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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]

Violaxanthin is a plant-derived orange xanthophyll with remarkable antioxidant activity that has wide applications in various industries, such as food, agriculture, and cosmetics. In addition, it is the key precursor of important substances such as abscisic acid and fucoxanthin. Saccharomyces cerevisiae , as a GRAS (generally regarded as safe) chassis, provides a good platform for producing violaxanthin production with a yield of 7.3 mg/g DCW, which is far away from commercialization. Herein, an integrated strategy involving zeaxanthin epoxidase (ZEP) source screening, cytosol redox state engineering, and nicotinamide adenine dinucleotide phosphate (NADPH) regeneration was implemented to enhance violaxanthin production in S. cerevisiae . 58aa-truncated ZEP from Vitis vinifera exhibited optimal efficiency in an efficient zeaxanthin-producing strain. The titer of violaxanthin gradually increased by 17.9-fold (up to 119.2 mg/L, 15.19 mg/g DCW) via cytosol redox state engineering and NADPH supplementation. Furthermore, balancing redox homeostasis considerably improved the zeaxanthin concentration by 139.3% (up to 143.9 mg/L, 22.06 mg/g DCW). Thus, the highest reported titers of violaxanthin and zeaxanthin in S. cerevisiae were eventually achieved. This study not only builds an efficient platform for violaxanthin biosynthesis but also serves as a useful reference for the microbial production of xanthophylls.

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]