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The liver has a solid inbuilt antioxidant defense system to regulate oxidative stress. However, exposure to an excessive level of ROS causes liver injury. This study examined the cytoprotective effect of neoxanthin, a xanthophyll antioxidant molecule isolated from Solanum trilobatum in stress-induced HepG2 cells. The cytotoxic effect of H2O2 and cytoprotective potential of β-carotene, lutein, and neoxanthin was analyzed by WST-1 assay. The intracellular ROS level and mitochondrial membrane potential (MMP) were measured using DCFH-DA (2′, 7′-dichlorofluorescin diacetate) and JC-10 MMP assay. The expression of anti-oxidant and apoptotic markers was measured by western blot analysis. Neoxanthin pretreatment exhibited better protection than β-carotene and lutein against cell death caused by H2O2. It significantly arrested H2O2-mediated elevation of intracellular ROS levels and protected MMP. The intracellular antioxidant enzymes HO-1 and SOD-2 were upregulated by neoxanthin pretreatment. Neoxanthin also activated the protein expression of redox-sensitive transactivation factors, Nrf2 and NF-kB. The cytoprotective effect of neoxanthin was associated with increased expression of the anti-apoptotic protein, Bcl-2 and decreased pro-apoptotic protein Bax. For the first time, our results demonstrate that neoxanthin offers adequate protection against stress-mediated cytotoxicity in hepatocytes by activating the intracellular antioxidant defense system and blocking apoptosis.

The physicochemical and antioxidant properties of seven carotenoids: antheraxanthin, β-carotene, neoxanthin, peridinin, violaxanthin, xanthrophyll and zeaxanthin were studied by theoretical means. Then the Optoelectronic properties and interaction of chlorophyll-carotenoid complexes are analysed by TDDFT and IGMPLOT. Global reactivity descriptors for carotenoids and chlorophyll (Chla, Chlb) are calculated via conceptual density functional theory (CDFT). The higher HOMO–LUMO (HL) gap indicated structural stability of carotenoid, chlorophyll and chlorophyll-carotenoid complexes. The chemical hardness for carotenoids and Chlorophyll is found to be lower in the solvent medium than in the gas phase. Results showed that carotenoids can be used as good reactive nucleophile due to lower µ and ω. As proton affinities (PAs) are much lower than the bond dissociation enthalpies (BDEs), it is anticipated that direct antioxidant activity in these carotenoids is mainly due to the sequential proton loss electron transfer (SPLET) mechanism with dominant solvent effects. Also lower PAs of carotenoid suggest that antioxidant activity by the SPLET mechanism should be a result of a balance between proclivities to transfer protons. Reaction rate constant with Transition-State Theory (TST) were estimated for carotenoid-Chlorophyll complexes in gas phase. Time dependent Density Functional Theory (TDDFT) showed that all the chlorophyll (Chla, Chlb)–carotenoid complexes show absorption wavelength in the visible region. The lower S1–T1 adiabatic energy gap indicated ISC transition from S1 to T1 state.

Allelopathic activities of three carotenoids of a natural pigment group, neoxanthin, crocin and β-carotene, were assayed by the protoplast co-culture method with digital image analysis (DIA-PP method). Effects on three different growth stages of lettuce protoplasts, i.e., cell wall formation, cell division, and yellow pigment accumulation, were investigated using 96-well culture plates. Cell division was inhibited 65–95% by all three carotenoids at 33–100 µM. Inhibition of cell division stage was stronger than at the cell wall formation stage in neoxanthin, and the water-soluble carotenoid, crocin, whose yellow pigment was incorporated into the vacuole of lettuce protoplasts. Neoxanthin at 33 µM and crocin at higher than 100 µM inhibited more than 100% of the yellow pigment accumulation. By contrast, at low concentrations (0.01–1 µM) β-carotene stimulated growth at the cell division stage. At high concentrations of β-carotene (100–500 µM), inhibition was prominent at all three stages, and also in neighboring wells of zero control, which suggested emission of a volatile compound by β-carotene. They were compared with the report of the volatile compound, tulipalin A. Differences in patterns of inhibition of carotenoids on lettuce protoplast growth were compared with reports of another natural pigment, anthocyanin, and anthocyanin-containing red callus cultured in the light, and with that of neoxanthin-containing yellow callus cultured in the dark.

Potato tubers are an important agricultural crop containing carotenoids, an important group of secondary plant metabolites showing positive health benefits. This paper examines nine carotenoids at weekly intervals in three cultivars with different flesh color during the tuber maturation process, as analyzed by HPLC-DAD. Total carotenoids showed a bell-shaped distribution with maximum values in the middle of the tuber maturation process rather than in ready-to-harvest tubers. Xanthophylls (viola-, anthera-, neoxanthin and lutein) were the predominant carotenoid forms found in the cultivars. Red and purple-fleshed cultivars contained only a quarter of the total carotenoids of a commercial cultivar with yellow flesh. Weather conditions moderately influenced the carotenoids of the xanthophyll cycle. In red or purple-fleshed tubers no relationship was found between total carotenoids and total anthocyanidins.

Neoxanthin (Neo), which is only bound to the peripheral antenna proteins of photosystem (PS) II, is a conserved carotenoid in all green plants. It has been demonstrated that Neo plays an important role in photoprotection and its deficiency fails to impact LHCII stability in vitro and indoor plant growth in vivo. Whether Neo is involved in maintaining the PSII complex structure or adaptive mechanisms for the everchanging environment has not yet been elucidated. In this study, the role of Neo in maintaining the structure and function of the PSII-LHCII supercomplexes was studied using Neo deficient Arabidopsis mutants. Our results show that Neo deficiency had little effect on the electron transport capacity and the plant fitness, but the PSII-LHCII supercomplexes were significantly impacted by the lack of Neo. In the absence of Neo, the M-type LHCII trimer cannot effectively associate with the C2 S2 -type PSII-LHCII supercomplexes even in moderate light conditions. Interestingly, Neo deficiency also leads to decreased PSII protein phosphorylation but rapid transition from state 1 to state 2. We suggest that Neo might enforce the interactions between LHCII and the minor antennas and that the absence of Neo makes M-type LHCII disassociate from the PSII complex, leading to the disassembly of the PSII-LHCII C2 S2 M2 supercomplexes, which results in alterations in the phosphorylation patterns of the thylakoid photosynthetic proteins and the kinetics of state transition.

Marigold (Tagetes erecta L.) is an important ornamental plant with a wide variety of flower colors. Despite its economic value, few biochemical and molecular studies have explored the generation of flower color in this species. To study the mechanism underlying marigold petal color, we performed a metabolite analysis and de novo cDNA sequencing on the inbred line ‘V-01’ and its petal color mutant ‘V-01M’ at four flower developmental stages. A total of 49,217 unigenes were identified from 24 cDNA libraries. Based on our metabolites and transcriptomic analyses, we present an overview of carotenoid biosynthesis, degradation, and accumulation in marigold flowers. The carotenoid content of the yellow mutant ‘V-01M’ was higher than that of the orange inbred line ‘V-01’, and the abundances of the yellow compounds lutein, neoxanthin, violaxanthin, zeaxanthin, and antheraxanthin were significantly higher in the mutant. During flower development, the carotenoid biosynthesis genes were upregulated in both ‘V-01’ and ‘V-01M’, with no significant differences between the two lines. By contrast, the carotenoid degradation genes were dramatically downregulated in the yellow mutant ‘V-01M’. We therefore speculate that the carotenoid degradation genes are the key factors regulating the carotenoid content of marigold flowers. Our research provides a large amount of transcriptomic data and insights into the marigold color metabolome.

Leaves are primary plant organs harbouring chloroplasts that enable the sensing of changes in environmental conditions and contribute to maintaining cellular homeostasis. Carotenoids, carotenoid-derived phytohormones and apocarotenoid metabolites facilitate photosynthesis, photoprotection and stress acclimation in leaves. The thylakoids in the chloroplasts from the photosynthetically functional leaves accumulate a specific composition of lutein, β-carotene, violaxanthin, and neoxanthin that act as accessory pigments to chlorophyll and maintain optimal photosynthetic performance. The developmental state of leaves and environmental conditions can affect carotenoid metabolism and, hence, chloroplast functions in leaves. The etioplasts in the cotyledons from the germinating seedlings abundantly accumulate lutein. The light exposure of cotyledons triggers ultrastructural development of chloroplast accumulating all primary foliar carotenoids and chlorophylls. The accumulation of zeaxanthin and antheraxanthin during intense light and high temperature maintains the functional integrity of thylakoid membranes and chloroplasts. Carotenoids are continuously synthesised and degraded in leaves, yet their composition remains tightly maintained. The disintegration of chloroplast, such as during the leaf senescence, ceases the carotenoid biosynthesis and triggers carotenoid degradation. This review sheds light on the fundamental aspects of carotenoid metabolism. The effects of altered light regimes, carbon dioxide (CO2), temperature, drought, and soil nutrient levels are discussed in the context of carotenoid metabolism in leaves. The outstanding questions on how environmental perturbations can alter carotenoid metabolism and chloroplast development in leaves are highlighted. This review provides further insights on how carotenoid regulatory networks could be engineered to enrich carotenoids in the leaves of food and horticultural crops.

In this study, the microalga Chlorococum humicula was evaluated as a potential source of carotenoids and chlorophylls, and extraction of these pigments with liquefied dimethyl ether (DME) as an alternative green solvent was investigated. Carotenoids and chlorophylls in the algae were identified by spectral and comparative band ratio analyses. The liquefied DME extraction was shown to be a rather fast process for the extraction of pigments from C. humicula at relatively high yields. The effects of the extraction solvent: sample ratio, time, and temperature in the liquefied DME extraction of C. humicola on the yields of neoxanthin, violaxanthin, lutein, chlorophyll-b, chlorophyll-a, as well as the total carotenoids and chlorophylls, were determined. The highest total carotenoids yield (4.14 mg/g dry weight algae) and total chlorophylls yield (8.45 mg/g dry weight algae) were obtained using a 45:1 (w/w) liquefied DME: algae wet weight ratio for 20 min at 41 °C. At this condition, extraction of 91 % and 35 % of the total carotenoids and chlorophylls, respectively, was attained. In contrast, the extraction yield and percentage recovery of carotenoids obtained with acetone, a conventional solvent, was found to be lower than that of the liquefied DME, suggesting that liquefied DME has a higher selectivity for the less polar carotenoids than that of acetone.

Dynamics of chlorophyll a fluorescence changes (including JIP test parameters) and photosynthetic pigments were studied in oat (Avena sativa L.) leaves of late summer sowing during autumnal cooling. As the average daily temperature decreased from 8 to 2°С, the chlorophyll content decreased by 40%, while the chlorophyll a/b ratio ranged between 2.2 and 2.4. The total pool of carotenoids and the relative content of β‑carotene, lutein, neoxanthin, and pigments of the violaxanthin cycle changed insignificantly. The relative number of QB-nonreducing reaction centers (RC) of photosystem II (PSII) and the quantum yield of energy-dependent component qE of nonphotochemical quenching in the PSII antenna (φNPQ parameter) were found to increase during the seasonal cooling. The values of Fv/Fm parameter characterizing the maximal photochemical efficiency of PSII complexes were 0.72–0.76. At temperatures around 0°C, Fv/Fm decreased to 0.62. At the same time, the nonregulated (light-independent) thermal dissipation (photoinhibitory component qI of nonphotochemical quenching) was enhanced, which was manifested in the increase in parameters φDo and DI0/RC. It is concluded that, under seasonal cooling, the PSII reaction centers partly lose photochemical activity and start functioning as dissipative centers of excitation energy. In a significant part of inactivated RC, the loss of QA-reduction was reversible, which implies the formation of photoinactivated forms of PSII complexes stabilized at low temperatures. The decrease in daily average temperatures to −3°С with nighttime low temperatures from −7 to −8°С led to plant death within 3 days.

The present study investigated how different storage conditions affect antioxidant levels in three scab-resistant and powdery mildew-tolerant apple cultivars. The contents of carotenoids, along with free and glycosylated phenolic acids, were quantified in harvested apples and apples stored in boxes with controlled atmospheric conditions (1.2% O2 and 2.2% CO2) and temperature (1 °C), boxes with regulated temperature (1 °C) and storage rooms with a temperature between 1 and 4 °C. All three cultivars demonstrated a continuous decrease in total carotenoid content (sum of s-carotene, lutein, neoxanthin, violaxanthin, zeaxanthin and antheraxanthin) during the storage period. However, apples stored under controlled atmospheric conditions showed significantly higher carotenoid levels than apples stored under other approaches over 3 and 5 months of storage. Although the storage approach clearly affected carotenoid levels across the three tested cultivars, the ratio between the levels of individual carotenoids was neither affected by storage approach nor cultivar. Free phenolic acid contents were found to be cultivar specific; whereas, all three cultivars demonstrated similar glycosylated phenolic acid contents. Chlorogenic acid was the most common free phenolic acid in all three cultivars, while protocatechuic and caffeic acid were the dominant glycosylated phenolic acids identified from the cultivars. Interestingly, stored apples showed higher free phenolic acid levels than harvested apples; while, both harvested and stored apples showed similar concentrations of glycosylated phenolic acids. As such, none of the three tested storage conditions had a pronounced effect on either group of phenolic acids (total content or concentrations of individual compounds).

In this study, two carotenoid antioxidants lutein and neoxanthin were investigated for their ability to prevent oxidatively induced DNA damage. A Chlamydomonas reinhardtii alga was used as the natural source of lutein and neoxanthin. Analysis using HPLC with Diode Array Detector (DAD) showed that lutein and neoxanthin were the most abundant carotenoids in the extract of C. reinhardtii. An analysis using GC–MS/MS was performed to measure the damage induced by Fenton reaction to DNA and to evaluate the ability of lutein, neoxanthin and C. reinhardtii extract to inhibit such damage. The results showed that lutein and neoxanthin have the ability to reduce oxidatively induced DNA base damage to less than half of its magnitude. Extract of C. reinhardtii inhibited about 76% of the damage in comparison to control sample. Neoxanthin in lower concentrations than those of lutein proved to be better inhibitor of oxidatively induced DNA damage.

The accumulation of carotenoids (carotenogenesis) in the cells of Haematococcus pluvialis (strain IBCE H-17) upon cultivation in nutrient media deficient in nutrient elements (N and K + P) is shown. K and P deficiency in nutrient medium led to accumulation of astaxanthin, β-carotene, anteraxanthin, zeaxanthin and violaxanthin after 18 days of cultivation, while the amount of neoxanthin and lutein was at the control level. Carotenogenesis under such conditions was accompanied by high levels of chlorophyll (a + b). The use of nutrient medium deficient in N led to the accumulation of astaxanthin and, to a lesser extent, β-carotene in haematococcus cells, while lutein, anteraxanthin, zeaxanthin, violaxanthin and neoxanthin levels were lower or the same as control. N deficiency caused the decrease in amount of chlorophyll (a + b). Nitrogen deficiency was 4.3 times more effective than control medium and 1.8 times more effective than combined potassium and phosphorus deficiency in promoting astaxanthin accumulation.

We present the results of experiments on the influence of feeding the marine bivalve mollusk Cerastoderma glaucum (Bruguiere, 1789) with a monoculture of the green algae Tetraselmis viridis on the composition of carotenoids in its tissues. The carotenoid repertoire of C. glaucum was found to include β-carotene, alloxanthin, lutein, diadinoxanthin, neoxanthin, fucoxanthin, heteroxanthin, mactraxanthin and four carotenoid esters, with mactraxanthin being specific to C. glaucum. In T. viridis, carotenoids were represented by β, e -carotene, lutein, zeaxanthin, violaxanthin, neoxanthin. A 6-day microalgal mono-diet led to an increase in the total carotenoid content in mollusk tissues, mainly at the expense of pigments characteristic of T. viridis. The process was accompanied by a decrease in the concentration of other plant carotenoids and mactraxanthin isomerization. Despite the changes in the accumulation and transformation of carotenoids, the experiment led to a 20% death rate among individuals, indicating an adverse effect of a monotonous diet on mollusks.

The effects of different protective structures on horticultural and nutritional yield of amaranth and water spinach were studied in three seasons of 2020–2021 in Taiwan. The number of people that can receive recommended dietary intake of iron and β-Carotene from vegetables grown under different production conditions was also estimated. The yield of white and red amaranths was consistently better (7.68–19.70 t/ha) under pink poly-net house in all the seasons, but the yield of water spinach was consistently better under white poly-net house (16.25–20.88 t/ha). Spider mite (fall & spring) and aphid (winter) infestation was mostly observed on all crops under poly-net houses. Neoxanthin, lutein and β-carotene were almost two-fold higher in red amaranth harvested from poly-net houses than open field. Based on the RDI values, β-Carotene supply to both men and women (14+) was consistently higher in all crops produced under pink ploy-net houses in all seasons, except for white amaranth produced under white poly-net house during winter. Its supply to 64,788 more men and 83,298 more women was estimated for red amaranth harvested from pink poly-net house than other production conditions. α-carotene was 2–3 fold higher in amaranths and water spinach harvested from poly-net houses than open field. The iron content of the amaranths was lower in poly-net houses (234.50–574.04 g/ha) than open field (645.42–881.67 g/ha) in the fall, but its supply from pink poly-net house was comparable with open field in the winter. However, pink poly-net house was the highest iron supplier from water spinach (323.90 g/ha) in the winter, which was estimated to provide iron to 19,450–22,939 more men and women than other production conditions. Both poly-net houses were the sole supplier of iron through amaranths in the spring, with pink poly-net house supplying iron to 2,000–5,000 more men and women. Thus, protected cultivation not only leads to more marketable yields but also results in higher quantities of health promoting nutrients. Hence, pink poly-net house may be considered to produce more nutritious vegetables, especially during the off-season to bridge the gaps in the seasonal variations in vegetable consumption, besides providing better income opportunities to the smallholder farmers.

The oil yield, measured in oil extraction rate per hectare in the palm oil industry, is directly affected by the ripening levels of the oil palm fresh fruit bunches at the point of harvesting. A rapid, non-invasive and reliable method in assessing the maturity level of oil palm harvests will enable harvesting at an optimum time to increase oil yield. This study shows the potential of using Raman spectroscopy to assess the ripeness level of oil palm fruitlets. By characterizing the carotene components as useful ripeness features, an automated ripeness classification model has been created using machine learning. A total of 46 oil palm fruit spectra consisting of 3 ripeness categories; under ripe, ripe, and over ripe, were analyzed in this work. The extracted features were tested with 19 classification techniques to classify the oil palm fruits into the three ripeness categories. The Raman peak averaging at 1515 cm−1 is shown to be a significant molecular fingerprint for carotene levels, which can serve as a ripeness indicator in oil palm fruits. Further signal analysis on the Raman peak reveals 4 significant sub bands found to be lycopene (ν1a), β-carotene (ν1b), lutein (ν1c) and neoxanthin (ν1d) which originate from the C=C stretching vibration of carotenoid molecules found in the peel of the oil palm fruit. The fine KNN classifier is found to provide the highest overall accuracy of 100%. The classifier employs 6 features: peak intensities of bands ν1a to ν1d and peak positions of bands ν1c and ν1d as predictors. In conclusion, the Raman spectroscopy method has the potential to provide an accurate and effective way in determining the ripeness of oil palm fresh fruits.

Light is the most essential environmental factor that plants require for the growth and development. However, light is highly heterogeneous in natural condition, and plants have to evolve a series of strategies to acclimate the dynamic light since it is sessile. The photosynthetic performance and regulatory mechanisms involved in acclimation were elucidated in shade-tolerant plant Amorphophallus xiei under steady state and fluctuating high light. The dissipation and allocation of light energy, photosynthetic capacity, and antioxidant defense were examined in A. xiei cultivated under 4%, 17%, and 100% of full sunlight. High-light-grown plants exhibited reduced photosynthesis and the slowest response to simulated sunflecks than the other two treatments. Maximum and actual efficiency of PSII photochemistry (Fv′/Fm′ and ΦPSII) and electron transport rate (ETR) were lowest, and non-photochemical quenching (NPQ) were highest in high-light-grown plants than in low- and intermediate-light-grown plants subjected to different photon flux densities (PFD) and intercellular CO2 concentrations (Ci). Fv′/Fm′, ΦPSII, ETR and NPQ were lowest in high-light-grown A. xiei than others, when plants were exposed to simulated sunflecks. In fully light-induced leaves, high-light-grown plants showed a maximum value in quantum efficiency of light-dependent thermal dissipation (ΦNPQ), and a minimum value in ΦPSII was recorded. Low-light-grown plants subjected to simulated sunflecks, a maximum value in ΦNPQ and ΦPSII were observed. Mass-based nitrogen content (Narea), specific leaf area (SLA), superoxide dismutase (SOD) and catalase (CAT) activities significantly declined with the increase of growth irradiance. The high-light-grown plants showed higher xanthophyll de-epoxidation and ascorbate–glutathione cycle activity, while low-light-grown plants showed higher neoxanthin and β-carotene contents. In general, high-light-grown A. xiei generally show a depressed photosynthetic capacity, and are efficient in dissipating excess light energy by NPQ under a steady-state light but not under a highly fluctuating light. Low-light-grown plants show a rapid photosynthetic assimilation response to sunflecks and then a rapid activation of the energy-dependent quenching (qE) component of NPQ under the sunflecks. In addition, high-light-grown plants, although using various strategies to reduce light absorption and to scavenge reactive oxygen species, have less efficient protection against photodamage.

Chinese kale (Brassica oleracea var. alboglabra) is rich in carotenoids, and neoxanthin is one of the most important carotenoids in Chinese kale. In this study, the function of the neoxanthin synthase gene (BoaNXS) in Chinese kale was investigated. BoaNXS, which had a 699-bp coding sequence, was cloned from the white flower cultivar of Chinese kale and was expressed in all developmental stages and organs of Chinese kale, its expression was highest in young seeds. The subcellular localization indicated that BoaNXS was localized in the chloroplast. BoaNXS-overexpressed plants were obtained via Agrobacterium-mediated transient overexpression methodology, and the gene overexpression efficiencies ranged from 2.10- to 4.24-fold. The color in the leaves of BoaNXS-overexpressed plants changed from green to yellow-green, the content of total and individual carotenoids, such as neoxanthin, violaxanthin, and lutein, was significantly increased, and the expression levels of most carotenoid biosynthetic genes were notably increased. These findings indicated that BoaNXS is of vital importance in carotenoid biosynthesis in Chinese kale and could be used as a candidate gene for enriching the carotenoid accumulation and color of Chinese kale and other Brassica vegetables.

The dietary needs of humans for provitamin A carotenoids arise from their inability to synthesize vitamin A de novo. To improve the status of this essential micronutrient, special attention has been given to biofortification of staple foods, such as wheat grains, which are consumed in large quantities but contain low levels of provitamin A carotenoids. However, there remains an unclear contribution of metabolic genes and homoeologs to the turnover of carotenoids in wheat grains. To better understand carotenoid catabolism in tetraploid wheat, Targeting Induced Local Lesions in Genomes (TILLING) mutants of CCD4, encoding a Carotenoid Cleavage Dioxygenase (CCD) that cleaves carotenoids into smaller apocarotenoid molecules, were isolated and characterized. Our analysis showed that ccd4 mutations co-segregated with Poltergeist-like (pll) mutations in the TILLING mutants of A and B subgenomes, hence the ccd-A4 pll-A, ccd-B4 pll-B, and ccd-A4 ccd-B4 pll-A pll-B mutants were analyzed in this study. Carotenoid profiles are comparable in mature grains of the mutant and control plants, indicating that CCD4 homoeologs do not have a major impact on carotenoid accumulation in grains. However, the neoxanthin content was increased in leaves of ccd-A4 ccd-B4 pll-A pll-B relative to the control. In addition, four unidentified carotenoids showed a unique presence in leaves of ccd-A4 ccd-B4 pll-A pll-B plants. These results suggested that CCD4 homoeologs may contribute to the turnover of neoxanthin and the unidentified carotenoids in leaves. Interestingly, abnormal spike, grain, and seminal root phenotypes were also observed for ccd-A4 pll-A, ccd-B4 pll-B, and ccd-A4 ccd-B4 pll-A pll-B plants, suggesting that CCD4 and/or PLL homoeologs could function toward these traits. Overall, this study not only reveals the role of CCD4 in cleavage of carotenoids in leaves and grains, but also uncovers several critical growth traits that are controlled by CCD4, PLL, or the CCD4-PLL interaction.

Tetraselmis suecica is a green microalga that thrives under a wide range of conditions, used in the commercial culture of fish, mollusk, and crustacean larvae for supplementing the demand for fertilizers. Its pigments have applications in human health care as drug products, vitamins, and cosmetics. Growth and pigment concentration of T. suecica were evaluated in experimental cultures with different nutrient concentrations and light intensities to determine the most appropriate culture conditions to optimize the production of biomass and pigments. Chlorophyll-a, chlorophyll-b, lutein, violaxanthin, α, β-carotene, and neoxanthin concentrations were evaluated under three different nutrient conditions (441.5/18.1, 883/36.3, and 1766/76.2 μM of NaNO3/NaH2PO4) and four light intensities (50, 150, 300, and 750 μmol quanta m−2 s−1). Increases in either or both of these factors lead to increases in the concentration of all pigments. Chlorophyll-a reached up to 5×103 mg m−3, chlorophyll-b up to 2500 mg m−3, lutein 600 mg m−3, violaxanthin 300 mg m−3, α, β-carotene 500 mg m−3, and neoxanthin 400 mg m−3. Growth rate (μ) attained values of 1.6 d−1. An index to evaluate the efficiency of pigment production by light intensity (called LER) was computed. The highest LER was recorded at 50 μmol quanta m−2 s−1 and a nutrient concentration of 1766/76.2 μM (NaNO3/NaH2PO4); this treatment optimizes pigment production with the lowest light intensity. Our results show that the optimum light intensity should be selected according to the objective of the culture, either maximizing pigment concentration for harvesting at higher concentrations or reducing production costs regarding light consumption.

Persistent pollutants such as pharmaceuticals, pesticides, musk fragrances, and dyes are frequently detected in different environmental compartments and negatively impact the environment and humans. Understanding the impacts of diffuse environmental pollutants on plants is still limited, especially at realistic environmental concentrations of contaminants. We studied the effects of key representatives of two major classes of environmental pollutants (nine different antibiotics and six different textile dyes) on the leaf carotenoid (violaxanthin and neoxanthin) content in wheat (Triticum aestivum L.) using different pollutant concentrations and application times. The wheat plants were watered with solutions of selected environmental pollutants in two different concentrations of 0.5 mg L−1 and 1.5 mg L−1 for one week (0.5 L) and two weeks (1 L). Both categories of pollutants selected for this study negatively influenced the content of violaxanthin and neoxanthin, whereas the textile dyes represented more severe stress to the wheat plants. The results demonstrate that chronic exposure to common diffusively spread environmental contaminants constitutes significant stress to the plants.

To reveal the molecular mechanism underlying peel colouration, carotenoid metabolites and the transcriptome were jointly analysed in zucchini peels with three different colours: light green (Lg), yellow (Y), and orange (O). Our results showed that the carotenoid levels in O (157.075 μg/g) and Y (22.734 μg/g) were both significantly higher than in Lg (7.435 μg/g), while the chlorophyll content was highest in Lg (32.326 μg/g), followed by O (7.294 μg/g) and Y (4.617 μg/g). A total of 14 carotenoids were detected in zucchini peels, primarily lutein (103.167 μg/g in Lg, 509.667 μg/g in Y, and 1543.333 μg/g in O). In particular, significant accumulation of antheraxanthin, zeaxanthin, neoxanthin, and β-cryptoxanthin was first reported in orange zucchini in this study. Furthermore, two modules with hub genes related to carotenoid or chlorophyll content were identified through weighted gene coexpression network analysis. Additionally, the transcription level of some hub genes (PIF4, APRR2, bHLH128, ERF4, PSY1, LCYE2, and RCCR3) was highly correlated with pigment content in the peel, which may be responsible for carotenoid accumulation and chlorophyll degradation in the Y and O varieties. Taken together, the results obtained in this study help to provide a novel mechanism underlying peel colouration in zucchini.

RATIONALE Liquid chromatography/photodiode array atmospheric pressure chemical ionization mass spectrometry (LC/PDA-APCI-MS) is used for the analysis of various carotenoid pigments in plants. Among them, it is difficult to distinguish between the isomeric violaxanthin/neoxanthin esters. METHODS The yellow pigments of tomato petals were extracted with acetone, and the extracts were kept at -30°C to allow the contaminating triacylglycerols to settle out physically. The supernatants were analyzed using LC/PDA-APCI-MS with a high-resolution orbitrap mass spectrometer for their exact masses. The expected carotenoid esters were calculated with the combination of carotenoids and fatty acids, and they were matched with the experimental exact masses. The fatty acid structures in the carotenoid esters were also identified using collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). The isomeric violaxanthin/neoxanthin esters were distinguished using CID MS/MS from their in-source dehydrated product ions as pseudoprecursor ions. RESULTS The in-source dehydrated ions [M - H2 O + H]+ of neoxanthin diesters predominated over their protonated molecules [M + H]+ in LC/MS. By contrast, the protonated molecules of violaxanthin diesters predominated. The 92 u loss product ions [M - H2 O - C7 H8 + H]+ were observed from the dehydrated violaxanthin diesters, but they were not generated from the dehydrated neoxanthin diesters in the CID MS/MS of their dehydrated pseudoprecursor ion [M - H2 O + H]+ . CONCLUSIONS The allene allyl carbocation in neoxanthin diesters was generated from dehydration after preferential protonation at the hydroxy group. The epoxide group of violaxanthin diesters opens easily after protonation; however, the dehydration did not proceed at this stage. The 92 u loss of C7 H8 was explained by an intramolecular [2 + 2] cycloaddition, which proceeded preferentially in dehydrated violaxanthin diesters because the carbocations in the dehydrated species were conjugated to the polyene and those double bonds were depolarized during CID MS/MS. Therefore, the isomeric neoxanthin/violaxanthin diesters were distinguished using LC/PDA-APCI-MS and MS/MS. This method was a practical and useful method of profiling the carotenoid esters of the yellow petals.

Rumengan AP, Mandiangan ES, Tanod WA, Paransa DSJ, Paruntu CP, Mantiri DMH. 2021. Identification of pigment profiles and antioxidant activity of Rhizophora mucronata mangrove leaves origin Lembeh, North Sulawesi, Indonesia. Biodiversitas 22: 2805-2816. Mangroves plants contain unique pigments which can serve both nutraceutical and pharmaceutical purpose. Therefore, this preliminary study aims to identify the pigment profiles of Rhizophora mucronata mangrove leaves (HPLC method) and to computationally evaluate the antioxidant mechanism of the pigments (PASS and STITCH analysis). Furthermore, it evaluated the antioxidant capacity of R. mucronata leaves extracts (DPPH method), and the pigment profiles detected using HPLC were chlorophyll a (68.61%), chlorophyll b (27.69%), lutein (29.94%), beta-carotene (14.05%), pheophytin a (8.72), violaxanthin (5.19%), and neoxanthin (3.65%). Beta-carotene, lutein, neoxanthin, and violaxanthin were predicted to have potential as antioxidants properties using PASS analysis. While neoxanthin and violaxanthin were predicted as free radical scavengers, beta-carotene was an Nrf-2 stimulant. The STITCH analysis showed that the pigments contained in the leaves interacted synergistically by activating as antioxidant enzymes and inhibiting the expression of oxidative stress proteins. The ethanol extract of R. mucronata leaves may be a potent antioxidant with an IC50 20.99 ± 0.33 µg/mL. Therefore, the pigment contained in R. mucronata leaves is a potential source of antioxidants.

Seasonal accumulation of hormonal and photoprotective isoprenoids, particularly α-tocopherol, carotenoids and abscisic acid, indicate their important role in protecting Cistus albidus plants from environmental stress during a Mediterranean winter. The high diurnal amounts of α-tocopherol and xanthophylls 3 h before maximum light intensity suggest a photoprotective response against the prevailing diurnal changes. The timing to modulate acclimatory/defense responses under changing environmental conditions is one of the most critical points for plant fitness and stress tolerance. Here, we report seasonal and diurnal changes in the contents of isoprenoids originated from the methylerythritol phosphate pathway, including chlorophylls, carotenoids, tocochromanols, and phytohormones (abscisic acid, cytokinins, and gibberellins) in C. albidus during a Mediterranean winter. Plants were subjected not only to typically low winter temperatures but also to drought, as shown by a mean plant water status of 54% during the experimental period. The maximum PSII efficiency, however, remained consistently high (Fv/Fm > 0.8), proving that C. albidus had efficient mechanisms to tolerate combined stress conditions during winter. While seasonal α-tocopherol contents remained high (200–300 µg/g DW) during the experimental period, carotenoid contents increased during winter attaining maximum levels in February (minimum air temperature ≤ 5 °C for 13 days). Following the initial transient increases of bioactive trans-zeatin (about fivefold) during winter, the increased abscisic acid contents proved its important role during abiotic stress tolerance. Diurnal amounts of α-tocopherol and xanthophylls, particularly lutein, zeaxanthin and neoxanthin including the de-epoxidation state, reached maximum levels as early as 2 h after dawn, when solar intensity was 68% lower than the maximum solar radiation at noon. It is concluded that (1) given their proven antioxidant properties, both α-tocopherol and carotenoids seem to play a crucial role protecting the photosynthetic apparatus under severe stress conditions; (2) high seasonal amounts of abscisic acid indicate its important role in abiotic stress tolerance within plant hormones, although under specific environmental conditions, accumulation of bioactive cytokinins appears to be involved to enhance stress tolerance; (3) the concerted diurnal adjustment of α-tocopherol and xanthophylls as early as 3 h before maximum light intensity suggests that plants anticipated the predictable diurnal changes in the environment to protect the photosynthetic apparatus.

For winter survival, evergreens not only need cold acclimation (CA), but also photoprotection. This study was conducted to examine how two elevational ecotypes of Rhododendron catawbiense respond to cold and light stresses in a common garden. Seasonal changes in leaf freezing tolerance (LFT), photosystem II efficiency, photosynthesis, carotenoids (xanthophyll cycle pigments, lutein, beta-carotene, neoxanthin) were investigated from summer to winter. Parallel changes in the expression of violaxanthin de-epoxidase (VDE) gene were examined to explore its association with violaxanthin de-epoxidation. High elevation (HE) ecotype attained overall greater LFT while low elevation (LE) ecotype was more responsive to short-days for the first stage of CA. In summer, both ecotypes predominantly employed reversible xanthophyll cycle (RXC) to dissipate excess light energy, but LE used it more robustly. In autumn, only HE exhibited slight damage to the reaction centers (RCs), i.e. photosystem II degradation (PSD). In winter, both ecotypes suspended photosynthesis, were photoinhibited, and employed primarily sustained de-epoxidation (SD) for photoprotection; but LE still used RXC more effectively than HE. Overall, LE employed xanthophyll cycle more robustly in summer or winter. Results suggest that microclimates of R. catawbiense ecotypes inform their differential physiological response to cold and light stresses. VDE expression data indicate violaxanthin de-epoxidation in R. catawbiense may not be solely transcriptionally regulated.

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

Fourteen watermelon cultivars with different fruit flesh colors (red, salmon yellow, orange, and canary yellow) were analyzed for carotenoid contents (prolycopene, lycopene, β-carotene, ζ-carotene, and neoxanthin). Genes encoding the carotenoid biosynthesis enzymes carotenoid isomerase (encoded by CRTISO), which catalyzes the isomerization of prolycopene to lycopene, and β-carotene hydroxylase (CHYB), which catalyzes the conversion of β-carotene to xanthophyll, were also analyzed. High-performance liquid chromatography showed that the salmon yellow and orange flesh accumulated either prolycopene (orange-P flesh) or β-carotene (orange-β flesh), whereas lycopene and neoxanthin were the main carotenoids in the red and canary yellow flesh, respectively. Quantitative real-time polymerase chain reaction indicated that CRTISO and CHYB were mainly expressed during fruit maturation, regardless of the flesh color, and there was no significant association between differential gene expression and flesh color. Importantly, transcript sequencing revealed a non-synonymous single-nucleotide mismatch (T > C1976) in exon 13 of CRTISO between orange-P-fleshed and other cultivars, suggesting CRTISO as a candidate gene for high prolycopene accumulation. However, in β-carotene-accumulating cultivars, there were no mutations in CHYB transcripts. A cleaved amplified polymorphic sequence marker was developed for T > C1976, and its applicability for marker-assisted selection of orange-P flesh was validated in 105 watermelon accessions.

We tested the potential of Raman microspectroscopy to determine carotenoid pigments — both primary (lutein, beta-carotene) and secondary (astaxanthin) carotenoids — in the different species and life-cycle stages of snow algae from the order Chlamydomonadales (Chlorophyta). We compared the performance of Raman spectrometry to a reference method of biological pigment analysis, high-performance liquid chromatography (HPLC). The three main carotenoid Raman bands of the astaxanthin-rich red cysts were located at 1520, 1156 and 1006 cm−1. The shifts (orange aplanozygotes and green motile cells with flagella) in the position of the ν1(C C) Raman band of the polyenic chain is consistent with the expected changes in the ratios of the various carotenoid pigments. Flagellated green cells commonly contain lutein as a major carotenoid, together with minor amounts of β‑carotene and varying amounts of antheraxanthin, violaxanthin and neoxanthin. Aplanozygotes contain mixtures of both primary and secondary carotenoids. In most cases, the ν1(C C) band is an overlapping set of bands, which is due to the signal of all carotenoid pigments in the sample, and a deconvolution along with the band position shifts (mainly ν1) could be used to characterize the mixture of carotenoids. However, the ability of Raman spectroscopy to discriminate between structurally slightly differing carotenoid pigments or several carotenoids in an admixture in an unknown biological system remains limited.

Some carotenoids are found in the Euglena gracilis, including β-carotene, diadinoxanthin, diatoxanthins, and neoxanthin as the major species; however, the molecular mechanism underlying carotenoid biosynthesis in E. gracilis is not well understood. To clarify the pathway and regulation of carotenoid biosynthesis in this alga, we functionally characterized the cytochrome P450 (CYP)-type carotene hydroxylase gene EgCYP97H1. Heterologous in vivo enzyme assay in E. coli indicated that EgCYP97H1 hydroxylated β-carotene to β-cryptoxanthin. E. gracilis cells suppressing EgCYP97H1 resulted in marked growth inhibition and reductions in total carotenoid and chlorophyll contents. Analysis of carotenoid composition revealed that suppression of EgCYP97H1 resulted in higher level of β-carotene, suggesting that EgCYP97H1 is physiologically essential for carotenoid biosynthesis and thus normal cell growth. To our knowledge, this is the first time EgCYP97H1 has been suggested to be β-carotene monohydroxylase, but not β-carotene dihydroxylase. Moreover, during light adaptation of dark-grown E. gracilis, transcript levels of the carotenoid biosynthetic genes (EgCYP97H1, geranylgeranyl pyrophosphate synthase EgcrtE, and phytoene synthase EgcrtB) remained virtually unchanged. In contrast, carotenoid accumulation in E. gracilis grown under the same conditions was inhibited by treatment with a translational inhibitor but not a transcriptional inhibitor, indicating that photo-responsive carotenoid biosynthesis is regulated post-transcriptionally in this alga.

An interesting population of ulvacean green algae was collected from a linmo-rheocrenic, thermal, mineral (chloride, sodium, sulphate) spring known as "Ain Abu Sherouf" in the Siwa Oasis, the Western Desert of Egypt. A detailed combined investigation on its morphotaxonomy, autecology, and a multilocus sequence data set including the chloroplast-encoded rbcL gene and the nuclear-encoded nrDNA SSU and ITS allowed us to identify this population as Ulva ftexuosa subsp. paradoxa (syn. Ulva paradoxa, Ulvales, Chlorophyta), although the algal thalli were in average narrower than usual in this taxon. Analysis of the molecular rbcL-ITS sequencing data demonstrated a close phylogenetic relationship of the studied population to two Ulva isolates from Japan and China and their taxonomic status was further discussed. The pigment profiling confirmed chlorophylls and carotenoids typical of the Ulvaceae (lutein, alpha/beta-carotenes, violaxanthin, and neoxanthin). Lipidomic analysis revealed the presence of the monogalactosyl diacylglycerols (MGDG), digalactosyl diacylglycerols (DGDG), sulfoquinovosyl diacylglycerols (SQDG), and diacylglyceryl N,N,N-trimethylhomoserine (DGTS) lipid classes. Presence of the plasma membrane DGTS lipids in remarkable proportion might be an adaptation to this nutrient-poor mineral spring habitat, with relatively low phosphorus values. The lower unsaturation index (UI) values of the plastidial DGDG and SQDG lipids are likely to reflect the need to maintain adequate membrane fluidity in this thermal mineral spring system. This polyphasic study not only extended our little knowledge on the distribution, autecology, and adaptive mechanisms of U. flexuosa subsp. paradoxa in the Saharan habitats of Africa, but also re-assessed and refined the taxonomic and phylogenetic affiliation of previously sampled close relatives from East Asia.

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 IC50 values of 3.8 and 7.5 μM, respectively.

The present study aimed to investigate the effect of young leaves on fat accumulation in differentiated 3T3-L1 adipocytes. A potent preventive effect on fat accumulation was observed in fractions of young leaves of spinach, beet, and arugula extracted with a low-polarity solvent (hexane:acetone:ethanol:toluene = 10:6:7:6). This effect was seemingly associated with the leaf carotenoid content, including lutein, β-carotene, and neoxanthin. Among these, only neoxanthin, with the characteristic structure of 5,6-monoepoxide and an allenic bond, significantly prevented fat accumulation in a dose-dependent manner. The preventive effect and carotenoid content, including neoxanthin, of these young leaves did not differ from those of the corresponding adult leaves. Therefore, our study demonstrated that young vegetable leaves, such as spinach, beet, and arugula leaves, contained neoxanthin, which prevented fat accumulation in adipocytes in vitro. In the future, the effectiveness of such young leaves and neoxanthin should be investigated in vivo.

Summary • Here, the effects of salinity (0–400 mM NaCl) on photosynthesis, photosystem II (PSII) efficiency and the xanthophyll cycle were investigated in the halophyte Atriplex centralasiatica grown under outdoor conditions. • Leaf sodium and chloride in leaves increased considerably whereas CO2 assimilation rate decreased. PSII efficiency (ΦPSII) and the efficiency of excitation energy capture by open PSII reaction centres decreased whereas nonphotochemical quenching (NPQ) increased significantly. There was no change in photochemical quenching (qP) in salt-adapted plants. Salinity induced no changes in the maximum efficiency of PSII photochemistry (Fv/Fm) measured either at midday or at predawn. However, Fv/Fm values were c. 20% lower at midday than at predawn. • Contents of chlorophyll (a + b), neoxanthin, lutein and β-carotene were unchanged with increasing salt concentration, but zeaxanthan increased significantly, at the expense of violaxanthin. There was a linear relationship between the de-epoxidation state of the xanthophyll cycle and ΦPSII, , and NPQ. • Our results suggest that A. centralasiatica shows high tolerance to both high salinity and photoinhibition and that the xanthophyll cycle played an important role in protecting photosynthetic apparatus from photoinhibitory damage. Tolerance of PSII to salinity and photoinhibition can be viewed as an important strategy for A. centralasiatica to grow in very high saline soil during the summer season with high irradiance.

In this study, various ratios of combined red, blue, and amber light-emitting diodes (rbaLEDs) were investigated for their effect on the expression of carotenoid biosynthetic genes and carotenoid accumulation in eight Brassica microgreens. Total and individual (β-carotene, lutein, α-carotene, neoxanthin, and violaxanthin) carotenoids were increased 20-44 and 10-55%, respectively, under dose-dependent increasing amber-blue light and decreasing red in most microgreens. Lipophilic 2,2-diphenyl-1-picrylhydrazyl and ferric reducing antioxidant power antioxidant activities were significantly increased under higher amber and blue light fractions, while oxygen radical absorbance capacity was generally decreased. Under rbaLED in mizuna (B. rapa) microgreens, the lycopene epsilon cyclase (LYCe) expression was 10-15-fold higher, which resulted in downstream accumulation of α-carotene and lutein. Lycopene beta cyclase (LYCβ) was not significantly changed, suggesting that β-carotene, violaxanthin and neoxanthin were mainly controlled by upstream phytoene synthase and branch-point LYCe. Increased beta-ring carotenoid hydroxylase (CHXβ) expression was also consistent with lutein accumulation. This study demonstrated for the first time that amber LED was involved in the regulatory mechanism of carotenoid biosynthesis, thus a potential novel approach to production of antioxidant-rich microgreens.