beta-carotene and antheraxanthin

Carotenoids possess important biological functions that make them essential components of the human diet. β-Carotene and some other carotenoids have vitamin A activity while lutein and zeaxanthin, typically referred to as the macular pigments, are involved in good vision and in delaying the onset of age-related eye diseases. In order to create a zeaxanthin-producing tomato fruit, two transgenic lines, one with a high β-carotene cyclase activity and the other with a high β-carotene hydroxylase activity, have been genetically crossed. Ripe fruits from the resulting progeny contained significant levels of violaxanthin, antheraxanthin, and xanthophyll esters. However, their zeaxanthin content was not as high as expected, and the total level of carotenoids was only 25% of the carotenoids found in ripe fruits of the comparator line. Targeted transcript analysis and apocarotenoids determinations indicated that transcriptional regulation of the pathway or degradation of synthesized carotenoids were not responsible for the low carotenoid content of hybrid fruits which instead appeared to result from a substantial reduction of carotenoid biosynthesis. Notably, the content of an unidentified hydroxylated cyclic (C13) apocarotenoid was 13 times higher in the hybrid fruits than in the control fruits. Furthermore, a GC-MS-based metabolite profiling demonstrated a perturbation of carotenogenesis in ripening hybrid fruits compatible with a block of the pathway. Moreover, carotenoid profiling on leaf, fruit, and petal samples from a set of experimental lines carrying the hp3 mutation, in combination with the two transgenes, indicated that the carotenoid biosynthesis in petal and fruit chromoplasts could be regulated. Altogether the data were consistent with the hypothesis of the regulation of the carotenoid pathway in tomato chromoplasts through a mechanism of feedback inhibition mediated by a xanthophyll-derived apocarotenoid. This chromoplast-specific post-transcriptional mechanism was disclosed in transgenic fruits of HU hybrid owing to the abnormal production of zeaxanthin and antheraxanthin, the more probable precursors of the apocarotenoid signal. A model describing the regulation of carotenoid pathway in tomato chromoplasts is presented.

Topics: beta Carotene; Carotenoids; Fruit; Gene Expression Regulation, Plant; Humans; Lutein; Plastids; Solanum lycopersicum; Xanthophylls; Zeaxanthins

Carotenoid-rich foods are associated with antioxidant activity and the ability to alleviate chronic diseases.. The present study investigated the effect of processing on the content and bioaccessibility of carotenoids from 13 cultivars of red chili pepper (Capsicum annuum).. Carotenoids in chili peppers were analyzed before an in vitro digestion process. The portion of carotenoid transferred to the micelle fraction (bioaccessibility) was also quantified.. β-Carotene, β-cryptoxanthin, capsanthin and antheraxanthin were the most abundant carotenoids. Zeaxanthin, violaxanthin, neoxanthin and lutein were detected at lower concentrations. In general, freezing and boiling reduced carotenoid contents. Capsanthin and zeaxanthin had the highest bioaccessibility at an average value from 36 to 40%, followed by antheraxanthin (26%). Bioaccessibility of β-cryptoxanthin, violaxanthin and β-carotene was lower, averaging 6.1, 4.8 and 4.0%, respectively. Neoxanthin and lutein were not detected in micelles. Freezing increased the bioaccessibility of capsanthin, zeaxanthin, antheraxanthin, β-cryptoxanthin and violaxanthin; β-cryptoxanthin bioaccessibility increased and capsanthin and zeaxanthin bioaccessibility decreased following boiling.. Differences in the contents and bioaccessibility of carotenoids in 13 C. annuum cultivars and between the processed methods were herein evidenced.

Topics: beta Carotene; Biological Availability; Capsicum; Carotenoids; Cryptoxanthins; Digestion; Food Handling; Freezing; Hot Temperature; In Vitro Techniques; Species Specificity; Xanthophylls; Zeaxanthins

Carotenoids are natural compounds whose nutritional importance comes from the provitamin A activity of some of them and their protection against several serious human disorders. The degradation of carotenoids was investigated during apricot drying by microwave and convective hot-air at 60 and 70 °C. Seven carotenoids were identified: antheraxanthin, lutein, zeaxanthin, β-cryptoxanthin, 13-cis-β-carotene, all-trans-β-carotene and 9-cis-β-carotene; among these, all-trans-β-carotene was found to be about 50 % of total carotenoids. First-order kinetic models were found to better describe all-trans-β-carotene reduction during drying, with a degradation rate constant (k1) that increased two folds when temperatures increased by 10 °C, in both methods. No differences were found in k1 between apricots dried by hot air at 70 °C (k1 = 0.0340 h(-1)) and by microwave at 60 °C. The evolution of total carotenoids (117.1 mg/kg on dry basis) during drying highlighted a wider decrease (about 50%) when microwave heating was employed, for both set temperatures. Antheraxantin was found to be the carotenoid most susceptible to heat, disappearing at 6 h during both trials with microwave as well as during convective hot-air at 70 °C. For this reason, antheraxanthin could be a useful marker for the evaluation of thermal damage due to the drying process. Also the degree of isomerization of all-trans-β-carotene could be a useful marker for the evaluation of the drying process.

Topics: beta Carotene; Biomarkers; Carotenoids; Cryptoxanthins; Desiccation; Drug Stability; Food Handling; Fruit; Hot Temperature; Isomerism; Kinetics; Lutein; Nutritive Value; Prunus; Xanthophylls; Zeaxanthins

The carotenoid composition of sarsaparilla ( Smilax aspera L.) berries has been analyzed for the first time. Lycopene was found to be the main carotenoid (242.44 μg/g fresh wt) in the pulp, followed by β-carotene (65.76 μg/g fresh wt) and β-cryptoxanthin (42.14 μg/g fresh wt; including the free and esterified forms). Other minor carotenoids were lycophyll (13.70 μg/g fresh wt), zeaxanthin (8.56 μg/g fresh wt; including the free and esterified forms), lutein (0.94 μg/g fresh wt), and antheraxanthin (0.58 μg/g fresh wt). β-Cryptoxanthin and zeaxanthin were present in free and esterified forms. β-Cryptoxanthin was mainly esterified with saturated fatty acids (capric, lauric, myristic, palmitic, and stearic), although a low amount of β-cryptoxanthin oleate was also detected. In the case of zeaxanthin, only a monoester with myristic acid (zeaxanthin monomyristate) was identified. The diverse carotenoid profile, some with provitamin A activity, together with the relatively high content, up to 375 μg/g fresh wt, makes sarsaparilla berries a potential source of carotenoids for the food, animal feed, and pharmaceutical industries.

Topics: beta Carotene; Carotenoids; Chromatography, Gas; Chromatography, High Pressure Liquid; Cryptoxanthins; Fatty Acids; Fruit; Lutein; Lycopene; Mass Spectrometry; Smilax; Xanthophylls; Zeaxanthins

This study focused on the bioaccessibility of carotenoids from Chlorella ellipsoidea using a simulated digestion model. To increase the bioaccessibility of carotenoids, C. ellipsoidea was pulverized by microfluidization at pressures up to 20000 psi. The microfluidization treatment significantly reduced mean microalga particle size from 2463 to 361 nm. The major carotenoid in C. ellipsoidea was zeaxanthin, with two minor carotenoids, antheraxanthin and β-carotene. After microfluidization, the zeaxanthin and β-carotene contents in C. ellipsoidea were not changed in comparison to the untreated group, whereas the antheraxanthin content was significantly reduced. The bioaccessibility of carotenoids in untreated C. ellipsoidea was very low (zeaxanthin, 2.60%; β-carotene, 1.69%). Approximately 95% of total C. ellipsoidea carotenoids could not be released and micellized by simulated digestion. The microfluidized microalga (at 20000 psi) was 10 times more effective for zeaxanthin and β-carotene micelle formation compared with untreated C. ellipsoidea, showing higher bioaccessibility of carotenoids (zeaxanthin, 32.60%; β-carotene, 18.19%). These results indicate that microfluidization may be useful for disrupting C. ellipsoidea cell walls and improving zeaxanthin and β-carotene bioaccessibility from C. ellipsoidea during the digestion process.

Topics: alpha-Amylases; beta Carotene; Bile Acids and Salts; Biological Availability; Carotenoids; Chlorella; Digestion; Hydrogen-Ion Concentration; In Vitro Techniques; Lipase; Micelles; Particle Size; Pressure; 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

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

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

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

Nonlinear regression analysis (NLR) is applied to quantify the dynamic response of non-photochemical fluorescence quenching (NPQ) of Trifolium repens cv. Regal upon dark to light transition. Commonly, only steady-state levels of NPQ are evaluated, ignoring transient kinetics. Experimental NPQ kinetics are fitted best with a sum of two functions: a sigmoidal Hill function plus a transient logarithmic normal function. It is shown that not only steady-state level of NPQ, but also the speed at which steady state is reached, increased with light intensity. The question is raised which biological processes cause the induction of the components of NPQ kinetics. The NPQ kinetics are found to resemble the kinetics of antheraxanthin and zeaxanthin formation during a dark to light transition. Furthermore, both molecules are known to induce NPQ. The hypothesis is put forward that a transient phase of NPQ (0-2 min after transition) is dependent upon concentrations of antheraxanthin, whereas the saturating phase corresponds with the production of zeaxanthin. A mathematical model, based on the presented hypothesis, predicts the effect of increasing light intensity on concentrations of antheraxanthin and zeaxanthin which correspond with experimental results. Implications of the hypothesis are discussed as well as the role of NLR in evaluating chlorophyll a fluorescence kinetics.

Topics: Adaptation, Ocular; beta Carotene; Chlorophyll; Chlorophyll A; Fluorescence; Models, Biological; Photosynthesis; Regression Analysis; Trifolium; Xanthophylls; Zeaxanthins

Light-dependent conversion of violaxanthin to zeaxanthin, the so-called xanthophyll cycle, was shown to serve as a major, short-term light acclimation mechanism in higher plants. The role of xanthophylls in thermal dissipation of surplus excitation energy was deduced from the linear relationship between zeaxanthin formation and the magnitude of non-photochemical quenching. Unlike in higher plants, the role of the xanthophyll cycle in green algae (Chlorophyta) is ambiguous, since its contribution to energy dissipation can significantly vary among species. Here, we have studied the role of the xanthophyll cycle in the adaptation of several species of green algae (Chlorella, Scenedesmus, Haematococcus, Chlorococcum, Spongiochloris) to high irradiance. The xanthophyll cycle has been found functional in all tested organisms; however its contribution to non-photochemical quenching is not as significant as in higher plants. This conclusion is supported by three facts: (i) in green algae the content of zeaxanthin normalized per chlorophyll was significantly lower than that reported from higher plants, (ii) antheraxanthin + zeaxanthin content displayed different diel kinetics from NPQ and (iii) in green algae there was no such linear relationship between NPQ and Ax + Zx, as found in higher plants. We assume that microalgae rely on other dissipation mechanism(s), which operate along with xanthophyll cycle-dependent quenching.

Topics: Acclimatization; beta Carotene; Chlorophyll; Chlorophyta; Light; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Temperature; 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

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

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

Topics: beta Carotene; Chlorophyta; Cyclization; Hydrogen-Ion Concentration; Light; Oxidoreductases; Spinacia oleracea; Substrate Specificity; Xanthophylls

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 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

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 xanthophyll cycle-dependent dissipation of excitation energy in higher plants is one of the most important regulatory and photoprotective mechanisms in photosynthesis. Using parallel time-resolved and pulse-amplitude modulation fluorometry, we studied the influence of the intrathylakoid pH and the xanthophyll cycle carotenoids on the PSII chlorophyll (Chl) a fluorescence yield in thylakoids of Arabidopsis, spinach, and barley. Increases in concentrations of dithiothreitol in thylakoids, which have a trans-thylakoid membrane pH gradient and are known to have decreased conversion of violaxanthin (V) to zeaxanthin (Z), lead to (1) decreases in the fractional intensity of the approximately 0.5 ns Chl a fluorescence lifetime (tau) distribution component and simultaneous increases in a 1.6-1.8 ns fluorescence component and (2) increases in the maximal fluorescence intensity. These effects disappear when the pH gradient is eliminated by the addition of nigericin. To quantitatively explain these results, we present a new mathematical model that describes the simultaneous effects of the chloroplast trans-thylakoid membrane pH gradient and xanthophyll cycle pigments on the PSII Chl a fluorescence tau distributions and intensity. The model assumes that (1) there exists a specific binding site for Z (or antheraxanthin, A) among or in an inner antenna complex (primarily CP29), (2) this binding site is activated by a low intrathylakoid pH (pK approximately 4.5) that increases the affinity for Z (or A), (3) about one Z or A molecule binds to the activated site, and (4) this binding effectively "switches" the fluorescence tau distribution of the PSII unit to a state with a decreased fluorescence tau and emission intensity (a "dimmer switch" concept). This binding is suggested to cause the formation of an exciton trap with a rapid intrinsic rate constant of heat dissipation. Statistical analysis of the data yields an equilibrium association constant, Ka, that ranges from 0.7 to 3.4 per PSII for the protonated/activated binding site for Z (or A). The model explains (1) the relative fraction of the approximately 0.5 ns fluorescence component as a function of both Z and A concentration and intrathylakoid pH, (2) the dependence of the ratio of F'm/Fm on the fraction of the 0.5 ns fluorescence tau component (where F'm and Fm are maximal fluorescence intensities in the presence and the absence of a pH gradient), and (3) the dependence of the ratio of F'm/Fm on the concentra

Topics: beta Carotene; Carotenoids; Chlorophyll; Chlorophyll A; Chloroplasts; Fluorescence Polarization; Hordeum; Hydrogen-Ion Concentration; Intracellular Membranes; Lutein; Photochemistry; Pigments, Biological; Spectrometry, Fluorescence; Spinacia oleracea; 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

The xanthophyll cycle apparently aids the photoprotection of photosystem II by regulating the nonradiative dissipation of excess absorbed light energy as heat. However, it is a controversial question whether the resulting nonphotochemical quenching is soley dependent on xanthophyll cycle activity or not. The xanthophyll cycle consists of two enzymic reactions, namely deepoxidation of the diepoxide violaxanthin to the epoxide-free zeaxanthin and the much slower, reverse process of epoxidation. While deepoxidation requires a transthylakoid pH gradient (delta pH), epoxidation can proceed irrespective of a delta pH. Herein, we compared the extent and kinetics of deepoxidation and epoxidation to the changes in fluorescence in the presence of a light-induced thylakoid delta pH. We show that epoxidation reverses fluorescence quenching without affecting thylakoid delta pH. These results suggest that epoxidase activity reverses quenching by removing deepoxidized xanthophyll cycle pigments from quenching complexes and converting them to a nonquenching form. The transmembrane organization of the xanthophyll cycle influences the localization and the availability of deepoxidized xanthophylls is to support nonphotochemical quenching capacity. The results confirm the view that rapidly reversible nonphotochemical quenching is dependent on deepoxidized xanthophyll.

Topics: beta Carotene; Carotenoids; Chlorophyll; Chlorophyll A; Chloroplasts; Epoxy Compounds; Hydrogen-Ion Concentration; Light-Harvesting Protein Complexes; Membrane Potentials; NADP; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Spectrometry, Fluorescence; Xanthophylls; Zeaxanthins