the negatively charged lipids, SQDG and PG strongly suppressed the de-epoxidation of diadinoxanthin in artificial membrane systems. In in vitro assays employing the isolated diadinoxanthin cycle enzyme diadinoxanthin de-epoxidase, no or only a very weak de-epoxidation reaction was observed in the presence of SQDG or PG, respectively. In binary mixtures of the inverted hexagonal phase forming lipid MGDG with the negatively charged bilayer lipids, comparable suppression took place. This is in contrast to binary mixtures of MGDG with the neutral bilayer lipids DGDG and PC, where rapid and efficient de-epoxidation was observed. In complex lipid mixtures resembling the lipid composition of the native diatom thylakoid membrane, we again found strong suppression of diadinoxanthin de-epoxidation due to the presence of SQDG or PG. We conclude that, in the native thylakoids of diatoms, a strict separation of the MGDG and SQDG domains must occur; otherwise, the rapid diadinoxanthin de-epoxidation observed in intact cells upon illumination would not be possible.     

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     

Role of hexagonal structure-forming lipids in diadinoxanthin and violaxanthin solubilization and de-epoxidation

In this study, we have examined the influence of different lipids on the solubility of the xanthophyll cycle pigments diadinoxanthin (Ddx) and violaxanthin (Vx) and on the efficiency of Ddx and Vx de-epoxidation by the enzymes Vx de-epoxidase (VDE) from wheat and Ddx de-epoxidase (DDE) from the diatom Cyclotella meneghiniana, respectively. Our results show that the lipids MGDG and PE are able to solubilize both xanthophyll cycle pigments in an aqueous medium. Substrate solubilization is essential for de-epoxidase activity, because in the absence of MGDG or PE Ddx and Vx are present in an aggregated form, with limited accessibility for DDE and VDE. Our results also show that the hexagonal structure-forming lipids MGDG and PE are able to solubilize Ddx and Vx at much lower lipid concentrations than bilayer-forming lipids DGDG and PC. We furthermore found that, in the presence of MGDG or PE, Ddx is much more solubilizable than Vx. This substantial difference in Ddx and Vx solubility directly affects the respective de-epoxidation reactions. Ddx de-epoxidation by the diatom DDE is saturated at much lower MGDG or PE concentrations than Vx de-epoxidation by the higher-plant VDE. Another important result of our study is that bilayer-forming lipids DGDG and PC are not able to induce efficient xanthophyll de-epoxidation. Even in the presence of high concentrations of DGDG or PC, where Ddx and Vx are completely solubilized, a strongly inhibited Ddx de-epoxidation is observed, while Vx de-epoxidation by VDE is completely absent. This indicates that the inverted hexagonal phase domains provided by lipid MGDG or PE are essential for de-epoxidase activity. We conclude that in the natural thylakoid membrane MGDG serves to solubilize the xanthophyll cycle pigments and furthermore provides inverted hexagonal structures associated with the membrane bilayer, which are essential for efficient xanthophyll de-epoxidase activity.     

The super-excess energy dissipation in diatom algae: comparative analysis with higher plants

When grown at intermittent light regime, diatom alga Phaeodactylum tricornutum is able to form photoprotective non-photochemical chlorophyll fluorescence quenching (NPQ) three to five times larger than that observed in the higher plants. This quenching is sustained in the dark for 5 to 10 min, reverses completely within approximately 1 h and seems to be very tightly related to the presence of the zeaxanthin analogue, diatoxanthin. Addition of the uncoupler NH4Cl before illumination can completely abolish formation of NPQ, revealing the ΔpH-dependency of the xanthophyll cycle activity. Once established, NPQ can also be almost completely reversed by the uncoupler. However, the higher NPQ is formed the more time is required for its reversal. At the point when the fluorescence was approximately 90% recovered the level of illumination-induced diatoxanthin was found to be only partially reduced. This indicates that the proton gradient is a key triggering factor of NPQ. It was also noticed that NPQ in Phaeodactylum cells was absent even when majority of reaction centers were closed and the plastoquinone pool was significantly reduced. The absence of NPQ at these conditions could be due to very low levels of ΔpH. It is likely that in diatoms alternative sources of protons such as the PS I cyclic electron transfer and/or chlororespiration are important in generating the proton gradient sufficient to trigger NPQ. Absorption changes associated with the xanthophyll cycle activity were found to be larger than those for higher plants. The position of the positive maximum in the difference spectrum illuminated-minus-dark was 512–514 nm in comparison to the 505–508 nm for leaves. The 535 nm band associated with NPQ in plants is absent in Phaeodactylum. An uncoupler-sensitive absorption change at 522 nm was discovered. Kinetics of NPQ showed linear correlation with the 522 nm absorption change. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

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.     

SHORT-TERM RESPONSE OF THE DIADINOXANTHIN CYCLE AND FLUORESCENCE YIELD TO HIGH IRRADIANCE IN CHAETOCEROS MUELLERI (BACILLARIOPHYCEAE)1

The relationship between the diadinoxanthin cycle and changes in fluorescence yield in the diatom Chaetoceros muelleri Lemm. (clone CH10, Amorient Aquafarm, Inc., Hawaii) was investigated. High-light-induced changes in fluorescence yield and xanthophyll de-epoxidation occurred very rapidly (first order rate constant 1.60 min^?1). The observed light-induced changes in diatoxanthin and diadinoxanthin concentration were consistent with a two-pool scheme for diadinoxanthin, one of which does not undergo de-epoxidation. Changes in xanthophyll concentration correlated with changes in in vivo absorbance indicating that diadinoxanthin cycle activity in vivo can be monitored spectrophotometrically. However, changes in cell absorbance were small relative to total optical absorption cross section. Increases in the concentration of diatoxanthin were linearly correlated with increases in the rate constant for thermal de-excitation in the antenna of photosystem II (PSII). Antenna quenching produced or mediated by diatoxanthin may, thus, protect the PSII reaction center in diatoms. Changes in the maximum fluorescence yield suggested that changes in the reaction center also contributed to nonphotochemical quenching of fluorescence. Thus, reaction center quenching affected the relationship between antenna quenching and changes in photochemical efficiency producing the effect of a decrease in fluorescence yield without a decrease in photochemical efficiency.     

Supplementary ultraviolet-B radiation induces a rapid reversal of the diadinoxanthin cycle in the strong light-exposed diatom Phaeodactylum tricornutum

A treatment of the diatom Phaeodactylum tricornutum with high light (HL) in the visible range led to the conversion of diadinoxanthin (Dd) to diatoxanthin (Dt). In a following treatment with HL plus supplementary ultraviolet (UV)-B, the Dt was rapidly epoxidized to Dd. Photosynthesis of the cells was inhibited under HL + UV-B. This is accounted for by direct damage by UV-B and damage because of the UV-B-induced reversal of the Dd cycle and the associated loss of photoprotection. The reversal of the Dd cycle by UV-B was faster in the presence of dithiothreitol, an inhibitor of the Dd de-epoxidase. Our results imply that the reversal of the Dd cycle by HL + UV-B was caused by an increase in the rate of gross Dt epoxidation, whereas the de-epoxidation of Dd was unaffected by UV-B. This is further supported by our finding that the in vitro de-epoxidation activity and the affinity toward the cosubstrate ascorbic acid of the Dd de-epoxidase were both unaffected by UV-B pretreatment of intact cells. We provide evidence that Dt epoxidation is normally down-regulated by a high pH gradient under HL. It is proposed that supplementary UV-B affected the pH gradient across the thylakoid membrane, which disrupted the down-regulation of Dt epoxidation and led to the observed increase in the rate of Dt epoxidation.     

Xanthophyll synthesis in diatoms: quantification of putative intermediates and comparison of pigment conversion kinetics with rate constants derived from a model

Recently, we reported the presence of the violaxanthin-antheraxanthin-zeaxanthin cycle in diatoms, and showed that violaxanthin is the putative precursor of both diadinoxanthin and fucoxanthin in the diatom Phaeodactylum tricornutum Bohlin (M. Lohr and C. Wilhelm, 1999, Proc. Natl. Acad. Sci. USA 96: 8784–8789). In the present study, two possible intermediates in the synthesis of violaxanthin from β-carotene were identified in P. tricornutum, namely β-cryptoxanthin and β-cryptoxanthin epoxide. In low light, the latter pigment prevails, but in high light β-cryptoxanthin accumulates, probably as the result of an increased activity of the xantophyll-cycle de-epoxidase. The apparent kinetics of several xanthophyll conversion steps were determined for P. tricornutum and Cyclotella meneghiniana Kützing. The experimentally determined conversion rates were used to evaluate the hypothetical pathway of xanthophyll synthesis in diatoms. For this purpose a mathematical model was developed which allows the calculation of theoretical rates of pigment conversion for microalgae under steady-state growth conditions. A comparison between measured and calculated conversion rates agreed well with the proposal of a sequential synthesis of fucoxanthin via violaxanthin and diadinoxanthin. The postulation of zeaxanthin as an obligatory intermediate in the synthesis of violaxanthin, however, resulted in large discrepancies between the measured and calculated rates of its epoxidation. Instead of zeaxanthin, β-cryptoxanthin epoxide may be involved in the biosynthesis of violaxanthin in diatoms. Received: 16 March 2000 / Accepted: 30 June 2000     

Indole-3-acetic acid attenuates the fungal lesions in infected potato tubers

In this study, the enzyme activity of partially purified diadinoxanthin de-epoxidase (DDE) from the diatom Cyclotella meneghiniana was investigated at different ascorbate concentrations and pH values. In comparison with spinach violaxanthin de-epoxidase (VDE), we found a much higher affinity of the enzyme for the co-substrate ascorbate. The K_m value of DDE at pH 5 (0.7 m M ) was significantly lower than that observed for VDE (2.3 m M ). The pH-optimum of DDE activity was found at pH 5 at low ascorbate concentrations. At high ascorbate concentrations, we observed a strong shift of the pH optimum towards higher pH values, and significant DDE activity was still present at almost neutral pH values. This is in contrast to VDE, where despite a slight shift towards higher pH values, enzyme activity was never observed above pH 6.5. The pH optimum of VDE was always found in a narrow range between pH 5 and 5.2, irrespective of the presence of high or low ascorbate concentrations. The high affinity of DDE for ascorbate indicates that, even at a limited availability of reduced ascorbate, high enzyme activity is possible at low pH values. At high ascorbate concentrations, on the other hand, DDE activity can be shifted towards neutral pH values, thereby facilitating a very fast and strong response to small pH changes in the thylakoid lumen. The importance of the high ascorbate affinity of DDE for the physiology of intact diatom cells is discussed.     

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     

Violaxanthin de-epoxidase. Lipid composition and substrate specificity.

Violaxanthin de-epoxidase isolated from lettuce chloroplasts (Lactuca sativa var. Romaine) contained a single lipid component, monogalactosyldiglyceride (MG) at about 8 g per 100 g protein. The effects of MG on activation of solvent-extracted enzyme and on K_m suggest that MG has two roles, namely, as a functional component of the binding site and as a substrate-solubilizing agent whose structure satisfies binding site requirements. Substrate specificity examined with various naturally occurring and semisynthetic epoxy carotenoids with known chirality showed violaxanthin de-epoxidase to be stereospecific for 3-hydroxy, 5,6-epoxy carotenoids which are in a 3S, 5R, 6S configuration. Although monoepoxides with the above configuration were active, their rates varied, apparently due to the influence of structural differences in the nonepoxide end groups. Hence while all-trans neoxanthin showed low rates, the de-epoxidation rate of antheraxanthin was 5-fold higher than violaxanthin. Neoxanthin and violeoxanthin, both naturally occurring pigments with 9-cis configurations in the acyclic polyene chain, were inactive. These effects support the view that violaxanthin de-epoxidase is a mono de-epoxidase and that the stereospecific active center is situated in a narrow well-like cavity which favors an all-trans configuration of the polyene chain. The 3-hydroxy, 5,6-epoxy group of the naturally occurring pigments, diadinoxanthin, antheraxanthin, and β-cryptoxanthin epoxide are assumed to be the 3S, 5R, 6S configuration based on their reactivity with violaxanthin de-epoxidase.     

Regulation of violaxanthin de-epoxidase activity by pH and ascorbate concentration

The activity of violaxanthin de-epoxidase has been studied both in isolated thylakoids and after partial purification, as a function of pH and ascorbate concentration. We demonstrate that violaxanthin de-epoxidase has a K_m for ascorbate that is strongly dependent on pH, with values of 10, 2.5, 1.0 and 0.3 mM at pH 6.0, 5.5, 5.0 and 4.5, respectively. These values can be expressed as a single K_m±0.1±0.02 mM for the acid form of ascorbate. Release of the protein from the thylakoids by sonication was also found to be strongly pH dependent with a cooperativity of 4 with respect to protons and with an inflexion point at pH 6.7. These results can explain some of the discrepancies reported in the literature and provide a more consistent view of zeaxanthin formation in vivo.     

The light-harvesting antenna of the diatom Phaeodactylum tricornutum. Evidence for a diadinoxanthin-binding subcomplex

Diatoms differ from higher plants by their antenna system, in terms of both polypeptide and pigment contents. A rapid isolation procedure was designed for the membrane-intrinsic light harvesting complexes (LHC) of the diatom Phaeodactylum tricornutum to establish whether different LHC subcomplexes exist, as well to determine an uneven distribution between them of pigments and polypeptides. Two distinct fractions were separated that contain functional oligomeric complexes. The major and more stable complex ( approximately 75% of total polypeptides) carries most of the chlorophyll a, and almost only one type of carotenoid, fucoxanthin. The minor complex, carrying approximately 10-15% of the total antenna chlorophyll and only a little chlorophyll c, is highly enriched in diadinoxanthin, the main xanthophyll cycle carotenoid. The two complexes also differ in their polypeptide composition, suggesting specialized functions within the antenna. The diadinoxanthin-enriched complex could be where the de-epoxidation of diadinoxanthin into diatoxanthin mostly occurs.     

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.     

Influence of Ca2+ on the thylakoid lumen violaxanthin de-epoxidase activity through Ca2+ gating of H+ flux at the CFo 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 (Δ~μ_H+) energy coupling. The most clearly identified factor regulating switching between localized and delocalized energy coupling modes is Ca²⁺ binding to the lumen side of the thylakoid membrane; in particular, Ca²⁺ 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 Ca²⁺ from the CF_o H⁺ channel and thus to modulate the extent of lumenal acidification while maintaining a fairly constant rate of ATP formation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Stimulation of the diadinoxanthin cycle by UV-B radiation in the diatom Phaeodactylum tricornutum

In this study we show that the diadinoxanthin cycle in the diatom Phaeodactylum tricornutum is stimulated by mild UV-B radiation. High steady state concentrations of diatoxanthin established during a period of strong actinic illumination with white light (300 mol photons m-2 s-1 PAR) are further increased if weak UV-B (3 mol photons m-2 s-1) is additionally applied. Short term increases in the diatoxanthin concentration caused by UV-B strongly correlate with a stoichiometric decrease in diadinoxanthin. The UV-B dependent increase in diatoxanthin is correlated with a concommitant enhancement of non-photochemical quenching of chlorophyll fluorescence and a decrease in the quantum efficiency of oxygen evolution. This indicates that UV-B induced diatoxanthin functions in thermal energy dissipation. Possible scenarios for a stimulation of the diadinoxanthin cycle by UV-B are discussed.