Diurnal dynamics of nonphotochemical quenching in Arabidopsis npq mutants assessed by solar-induced fluorescence and reflectance measurements in the field.

Solar-induced fluorescence (SIF) is highly relevant in mapping photosynthesis from remote-sensing platforms. This requires linking SIF to photosynthesis and understanding the role of nonphotochemical quenching (NPQ) mechanisms under field conditions. Hence, active and passive fluorescence were measured in Arabidopsis with altered NPQ in outdoor conditions. Plants with mutations in either violaxanthin de-epoxidase (npq1) or PsbS protein (npq4) exhibited reduced NPQ capacity. Parallel measurements of NPQ, photosystem II efficiency, SIF and spectral reflectance (ρ) were conducted diurnally on one sunny summer day and two consecutive days during a simulated cold spell. Results showed that both npq mutants exhibited higher levels of SIF compared to wild-type plants. Changes in reflectance were related to changes in the violaxanthin-antheraxanthin-zeaxanthin cycle and not to PsbS-mediated conformational changes. When plants were exposed to cold temperatures, rapid onset of photoinhibition strongly quenched SIF in all lines. Using well-characterized Arabidopsis npq mutants, we showed for the first time the quantitative link between SIF, photosynthetic efficiency, NPQ components and leaf reflectance. We discuss the functional potential and limitations of SIF and reflectance measurements for estimating photosynthetic efficiency and NPQ in the field.

Violaxanthin: natural function and occurrence, biosynthesis, and heterologous production.

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, ß-carotene, lutein, zeaxanthin, ß-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.

A functional zeaxanthin epoxidase from red algae shedding light on the evolution of light-harvesting carotenoids and the xanthophyll cycle in photosynthetic eukaryotes.

The epoxy-xanthophylls antheraxanthin and violaxanthin are key precursors of light-harvesting carotenoids and participate in the photoprotective xanthophyll cycle. Thus, the invention of zeaxanthin epoxidase (ZEP) catalyzing their formation from zeaxanthin has been a fundamental step in the evolution of photosynthetic eukaryotes. ZEP genes have only been found in Viridiplantae and chromalveolate algae with secondary plastids of red algal ancestry, suggesting that ZEP evolved in the Viridiplantae and spread to chromalveolates by lateral gene transfer. By searching publicly available sequence data from 11 red algae covering all currently recognized red algal classes we identified ZEP candidates in three species. Phylogenetic analyses showed that the red algal ZEP is most closely related to ZEP proteins from photosynthetic chromalveolates possessing secondary plastids of red algal origin. Its enzymatic activity was assessed by high performance liquid chromatography (HPLC) analyses of red algal pigment extracts and by cloning and functional expression of the ZEP gene from Madagascaria erythrocladioides in leaves of the ZEP-deficient aba2 mutant of Nicotiana plumbaginifolia. Unlike other ZEP enzymes examined so far, the red algal ZEP introduces only a single epoxy group into zeaxanthin, yielding antheraxanthin instead of violaxanthin. The results indicate that ZEP evolved before the split of Rhodophyta and Viridiplantae and that chromalveolates acquired ZEP from the red algal endosymbiont and not by lateral gene transfer. Moreover, the red algal ZEP enables engineering of transgenic plants incorporating antheraxanthin instead of violaxanthin in their photosynthetic machinery.

Carotenoid Profiling of a Red Seaweed Pyropia yezoensis: Insights into Biosynthetic Pathways in the Order Bangiales.

Carotenoids are natural pigments that contribute to light harvesting and photo-protection in photosynthetic organisms. In this study, we analyzed the carotenoid profiles, including mono-hydroxy and epoxy-carotenoids, in the economically valuable red seaweed Pyropia yezoensis, to clarify the detailed biosynthetic and metabolic pathways in the order Bangiales. P. yezoensis contained lutein, zeaxanthin, α-carotene, and ß-carotene, as major carotenoids in both the thallus and conchocelis stages. Monohydroxy intermediate carotenoids for the synthesis of lutein with an ε-ring from α-carotene, α-cryptoxanthin (ß,ε-caroten-3'-ol), and zeinoxanthin (ß,ε-caroten-3-ol) were identified. In addition, ß-cryptoxanthin, an intermediate in zeaxanthin synthesis from ß-carotene, was also detected. We also identified lutein-5,6-epoxide and antheraxanthin, which are metabolic products of epoxy conversion from lutein and zeaxanthin, respectively, by LC-MS and ¹H-NMR. This is the first report of monohydroxy-carotenoids with an ε-ring and 5,6-epoxy-carotenoids in Bangiales. These results provide new insights into the biosynthetic and metabolic pathways of carotenoids in red seaweeds.

Carotenogenesis diversification in phylogenetic lineages of Rhodophyta.

Carotenoid composition is very diverse in Rhodophyta. In this study, we investigated whether this variation is related to the phylogeny of this group. Rhodophyta consists of seven classes, and they can be divided into two groups on the basis of their morphology. The unicellular group (Cyanidiophyceae, Porphyridiophyceae, Rhodellophyceae, and Stylonematophyceae) contained only ß-carotene and zeaxanthin, "ZEA-type carotenoids." In contrast, within the macrophytic group (Bangiophyceae, Compsopogonophyceae, and Florideophyceae), Compsopogonophyceae contained antheraxanthin in addition to ZEA-type carotenoids, "ANT-type carotenoids," whereas Bangiophyceae contained α-carotene and lutein along with ZEA-type carotenoids, "LUT-type carotenoids." Florideophyceae is divided into five subclasses. Ahnfeltiophycidae, Hildenbrandiophycidae, and Nemaliophycidae contained LUT-type carotenoids. In Corallinophycidae, Hapalidiales and Lithophylloideae in Corallinales contained LUT-type carotenoids, whereas Corallinoideae in Corallinales contained ANT-type carotenoids. In Rhodymeniophycidae, most orders contained LUT-type carotenoids; however, only Gracilariales contained ANT-type carotenoids. There is a clear relationship between carotenoid composition and phylogenetics in Rhodophyta. Furthermore, we searched open genome databases of several red algae for references to the synthetic enzymes of the carotenoid types detected in this study. ß-Carotene and zeaxanthin might be synthesized from lycopene, as in land plants. Antheraxanthin might require zeaxanthin epoxydase, whereas α-carotene and lutein might require two additional enzymes, as in land plants. Furthermore, Glaucophyta contained ZEA-type carotenoids, and Cryptophyta contained ß-carotene, α-carotene, and alloxanthin, whose acetylenic group might be synthesized from zeaxanthin by an unknown enzyme. Therefore, we conclude that the presence or absence of the four enzymes is related to diversification of carotenoid composition in these three phyla.

Antheraxanthin: Insights delving from biosynthesis to processing effects.

Antheraxanthin (C40H56O3) is one of fat-soluble carotenoids belonging to natural pigments. Its chemical structure is based on the unsaturated polyene chain skeleton, with a hydroxy-ß-ionone ring and an epoxy-ß-ionone ring on each side of the skeleton. It is found in a wide range of plants and photosynthetic bacteria, and external stimuli (high temperature, drought, ozone treatment, etc.) can significantly affect its synthesis. It also, like other carotenoids, exhibits a diverse potential pharmacological profile as well as nutraceutical values. However, it is worth noting that various food processing methods (extrusion, puffing, baking, etc.) and storage conditions for fruits and vegetables have distinct impacts on the bioaccessibility and retention of antheraxanthin. This compilation of antheraxanthin includes sources, biosynthesis, chemical analysis, and processing effects.

Combined dynamics of the 500-600 nm leaf absorption and chlorophyll fluorescence changes in vivo: Evidence for the multifunctional energy quenching role of xanthophylls.

Carotenoids (Cars) regulate the energy flow towards the reaction centres in a versatile way whereby the switch between energy harvesting and dissipation is strongly modulated by the operation of the xanthophyll cycles. However, the cascade of molecular mechanisms during the change from light harvesting to energy dissipation remains spectrally poorly understood. By characterizing the in vivo absorbance changes (ΔA) of leaves from four species in the 500-600 nm range through a Gaussian decomposition, while measuring passively simultaneous Chla fluorescence (F) changes, we present a direct observation of the quick antenna adjustments during a 3-min dark-to-high-light induction. Underlying spectral behaviours of the 500-600 nm ΔA feature can be characterized by a minimum set of three Gaussians distinguishing very quick dynamics during the first minute. Our results show the parallel trend of two Gaussian components and the prompt Chla F quenching. Further, we observe similar quick kinetics between the relative behaviour of these components and the in vivo formations of antheraxanthin (Ant) and zeaxanthin (Zea), in parallel with the dynamic quenching of singlet excited chlorophyll a (1Chla*) states. After these simultaneous quick kinetical behaviours of ΔA and F during the first minute, the 500-600 nm feature continues to increase, indicating a further enhanced absorption driven by the centrally located Gaussian until 3 min after sudden light exposure. Observing these precise underlying kinetic trends of the spectral behaviour in the 500-600 nm region shows the large potential of in vivo leaf spectroscopy to bring new insights on the quick redistribution and relaxation of excitation energy, indicating a key role for both Ant and Zea.

Carotenoid content of extruded and puffed products made of colored-grain wheats.

Wheat is a relevant source not only of essential macronutrients but also of many other health-promoting phytochemicals (carotenoids, anthocyanins, tocols, phenolic acids, etc.). Colored-grain wheats were used for extrusion and kernel puffing. The total content of carotenoids (sum of lutein, zeaxanthin, antheraxanthin, α- and ß-carotene, and xanthophyll esters) decreased significantly due to extrusion (to 25.7%) and puffing (to 31.6%), compared to the content in the raw material. Zeaxanthin was shown to be the most stable among all detected carotenoids (30.8 and 48.7% was preserved). The results of the performed analyses have not confirmed greater stability of xanthophyll esters against higher temperatures (decrease to 29.5 and 22.1%). Both technologies induced E-to Z-isomerization of all-E-lutein and puffing also of all-E-zeaxanthin. Higher concentrations of 13-Z- and 9-Z-zeaxanthin were identified in puffed grains (2× and 37× on average). To preserve more carotenoids, it is appropriate to look for a more suitable food processing technology.

Integrative analysis of metabolome and transcriptome reveals the mechanism of color formation in pepper fruit (Capsicum annuum L.).

To understand the mechanism of the color formation of pepper fruit, integrative analysis of the metabolome and transcriptome profiles was performed in pepper varieties with 4 different fruit colors. A total of 188 flavonoids were identified, and most of the anthocyanins, flavonols and flavones showed markedly higher abundances in purple variety than in other varieties, which was linked to the high expression of flavonoid synthesis and regulatory genes. Using weighted gene co-expression network analyses, modules related to flavonoid synthesis and candidate genes that regulate flavonoid synthesis and transport were identified. Furthermore, the analysis of 12 carotenoids showed that the content of xanthophylls at 50 days after anthesis was significantly different between the four pepper varieties, which was resulted from the differential expressions of genes downstream of the carotenoid pathway. Our results provide new insights into the understanding of the synthesis and accumulation of flavonoids and carotenoids in pepper fruit.

Characterization and the impact of in vitro simulated digestion on the stability and bioaccessibility of carotenoids and their esters in two Pouteria lucuma varieties.

Lucuma is a starchy orange-yellow fruit native to the Andean region. It is widely consumed in Latin America and has been recently adapted to the agronomical characteristics of the south region of Spain. However, its carotenoid profile has never been reported. The aim of this study was to characterize the carotenoid and carotenoid ester composition of lucuma pulps (var. Molina and Beltran) and assess their bioaccessibility with an in vitro simulated gastrointestinal digestion according to the INFOGEST® methodology. The carotenoid profile in lucuma pulps revealed a high qualitative diversity composed of 33 compounds, corresponding to 9 free xanthophylls, 9 hydrocarbon carotenes and 15 xanthophyll esters. (13Z)-violaxanthin, (all-E)-violaxanthin and (all-E)-antheraxanthin were the most abundant carotenoids in lucuma fruits and were naturally present as xanthophyll esters: (all-E)-antheraxanthin 3-O-palmitate, (all-E)-violaxanthin laurate and (all-E)-violaxanthin palmitate. Carotenoids were stable during in vitro digestion; however, their release from the food matrix was limited which contributed to their low bioaccessibility.

Genuine Carotenoid Profiles in Sweet Orange [Citrus sinensis (L.) Osbeck cv. Navel] Peel and Pulp at Different Maturity Stages.

The carotenogenesis in the endocarp and flavedo of Navel oranges over four consecutive maturity stages was assessed by high-performance liquid chromatography-diode array detection-atmospheric pressure chemical ionization-multistage mass spectrometry. After optimization of the extraction method, 77 carotenoids, including 26 monoesters and 33 diesters of violaxanthin, ß-citraurin, and antheraxanthin, were characterized. Whereas chloroplast-specific pigments, such as (all-E)-lutein and (all-E)-ß-carotene, predominated in the flavedo of green-ripe fruit, a highly complex pattern of xanthophyll esters was found in the mature oranges. Total carotenoid contents of flavedo were approximately 9-fold higher [12 605 µg/100 g of fresh weight (FW)] than those in the endocarp (1354 µg/100 g of FW) at the fully mature stage. The mature endocarp abundantly contained violaxanthin mono- and diesters, in addition to diverse antheraxanthin esters, which were exclusively detected in this fruit fraction. Likewise, ß-citraurin esters were found to be unique flavedo constituents of mature fruit. Therefore, they may support the detection of fraudulent use of peel fractions during orange juice production.

Carotenoid changes of colored-grain wheat flours during bun-making.

Colored-grain wheat genotypes were used in the preparation of flour, dough, buns, and buns stored for a short period of time. The main carotenoid in all genotypes was lutein, followed by its esters, zeaxanthin, and ß-carotene, while antheraxanthin and α-carotene occurred only at negligible levels. The highest carotenoid contents were observed in yellow- and purple-grained genotypes. After the preparation of dough, total carotenoid content (TCC) decreased significantly by an average of 61.5%. Zeaxanthin was shown to be stable, whereas α-carotene was destroyed. In baked buns, the average decrease of TCC and all-E-lutein was lower than in unbaked dough. Greater decreases were recorded for esters, antheraxanthin, and ß-carotene. After storing buns for 24 h at room temperature, approximately one-quarter of TCC observed in the original flour was preserved. Z-Isomers of lutein occurred in minor concentrations, but the degradation of this component, and that of zeaxanthin, was low, suggesting E- to Z-isomerization.

Antioxidant Activities of the Antheraxanthin-related Carotenoids, Antheraxanthin, 9-cis-Antheraxanthin, and Mutatoxanthins.

In this study, we investigated the antioxidant activities of antheraxanthin-related carotenoids. Antheraxanthin and 9-cis-antheraxanthin were prepared from persimmon and orange fruit, respectively, and converted to other carotenoids under acidic conditions. Resulting carotenoids were purified using preparative silica gel HPLC, and their structures were analyzed in detail by NMR spectra. Both antheraxanthin and 9-cis-antheraxanthin were found to be converted to (8R)- and (8S)-mutatoxanthin at an approximate ratio of 3:2. High antioxidant activities were observed for antheraxanthin, 9-cis-antherxanthin, (8R)-mutatoxanthin, and (8S)-mutatoxanthin, with potent lipid peroxidation inhibitory and moderate 1O2-quenching activities.

Free and esterified carotenoids in pigmented wheat, tritordeum and barley grains.

Carotenoids are important phytonutrients responsible for the yellow endosperm color in cereal grains. Five carotenoids, namely lutein, zeaxanthin, antheraxanthin, α- and ß-carotene, were quantified by HPLC-DAD-MS in fourteen genotypes of wheat, barley and tritordeum harvested in Czechia in 2014 and 2015. The highest carotenoid contents were found in yellow-grained tritordeum HT 439 (12.16µg/gDW), followed by blue-grained wheat V1-131-15 (7.46µg/gDW), and yellow-grained wheat TA 4024 (7.04µg/gDW). Comparing carotenoid contents, blue varieties had lower whereas purple ones had the same or higher levels than conventional bread wheat. Lutein was the main carotenoid found in wheat and tritordeum while zeaxanthin dominated in barley. The majority of cereals contained considerable levels of esterified forms (up to 61%) of which lutein esters prevailed. It was assessed that cereal genotype determines the proportion of free and esterified forms. High temperatures and drought during the growing season promoted carotenoid biosynthesis.

Carotenoid profiling of yams: Clarity, comparisons and diversity.

Screening carotenoids of elite accessions of yam (Dioscorea spp.) used in the global yam breeding program has been conducted to quantitatively determine the carotenoid composition of the crop. Comparisons to previous data reporting cerotenoid levels in yam has been made, in order to deduce greater perspectives across multiple studies. Characterisation of complex species and accession -specific profiles have shown a rich base of diversity that can inform breeding strategies. Key findings include; (i) the identification of accessions rich in ß-carotene which can aid provitamin A biofortification, (ii) Data disputing the commonly held belief that yellow Guinea yam (D. cayennensis) has higher ß-carotene content than that of white Guinea yam (D. rotundata), and (iii) the tentative identification of C25-epoxy-apocarotenoid persicaxanthin with potential implications for tuber dormancy.

Characterization of carotenoid profile of Spanish Sanguinos and Verdal prickly pear (Opuntia ficus-indica, spp.) tissues.

Carotenoid profiles of different tissues (peel, pulp and whole fruit) of Spanish Sanguinos (red) and Verdal (orange) prickly pears (Opuntia ficus-indica spp.) have been characterized in detail and quantified for the first time. Carotenoids were determined by HPLC-PDA-MS (APCI+), using a reverse phase C30 column. A total of 9 xantophylls and 4 hydrocarbon carotenes were identified. Also, minor amounts of chlorophyll a, a' and b can be observed in Opuntia peel extracts. All carotenoids were found to be present in their free form (no carotenoid esters were detected). The RAE was highest in Opuntia peels, showing values from 19.20 to 16.48µg/100g fresh weigth, for Sanguinos and Verdal Opuntia fruits, respectively. The main carotenoid in Opuntia peel extracts was (all-E)-lutein with 1132.51 and 767.98µg/100g fresh weigth, followed by (all-E)-ß-carotene with 200.40 and 173.50µg/100g fresh weigth for Sanguinos and Verdal varieties of Opuntia fruits, respectively.

Carotenoid profile and retention in yellow-, purple- and red-fleshed potatoes after thermal processing.

This research aimed to investigate the effect of thermal processing on carotenoid profile, quantity and stability in 22 colour-fleshed potato cultivars grown in the Czech Republic. The total of nine carotenoids was analysed by HPLC using a C30 column and PDA detection. The total carotenoid content for all cultivars ranged from 1.44 to 40.13 µg/g DM. Yellow cultivars showed a much higher average total carotenoid content (26.22 µg/g DM) when compared to red/purple-fleshed potatoes (5.69 µg/g DM). Yellow cultivars were dominated by antheraxanthin, whereas neoxanthin was the main carotenoid in red/purple cultivars. Thermal processing significantly impacted all potato cultivars. Boiling decreased the total carotenoids by 92% compared to baking (88%). Lutein was the most stable carotenoid against thermal processing (decreased by 24-43%) followed by ß-carotene (decreased by 78-83%); other carotenoids were degraded nearly completely. Increased formation of (Z)-isomers by thermal processing has not been confirmed.

Carotenoid composition of strawberry tree (Arbutus unedo L.) fruits.

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

Colour and carotenoid changes of pasteurised orange juice during storage.

The correlation of carotenoid changes with colour degradation of pasteurised single strength orange juice was investigated at 20, 28, 35 and 42°C for a total of 32 weeks of storage. Changes in colour were assessed using the CIELAB system and were kinetically described by a zero-order model. L(∗), a(∗), b(∗), ΔE(∗), Cab(∗) and hab were significantly changed during storage (p<0.05). Activation energies for all colour parameters were 64-73 kJ mol(-1). Several carotenoids showed important changes and appeared to have different susceptibilities to storage. A decrease of ß-cryptoxanthin was observed at higher temperatures, whereas antheraxanthin started to decrease at lower temperatures. Depending on the time and temperature, changes in carotenoids could be due to isomerisation reactions, which may lead to a perceptible colour change. Although the contribution of carotenoids was recognised to some extent, other reactions seem of major importance for colour degradation of orange juice during storage.

Xanthophyll cycle pool size and composition in several Cosmarium strains (Zygnematophyceae, Streptophyta) are related to their geographic distribution patterns.

The photosynthetic behaviour and composition of photosynthetic pigments of four Cosmarium strains collected from different geographic areas were examined under moderate and photoinhibitory white light by means of PAM fluorometry and high-performance liquid chromatography. Generally, all of the Cosmarium strains displayed the photosynthetic performance and the composition of xanthophyll cycle pigments corresponding to that of high-light adapted plants and algae, when grown under the standard laboratory conditions. However, photoinhibitory treatments provoked several strain- and species-specific characteristics despite the long-term cultivation in laboratory conditions. The typical arctic taxon, C. crenatum var. boldtianum, displayed an incomplete violaxanthin cycle yielding an accumulation of antheraxanthin during high light stress, which is considered as an adaptation to occasional high irradiances in the polar zone due to the albedo. So far, the violaxanthin/antheraxanthin turnover was known only in some prasinophycean algae. Antheraxanthin actively participated in the heat dissipation from PSII centres in C. crenatum, as concluded from a significant positive correlation between non-photochemical quenching (NPQ) and the quantity of antheraxanthin. In contrast, all the other Cosmarium strains displayed a complete violaxanthin de-epoxidase action during the high light treatments, as judged from the relatively high production of zeaxanthin which participated in thermal dissipation of excess energy.