zeaxanthin and violaxanthin

Violaxanthin is biosynthesized from zeaxanthin with zeaxanthin epoxidase (ZEP) by way of antheraxanthin only in photosynthetic eukaryotes including higher plants and involved in the xanthophyll cycle to eliminate excessive light energy. Violaxanthin and antheraxanthin have commercially been unavailable, in contrast to commercial production of other carotenoids contained in higher plants, e.g., lycopene, β-carotene, lutein, zeaxanthin, β-cryptoxanthin, and capsanthin. One of the reasons is considered that resource plants or other resource organisms do not exist for enabling efficient supply of the epoxy-carotenoids, which are expected to be produced through (metabolic) pathway engineering with heterologous microbial hosts such as Escherichia coli and Saccharomyces cerevisiae. In this Mini-Review, we show heterologous production of violaxanthin with the two microorganisms that have exhibited significant advances these days. We further describe natural function and occurrence, and biosynthesis involving violaxanthin, antheraxanthin, and their derivatives that include auroxanthin and mutatoxanthin. KEY POINTS: • A comprehensive review on epoxy-carotenoids violaxanthin and antheraxanthin. • Pathway engineering for the epoxy-carotenoids in heterologous microbes. • Our new findings on violaxanthin production with the budding yeast.

Topics: Carotenoids; Lutein; Xanthophylls; Zeaxanthins

Carotenoids are bioactive compounds with remarkably special properties produced by plants in response to internal and external stresses. In this review paper, we focus on the subject of carotenoid biosynthesis and several factors that have been reported to significantly enhance or reduce carotenoid accumulation in studied plant species. These factors include varietal aspects, location, growing season, and type of stress experienced by a plant. In addition, we propose that there are three stress resistance mechanisms in plants: avoidance, tolerance, and acclimation. Better understanding of the environmental factors affecting carotenoid biosynthesis will help researchers to develop methods for enhancing the production of carotenoids and other pigments to desired concentrations in plant crops.

Topics: Carotenoids; Environment; Oxidation-Reduction; Photosynthesis; Plants; Stress, Physiological; Xanthophylls; Zeaxanthins

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

Topics: Light-Harvesting Protein Complexes; Membrane Lipids; Plants; Thylakoids; Xanthophylls; Zeaxanthins

Life on earth depends on photosynthetic primary producers that exploit sunlight to fix CO

Topics: Biomass; Microalgae; Xanthophylls; Zeaxanthins

Carotenoids represent the first line of defence of photosystems against singlet oxygen (

Topics: Arabidopsis; Carotenoids; Chlorophyll; Energy Transfer; Light-Harvesting Protein Complexes; Lutein; Photosystem II Protein Complex; Xanthophylls; Zeaxanthins

The suppression of the HYD-1 gene by a TILLING approach increases the amount of β-carotene in durum wheat kernel. Vitamin A deficiency is a major public health problem that affects numerous countries in the world. As humans are not able to synthesize vitamin A, it must be daily assimilated along with other micro- and macronutrients through the diet. Durum wheat is an important crop for Mediterranean countries and provides a discrete amount of nutrients, such as carbohydrates and proteins, but it is deficient in some essential micronutrients, including provitamin A. In the present work, a targeting induced local lesions in genomes strategy has been undertaken to obtain durum wheat genotypes biofortified in provitamin A. In detail, we focused on the suppression of the β-carotene hydroxylase 1 (HYD1) genes, encoding enzymes involved in the redirection of β-carotene toward the synthesis of the downstream xanthophylls (neoxanthin, violaxanthin and zeaxanthin). Expression analysis of genes involved in carotenoid biosynthesis revealed a reduction of the abundance of HYD1 transcripts greater than 50% in mutant grain compared to the control. The biochemical profiling of carotenoid in the wheat mutant genotypes highlighted a significant increase of more than 70% of β-carotene compared to the wild-type sibling lines, with no change in lutein, α-carotene and zeaxanthin content. This study sheds new light on the molecular mechanism governing carotenoid biosynthesis in durum wheat and provides new genotypes that represent a good genetic resource for future breeding programs focused on the provitamin A biofortification through non-transgenic approaches.

Topics: Carotenoids; Edible Grain; Food, Fortified; Gene Knockout Techniques; Genotype; Metabolic Engineering; Mixed Function Oxygenases; Phylogeny; Plant Breeding; Provitamins; Seeds; Triticum; Vitamin A; Xanthophylls; Zeaxanthins

Plants use energy from the sun yet also require protection against the generation of deleterious photoproducts from excess energy. Photoprotection in green plants, known as nonphotochemical quenching (NPQ), involves thermal dissipation of energy and is activated by a series of interrelated factors: a pH drop in the lumen, accumulation of the carotenoid zeaxanthin (Zea), and formation of arrays of pigment-containing antenna complexes. However, understanding their individual contributions and their interactions has been challenging, particularly for the antenna arrays, which are difficult to manipulate in vitro. Here, we achieved systematic and discrete control over the array size for the principal antenna complex, light-harvesting complex II, using near-native in vitro membranes called nanodiscs. Each of the factors had a distinct influence on the level of dissipation, which was characterized by measurements of fluorescence quenching and ultrafast chlorophyll-to-carotenoid energy transfer. First, an increase in array size led to a corresponding increase in dissipation; the dramatic changes in the chlorophyll dynamics suggested that this is due to an allosteric conformational change of the protein. Second, a pH drop increased dissipation but exclusively in the presence of protein-protein interactions. Third, no Zea dependence was identified which suggested that Zea regulates a distinct aspect of NPQ. Collectively, these results indicate that each factor provides a separate type of control knob for photoprotection, which likely enables a flexible and tunable response to solar fluctuations.

Topics: Carotenoids; Chlorophyll; Energy Transfer; Hydrogen-Ion Concentration; Light; Light-Harvesting Protein Complexes; Nanostructures; Protein Binding; Protein Multimerization; Spinacia oleracea; Thylakoids; Xanthophylls; Zeaxanthins

Life on Earth depends on photosynthesis, the conversion of light energy into chemical energy. Plants collect photons by light harvesting complexes (LHC)-abundant membrane proteins containing chlorophyll and xanthophyll molecules. LHC-like proteins are similar in their amino acid sequence to true LHC antennae, however, they rather serve a photoprotective function. Whether the LHC-like proteins bind pigments has remained unclear. Here, we characterize plant LHC-like proteins (LIL3 and ELIP2) produced in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). Both proteins were associated with chlorophyll a (Chl) and zeaxanthin and LIL3 was shown to be capable of quenching Chl fluorescence via direct energy transfer from the Chl Q

Topics: Arabidopsis Proteins; Carotenoids; Chlorophyll; Chloroplast Proteins; Energy Transfer; Mutation; Protein Binding; Protein Folding; Protein Multimerization; Synechocystis; Xanthophylls; Zeaxanthins

The xanthophyll cycle is a regulatory mechanism operating in the photosynthetic apparatus of plants. It consists of the conversion of the xanthophyll pigment violaxanthin to zeaxanthin, and vice versa, in response to light intensity. According to the current understanding, one of the modes of regulatory activity of the cycle is associated with the influence on a molecular organization of pigment-protein complexes. In the present work, we analyzed the effect of violaxanthin and zeaxanthin on the molecular organization of the LHCII complex, in the environment of membranes formed with chloroplast lipids. Nanoscale imaging based on atomic force microscopy (AFM) showed that the presence of exogenous xanthophylls promotes the formation of the protein supramolecular structures. Nanoscale infrared (IR) absorption analysis based on AFM-IR nanospectroscopy suggests that zeaxanthin promotes the formation of LHCII supramolecular structures by forming inter-molecular β-structures. Meanwhile, the molecules of violaxanthin act as "molecular spacers" preventing self-aggregation of the protein, potentially leading to uncontrolled dissipation of excitation energy in the complex. This latter mechanism was demonstrated with the application of fluorescence lifetime imaging microscopy. The intensity-averaged chlorophyll a fluorescence lifetime determined in the LHCII samples without exogenous xanthophylls at the level of 0.72 ns was longer in the samples containing exogenous violaxanthin (2.14 ns), but shorter under the presence of zeaxanthin (0.49 ns) thus suggesting a role of this xanthophyll in promotion of the formation of structures characterized by effective excitation quenching. This mechanism can be considered as a representation of the overall photoprotective activity of the xanthophyll cycle.

Topics: Chlorophyll A; Intracellular Membranes; Light-Harvesting Protein Complexes; Narcissus; Protein Structure, Secondary; Spinacia oleracea; Xanthophylls; Zeaxanthins

The violaxanthin (V)-antheraxanthin (A)-zeaxanthin (Z) (VAZ) cycle was deemed a non-second-scale process of photoprotection in higher plants and microalgae, but the validity of this view has not been confirmed. To test this view, we explored responses of the VAZ cycle and the relationship between the VAZ cycle and non-photochemical quenching (NPQ) under highlight at second and minute scales in Heterosigma akashiwo and Platymonas sp. Both A and Z were generated in H. akashiwo during 15 s of light exposure, whereas only A rapidly accumulated within 15 s of exposure in Platymonas sp. The above results, together with a time-dependent sigmoidal relationship between the VAZ cycle (de-epoxidation state, A/Chl a, and Z/Chl a) and NPQ, proved that the VAZ cycle was a second-scale process related to NPQ. In addition, we found that not all NPQ was dependent on the VAZ cycle and suggested that NPQ model should be carefully modified due to the species-specific proportions of de-epoxidation-dependent NPQ.

Topics: Chlorophyceae; Light; Microalgae; Xanthophylls; Zeaxanthins

Antenna complexes in photosystems of plants and green algae are able to switch between a light-harvesting unquenched conformation and a quenched conformation so to avoid photodamage. When the switch is activated, nonphotochemical quenching (NPQ) mechanisms take place for an efficient deactivation of excess excitation energy. The molecular details of these mechanisms have not been fully clarified but different hypotheses have been proposed. Among them, a popular one involves excitation energy transfer (EET) from the singlet excited Chls to the lowest singlet state (S

Topics: Chlorophyll; Energy Transfer; Light-Harvesting Protein Complexes; Photosynthesis; Photosystem II Protein Complex; Plant Proteins; Plants; Xanthophylls; Zeaxanthins

Nonphotochemical quenching (NPQ) is a mechanism of regulating light harvesting that protects the photosynthetic apparatus from photodamage by dissipating excess absorbed excitation energy as heat. In higher plants, the major light-harvesting antenna complex (LHCII) of photosystem (PS) II is directly involved in NPQ. The aggregation of LHCII is proposed to be involved in quenching. However, the lack of success in isolating native LHCII aggregates has limited the direct interrogation of this process. The isolation of LHCII in its native state from thylakoid membranes has been problematic because of the use of detergent, which tends to dissociate loosely bound proteins, and the abundance of pigment-protein complexes (e.g. PSI and PSII) embedded in the photosynthetic membrane, which hinders the preparation of aggregated LHCII. Here, we used a novel purification method employing detergent and amphipols to entrap LHCII in its natural states. To enrich the photosynthetic membrane with the major LHCII, we used Arabidopsis thaliana plants lacking the PSII minor antenna complexes (NoM), treated with lincomycin to inhibit the synthesis of PSI and PSII core proteins. Using sucrose density gradients, we succeeded in isolating the trimeric and aggregated forms of LHCII antenna. Violaxanthin- and zeaxanthin-enriched complexes were investigated in dark-adapted, NPQ, and dark recovery states. Zeaxanthin-enriched antenna complexes showed the greatest amount of aggregated LHCII. Notably, the amount of aggregated LHCII decreased upon relaxation of NPQ. Employing this novel preparative method, we obtained a direct evidence for the role of in vivo LHCII aggregation in NPQ.

Topics: Arabidopsis; Kinetics; Light-Harvesting Protein Complexes; Lincomycin; Plants, Genetically Modified; Protein Multimerization; Spectrometry, Fluorescence; Thylakoids; Ultracentrifugation; Xanthophylls; Zeaxanthins

The influence of six different light regimes throughout the photosynthetically active radiation range (from 400 to 700 nm, including blue, green, yellow, red-orange, red, and white) at two intensities (100 and 300 µmol photons m

Topics: beta Carotene; Chlorophyll; Diatoms; Light; Photosynthesis; Pigments, Biological; Xanthophylls; Zeaxanthins

The consumption of zeaxanthin (Z) through a vegetable-rich diet is recommended to reduce the progression of age-related macular degeneration. Due to Z's intrinsic dynamic character that results from its participation in the photoprotective xanthophyll cycle involving the carotenoids violaxanthin, antheraxanthin and zeaxanthin (VAZ), post-harvest handling practices and storage usually retain low amounts of this bioactive compound (compared to the rest of phytochemicals that are, in general, more stable). Thus, the aim of this work was to investigate in important consumed leafy vegetables the effects of different storage conditions on carotenoids (mainly Z) including i) packaging under three modified atmospheres (MAs), ii) light refrigerated supermarket storage and iii) dark refrigerated domestic storage. The results showed that an MA with low O

Topics: Atmosphere; Carotenoids; Food Packaging; Food Storage; Humans; Nutritive Value; Plant Leaves; Refrigeration; Vegetables; Xanthophylls; Zeaxanthins

Topics: Beta-Cryptoxanthin; Calendula; Carotenoids; Cryptoxanthins; Esters; Fatty Acids; Flowers; Lutein; Tandem Mass Spectrometry; Xanthine; Xanthophylls; Zeaxanthins

Plants have developed multiple self-regulatory mechanisms to efficiently function under varying sunlight conditions. At high light intensities, non-photochemical quenching (NPQ) is activated on a molecular level, safely dissipating an excess excitation as heat. The exact molecular mechanism for NPQ is still under debate, but it is widely agreed that the direct participation of the carotenoid pigments is involved, one of the proposed candidate being the zeaxanthin. In this work, we performed fluorescence measurements of violaxanthin- and zeaxanthin-enriched major light-harvesting complexes (LHCII), in ensemble and at the single pigment-protein complex level, where aggregation is prevented by immobilization of LHCIIs onto a surface. We show that a selective enrichment of LHCII with violaxanthin or zeaxanthin affects neither the ability of LHCII to switch into a dissipative conformation nor the maximal level of induced quenching. However, the kinetics of the fluorescence decrease due to aggregation on the timescale of seconds are different, prompting towards a modulatory effect of zeaxanthin in the dynamics of quenching.

Topics: Adaptation, Physiological; Hydrogen-Ion Concentration; Light; Light-Harvesting Protein Complexes; Plant Leaves; Protein Conformation; Spectrometry, Fluorescence; Spinacia oleracea; Thylakoids; Xanthophylls; Zeaxanthins

We investigated the transcriptional regulation of six genes involved in carotenoid biosynthesis, together with the carotenoid accumulation during postharvest ripening of three different papaya genotypes of contrasting pulp color. Red-pulp genotype (RPG) showed the lowest content of yellow pigments (YP), such as β-cryptoxanthin, zeaxanthin, and violaxanthin, together with the lowest relative expression levels (REL) of CpLCY-β2 and CpCHX-β genes. On the contrary, the yellow-pulp genotype (YPG) showed the highest content of YP and the highest REL of CpLCY-β2 and CpCHX-β genes. Interestingly, the orange-pulp genotype (OPG) showed intermediate content of YP and intermediate REL of CpLCY-β2 and CpCHX-β genes. The highest content of β-carotene shown by OPG despite having an intermediate REL of the CpLCY-β2 genes, suggests a post-transcriptional regulation. Thus, the transcriptional level of the genes, directing the carotenoid biosynthesis pathway, can partially explain the accumulation of carotenoids during the postharvest ripening in C. papaya genotypes of contrasting pulp color.

Topics: beta Carotene; Beta-Cryptoxanthin; Carica; Carotenoids; Citrus sinensis; Color; Fruit; Gene Expression Regulation, Plant; Genes, Plant; Genotype; Lycopene; Pigmentation; Plant Proteins; RNA, Plant; Xanthophylls; Zeaxanthins

A special domain of the thylakoid membrane of higher plants has been isolated which carries out the de-epoxidation of the xanthophyll cycle pigment violaxanthin to zeaxanthin. Recent models indicate that in the chloroplast of higher plants, the violaxanthin (V) cycle takes place within specialized domains in the thylakoid membrane. Here, we describe a new procedure to directly isolate such a domain in functional state. The procedure consists of a thylakoid membrane isolation at a pH value of 5.2 which realizes the binding of the enzyme V de-epoxidase (VDE) to the membrane throughout the preparation process. Isolated thylakoid membranes are then solubilized with the very mild detergent n-dodecyl α-D-maltoside and the pigment-protein complexes are separated by sucrose gradient ultracentrifugation. The upper main fraction of the sucrose gradient represents a V cycle domain which consists of the major light-harvesting complex of photosystem II (LHCII), a special lipid composition with an enrichment of the galactolipid monogalactosyldiacylglycerol (MGDG) and the VDE. The domain is isolated in functional state as evidenced by the ability to convert the LHCII-associated V to zeaxanthin. The direct isolation of a V cycle domain proves the most important hypotheses concerning the de-epoxidation reaction in intact thylakoid membranes. It shows that the VDE binds to the thylakoid membrane at low pH values of the thylakoid lumen, that it binds to membrane regions enriched in LHCII, and that the domain contains high amounts of MGDG. The last point is in line with the importance of the galactolipid for V solubilisation and, by providing inverted hexagonal lipid structures, for VDE activity.

Topics: Blotting, Western; Centrifugation, Density Gradient; Electrophoresis, Polyacrylamide Gel; Oxidoreductases; Plant Leaves; Spectrometry, Fluorescence; Spinacia oleracea; Thylakoids; Xanthophylls; Zeaxanthins

The effect of violaxanthin and zeaxanthin, two main carotenoids of the xanthophyll cycle, on molecular organization of LHCII, the principal photosynthetic antenna complex of plants, was studied in a model system based on lipid-protein membranes, by means of analysis of 77 K chlorophyll a fluorescence and "native" electrophoresis. Violaxanthin was found to promote trimeric organization of LHCII, contrary to zeaxanthin which was found to destabilize trimeric structures. Moreover, violaxanthin was found to induce decomposition of oligomeric LHCII structures formed in the lipid phase and characterized by the fluorescence emission band at 715 nm. Both pigments promoted formation of two-component supramolecular structures of LHCII and xanthophylls. The violaxanthin-stabilized structures were composed mostly of LHCII trimers while, the zeaxanthin-stabilized supramolecular structures of LHCII showed more complex organization which depended periodically on the xanthophyll content. The effect of the xanthophyll cycle pigments on molecular organization of LHCII was analyzed based on the results of molecular modeling and discussed in terms of a physiological meaning of this mechanism. Supramolecular structures of LHCII stabilized by violaxanthin, prevent uncontrolled oligomerization of LHCII, potentially leading to excitation quenching, therefore can be considered as structures protecting the photosynthetic apparatus against energy loses at low light intensities.

Topics: Binding Sites; Light; Light-Harvesting Protein Complexes; Molecular Docking Simulation; Photosystem II Protein Complex; Protein Binding; Protein Conformation; Radiation Dosage; Xanthophylls; Zeaxanthins

The photosynthetic apparatus of higher plants acclimates to irradiance. Among the features which are changing is the pool size of the pigments belonging to the violaxanthin cycle, in which zeaxanthin is formed. In high light grown leaves, the violaxanthin cycle pool size is up to five times larger than in low light. The changes are reversible on a time scale of several days. Since it has been published that violaxanthin cycle pigments do not transfer absorbed energy to chlorophyll, we hypothesized that excitation of chlorophyll fluorescence in the blue spectral region may be reduced in high light-acclimated leaves. Fluorescence excitation spectra of leaves of the Arabidopsis thaliana tt3 mutant showed strong differences between high and low light-acclimated plants from 430 to 520 nm. The resulting difference spectrum was similar to carotenoids but shifted by about 20 nm to higher wavelengths. A good correlation was observed between the fluorescence excitation ratio F 470/F 660 and the violaxanthin cycle pool size when leaves were acclimated to a range of irradiances. In parallel to the decline of F 470/F 660 with high light acclimation also the quantum yield of photosynthetic oxygen evolution in blue light decreased. The data confirm that violaxanthin cycle carotenoids do not transfer absorbed light to chlorophyll. It is proposed to use the ratio F 470/F 660 as an indicator for the light acclimation status of the chloroplasts in a leaf.

Topics: Acclimatization; Arabidopsis; Carotenoids; Chlorophyll; Energy Transfer; Fluorescence; Light; Mutation; Photosynthesis; Pigmentation; Plant Leaves; Xanthophylls; Zeaxanthins

Photosynthetic organisms need protection against excessive light. By using non-photochemical quenching, where the excess light is converted into heat, the organism can survive at higher light intensities. This process is partly initiated by the formation of zeaxanthin, which is achieved by the de-epoxidation of violaxanthin and antheraxanthin to zeaxanthin. This reaction is catalyzed by violaxanthin de-epoxidase (VDE). VDE consists of three domains of which the central lipocalin-like domain has been the most characterized. By truncating the domains surrounding the lipocalin-like domain, we show that VDE activity is possible without the C-terminal domain but not without the N-terminal domain. The N-terminal domain shows no VDE activity by itself but when separately expressed domains are mixed, VDE activity is possible. This shows that these domains can be folded separately and could therefore be studied separately. An increase of the hydrodynamic radius of wild-type VDE was observed when pH was lowered toward the pH required for activity, consistent with a pH-dependent oligomerization. The C-terminally truncated VDE did not show such an oligomerization, was relatively more active at higher pH but did not alter the KM for ascorbate. Circular dichroism measurements revealed the presence of α-helical structure in both the N- and C-terminal domains. By measuring the initial formation of the product, VDE was found to convert a large number of violaxanthin molecules to antheraxanthin before producing any zeaxanthin, favoring a model where violaxanthin is bound non-symmetrically in VDE.

Topics: Escherichia coli; Gene Expression; Hydrodynamics; Hydrogen-Ion Concentration; Oxidation-Reduction; Oxidoreductases; Plant Proteins; Protein Domains; Sequence Deletion; Spinacia oleracea; Xanthophylls; Zeaxanthins

Violaxanthin de-epoxidase (VDE) is a conditionally soluble enzyme located in the thylakoid lumen and catalyses the conversion of violaxanthin to antheraxanthin and zeaxanthin, which are located in the thylakoid membrane. These reactions occur when the plant or algae are exposed to saturating light and the zeaxanthin formed is involved in the process of non-photochemical quenching that protects the photosynthetic machinery during stress. Oversaturation by light results in a reduction of the pH inside the thylakoids, which in turn activates VDE and the de-epoxidation of violaxanthin. To elucidate the structural events responsible for the pH-dependent activation of VDE, full length and truncated forms of VDE were studied at different pH using circular dichroism (CD) spectroscopy, crosslinking and small angle X-ray scattering (SAXS). CD spectroscopy showed the formation of α-helical coiled-coil structure, localised in the C-terminal domain. Chemical crosslinking of VDE showed that oligomers were formed at low pH, and suggested that the position of the N-terminal domain is located near the opening of lipocalin-like barrel, where violaxanthin has been predicted to bind. SAXS was used to generate models of monomeric VDE at high pH and also a presumably dimeric structure of VDE at low pH. For the dimer, the best fit suggests that the interaction is dominated by one of the domains, preferably the C-terminal domain due to the lost ability to oligomerise at low pH, shown in earlier studies, and the predicted formation of coiled-coil structure.

Topics: Hydrogen-Ion Concentration; Light; Models, Molecular; Oxidoreductases; Photosynthesis; Plant Proteins; Polymerization; Scattering, Small Angle; Spinacia oleracea; Thylakoids; X-Ray Diffraction; Xanthophylls; Zeaxanthins

Plants harvest photons for photosynthesis using light-harvesting complexes (LHCs)-an array of chlorophyll proteins that can reversibly switch from harvesting to energy-dissipation mode to prevent over-excitation and damage of the photosynthetic apparatus. In unicellular algae and lower plants this process requires the LHCSR proteins which senses over-acidification of the lumen trough protonatable residues exposed to the thylakoid lumen to activate quenching reactions. Further activation is provided by replacement of the violaxanthin ligand with its de-epoxidized product, zeaxanthin, also induced by excess light. We have produced the ppLHCSR1 protein from Physcomitrella patens by over-expression in tobacco and purified it in either its violaxanthin- or the zeaxanthin-binding form with the aim of analyzing their spectroscopic properties at either neutral or acidic pH. Using femtosecond spectroscopy, we demonstrated that the energy dissipation is achieved by two distinct quenching mechanism which are both activated by low pH. The first is present in both ppLHCSR1-Vio and ppLHCSR1-Zea and is characterized by 30-40ps time constant. The spectrum of the quenching product is reminiscent of a carotenoid radical cation, suggesting that the pH-induced quenching mechanism is likely electron transfer from the carotenoid to the excited Chl a. In addition, a second quenching channel populating the S

Topics: Bryopsida; Chlorophyll; Electron Transport; Energy Transfer; Hydrogen-Ion Concentration; Kinetics; Light-Harvesting Protein Complexes; Models, Biological; Nicotiana; Photosynthesis; Plants, Genetically Modified; Protein Binding; Spectrum Analysis; Xanthophylls; Zeaxanthins

Aggregation induced conformational change of light harvesting antenna complexes is believed to constitute one of the pathways through which photosynthetic organisms can safely dissipate the surplus of energy while exposed to saturating light. In this study, Stark fluorescence (SF) spectroscopy is applied to minor antenna complexes (CP24, CP26 and CP29) both in their light-harvesting and energy-dissipating states to trace and characterize different species generated upon energy dissipation through aggregation (in-vitro) induced conformational change. SF spectroscopy could identify three spectral species in the dissipative state of CP24, two in CP26 and only one in CP29. The comprehensive analysis of the SF spectra yielded different sets of molecular parameters for the multiple spectral species identified in CP24 or CP26, indicating the involvement of different pigments in their formation. Interestingly, a species giving emission around the 730nm spectral region is found to form in both CP24 and CP26 following transition to the energy dissipative state, but not in CP29. The SF analyses revealed that the far red species has exceptionally large charge transfer (CT) character in the excited state. Moreover, the far red species was found to be formed invariably in both Zeaxanthin (Z)- and Violaxathin (V)-enriched CP24 and CP26 antennas with identical CT character but with larger emission yield in Z-enriched ones. This suggests that the carotenoid Z is not directly involved but only confers an allosteric effect on the formation of the far red species. Similar far red species with remarkably large CT character were also observed in the dissipative state of the major light harvesting antenna (LHCII) of plants [Wahadoszamen et al. PCCP, 2012], the fucoxanthin-chlorophyll protein (FCP) of brown algae [Wahadoszamen et al. BBA, 2014] and cyanobacterial IsiA [Wahadoszamen et al. BBA, 2015], thus pointing to identical sites and pigments active in the formation of the far red quenching species in different organisms.

Topics: Chlorophyll; Energy Transfer; Light; Light-Harvesting Protein Complexes; Photosynthesis; Protein Conformation; Species Specificity; Spectrometry, Fluorescence; Spinacia oleracea; Structure-Activity Relationship; Xanthophylls; Zeaxanthins

The main physiological function of LHCII (light-harvesting pigment-protein complex of photosystem II), the largest photosynthetic antenna complex of plants, is absorption of light quanta and transfer of excitation energy toward the reaction centers, to drive photosynthesis. However, under strong illumination, the photosynthetic apparatus faces the danger of photodegradation and therefore excitations in LHCII have to be down-regulated, e.g., via thermal energy dissipation. One of the elements of the regulatory system, operating in the photosynthetic apparatus under light stress conditions, is a conversion of violaxanthin, the xanthophyll present under low light, to zeaxanthin, accumulated under strong light. In the present study, an effect of violaxanthin and zeaxanthin on the molecular organization and the photophysical properties of LHCII was studied in a monomolecular layer system with application of molecular imaging (atomic force microscopy, fluorescence lifetime imaging microscopy) and spectroscopy (UV-Vis absorption, FTIR, fluorescence spectroscopy) techniques. The results of the experiments show that violaxanthin promotes the formation of supramolecular LHCII structures preventing dissipative excitation quenching while zeaxanthin is involved in the formation of excitonic energy states able to quench chlorophyll excitations in both the higher (B states) and lower (Q states) energy levels. The results point to a strategic role of xanthophylls that are not embedded in a protein environment, in regulation of the photosynthetic light harvesting activity in plants.

Topics: Air; Kinetics; Light-Harvesting Protein Complexes; Lycium; Narcissus; Photosynthesis; Spectrometry, Fluorescence; Spinacia oleracea; Surface Properties; Thermodynamics; Thylakoids; Water; Xanthophylls; Zeaxanthins

Antarctic algae play a fundamental role in polar ecosystem thanks to their ability to grow in an extreme environment characterized by low temperatures and variable illumination. Here, for prolonged periods, irradiation is extremely low and algae must be able to harvest light as efficiently as possible. On the other side, at low temperatures even dim irradiances can saturate photosynthesis and drive to the formation of reactive oxygen species. Colonization of this extreme environment necessarily required the optimization of photosynthesis regulation mechanisms by algal organisms. In order to investigate these adaptations we analyzed the time course of physiological and morphological responses to different irradiances in Koliella antarctica, a green microalga isolated from Ross Sea (Antarctica). Koliella antarctica not only modulates cell morphology and composition of its photosynthetic apparatus on a long-term acclimation, but also shows the ability of a very fast response to light fluctuations. Koliella antarctica controls the activity of two xanthophyll cycles. The first, involving lutein epoxide and lutein, may be important for the growth under very low irradiances. The second, involving conversion of violaxanthin to antheraxanthin and zeaxanthin, is relevant to induce a fast and particularly strong non-photochemical quenching, when the alga is exposed to higher light intensities. Globally K. antarctica thus shows the ability to activate a palette of responses of the photosynthetic apparatus optimized for survival in its natural extreme environment.

Topics: Acclimatization; Adaptation, Physiological; Cold Temperature; Environment; Light; Lutein; Photosynthesis; Streptophyta; Xanthophylls; Zeaxanthins

Violaxanthin de-epoxidase (VDE) catalyses the conversion of violaxanthin to zeaxanthin at the lumen side of the thylakoids during exposure to intense light. VDE consists of a cysteine-rich N-terminal domain, a lipocalin-like domain and a negatively charged C-terminal domain. That the cysteines are important for the activity of VDE is well known, but in what way is less understood. In this study, wild-type spinach VDE was expressed in E. coli as inclusion bodies, refolded and purified to give a highly active and homogenous preparation. The metal content (Fe, Cu, Ni, Mn, Co and Zn) was lower than 1 mol% excluding a metal-binding function of the cysteines. To investigate which of the 13 cysteines that could be important for the function of VDE, we constructed mutants where the cysteines were replaced by serines, one by one. For 12 out of 13 mutants the activity dropped by more than 99.9%. A quantification of free cysteines showed that only the most N-terminal of these cysteines was in reduced form in the native VDE. A disulphide pattern in VDE of C9-C27, C14-C21, C33-C50, C37-C46, C65-C72 and C118-C284 was obtained after digestion of VDE with thermolysin followed by mass spectroscopy analysis of reduced versus non-reduced samples. The residual activity found for the mutants showed a variation that was consistent with the results obtained from mass spectroscopy. Reduction of the disulphides resulted in loss of a rigid structure and a decrease in thermal stability of 15 °C.

Topics: Amino Acid Sequence; Cystine; Disulfides; Escherichia coli; Hot Temperature; Molecular Sequence Data; Mutation; Oxidoreductases; Protein Stability; Spinacia oleracea; Thylakoids; Xanthophylls; Zeaxanthins

The xanthophyll cycle is involved in dissipating excess light energy to protect the photosynthetic apparatus in a process commonly assessed from non-photochemical quenching (NPQ) of chlorophyll fluorescence. Here, it is shown that the xanthophyll cycle is modulated by the necrotrophic pathogen Sclerotinia sclerotiorum at the early stage of infection. Incubation of Sclerotinia led to a localized increase in NPQ even at low light intensity. Further studies showed that this abnormal change in NPQ was closely correlated with a decreased pH caused by Sclerotinia-secreted oxalate, which might decrease the ATP synthase activity and lead to a deepening of thylakoid lumen acidification under continuous illumination. Furthermore, suppression (with dithiothreitol) or a defect (in the npq1-2 mutant) of violaxanthin de-epoxidase (VDE) abolished the Sclerotinia-induced NPQ increase. HPLC analysis showed that the Sclerotinia-inoculated tissue accumulated substantial quantities of zeaxanthin at the expense of violaxanthin, with a corresponding decrease in neoxanthin content. Immunoassays revealed that the decrease in these xanthophyll precursors reduced de novo abscisic acid (ABA) biosynthesis and apparently weakened tissue defense responses, including ROS induction and callose deposition, resulting in enhanced plant susceptibility to Sclerotinia. We thus propose that Sclerotinia antagonizes ABA biosynthesis to suppress host defense by manipulating the xanthophyll cycle in early pathogenesis. These findings provide a model of how photoprotective metabolites integrate into the defense responses, and expand the current knowledge of early plant-Sclerotinia interactions at infection sites.

Topics: Abscisic Acid; Arabidopsis; Ascomycota; Glucans; Host-Pathogen Interactions; Hydrogen-Ion Concentration; Mutation; Oxalates; Oxidoreductases; Photosynthesis; Plant Immunity; Plant Leaves; Plant Proteins; Plants, Genetically Modified; Reactive Oxygen Species; Reducing Agents; Thylakoids; Xanthophylls; Zeaxanthins

Assessing photosynthesis rates at the ecosystem scale and over large regions is important for tracking the global carbon cycle and remote sensing has provided new and useful approaches for performing this assessment. The photochemical reflectance index (PRI) is a good estimator of short-term light-use efficiency (LUE) at the leaf scale; however, confounding factors appear at larger temporal and spatial scales. In this study, canopy-scale PRI variability was investigated for three species (Fagus sylvatica L., Quercus robur L. and Pinus sylvestris L.) growing under contrasting soil moisture conditions. Throughout the growing season, no significant differences in chlorophyll content and in violaxanthin, antheraxanthin and zeaxanthin were found between species or treatments. The daily PRI vs PAR (photosynthetically active radiation) relationships were determined using continuous measurements obtained at high frequency throughout the entire growing season, from early spring budburst to later autumn senescence, and were used to deconvolute the physiological PRI variability related to LUE variations due to phenological variability and related to temporal changes in the biochemical and structural canopy attributes. The PRI vs PAR relationship is used to show that the canopy-scale PRI measured at low radiation depends on the chlorophyll content of the canopy. The range of PRI variations at an intra-daily scale and the dynamics of the xanthophyll pool do not vary between days, which suggests that the PRI responds to a xanthophyll ratio. The PAR values at PRI saturation are mainly related to the canopy chlorophyll content during budburst and senescence and to the soil moisture content when the chlorophyll content is no longer a limiting factor. This parameter is significantly lower in the oak species that experience less stress from variations in soil moisture and is species dependant. These results provide new insights regarding the analysis and the meaning of PRI variability as a proxy for LUE at the canopy scale.

Topics: Chlorophyll; Fagus; Photochemical Processes; Photosynthesis; Pinus; Plant Leaves; Quercus; Seasons; Soil; Xanthophylls; Zeaxanthins

Photosynthetic organisms protect themselves from high-light stress by dissipating excess absorbed energy as heat in a process called non-photochemical quenching (NPQ). Zeaxanthin is essential for the full development of NPQ, but its role remains debated. The main discussion revolves around two points: where does zeaxanthin bind and does it quench? To answer these questions we have followed the zeaxanthin-dependent quenching from leaves to individual complexes, including supercomplexes. We show that small amounts of zeaxanthin are associated with the complexes, but in contrast to what is generally believed, zeaxanthin binding per se does not cause conformational changes in the complexes and does not induce quenching, not even at low pH. We show that in NPQ conditions zeaxanthin does not exchange for violaxanthin in the internal binding sites of the antennas but is located at the periphery of the complexes. These results together with the observation that the zeaxanthin-dependent quenching is active in isolated membranes, but not in functional supercomplexes, suggests that zeaxanthin is acting in between the complexes, helping to create/participating in a variety of quenching sites. This can explain why none of the antennas appears to be essential for NPQ and the multiple quenching mechanisms that have been observed in plants.

Topics: Arabidopsis; Circular Dichroism; Kinetics; Light; Photosynthesis; Photosystem II Protein Complex; Plant Leaves; Thylakoids; Xanthophylls; Zeaxanthins

It is proposed that xanthophylls, and carotenoids in general, may assist in energy transfer from the chlorophyll Soret band to the Q band. Ground-state (1Ag ) and excited-state (1Bu ) optimizations of violaxanthin (Vx) and zeaxanthin (Zx) are performed in an environment mimicking the light-harvesting complex II (LHCII), including the closest chlorophyll b molecule (Chl). Time-dependent density functional theory (TD-DFT, CAM-B3LYP functional) is used in combination with a semi-empirical description to obtain the excited-state geometries, supported by additional DFT/multireference configuration interaction calculations, with and without point charges representing LHCII. In the ground state, Vx and Zx show similar properties. At the 1Bu minimum, the energy of the Zx 1Bu state is below the Chl Q band, in contrast to Vx. Both Vx and Zx may act as acceptors of Soret-state energy; transfer to the Q band seems to be favored for Vx. These findings suggest that carotenoids may generally mediate Soret-to-Q energy flow in LHCII.

Topics: Carotenoids; Chlorophyll; Energy Transfer; Light-Harvesting Protein Complexes; Models, Molecular; Quantum Theory; Thermodynamics; Xanthophylls; Zeaxanthins

PeVDE was expressed primarily in bamboo leaves, which was up-regulated under high light. The protein encoded by PeVDE had enzyme activity of catalyzing violaxanthin (V) to zeaxanthin (Z) through antheraxanthin (A) as assay shown in vitro. Violaxanthin de-epoxidase (VDE), a key enzyme of xanthophyll cycle, catalyzes conversion from violaxanthin (V) to zeaxanthin (Z) through antheraxanthin (A) to protect photosynthesis apparatus. A cDNA, PeVDE, encoding a VDE was isolated from bamboo (Phyllostachys edulis) by RT-PCR and RACE methods. PeVDE is 1,723 bp and contains an ORF encoding 451 amino acids, with a transit peptide of 103 amino acids. The mature protein is deduced to have 348 amino acids with a calculated molecular weight of 39.6 kDa and a theoretic isoelectric point of 4.5. Semi-quantitative RT-PCR assay indicated that the highest expression level of PeVDE was in leaf, which agreed with the accumulation pattern of PeVDE protein. Real time PCR results showed that PeVDE was up-regulated and reached the highest level after the treatment (1,200 μmo1 m(-2) s(-1)) for 2 h, then decreased and kept at the level similar to that of 0.5 h after treatment for 8 h. To investigate the function of PeVDE, mature protein was heterologously expressed in Escherichia coli and the enzymatic activity assay was carried out using V as substrate. The pigments that formed in the reaction mixture were extracted and analyzed by HPLC method. Besides V, A and Z were detected in the reaction mixture, which indicated that the recombinant protein exhibited enzymatic activity of catalyzing V into Z through A. This study indicates that PeVDE functions through regulating the components of xanthophyll cycle, which might be one of the critical factors that contribute to the growth of bamboo under naturally varying light conditions.

Topics: Amino Acid Sequence; Base Sequence; DNA, Complementary; DNA, Plant; Gene Expression Regulation, Plant; Light; Molecular Sequence Data; Oxidoreductases; Photosynthesis; Plant Leaves; Plant Proteins; Poaceae; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Xanthophylls; Zeaxanthins

It has long been suspected that photoprotective mechanisms in green algae are similar to those in seed plants. However, exceptions have recently surfaced among aquatic and marine green algae in several taxonomic classes. Green algae are highly diverse genetically, falling into 13 named classes, and they are diverse ecologically, with many lineages including members from freshwater, marine, and terrestrial habitats. Genetically similar species living in dramatically different environments are potentially a rich source of information about variations in photoprotective function. Using aquatic and desert-derived species from three classes of green algae, we examined the induction of photoprotection under high light, exploring the relationship between nonphotochemical quenching and the xanthophyll cycle. In liquid culture, behavior of aquatic Entransia fimbriata (Klebsormidiophyceae) generally matched patterns observed in seed plants. Nonphotochemical quenching was lowest after overnight dark adaptation, increased with light intensity, and the extent of nonphotochemical quenching correlated with the extent of deepoxidation of xanthophyll cycle pigments. In contrast, overnight dark adaptation did not minimize nonphotochemical quenching in the other species studied: desert Klebsormidium sp. (Klebsormidiophyceae), desert and aquatic Cylindrocystis sp. (Zygnematophyceae), and desert Stichococcus sp. (Trebouxiophyceae). Instead, exposure to low light reduced nonphotochemical quenching below dark-adapted levels. De-epoxidation of xanthophyll cycle pigments paralleled light-induced changes in nonphotochemical quenching for species within Klebsormidiophyceae and Trebouxiophyceae, but not Zygnematophyceae. Inhibition of violaxanthin-zeaxanthin conversion by dithiothreitol reduced high-light-associated nonphotochemical quenching in all species (Zygnematophyceae the least), indicating that zeaxanthin can contribute to photoprotection as in seed plants but to different extents depending on taxon or lineage.

Topics: Adaptation, Physiological; Aquatic Organisms; Chlorophyll; Chlorophyta; Desert Climate; Fluorescence; Light; Molecular Sequence Data; Phylogeny; Xanthophylls; Zeaxanthins

Nonphotochemical quenching (NPQ) dissipates excess energy to protect the photosynthetic apparatus from excess light. The moss Physcomitrella patens exhibits strong NPQ by both algal-type light-harvesting complex stress-related (LHCSR)-dependent and plant-type S subunit of Photosystem II (PSBS)-dependent mechanisms. In this work, we studied the dependence of NPQ reactions on zeaxanthin, which is synthesized under light stress by violaxanthin deepoxidase (VDE) from preexisting violaxanthin. We produced vde knockout (KO) plants and showed they underwent a dramatic reduction in thermal dissipation ability and enhanced photoinhibition in excess light conditions. Multiple mutants (vde lhcsr KO and vde psbs KO) showed that zeaxanthin had a major influence on LHCSR-dependent NPQ, in contrast with previous reports in Chlamydomonas reinhardtii. The PSBS-dependent component of quenching was less dependent on zeaxanthin, despite the near-complete violaxanthin to zeaxanthin exchange in LHC proteins. Consistent with this, we provide biochemical evidence that native LHCSR protein binds zeaxanthin upon excess light stress. These findings suggest that zeaxanthin played an important role in the adaptation of modern plants to the enhanced levels of oxygen and excess light intensity of land environments.

Topics: Adaptation, Physiological; Biosynthetic Pathways; Bryopsida; Chlorophyll; Gene Knockout Techniques; Light; Light-Harvesting Protein Complexes; Photosystem II Protein Complex; Thylakoids; Xanthophylls; Zeaxanthins

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

Topics: Chlorophyll; Chloroplasts; Electron Transport; Light; Oxidation-Reduction; Oxidoreductases; Photoperiod; Protons; Reactive Oxygen Species; Ulva; Xanthophylls; Zeaxanthins

Nonphotochemical quenching (NPQ) of Photosystem II fluorescence is one of the most important photoprotection responses of phototropic organisms. NPQ in Macrocystis pyrifera is unique since the fast induction of this response, the energy dependent quenching (qE), is not present in this alga. In contrast to higher plants, NPQ in this organism is much more strongly related to xanthophyll cycle (XC) pigment interconversion. Characterization of how NPQ is controlled when qE is not present is important as this might represent an ancient response to light stress. Here, we describe the influence of the XC pigment pool (ΣXC) size on NPQ induction in M. pyrifera. The sum of violaxanthin (Vx) plus antheraxanthin and zeaxanthin (Zx) represents the ΣXC. This pool was three-fold larger in blades collected at the surface of the water column (19molmol(-1) Chl a×100) than in blades collected at 6m depth. Maximum NPQ was not different in samples with a ΣXC higher than 12molmol(-1) Chl a×100; however, NPQ induction was faster in blades with a large ΣXC. The increase in the NPQ induction rate was associated with a faster Vx to Zx conversion. Further, we found that NPQ depends on the de-epoxidation state of the ΣXC, not on the absolute concentration of Zx and antheraxanthin. Thus, there was an antagonist effect between Vx and de-epoxidated xanthophylls for NPQ. These results indicate that in the absence of qE, a large ΣXC is needed in M. pyrifera to respond faster to light stress conditions.

Topics: Epoxy Compounds; Light; Macrocystis; Xanthophylls; Zeaxanthins

Under excess light, the efficient PSII light-harvesting antenna is switched into a photoprotected state in which potentially harmful absorbed energy is thermally dissipated. Changes occur rapidly and reversibly, enhanced by de-epoxidation of violaxanthin (V) to zeaxanthin (Z). This process is usually measured as non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence. Using instrumentation for instantaneous leaf freezing, NPQ, spectral reflectance, and interconversions within the xanthophyll cycle with time resolution of seconds were recorded from Quercus coccifera leaves during low light (LL) to high light (HL) transitions, followed by relaxation at LL. During the first 30 s of both the LL to HL and HL to LL transitions, no activity of the xanthophyll cycle was detected, whereas 70-75% of the NPQ was formed and relaxed, respectively, by that time, the latter being traits of a rapidly reversible photoprotective energy dissipation. Three different Z pools were identified, which play different roles in energy dissipation and photoprotection. In conclusion, ΔpH was crucial to NPQ formation and relaxation in Q. coccifera during light transitions. Only a minor fraction of Z was associated to quenching, whereas the largest Z pool was not related to thermal dissipation. The latter is proposed to participate in photoprotection acting as antioxidant.

Topics: Antioxidants; Chlorophyll; Energy Transfer; Hydrogen-Ion Concentration; Light; Oxidation-Reduction; Photochemical Processes; Photosystem II Protein Complex; Plant Leaves; Quercus; Time Factors; Xanthophylls; Zeaxanthins

The spectroscopic properties and energy transfer dynamics of the protein-bound chlorophylls and xanthophylls in monomeric, major LHCII complexes, and minor Lhcb complexes from genetically altered Arabidopsis thaliana plants have been investigated using both steady-state and time-resolved absorption and fluorescence spectroscopic methods. The pigment-protein complexes that were studied contain Chl a, Chl b, and variable amounts of the xanthophylls, zeaxanthin (Z), violaxanthin (V), neoxanthin (N), and lutein (L). The complexes were derived from mutants of plants denoted npq1 (NVL), npq2lut2 (Z), aba4npq1lut2 (V), aba4npq1 (VL), npq1lut2 (NV), and npq2 (LZ). The data reveal specific singlet energy transfer routes and excited state spectra and dynamics that depend on the xanthophyll present in the complex.

Topics: Arabidopsis; Arabidopsis Proteins; Energy Transfer; Light-Harvesting Protein Complexes; Lutein; Mutation; Spectrometry, Fluorescence; Xanthophylls; Zeaxanthins

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.

Topics: Carotenoids; Lutein; Photosystem II Protein Complex; Plants; Seasons; Tracheophyta; Xanthophylls; Zeaxanthins

Zeaxanthin epoxidase (ZE) is an enzyme operating in the violaxanthin cycle, which is involved in photoprotective mechanisms. In this work model systems to study zeaxanthin (Zx) epoxidation were developed. Two assay systems are presented in which epoxidation of Zx was observed. In these assays two mutants of Arabidopsis thaliana which have active only one of the two xanthophyll cycle enzymes were used. The npq1 mutant possesses an active ZE and is thus able to convert Zx to violaxanthin (Vx) but the violaxanthin de-epoxidase (VDE) is inactive, so that Vx cannot be converted to Zx. The other mutant, npq2, possesses an active VDE and can convert exogenous Vx to Zx under strong light conditions but reverse reaction is not possible. The first assay containing thylakoids from npq1 and npq2 mutants of A. thaliana gave positive results and high efficiency of epoxidation reaction was observed. The amount of Zx was reduced by 25%. To optimize high efficiency of epoxidation reaction additional factors facilitating both fusion of the two types of thylakoids and incorporation of Zx to their membranes were also studied. The second kind of assay contained npq1 mutant thylakoids of A. thaliana supplemented with exogenous Zx and monogalactosyldiacylglycerol (MGDG). Experiments with different proportions of Zx and MGDG showed that their optimal ratio is 1:60. In such system, due to epoxidation, the amount of Zx was reduced by 38% of its initial level. The in vitro systems of Zx epoxidation described in this paper enable analysis some properties of the ZE without necessity of its isolation.

Topics: Arabidopsis; Galactolipids; Oxidoreductases; Xanthophylls; Zeaxanthins

Photoprotective nonphotochemical quenching (NPQ) in higher plants is the result of the formation of energy-quenching traps in the light-harvesting antenna of photosystem II (PSII). The primary driving forces behind NPQ are the protonation of the thylakoid lumen and the de-epoxidation of the xanthophyll violaxanthin to zeaxanthin in the antenna. There is currently some disagreement over whether de-epoxidation occurs only at the peripheral, V1, binding site of the major LHCII or also at the internal, L2, site of the minor antenna CP29 complex of PSII. We have used density functional theory (DFT) to study of hydrogen bonding between xanthophylls and the protein scaffold of LHCII and CP29. We argue that a lack of hydrogen bonding for violaxanthin in LHCII is consistent with it being weakly bound and accessible for de-epoxidation. Conversely, the strong violaxanthin-protein hydrogen bonding at the L2 site of CP29 is consistent with evidence that it is not readily accessible for de-epoxidation and therefore quenching by zeaxanthin at the L2 of CP29 is an unlikely candidate for in vivo NPQ.

Topics: Binding Sites; Hydrogen Bonding; Models, Theoretical; Photosystem II Protein Complex; Xanthophylls; Zeaxanthins

Nonradioactive dissipation of excitation energy is the most important photoprotective mechanism in plants. In high light, the trans-thylakoid ΔpH can activate the xanthophyll cycle and protonate the PsbS protein, which are important in regulating the process of non-photochemical quenching. Despite it has been proven that the ΔpH can directly switch the major light harvesting complex (LHCIIb) from the light harvesting state to the energy dissipating state both in vitro and in vivo, the region responding to ΔpH change in LHCIIb remains obscure. In the present study, we proposed that Asp211 and Asp215 should be the amino acids that respond to the change of the trans-thylakoid ΔpH on the basis of pK(a) calculations of LHCIIb. Furthermore, Asp215 is the outmost acidic amino acid in trimeric LHCIIb, and its pK(a) value is sensitive to the protein environment, suggesting that the regulation of the protonation state of Asp215 may be important for the aggregation of LHCIIb occurred in the process of qE in vivo and in vitro.

Topics: Hydrogen-Ion Concentration; Light-Harvesting Protein Complexes; Models, Molecular; Xanthophylls; Zeaxanthins

Plants are often exposed to saturating light conditions, which can lead to oxidative stress. The carotenoid zeaxanthin, synthesized from violaxanthin by Violaxanthin De-Epoxidase (VDE) plays a major role in the protection from excess illumination. VDE activation is triggered by a pH reduction in the thylakoids lumen occurring under saturating light. In this work the mechanism of the VDE activation was investigated on a molecular level using multi conformer continuum electrostatic calculations, site directed mutagenesis and molecular dynamics. The pK(a) values of residues of the inactive VDE were determined to identify target residues that could be implicated in the activation. Five such target residues were investigated closer by site directed mutagenesis, whereas variants in four residues (D98, D117, H168 and D206) caused a reduction in enzymatic activity indicating a role in the activation of VDE while D86 mutants did not show any alteration. The analysis of the VDE sequence showed that the four putative activation residues are all conserved in plants but not in diatoms, explaining why VDE in these algae is already activated at higher pH. Molecular dynamics showed that the VDE structure was coherent at pH 7 with a low amount of water penetrating the hydrophobic barrel. Simulations carried out with the candidate residues locked into their protonated state showed instead an increased amount of water penetrating the barrel and the rupture of the H121-Y214 hydrogen bond at the end of the barrel, which is essential for VDE activation. These results suggest that VDE activation relies on a robust and redundant network, in which the four residues identified in this study play a major role.

Topics: Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; Enzyme Activation; Hydrogen Bonding; Hydrogen-Ion Concentration; Molecular Dynamics Simulation; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Oxidation-Reduction; Oxidoreductases; Protein Conformation; Static Electricity; Thylakoids; Xanthophylls; Zeaxanthins

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.

Topics: Acclimatization; Bryopsida; Chloroplasts; Droughts; Light; Osmotic Pressure; Oxidative Stress; Plant Proteins; Salt Tolerance; Sodium Chloride; Sorbitol; Thylakoid Membrane Proteins; Thylakoids; Xanthophylls; Zeaxanthins

In the violaxanthin cycle, the violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the inter-conversion between violaxanthin and zeaxanthin in both plants and green algae. The zeaxanthin epoxidase gene from the green microalga Chlorella zofingiensis (Czzep) has been isolated. This gene encodes a polypeptide of 596 amino acids. A single copy of Czzep has been found in the C. zofingiensis genome by Southern blot analysis. qPCR analysis has shown that transcript levels of Czzep were increased after zeaxanthin formation under high light conditions. The functionality of Czzep gene by heterologous genetic complementation in the Chlamydomonas mutant npq2, which lacks zeaxanthin epoxidase (ZEP) activity and accumulates zeaxanthin in all conditions, was analyzed. The Czzep gene was adequately inserted in the pSI105 vector and expressed in npq2. The positive transformants were able to efficiently convert zeaxanthin into violaxanthin, as well as to restore their maximum quantum efficiency of the PSII (Fv/Fm). These results show that Chlamydomonas can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications.

Topics: Chlamydomonas reinhardtii; Chlorella; Chlorophyta; Genetic Complementation Test; Nitrogen; Oxidoreductases; Transformation, Genetic; Xanthophylls; Zeaxanthins

The isolation and characterization of the lycopene ε-cyclase gene from the green microalga Chlorella (Chromochloris) zofingiensis (Czlcy-e) was performed. This gene is involved in the formation of the carotenoids α-carotene and lutein. Czlcy-e gene encoded a polypeptide of 654 amino acids. A single copy of Czlcy-e was found in C. zofingiensis. Functional analysis by heterologous complementation in Escherichia coli showed the ability of this protein to convert lycopene to δ-carotene. In addition, the regulation of the carotenogenic pathway by light and nitrogen was also studied in C. zofingiensis. High irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and β-carotene. Nitrogen starvation per se enhanced mRNA levels of all genes considered, except lcy-e and pds, but did not trigger the synthesis of astaxanthin, canthaxanthin nor zeaxanthin. The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress.

Topics: beta Carotene; Canthaxanthin; Carotenoids; Chlorella; Escherichia coli; Intramolecular Lyases; Light; Lutein; Microalgae; Nitrogen; RNA, Messenger; Stress, Physiological; Transcription, Genetic; Xanthophylls; Zeaxanthins

To prevent photo-oxidative damage to the photosynthetic membrane in strong light, plants dissipate excess absorbed light energy as heat in a mechanism known as non-photochemical quenching (NPQ). NPQ is triggered by the trans-membrane proton gradient (ΔpH), which causes the protonation of the photosystem II light-harvesting antenna (LHCII) and the PsbS protein, as well as the de-epoxidation of the xanthophyll violaxanthin to zeaxanthin. The combination of these factors brings about formation of dissipative pigment interactions that quench the excess energy. The formation of NPQ is associated with certain absorption changes that have been suggested to reflect a conformational change in LHCII brought about by its protonation. The light-minus-dark recovery absorption difference spectrum is characterized by a series of positive and negative bands, the best known of which is ΔA(535). Light-minus-dark recovery resonance Raman difference spectra performed at the wavelength of the absorption change of interest allows identification of the pigment responsible from its unique vibrational signature. Using this technique, the origin of ΔA(535) was previously shown to be a subpopulation of red-shifted zeaxanthin molecules. In the absence of zeaxanthin (and antheraxanthin), a proportion of NPQ remains, and the ΔA(535) change is blue-shifted to 525 nm (ΔA(525)). Using resonance Raman spectroscopy, it is shown that the ΔA(525) absorption change in Arabidopsis leaves lacking zeaxanthin belongs to a red-shifted subpopulation of violaxanthin molecules formed during NPQ. The presence of the same ΔA(535) and ΔA(525) Raman signatures in vitro in aggregated LHCII, containing zeaxanthin and violaxanthin, respectively, leads to a new proposal for the origin of the xanthophyll red shifts associated with NPQ.

Topics: Absorption; Arabidopsis; Binding Sites; Kinetics; Light; Light-Harvesting Protein Complexes; Protein Kinases; Protein Multimerization; Protein Structure, Quaternary; Spectrum Analysis, Raman; Xanthophylls; Zeaxanthins

The efficiency of photosystem II antenna complexes (LHCs) in higher plants must be regulated to avoid potentially damaging overexcitation of the reaction centre in excess light. Regulation is achieved via a feedback mechanism known as non-photochemical quenching (NPQ), triggered the proton gradient (ΔpH) causing heat dissipation within the LHC antenna. ΔpH causes protonation of the LHCs, the PsbS protein and triggers the enzymatic de-epoxidation of the xanthophyll, violaxanthin, to zeaxanthin. A key step in understanding the mechanism is to decipher whether PsbS and zeaxanthin cooperate to promote NPQ. To obtain clues about their respective functions we studied the effects of PsbS and zeaxanthin on the rates of NPQ formation and relaxation in wild-type Arabidopsis leaves and those overexpressing PsbS (L17) or lacking zeaxanthin (npq1). Overexpression of PsbS was found to increase the rate of NPQ formation, as previously reported for zeaxanthin. However, PsbS overexpression also increased the rate of NPQ relaxation, unlike zeaxanthin, which is known decrease the rate. The enhancement of PsbS levels in plants lacking zeaxanthin (npq1) by either acclimation to high light or crossing with L17 plants showed that the effect of PsbS was independent of zeaxanthin. PsbS levels also affected the kinetics of the 535 nm absorption change (ΔA535), which monitors the formation of the conformational state of the LHC antenna associated with NPQ, in an identical way. The antagonistic action of PsbS and zeaxanthin with respect to NPQ and ΔA535 relaxation kinetics suggests that the two molecules have distinct regulatory functions.

Topics: Acclimatization; Arabidopsis; Arabidopsis Proteins; Feedback, Physiological; Genetic Variation; Light; Light-Harvesting Protein Complexes; Mutation; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Plant Leaves; Xanthophylls; Zeaxanthins

Although the wheat hybrids have often shown higher grain yields, the physiological basis of the higher yields remains unknown. Previous studies suggest that tolerance to photoinhibition in the hybrid may be one of the physiological bases (Yang et al., 2006, Plant Sci 171:389-97). The objective of this study was to further investigate the possible mechanism responsible for tolerance to photoinhibition in the hybrid. Photosystem II (PSII) photochemistry, the xanthophyll cycle, and antioxidative defense system were compared between the hybrid and its parents subjected to high light stress (1500μmolm(-2)s(-1)). The analyses of oxygen-evolving activity, chlorophyll fluorescence, and protein blotting demonstrated that the higher tolerance in the hybrid than in its parents was associated with its higher tolerance of PSII to photoinhibition. High light induced an increase in non-photochemical quenching, and this increase was greater in the hybrid than in its parents. There were no differences in the pool size of the xanthophyll cycle between the hybrid and its parents. The content of violaxanthin decreased significantly, whereas the content of zeaxanthin+antherxanthin increased considerably during high light treatments. However, the decrease in violaxanthin content and the increase in zeaxanthin+antherxanthin content were greater in the hybrid than in its parents. High light resulted in a significant accumulation of H(2)O(2), O(2)(-) and catalytic Fe, and this accumulation was less in the hybrid than in its parents. High light induced a significant increase in the activities of superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, and monodehydroascorbate reductase, and these increases were greater in the hybrid than its parents. These results suggest that the higher tolerance to photoinhibition in the hybrid may be associated with its higher capacity for antioxidative defense metabolism and the xanthophyll cycle.

Topics: Adaptation, Physiological; Antioxidants; Chimera; Chlorophyll; Light; Oxygen; Photochemistry; Photosystem II Protein Complex; Plant Leaves; Stress, Physiological; Time Factors; Triticum; Xanthophylls; Zeaxanthins

Using freeze-fracture electron microscopy we have recently shown that non-photochemical quenching (NPQ), a mechanism of photoprotective energy dissipation in higher plant chloroplasts, involves a reorganization of the pigment-protein complexes within the stacked grana thylakoids. Photosystem II light harvesting complexes (LHCII) are reorganized in response to the amplitude of the light driven transmembrane proton gradient (ΔpH) leading to their dissociation from photosystem II reaction centers and their aggregation within the membrane1. This reorganization of the PSII-LHCII macrostructure was found to be enhanced by the formation of zeaxanthin and was associated with changes in the mobility of the pigment-protein complexes therein1. We suspected that the structural changes we observed were linked to the ΔpH-induced changes in thylakoid membrane thickness that were first observed by Murikami and Packer. Here using thin-section electron microscopy we show that the changes in thylakoid membrane thickness do not correlate with ΔpH per se but rather the amplitude of NPQ and is thus affected by the de-epoxidation of the LHCII bound xanthophyll violaxanthin to zeaxanthin. We thus suggest that the change in thylakoid membrane thickness occurring during NPQ reflects the conformational change within LHCII proteins brought about by their protonation and aggregation within the membrane

Topics: Arabidopsis; Chloroplasts; Light-Harvesting Protein Complexes; Microscopy, Electron, Transmission; Photosystem II Protein Complex; Plant Proteins; Spinacia oleracea; Thylakoids; Xanthophylls; Zeaxanthins

In the violaxanthin (V) cycle, V is de-epoxidized to zeaxanthin (Z) when strong light or light combined with other stressors lead to an overexcitation of photosystems. However, plants can also suffer stress in darkness and recent reports have shown that dehydration triggers V-de-epoxidation in the absence of light. In this study, we used the highly stress-tolerant brown alga Pelvetia canaliculata as a model organism, due to its lack of lutein and its non-photochemical quenching independent of the transthylakoidal-ΔpH, to study the triggering of the V-cycle in darkness induced by abiotic stressors.. We have shown that besides desiccation, other factors such as immersion, anoxia and high temperature also induced V-de-epoxidation in darkness. This process was reversible once the treatments had ceased (with the exception of heat, which caused lethal damage). Irrespective of the stressor applied, the resulting de-epoxidised xanthophylls correlated with a decrease in Fv/Fm, suggesting a common function in the down-regulation of photosynthetical efficiency. The implication of the redox-state of the plastoquinone-pool and of the differential activity of V-cycle enzymes on V-de-epoxidation in darkness was also examined. Current results suggest that both violaxanthin de-epoxidase (VDE) and zeaxanthin-epoxidase (ZE) have a basal constitutive activity even in darkness, being ZE inhibited under stress. This inhibition leads to Z accumulation.. This study demonstrates that V-cycle activity is triggered by several abiotic stressors even when they occur in an absolute absence of light, leading to a decrease in Fv/Fm. This finding provides new insights into an understanding of the regulation mechanism of the V-cycle and of its ecophysiological roles.

Topics: Darkness; Desiccation; Hot Temperature; Oxidation-Reduction; Oxidoreductases; Oxygen; Phaeophyceae; Stress, Physiological; Xanthophylls; Zeaxanthins

Lhcb6 (CP24) is a monomeric antenna protein of photosystem II, which has been shown to play special roles in photoprotective mechanisms, such as the Non-Photochemical Quenching and reorganization of grana membranes in excess light conditions. In this work we analyzed Lhcb6 in vivo and in vitro: we show this protein, upon activation of the xanthophyll cycle, accumulates zeaxanthin into inner binding sites faster and to a larger extent than any other pigment-protein complex. By comparative analysis of Lhcb6 complexes violaxanthin or zeaxanthin binding, we demonstrate that zeaxanthin not only down-regulates chlorophyll singlet excited states, but also increases the efficiency of chlorophyll triplet quenching, with consequent reduction of singlet oxygen production and significant enhancement of photo-stability. On these bases we propose that Lhcb6, the most recent addition to the Lhcb protein family which evolved concomitantly to the adaptation of photosynthesis to land environment, has a crucial role in zeaxanthin-dependent photoprotection.

Topics: Arabidopsis; Arabidopsis Proteins; Chlorophyll; Chlorophyll Binding Proteins; Epoxy Compounds; Kinetics; Light; Light-Harvesting Protein Complexes; Photobleaching; Photosystem II Protein Complex; Plant Leaves; Protein Binding; Singlet Oxygen; Spectrum Analysis; Thylakoids; Xanthophylls; Zeaxanthins

The influence of excess Cu ions and high light treatment on the function of photosystem II was investigated in order to examine how this heavy metal modifies the photoprotective mechanisms operating at the molecular level in Secale cereale plants. Thus, non-treated plants and those treated with 5 or 50 microM Cu, simultaneously illuminated with 150 micromol m(-2) s(-1) or 1200 micromol m(-2) s(-1) light intensity, were studied. To analyze the PSII reaction to the stress conditions, Chl a fluorescence induction was applied. An increase in the value of Phi(PSII) and R(fd) parameters indicated that the photosynthetic apparatus adapted to the high light condition by effective utilization of excitation energy in the light and dark phases of photosynthesis. This phenomenon was accompanied by dissipation of excitation energy within the antenna complexes. The xanthophyll cycle pigments in Secale cereale leaves were separated and quantified by the HPLC technique. The results showed that, under high light irradiance, both 5 and 50 microM Cu induced the process of violaxanthin de-epoxidation and zeaxanthin accumulation. The significant zeaxanthin accumulation was found to be involved in photoprotective energy dissipation as heat, which was supported by correlation between the rate of violaxanthin de-epoxidation and the value of SV parameters. Interestingly, Cu treatment caused violaxanthin isomerization from its trans to 15-, 13- and 9-cis forms in proportional correlation to the metal concentration. This phenomenon was confirmed by a study of Cu-induced violaxanthin isomerization in vitro, which suggests a direct metal-pigment molecule interaction. We also observed that the violaxanthin trans-cis isomerization increased simultaneously with anteraxanthin content. On the basis of these findings, it can be speculated that violaxanthin isomerization is the basic process responsible for the xanthophyll cycle operation.

Topics: Copper; Isomerism; Light; Photosystem II Protein Complex; Plant Leaves; Secale; Xanthophylls; Zeaxanthins

Spectroscopic measurements of Arabidopsis leaves have shown that the energy-dependent component of non-photochemical quenching (NPQ), known as qE, is associated with an absorption change at 535 nm (ΔA(535)). Identical measurements on the zeaxanthin-deficient mutant npq1 reveal a similar spectroscopic signature at 525 nm (ΔA(525)). We investigated whether these red-shifts may arise from excitonic interactions among homodimers of xanthophylls, zeaxanthin, and violaxanthin, bound at the peripheral V1 binding site on adjacent light-harvesting complex II (LHCII) trimers. Estimates of the relative geometries of these pigment pairs were obtained from the structure of LHCII. The excitonic couplings of zeaxanthin and violaxanthin dimers were probed using the time-dependent density functional theory method (TD-DFT). Calculations indicated that dimers formed between zeaxanthin or violaxanthin molecules using the published LHCII structure resulted in absorption blue shifts, typical of an H-type (parallel) geometry. In contrast, if the volume of the LHCII structure was modified to reflect the change in membrane thickness that occurs upon ΔpH formation, then both zeaxanthin and violaxanthin dimers adopted a J-type (collinear) geometry, and the resulting spectral shift was to the red region. The magnitudes of these predicted red-shifts are in good agreement with the experimental magnitudes. We therefore conclude that the observed xanthophyll red-shift results from the combination of both LHCII aggregation and changes in membrane thickness during qE. ΔA(535) may therefore be considered a "marker of aggregation" between LHCII trimers upon qE formation.

Topics: Arabidopsis; Dimerization; Light-Harvesting Protein Complexes; Models, Molecular; Models, Theoretical; Photosynthesis; Plant Proteins; Protein Conformation; Quantum Theory; Thylakoids; Xanthophylls; Zeaxanthins

The photoprotective role of the universal violaxanthin cycle that interconverts violaxanthin (V), antheraxanthin (A), and zeaxanthin (Z) is well established, but functions of the analogous conversions of lutein-5,6-epoxide (Lx) and lutein (L) in the selectively occurring Lx cycle are still unclear. We investigated carotenoid pools in Lx-rich leaves of avocado (Persea americana) during sun or shade acclimation at different developmental stages. During sun exposure of mature shade leaves, an unusual decrease in L preceded the deepoxidation of Lx to L and of V to A+Z. In addition to deepoxidation, de novo synthesis increased the L and A+Z pools. Epoxidation of L was exceptionally slow, requiring about 40 d in the shade to restore the Lx pool, and residual A+Z usually persisted overnight. In young shade leaves, the Lx cycle was reversed initially, with Lx accumulating in the sun and declining in the shade. De novo synthesis of xanthophylls did not affect alpha- and beta-carotene pools on the first day, but during long-term acclimation alpha-carotene pools changed noticeably. Nonetheless, the total change in alpha- and beta-branch carotenoid pools was equal. We discuss the implications for regulation of metabolic flux through the alpha- and beta-branches of carotenoid biosynthesis and potential roles for L in photoprotection and Lx in energy transfer to photosystem II and explore physiological roles of both xanthophyll cycles as determinants of photosystem II efficiency.

Topics: Acclimatization; Chlorophyll; Kinetics; Lutein; Persea; Plant Leaves; Sunlight; Xanthophylls; Zeaxanthins

Nonphotochemical quenching (NPQ) is a fundamental mechanism in photosynthesis by which plants protect themselves against excess excitation energy and which is thus of crucial importance for plant survival and fitness. Recently, carotenoid radical cation (Car(*+)) formation has been discovered to be a key step in the feedback deexcitation quenching component (qE) of NPQ, whose molecular mechanism and location remains elusive. A recent model for qE suggests that the replacement of violaxanthin (Vio) by zeaxanthin (Zea) in photosynthetic pigment binding pockets can in principle result in qE via the so-called "gear-shift" or electron transfer quenching mechanisms. We performed pump-probe measurements on individual antenna complexes of photosystem II (CP24, CP26 and CP29) upon excitation of the chlorophylls (Chl) into their first excited Q(y) state at 660 nm when either Vio or Zea was bound to those complexes. The Chl lifetime was then probed by measuring the decay kinetics of the Chl excited state absorption (ESA) at 900 nm. The charge-transfer quenching mechanism, which is characterized by a spectral signature of the transiently formed Zea radical cation (Zea(*+)) in the near-IR, has also been addressed, both in solution and in light-harvesting complexes of photosystem II (LHC-II). Applying resonant two-photon two-color ionization (R2P2CI) spectroscopy we show here the formation of beta-Car(*+) in solution, which occurs on a femtosecond time-scale by direct electron transfer to the solvent. The beta-Car(*+) maxima strongly depend on the solvent polarity. Moreover, our two-color two-photon spectroscopy on CP29 reveals the spectral position of Zea(*+) in the near-IR region at 980 nm.

Topics: Arabidopsis Proteins; Cations, Monovalent; Chlorophyll; Chlorophyll Binding Proteins; Electron Transport; Free Radicals; Light-Harvesting Protein Complexes; Models, Biological; Photochemical Processes; Photosystem II Protein Complex; Plant Proteins; Recombinant Proteins; Spectrophotometry; Xanthophylls; Zeaxanthins

Zeaxanthin epoxidase (ZE, E.C. 1.14.13.90), an enzyme belonging to the lipocalin superfamily, catalyses the conversion of zeaxanthin to antheraxanthin and violaxanthin. These reactions are part of the xanthophyll biosynthetic pathway and the xanthophyll cycle. The role of carotenoids in the dissipation of excessive light energy has been widely studied using mutants with a disabled carotenoid biosynthetic pathway. In this paper, the transgenic line MaZEP7 with partially disabled ZE activity is described and compared with wild-type plants and npq2 mutant lacking active ZE. We examined the presence and the abundance of aba1 transcripts, measured pigment composition, xanthophyll cycle functioning and chlorophyll fluorescence in all three lines. The MaZEP7 line contains additional copies of the aba1 gene introduced by agroinfiltration, but no enhanced aba1 transcript level was observed. In addition, ZE activity in MaZEP7 was impaired, resulting in an altered xanthophyll profile. In dark-adapted plants, violaxanthin and neoxanthin levels were lower than in wild-type plants, whereas antheraxanthin and zeaxanthin levels were considerably higher. The presence of lutein epoxide was also observed. Violaxanthin levels changed only minimally during light exposition, whereas antheraxanthin was converted to zeaxanthin and there was no epoxidation during the course of the experiment indicating disturbed xanthophyll cycle functioning. The amounts of carotenoids and chlorophylls on a dry weight basis and chl a/chl b ratio were similar in all lines. The presence of epoxidated pigments in MaZEP7 plants indicates that epoxidation occurs, but it is likely very slow. Chlorophyll fluorescence measurements showed that the dependence of electron transport rates on light intensity for the MaZEP7 line resembled the npq2 mutant. Kinetic measurements showed that the MaZEP7 line exhibited very rapid induction and a high steady-state value of non-photochemical quenching.

Topics: Arabidopsis; Carotenoids; Chlorophyll; Gene Expression Regulation, Plant; Kinetics; Light; Oxidoreductases; Photochemistry; Plants, Genetically Modified; Xanthophylls; Zeaxanthins

We attempted to establish a high-speed and high-resolution profiling method for a carotenoid mixture as a highly selective and highly sensitive detection method; the analysis was carried out by supercritical fluid chromatography (SFC) coupled with mass spectrometry (MS). When an octadecyl-bonded silica (ODS) particle-packed column was used for separation, seven carotenoids including structural isomers were successfully separated within 15 min. This result indicated not only improved separation but also improved throughput compared to the separation and throughput in RP-HPLC. The use of a monolithic ODS column resulted in additional improvement in both the resolution and the throughput; the analysis time was reduced to 4 min by increasing the flow rate. Furthermore, carotenoids in biological samples containing the complex matrices were separated effectively by using several monolithic columns whose back pressure was very low. The mass spectrometer allowed us to perform a more sensitive analysis than UV detection; the detection limit of each carotenoid was 50 pg or below. This is the first report of carotenoid analysis carried out by SFC-MS. The profiling method developed in this study will be a powerful tool for carrying out accurate profiling of biological samples.

Topics: Animals; beta Carotene; Carotenoids; Chlamydomonas reinhardtii; Chromatography, Supercritical Fluid; Lutein; Lycopene; Mass Spectrometry; Pressure; Rheology; Sensitivity and Specificity; Silicon Dioxide; Xanthophylls; Zeaxanthins

Light harvesting complexes have been identified in all chlorophyll-based photosynthetic organisms. Their major function is the absorption of light and its transport to the reaction centers, however, they are also involved in excess energy quenching, the so-called non-photochemical quenching (NPQ). In particular, electron transfer and the resulting formation of carotenoid radical cations have recently been discovered to play an important role during NPQ in green plants. Here, the results of our theoretical investigations of carotenoid radical cation formation in the major light harvesting complex LHC-II of green plants are reported. The carotenoids violaxanthin, zeaxanthin and lutein are considered as potential quenchers. In agreement with experimental results, it is shown that zeaxanthin cannot quench isolated LHC-II complexes. Furthermore, subtle structural differences in the two lutein binding pockets lead to substantial differences in the excited state properties of the two luteins. In addition, the formation mechanism of carotenoid radical cations in light harvesting complexes LH2 and LH1 of purple bacteria is studied. Here, the energetic position of the S(1) state of the involved carotenoids neurosporene, spheroidene, spheroidenone and spirilloxanthin seems to determine the occurrence of radical cations in these LHCs upon photo-excitation. An elaborate pump-deplete-probe experiment is suggested to challenge the proposed mechanism.

Topics: Bacterial Proteins; Carotenoids; Cations, Monovalent; Energy Transfer; Free Radicals; Light-Harvesting Protein Complexes; Lutein; Models, Biological; Models, Molecular; Photochemical Processes; Plant Proteins; Plants; Proteobacteria; Xanthophylls; Zeaxanthins

In the Crassulacean acid metabolism (CAM) plants Clusia alata Triana and Planch., decarboxylation of citrate during phase III of CAM took place later than malate decarboxylation. The interdependence of these two CO(2) and NADPH sources is discussed. High light accelerated malate decarboxylation during the day and lowered citrate levels. Strong light stress also activated mechanisms that can protect the plant against oxidative stress. Upon transfer from low light (200micromol m(-2)s(-1)) to high light (650-740micromol m(-2)s(-1)), after 2 days, there was a transient increase of non-photochemical quenching (NPQ) of fluorescence of chlorophyll a of photosystem II. This indicated acute photoinhibition, which declined again after 7 days of exposure. Conversely, after 1 week exposure to high light, the mechanisms of interconversion of violaxanthin (V), antheraxanthin (A), zeaxanthin (Z) (epoxydation/de-epoxydation) were activated. This was accompanied by an increase in pigment levels at dawn and dusk.

Topics: Adaptation, Physiological; Chlorophyll; Chlorophyll A; Citric Acid; Clusia; Decarboxylation; Fluorescence; Light; Malates; Photosynthesis; Photosystem II Protein Complex; Stress, Physiological; Xanthophylls; Zeaxanthins

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

Topics: Amino Acid Sequence; Arabidopsis; Ascorbic Acid; Crystallography, X-Ray; Gene Expression Regulation, Plant; Hydrogen-Ion Concentration; Models, Molecular; Molecular Sequence Data; Oxidoreductases; Photosynthesis; Protein Multimerization; Protein Structure, Secondary; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Substrate Specificity; Thylakoids; Xanthophylls; Zeaxanthins

We performed transient absorption (TA) measurements on CP29 minor light-harvesting complexes that were reconstituted in vitro with either violaxanthin (Vio) or zeaxanthin (Zea) and demonstrate that the Zea-bound CP29 complexes exhibit charge-transfer (CT) quenching that has been correlated with the energy-dependent quenching (qE) in higher plants. Simulations of the difference TA kinetics reveal two-phase kinetics for intracomplex energy transfer to the CT quenching site in CP29 complexes, with a fast <500 fs component and a approximately 6 ps component. Specific chlorophyll sites within CP29 are identified as likely locations for CT quenching. We also construct a kinetic model for CT quenching during qE in an intact system that incorporates CP29 as a CT trap and show that the model is consistent with previous in vivo measurements on spinach thylakoid membranes. Finally, we compare simulations of CT quenching in thylakoids with those of the individual CP29 complexes and propose that CP29 rather than LHCII is a site of CT quenching.

Topics: Adsorption; Electron Transport; Energy Transfer; Kinetics; Light; Light-Harvesting Protein Complexes; Models, Chemical; Photosystem II Protein Complex; Spectrophotometry, Infrared; Xanthophylls; Zeaxanthins

The response of Norway spruce saplings (Picea abies [L.] Karst.) was monitored continuously during short-term exposure (10 days) to high irradiance (HI; 1000micromol m(-2)s(-1)). Compared with plants acclimated to low irradiance (100micromol m(-2)s(-1)), plants after HI exposure were characterized by a significantly reduced CO(2) assimilation rate throughout the light response curve. Pigment contents varied only slightly during HI exposure, but a rapid and strong response was observed in xanthophyll cycle activity, particularly within the first 3 days of the HI treatment. Both violaxanthin convertibility under HI and the amount of zeaxanthin pool sustained in darkness increased markedly under HI conditions. These changes were accompanied by an enhanced non-radiative dissipation of absorbed light energy (NRD) and the acceleration of induction of both NRD and de-epoxidation of the xanthophyll cycle pigments. We found a strong negative linear correlation between the amount of sustained de-epoxidized xanthophylls and the photosystem II (PSII) photochemical efficiency (F(V)/F(M)), indicating photoprotective down-regulation of the PSII function. Recovery of F(V)/F(M) at the end of the HI treatment revealed that Norway spruce was able to cope with a 10-fold elevated irradiance due particularly to an efficient NRD within the PSII antenna that was associated with enhanced violaxanthin convertibility and a light-induced accumulation of zeaxanthin that persisted in darkness.

Topics: Absorption; Carbon Dioxide; Chlorophyll; Chlorophyll A; Electron Transport; Epoxy Compounds; Fluorescence; Light; Lutein; Norway; Photosynthesis; Photosystem II Protein Complex; Picea; Time Factors; Xanthophylls; Zeaxanthins

The accumulation of carotenoids in higher plants is regulated by the environment, tissue type and developmental stage. In Brassica napus leaves, beta-carotene and lutein were the main carotenoids present while petals primarily accumulated lutein and violaxanthin. Carotenoid accumulation in seeds was developmentally regulated with the highest levels detected at 35-40 days post anthesis. The carotenoid biosynthesis pathway branches after the formation of lycopene. One branch forms carotenoids with two beta rings such as beta-carotene, zeaxanthin and violaxanthin, while the other introduces both beta- and epsilon-rings in lycopene to form alpha-carotene and lutein. By reducing the expression of lycopene epsilon-cyclase (epsilon-CYC) using RNAi, we investigated altering carotenoid accumulation in seeds of B. napus. Transgenic seeds expressing this construct had increased levels of beta-carotene, zeaxanthin, violaxanthin and, unexpectedly, lutein. The higher total carotenoid content resulting from reduction of epsilon-CYC expression in seeds suggests that this gene is a rate-limiting step in the carotenoid biosynthesis pathway. epsilon-CYC activity and carotenoid production may also be related to fatty acid biosynthesis in seeds as transgenic seeds showed an overall decrease in total fatty acid content and minor changes in the proportions of various fatty acids.

Topics: beta Carotene; Blotting, Southern; Brassica napus; Carotenoids; Chromatography, Gas; Chromatography, High Pressure Liquid; Down-Regulation; Fatty Acids; Gene Expression Regulation, Developmental; Gene Expression Regulation, Plant; Intramolecular Lyases; Lutein; Plants, Genetically Modified; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Plant; RNA, Small Interfering; Seeds; Xanthophylls; Zeaxanthins

Laurdan (6-lauroyl-2-dimethylaminonaphthalene) fluorescence spectroscopy has been applied to probe the physical status of the thylakoid membrane upon conversion of violaxanthin to zeaxanthin. So far, only phospholipid-dominated membranes have been studied by this method and hereby we report the first use of laurdan in mono- and digalactosyldiacylglycerol-dominated membrane systems. The generalised polarisation (GP) of laurdan was used as a measure of the structural effect of xanthophyll cycle pigments in isolated spinach (Spinacia oleracea) thylakoids and in model membrane vesicles composed of chloroplast galactolipids. Higher GP values indicate a membrane in a more ordered structure, whereas lower GP values point to a membrane in a less ordered fluid phase. The method was used to probe the effect of violaxanthin and zeaxanthin in thylakoid membranes at different temperatures. At 4, 25 and 37 degrees C the GP values for dark-adapted thylakoids in the violaxanthin-form were 0.55, 0.28 and 0.26. After conversion of violaxanthin to zeaxanthin, at the same temperatures, the GP values were 0.62, 0.36 and 0.34, respectively. GP values increased gradually upon conversion of violaxanthin to zeaxanthin. Similar results were obtained in the liposomal systems in the presence of these xanthophyll cycle pigments. We conclude from these results that the conversion of violaxanthin to zeaxanthin makes the thylakoid membrane more ordered.

Topics: 2-Naphthylamine; Fatty Acids; Galactolipids; Laurates; Lipid Bilayers; Liposomes; Spectrometry, Fluorescence; Spinacia oleracea; Temperature; Thylakoids; Xanthophylls; Zeaxanthins

Recently, a mechanism for the energy-dependent component (qE) of non-photochemical quenching (NPQ), the fundamental photo-protection mechanism in green plants, has been suggested. Replacement of violaxanthin by zeaxanthin in the binding pocket of the major light harvesting complex LHC-II may be sufficient to invoke efficient chlorophyll fluorescence quenching. Our quantum chemical calculations, however, show that the excited state energies of violaxanthin and zeaxanthin are practically identical when their geometry is constrained to the naturally observed structure of violaxanthin in LHC-II. Therefore, since violaxanthin does not quench LHC-II, zeaxanthin should not either. This theoretical finding is nicely in agreement with experimental results obtained by femtosecond spectroscopy on LHC-II complexes containing violaxanthin or zeaxanthin.

Topics: Chlorophyll; Computer Simulation; Fluorescence; Light-Harvesting Protein Complexes; Models, Molecular; Molecular Structure; Protein Structure, Secondary; Xanthophylls; Zeaxanthins

Pulsed electron nuclear double resonance (ENDOR) and two-dimensional (2D)-hyperfine sublevel correlation spectroscopy (HYSCORE) studies in combination with density functional theory (DFT) calculations revealed that photo-oxidation of natural zeaxanthin (ex Lycium halimifolium) and violaxanthin (ex Viola tricolor) on silica-alumina produces the carotenoid radical cations (Car*+) and also the neutral carotenoid radicals (#Car*) as a result of proton loss (indicated by #) from the C4(4') methylene position or one of the methyl groups at position C5(5'), C9(9'), or C13(13'), except for violaxanthin where the epoxide at positions C5(5')-C6(6') raises the energy barrier for proton loss, and the neutral radicals #Car*(4) and #Car*(5) are not observed. DFT calculations predict the largest isotropic beta-methyl proton hyperfine couplings to be 8 to 10 MHz for Car*+, in agreement with previously reported hyperfine couplings for carotenoid pi-conjugated radicals with unpaired spin density delocalized over the whole molecule. Anisotropic alpha-proton hyperfine coupling tensors determined from the HYSCORE analysis were assigned on the basis of DFT calculations with the B3LYP exchange-correlation functional and found to arise not only from the carotenoid radical cation but also from carotenoid neutral radicals, in agreement with the analysis of the pulsed ENDOR data. The formation of the neutral radical of zeaxanthin should provide another effective nonphotochemical quencher of the excited state of chlorophyll for photoprotection in the presence of excess light.

Topics: Aluminum Oxide; Anisotropy; Electron Spin Resonance Spectroscopy; Free Radicals; Models, Molecular; Molecular Conformation; Oxidation-Reduction; Photochemistry; Protons; Silicon Dioxide; Spectrophotometry, Ultraviolet; Xanthophylls; Zeaxanthins

The epoxidation of zeaxanthin (Zx) to violaxanthin after exposure to different light stress conditions has been studied in Arabidopsis (Arabidopsis thaliana). Formation of Zx was induced by illumination of intact leaves for up to 8 h at different light intensities and temperatures. The kinetics of epoxidation was found to be gradually retarded with increasing light stress during pre-illumination, indicating a gradual down-regulation of the Zx epoxidase activity. Retardation of the epoxidation rates by a factor of up to 10 was inducible either by increasing the light intensity or by extending the illumination time or by decreasing the temperature during pre-illumination. The retardation of the epoxidation kinetics was correlated with a decrease of the PSII quantum efficiency after the pre-illumination treatment. Experiments with the stn7/stn8 mutant of Arabidopsis indicated that the thylakoid protein kinases STN7 and STN8, which are required for the phosphorylation of PSII proteins, are not involved in the short-term down-regulation of Zx epoxidation. However, the retardation of Zx epoxidation was maintained in thylakoids isolated from pre-illuminated leaves, indicating that a direct modification of the Zx epoxidase is most likely involved in the light-induced down-regulation.

Topics: Arabidopsis; Dose-Response Relationship, Radiation; Immunoblotting; Light; Oxidation-Reduction; Oxidative Stress; Oxidoreductases; Photosystem II Protein Complex; Time Factors; Xanthophylls; Zeaxanthins

Nonphotochemical quenching (NPQ) is the photoprotective dissipation of energy in photosynthetic membranes. The hypothesis that the DeltapH-dependent component of NPQ (qE) component of non-photochemical quenching is controlled allosterically by the xanthophyll cycle has been tested using Arabidopsis mutants with different xanthophyll content and composition of Lhcb proteins. The titration curves of qE against DeltapH were different in chloroplasts containing zeaxanthin or violaxanthin, proving their roles as allosteric activator and inhibitor, respectively. The curves differed in mutants deficient in lutein and specific Lhcb proteins. The results show that qE is determined by xanthophyll occupancy and the structural interactions within the antenna that govern allostericity.

Topics: Allosteric Regulation; Arabidopsis; Arabidopsis Proteins; Chlorophyll Binding Proteins; Hydrogen-Ion Concentration; Light; Light-Harvesting Protein Complexes; Mutation; Photochemistry; Photosynthesis; Proton-Motive Force; Xanthophylls; Zeaxanthins

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 NPQ approximately 0.9) in barley leaves treated with 10 microM methyl viologen (MV) under white light (100 micromol photonsm(-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.

Topics: Carbon Dioxide; Dose-Response Relationship, Radiation; Electron Transport; Gene Expression Regulation, Plant; Hordeum; Light; NADP; Oxidative Stress; Paraquat; Photosystem I Protein Complex; Photosystem II Protein Complex; Pigments, Biological; Plant Leaves; Xanthophylls; Zeaxanthins

Two mutants of Arabidopsis thaliana deficient in lutein have been investigated with respect to their responses to growth under a range of suboptimal conditions. The first mutant, lut1, was enriched in violaxanthin, antheraxanthin, zeaxanthin and zeinoxanthin compared with the wild-type (WT). In the second mutant, lut2, the lack of lutein was compensated for only by an increase in xanthophyll cycle (XC) carotenoids. Upon transfer of plants grown under optimal conditions to high light (HL), drought or HL + drought, both mutants acclimated during several days to the new conditions to the same extent as the WT. In contrast, transfer to chilling conditions (6 degrees C) for 6 days induced responses that were different between WT and mutants and between the mutants themselves. In contrast to the WT, the lut2 mutant in particular exhibited a large increase in the Chl a/b ratio and the XC pool size, extensive de-epoxidation and an enhanced extent of non-photochemical quenching. It is suggested that although the role of lutein in the structure and organisation of the light-harvesting complexes can be fulfilled by other xanthophylls under excess light conditions at optimal temperatures, this is not the case at low temperature.

Topics: Acclimatization; Arabidopsis; beta Carotene; Chloroplasts; Cryptoxanthins; Droughts; Intracellular Membranes; Light; Lutein; Mutation; Xanthophylls; Zeaxanthins

Energy-dependent quenching of excess absorbed light energy (qE) is a vital mechanism for regulating photosynthetic light harvesting in higher plants. All of the physiological characteristics of qE have been positively correlated with charge transfer between coupled chlorophyll and zeaxanthin molecules in the light-harvesting antenna of photosystem II (PSII). We found evidence for charge-transfer quenching in all three of the individual minor antenna complexes of PSII (CP29, CP26, and CP24), and we conclude that charge-transfer quenching in CP29 involves a delocalized state of an excitonically coupled chlorophyll dimer. We propose that reversible conformational changes in CP29 can "tune" the electronic coupling between the chlorophylls in this dimer, thereby modulating the energy of the chlorophyll-zeaxanthin charge-transfer state and switching on and off the charge-transfer quenching during qE.

Topics: Arabidopsis Proteins; Chlorophyll; Chlorophyll A; Chlorophyll Binding Proteins; Chloroplast Proteins; Electron Transport; Electrophysiology; Light; Light-Harvesting Protein Complexes; Lutein; Models, Molecular; Photosystem II Protein Complex; Protein Conformation; Recombinant Proteins; Ribonucleoproteins; Structure-Activity Relationship; Xanthophylls; Zeaxanthins

To evaluate the role of specific xanthophylls in light utilization, wild-type and xanthophyll mutant plants (npq1, npq2, lut2, lut2npq1 and lut2npq2) from Arabidopsis thaliana were grown under three different light regimes: 30 (low light, LL), 150 (medium light, ML) and 450 (high light, HL) mumol photons m(-2) s(-1). We studied the pigment content, growth rate, xanthophyll cycle activity, chlorophyll fluorescence parameters and the response to photoinhibition. All genotypes differed strongly in the growth rates and the resistance against photoinhibition. In particular, replacement of lutein (Lut) by violaxanthin (Vx) in the lut2npq1 mutant did not affect the growth at non-saturating light intensities (LL and ML), but led to a pronounced reduction of growth under HL conditions, indicating an important photoprotective role of Lut. This was further supported by a much higher sensitivity of all Lut-deficient plants to photoinhibition in comparison with the wild type. In contrast, replacement of Lut by zeaxanthin (Zx) in lut2npq2 led to a pronounced reduction of growth under all light regimes, most likely related to the permanent non-photochemical dissipation of excitation energy by Zx at Vx-binding sites and the destabilization of antenna proteins by binding of Zx to Lut-binding sites. The high susceptibility of lut2npq2 to photoinhibition in comparison with npq2 further indicated that the photoprotective function of Zx is abolished in the absence of Lut. Thus, it can be concluded from our work that neither Vx nor Zx is able to fulfil the essential photoprotective function at Lut-binding sites under in vivo conditions.

Topics: Arabidopsis; Light; Mutation; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Time Factors; Xanthophylls; Zeaxanthins

Nonphotochemical quenching (NPQ) is a fundamental mechanism in photosynthesis which protects plants against excess excitation energy and is of crucial importance for their survival and fitness. Recently, carotenoid radical cation (Car*+) formation has been discovered to be a key step for the feedback deexcitation quenching mechanism (qE), a component of NPQ, of which the molecular mechanism and location is still unknown. We have generated and characterized carotenoid radical cations by means of resonant two color, two photon ionization (R2C2PI) spectroscopy. The Car*+ bands have maxima located at 830 nm (violaxanthin), 880 nm (lutein), 900 nm (zeaxanthin), and 920 nm (beta-carotene). The positions of these maxima depend strongly on solution conditions, the number of conjugated C=C bonds, and molecular structure. Furthermore, R2C2PI measurements on the light-harvesting complex of photosystem II (LHC II) samples with or without zeaxanthin (Zea) reveal the violaxanthin (Vio) radical cation (Vio*+) band at 909 nm and the Zea*+ band at 983 nm. The replacement of Vio by Zea in the light-harvesting complex II (LHC II) has no influence on the Chl excitation lifetime, and by exciting the Chls lowest excited state, no additional rise and decay corresponding to the Car*+ signal observed previously during qE was detected in the spectral range investigated (800-1050 nm). On the basis of our findings, the mechanism of qE involving the simple replacement of Vio with Zea in LHC II needs to be reconsidered.

Topics: beta Carotene; Cations; Free Radicals; Lutein; Oxidation-Reduction; Photosynthesis; Photosystem II Protein Complex; Pisum sativum; Plant Leaves; Quantum Theory; Solutions; Spectrum Analysis; Thylakoids; Xanthophylls; Zeaxanthins

The xanthophyll cycle has a major role in protecting plants from photooxidative stress, although the mechanism of its action is unclear. Here, we have investigated Arabidopsis plants overexpressing a gene encoding beta-carotene hydroxylase, containing nearly three times the amount of xanthophyll cycle carotenoids present in the wild-type. In high light at low temperature wild-type plants exhibited symptoms of severe oxidative stress: lipid peroxidation, chlorophyll bleaching, and photoinhibition. In transformed plants, which accumulate over twice as much zeaxanthin as the wild-type, these symptoms were significantly ameliorated. The capacity of non-photochemical quenching is not significantly different in transformed plants compared with wild-type and therefore an enhancement of this process cannot be the cause of the stress tolerant phenotype. Rather, it is concluded that it results from the antioxidant effect of zeaxanthin. 80-90% of violaxanthin and zeaxanthin in wild-type and transformed plants was localized to an oligomeric LHCII fraction prepared from thylakoid membranes. The binding of these pigments in intact membranes was confirmed by resonance Raman spectroscopy. Based on the structural model of LHCII, we suggest that the protein/lipid interface is the active site for the antioxidant activity of zeaxanthin, which mediates stress tolerance by the protection of bound lipids.

Topics: Antioxidants; Apoproteins; Arabidopsis; Light; Lipids; Oxidants; Oxidative Stress; Photochemistry; Photosystem II Protein Complex; Plant Leaves; Plant Proteins; Protein Binding; Spectrum Analysis, Raman; Temperature; Thylakoids; Xanthophylls; Zeaxanthins

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 degrees C, 25 degrees C and 37 degrees 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(alpha)), such as alpha-tocopherol and 8-cetylether (C(16)EO(8)), only 55% and 35% of the violaxanthin was converted at 25 degrees 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.

Topics: alpha-Linolenic Acid; alpha-Tocopherol; Ethers; Kinetics; Lipids; Mechanotransduction, Cellular; Membrane Fluidity; Metabolic Clearance Rate; Signal Transduction; Stress, Mechanical; Temperature; Thylakoids; Xanthophylls; Zeaxanthins

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 Delta pH-dependent conformational changes at 535 nm (DeltaA (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.

Topics: Absorption; Arabidopsis; Arabidopsis Proteins; Chlorophyll; Chromosome Mapping; Electron Transport; Fluorescence; Hydrogen-Ion Concentration; Light; Light-Harvesting Protein Complexes; Mutation; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Plant Leaves; Sequence Analysis, DNA; Thylakoids; Xanthophylls; Zeaxanthins

We compared the metabolic responses of leaves and roots of two Eucalyptus globulus Labill. clones differing in drought sensitivity to a slowly imposed water deficit. Responses measured included changes in concentrations of soluble and insoluble sugars, proline, total protein and several antioxidant enzymes. In addition to the general decrease in growth caused by water deficit, we observed a decrease in osmotic potential when drought stress became severe. In both clones, the decrease was greater in roots than in leaves, consistent with the observed increases in concentrations of soluble sugars and proline in these organs. In roots of both clones, glutathione reductase activity increased significantly in response to water deficit, suggesting that this enzyme plays a protective role in roots during drought stress by catalyzing the catabolism of reactive oxygen species. Clone CN5 has stress avoidance mechanisms that account for its lower sensitivity to drought compared with Clone ST51.

Topics: Adaptation, Physiological; Ascorbate Peroxidases; beta Carotene; Biomass; Carbohydrate Metabolism; Carbohydrates; Catalase; Dehydration; Disasters; Eucalyptus; Glutathione Reductase; Osmosis; Peroxidases; Plant Leaves; Plant Proteins; Plant Roots; Proline; Superoxide Dismutase; Water; Xanthophylls; Zeaxanthins

The content and relative variations of individual carotenoids during the leaflet development stages (I, II, III, A and P) of six species of Ceratozamia (Cycads) were investigated. There is an unusual, transitory and marked presence of six red stroma keto-carotenoids in the first development stages, while the thylakoidal carotenoids showed a low concentration during the same period. As no official A1cm1% coefficients were available, it was necessary to calculate these for the following stroma carotenoids: semi-beta-carotenone (major component), triphasiaxanthin, ceratoxanthin, ceratozamiaxanthin, kuesteriaxanthin and ceratoxanthone. The stroma keto-carotenoids, which reached the highest content in the first development stage (average of 78% of total carotenoids), were always present in the five species: C. fuscoviridis, C. robusta, C. spinosa, C. kuesteriana and C. hildae, but never in C. mexicana. From stage II, the stroma keto-carotenoids decreased and finally disappeared in the green adult leaflets. The thylakoidal carotenoids showed a minimum at stage III, and then increased to a maximum in the perennial leaflets. Among these, beta-carotene showed an anomalous and characteristic behaviour, being a minor component during leaflet development (from stage I to A). In stage P it was markedly exceeded not only by lutein but also by alpha-carotene, neoxanthin and violaxanthin, and in C. robusta also by lutein-5,6-epoxide. Additionally, the alpha/beta ratio in these species is unusual: it increased from 0.3-0.5 to 1.5-3.0 during leaflet development. Moreover, antheraxanthin amounts are very small, while zeaxanthin was present only in the evergreen leaflets of C. mexicana in small quantities. Lutein-5,6-epoxide represented more than 5% of thylakoidal carotenoids in the leaflets of all the species. A revision of the taxonomic rank of C. fuscoviridis is recommended.

Topics: Carotenoids; Chloroplasts; Lutein; Plant Leaves; Thylakoids; Xanthophylls; Zamiaceae; Zeaxanthins

In the present study, we investigated the epoxidation reaction of the violaxanthin (Vx) cycle in intact cells of Chlorella vulgaris. Our results show that the overall epoxidation is slightly slower in darkness compared to the epoxidation during high light (HL) illumination. The calculation of the rate constants of the two epoxidation steps revealed that, for both conditions, the first epoxidation step from zeaxanthin (Zx) to antheraxanthin (Ax) is faster than the second epoxidation step from Ax to Vx. However, the most noteworthy result of our present study is that Ax, which is transiently formed during the epoxidation reaction, participates in non-photochemical quenching of chlorophyll fluorescence (NPQ). A correlation between NPQ and the de-epoxidized xanthophyll cycle pigments during the time-course of the epoxidation reaction can only be achieved when NPQ is plotted versus the sum of Zx and Ax. The accumulation of significant amounts of Ax during the epoxidation reaction further indicates that Ax-dependent quenching proceeds with a similar efficiency compared to the Zx-mediated NPQ. As the xanthophyll-dependent NPQ relies on the presence of de-epoxidized xanthophylls in the PS II antenna, Ax-dependent NPQ is only possible under the assumption that Ax rebinds to the light-harvesting complex (LHC) II during the epoxidation reaction.

Topics: Chlorella vulgaris; Chlorophyll; Darkness; Fluorescence; Kinetics; Light; Light-Harvesting Protein Complexes; Oxidoreductases; Xanthophylls; Zeaxanthins

The xanthophyll cycle pigments, violaxanthin and zeaxanthin, present outside the light-harvesting pigment-protein complexes of Photosystem II (LHCII) considerably enhance specific aggregation of proteins as revealed by analysis of the 77 K chlorophyll a fluorescence emission spectra. Analysis of the infrared absorption spectra in the Amide I region shows that the aggregation is associated with formation of intermolecular hydrogen bonding between the alpha helices of neighboring complexes. The aggregation gives rise to new electronic energy levels, in the Soret region (530 nm) and corresponding to the Q spectral region (691 nm), as revealed by analysis of the resonance light scattering spectra. New electronic energy levels are interpreted in terms of exciton coupling of protein-bound photosynthetic pigments. The energy of the Q excitonic level of chlorophyll is not high enough to drive the light reactions of Photosystem II but better suited to transfer excitation energy to Photosystem I, which creates favourable energetic conditions for the state I-state II transition. The lack of fluorescence emission from this energy level, at physiological temperatures, is indicative of either very high thermal energy conversion rate or efficient excitation quenching by carotenoids. Chlorophyll a fluorescence was quenched up to 61% and 34% in the zeaxanthin- and violaxanthin-containing samples, respectively, as compared to pure LHCII. Enhanced aggregation of LHCII, observed in the presence of the xanthophyll cycle pigments, is discussed in terms of the switch between light-harvesting and energy dissipation systems.

Topics: Carotenoids; Chloroplasts; Energy Transfer; Light; Light-Harvesting Protein Complexes; Lycium; Photosystem II Protein Complex; Scattering, Radiation; Spectrometry, Fluorescence; Spectrophotometry, Infrared; Viola; Xanthophylls; Zea mays; Zeaxanthins

Xanthophylls are a major class of photosynthetic pigments that participate in an adaptation mechanism by which higher plants protect themselves from high light stress. In the present work, an ultrafast time-resolved spectroscopic investigation of all the major xanthophyll pigments from spinach has been performed. The molecules are zeaxanthin, lutein, violaxanthin, and neoxanthin. beta-Carotene was also studied. The experimental data reveal the inherent spectral properties and ultrafast dynamics including the S(1) state lifetimes of each of the pigments. In conjunction with quantum mechanical computations the results address the molecular features of xanthophylls that control the formation and decay of the S* state in solution. The findings provide compelling evidence that S* is an excited state with a conformational geometry twisted relative to the ground state. The data indicate that S* is formed via a branched pathway from higher excited singlet states and that its yield depends critically on the presence of beta-ionylidene rings in the polyene system of pi-electron conjugated double bonds. The data are expected to be beneficial to researchers employing ultrafast time-resolved spectroscopic methods to investigate the mechanisms of both energy transfer and nonphotochemical quenching in higher plant preparations.

Topics: beta Carotene; Electrons; Models, Molecular; Photochemistry; Pigments, Biological; Quantum Theory; Spectrophotometry; Time Factors; Xanthophylls; Zeaxanthins

Plants are exposed to sun light intensities that vary rapidly over several orders of magnitude during a typical day. It is known that the regulation of photosynthetic activity under these circumstances is essential for the survival and fitness of natural and gene modified plants. A quick balancing between utilization and dissipation of absorbed light energy ensures optimized levels of CO(2) fixation and protection from photo damage by excessive light-irradiation. Despite intensive investigations the biophysical mechanisms of these regulation processes are still poorly understood. Potentially involved singlet states of carotenoids are optically "dark" and so far it was impossible to investigate their role directly in living plants by conventional absorption or fluorescence spectroscopy. Here, we show by selective two-photon excitation of the carotenoid dark states in plant that a dominant part of the regulation is correlated with a substantial change in the energy transfer between these states and the chlorophylls (Chl). The results support a considerable role of the molecular gear shift model in which a reversible and step-wise enzymatic modification of the electronic structure of xanthophyll carotenoids enables a switching between carotenoid-to-Chl light-harvesting and Chl-to-carotenoid quenching. The shifting can be observed in real time in any plant. Treatment with the xanthophyll cycle inhibitor dithiothreitol slowed down both the light adaptation and the carotenoid-Chl energy flow changes to the same extent. Based on these results, we propose a biophysical quenching model in which both carotenoid dark states and radical cations contribute to the dissipation of excessive excitation energy.

Topics: Arabidopsis; Carotenoids; Darkness; Energy Transfer; Photons; Photosynthesis; Poaceae; Spectrometry, Fluorescence; Time Factors; Xanthophylls; Zeaxanthins

This paper reports the chemical evidence of the balance between radical molecular ions and protonatedmolecules of xanthophylls (an oxygen-containing carotenoid) with a conjugated pi-system (polyene) and oxygen as a heteroatom in ESI and HRESI mass spectrometry. The ionization energy of neutral xanthophylls was calculated by semi-empirical methods. The results were compared with those previously published for carotenoids and retinoids, which have also been shown in ESI-MS to form M(+*) and [M + H](+), respectively. This study demonstrates, for the first time, the correlation of an extended conjugation and the presence of oxygen in the formation and balance of M(+*) or [M + H](+) for the carotenoids, neoxanthin, lutein, violaxanthin and zeaxanthin.

Topics: beta Carotene; Ions; Lutein; Molecular Structure; Protons; Spectrometry, Mass, Electrospray Ionization; Xanthophylls; Zeaxanthins

Carotenoids are the effective modulators of physical properties of model and natural membranes. To demonstrate the relationship between the structure of carotenoids and their effect on the molecular dynamics of membranes, we have investigated the influence of five structurally different carotenoids: beta-carotene, lycopene, lutein, violaxanthin, zeaxanthin and additionally carotane--a fully saturated derivative of beta-carotene, on thermotropic phase behaviour of dipalmitoylphosphatidylcholine (DPPC) multilamellar vesicles by means of differential scanning calorimetry (DSC). The results obtained indicate that the carotenoids used modulated the thermotropic properties of multibilayers to various extents, broadening the pretransition and the main phase transition peaks and shifting them to lower temperatures. Pronounced decrease of pretransition enthalpy (DeltaH(p)) proves that carotenoids very strongly alter the membrane properties in its gel phase. Comparison of the influence of several carotenoids shows that a rigid, polyisoprenoid chain plays a basic role in altering the thermotropic properties of such membranes and the presence of rings without oxygen-containing groups has a minor significance for the observed interactions. Carotenoids containing epoxy and/or hydroxy groups attached to their rings modify the thermotropic phase behaviour of DPPC multilamellar vesicles stronger than carotenes--a result of their orientation in the DPPC bilayer.

Topics: 1,2-Dipalmitoylphosphatidylcholine; beta Carotene; Calorimetry, Differential Scanning; Carotenoids; Lipid Bilayers; Lutein; Lycopene; Structure-Activity Relationship; Temperature; Thermodynamics; Xanthophylls; Zeaxanthins

Lutein and zeaxanthin are dihydroxy xanthophylls that are produced from their corresponding carotene precursors by the action of beta- and epsilon -ring carotenoid hydroxylases. Two genes that encode beta-ring hydroxylases (beta-hydroxylases 1 and 2) have been identified in the Arabidopsis genome and are highly active toward beta-rings but only weakly active toward epsilon -rings. A third distinct activity required for epsilon -ring hydroxylation has been defined by mutation of the LUTEIN1 (LUT1) locus, but LUT1 has not yet been cloned. To address the individual and overlapping functions of the three Arabidopsis carotenoid hydroxylase activities in vivo, T-DNA knockout mutants corresponding to beta-hydroxylases 1 and 2 (b1 and b2, respectively) were isolated and all possible hydroxylase mutant combinations were generated. beta-Hydroxylase single mutants do not exhibit obvious growth defects and have limited impact on carotenoid composition relative to the wild type, suggesting that the encoded proteins have a significant degree of functional redundancy in vivo. Surprisingly, the b1 b2 double mutant, which lacks both known beta-hydroxylase enzymes, still contains significant levels of beta-carotene-derived xanthophylls, suggesting that additional beta-ring hydroxylation activity exists in vivo. The phenotype of double and triple hydroxylase mutants indicates that at least a portion of this activity resides in the LUT1 gene product. Despite the severe reduction of beta-carotene-derived xanthophylls (up to 90% in the lut1 b1 b2 triple mutant), the double and triple hydroxylase mutants still contain at least 50% of the wild-type amount of hydroxylated beta-rings. This finding suggests that it is the presence of minimal amounts of hydroxylated beta-rings, rather than minimal amounts of specific beta-carotene-derived xanthophylls, that are essential for light-harvesting complex II assembly and function in vivo. The carotenoid profiles in wild-type seeds and the effect of single and multiple hydroxylase mutations are distinct from those in photosynthetic tissues, indicating that the activities of each gene product differ in the two tissues. Overall, the hydroxylase mutants provide insight into the unexpected overlapping activity of carotenoid hydroxylases in vivo.

Topics: Arabidopsis; beta Carotene; Carotenoids; DNA, Bacterial; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Isoenzymes; Light; Mixed Function Oxygenases; Mutagenesis, Insertional; Mutation; Photosynthesis; Plant Leaves; Seeds; Xanthophylls; Zeaxanthins

The carotenoid pattern of four yellow- and four white-fleshed potato cultivars (Solanum tuberosum L.), common on the German market, was investigated using HPLC and LC(APCI)-MS for identification and quantification of carotenoids. In each case, the carotenoid pattern was dominated by violaxanthin, antheraxanthin, lutein, and zeaxanthin, which were present in different ratios, whereas neoxanthin, beta-cryptoxanthin, and beta,beta-carotene generally are only minor constituents. In contrast to literature data, antheraxanthin was found to be the only carotenoid epoxide present in native extracts. The total concentration of the four main carotenoids reached 175 microg/100 g, whereas the sum of carotenoid esters accounted for 41-131 microg/100 g. Therefore, carotenoid esters are regarded as quantitatively significant compounds in potatoes. For LC(APCI)-MS analyses of carotenoid esters, a two-stage cleanup procedure was developed, involving column chromatography on silica gel and enzymatic cleavage of residual triacylglycerides by lipases. This facilitated the direct identification of several potato carotenoid esters without previous isolation of the compounds. Although the unequivocal identification of all parent carotenoids was not possible, the cleanup procedure proved to be highly efficient for LC(APCI)-MS analyses of very low amounts of carotenoid esters.

Topics: beta Carotene; Carotenoids; Chromatography, High Pressure Liquid; Chromatography, Liquid; Epoxy Compounds; Esters; Lipase; Lutein; Mass Spectrometry; Solanum tuberosum; Triglycerides; Xanthophylls; Zeaxanthins

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

Topics: Arabidopsis; beta Carotene; Biological Transport; Electron Transport; Hydrogen-Ion Concentration; Light; Mutation; Oxidoreductases; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Temperature; Thylakoids; Xanthophylls; Zeaxanthins

Zeaxanthin is an important dietary carotenoid but its abundance in our food is low. In order to provide a better supply of zeaxanthin in a staple crop, two different potato (Solanum tuberosum L.) varieties were genetically modified. By transformation with sense and antisense constructs encoding zeaxanthin epoxidase, zeaxanthin conversion to violaxanthin was inhibited. Both approaches (antisense and co-suppression) yielded potato tubers with higher levels of zeaxanthin. Depending on the transgenic lines and tuber development, zeaxanthin content was elevated 4 to 130-fold reaching values up to 40 microg/g dry weight. As a consequence of the genetic manipulation, the amount of violaxanthin was diminished dramatically and in some cases the monoepoxy intermediate antheraxanthin accumulated. Between one and eight copies of the sense or antisense epoxidase gene fragments were integrated into the genome. In addition, most of the transformants with higher zeaxanthin levels showed also increased total carotenoid contents (up to 5.7-fold) and some of them exhibited reduced amounts of lutein. The increase in total carotenoids suggests that the genetic modification affects the regulation of the whole carotenoid biosynthetic pathway in potato tubers. Northern blot analysis demonstrated that upregulation of carotenogenesis in the transgenics is accompanied by substantial higher phytoene synthase transcript levels in 6-week-old tubers and a very slight increase of the beta-carotene hydroxylase transcript. The amount of the deoxyxylulose 5-phosphate synthase mRNA was very similar in wild type and transformed tubers. Abscisic acid content of tubers remained unchanged whereas alpha-tocopherol was 2 to 3 fold elevated in the transformants.

Topics: beta Carotene; Carotenoids; Down-Regulation; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Genetic Engineering; Oligonucleotides, Antisense; Oxidoreductases; Plant Tubers; Plants, Genetically Modified; Quality Control; Solanum tuberosum; Transcription, Genetic; Xanthophylls; Zeaxanthins

The carotenoid species lutein, violaxanthin, and zeaxanthin are crucial in the xanthophyll-dependent nonphotochemical quenching occurring in photosynthetic systems of higher plants, since they are involved in dissipation of excess energy and thus protect the photosynthetic machinery from irreversible inhibition. Nonetheless, important properties of the xanthophyll cycle carotenoids, such as the energy of their S(1) electronic states, are difficult to study and were only recently determined in organic solvents [Polívka, T. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 4914. Frank, H. A. (2000) Biochemistry 39, 2831]. In the present study, we have determined the S(1) energies of three carotenoid species, violaxanthin, lutein, and zeaxanthin, in their LHCII (peripheral light-harvesting complex of photosystem II) protein environment by constructing recombinant Lhcb1 (Lhc = light-harvesting complex) proteins containing single carotenoid species. Within experimental error the S(1) energy is the same for all three carotenoids in the monomeric LHCII, 13,900 +/- 300 cm(-1) (720 +/- 15 nm), thus well below the Q(y)() transitions of chlorophylls. In addition, we have found that, although the S(1) lifetimes of violaxanthin, lutein, and zeaxanthin differ substantially in solution, when incorporated into the LHCII protein, their S(1) states have in fact the same lifetime of about 11 ps. Despite the similar spectroscopic properties of the carotenoids bound to the LHCII, we observed a maximal fluorescence quenching when zeaxanthin was present in the LHCII complex. On the basis of these observations, we suggest that, rather than different photochemical properties of individual carotenoid species, changes in the protein conformation induced by binding of carotenoids with distinct molecular structures are involved in the quenching phenomena associated with Lhc proteins.

Topics: beta Carotene; Carotenoids; Electrophysiology; Kinetics; Light; Lutein; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Recombinant Proteins; Spectrometry, Fluorescence; Spectrophotometry; Time Factors; Xanthophylls; Zeaxanthins

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

Topics: Arabidopsis; beta Carotene; Binding Sites; Escherichia coli; Gene Expression Regulation, Enzymologic; Hydrogen-Ion Concentration; Light; Lipid Metabolism; Mutation; Nicotiana; Oxidoreductases; Plants, Genetically Modified; Protein Binding; Sequence Deletion; Thylakoids; Xanthophylls; Zeaxanthins

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

Topics: Arabidopsis; Ascorbic Acid; beta Carotene; Electron Transport; Hydrogen-Ion Concentration; Light; Models, Chemical; Mutation; Oxidoreductases; Photochemistry; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Plant Leaves; Thylakoids; Xanthophylls; Zeaxanthins

Photosystem I (PSI) of higher plants contains 18 subunits. Using Arabidopsis En insertion lines, we have isolated knockout alleles of the genes psaG, psaH2, and psaK, which code for PSI-G, -H, and -K. In the mutants psak-1 and psag-1.4, complete loss of PSI-K and -G, respectively, was confirmed, whereas the residual H level in psah2-1.4 is due to a second gene encoding PSI-H, psaH1. Double mutants, lacking PSI-G, and also -K, or a fraction of -H, together with the three single mutants were characterized for their growth phenotypes and PSI polypeptide composition. In general, the loss of each subunit has secondary, in some cases additive, effects on the abundance of other PSI polypeptides, such as D, E, H, L, N, and the light-harvesting complex I proteins Lhca2 and 3. In the G-less mutant psag-1.4, the variation in PSI composition suggests that PSI-G stabilizes the PSI-core. Levels of light-harvesting complex I proteins in plants, which lack simultaneously PSI-G and -K, indicate that PSI subunits other than G and K can also bind Lhca2 and 3. In the same single and double mutants, psag-1.4, psak-1, psah2-1.4, psag-1.4/psah2-1.4, and psag-1.4/psak-1 photosynthetic electron flow and excitation energy quenching were analyzed to address the roles of the various subunits in P700 reduction (mediated by PSI-F and -N) and oxidation (PSI-E), and state transitions (PSI-H). Based on the results, we also suggest for PSI-K a role in state transitions.

Topics: Alleles; Arabidopsis; Base Sequence; beta Carotene; Blotting, Western; Chlorophyll; Light-Harvesting Protein Complexes; Lutein; Mutation; Oxidation-Reduction; Oxygen; Photosynthetic Reaction Center Complex Proteins; Photosystem I Protein Complex; Pigments, Biological; Plant Leaves; Plant Proteins; Reactive Oxygen Species; Sequence Homology, Nucleic Acid; Thylakoids; Xanthophylls; Zeaxanthins

Guard cells of the orchid genus, Paphiopedilum have been reported to lack developed chloroplasts and detectable chlorophyll a autofluorescence. Paphiopedilum stomata lack a photosynthesis-dependent opening response but have a blue light-specific opening. The present study found that low fluence rate green and red light elicited stomatal opening in Paphiopedilum and this opening was reversed by far red light, indicating the presence of a phytochrome-mediated opening response. Phytochrome-dependent, red light-stimulated opening was largest under low fluence rates and decreased to near zero as fluence rate increased. A recently discovered green light reversibility of blue light-specific stomatal opening was used to probe the properties of the blue light response in Paphiopedilum stomata. Blue light-stimulated opening was completely reversed by green light in the presence of far red light. Red light enhanced the blue light response of Paphiopedilum guard cells when given as a pretreatment or together with blue light. Analysis of guard cell pigments showed that guard cells have small amounts of chlorophyll a and b, zeaxanthin, violaxanthin, antheraxanthin and lutein. Zeaxanthin content increased in response to blue light or ascorbate and declined in the dark or under illumination in the presence of dithiothreitol, indicating the presence of an active xanthophyll cycle. Thus Paphiopedilum stomata possess both a blue light-mediated opening response with characteristics similar to species with normal chloroplast development and a novel phytochrome-mediated opening response.

Topics: Adaptation, Physiological; Ascorbic Acid; beta Carotene; Chlorophyll; Chlorophyll A; Dithiothreitol; Light; Lutein; Orchidaceae; Photosynthesis; Phytochrome; Pigments, Biological; Plant Epidermis; Xanthophylls; Zeaxanthins

Nonradiative dissipation of excitation energy is the major photoprotective mechanism in plants. The formation of zeaxanthin in the antenna of photosystem II has been shown to correlate with the onset of nonphotochemical quenching in vivo. We have used recombinant CP29 protein, over-expressed in Escherichia coli and refolded in vitro with purified pigments, to obtain a protein indistinguishable from the native complex extracted from thylakoids, binding either violaxanthin or zeaxanthin together with lutein. These recombinant proteins and the native CP29 were used to measure steady-state chlorophyll fluorescence emission and fluorescence decay kinetics. We found that the presence of zeaxanthin bound to CP29 induces a approximately 35% decrease in fluorescence yield with respect to the control proteins (the native and zeaxanthin-free reconstituted proteins). Fluorescence decay kinetics showed that four components are always present but lifetimes (tau) as well as relative fluorescence quantum yields (rfqy) of the two long-lived components (tau3 and tau4) are modified by the presence of zeaxanthin. The most relevant changes are observed in the rfqy of tau3 and in the average lifetime ( approximately 2.4 ns with zeaxanthin and 3.2-3.4 ns in the control proteins). When studied in vitro, no significant effect of acidic pH (5.2-5.3) is observed on chlorophyll A fluorescence yield or kinetics. The data presented show that recombinant CP29 is able to bind zeaxanthin and this protein-bound zeaxanthin induces a significant quenching effect.

Topics: beta Carotene; Carotenoids; Circular Dichroism; Escherichia coli; Light-Harvesting Protein Complexes; Lutein; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Protein Folding; Recombinant Proteins; Spectrometry, Fluorescence; Xanthophylls; Zeaxanthins

The steady state absorption and fluorescence spectroscopic properties of the xanthophylls, violaxanthin, zeaxanthin, and lutein, and the efficiencies of singlet energy transfer from the individual xanthophylls to chlorophyll have been investigated in recombinant CP26 protein overexpressed in Escherichia coli and then refolded in vitro with purified pigments. Also, the effect of the different xanthophylls on the extents of static and dynamic quenching of chlorophyll fluorescence has been investigated. Absorption, fluorescence, and fluorescence excitation demonstrate that the efficiency of light harvesting from the xanthophylls to chlorophyll a is relatively high and insensitive to the particular xanthophyll that is present. A small effect of the different xanthophylls is observed on the extent of quenching of Chl fluorescence. The data provide the precise wavelengths of the absorption and fluorescence features of the bound pigments in the highly congested spectral profiles from these light-harvesting complexes. This information is important in assessing the mechanisms by which higher plants dissipate excess energy in light-harvesting proteins.

Topics: beta Carotene; Chlorophyll; Chlorophyll A; Energy Transfer; Escherichia coli; Light-Harvesting Protein Complexes; Lutein; Photochemistry; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Pigments, Biological; Recombinant Proteins; Spectrometry, Fluorescence; Spinacia oleracea; Xanthophylls; Zeaxanthins

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 beta-carotene were identified in P. tricornutum, namely beta-cryptoxanthin and beta-cryptoxanthin epoxide. In low light, the latter pigment prevails, but in high light beta-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 Kuitzing. 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, beta-cryptoxanthin epoxide may be involved in the biosynthesis of violaxanthin in diatoms.

Topics: Antioxidants; beta Carotene; Carotenoids; Chromatography, High Pressure Liquid; Cryptoxanthins; Diatoms; Dithioerythritol; Epoxy Compounds; Herbicides; Light; Lutein; Models, Biological; Pigments, Biological; Pyridazines; Sulfhydryl Reagents; Xanthophylls; Zeaxanthins

Resonance Raman excitation spectroscopy combined with ultra low temperature absorption spectral analysis of the major xanthophylls of higher plants in isolated antenna and intact thylakoid membranes was used to identify carotenoid absorption regions and study their molecular configuration. The major electronic transitions of the light-harvesting complex of photosystem II (LHCIIb) xanthophylls have been identified for both the monomeric and trimeric states of the complex. One long wavelength state of lutein with a 0-0 transition at 510 nm was detected in LHCIIb trimers. The short wavelength 0-0 transitions of lutein and neoxanthin were located at 495 and 486 nm, respectively. In monomeric LHCIIb, both luteins absorb around 495 nm, but slight differences in their protein environments give rise to a broadening of this band. The resonance Raman spectra of violaxanthin and zeaxanthin in intact thylakoid membranes was determined. The broad 0-0 absorption transition for zeaxanthin was found to be located in the 503-511 nm region. Violaxanthin exhibited heterogeneity, having two populations with one absorbing at 497 nm (0-0), 460 nm (0-1), and 429 nm (0-2), and the other major pool absorbing at 488 nm (0-0), 452 nm (0-1), and 423 nm (0-2). The origin of this heterogeneity is discussed. The configuration of zeaxanthin and violaxanthin in thylakoid membranes was different from that of free pigments, and both xanthophylls (notably, zeaxanthin) were found to be well coordinated within the antenna proteins in vivo, arguing against the possibility of their free diffusion in the membrane and supporting our recent biochemical evidence of their association with intact oligomeric light-harvesting complexes (Ruban, A. V., Lee, P. J., Wentworth, M., Young, A. J., and Horton, P. (1999) J. Biol. Chem. 274, 10458-10465).

Topics: beta Carotene; Carotenoids; Lutein; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Plants; Spectrophotometry, Atomic; Spectrum Analysis, Raman; Temperature; Thylakoids; Xanthophylls; Zeaxanthins

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

Topics: beta Carotene; Bioreactors; Cell Division; Enzyme Inhibitors; Eukaryota; Hydrogen-Ion Concentration; Iodoacetamide; Lighting; Lutein; Photosynthesis; Temperature; Xanthophylls; Zeaxanthins

The relationship between the degradation rate and structure of each pigment of the pepper carotenoid profile was studied in mixtures of dehydrated fruit with lipid substrates of differing degrees of unsaturation and in different proportions (20 and 40%). The differences in structural nature of the carotenoids present in the pepper fruit produce a variable rate of oxidation, resulting in nonuniform degradation. The yellow xanthophylls and beta-carotene have the highest rates of oxidation, with the ketocarotenoids and violaxanthin degrading at lower rates. Autoxidation is greater or lesser depending on the functional groups, which stabilize the radical intermediaries of the reaction. The behavior of capsanthin and capsorubin is that expected of carotenoids having structures that include keto groups: a markedly greater stability to autoxidation processes. This increases their antioxidant capacity, adding to their beneficial impact by reducing the proliferation of radical processes, which are detrimental to health.

Topics: beta Carotene; Capsicum; Carotenoids; Chromatography, High Pressure Liquid; Cryptoxanthins; Esterification; Kinetics; Molecular Structure; Oxidation-Reduction; Structure-Activity Relationship; Xanthophylls; Zeaxanthins

Increased intake of fruits and vegetables is associated with reduced risk of cancer and other chronic diseases. Epoxycarotenoids are widely distributed in nature and constitute major dietary carotenoids in a number of fruits and vegetables. Previous studies have shown that beta-carotene 5,6-epoxide was absorbed well by humans, and was much more active than beta-carotene in inducing the differentiation of NB4 cells. Xanthophyll epoxides such as neoxanthin, violaxanthin and lutein 5,6-epoxide, are more abundant than epoxy-hydrocarbon carotenes in a number of vegetables and fruits that humans consume. To determine whether xanthophyll epoxides are also absorbed by humans, lutein 5,6-epoxide (taraxanthin) and zeaxanthin 5,6,5'6'-diepoxide (violaxanthin) were chemically prepared, dissolved in corn oil and orally administered to three human subjects. Analysis of plasma for carotenoids within 9 h after a single oral dose of either violaxanthin or taraxanthin failed to show any violaxanthin, taraxanthin or any of their metabolites.

Topics: Absorption; beta Carotene; Carotenoids; Chromatography, High Pressure Liquid; Epoxy Compounds; Female; Fruit; Humans; Lutein; Male; Middle Aged; Vegetables; Xanthophylls; Zeaxanthins

The xanthophyll cycle is an enzymatic, reversible process through which the carotenoids violaxanthin, antheraxanthin, and zeaxanthin are interconverted in response to the need to balance light absorption with the capacity to use the energy to drive the reactions of photosynthesis. The cycle is thought to be one of the main avenues for safely dissipating excitation energy absorbed by plants in excess of that needed for photosynthesis. One of the key factors needed to elucidate the molecular mechanism by which the potentially damaging excess energy is dissipated is the energy of the lowest excited singlet (S(1)) state of the xanthophyll pigments. Absorption from the ground state (S(0)) to S(1) is forbidden by symmetry, making a determination of the S(1) state energies of these molecules by absorption spectroscopy very difficult. Fluorescence spectroscopy is potentially the most direct method for obtaining the S(1) state energies. However, because of problems with sample purity, low emission quantum yields, and detection sensitivity, fluorescence spectra from these molecules, until now, have never been reported. In this work these technical obstacles have been overcome, and S(1) --> S(0) fluorescence spectra of violaxanthin and zeaxanthin are presented. The energies of the S(1) states deduced from the fluorescence spectra are 14 880 +/- 90 cm(-)(1) for violaxanthin and 14 550 +/- 90 cm(-)(1) for zeaxanthin. The results provide important insights into the mechanism of nonphotochemical dissipation of excess energy in plants.

Topics: beta Carotene; Chlorophyll; Chlorophyll A; Energy Transfer; Lutein; Mathematical Computing; Normal Distribution; Photochemistry; Spectrometry, Fluorescence; Spinacia oleracea; Xanthophylls; Zeaxanthins

Non-photochemical quenching of chlorophyll fluorescence in plants occurs in the light harvesting antenna of photosystem II and is regulated by the xanthophyll cycle. A new in vitro model for this process has been developed. Purified light harvesting complexes above the detergent critical micelle concentration have a stable high fluorescence yield but a rapidly inducible fluorescence quenching occurs upon addition of zeaxanthin. Violaxanthin was without effect, lutein and antheraxanthin induced a marginal response, whereas the violaxanthin analogue, auroxanthin, induced strong quenching. Quenching was not caused by aggregation of the complexes but was accompanied by a spectral broadening and red shift, indicating a zeaxanthin-dependent alteration in the chlorophyll environment.

Topics: beta Carotene; Chlorophyll; Fluorescence; Light-Harvesting Protein Complexes; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Spinacia oleracea; Xanthophylls; Zeaxanthins

Infiltrating detached maize (Zea mays L.) leaves with L-galactono-1,4-lactone (L-GAL) resulted in a 4-fold increase in the content of leaf ascorbate. Upon exposure to high irradiance (1000 mumol photons m-2 s-1) at 5 degrees C, L-GAL leaves de-epoxidized the xanthophyll-cycle pigments faster than the control leaves; the maximal ratio of de-epoxidized xanthophyll-cycle pigments to the whole xanthophyll-cycle pool was the same in both leaf types. The elevated ascorbate content, together with the faster violaxanthin de-epoxidation, did not affect the degree of photoinhibition and the kinetics of the recovery from photoinhibition, assayed by monitoring the maximum quantum efficiency of photosystem II primary photochemistry (Fv/Fm). Under the experimental conditions, the thermal energy dissipation seems to be zeaxanthin-independent since, in contrast to the de-epoxidation, the decrease in the efficiency of excitation-energy capture by open photosystem II reaction centers (F'v/F'm) during the high-irradiance treatment at low temperature showed the same kinetic in both leaf types. This was also observed for the recovery of the maximal fluorescence after stress. Furthermore, the elevated ascorbate content did not diminish the degradation of pigments or alpha-tocopherol when leaves were exposed for up to 24 h to high irradiance at low temperature. Moreover, a higher content of ascorbate appeared to increase the requirement for reduced glutathione.

Topics: Ascorbic Acid; beta Carotene; Chlorophyll; Chlorophyll A; Cold Temperature; Energy Metabolism; Light; Light-Harvesting Protein Complexes; Oxidative Stress; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Pigments, Biological; Plant Leaves; Sugar Acids; Xanthophylls; Zea mays; Zeaxanthins

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.

Topics: beta Carotene; Carotenoids; Lutein; Models, Chemical; Oxidoreductases; Plants; Xanthophylls; Zeaxanthins

Carotenoids are involved in a variety of biological functions, yet the underlying mechanisms are poorly understood, in part because of the long-standing difficulty in assigning the location of the first excited (S1) state. Here, we present a method for determining the energy of the forbidden S1 state, on the basis of ultrafast spectroscopy of the short lived level. Femtosecond transient absorption spectra and kinetics of the S1 --> S2 transition revealed the location of the intermediate level in two carotenoid species involved in the xanthophyll cycle, zeaxanthin and violaxanthin, and yielded surprising implications regarding the mechanism of photoregulation in photosynthesis.

Topics: Animals; beta Carotene; Carotenoids; Energy Transfer; Spectrum Analysis; Xanthophylls; Zeaxanthins

When light energy absorbed by plants becomes excessive relative to the capacity of photosynthesis, the xanthophyll violaxanthin is reversibly deepoxidized to zeaxanthin (violaxanthin cycle). The protective function of this phenomenon was investigated in a mutant of Arabidopsis thaliana, npq1, that has no functional violaxanthin deepoxidase. Two major consequences of the npq1 mutation are the absence of zeaxanthin formation in strong light and the partial inhibition of the quenching of singlet excited chlorophylls in the photosystem II light-harvesting complexes. Prolonged exposure of whole plants to bright light resulted in a limited photoinhibition of photosystem II in both npq1 and wild-type leaves, although CO(2) fixation and the linear electron transport in npq1 plants were reduced substantially. Lipid peroxidation was more pronounced in npq1 compared with the wild type, as measured by chlorophyll thermoluminescence, ethane production, and the total hydroperoxy fatty acids content. Lipid peroxidation was amplified markedly under chilling stress, and photooxidative damage ultimately resulted in leaf bleaching and tissue necrosis in npq1. The npq4 mutant, which possesses a normal violaxanthin cycle but has a limited capacity of quenching singlet excited chlorophylls, was rather tolerant to lipid peroxidation. The double mutant, npq4 npq1, which differs from npq4 only by the absence of the violaxanthin cycle, exhibited an increased susceptibility to photooxidative damage, similar to that of npq1. Our results demonstrate that the violaxanthin cycle specifically protects thylakoid membrane lipids against photooxidation. Part of this protection involves a mechanism other than quenching of singlet excited chlorophylls.

Topics: Arabidopsis; beta Carotene; Carotenoids; Chlorophyll; Ethane; Light; Light-Harvesting Protein Complexes; Lipid Peroxidation; Membrane Lipids; Oxidative Stress; Oxidoreductases; Photons; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Vitamin E; Xanthophylls; Zeaxanthins

Plants of Nicotiana tabacum (O3-tolerant cv Bel-B and O3-sensitive cv Bel-W3) were exposed to 150 ppb of ozone for 5 h; the fumigation produced visual injury in mature leaves, particularly in Bel-W3. After O3-treatment the pigments of the xanthophyll cycle pool decreased in both cvs, with a strong reduction in violaxanthin content, while antheraxanthin and zeaxanthin increased slightly. Under these conditions the content of leaf abscisic acid (ABA) markedly increased, particularly in O3-sensitive cv, indicating that the violaxanthin may have been partially converted into ABA. The control plants of Bel-B showed an ascorbic acid content four times greater than Bel-W3 and the ozone treatment did not produce significant differences in the ascorbic acid content and in the redox state. The two tobacco cvs were found to have similar total glutathione content, however the redox state was lower in O3-sensitive cv and decreased after ozone exposure. Ozone fumigation caused an increase in oxidized glutathione, particularly in Bel-W3, associated with a reduced glutathione reductase (GR) activity and a reduced GR protein content.

Topics: Ascorbic Acid; beta Carotene; Carotenoids; Free Radicals; Glutathione; Glutathione Reductase; Lutein; Nicotiana; Oxidative Stress; Ozone; Pigments, Biological; Plant Leaves; Plants, Toxic; Xanthophylls; Zeaxanthins

The carotenoid composition was investigated during enhanced D1 protein turnover in Chlamydomonas reinhardtii exposed to high light. After 2 h of high light there was no loss of the D1 protein yet. However, the beta-carotene content was significantly reduced. In parallel, an increase of the zeaxanthin content was found, which was higher than can be accounted for by the light-induced de-epoxidation of violaxanthin in the xanthophyll cycle reactions. We therefore assume that beta-carotene of photosystem II (PS II) is hydroxylated to zeaxanthin under high light stress. Inhibitors of carotene biosynthesis led to the loss of both PS II activity and D1 protein, indicating the requirement of beta-carotene synthesis for the reassembly of PS II in high light. Diuron blocked D1 protein as well as beta-carotene turnover. In the presence of chloramphenicol -- which allows just one turnover of the D1 protein -- 15% of the total beta-carotene was lost, calculated to be two beta-carotene.

Topics: Animals; beta Carotene; Chlamydomonas reinhardtii; Chloramphenicol; Diuron; Ethylamines; Light; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Xanthophylls; Zeaxanthins

A conserved regulatory mechanism protects plants against the potentially damaging effects of excessive light. Nearly all photosynthetic eukaryotes are able to dissipate excess absorbed light energy in a process that involves xanthophyll pigments. To dissect the role of xanthophylls in photoprotective energy dissipation in vivo, we isolated Arabidopsis xanthophyll cycle mutants by screening for altered nonphotochemical quenching of chlorophyll fluorescence. The npq1 mutants are unable to convert violaxanthin to zeaxanthin in excessive light, whereas the npq2 mutants accumulate zeaxanthin constitutively. The npq2 mutants are new alleles of aba1, the zeaxanthin epoxidase gene. The high levels of zeaxanthin in npq2 affected the kinetics of induction and relaxation but not the extent of nonphotochemical quenching. Genetic mapping, DNA sequencing, and complementation of npq1 demonstrated that this mutation affects the structural gene encoding violaxanthin deepoxidase. The npq1 mutant exhibited greatly reduced nonphotochemical quenching, demonstrating that violaxanthin deepoxidation is required for the bulk of rapidly reversible nonphotochemical quenching in Arabidopsis. Altered regulation of photosynthetic energy conversion in npq1 was associated with increased sensitivity to photoinhibition. These results, in conjunction with the analysis of npq mutants of Chlamydomonas, suggest that the role of the xanthophyll cycle in nonphotochemical quenching has been conserved, although different photosynthetic eukaryotes rely on the xanthophyll cycle to different extents for the dissipation of excess absorbed light energy.

Topics: Amino Acid Sequence; Arabidopsis; Base Sequence; beta Carotene; Chromosome Mapping; Energy Metabolism; Ethyl Methanesulfonate; Fast Neutrons; Genes, Plant; Kinetics; Light; Lutein; Mutagenesis; Oxidoreductases; Photosynthesis; Point Mutation; Polymorphism, Genetic; Xanthophylls; Zeaxanthins

Under many environmental conditions, plants are exposed to levels of sunlight in excess of those required for photosynthesis. Then, a regulated increase in the rate of nonradiative dissipation of excess excitation energy in the thylakoid membrane correlates with the conversion of the carotenoid violaxanthin into zeaxanthin and provides protection from the damaging effects of excessive irradiation. The hypothesis that these carotenoids specifically control the oligomerization of the light harvesting complexes of photosystem II was tested by investigating the effects of violaxanthin and zeaxanthin on the behavior of the major complex, LHCIIb, on sucrose gradients; it was found that zeaxanthin stimulated the formation of LHCIIb aggregates with reduced chlorophyll fluorescence yield whereas violaxanthin caused the inhibition of such aggregation and an elevation of fluorescence. Measurements of 77 K fluorescence indicated that zeaxanthin was not exerting an additional direct quenching of chlorophyll fluorescence. These effects can explain the physiological control of the light harvesting system by the xanthophyll cycle.

Topics: beta Carotene; Biopolymers; Fluorescence; Light-Harvesting Protein Complexes; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Plants; Xanthophylls; Zeaxanthins

In higher plants non-photochemical dissipation of excess light, trapped by the pigment pool of photosystem II, prevents photodamage to the photosynthetic apparatus. We report here that an algal virus infecting Chlorella strain Pbi induces non-photochemical quenching of photosystem II fluorescence, indicating enhanced loss of absorbed light energy from photosystem II. This phenomenon occurs soon after the establishment of the virus infection cycle and is observed at low irradiance (20 micromol quanta m-2 s-1). At low light, infection associated non-photochemical quenching is not linked to extensive conversion of violaxanthin to antheraxanthin and zeaxanthin. However, such conversion occurs rapidly (2-10 min) in infected cells under conditions of high irradiance (100-300 micromol quanta m-2 s-1). Under similar conditions uninfected Chlorella cells do not display significant changes in non-photochemical quenching.

Topics: beta Carotene; Carotenoids; Chlorella; Chlorophyll; Cycloheximide; Dithiothreitol; Epoxy Compounds; Fluorescence; Genes, Viral; Light; Light-Harvesting Protein Complexes; Lutein; Paraquat; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Phycodnaviridae; Pigments, Biological; Xanthophylls; Zeaxanthins

Changes in carotenoid composition, CO2 assimilation and chlorophyll fluorescence due to photoinhibition at 5 degrees C and 20 degrees C were studied in 12 day and 30 day old sorghum leaves. The old leaves had a higher violaxanthin (V) content and less beta-carotene. Photoinhibition at both temperatures caused significant increases in zeaxanthin (Z) and decreases in violaxanthin. However, in young leaves the increase in zeaxanthin was greater than the decrease in violaxanthin. In young leaves the V + A + Z pool size (A = antheraxanthin) almost doubled under photoinhibitory conditions (compared to controls) while in old leaves the V + A + Z pool remained approximately constant. After photoinhibition treatment changes in the levels of the xanthophylls were restored during a recovery period both in young and old leaves. When rephotoinhibited after a 48 hr recovery period, the young plants showed better protection against photoinhibition. We suggest that in young leaves zeaxanthin is newly synthesized under photoinhibitory conditions besides being de-epoxidized from violaxanthin and that the synthesis of V + A + Z pool is higher at 20 degrees C than at 5 degrees C in both young and old leaves.

Topics: beta Carotene; Carbon Dioxide; Carotenoids; Chlorophyll; Chloroplasts; Fluorescence; Light; Lutein; Pigments, Biological; Plant Leaves; Temperature; Time Factors; Xanthophylls; Zeaxanthins

The rapid conversion of the carotenoid violaxanthin to zeaxanthin via antheraxanthin (xanthophyll cycle) in potato leaves exposed at 23 degrees C to a strong white light of 2000 microE.m-2.s-1 was associated with a slight inhibition of photosynthetic electron transport (as estimated from chlorophyll fluorescence measurements) and a low lipid peroxidation (as estimated from ethane measurements). When the xanthophyll cycle was blocked by dithiothreitol (3 mM) or low temperature (3 degrees C), photoinhibition of electron transport was exacerbated and pronounced lipid peroxidation occurred concomitantly. Accumulation of zeaxanthin and antheraxanthin in potato leaves by a non-photoinhibitory light treatment at 23 degrees C (900 microE.m-2.s-1 for 1 h) considerably reduced the level of lipid peroxidation during subsequent light stress at 3 degrees C. The presented results indicate that one of the functions of the xanthophyll cycle could be the protection of thylakoid membranes against lipid peroxidation, suggesting that zeaxanthin and antheraxanthin synthesized in strong light are present as free pigments in the membrane lipid bilayer.

Topics: beta Carotene; Carotenoids; Cold Temperature; Dithiothreitol; Electron Transport; Light; Lipid Peroxidation; Lutein; Photosynthesis; Plant Leaves; Solanum tuberosum; Xanthophylls; Zeaxanthins

Chlorella pyrenoidosa was grown in steady-state continuous cultures in either high or low light. Samples of these cultures were incubated in darkness (violaxanthin state) or in saturating light (zeaxanthin state). These samples were kept in the respective preadapted states throughout the entire photodamage treatment. Photodamage involved exposure to single-turnover flashes fired at a low (non-actinic) frequency. The damage caused by the light stress thus applied was monitored by changes in photosynthetic properties and pigment composition. Cells preadapted in the light resisted photodamage better than those kept in darkness. The low light grown cells were more vulnerable to photodamage than the high light grown cells. Our experimental approach permitted the equilibria between the components that participate in the xanthophyll cycle to be set without addition of inhibitors. Regardless of the total amount of violaxanthin being present, its conversion to anthera- and zeaxanthin is a prerequisite for protection. The protection is most effective for photosystem II. It appeared that antheraxanthin accumulates as a result of photodamaging flashes provided that these are fired in the presence of background light, i.e. with zeaxanthin present. From this, it is newly derived that the xanthophyll cycle operates in full in the light, including epoxidation of zeaxanthin. The latter conversion was also demonstrated in vitro, via nonenzymatic oxygen-dependent turnover of zeaxanthin into violaxanthin.

Topics: beta Carotene; Carotenoids; Chlorella; Light; Lutein; Photosynthesis; Pigments, Biological; Xanthophylls; Zeaxanthins

The oxygen diffusion-concentration product was determined in phosphatidylcholine (PC) bilayers from oxygen broadening of the spin label EPR spectra. The use of fatty acid spin labels makes it possible to do structural and oximetric measurements with the same sample. We find that polar carotenoids, zeaxanthin and violaxanthin, increase ordering of hydrocarbon chains in saturated (dimyristoyl-PC) and unsaturated (egg yolk PC) membranes and also significantly decrease the oxygen diffusion-concentration product in the hydrocarbon region of these membranes. At 25 degrees C in the presence of 10 mol% of carotenoids, the product is about 30% smaller than in pure PC membranes. Intercalation of carotenoids decreases the oxygen diffusion-concentration product in the central part of the bilayer and has little effect on the product in the polar head group region. In contrast, cholesterol molecules significantly reduce the product on and near the membrane surface, and do not change it (saturated PC) or increase it (unsaturated PC) in the middle of the bilayer (Subczynski, W.K., Hyde, J.S. and Kusumi, A. (1989) Proc. Natl. Acad. Sci. USA 86, 4474-4478). The decrease of oxygen diffusion-concentration product may be a mechanism of carotenoid protective activity, which should be effective in plant and animal cells in the light as well as in the dark.

Topics: beta Carotene; Carotenoids; Diffusion; Dimyristoylphosphatidylcholine; Electron Spin Resonance Spectroscopy; Fatty Acids; Glycolipids; Lipid Bilayers; Mathematics; Membranes, Artificial; Models, Molecular; Oximetry; Oxygen; Phosphatidylcholines; Xanthophylls; Zeaxanthins

A new kinetic model of the xanthophyll cycle is proposed. The model is based on the assumption that the light-dependent interconversion of the so-called available and unavailable violaxanthin constitutes the rate-limiting process of the cycle at intermediate, non-saturating light intensities. This assumption, together with the known properties of violaxanthin de-epoxidase, explains all specific features of the experimental facts.

Topics: beta Carotene; Carotenoids; Chloroplasts; Computer Simulation; Kinetics; Lutein; Spectrum Analysis; Xanthophylls; Zeaxanthins

Oriented multibilayers of dimyristoyl phosphatidylcholine (DMPC) modified with violaxanthin or zeaxanthin were examined by X-ray diffractometry and linear dichroism. It appears that pigment molecules and the normal to the bilayer plane form an angle of 24-25 degrees. It was also observed that rather small concentrations of added xanthophylls (molar fraction up to 3%) increase the pigmented bilayer thickness by a value of about 2 A as compared with that of the pure DMPC bilayer. The observed nonzero linear dichroism at normal incidence of light suggests the possibility of nonhomogeneous orientation of transition dipoles in the plane of the bilayer.

Topics: beta Carotene; Carotenoids; Dimyristoylphosphatidylcholine; Light; Lipid Bilayers; Lutein; Mathematics; Membrane Lipids; X-Ray Diffraction; Xanthophylls; Zeaxanthins