chlorophyll-a and neoxanthin

Carotenoids are essential components of photosynthetic organisms including land plants, algae, cyanobacteria, and photosynthetic bacteria. Although the light-mediated regulation of carotenoid biosynthesis, including the light/dark cycle as well as the dependence of carotenoid biosynthesis-related gene translation on light wavelength, has been investigated in land plants, these aspects have not been studied in microalgae. Here, we investigated carotenoid biosynthesis in Euglena gracilis and found that zeaxanthin accumulates in the dark. The major carotenoid species in E. gracilis, namely β-carotene, neoxanthin, diadinoxanthin and diatoxanthin, accumulated corresponding to the duration of light irradiation under the light/dark cycle, although the translation of carotenoid biosynthesis genes hardly changed. Irradiation with either blue or red-light (3 μmol photons m

Topics: Acclimatization; beta Carotene; Chlorophyll; Euglena gracilis; Gene Expression Regulation; Light; Photosystem II Protein Complex; Xanthophylls; Zeaxanthins

The aim of this study was to evaluate the impact of selected types of LED (light emitting diodes) lighting on the quality of alfalfa sprouts. In the experiment, cold white, warm white and multicolour: (red, green, blue-RGB) LEDs were applied, and dispersed sunlight was used as a control. The product was examined for the yield and the contents of dry matter, total polyphenols, ascorbic acid, chlorophylls, β-carotene, lutein, neoxanthin and violaxanthin. Cotyledons' mass in the whole plant increased under LED illumination and was up to 50% greater for sprouts grown in RGB light compared to those cultivated in dispersed sunlight. The highest chlorophyll and carotenoid pigment contents in cotyledons were observed under RGB LED and cold white treatments. Similarly, RGB LEDs allows one to obtain the product with the highest level of total phenolic compounds. The highest ascorbic acid content was observed in sprouts growing under sunlight, followed by RGB.

Topics: beta Carotene; Chlorophyll; Chromatography, High Pressure Liquid; Germination; Light; Lutein; Medicago sativa; Polyphenols; Seedlings; Xanthophylls

In the photic zone, phytoplankton experience diurnal variation in light intensity. However, prolonged exposure to aphotic condition influences their physiological state. Pigment composition is a useful biomarker to decipher cells physiological state and adaptive response to changing environmental conditions. Chlorophyll, a natural pigment, is biosynthesised even in darkness and studies have shown this ability is determined by genetic characteristics of an organism. The purpose of this study was to examine the influence of darkness on pigments and chlorophyll autofluorescence of Tetraselmis indica. Dark exposure (up to 6 months) had no significant impact on chlorophyll a and b concentration, whereas carotenoids were enhanced. Upon re-illumination pigments gradually recovered to pre-dark phase condition. These adaptive survival strategies of T. indica by altering pigment concentration in response to prolonged darkness are interesting. The absence of loroxanthin and loroxanthin esters in T. indica is reported in a first Tetraselmis species so far. In addition, the evaluation of autofluorescence and cellular chlorophyll concentration pointed out that they are not interdependent in this species. Hence, careful consideration of these two factors is needed when either of them is used as a proxy for other. The results obtained encourage a thorough study of pigment analysis, especially when subjected to darkness, to elucidate potential role in the evolution, chemotaxonomy, and survivability of species.

Topics: Carotenoids; Chlorophyll; Chlorophyll A; Chlorophyta; Chromatography, High Pressure Liquid; Darkness; Pigments, Biological; Spectrometry, Fluorescence; Time Factors; Xanthophylls

The light-harvesting chlorophyll

Topics: Arabidopsis; Arabidopsis Proteins; Chlorophyll; Light; Light-Harvesting Protein Complexes; Mutation; Photosynthesis; Photosystem II Protein Complex; Plant Leaves; Protein Binding; Spectrophotometry; Xanthophylls

The ecological importance and diversity of pico/nanoplanktonic algae remains poorly studied in marine waters, in part because many are tiny and without distinctive morphological features. Amongst green algae, Mamiellophyceae such as Micromonas or Bathycoccus are dominant in coastal waters while prasinophytes clade VII, yet not formerly described, appear to be major players in open oceanic waters. The pigment composition of 14 strains representative of different subclades of clade VII was analyzed using a method that improves the separation of loroxanthin and neoxanthin. All the prasinophytes clade VII analyzed here showed a pigment composition similar to that previously reported for RCC287 corresponding to pigment group prasino-2A. However, we detected in addition astaxanthin for which it is the first report in prasinophytes. Among the strains analyzed, the pigment signature is qualitatively similar within subclades A and B. By contrast, RCC3402 from subclade C (Picocystis) lacks loroxanthin, astaxanthin, and antheraxanthin but contains alloxanthin, diatoxanthin, and monadoxanthin that are usually found in diatoms or cryptophytes. For subclades A and B, loroxanthin was lowest at highest light irradiance suggesting a light-harvesting role of this pigment in clade VII as in Tetraselmis.

Topics: Carotenoids; Chlorophyll; Chlorophyll A; Chlorophyta; Light; Lutein; Oceans and Seas; Photosynthesis; Pigments, Biological; Xanthophylls; Zeaxanthins

Resonance Raman spectroscopy was used to evaluate the structure of light-harvesting chlorophyll (Chl) a/b complexes of photosystem II (LHCII), reconstituted from wild-type (WT) and mutant apoproteins over-expressed in Escherichia coli. The point mutations involved residue S123, exchanged for either P (S123P) or G (S123G). In all reconstituted proteins, lutein 2 displayed a distorted conformation, as it does in purified LHCII trimers. Reconstituted WT and S123G also exhibited a conformation of bound neoxanthin (Nx) molecules identical to the native protein, while the S123P mutation was found to induce a change in Nx conformation. This structural change of neoxanthin is accompanied by a blue shift of the absorption of this carotenoid molecule. The interactions assumed by (and thus the structure of the binding sites of) the bound Chls b were found identical in all the reconstituted proteins, and only marginally perturbed as compared to purified LHCII. The interactions assumed by bound Chls a were also identical in purified LHCII and the reconstituted WT. However, the keto carbonyl group of one Chl a, originally free-from-interactions in WT LHCII, becomes involved in a strong H-bond with its environment in LHCII reconstituted from the S123P apoprotein. As the absorption in the Qy region of this protein is identical to that of the LHCII reconstituted from the WT apoprotein, we conclude that the interaction state of the keto carbonyl of Chl a does not play a significant role in tuning the binding site energy of these molecules.

Topics: Binding Sites; Chlorophyll; Chlorophyll A; Light-Harvesting Protein Complexes; Lutein; Mutation; Photosystem II Protein Complex; Spectrum Analysis, Raman; Xanthophylls

The antioxidant activity and individual bioactive compounds of lettuce, cultivated with 2.5-30% (v/v) of fresh or composted espresso spent coffee grounds, were assessed. A progressive enhancement of lettuce's antioxidant capacity, evaluated by radical scavenging effect and reducing power, was exhibited with the increment of fresh spent coffee amounts, while this pattern was not so clear with composted treatments. Total reducing capacity also improved, particularly for low spent coffee concentrations. Additionally, very significant positive correlations were observed for all carotenoids in plants from fresh spent coffee treatments, particularly for violaxanthin, evaluated by HPLC. Furthermore, chlorophyll a was a good discriminating factor between control group and all spent coffee treated samples, while vitamin E was not significantly affected. Espresso spent coffee grounds are a recognised and valuable source of bioactive compounds, proving herein, for the first time, to potentiate the antioxidant pool and quality of the vegetables produced.

Topics: Antioxidants; Carotenoids; Chlorophyll; Chlorophyll A; Chromatography, High Pressure Liquid; Coffee; Crops, Agricultural; Fertilizers; Lactuca; Lutein; Plant Extracts; Soil; Tocopherols; Xanthophylls

Measurement of the second hyperpolarizability (γ) values of compounds can provide insight into the molecular structural requirements for enhancement of third harmonic generation (THG) signal. A convenient method for measuring the γ of compounds in solutions was developed by implementing the THG ratio method which is based on measuring the THG intensity from two interfaces using a nonlinear optical microscope while accounting for the refractive index of solutions at the fundamental and third harmonic wavelengths. We demonstrated that the difference in refractive index at both wavelengths strongly influenced the calculation of γ values when compounds have absorption near the third harmonic or fundamental wavelength. To this end, a refractometer with the wavelength tuning range from UV to near IR was constructed, and the measured refractive indices were used to extract the γ values. The γ values of carotenoids and chlorophylls found in photosynthetic pigment-protein complexes were explored. Large differences in the refractive index at third harmonic and fundamental wavelengths for chlorophylls result in γ values that are more than two orders of magnitude larger than γ values for carotenoids as well as the sign of chlorophylls'γ values is negative while carotenoids have positive γ values.

Topics: Absorption; Carotenoids; Chlorophyll; Chlorophyll A; Microscopy; Refractometry; Solutions; Xanthophylls

In photosynthetic organisms, feedback dissipation of excess absorbed light energy balances harvesting of light with metabolic energy consumption. This mechanism prevents photodamage caused by reactive oxygen species produced by the reaction of chlorophyll (Chl) triplet states with O₂. Plants have been found to perform the heat dissipation in specific proteins, binding Chls and carotenoids (Cars), that belong to the Lhc family, while triggering of the process is performed by the PsbS subunit, needed for lumenal pH detection. PsbS is not found in algae, suggesting important differences in energy-dependent quenching (qE) machinery. Consistent with this suggestion, a different Lhc-like gene product, called LhcSR3 (formerly known as LI818) has been found to be essential for qE in Chlamydomonas reinhardtii. In this work, we report the production of two recombinant LhcSR isoforms from C. reinhardtii and their biochemical and spectroscopic characterization. We found the following: (i) LhcSR isoforms are Chl a/b- and xanthophyll-binding proteins, contrary to higher plant PsbS; (ii) the LhcSR3 isoform, accumulating in high light, is a strong quencher of Chl excited states, exhibiting a very fast fluorescence decay, with lifetimes below 100 ps, capable of dissipating excitation energy from neighbor antenna proteins; (iii) the LhcSR3 isoform is highly active in the transient formation of Car radical cation, a species proposed to act as a quencher in the heat dissipation process. Remarkably, the radical cation signal is detected at wavelengths corresponding to the Car lutein, rather than to zeaxanthin, implying that the latter, predominant in plants, is not essential; (iv) LhcSR3 is responsive to low pH, the trigger of non-photochemical quenching, since it binds the non-photochemical quenching inhibitor dicyclohexylcarbodiimide, and increases its energy dissipation properties upon acidification. This is the first report of an isolated Lhc protein constitutively active in energy dissipation in its purified form, opening the way to detailed molecular analysis. Owing to its protonatable residues and constitutive excitation energy dissipation, this protein appears to merge both pH-sensing and energy-quenching functions, accomplished respectively by PsbS and monomeric Lhcb proteins in plants.

Topics: Absorption; Algal Proteins; Amino Acid Sequence; Chlamydomonas reinhardtii; Chlorophyll; Feedback, Physiological; Fluorescence; Light; Light-Harvesting Protein Complexes; Lutein; Molecular Sequence Data; Protein Binding; Protein Isoforms; Protein Processing, Post-Translational; Protein Refolding; Protein Structure, Tertiary; Recombinant Proteins; Sequence Alignment; Thermodynamics; Thylakoids; Xanthophylls

A recently developed technique for dilution of the naturally high protein packing density in isolated grana membranes was applied to study the dependence of the light harvesting efficiency of photosystem (PS) II on macromolecular crowding. Slight dilution of the protein packing from 80% area fraction to the value found in intact grana thylakoids (70%) leads to an improved functionality of PSII (increased antenna size, enhanced connectivity between reaction centers). Further dilution induces a functional disconnection of light-harvesting complex (LHC) II from PSII. It is concluded that efficient light harvesting by PSII requires an optimal protein packing density in grana membranes that is close to 70%. We hypothesize that the decreased efficiency in overcrowded isolated grana thylakoids is caused by excited state quenching in LHCII, which has previously been correlated with neoxanthin distortion. Resonance Raman spectroscopy confirms this increase in neoxanthin distortion in overcrowded grana as compared with intact thylakoids. Furthermore, analysis of the changes in the antenna size in highly diluted membranes indicates a lipid-induced dissociation of up to two trimeric LHCII from PSII, leaving one trimer connected. This observation supports a hierarchy of LHCII-binding sites on PSII.

Topics: Binding Sites; Chlorophyll; Freeze Fracturing; Light; Lipids; Models, Biological; Photosynthesis; Photosystem II Protein Complex; Plant Proteins; Spectrometry, Fluorescence; Spectrum Analysis, Raman; Spinacia oleracea; Temperature; Thylakoids; Xanthophylls

Carotenoids are naturally occurring pigments that absorb light in the spectral region in which the sun irradiates maximally. These molecules transfer this energy to chlorophylls, initiating the primary photochemical events of photosynthesis. Carotenoids also regulate the flow of energy within the photosynthetic apparatus and protect it from photoinduced damage caused by excess light absorption. To carry out these functions in nature, carotenoids are bound in discrete pigment-protein complexes in the proximity of chlorophylls. A few three-dimensional structures of these carotenoid complexes have been determined by X-ray crystallography. Thus, the stage is set for attempting to correlate the structural information with the spectroscopic properties of carotenoids to understand the molecular mechanism(s) of their function in photosynthetic systems. In this Account, we summarize current spectroscopic data describing the excited state energies and ultrafast dynamics of purified carotenoids in solution and bound in light-harvesting complexes from purple bacteria, marine algae, and green plants. Many of these complexes can be modified using mutagenesis or pigment exchange which facilitates the elucidation of correlations between structure and function. We describe the structural and electronic factors controlling the function of carotenoids as energy donors. We also discuss unresolved issues related to the nature of spectroscopically dark excited states, which could play a role in light harvesting. To illustrate the interplay between structural determinations and spectroscopic investigations that exemplifies work in the field, we describe the spectroscopic properties of four light-harvesting complexes whose structures have been determined to atomic resolution. The first, the LH2 complex from the purple bacterium Rhodopseudomonas acidophila, contains the carotenoid rhodopin glucoside. The second is the LHCII trimeric complex from higher plants which uses the carotenoids lutein, neoxanthin, and violaxanthin to transfer energy to chlorophyll. The third, the peridinin-chlorophyll-protein (PCP) from the dinoflagellate Amphidinium carterae, is the only known complex in which the bound carotenoid (peridinin) pigments outnumber the chlorophylls. The last is xanthorhodopsin from the eubacterium Salinibacter ruber. This complex contains the carotenoid salinixanthin, which transfers energy to a retinal chromophore. The carotenoids in these pigment-protein complexes transfer

Topics: Carotenoids; Chlorophyll; Dinoflagellida; Energy Transfer; Eukaryota; Glucosides; Glycosides; Light; Light-Harvesting Protein Complexes; Lutein; Photosynthesis; Rhodopseudomonas; Thylakoids; Xanthophylls

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

The sun-exposed peel of 'Gala' apple with or without sunburn was compared in terms of photooxidation and photoprotection, and a controlled experiment was conducted to probe the initial responses of PSII to high light and high temperature. The content of carotenoids, lutein and xanthophylls on a chlorophyll basis was higher in the sunburned peel although they were lower expressed on a peel area basis. Significant loss of beta-carotene and neoxanthin was observed relative to chlorophylls in the sunburned peel. O(2) evolution rates and the activity of key enzymes in the Calvin cycle were lower in the sunburned peel, but the activity of these enzymes decreased to a lesser extent than the O(2) evolution rates. The activity of antioxidant enzymes in the ascorbate-glutathione cycle and the level of total ascorbate, total glutathione, and reduced glutathione were higher in the sunburned peel. However, the sunburned peel had higher H(2)O(2) and malondialdehyde contents. Fruit peels treated with high temperature (45 degrees C) alone showed a clear "K" step in their chlorophyll fluorescence transients whereas high temperature coupled with high light (1,600 micromol m(-2) s(-1)) led to the disappearance of the "K" step and a further decrease in F (V)/F (M) (similar to what was observed in the sunburned peel). We conclude that high temperature coupled with high light damages the PSII complexes at both the donor and acceptor sides. Although both the xanthophyll cycle and the antioxidant system are up-regulated in response to the photooxidative stress, this up-regulation does not provide enough protection against the photooxidation.

Topics: Ascorbic Acid; Carotenoids; Chlorophyll; Fruit; Glutathione; Hot Temperature; Light; Lutein; Malondialdehyde; Malus; Oxidation-Reduction; Xanthophylls

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

In this work the photoprotective role of all xanthophylls in LHCII, Lhcb4, and Lhcb5 is investigated by laser-induced Triplet-minus-Singlet (TmS) spectroscopy. The comparison of native LHCII trimeric complexes with different carotenoid composition shows that the xanthophylls in sites V1 and N1 do not directly contribute to the chlorophyll triplet quenching. The largest part of the triplets is quenched by the lutein bound in site L1, which is located in close proximity to the chlorophylls responsible for the low energy state of the complex. The lutein in the L2 site is also active in triplet quenching, and it shows a longer triplet lifetime than the lutein in the L1 site. This lifetime difference depends on the occupancy of the N1 binding site, where neoxanthin acts as an oxygen barrier, limiting the access of O(2) to the inner domain of the Lhc complex, thereby strongly contributing to the photostability. The carotenoid triplet decay of monomeric Lhcb1, Lhcb4, and Lhcb5 is mono-exponential, with shorter lifetimes than observed for trimeric LHCII, suggesting that their inner domains are more accessible for O(2). As for trimeric LHCII, only the xanthophylls in sites L1 and L2 are active in triplet quenching. Although the chlorophyll to carotenoid triplet transfer is efficient (95%) in all complexes, it is not perfect, leaving 5% of the chlorophyll triplets unquenched. This effect appears to be intrinsically related to the molecular organization of the Lhcb proteins.

Topics: Arabidopsis; Arabidopsis Proteins; Chlorophyll; Chlorophyll Binding Proteins; Light-Harvesting Protein Complexes; Lutein; Oxygen; Photosystem II Protein Complex; Xanthophylls

In the present study, xanthophyll composition of eight parasitic Cuscuta species under different light conditions was investigated. Neoxanthin was not detected in four of the eight species examined, while in others it occurred at the level of several percent of total xanthophylls. In C. gronovii and C. lupuliformis it was additionally found that the neoxanthin content was considerably stimulated by strong light. In dark-adapted plants, lutein epoxide level amounted to 10-22% of total xanthophylls in only three species, the highest being for C. lupuliformis, while in others it was below 3%, indicating that the lutein epoxide cycle is limited to only certain Cuscuta species. The obtained data also indicate that the presence of the lutein epoxide cycle and of neoxanthin is independent and variable among the Cuscuta species. The xanthophyll cycle carotenoids violaxanthin, antheraxanthin and zeaxanthin were identified in all the examined species and occurred at the level found in other higher plants. The xanthophyll and lutein epoxide cycle pigments showed typical response to high light stress. The obtained results also suggest that the ability of higher plants to synthesize lutein epoxide probably does not depend on the substrate specificity of zeaxanthin epoxidase but on the availability of lutein for the enzyme.

Topics: Animals; Carotenoids; Chlorophyll; Chromatography, High Pressure Liquid; Cuscuta; Epoxy Compounds; Light; Lutein; Pigmentation; Substrate Specificity; Time Factors; Xanthophylls

The aba4-1 mutant completely lacks neoxanthin but retains all other xanthophyll species. The missing neoxanthin in light-harvesting complex (Lhc) proteins is compensated for by higher levels of violaxanthin, albeit with lower capacity for photoprotection compared with proteins with wild-type levels of neoxanthin. Detached leaves of aba4-1 were more sensitive to oxidative stress than the wild type when exposed to high light and incubated in a solution of photosensitizer agents. Both treatments caused more rapid pigment bleaching and lipid oxidation in aba4-1 than wild-type plants, suggesting that neoxanthin acts as an antioxidant within the photosystem II (PSII) supercomplex in thylakoids. While neoxanthin-depleted Lhc proteins and leaves had similar sensitivity as the wild type to hydrogen peroxide and singlet oxygen, they were more sensitive to superoxide anions. aba4-1 intact plants were not more sensitive than the wild type to high-light stress, indicating the existence of compensatory mechanisms of photoprotection involving the accumulation of zeaxanthin. However, the aba4-1 npq1 double mutant, lacking zeaxanthin and neoxanthin, underwent stronger PSII photoinhibition and more extensive oxidation of pigments than the npq1 mutant, which still contains neoxanthin. We conclude that neoxanthin preserves PSII from photoinactivation and protects membrane lipids from photooxidation by reactive oxygen species. Neoxanthin appears particularly active against superoxide anions produced by the Mehler's reaction, whose rate is known to be enhanced in abiotic stress conditions.

Topics: Arabidopsis; Arabidopsis Proteins; Binding Sites; Centrifugation, Density Gradient; Chlorophyll; Energy Metabolism; Free Radical Scavengers; Light; Light-Harvesting Protein Complexes; Models, Molecular; Molecular Sequence Data; Mutation; Oxidants; Oxidative Stress; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Plant Leaves; Protein Binding; Reactive Oxygen Species; Temperature; Xanthophylls

The parasitic angiosperm Cuscuta reflexa has a highly unusual carotenoid composition in that it does not contain neoxanthin, an otherwise ubiquitous component of the major light-harvesting complex protein (LHCIIb) in all other higher plant species studied to date. Combined HPLC and mass spectrometric analysis has enabled us to detect in tissues of C. reflexa two new types of xanthophylls: lutein-5,6-epoxide and 9-cis-violaxanthin. We have isolated the LHCIIb complex from thylakoids and analyzed chlorophyll and carotenoid composition. The data show that the 9-cis-violaxanthin is present in amounts similar to that of neoxanthin in most plants. On the other hand, lutein-5,6-epoxide was found to be in substoichiometric quantities, suggesting a peripheral location similar to the loosely-associated all-trans-violaxanthin and also enabling suitable accessibility for the de-epoxidase (VDE). Absorption spectroscopy revealed close similarities of the excited state energies of neoxanthin and 9-cis-violaxanthin in vitro and in intact LHCIIb complex. Resonance Raman analysis clearly indicates a cis conformation of violaxanthin in the complex, confirming the pigment analysis data and proving that not only does violaxanthin replace neoxanthin as an intrinsic component of LHCIIb in C. reflexa but it also adopts the same 9-cis conformation of neoxanthin. These results suggest that the N1 binding site of LHCIIb preferentially binds 9-cis-5,6-epoxy carotenoids, which has implications for the features of this binding site and its role in the photosystem II antenna assembly and stability.

Topics: Binding Sites; Carotenoids; Chlorophyll; Chromatography, High Pressure Liquid; Cuscuta; Light-Harvesting Protein Complexes; Magnoliopsida; Mass Spectrometry; Models, Chemical; Photosystem II Protein Complex; Plant Proteins; Protein Binding; Protein Conformation; Spectrophotometry; Spectrum Analysis, Raman; Spinacia oleracea; Thylakoids; Time Factors; Xanthophylls

The main light harvesting complex of photosystem II in plants, LHCII, exists in a trimeric state. To understand the biological significance of trimerization, a comparison has been made been LHCII trimers and LHCII monomers prepared by treatment with phospholipase. The treatment used caused no loss of chlorophyll, but there was a difference in carotenoid composition, together with the previously observed alterations in absorption spectrum. It was found that, when compared to monomers, LHCII trimers showed increased thermal stability and a reduced structural flexibility as determined by the decreased rate and amplitude of fluorescence quenching in low-detergent concentration. It is suggested that LHCII should be considered as having two interacting domains: the lutein 1 domain, the site of fluorescence quenching [Wentworth et al. (2003) J. Biol. Chem. 278, 21845-21850], and the lutein 2 domain. The lutein 2 domain faces the interior of the trimer, the differences in absorption spectrum and carotenoid binding in trimers compared to monomers indicating that the trimeric state modulates the conformation of this domain. It is suggested that the lutein 2 domain controls the conformation of the lutein 1 domain, thereby providing allosteric control of fluorescence quenching in LHCII. Thus, the pigment configuration and protein conformation in trimers is adapted for efficient light harvesting and enhanced protein stability. Furthermore, trimers exhibit the optimum level of control of energy dissipation by modulating the development of the quenched state of the complex.

Topics: Chlorophyll; Hot Temperature; Light-Harvesting Protein Complexes; Phospholipases A; Photosystem II Protein Complex; Plant Leaves; Protein Denaturation; Spectrometry, Fluorescence; Spinacia oleracea; Thermodynamics; Xanthophylls

The electric-field induced absorption changes (Stark effect) of reconstituted light-harvesting complex II (LHCII) in different oligomerisation states-monomers and trimers-with different xanthophyll content have been probed at 77 K. The Stark spectra of the reconstituted control samples, containing the xanthophylls lutein and neoxanthin, are very similar to previously reported spectra of native LHCII. Reconstituted LHCII, containing lutein but no neoxanthin, shows a similar electrooptical response in the Chl a region, but the Stark signal of Chl b around 650 nm amounts to at most approximately 25% of that of the control samples. We conclude that neoxanthin strongly modifies the electronic states of the nearby Chl b molecules causing a large electrooptical response at 650 nm stemming from one or more Chls b in the control samples. Ambiguities about the assignment of several bands in the Soret region [Biochim. Biophys. Acta 1605 (2003) 83] are resolved and the striking difference in electric field response between the two lutein molecules is confirmed. The Stark effect in the carotenoid spectral region in both control and neoxanthin-deficient samples is almost identical, showing that the neoxanthin Stark signal is small and much less intense than the lutein Stark signal.

Topics: Carotenoids; Chlorophyll; Light-Harvesting Protein Complexes; Lutein; Photosystem II Protein Complex; Plant Proteins; Plants; Recombinant Proteins; Spectrum Analysis; Xanthophylls

Six plant pigments separated by paper chromatography have been measured by photoacoustic spectroscopy (PAS). Their PA spectra are compared with the corresponding absorption spectra, which indicate that PAS is a convenient and effective method for identifying plant pigment. The sorts of marine algae are numerous and pigments in marine algae have certain characteristics. The PA spectra of green, red and brown algae have been reported. Pigment characteristics in these algae are identified by their second derivative PA spectra.

Topics: Acoustics; beta Carotene; Chlorophyll; Chlorophyll A; Chromatography, Paper; Eukaryota; Light; Lutein; Photochemistry; Pigments, Biological; Plants; Spectrum Analysis; Xanthophylls

To cope with environmental stress, plants are equipped with antioxidative (e.g., ascorbate, glutathione and alpha-tocopherol) and photoprotective (e.g., xanthophyll cycle pigments) defense systems. We investigated the defense capacities of three tree age classes (mature, sapling and seedling) of Norway spruce (Picea abies (L.) Karst.) at a field site near the timberline. Biochemical data were expressed on both a needle dry mass and a surface area basis. Compared with current-year needles, previous-year needles contained higher mass- and area-based concentrations of chlorophylls and alpha-tocopherol, and a larger xanthophyll cycle pool that was in a more epoxidized state. Total glutathione concentration was lower, the glutathione pool was more reduced and the ascorbate pool was more oxidized in previous-year needles than in current-year needles. Needle concentrations of glutathione and alpha-tocopherol increased and chlorophyll concentration decreased with increasing tree age when expressed on a surface area basis. On a dry mass basis, these trends were reversed or nonexistent. The ascorbate pool was more reduced and the glutathione pool was more oxidized in needles of mature trees than in needles of saplings and seedlings. The proportion of protective xanthophyll cycle pigments decreased and the de-epoxidation state increased with increasing tree age. We conclude that tree age and the basis of expression of antioxidant concentration--surface area or dry mass--are important in scaling from seedlings to large trees.

Topics: alpha-Tocopherol; Antioxidants; Ascorbic Acid; beta Carotene; Carotenoids; Chlorophyll; Glutathione; Picea; Pigments, Biological; Plant Leaves; Trees; Xanthophylls

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

Chlamydomonas reinhardtii double mutant npq2 lor1 lacks the beta, epsilon-carotenoids lutein and loroxanthin as well as all beta,beta-epoxycarotenoids derived from zeaxanthin (e.g. violaxanthin and neoxanthin). Thus, the only carotenoids present in the thylakoid membranes of the npq2 lor1 cells are beta-carotene and zeaxanthin. The effect of these mutations on the photochemical apparatus assembly and function was investigated. In cells of the mutant strain, the content of photosystem-II (PSII) and photosystem-I (PSI) was similar to that of the wild type, but npq2 lor1 had a significantly smaller PSII light-harvesting Chl antenna size. In contrast, the Chl antenna size of PSI was not truncated in the mutant. SDS-PAGE and Western blot analysis qualitatively revealed the presence of all LHCII and LHCI apoproteins in the thylakoid membrane of the mutant. The results showed that some of the LHCII and most of the LHCI were assembled and functionally connected with PSII and PSI, respectively. Photon conversion efficiency measurements, based on the initial slope of the light-saturation curve of photosynthesis and on the yield of Chl a fluorescence in vivo, showed similar efficiencies. However, a significantly greater light intensity was required for the saturation of photosynthesis in the mutant than in the wild type. It is concluded that zeaxanthin can successfully replace lutein and violaxanthin in most of the functional light-harvesting antenna of the npq2 lor1 mutant.

Topics: Animals; beta Carotene; Chlamydomonas; Chlorophyll; Chloroplasts; Darkness; Light; Light-Harvesting Protein Complexes; Lutein; Mutation; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Photosystem I Protein Complex; Photosystem II Protein Complex; Plants; Proteins; Thylakoids; Xanthophylls

The major light-harvesting complex of photosystem II can be reconstituted in vitro from its bacterially expressed apoprotein with chlorophylls a and b and neoxanthin, violaxanthin, lutein, or zeaxanthin as the only xanthophyll. Reconstitution of these one-carotenoid complexes requires low-stringency conditions during complex formation and isolation. Neoxanthin complexes (containing 30-50% of the all-trans isomer) disintegrate during electrophoresis, exhibit a largely reduced resistance against proteolytic attack; in addition, energy transfer from Chl b to Chl a is easily disrupted at elevated temperature. Complexes reconstituted in the presence of either zeaxanthin or lutein contain nearly two xanthophylls per 12 chlorophylls and are more resistant against trypsin. Lutein-LHCIIb also exhibits an intermediate maintenance of energy transfer at higher temperature. Violaxanthin complexes approach a xanthophyll/12 chlorophyll ratio of 3, similar to the ratio in recombinant LHCIIb containing all xanthophylls. On the other hand, violaxanthin-LHCIIb exhibits a low thermal stability like neoxanthin complexes, but an intermediate accessibility towards trypsin, similar to lutein-LHCIIb and zeaxanthin-LHCIIb. Binary competition experiments were performed with two xanthophylls at varying ratios in the reconstitution. Analysis of the xanthophyll contents in the reconstitution products yielded information about relative carotenoid affinities of three assumed binding sites. In lutein/neoxanthin competition experiments, two binding sites showed a strong preference (> 200-fold) for lutein, whereas the third binding site had a higher affinity (25-fold) to neoxanthin. Competition between lutein and violaxanthin gave a similar result, although the specificities were lower: two binding sites have a 36-fold preference for lutein and one has a fivefold preference for violaxanthin. The lowest selectivity was between lutein and zeaxanthin: two binding sites had a fivefold higher affinity for lutein and one has a threefold higher affinity to zeaxanthin.

Topics: Apoproteins; beta Carotene; Binding Sites; Binding, Competitive; Carotenoids; Chlorophyll; Chlorophyll A; Electrophoresis, Polyacrylamide Gel; Energy Transfer; Light-Harvesting Protein Complexes; Lutein; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Pigments, Biological; Plant Proteins; Plants; Protein Precursors; Substrate Specificity; Trypsin; Xanthophylls; Zeaxanthins

Fourteen different revertants of Chlamydomonas reinhardtii recovered with ability of biosynthesis chlorophyll b were hybridized with wile-type strain, and tetrad analysis with random sampling was performed. It appeared that sub genes resulting in cbnI gene to reverse mutation, and localize on the first chromosome. According to its linkage that differences, 5 strains carrying various mutant alleles of suppressor genes were determined. Forward hybridological analysis demonstrated that the sub genes were absent of allelic specificity and had a single genic character in response to suppression. Phenotypic analysis of the sub/Sub diplontic hybrid have verified the dominant character of mutant sub genes. The phenomenon of present various allelic sub genes and all its characters revealed that the possibility of several ways or various regulatory means exists in biosynthesis of chlorophyll b.

Topics: Animals; Carotenoids; Chlamydomonas reinhardtii; Chlorophyll; Genes, Suppressor; Mutation; Xanthophylls

The localisation of the xanthophyll neoxanthin within the structure of the major light harvesting complex (LHCII) of higher plants has been investigated by site-directed mutagenesis and spectroscopic methods. Mutation analysis performed on pigment binding sites in different helix domains leads to selective loss of neoxanthin for mutations on helix C thus localising this pigment between the helix C and helix A/B domains. Recombinant proteins binding two lutein molecules per polypeptide but lacking neoxanthin have been used in order to determine the contribution of neoxanthin to the absorption and linear dichroism spectra. The data were used to derive the orientation of the neoxanthin transition moment, lying in the polyene chain, which was thus determined to form an angle of 57 +/- 1.5 degrees with respect to the normal to the membrane plane where the protein is inserted. On the basis of these results we propose a model for the localisation of the carotenoid site in the LHCII structure which is still unresolved.

Topics: Binding Sites; Carotenoids; Chlorophyll; Chromatography, High Pressure Liquid; Light-Harvesting Protein Complexes; Lutein; Models, Molecular; Mutation; Photosynthetic Reaction Center Complex Proteins; Protein Conformation; Recombinant Proteins; Spectrum Analysis; Xanthophylls

Singlet energy transfer between the carotenoids (Cars) and chlorophylls (Chls) in the light-harvesting complex II (LHC II) from higher plants has been studied using ultrafast transient absorption spectroscopy by exciting the Cars directly in the 475-515 nm wavelength range. LHC II trimers from Arabidopsis thaliana with well-defined Car compositions have been used. From HPLC, the wild type (WT) monomer contains two luteins (Ls), one neoxanthin (N), and a trace of violaxanthin (V) per 12 Chls. The ABA-3 mutant contains 1.4 Ls and 0.6 zeaxanthin (Z) per monomer. Though exploitation of the difference in Car constitution and exciting the WT at 475 and 490 nm, and the ABA-3 mutant at 490 and 515 nm, the different Car contributions to energy transfer have been probed. Evidence for energy transfer mainly from the Car to Chl b is observed in the WT. In the mutant, additional transfer from Car to Chl a correlates with the presence of Z. The results imply predominant energy transfer from the central Ls to Chl b which requires a modification of the currently accepted arrangement of Chl pigments in LHC II.

Topics: Arabidopsis; beta Carotene; Carotenoids; Chlorophyll; Energy Transfer; Kinetics; Light-Harvesting Protein Complexes; Lutein; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Spectrophotometry; Time Factors; Xanthophylls