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Abstract Background Xanthophylls are a large class of carotenoids that are found in a variety of organisms and play particularly important roles in the light-harvesting and photoprotection processes of plants and algae. Violaxanthin is an important plant-derived xanthophyll with wide potential applications in medicines, foods, and cosmetics because of its antioxidant activity and bright yellow color. To date, however, violaxanthins have not been produced using metabolically engineered microbes on a commercial scale. Metabolic engineering for microbial production of violaxanthin is hindered by inefficient synthesis pathway in the heterologous host. We systematically optimized the carotenoid chassis and improved the functional expression of key enzymes of violaxanthin biosynthesis in Escherichia coli. Results Co-overexpression of crtY (encoding lycopene β-cyclase), crtZ (encoding β-carotene 3-hydroxylase), and ZEP (encoding zeaxanthin epoxidase) had a notable impact on their functions, resulting in the accumulation of intermediate products, specifically lycopene and β-carotene. A chassis strain that did not accumulate the intermediate was optimized by several approaches. A promoter library was used to optimize the expression of crtY and crtZ. The resulting strain DZ12 produced zeaxanthin without intermediates. The expression of ZEP was further systematically optimized by using DZ12 as the chassis host. By using a low copy number plasmid and a modified dithiol/disulfide system, and by co-expressing a full electron transport chain, we generated a strain producing violaxanthin at about 25.28 ± 3.94 mg/g dry cell weight with decreased byproduct accumulation. Conclusion We developed an efficient metabolically engineered Escherichia coli strain capable of producing a large amount of violaxanthin. This is the first report of a metabolically engineered microbial platform that could be used for the commercial production of violaxanthin.

Background: Xanthophylls are a large class of carotenoids that are found in a variety of organisms and play particularly important roles in the light-harvesting and photoprotection processes of plants and algae. Violaxanthin is an important plant-derived xanthophyll with wide potential applications in medicines, foods, and cosmetics because of its antioxidant activity and bright yellow color. To date, however, violaxanthins have not been produced using metabolically engineered microbes on a commercial scale. Metabolic engineering for microbial production of violaxanthin is hindered by inefficient synthesis pathway in the heterologous host. We systematically optimized the carotenoid chassis and improved the functional expression of key enzymes of violaxanthin biosynthesis in Escherichia coli. Results: Co-overexpression of crtY (encoding lycopene β-cyclase), crtZ (encoding β-carotene 3-hydroxylase), and ZEP (encoding zeaxanthin epoxidase) had a notable impact on their functions, resulting in the accumulation of intermediate products, specifically lycopene and β-carotene. A chassis strain that did not accumulate the intermediate was optimized by several approaches. A promoter library was used to optimize the expression of crtY and crtZ. The resulting strain DZ12 produced zeaxanthin without intermediates. The expression of ZEP was further systematically optimized by using DZ12 as the chassis host. By using a low copy number plasmid and a modified dithiol/disulfide system, and by co-expressing a full electron transport chain, we generated a strain producing violaxanthin at about 25.28 ± 3.94 mg/g dry cell weight with decreased byproduct accumulation. Conclusion: We developed an efficient metabolically engineered Escherichia coli strain capable of producing a large amount of violaxanthin. This is the first report of a metabolically engineered microbial platform that could be used for the commercial production of violaxanthin. [ABSTRACT FROM AUTHOR]

Nannochloropsis has been considered as a promising feedstock for the industrial production of violaxanthin. However, a rational breeding strategy for the enhancement of violaxanthin content in this microalga is still vacant, thereby limiting its industrial application. All-trans-lycopene locates in the first branch point of carotenogenesis. The carotenoid isomerase (CRTISO), catalyzing the lycopene formation, is thus regarded as a key enzyme for carotenogenesis. Phaeodactylum tricornutum can accumulate high-level carotenoids under optimal conditions. Therefore, it is feasible to improve violaxanthin level in Nannochloropsis by overexpression of PtCRTISO. Protein targeting analysis of seven PtCRTISO candidates (PtCRTISO1-6 and PtCRTISO-like) demonstrated that PtCRTISO4 was most likely the carotenoid isomerase of P. tricornutum. Moreover, the transcriptional pattern of PtCRTISO4 at different cultivation periods was quite similar to other known carotenogenesis genes. Thus, PtCRTISO4 was transformed into N. oceanica. Compared to the wild type (WT), all three transgenic lines (T1-T3) of N. oceanica exhibited higher levels of total carotenoid and violaxanthin. Notably, T3 exhibited the peak violaxanthin content of 4.48 mg g-1 dry cell weight (DCW), which was 1.68-folds higher than WT. Interestingly, qRT-polymerase chain reaction (PCR) results demonstrated that phytoene synthase (NoPSY) rather than z-carotene desaturase (NoZDS) and lycopene b-cyclase (NoLCYB) exhibited the highest upregulation, suggesting that PtCRTISO4 played an additional regulatory role in terms of carotenoid accumulation. Moreover, PtCRTISO4 overexpression increased C18:1n-9 but decreased C16:1n-7, implying that C18:1 may serve as a main feedstock for xanthophyll esterification in Nannochloropsis. Our results will provide valuable information for the violaxanthin production from Nannochloropsis. [ABSTRACT FROM AUTHOR]

The summer climate in Maritime Antarctica is characterised by high humidity and cloudiness with slightly above zero temperatures. Under such conditions, photosynthetic activity is temperature-limited and plant communities are formed by a few species. These conditions could prevent the operation of the photoprotective xanthophyll (VAZ) cycle as low irradiance reduces the excess of energy and low temperatures limit enzyme activity. The VAZ cycle regulates the dissipation of the excess of absorbed light as heat, which is the main mechanism of photoprotection in plants. To test whether this mechanism operates dynamically in Antarctic plant communities, we characterised pigment dynamics under natural field conditions in two representative species: the moss Polytrichum juniperinum and the grass Deschampsia antarctica. Pigment analyses revealed that the total VAZ pool was in the upper range of the values reported for most plant species, suggesting that they are exposed to a high degree of environmental stress. Despite cloudiness, there was a strong conversion of violaxanthin (V) to zeaxanthin (Z) during daytime. Conversely, the dark-induced enzymatic epoxidation back to V was not limited by nocturnal temperatures. In contrast with plants from other cold ecosystems, we did not find any evidence of overnight retention of Z or sustained reductions in photochemical efficiency. These results are of interest for modelling, remote sensing and upscaling of the responses of Antarctic vegetation to environmental challenges. [ABSTRACT FROM AUTHOR]

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Abstract Background Nannochloropsis is a marine microalga that has been extensively studied. The major carotenoid produced by this group of microalgae is violaxanthin, which exhibits anti-inflammatory, anti-photoaging, and antiproliferative activities. Therefore, it has a wide range of potential applications. However, large-scale production of this pigment has not been much studied, thereby limiting its industrial application. Results To develop a novel strain producing high amount of violaxanthin, various Nannochloropsis species were isolated from seawater samples and their violaxanthin production potential were compared. Of the strains tested, N. oceanica WS-1 exhibited the highest violaxanthin productivity; to further enhance the violaxanthin yield of WS-1, we performed gamma-ray-mediated random mutagenesis followed by colorimetric screening. As a result, Mutant M1 was selected because of its significant higher violaxanthin content and biomass productivity than WS-1 (5.21 ± 0.33 mg g− 1 and 0.2101 g L− 1 d− 1, respectively). Subsequently, we employed a 10 L-scale bioreactor to confirm the large-scale production potential of M1, and the results indicated a 43.54 % increase in violaxanthin production compared with WS-1. In addition, comparative transcriptomic analysis performed under normal light condition identified possible mechanisms associated with remediating photo-inhibitory damage and other key responses in M1, which seemed to at least partially explain enhanced violaxanthin content and delayed growth. Conclusions Nannochloropsis oceanica mutant (M1) with enhanced violaxanthin content was developed and its physiological characteristics were investigated. In addition, enhanced production of violaxanthin was demonstrated in the large-scale cultivation. Key transcriptomic responses that are seemingly associated with different physiological responses of M1 were elucidated under normal light condition, the details of which would guide ongoing efforts to further maximize the industrial potential of violaxanthin producing strains.

To determine the optimum parameters for extracting three carotenoids including zeaxanthin, lutein epoxide, and violaxanthin from pepper leaves by response surface methodology (RSM), a solvent of acetone and ethyl acetate (1:2) was used to extract carotenoids with four independent factors: ultrasound time (20–60 min); ratio of sample to solvent (1:12–1:4); saponification time (10–50 min); and concentration of saponification solution (KOH–methanol) (10–30%). A second-order polynomial model produced a satisfactory fitting of the experimental data with regard to zeaxanthin (R² = 75.95%, p < 0.0197), lutein epoxide (R² = 90.24%, p < 0.0001), and violaxanthin (R² = 73.84%, p < 0.0809) content. The optimum joint extraction conditions of zeaxanthin, lutein epoxide, and violaxanthin were 40 min, 1:8, 32 min, and 20%, respectively. The optimal predicted contents for zeaxanthin (0.823022 μg/g DW), lutein epoxide (4.03684 μg/g dry; DW—dry weight), and violaxanthin (16.1972 μg/g DW) in extraction had little difference with the actual experimental values obtained under the optimum extraction conditions for each response: zeaxanthin (0.8118 μg/g DW), lutein epoxide (3.9497 μg/g DW), and violaxanthin (16.1590 μg/g DW), which provides a theoretical basis and method for cultivating new varieties at low temperatures and weak light resistance. [ABSTRACT FROM AUTHOR]

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

Summary: Xanthophyll cycles are broadly important in photoprotection, and the reversible de‐epoxidation of xanthophylls typically occurs in excess light conditions. However, as presented in this review, compiling evidence in a wide range of photosynthetic eukaryotes shows that xanthophyll de‐epoxidation also occurs under diverse abiotic stress conditions in darkness. Light‐driven photochemistry usually leads to the pH changes that activate de‐epoxidases (e.g. violaxanthin de‐epoxidase), but in darkness alternative electron transport pathways and luminal domains enriched in monogalactosyl diacyl glycerol (which enhance de‐epoxidase activity) likely enable de‐epoxidation. Another 'dark side' to sustaining xanthophyll de‐epoxidation is inactivation and/or degradation of epoxidases (e.g. zeaxanthin epoxidase). There are obvious benefits of such activity regarding stress tolerance, and indeed this phenomenon has only been reported in stressful conditions. However, more research is required to unravel the mechanisms and understand the physiological roles of dark‐induced formation of zeaxanthin. Notably, the de‐epoxidation of violaxanthin to antheraxanthin and zeaxanthin in darkness is still a frequently ignored process, perhaps because it questions a previous paradigm. With that in mind, this review seeks to shed some light on the dark side of xanthophyll de‐epoxidation, and point out areas for future work. [ABSTRACT FROM AUTHOR]

Background: Nannochloropsis is a marine microalga that has been extensively studied. The major carotenoid produced by this group of microalgae is violaxanthin, which exhibits anti-inflammatory, anti-photoaging, and antiproliferative activities. Therefore, it has a wide range of potential applications. However, large-scale production of this pigment has not been much studied, thereby limiting its industrial application. Results: To develop a novel strain producing high amount of violaxanthin, various Nannochloropsis species were isolated from seawater samples and their violaxanthin production potential were compared. Of the strains tested, N. oceanica WS-1 exhibited the highest violaxanthin productivity; to further enhance the violaxanthin yield of WS-1, we performed gamma-ray-mediated random mutagenesis followed by colorimetric screening. As a result, Mutant M1 was selected because of its significant higher violaxanthin content and biomass productivity than WS-1 (5.21 ± 0.33 mg g− 1 and 0.2101 g L− 1 d− 1, respectively). Subsequently, we employed a 10 L-scale bioreactor to confirm the large-scale production potential of M1, and the results indicated a 43.54 % increase in violaxanthin production compared with WS-1. In addition, comparative transcriptomic analysis performed under normal light condition identified possible mechanisms associated with remediating photo-inhibitory damage and other key responses in M1, which seemed to at least partially explain enhanced violaxanthin content and delayed growth. Conclusions: Nannochloropsis oceanica mutant (M1) with enhanced violaxanthin content was developed and its physiological characteristics were investigated. In addition, enhanced production of violaxanthin was demonstrated in the large-scale cultivation. Key transcriptomic responses that are seemingly associated with different physiological responses of M1 were elucidated under normal light condition, the details of which would guide ongoing efforts to further maximize the industrial potential of violaxanthin producing strains. [ABSTRACT FROM AUTHOR]

Pigments are a fundamental part of the microalgal cell, with chlorophylls at the center of photosynthesis and carotenoids as accessory pigments. The characteristic pigment profile of the Eustigmatophyceae was one of the evidences which contributed to the segregation of this class of organisms from the Xanthophyceae. Recent findings indicate that eustigmatophyte carotenoids are interesting compounds with laboratory-scale proven health-promoting effects but only a few studies dedicate to the characterization of the pigment profile of the class. In the present work, extracts of 27 eustigmatophyte strains held at the Coimbra Collection of Algae (ACOI) were prepared and analyzed by HPLC-DAD. Results showed a typical eustigmatophyte pigment profile present in all strains, namely, chlorophyll a, violaxanthin, vaucheriaxanthin diester, β,β-carotene, and other minor carotenoids. Violaxanthin was the most abundant carotenoid, achieving nearly half the total pigment content in Monodopsis unipapilla ACOI 2938, and also in two studied Vischeria strains, representing ca. 70% of carotenoids in both strains. The second major carotenoid was β,β-carotene, with the highest production of this commercially important carotenoid detected in Pseudostaurastrum sp. ACOI 3413 (24% of total pigments. Characiopsis cf. saccata ACOI 481 and Characiopsis minuta ACOI 2423 had the highest amount of vaucheriaxanthin diester (19% of total pigments). To our best knowledge, this represents the first report where the pigment profile of eustigmatophytes with stipitate Characiopsis-like morphology is characterized. The results reveal the high potential use of eustigmatophytes as sources of naturally derived carotenoids. [ABSTRACT FROM AUTHOR]

The role of non-bilayer lipids and non-lamellar lipid phases in biological membranes is an enigmatic problem of membrane biology. Non-bilayer lipids are present in large amounts in all membranes; in energy-converting membranes they constitute about half of their total lipid content—yet their functional state is a bilayer. In vitro experiments revealed that the functioning of the water-soluble violaxanthin de-epoxidase (VDE) enzyme of plant thylakoids requires the presence of a non-bilayer lipid phase. 31P-NMR spectroscopy has provided evidence on lipid polymorphism in functional thylakoid membranes. Here we reveal reversible pH- and temperature-dependent changes of the lipid-phase behaviour, particularly the flexibility of isotropic non-lamellar phases, of isolated spinach thylakoids. These reorganizations are accompanied by changes in the permeability and thermodynamic parameters of the membranes and appear to control the activity of VDE and the photoprotective mechanism of non-photochemical quenching of chlorophyll-a fluorescence. The data demonstrate, for the first time in native membranes, the modulation of the activity of a water-soluble enzyme by a non-bilayer lipid phase. [ABSTRACT FROM AUTHOR]

Microbial production of carotenoids has mainly focused towards a few products, such as β-carotene, lycopene and astaxanthin. However, other less explored carotenoids, like violaxanthin, have also shown unique properties and promissory applications. Violaxanthin is a plant-derived epoxidated carotenoid with strong antioxidant activity and a key precursor of valuable compounds, such as fucoxanthin and β-damascenone. In this study, we report for the first time the heterologous production of epoxycarotenoids in yeast. We engineered the yeast Saccharomyces cerevisiae following multi-level strategies for the efficient accumulation of violaxanthin. Starting from a β-carotenogenic yeast strain, we first evaluated the performance of several β-carotene hydroxylases (CrtZ), and zeaxanthin epoxidases (ZEP) from different species, together with their respective N-terminal truncated variants. The combined expression of CrtZ from Pantoea ananatis and truncated ZEP of Haematococcus lacustris showed the best performance and led to a yield of 1.6 mg/g DCW of violaxanthin. Further improvement of the epoxidase activity was achieved by promoting the transfer of reducing equivalents to ZEP by expressing several redox partner systems. The co-expression of the plant truncated ferredoxin-3, and truncated root ferredoxin oxidoreductase-1 resulted in a 2.2-fold increase in violaxanthin yield (3.2 mg/g DCW). Finally, increasing gene copy number of carotenogenic genes enabled reaching a final production of 7.3 mg/g DCW in shake flask cultures and batch bioreactors, which is the highest yield of microbially produced violaxanthin reported to date. • First report on the engineering of Saccharomyces cerevisiae for the efficient production of violaxanthin. • Optimal violaxanthin pathway was constructed after screening several CrtZ and ZEP genes. • The impact of the extent of gene truncation on the catalytic activity of the translated enzymes was elucidated. • The data suggest that ZEP requires membrane association and accessory redox partners to efficiently epoxidate zeaxanthin. • The highest producing strain required the expression of 10 heterologous genes to reach a yield of 7.3 mg/g DCW of violaxanthin. [ABSTRACT FROM AUTHOR]

The role of α-carbonic anhydrase 4 (α-CA4) in photosynthetic machinery functioning in thylakoid membranes was studied, using Arabidopsis thaliana wild type plants (WT) and the plants with knockout of At4g20990 gene encoding α-CA4 (αCA4-mut) grown both in low light (LL, 80 μmol quanta m−2 s−1) or in high light (HL, 400 μmol quanta m−2 s−1). It was found that a content of PsbS protein, one of determinants of non-photochemical quenching of chlorophyll fluorescence, increased in mutants by 30% and 100% compared with WT plants in LL and in HL, respectively. Violaxanthin cycle pigments content and violaxanthin deepoxidase activity in HL were also higher in αCA4-mut than in WT plants. The content of PSII core protein, D1, when adapting to HL, decreased in WT plants and remained unchanged in mutants. This indicates, that the decrease in the content of Lhcb1 and Lhcb2 proteins in HL (Rudenko et al. Protoplasma 55(1):69-78, 2018) in WT plants resulted from decrease of both Photosystem II (PSII) complex content and content of these proteins in this complex, whereas in αCA4-mut plants from the latter process only. The absence of α-CA4 did not affect the rate of electron transport through Photosystem I (PSI) in thylakoids of mutant vs. WT, but led to 50–80% increase in the rate of electron transport from H2O to QA, evidencing the location of α-CA4 close to PSII. The latter difference may raise the question about its causal connection with the difference in the D1 protein content change during adapting to increased illumination in the presence and the absence of α-CA4. [ABSTRACT FROM AUTHOR]

Carotenoids are naturally synthesized in some species of bacteria, archaea, and fungi (including yeasts) as well as all photosynthetic organisms. Escherichia coli has been the most popular bacterial host for the heterologous production of a variety of carotenoids, including even xanthophylls unique to photosynthetic eukaryotes such as lutein, antheraxanthin, and violaxanthin. However, conversion efficiency of these epoxy-xanthophylls (antheraxanthin and violaxanthin) from zeaxanthin remained substantially low. We here examined several factors affecting their productivity in E. coli. Two sorts of plasmids were introduced into the bacterial host, i.e., a plasmid to produce zeaxanthin due to the presence of the Pantoea ananatis crtE, crtB, crtI, crtY, and crtZ genes in addition to the Haematococcus pluvialis IDI gene, and one containing each of zeaxanthin epoxidase (ZEP) genes originated from nine photosynthetic eukaryotes. It was consequently found that paprika (Capsicum annuum) ZEP (CaZEP) showed the highest conversion activity. Next, using the CaZEP gene, we performed optimization experiments in relation to E. coli strains as the production hosts, expression vectors, and ribosome-binding site (RBS) sequences. As a result, the highest productivity of violaxanthin (231 μg/g dry weight) was observed, when the pUC18 vector was used with CaZEP preceded by a RBS sequence of score 5000 in strain JM101(DE3). [ABSTRACT FROM AUTHOR]

Magnetic fields in biological systems is a promising research field; however, their application for microalgae has not been fully exploited. This work aims to measure the enzymatic activity and non-enzymatic activity of two microalgae species in terms of superoxide dismutase (SOD), catalase (CAT), and carotenoids, respectively, in response to static magnetic fields-induced stress. Two magnet configurations (north and south) and two exposure modes (continuous and pulse) were applied. Two microalgae species were considered, the Scenedesmus obliquus and Nannochloropsis gaditana. The SOD activity increased by up to 60% in S. obliquus under continuous exposure. This trend was also found for CAT in the continuous mode. Conversely, under the pulse mode, its response was hampered as the SOD and CAT were reduced. For N. gaditana, SOD increased by up to 62% with the south configuration under continuous exposure. In terms of CAT, there was a higher activity of up to 19%. Under the pulsed exposure, SOD activity was up to 115%. The CAT in this microalga was increased by up to 29%. For N. gaditana, a significant increase of over 40% in violaxanthin production was obtained compared to the control, when the microalgae were exposed to SMF as a pulse. Depending on the exposure mode and species, this methodology can be used to produce oxidative stress and obtain an inhibitory or enhanced response in addition to the significant increase in the production of antioxidant pigments.

Survival of phototrophic organisms depends on their ability to collect and convert enough light energy to support their metabolism. Phototrophs can extend their absorption cross section by using diverse pigments and by tuning the properties of these pigments via pigment–pigment and pigment–protein interaction. It is well known that some cyanobacteria can grow in heavily shaded habitats by utilizing far-red light harvested with far-red-absorbing chlorophylls d and f. We describe a red-shifted light-harvesting system based on chlorophyll a from a freshwater eustigmatophyte alga Trachydiscus minutus (Eustigmatophyceae, Goniochloridales). A comprehensive characterization of the photosynthetic apparatus of T. minutus is presented. We show that thylakoid membranes of T. minutus contain light-harvesting complexes of several sizes differing in the relative amount of far-red chlorophyll a forms absorbing around 700 nm. The pigment arrangement of the major red-shifted light-harvesting complex is similar to that of the red-shifted antenna of a marine alveolate alga Chromera velia. Evolutionary aspects of the algal far-red light-harvesting complexes are discussed. The presence of these antennas in eustigmatophyte algae opens up new ways to modify organisms of this promising group for effective use of far-red light in mass cultures. [ABSTRACT FROM AUTHOR]

This work highlights spectroscopic investigations on a new representative of photosynthetic antenna complexes in the LHC family, a putative violaxanthin/vaucheriaxanthin chlorophyll a (VCP) antenna complex from a freshwater Eustigmatophyte alga FP5. A representative VCP-like complex, named as VCP-B3 was studied with both static and time-resolved spectroscopies with the aim of obtaining a deeper understanding of excitation energy migration within the pigment array of the complex. Compared to other VCP representatives, the absorption spectrum of the VCP-B3 is strongly altered in the range of the chlorophyll a Qy band, and is substantially red-shifted with the longest wavelength absorption band at 707 nm at 77 K. VCP-B3 shows a moderate xanthophyll-to-chlorophyll a efficiency of excitation energy transfer in the 50–60% range, 20–30% lower from comparable VCP complexes from other organisms. Transient absorption studies accompanied by detailed data fitting and simulations support the idea that the xanthophylls that occupy the central part of the complex, complementary to luteins in the LHCII, are violaxanthins. Target analysis suggests that the primary route of xanthophyll-to-chlorophyll a energy transfer occurs via the xanthophyll S1 state. [ABSTRACT FROM AUTHOR]

Abstract Photosynthetic eukaryotes whose cells harbor plastids originating from secondary endosymbiosis of a red alga include species of major ecological and economic importance. Since utilization of solar energy relies on the efficient light-harvesting, one of the critical factors for the success of the red lineage in a range of environments is to be found in the adaptability of the light-harvesting machinery, formed by the proteins of the light-harvesting complex (LHC) family. A number of species are known to employ mainly a unique class of LHC containing red-shifted chlorophyll a (Chl a) forms absorbing above 690 nm. This appears to be an adaptation to shaded habitats. Here we present a detailed investigation of excitation energy flow in the red-shifted light-harvesting antenna of eustigmatophyte Trachydiscus minutus using time-resolved fluorescence and ultrafast transient absorption measurements. The main carotenoid in the complex is violaxanthin, hence this LHC is labeled the red-violaxanthin-Chl a protein, rVCP. Both the carotenoid-to-Chl a energy transfer and excitation dynamics within the Chl a manifold were studied and compared to the related antenna complex, VCP, that lacks the red-Chl a. Two spectrally defined carotenoid pools were identified in the red antenna, contributing to energy transfer to Chl a, mostly via S 2 and hot S 1 states. Also, Chl a triplet quenching by carotenoids is documented. Two separate pools of red-shifted Chl a were resolved, one is likely formed by excitonically coupled Chl a molecules. The structural implications of these observations are discussed. Highlights • Algal antenna with absorption >700 nm, binds Chl a, violaxanthin, vaucheriaxanthin. • Two pools of carotenoids, both transfer energy efficiently to red chlorophylls. • There are two pools of red Chl a, only one accepts excitation from carotenoids. • The lowest-energy Chl a states are formed by excitonically coupled pigments. [ABSTRACT FROM AUTHOR]

[Display omitted] • Enrichment with Nannochloropsis shows oxidative instability. • Enrichment with Phaeodactylum shows oxidative stability. • Impact of carotenoid content and type rather than presence only. • N–3 LC–PUFA analysis is a useful addition for detecting lipid oxidation. Microalgae rich in omega-3 long-chain polyunsaturated fatty acids (n–3 LC–PUFA) have already shown their potential for developing functional food rich in these healthy fatty acids. Not only could they offer a more sustainable alternative for the fish stock that is currently relied upon but is unable to keep up with the demand, enrichment with certain microalgae also leads to oxidatively stable products. Although the reason for this stability has been attributed to the presence of endogenous carotenoids, further insight into their antioxidative role is missing and would be clarifying for selecting the proper microalgae for food enrichment. In trying to further accomplish this, a storage experiment (4 weeks, 37 °C) was set up with the parallel analysis of both oxidation products (primary and secondary) and carotenoids of two aqueous model systems of different (promising) microalgae (Nannochloropsis and Phaeodactylum). The results showed a clear difference in oxidative stability despite both microalgae containing endogenous carotenoids: Nannochloropsis led to oxidatively unstable and Phaeodactylum to oxidatively stable products. This was clearly confirmed by the analysis of n–3 LC–PUFA throughout storage which showed a breakdown of half of the n–3 LC–PUFA for Nannochloropsis. All carotenoids (violaxanthin, zeaxanthin and β-carotene for Nannochloropsis , and fucoxanthin and β-carotene for Phaeodactylum) acted as an antioxidant as shown by their degradation throughout storage, but the difference in oxidative stability pointed out an impact of carotenoid content and (possibly) type. The presence of a sufficient amount of carotenoids seems to be an important factor for perceiving oxidative stability. Phaeodactylum has shown to be more potent for food enrichment. [ABSTRACT FROM AUTHOR]

The foremost problem in carotenoid research is the excessive cost and difficulty of maintaining pure carotenoid compounds. This work presents an economical, efficient, and simplified one-step scheme for the purification of four major xanthophyll carotenoids from lettuce by utilizing preparative thin layer chromatography on Hyflo-Super-Cel: MgO (Heavy): calcium sulfate hemihydrate (9:9:2 w/w) based adsorbent. The mobile phase of acetone: hexane (1:1) provided the perfect separation of major xanthophylls, resulting in 95–96% purity after just single-step separation, with no interference from chlorophylls or other minor carotenoids. The identity of carotenoids was confirmed by absorption spectroscopy, chemical tests and APCI + –MS/MS. The proposed scheme can be used to isolate the carotenoids at the analytical and preparative scale. In anticancer studies, among four xanthophylls, 9- Z -neoxanthin was found most potent for reduction of cell viability of cervical (HeLa) and lung cancer (A549) cells, with IC 50 values of 3.8 and 7.5 μM, respectively. [ABSTRACT FROM AUTHOR]

Plastidial isoprenoids, such as carotenoids and tocopherols, are important anti-oxidant metabolites synthesized in plastids from precursors generated by the methylerythritol 4-phosphate (MEP) pathway. In this study, we found that irradiation of Arabidopsis thaliana plants with UV-B caused a strong increase in the accumulation of the photoprotective xanthophyll zeaxanthin but also resulted in slightly higher levels of γ-tocopherol. Plants deficient in the MEP enzymes 1-deoxy-D-xylulose 5-phosphate synthase and 1-hydroxy-2-methyl-2-butenyl 4-diphosphate synthase showed a general reduction in both carotenoids and tocopherols and this was associated with increased DNA damage and decreased photosynthesis after exposure to UV-B. Genetic blockage of tocopherol biosynthesis did not affect DNA damage accumulation. In contrast, lut2 mutants that accumulate β,β-xanthophylls showed decreased DNA damage when irradiated with UV-B. Analysis of aba2 mutants showed that UV-B protection was not mediated by ABA (a hormone derived from β,β-xanthophylls). Plants accumulating β,β-xanthophylls also showed decreased oxidative damage and increased expression of DNA-repair enzymes, suggesting that this may be a mechanism for these plants to decrease DNA damage. In addition, in vitro experiments also provided evidence that β,β-xanthophylls can directly protect against DNA damage by absorbing radiation. Together, our results suggest that xanthophyll-cycle carotenoids that protect against excess illumination may also contribute to protection against UV-B. [ABSTRACT FROM AUTHOR]

Photoprotective non-photochemical quenching (NPQ) represents an effective way to dissipate the light energy absorbed in excess by most phototrophs. It is often claimed that NPQ formation/relaxation kinetics are determined by xanthophyll composition. We, however, found that, for the alveolate alga Chromera velia, this is not the case. In the present paper, we investigated the reasons for the constitutive high rate of quenching displayed by the alga by comparing its light harvesting strategies with those of a model phototroph, the land plant Spinacia oleracea. Experimental results and in silico studies support the idea that fast quenching is due not to xanthophylls, but to intrinsic properties of the Chromera light harvesting complex (CLH) protein, related to amino acid composition and protein folding. The p K a for CLH quenching was shifted by 0.5 units to a higher pH compared with higher plant antennas (light harvesting complex II; LHCII). We conclude that, whilst higher plant LHCIIs are better suited for light harvesting, CLHs are ‘natural quenchers’ ready to switch into a dissipative state. We propose that organisms with antenna proteins intrinsically more sensitive to protons, such as C. velia, carry a relatively high concentration of violaxanthin to improve their light harvesting. In contrast, higher plants need less violaxanthin per chlorophyll because LHCII proteins are more efficient light harvesters and instead require co-factors such as zeaxanthin and PsbS to accelerate and enhance quenching. [ABSTRACT FROM AUTHOR]

Persea Americana Mill. or Avocado is a tropical native American fruit. It belongs to the Lauraceae family. The name 'Avocado' has been derived from the Aztec word 'ahucatl'. 'Alligator pear' and 'butter fruit' are its' alternative names. It has been traditionally cultivated for food and medicinal purposes due to its high nutrition content as well as for its therapeutic properties. The predominant carotenoid in Avocado is Lutein. α-carotene, β-carotene, zeaxanthin, neoxanthin and violaxanthin are the other carotenoids present in small quantities in it. Avocado plant and their seed, fruit, pulp, avocado oil part use for various type of activity as cosmetic, refined cooking oil, weight management program, heart disease, stroke and cancer. [ABSTRACT FROM AUTHOR]

Abstract: Nannochloropsis oculata (Eustigmatophyceae) is a marine microalga of great biotechnological interest, mainly due to its large production of lipids containing polyunsaturated fatty acids. In addition, this species presents a wide range of commercial interest pigments, such as zeaxanthin, beta‐carotene, and other xanthophylls, with potential for several industrial applications. However, most of the research concerning pigment production by N. oculata has been conducted by employing high‐cost laboratorial growth media, which makes large‐scale pigment production using these microalgae impractical. Considering the high interest and commercial value in microalgae pigments, this study investigates the feasibility of producing pigments by N. oculata using five different low‐cost growth media (fertilizers and aquaculture effluents). Nutrient (ammonia, nitrite and phosphate) concentrations, cell abundance, biomass, and the concentration/composition of pigments were measured. The pigment profile of N. oculata showed chlorophyll‐a as the dominant pigment, along with violaxanthin, vaucheraxanthin, and lower concentrations of antheraxanthin, zeaxanthin and beta‐carotene. Although the highest biomass (516.4 ± 76.71 mg/L) and pigment content (0.98 mg/g) were achieved in the laboratory media (f/2), the low‐cost media (containing ammonium sulfate, calcium superphosphate and urea) revealed a great potential for the production of pigments, specially chlorophyll‐a, violaxanthin and zeaxanthin, due to the high pigment content per unit of biomass. [ABSTRACT FROM AUTHOR]

In the 20th century, annual mean temperatures in the European Alps rose by almost 1 K and are predicted to rise further, increasing the impact of temperature on alpine plants. The role of light in the heat hardening of plants is still not fully understood. Here, the alpine dwarf shrub Vaccinium gaultherioides was exposed in situ to controlled short‐term heat spells (150 min with leaf temperatures 43–49°C) and long‐term heat waves (7 days, 30°C) under different irradiation intensities. Lethal leaf temperatures (LT50) were calculated. Low solar irradiation [max. 250 photosynthetic photon flux density (PPFD)] during short‐term heat treatments mitigated the heat stress, shown by reduced leaf tissue damage and higher Fv/Fm (potential quantum efficiency of photosystem 2) than in darkness. The increase in xanthophyll cycle activity and ascorbate concentration was more pronounced under low light, and free radical scavenging activity increased independent of light conditions. During long‐term heat wave exposure, heat tolerance increased from 3.7 to 6.5°C with decreasing mean solar irradiation intensity (585–115 PPFD). Long‐term exposure to heat under low light enhanced heat hardening and increased photosynthetic pigment, dehydroascorbate and violaxanthin concentration. In conclusion, V. gaultherioides is able to withstand temperatures of around 50°C, and its heat hardening can be enhanced by low light during both short‐ and long‐term heat treatment. Data showing the specific role of light during short‐ and long‐term heat exposure and the potential risk of lethal damage in alpine shrubs as a result of rising temperature are discussed. [ABSTRACT FROM AUTHOR]

Asparagus represents a nutritious and refined food being very popular to consumers. The effects on the quality of spears cooked by conventional (boiling, steaming and microwaving) and different sous vide (using hot water or microwave) methods were analyzed. Physical, chemical and sensory traits were compared. Microwaving caused the largest weight change, the highest dry weight increase and the highest Total Color Difference in cooked samples compared to raw asparagus spears. Sous vide -microwaving showed an increase in greenness (the highest value of h°), better than the raw spears, and the lowest reduction in chlorophyll contents. After sous vide microwawing violaxanthin increased by about 42%, while after steaming neoaxanthin decreased by about 57%. Following sensory analysis, all cooked samples generally resulted acceptable (scores > 5 in a 1–9 hedonic scale), nevertheless sous vide -microwaved asparagus satisfied consumer acceptability more than the other cooked samples, especially when compared to steamed samples. Results indicate the sous vide -microwaving technique as optimal to preserve several traits, including the organoleptic ones, essential for the quality of cook-chilled asparagus spears. They also provide product-specific information usually required for cooking process strategies in the industrial area of ready-to-eat vegetables. [ABSTRACT FROM AUTHOR]

Non-photochemical quenching (NPQ) is the most important photoprotective system in higher plants. NPQ can be divided into several steps according to the timescale of relaxation of chlorophyll fluorescence after reaching a steady state (i.e., the fast phase, qE; middle phase, qZ or qT; and slow phase, qI). The dissipation of excess energy as heat during the xanthophyll cycle, a large component of NPQ, is detectable during the fast to middle phase (sec to min). Although thermal dissipation is primarily investigated using indirect methods such as chlorophyll a fluorescence measurements, such analyses require dark adaptation or the application of a saturating pulse during measurement, making it difficult to continuously monitor this process. Here, we designed an unconventional technique for real-time monitoring of changes in thylakoid lumen pH (as reflected by changes in xanthophyll pigment content) based on the photochemical reflectance index (PRI), which we estimated by measuring light-driven leaf reflectance at 531 nm. We analyzed two Arabidopsis thaliana mutants, npq1 (unable to convert violaxanthin to zeaxanthin due to inhibited violaxanthin de-epoxidase [VDE] activity) and npq4 (lacking PsbS protein), to uncover the regulator of the PRI. The PRI was variable in wild-type and npq4 plants, but not in npq1 , indicating that the PRI is related to xanthophyll cycle-dependent thermal energy quenching (qZ) rather than the linear electron transport rate or NPQ. In situ lumen pH substitution using a pH-controlled buffer solution caused a shift in PRI. These results suggest that the PRI reflects only xanthophyll cycle conversion and is therefore a useful parameter for monitoring thylakoid lumen pH (reflecting VDE activity) in vivo . [ABSTRACT FROM AUTHOR]

Main conclusion: Up to 40% of incident light was screened in red Berberis leaves in vivo by anthocyanins, resulting also in up to 40% reduction of light-limited photosynthesis. The biological function of anthocyanins in leaves has been strongly discussed, but the hypothesis of a screening function is favored by most authors. For an evaluation of the function as photoprotective pigments, a quantification of their screening of the mesophyll is important. Here, chlorophyll fluorescence excitation of leaves of a red and a green variety of Berberis thunbergii was used to estimate the extent of screening by anthocyanins at 545 nm and over the whole photosynthetically active wavelength range. Growth at high light (430 µmol m s) resulted in 90% screening at 545 nm corresponding to 40-50% screening over the whole wavelength range, depending on the light source. The concomitant reduction of photosynthetic quantum yield was of the same size as the calculated reduction of light reaching the chloroplasts. The induction of anthocyanins in the red variety also enhanced the epoxidation state of the violaxanthin cycle under growth conditions, indicating that red leaves were suffering less from excessive irradiance. Pool sizes of violaxanthin cycle carotenoids indicated a shade acclimation of the light harvesting complexes in red leaves. The observed reduction of internal light in anthocyanic leaves has by necessity a photoprotective effect. [ABSTRACT FROM AUTHOR]

Understanding the composition and content of carotenoids in red-flesh navel orange “Cara Cara” cultivated in different regions is valuable for its nutrition assessment and fruit processing control. Herein, the carotenoids of “Cara Cara” fruits, harvested from five typical citrus growing regions of China, were identified and quantified by HPLC-DAD coupled with HPLC-DAD-APCI-MS/MS. In saponified extracts, 19 free carotenoids including 8 carotenes and 11 xanthophylls were identified. In unsaponified extracts, 83.83–93.83% xanthophylls were esterified and 19 esters were inferred, with the 9- cis -violaxanthin ester confirmed as the dominant component. The carotenoid contents showed difference among the “Cara Cara” fruits from different growing regions, which might be attributed to the local environmental conditions, and the temperature was found directly correlated with total carotenoids content of “Cara Cara”. The data obtained in this study will facilitate the nutritional evaluation of “Cara Cara” and provide beneficial guidance for fruit processing. [ABSTRACT FROM AUTHOR]

Although sunlight provides the energy necessary for plants to survive and grow, light can also damage reaction centers of photosystem II (PSII) and reduce photochemical efficiency. To prevent damage, plants possess photoprotective mechanisms that dissipate excess excitation. A subset of these mechanisms is collectively referred to as NPQ, or nonphotochemical quenching of chlorophyll a fluorescence. The regulation of NPQ is intrinsically linked to the cycling of xanthophylls that affects the kinetics and extent of the photoprotective response. The violaxanthin cycle (VAZ cycle) and the lutein epoxide cycle (LxL cycle) are two xanthophyll cycles found in vascular plants. The VAZ cycle has been studied extensively, owing in large part to its presence in model plant species where mutants are available to aid in its characterization. In contrast, the LxL cycle is not found in model plants, and its role in photosynthetic processes has been more difficult to define. To address this challenge, we introduced the LxL cycle into Arabidopsis thaliana and functionally isolated it from the VAZ cycle. Using these plant lines, we showed an increase in dark-acclimated PSII efficiency associated with Lx accumulation and demonstrated that violaxanthin deepoxidase is responsible for the light-driven deepoxidation of Lx. Conversion of Lx to L was reversible during periods of low light and occurred considerably faster than rates previously described in nonmodel species. Finally, we present clear evidence of the LxL cycle’s role in modulating a rapid component of NPQ that is necessary to prevent photoinhibition in excess light. [ABSTRACT FROM AUTHOR]

Mustard, beet and parsley were grown to harvest time under selected LEDs: 638 + 660 + 731 + 0% 445 nm; 638 + 660 + 731 + 8% 445 nm; 638 + 660 + 731 + 16% 445 nm; 638 + 660 + 731 + 25% 445 nm; 638 + 660 + 731 + 33% 445 nm. From 1.2 to 4.3 times higher concentrations of chlorophylls a and b , carotenoids, α- and β-carotenes, lutein, violaxanthin and zeaxanthin was found under blue 33% treatment in comparison to lower blue light dosages. Meanwhile, the accumulation of metabolites, which were not directly connected with light reactions, such as tocopherols, was more influenced by lower (16%) blue light dosage, increasing about 1.3 times. Thus, microgreen enrichment of carotenoid and xanthophyll pigments may be achieved using higher (16–33%) blue light intensities. Changes in metabolite quantities were not the result of changes of other carotenoid concentration, but were more influenced by light treatment and depended on the species. Significant quantitative changes in response to blue light percentage were obtained for both directly and not directly light-dependent metabolite groups. [ABSTRACT FROM AUTHOR]

Measuring the kinetics of the violaxanthin cycle imposes an experimental challenge. Traditionally, carotenoid analysis was carried out laboriously with high-performance liquid chromatography. In this work, we present the first in vivo approach to directly measure the kinetics of the violaxanthin cycle, using resonance Raman spectroscopy in combination with baseline correction and principal component analysis. Applying the new approach allows measuring thousands of data points as opposed to the few possible with chemical analysis over the course of a violaxanthin cycle kinetics experiment. In vivo analysis of the violaxanthin cycle is necessary to fully understand adaptation kinetics to varying light conditions, the knowledge of which is especially important for assessing the stress tolerance of plants in the wake of the increasing climate change. Three experiments on the green alga Dunaliella salina were performed, featuring both the light-to-dark and dark-to-light transitions response of the algae. Copyright © 2017 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

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

Citrus fruit are an important reservoir of carotenoids. Numerous studies have been carried out to identify and profile the members of gene families involved in carotenoid biosynthetic pathway to explain the diversity of coloration in citrus fruit. It was found that gene expression analysis could not always explain the changes in carotenoid content and composition, indicating that other unknown genes and mechanisms should be operative. This review summarizes and updates the current knowledge on gene families involved in the citrus carotenoid biosynthetic pathway and their roles on the regulation of carotenoid biosynthesis, as well as provides insightful questions leading to future experimentation. [ABSTRACT FROM AUTHOR]

Dunaliella tertiolecta (DT) was chemically investigated to isolate molecules inhibiting cancer cell proliferation and inducing apoptosis in vitro. The potency to inhibit cell growth was used for the bio-guided fractionation and isolation of active compounds using chromatographic techniques. The DT dichloromethane extract exhibited a strong anti-proliferative activity on MCF-7 and LNCaP cells, and was further fractionated and sub-fractionated by RP-HPLC. High resolution mass spectrometry and spectrophotometric analysis unequivocally identified violaxanthin as the most antiproliferative molecule present in DT DCM extract. Violaxanthin purified from DT induced MCF-7 dose-dependent growth inhibition in continuous and discontinuous treatments, at concentrations as low as 0.1 µg·mL−1 (0.17 µM). Phosphatidylserine exposure, typical of early apoptosis, was observed after 48 h treatment at 8 µg·mL−1 (13.3 µM) but no DNA fragmentation, characteristic of late apoptosis steps, could be detected even after 72 h treatment at 40 µg·mL−1 (66.7 µM). Taken together, our results demonstrate the strong antiproliferative activity of violaxanthin on one human mammary cancer cell line, and suggest that studying the pharmacology of violaxanthin and pharmacomodulated derivatives on cancer cells may allow potent antiproliferative drugs to be obtained.

Violaxanthin is a major xanthophyll pigment in the microalga Eustigmatos cf. polyphem, but the amount produced after propagation can vary depending upon culture conditions. In this study, the effects of cultivation time, nitrogen concentration, light intensity, and culture mode on violaxanthin production were investigated. The results showed that this microalga vigorously grew and maintained a high level of violaxanthin in the fed-batch culture, and the highest violaxanthin productivity of 1.10 ± 0.03 mg L−1 d−1 was obtained under low light illumination with 18 mM of initial nitrogen supply for ten days. Additionally, violaxanthin was purified from E. cf. polyphem by silica gel chromatography and preparative high-performance liquid chromatography (PHPLC), and identified with high-resolution mass spectrometry (HRMS). The antioxidant activity of the purified violaxanthin was evaluated by three tests in vitro: reducing power assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2-azobis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical assays. The strongest inhibition of purified violaxanthin occurred during the scavenging of ABTS+ radicals, with EC50 of 15.25 μg mL−1. In conclusion, this is the first report to investigate the effects of different culture conditions on violaxanthin accumulation in E. cf. polyphem and provide a novel source for the production of violaxanthin that can be used for food and pharmaceutical applications.

• Carotenoid composition varies among different citrus varieties. • Carotenoid accumulation is highly regulated at the transcription level. • Carotenoid accumulation is affected by plant growth regulators. Citrus juice sacs, the major edible part of fruit, are one of the richest sources of carotenoids for human nutrition. In the juice sacs, high amounts of carotenoids are accumulated, and their contents and compositions vary greatly among different citrus varieties. In the past decades, the carotenoid in citrus fruit drew a lot of researcher's attention, and significant progress has been made in the elucidation of carotenoid accumulation in the juice sacs of citrus fruit. In this review, the current information on carotenoid accumulation in the juice sacs of citrus fruit was summarized, and the molecular mechanisms that regulated the carotenoid biosynthesis were reviewed. In addition, to improve the carotenoid accumulation in the juice sacs, the effects of different plant growth regulators and environmental conditions on carotenoid content and composition were evaluated. This review contributes to a better comprehension of carotenoid accumulation in the juice sacs of citrus fruit, which will provide new strategies to enhance the nutritional and commercial values of citrus fruit. [ABSTRACT FROM AUTHOR]

Resonance Raman spectroscopy was used to evaluate pigment structure in the FCP-like light-harvesting complex of Chromera velia ( Chromera light-harvesting complex or CLH). This antenna protein contains chlorophyll a , violaxanthin and a new isofucoxanthin-like carotenoid (called Ifx-l). We show that Ifx-l is present in two non-equivalent binding pockets with different conformations, having their (0,0) absorption maxima at 515 and 548 nm respectively. In this complex, only one violaxanthin population absorbing at 486 nm is observed. All the CLH-bound carotenoid molecules are in all-trans configuration, and among the two Ifx-l carotenoid molecules, the red one is twisted, as is the red-absorbing lutein in LHCII trimers. Analysis of the carbonyl stretching region for Chl a excitations indicates CLH binds up to seven Chl a molecules in five non-equivalent binding sites, in reasonable agreement with sequence analyses which have identified eight potential coordinating residues. The binding modes and conformations of CLH-bound pigments are discussed with respect to the known structures of LHCII and FCP. [ABSTRACT FROM AUTHOR]

Carotenoid contents of peel and pulp of local citrus fruits from Aceh were investigated using HPLC equipped with DAD detector and LC-MS. Most of the citrus samples investigated mainly contained lutein and zeaxanthin. Jeruk takengon ( Citrus nobilis) had a more complex carotenoid profile and the highest carotenoid contents compared to other citrus fruits studied here, and peel contained higher carotenoid contents than pulp. ( Z)-Violaxanthin and ( all- E)-violaxanthin were the main carotenoids of the peel, while ( all- E) β-cryptoxanthin and ( Z)-violaxanthin were the main carotenoids of the pulp of jeruk takengon. Determination of the lipophilic antioxidant capacity (L-TEAC) of the carotenoid extracts of jeruk takengon showed promising antioxidant potential. The peel extract showed stronger antioxidant capacity than the pulp extract, and saponification further improved the antioxidant capacity. Investigating the inhibition of α-amylase activity resulted in a moderate α-amylase inhibition by jeruk takengon due to the carotenoid contents and the contribution of some flavonoids. Pulp extracts exhibited better inhibition activity than peel extracts, and saponification led to a reduction of the α-amylase inhibition activity. Antibacterial activity tests showed that jeruk takengon could inhibit the growth of K. pneumonia and S. aureus, and saponification enhanced the antibacterial activities. In general, the peel extracts showed biological potential due to the relatively strong in vitro antioxidant, antibacterial and α-amylase inhibitory activity. In addition, these by-products of food processing may serve as potential carotenoid sources. [ABSTRACT FROM AUTHOR]

The photosynthetic responses to elevated CO concentration (EC) at ambient and ambient +4°C temperature were aßsessed in the second leaf of rice ( Oryza sativa L.) seedlings. The duration of different leaf developmental phases, as characterised by changes in photosynthetic pigment contents and photochemical potential, was protracted in the seedlings grown under EC. On the other hand, a temporal shift in the phases of development with an early onset of senescence was observed in the seedlings grown under EC at ambient +4°C temperature. The contents of carotenoids, ß-carotene, and xanthophyll cycle pigments revealed that EC downregulated the protective mechanism of photosynthetic apparatus against oxidative damages, whereas this mechanism assumed higher significance under EC at ambient +4°C temperature. We observed an enhancement in electron transport activity, photochemical potential, and net photosynthesis in spite of a loss in photostasis of photosynthesis under EC. On the other hand, the loss in photostasis of photosynthesis was exacerbated under EC at ambient +4°C temperature due to the decline in electron transport activity, photochemical potential, and net photosynthesis. [ABSTRACT FROM AUTHOR]

Dunaliella salina is a halotolerant marine microalga, known for the accumulation of β-carotene. The BCH-type β-carotene hydroxylase plays a major role in the conversion of β-carotene to xanthophylls. The β-carotene hydroxylase from the chlorophycean microalga, Chlamydomonas reinhardtii was amplified, and the sequence alignment analysis showed that the amplified gene belongs to the group of BCH-type hydroxylases. The amplified β-carotene hydroxylase gene with rubisco promoter and chloroplast transit peptide was introduced into the genome of the D. salina by Agrobacterium -mediated genetic transformation. The carotenoid analysis showed a 3-fold increase of violaxanthin at growth stage and a 2-fold increase of zeaxanthin under stress conditions in transformed D. salina . [ABSTRACT FROM AUTHOR]

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

We studied seasonal changes in the content and ratio between photosynthetic pigments in one-yearold needles of Scotch pine ( Pinus sylvestris L.) growing in Central Yakutia. Maximum accumulation of chlorophylls in developed young needles occurred in July when light and temperature conditions were favorable. In this period, the needles were notable for a relatively high level of β-carotene and neoxanthin and a reduced content of lutein and the pigments of violaxanthin cycle (VXC). In the course of autumn hardening, the content of chlorophylls decreased two times. Total content of carotenoids remained the same, but pigment composition considerably changed when plants progressed from a vegetating to frost-resistant state. We revealed time and temperature ranges of variation for individual carotenoids. In the beginning of hardening at reduced and low abovezero temperatures, the content of β-carotene in the needles decreased, the pigment-protein complexes (PPC) became enriched in lutein, the pigment pool of VXC gradually increased, and the content of neoxanthin transiently rose. When average daily air temperature further decreased to near- zero values, the content of zeaxanthin sharply rose. In winter, high levels of lutein and zeaxanthin were maintained. Main changes in pigment complex of the needles of P. sylvestris were completed before the coming of steady below-zero temperatures. The obtained data suggested that, upon seasonal decrease in temperature in early stages of hardening, a decrease in the level of chlorophyll promotes a reduction in the quantity of absorbed radiant energy. Apparently, this is accompanied by activation of the role of lutein and neoxanthin that perform specific photoprotective functions in antenna PPC associated with a gradual decrease in plants' ability to quench singlet energy of excited chlorophyll. Accumulation of zeaxanthin as a result of inhibition of back reaction of epoxidation at near-zero temperatures creates necessary prerequisites for turning on the mechanisms of steady dissipation of absorbed light energy, which do not depend on transmembrane proton gradient of thylakoids. At the same time, zeaxanthin can perform antioxidant functions both in PPC and in the lipid phase of thylakoid membranes. The obtained data point to an adaptive nature of the observed reactions and a specific role of individual pigments in structural and functional reorganization of photosynthetic machinery in the course of development of frost-resistance in the needles. [ABSTRACT FROM AUTHOR]

Photoprotection mechanisms protect photosynthetic organisms, especially under stress conditions, against photodamage that may inhibit photosynthesis. We investigated the effects of short-term immersion in hypo- and hypersalinity sea water on the photosynthesis and xanthophyll cycle in Sargassum fusiforme (Harvey) Setchell. The results indicated that under moderate light [110 μmol(photon) m s], the effective quantum yield of PSII was not reduced in S. fusiforme fronds after 1 h in hyposalinity conditions, even in fresh water, but it was significantly affected by extreme hypersalinity treatment (90‰ sea water). Under high light [HL, 800 μmol(photon) m s], photoprotective mechanisms operated efficiently in fronds immersed in fresh water as indicated by high reversible nonphotochemical quenching of chlorophyll fluorescence (NPQ) and de-epoxidation state; the quantum yield of PSII recovered during the subsequent relaxation period. In contrast, fronds immersed in 90‰ sea water did not withstand HL, barely developed reversible NPQ, and accumulated little antheraxanthin and zeaxanthin during HL, while recovery of the quantum yield of PSII was severely inhibited during the subsequent relaxation period. The data provided concrete evidence supporting the short-term tolerance of S. fusiforme to immersion in fresh water compared to hypersalinity conditions. The potential practical implications of these results were also discussed. [ABSTRACT FROM AUTHOR]

Resonance Raman analysis of the photosynthetic complex LHCII, immobilized in a polyacrylamide gel, reveals that one of the protein-bound xanthophylls, assigned as violaxanthin, undergoes light-induced molecular reconfiguration. The phototransformation is selectively observed in a trimeric structure of the complex and is associated with a pronounced twisting and a trans-cis molecular configuration change of the polyene chain of the carotenoid. Among several spectral effects accompanying the reconfiguration there are ones indicating a carotenoid triplet state. Possible physiological importance of the light-induced violaxanthin reconfiguration as a mechanism associated with making the pigment available for enzymatic deepoxidation in the xanthophyll cycle is discussed. [ABSTRACT FROM AUTHOR]

The carotenoid composition of strawberry tree ( Arbutus unedo ) fruits has been characterised in detail and quantified for the first time. According to the total carotenoid content (over 340 μg/g dw), mature strawberry tree berries can be classified as fruits with very high carotenoid content (>20 μg/g dw). (all- E )-Violaxanthin and 9 Z -violaxanthin were found to be the major carotenoid pigments, accounting for more than 60%, responsible for the bright colour of the flesh of ripe fruits. In addition other 5,6-epoxide carotenoids, such as (all- E )-neoxanthin, (9′ Z )-neoxanthin (all- E )-antheraxanthin and lutein 5,6-epoxide, together with (all- E )-lutein, (all- E )-zeaxanthin and (all- E )-β-carotene were found at high levels (>5–20 μg/g dw). The LC–MS (APCI+) analysis of the xanthophyll fraction in their native state (direct extract) revealed that most of them (>90%) were totally esterified with saturated fatty acids (capric, lauric, myristic, palmitic and stearic). Monoesters, homodiesters and heterodiesters of (all- E )-violaxanthin and 9 Z -violaxanthin were the major pigments. [ABSTRACT FROM AUTHOR]

Non-photochemical quenching ( NPQ) is a photoprotective mechanism in light-harvesting antennae. NPQ is triggered by chloroplast thylakoid lumen acidification and is accompanied by violaxanthin de-epoxidation to zeaxanthin, which further stimulates NPQ. In the present study, we show that violaxanthin can act in the opposite direction to zeaxanthin because an increase in the concentration of violaxanthin reduced NPQ in the light-harvesting antennae of Chromera velia. The correlation overlapped with a similar relationship between violaxanthin and NPQ as observed in isolated higher plant light-harvesting complex II. The data suggest that violaxanthin in C. velia can act as an inhibitor of NPQ, indicating that violaxanthin has to be removed from the vicinity of the protein to reach maximal NPQ. [ABSTRACT FROM AUTHOR]

Violaxanthin–chlorophyll a protein (VCP) from Nannochloropsis oceanica is a Chl a -only member of the LHC family of light-harvesting proteins. VCP binds carotenoids violaxanthin (Vio), vaucheriaxanthin (Vau), and vaucheriaxanthin-ester (Vau-ester). Here we report on energy transfer pathways in the VCP complex. The overall carotenoid-to-Chl a energy transfer has efficiency over 90%. Based on their energy transfer properties, the carotenoids in VCP can be divided into two groups; blue carotenoids with the lowest energy absorption band around 480 nm and red carotenoids with absorption extended up to 530 nm. Both carotenoid groups transfer energy efficiently from their S 2 states, reaching efficiencies of ~ 70% (blue) and ~ 60% (red). The S 1 pathway, however, is efficient only for the red carotenoid pool for which two S 1 routes characterized by 0.33 and 2.4 ps time constants were identified. For the blue carotenoids the S 1 -mediated pathway is represented only by a minor route likely involving a hot S 1 state. The relaxed S 1 state of blue carotenoids decays to the ground state within 21 ps. Presence of a fraction of non-transferring red carotenoids with the S 1 lifetime of 13 ps indicates some specific carotenoid-protein interaction that must shorten the intrinsic S 1 lifetime of Vio and/or Vau whose S 1 lifetimes in methanol are 26 and 29 ps, respectively. The VCP complex from N. oceanica is the first example of a light-harvesting complex binding only non-carbonyl carotenoids with carotenoid-to-chlorophyll energy transfer efficiency over 90%. [ABSTRACT FROM AUTHOR]

Oncidium 'Gower Ramsey' ( Onc. GR) is a popular cut flower, but its colour is limited to bright yellow. The β-ring carotene hydroxylase ( BCH2) gene is involved in carotenoid biogenesis for pigment formation. However, the role of BCH2 in Onc. GR is poorly understood. Here, we investigated the functions of three BCH2 genes, BCH-A2, BCH-B2 and BCH-C2 isolated from Onc. GR, to analyse their roles in flower colour. RT- PCR expression profiling suggested that BCH2 was mainly expressed in flowers. The expression of BCH-B2 remained constant while that of BCH-A2 gradually decreased during flower development. Using Agrobacterium tumefaciens to introduce BCH2 RNA interference ( RNAi), we created transgenic Oncidium plants with down-regulated BCH expression. In the transgenic plants, flower colour changed from the bright yellow of the wild type to light and white-yellow. BCH-A2 and BCH-B2 expression levels were significantly reduced in the transgenic flower lips, which make up the major portion of the Oncidium flower. Sectional magnification of the flower lip showed that the amount of pigmentation in the papillate cells of the adaxial epidermis was proportional to the intensity of yellow colouration. HPLC analyses of the carotenoid composition of the transgenic flowers suggested major reductions in neoxanthin and violaxanthin. In conclusion, BCH2 expression regulated the accumulation of yellow pigments in the Oncidium flower, and the down-regulation of BCH-A2 and BCH-B2 changed the flower colour from bright yellow to light and white-yellow. [ABSTRACT FROM AUTHOR]