diadinoxanthin and fucoxanthin

In the present study, low concentrations of the very mild detergent n-dodecyl-α-d-maltoside in conjunction with sucrose gradient ultracentrifugation were used to prepare fucoxanthin chlorophyll protein (FCP) complexes of the centric diatom Thalassiosira pseudonana. Two main FCP fractions were observed in the sucrose gradients, one in the upper part and one at high sucrose concentrations in the lower part of the gradient. The first fraction was dominated by the 18 kDa FCP protein band in SDS-gels. Since this fraction also contained other protein bands, it was designated as fraction enriched in FCP-A complex. The second fraction contained mainly the 21 kDa FCP band, which is typical for the FCP-B complex. Determination of the lipid composition showed that both FCP fractions contained monogalactosyl diacylglycerol as the main lipid followed by the second galactolipid of the thylakoid membrane, namely digalactosyl diacylglycerol. The negatively charged lipids sulfoquinovosyl diacylglycerol and phosphatidyl glycerol were also present in both fractions in pronounced concentrations. With respect to the pigment composition, the fraction enriched in FCP-A contained a higher amount of the xanthophyll cycle pigments diadinoxanthin (DD) and diatoxanthin (Dt), whereas the FCP-B fraction was characterized by a lower ratio of xanthophyll cycle pigments to the light-harvesting pigment fucoxanthin. Protein analysis by mass spectrometry revealed that in both FCP fractions the xanthophyll cycle enzyme diadinoxanthin de-epoxidase (DDE) was present. In addition, the analysis showed an enrichment of DDE in the fraction enriched in FCP-A but only a very low amount of DDE in the FCP-B fraction. In-vitro de-epoxidation assays, employing the isolated FCP complexes, were characterized by an inefficient conversion of DD to Dt. However, in line with the heterogeneous DDE distribution, the fraction enriched in FCP-A showed a more pronounced DD de-epoxidation compared with the FCP-B.

Topics: Chlorophyll Binding Proteins; Diatoms; Diglycerides; Xanthophylls

Integrated preparation of high-purity carotenoids from marine microalgae using green and efficient methods still faces enormous challenges. In this study, valorization of the economic Phaeodactylum tricornutum using integrated preparation of diadinoxanthin (Ddx) and fucoxanthin (Fx) was explored containing four steps including algae cultivation, solvent extraction, ODS open-column chromatography, and ethanol precipitation for the first time. Several essential key factors were optimized for simultaneously extracting Ddx and Fx from P. tricornutum. ODS open-column chromatography was used to isolate Ddx and Fx. Purification of Ddx and Fx was accomplished using ethanol precipitation. After optimization, the purity of Ddx and Fx was more than 95%, and the total recovery rates of Ddx and Fx were approximately 55% and 85%, respectively. The purified Ddx and Fx were identified as all-trans-diadinoxanthin and all-trans-fucoxanthin, respectively. The antioxidant capacity of the purified Ddx and Fx was assessed using two tests in vitro: DPPH and ABTS radical assays.

Topics: Diatoms; Dichlorodiphenyl Dichloroethylene; Ethanol; Xanthophylls

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

Diatoms are abundant photosynthetic organisms in aquatic environments and contribute 40% of its primary productivity. An important factor that contributes to the success of diatoms is their fucoxanthin chlorophyll a/c-binding proteins (FCPs), which have exceptional light-harvesting and photoprotection capabilities. Here, we report the crystal structure of an FCP from the marine diatom

Topics: Chlorophyll; Chlorophyll A; Chlorophyll Binding Proteins; Diatoms; Energy Transfer; Light; Photosynthesis; Protein Structure, Quaternary; Thylakoids; Xanthophylls

Fucoxanthin chlorophyll (Chl) a/ c-binding proteins (FCPs) are unique light-harvesting antennas in diatoms. Recent time-resolved fluorescence analysis of photosystem I with FCP associated (PSI-FCPI) has mainly shown excitation energy transfer among Chls a from FCPI to PSI in tens of picoseconds. However, it remains unclear how each pigment, especially carotenoids and Chl c, in the FCPI is functionally related to the energy transfer in a femtosecond time range. Here, we reveal ultrafast excitation energy transfer mechanism in the PSI-FCPI preparations isolated from a diatom, Chaetoceros gracilis, by means of femtosecond time-resolved fluorescence spectroscopy with an upconversion system. Compared with the fluorescence lifetime components of PSI core-like complexes, the energy transfer of Chl c → Chl a in the FCPI was observed within hundreds of femtoseconds, and the energy in the FCPI was transferred to PSI in ∼2 ps. The comparative fluorescence analyses provide physical insights into the energy transfer machinery within FCPI and from FCPI to PSI.

Topics: Carotenoids; Chlorophyll; Chlorophyll A; Chlorophyll Binding Proteins; Diatoms; Energy Transfer; Fluorescence; Photosystem I Protein Complex; Spectrometry, Fluorescence; Xanthophylls

High-throughput screening Raman spectroscopy (HTS-RS) with automated localization algorithms offers unsurpassed speed and sensitivity to investigate the effect of dithiothreitol on the diatom Phaedactylum tricornutum. The HTS-RS capability that was demonstrated for this model system can be transferred to unmet analytical applications such as kinetic in vivo studies of microalgal assemblages.

Topics: Algorithms; Diatoms; Dithiothreitol; High-Throughput Screening Assays; Light; Spectrum Analysis, Raman; Xanthophylls

Two marine microalgae, Nitzschia closterium and Isochrysis zhangjiangensis, treated for 9 days with photosynthetically active radiation (PAR), were subjected to 3 days of ultraviolet light band A at 365 nm (UVA365) followed by a 3 day post-UVA PAR treatment. Compared with the control that only had PAR treatment, UVA365 treatment significantly ( p < 0.05) increased the amounts of total xanthophylls in N. closterium and I. zhangjiangensis by up to 3.53- and 1.23-fold, respectively, after 3 days of UVA365 irradiation. In particular, UVA365 treatment markedly ( p < 0.05) increased two major bioactive xanthophyll pigments, fucoxanthin in N. closterium and diadinoxanthin in I. zhangjiangensis, by up to 1.97- and 1.52-fold, respectively. UVA365 treatment significantly ( p < 0.05) increased the accumulation of another bioactive microalgal metabolite, total mycosporine-like amino acids, in N. closterium and I. zhangjiangensis by up to 1.40- and 1.30-fold, respectively. UVA365 irradiation has potential as an efficient approach for enhancing the production of valuable microalgal metabolites.

Topics: Diatoms; Microalgae; Photosynthesis; Ultraviolet Rays; Xanthophylls

Phaeodactylum tricornutum like other diatoms synthesizes fucoxanthin and diadinoxanthin as major carotenoid end products. The genes involved have recently been assigned for early pathway steps. Beyond β-carotene, only gene candidates for β-carotene hydroxylase, zeaxanthin epoxidase and zeaxanthin de-epoxidase have been proposed from the available genome sequence. The two latter enzymes may be involved in the two different xanthophyll cycles which operate in P. tricornutum. The function of three putative zeaxanthin epoxidase genes (zep) was addressed by pathway complementation in the Arabidopsis thaliana Zep mutant npq2. Genes zep2 and zep3 were able to restore zeaxanthin epoxidation and a functional xanthophyll cycle but the corresponding enzymes exhibited different catalytic activities. Zep3 functioned as a zeaxanthin epoxidase whereas Zep2 exhibited a broader substrate specificity additionally converting lutein to lutein-5,6-epoxide. Although zep1 was transcribed and the protein could be identified after import into the chloroplast in A. thaliana, Zep1 was found not to be functional in zeaxanthin epoxidation. The non-photochemical quenching kinetics of wild type A. thaliana was only restored in transformant npq2-zep3.

Topics: Arabidopsis; Carotenoids; Diatoms; Genetic Complementation Test; Kinetics; Lutein; Mixed Function Oxygenases; Mutation; Oxidoreductases; Xanthophylls; Zeaxanthins

Fucoxanthin has been receiving ever-increasing interest due to its broad health beneficial effects. Currently, seaweeds are the predominant source of natural fucoxanthin. However, the disappointingly low fucoxanthin content has impeded their use, driving the exploration of alternative fucoxanthin producers. In the present study, thirteen diatom strains were evaluated with respect to growth and fucoxanthin production potential. Cyclotella cryptica (CCMP 333), which grew well for fucoxanthin production under both photoautotrophic and heterotrophic growth conditions, was selected for further investigation. The supply of nitrate and light individually or in combination were all found to promote growth and fucoxanthin accumulation. When transferring heterotrophic cultures to light, fucoxanthin responded differentially to light intensities and was impaired by higher light intensity with a concomitant increase in diadinoxanthin and diatoxanthin, indicative of the modulation of Diadinoxanthin Cycle to cope with the light stress. Taken together, we, for the first time, performed the screening of diatom strains for fucoxanthin production potential and investigated in detail the effect of nutritional and environmental factors on C. cryptica growth and fucoxanthin accumulation. These results provide valuable implications into future engineering of C. cryptica culture parameters for improved fucoxanthin production and C. cryptica may emerge as a promising microalgal source of fucoxanthin.

Topics: Diatoms; Light; Xanthophylls

Fucoxanthin, diadinoxanthin and diatoxanthin are carotenoids found in brown algae and most other heterokonts. These pigments are involved in photosynthetic and photoprotective reactions, and they have many potential health benefits. They can be extracted from diatom Phaeodactylum tricornutum by sonication, extraction with chloroform : methanol and preparative thin layer chromatography. We assessed the utility of an additional column chromatography step in purification of these pigments. This novel addition to the isolation protocol increased the purity of fucoxanthin and allowed for concentration of diadinoxanthin and diatoxanthin before HPLC separation. The enhanced protocol is useful for obtaining high purity pigments for biochemical studies.

Topics: Chloroform; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Diatoms; Liquid-Liquid Extraction; Methanol; Solvents; Sonication; Xanthophylls

The major light-harvesting pigment protein complex (fucoxanthin-chlorophyll-binding protein complex; FCP) was purified from a marine centric diatom, Chaetoceros gracilis, by mild solubilization followed by sucrose density gradient centrifugation, and then characterized. The dynamic light scattering measurement showed unimodality, indicating that the complex was highly purified. The amount of chlorophyll a (Chl a) bound to the purified FCP accounted for more than 60 % of total cellular Chl a. The complex was composed of three abundant polypeptides, although there are nearly 30 FCP-related genes. The two major components were identified as Fcp3 (Lhcf3)- and Fcp4 (Lhcf4)-equivalent proteins based on their internal amino acid sequences and a two-dimensional isoelectric focusing electrophoresis analysis developed in this work. Compared with the thylakoids, the FCP complex showed higher contents of fucoxanthin and chlorophyll c but lower contents of the xanthophyll cycle pigments diadinoxanthin and diatoxanthin. Fluorescence excitation spectra analyses indicated that light harvesting, rather than photosystem protection, is the major function of the purified FCP complex, which is associated with more than 60 % of total cellular Chl a. These findings suggest that the huge amount of Chl bound to the FCP complex composed of Lhcf3, Lhcf4, and an unidentified minor protein has a light-harvesting function to allow efficient photosynthesis under the dim-light conditions in the ocean.

Topics: Carrier Proteins; Chlorophyll; Chlorophyll A; Diatoms; Light; Light-Harvesting Protein Complexes; Photosystem II Protein Complex; Spectrometry, Fluorescence; Thylakoids; Xanthophylls

In this study, we demonstrate the selective in vivo detection of diadinoxanthin (DD) and diatoxanthin (DT) in intact Cyclotella cells using resonance Raman spectroscopy. In these cells, we were able to assess both the content of DD and DT carotenoids relative to chlorophyll and their conformation. In addition, the sensitivity and selectivity of the technique allow us to discriminate between different pools of DD on a structural basis, and to follow their fate as a function of the illumination conditions. We report that the additional DD observed when cells are grown in high-light conditions adopts a more twisted conformation than the lower levels of DD present when the cells are grown in low-light (LL) conditions. Thus, we conclude that this pool of DD is more tightly bound to a protein-binding site, which must differ from the one occupied by the DD present in LL conditions.

Topics: Binding Sites; Carotenoids; Chromatography, High Pressure Liquid; Diatoms; Light; Spectrum Analysis, Raman; Xanthophylls

The biosynthesis pathway to diadinoxanthin and fucoxanthin was elucidated in Phaeodactylum tricornutum by a combined approach involving metabolite analysis identification of gene function. For the initial steps leading to β-carotene, putative genes were selected from the genomic database and the function of several of them identified by genetic pathway complementation in Escherichia coli. They included genes encoding a phytoene synthase, a phytoene desaturase, a ζ-carotene desaturase, and a lycopene β-cyclase. Intermediates of the pathway beyond β-carotene, present in trace amounts, were separated by TLC and identified as violaxanthin and neoxanthin in the enriched fraction. Neoxanthin is a branching point for the synthesis of both diadinoxanthin and fucoxanthin and the mechanisms for their formation were proposed. A single isomerization of one of the allenic double bounds in neoxanthin yields diadinoxanhin. Two reactions, hydroxylation at C8 in combination with a keto-enol tautomerization and acetylation of the 3'-HO group results in the formation of fucoxanthin.

Topics: beta Carotene; Biosynthetic Pathways; Carotenoids; Diatoms; Escherichia coli; Genetic Complementation Test; Geranylgeranyl-Diphosphate Geranylgeranyltransferase; Intramolecular Lyases; Oxidoreductases; Phylogeny; Xanthophylls; zeta Carotene

In the present study we report that in the diatom Cyclotella meneghiniana the diatoxanthin-dependent non-photochemical quenching of chlorophyll fluorescence (NPQ) is heterogeneous and consists of three different components. (i) A transient NPQ component that generates immediately upon illumination, depends on the transthylakoid proton gradient as well as on the light intensity, and is modulated by the initial diatoxanthin content of the cells. It is located in the antenna complexes of C. meneghiniana and is comparable with the transient NPQ observed in vascular plants. (ii) A steady-state NPQ component is observed during later stages of the high-light illumination and depends on the diatoxanthin content formed by the light-activated diadinoxanthin cycle. (iii) A fast relaxing NPQ component is seen upon a transition of high-light-illuminated cells to complete darkness. This component relaxes within a time frame of tens of seconds and its extent is correlated with the amount of diatoxanthin formed during the phase of actinic illumination. It cannot be observed in dithiothreitol-treated cells where the de-epoxidation of diadinoxanthin to diatoxanthin is suppressed. The fast relaxing component can be interpreted as a relaxation of part of the steady-state NPQ. The different diatoxanthin-dependent components are characterized by different quenching efficiencies of diatoxanthin. Diatoxanthin involved in the transient NPQ exhibits a 2-fold higher quenching efficiency compared with diatoxanthin participating in the steady-state NPQ. It is proposed that the different quenching efficiencies of diatoxanthin are caused by the existence of different diatoxanthin pools within the antenna system of C. meneghiniana.

Topics: beta Carotene; Chlorophyll; Diatoms; Fluorescence; Light; Photosynthesis; Signal Transduction; Time Factors; Xanthophylls

Two different fucoxanthin-chlorophyll protein complexes (FCP) were purified from the centric diatom Cyclotella meneghiniana and characterized with regard to their polypeptide and pigment composition. Whereas the oligomeric FCPb complex is most probably composed of fcp5 gene products, the trimeric FCPa has subunits encoded by fcp1-3 and fcp6/7. The amount of the latter polypeptide is enhanced when FCPa is isolated from algae grown under HL conditions. This increase in Fcp6/7 polypeptides is accompanied by an increase in the pool of xanthophyll cycle pigments, diadinoxanthin and diatoxanthin, and a concomitant decrease in fucoxanthin content. In addition, the de-epoxidation ratio, i.e., the amount of diatoxanthin in relation to the pool of xanthophyll cycle pigments, is increased by a factor of 2. With regard to fluorescence yield, HL FCPa was quenched in comparison to LL FCPa. This is in accordance with the larger amount of diatoxanthin that is bound, which is supposed to act as a quencher like zeaxanthin in higher plants. Thus, we conclude that the enhanced content of diatoxanthin in FCPa plays a protective role, which is paralleled by a weakened light harvesting function due to a smaller amount of fucoxanthin.

Topics: Blotting, Western; Chlorophyll; Diatoms; Light-Harvesting Protein Complexes; Spectrometry, Fluorescence; Xanthophylls

The pigment composition of diatoms differs from that of green algae and plants. Diatoms contain chlorophyll (Chl(1)) c, fucoxanthin, and diadinoxanthin (DD). An intermittent light regime during growth induced a large increase in the DD content in the marine planktonic diatom Phaeodactylum tricornutum. Light-harvesting complex containing fucoxanthin (LHCF) subunits were purified on a sucrose gradient after treatment of thylakoid membranes with a mild detergent. DD was found in all the LHCF fractions: a "major" composite LHCF fraction and the two fractions where some LHCF was associated with photosystem centers. For cells enriched in DD, most of the additional DD molecules were bound to the major LHCF fraction. The DD enrichment of the major LHCF fraction was accompanied by a decrease in the fucoxanthin to Chl a ratio. Either some fucoxanthin molecules were replaced by DD or there could be a relative enrichment of subunits rich in DD at the expense of fucoxanthin/Chl c rich subunits. Under high light illumination, a higher degree of de-epoxidation of DD into DT was observed for the major LHCF of cells enriched in DD. This fraction has the higher DD content and the higher degree of de-epoxidation. These results show that the distal antennae, probably mostly isolated as the major LHCF fraction, play a crucial role in the formation of NPQ, its amplitude depending on the amount of DD bound and on the degree of de-epoxidation (Lavaud et al. (2002) Plant Physiol. 129, 1398-1406).

Topics: Darkness; Diatoms; Fluorescence; Photochemistry; Photoperiod; Photosynthesis; Spectrophotometry; Xanthophylls

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

According to general agreement, all photosynthetic organisms using xanthophyll cycling for photoprotection contain either the violaxanthin (Vx) cycle or the diadinoxanthin (Ddx) cycle instead. Here, we report the temporal accumulation of substantial amounts of pigments of the Vx cycle under prolonged high-light stress in several microalgae thought to possess only the Ddx cycle. In the diatom Phaeodactylum tricornutum, used as a model organism, these pigments also participate in xanthophyll cycling, and their accumulation depends on de novo synthesis of carotenoids and on deepoxidase activity. Furthermore, our data strongly suggest a biosynthetic sequence from Vx via Ddx to fucoxanthin in P. tricornutum. This gives experimental support to the long-stated hypothesis that Vx is a common precursor of all carotenoids with an allenic or acetylenic group, including the main light-harvesting carotenoids in most chlorophyll a/c-containing algae. Thus, another important function for xanthophyll cycling may be to optimize the biosynthesis of light-harvesting xanthophylls under fluctuating light conditions.

Topics: beta Carotene; Carotenoids; Cells, Cultured; Chromatography, High Pressure Liquid; Diatoms; Dithiothreitol; Enzyme Inhibitors; Epoxy Compounds; Eukaryota; Kinetics; Light; Lutein; Molecular Structure; Pigments, Biological; Xanthophylls