Enhancing the carotenoid content of <Emphasis Type="Italic">Brassica napus</Emphasis> seeds by downregulating lycopene epsilon cyclase

The accumulation of carotenoids in higher plants is regulated by the environment, tissue type and developmental stage. In Brassica napus leaves, beta-carotene and lutein were the main carotenoids present while petals primarily accumulated lutein and violaxanthin. Carotenoid accumulation in seeds was developmentally regulated with the highest levels detected at 35-40 days post anthesis. The carotenoid biosynthesis pathway branches after the formation of lycopene. One branch forms carotenoids with two beta rings such as beta-carotene, zeaxanthin and violaxanthin, while the other introduces both beta- and epsilon-rings in lycopene to form alpha-carotene and lutein. By reducing the expression of lycopene epsilon-cyclase (epsilon-CYC) using RNAi, we investigated altering carotenoid accumulation in seeds of B. napus. Transgenic seeds expressing this construct had increased levels of beta-carotene, zeaxanthin, violaxanthin and, unexpectedly, lutein. The higher total carotenoid content resulting from reduction of epsilon-CYC expression in seeds suggests that this gene is a rate-limiting step in the carotenoid biosynthesis pathway. epsilon-CYC activity and carotenoid production may also be related to fatty acid biosynthesis in seeds as transgenic seeds showed an overall decrease in total fatty acid content and minor changes in the proportions of various fatty acids.     

QTL and candidate genes phytoene synthase and ζ-carotene desaturase associated with the accumulation of carotenoids in maize

Carotenoids are a class of fat-soluble antioxidant vitamin compounds present in maize (Zea mays L.) that may provide health benefits to animals or humans. Four carotenoid compounds are predominant in maize grain: -carotene, -cryptoxanthin, zeaxanthin, and lutein. Although -carotene has the highest pro-vitamin A activity, it is present in a relatively low concentration in maize kernels. We set out to identify quantitative trait loci (QTL) affecting carotenoid accumulation in maize kernels. Two sets of segregating families were evaluated—a set of F_2:3 lines derived from a cross of W64a x A632, and their testcross progeny with AE335. Molecular markers were evaluated on the F_2:3 lines and a genetic linkage map created. High-performance liquid chromatography was performed to measure -carotene, -cryptoxanthin, zeaxanthin, and lutein on both sets of materials. Composite interval mapping identified chromosome regions with QTL for one or more individual carotenoids in the per se and testcross progenies. Notably QTL in the per se population map to regions with candidate genes, yellow 1 and viviparous 9, which may be responsible for quantitative variation in carotenoids. The yellow 1 gene maps to chromosome six and is associated with phytoene synthase, the enzyme catalyzing the first dedicated step in the carotenoid biosynthetic pathway. The viviparous 9 gene maps to chromosome seven and is associated with -carotene desaturase, an enzyme catalyzing an early step in the carotenoid biosynthetic pathway. If the QTL identified in this study are confirmed, particularly those associated with candidates genes, they could be used in an efficient marker-assisted selection program to facilitate increasing levels of carotenoids in maize grain.Communicated by P. Langridge     

Carotenoid Components in Soybean Seeds Varying with Seed Color and Maturation Stage

The difference in carotenoid components among various color types of soybean seeds, and the changes in carotenoid composition during seed development were examined by reverse-phase high-performance liquid chromatogrphy (HPLC). Lutein was the major carotenoid component in seed extracts from the common yellow soybean and from a variety having a black seed coat. Green soybean seeds contained several xanthophylls in addition to lutein. None of the mature soybean seeds contained β-carotene, a part from a trace amount being detected in a local variety of green soybean. The total carotenoid and lutein contents were higher in green soybeans than in the yellow types, and the estimated total amount of carotenoids correlates with that of chlorophylls. The thylakoid membrane residue in the plastids of green soybean had lost its functional lamella structure. Immature soybean seeds contained a green-vegetable type of carotenoids including α- and β-carotene. The amount of β-carotene decreased more rapidly than that of lutein and chlorophylls during seeds maturation. These results suggest that β-carotene, which acts as a photo-protective agent in developing seeds, is susceptible to degradation in the course of seed maturation.     

Lycopene and beta-carotene decompose more rapidly than lutein and zeaxanthin upon exposure to various pro-oxidants in vitro

Major carotenoids of human plasma and tissues were exposed to radical-initiated autoxidation conditions. The consumption of lutein and zeaxanthin, the only carotenoids in the retina, and lycopene and beta-carotene, the most effective quenchers of singlet oxygen in plasma, were compared. Under all conditions of free radical-initiated autoxidation of carotenoids which were investigated, the breakdown of lycopene and beta-carotene was much faster than that of lutein and zeaxanthin. Under the influence of UV light in presence of Rose Bengal, by far the highest breakdown rate was found for beta-carotene, followed by lycopene. Bleaching of carotenoid mixtures mediated by NaOCl, addition of azo-bis-isobutyronitril (AIBN), and the photoirradiation of carotenoid mixtures by natural sunlight lead to the following sequence of breakdown rates: lycopene > beta-carotene > zeaxanthin > lutein. The slow degradation of the xanthophylls zeaxanthin and lutein may be suggested to explain the majority of zeaxanthin and lutein in the retina of man and other species. In correspondence to that, the rapid degradation of beta-carotene and lycopene under the influence of natural sunlight and UV light is postulated to be the reason for the almost lack of those two carotenoids in the human retina. Nevertheless, a final proof of that theory is lacking.     

Decrease of serum carotenoids in Crohn's disease.

Crohn's disease (CD) is frequently complicated by various nutritional disturbances. Although it is important to correct these disturbances, the nutritional status of CD patients has been poorly documented, especially concerning vitamin status. The aims of this study were (a) to measure the serum concentrations of vitamin A and six other carotenoids (lutein, zeaxanthin, alpha-, beta-carotene, alpha-, beta-cryptoxanthin) in patients with CD and to compare them with those in healthy controls and (b) to follow the changes of serum carotenoid levels in CD patients during treatment. Twenty-eight patients with CD and 23 healthy persons were included in this study. The results of twelve patients were followed up through one year. The patients were free of any nutritional treatment. The serum concentrations of carotenoids were measured with high-pressure liquid chromatography (HPLC). The serum concentrations of five carotenoids were significantly lower in the patients than in the controls (vitamin A, zeaxanthin: P < 0.001; alpha-, beta-carotene: P < 0.01; lutein: P < 0.05). The carotenoid status of the followed patients advanced to the normal range, but this increase was not significant. These findings suggest that there is a deficiency of vitamin A and its provitamins in Crohn' s disease prior to treatment. However, because we did not evaluate the vitamin intake in this study, we could not conclude which of the factors--poor intake, increased requirement, or malabsorption--was more important in decreasing of carotenoid levels.     

Genetic control of grain yield and its related traits in bread wheat

Summary Genetic control of tiller number, grain number, grain weight, harvest index and grain yield in six generations, along with the biparentals, F₃s, F₂xparental progeny, and F₂xF₁ progeny were investigated in an intervarietal cross of bread wheat involving two highly competitive varieties, WL711 and HD 2009. The performance of F₁, B₁, B₂, F₂, × p₁, F₂ × P₂ and F₂ × F₁ progeny was midway between the parents involved with respect to all the evaluated characters. The biparental progeny excelled the mean performance of their corresponding F₂ and F₃ progeny in tiller number, seed weight and grain yield. The estimates of variance components obtained from the two models deployed were almost similar. Considerable additive genetic variance was observed for grains per spike, seed weight and grain yield while dominance variance was more pronounced for harvest index. The additive-dominance model was adequate for grains per spike and harvest index. Epistatic effects of additive × additive and additive × dominance type for tiller number and grain yield, and of additive × dominance type for seed weight were observed. The digenic epistatic model was inadequate for explaining the nature of gene action for tiller number, seed weight and grain yield. The studies indicated that non-allelic interactions should not be ignored in formulating wheat breeding programmes and that a biparental approach could be adopted as an extremely useful tool for enhancing genetic variability and the creation of transgressive segregants. The usefulness of breeding methodologies utilising a biparental approach is discussed.     

Validation of overweight children's fruit and vegetable intake using plasma carotenoids

Assessing dietary intake in children is difficult and limited validated tools exist. Plasma carotenoids are nutritional biomarkers of fruit and vegetable intake and therefore suitable to validate reported dietary intakes. The aim of this study was to examine the comparative validity of a food frequency questionnaire (FFQ), completed by parents reporting child fruit and vegetable intake compared to plasma carotenoid concentrations. A sample of children aged 5-12 years (n = 93) from a range of weight categories were assessed. Dietary intake was measured using a 137-item semi-quantitative FFQ. Plasma carotenoids were measured using reverse phase high-performance liquid chromatography. Pearson correlation coefficients between reported dietary intake of carotenoids and plasma carotenoid concentrations were strongest after adjustment for BMI (beta-carotene (r = 0.56, P < 0.05), alpha-carotene (r = 0.51, P < 0.001), cryptoxanthin (r = 0.32, P < 0.001)). Significantly lower levels (P < 0.05) of all plasma carotenoids, except lutein, were found among overweight and obese children when compared to healthy weight children. Parental report of children's carotenoid intakes, using a FFQ can be used to provide a relative validation of fruit and vegetable intake. The lower plasma carotenoid concentrations found in overweight and obese children requires further investigation.     

Carotenoid content in Anguilla anguilla (L.) individuals undertaking spawning migration

The authors investigated the carotenoid content in different parts of Anguilla anguilla (L.) undertaking spawning migration, in spring, summer and autumn. By means of column and thin-layer chromatography, the following carotenoids were found to be present: beta-carotene, epsilon-carotene, beta-cryptoxanthin, neothxanthin, lutein, tunaxanthin, zeaxanthin, lutein epoxide, 3'-hydroxyechincnone, canthaxanthin, idoxanthin, phoenicoxanthin, alpha-doradexanthin, beta-doradexanthin and astaxanthin. In the eel examined individuals a different carotenoid content was found in October. In winter when eels do not feed and therefore do not absorb carotenoids, carotenoid content decreases in the liver, the intestines, and particularly in the muscles. In spring when eels undertake active life carotenoid concentration increases rapidly in these organs within a month. In summer during intensive predation, carotenoid concentration in the muscles reaches a maximum.     

Genetic mapping of ovary colour and quantitative trait loci for carotenoid content in the fruit of <Emphasis Type="Italic">Cucurbita maxima</Emphasis> Duchesne

The high content of carotenoids, sugars, dry matter, vitamins and minerals makes the fruit of winter squash (Cucurbita maxima Duchesne) a valuable fresh-market vegetable and an interesting material for the food industry. Due to their nutritional value, long shelf-life and health protective properties, winter squash fruits have gained increased interest from researchers in recent years. Despite these advantages, the genetic and genomic resources available for C. maxima are still limited. The aim of this study was to use the genetic mapping approach to map the ovary colour locus and to identify the quantitative trait loci (QTLs) for high carotenoid content and flesh colour. An F₆ recombinant inbred line (RIL) mapping population was developed and used for evaluations of ovary colour, carotenoid content and fruit flesh colour. SSR markers and DArTseq genotyping-by-sequencing were used to construct an advanced genetic map that consisted of 1824 molecular markers distributed across linkage groups corresponding to 20 chromosomes of C. maxima. Total map length was 2208 cM and the average distance between markers was 1.21 cM. The locus affecting ovary colour was mapped at the end of chromosome 14. The identified QTLs for carotenoid content in the fruit and fruit flesh colour shared locations on chromosomes 2, 4 and 14. QTLs on chromosomes 2 and 4 were the most meaningful. A correlation was clearly confirmed between fruit flesh colour as described by the chroma value and carotenoid content in the fruit. A high-density genetic map of C. maxima with mapped loci for important fruit quality traits is a valuable resource for winter squash improvement programmes.     

Oxidation of carotenoids by heat and tobacco smoke

The stability to autoxidation of the polar carotenoids, lutein and zeaxanthin, was compared to that of the less polar carotenoids, beta-carotene and lycopene at physiologically or pathophysiologically relevant concentrations of 2 and 6 microM, after exposure to heat or cigarette smoke. Three methodological approaches were used: 1) Carotenoids dissolved in solvents with different polarities were incubated at 37 and 80 degrees C for different times. 2) Human plasma samples were subjected to the same temperature conditions. 3) Methanolic carotenoid solutions and plasma were also exposed to whole tobacco smoke from 1-5 unfiltered cigarettes. The concentrations of individual carotenoids in different solvents were determined spectrophotometrically. Carotenoids from plasma were extracted and analyzed using high performance liquid chromatography. Carotenoids were generally more stable at 37 than at 80 degrees C. In methanol and dichloromethane the thermal degradation of beta-carotene and lycopene was faster than that of lutein and zeaxanthin. However, in tetrahydrofuran beta-carotene and zeaxanthin degraded faster than lycopene and lutein. Plasma carotenoid levels at 37 degrees C did not change, but decreased at 80 degrees C. The decrease of beta-carotene and lycopene levels was higher than those for lutein and zeaxanthin. Also in the tobacco smoke experiments the highest autoxidation rates were found for beta-carotene and lycopene at 2 microM, but at 6 microM lutein and zeaxanthin depleted to the same extent as beta-carotene. These data support our previous studies suggesting that oxidative stress degrade beta-carotene and lycopene faster than lutein and zeaxanthin. The only exception was the thermal degradation of carotenoids solubilized in tetrahydrofuran, which favors faster breakdown of beta-carotene and zeaxanthin.     

The solubilisation pattern of lutein, zeaxanthin, canthaxanthin and beta-carotene differ characteristically in liposomes, liver microsomes and retinal epithelial cells

The incorporation efficiencies of lutein, zeaxanthin, canthaxanthin and beta-carotene into Retinal Pigment Epithelial (RPE) cells (the human RPE cell line D 407), liver microsomes and EYPC liposomes are investigated. In RPE cells the efficiency ratio of lutein and zeaxanthin compared to canthaxanthin and beta-carotene is higher than in the other membranes. The preferential interactions of lutein and zeaxanthin with RPE cells are discussed considering special protein binding properties. Incorporation yields were obtained from the UV-Vis spectra of the carotenoids. Membrane modulating effects of the carotenoids were obtained from the fluorescence spectra of co-incorporated Laurdan (6-dodecanoyl-2-dimethylaminonaphtalene). The Laurdan fluorescence quenching efficiencies of the membrane bound carotenoids offer an access to direct determinations of membrane carotenoid concentrations. Fetal calf serum as carrier for carotenoid incorporation appears superior to tetrahydrofuran.     

Development of an integrated intraspecific map of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) using two recombinant inbred line populations

A composite intraspecific linkage map of chickpea was developed by integrating individual maps developed from two F_8:9 RIL populations with one common parent. Different molecular markers viz. RAPD, ISSR, RGA, SSR and ASAP were analyzed along with three yield related traits: double podding, seeds per pod and seed weight. A total of 273 markers and 186 RILs were used to generate the map with eight linkage groups at a LOD score of ≥3.0 and maximum recombination fraction of 0.4. The map spanned 739.6 cM with 230 markers at an average distance of 3.2 cM between markers. The predominantly used SSR markers facilitated identification of homologous linkage groups from the previously published interspecific linkage map of chickpea and confirmed conservation of the SSR markers across the two maps as well as the variation in terms of marker distance and order. The double podding gene was tagged by the markers NCPGR33 and UBC249z at 2.0 and 1.1 cM, respectively. Whereas, seeds per pod, was tagged by the markers TA2x and UBC465 at 0.1 and 1.8 cM, respectively. Eight QTLs were identified that influence seed weight. The joint map approach allowed mapping a large number of markers with a moderate coverage of the chickpea genome and few linkage gaps. P. Radhika and S.J.M. Gowda contributed equally to this study.     

Seed-specific overexpression of an endogenous Arabidopsis phytoene synthase gene results in delayed germination and increased levels of carotenoids,chlorophyll, and abscisic acid

Phytoene synthase catalyzes the dimerization of two molecules of geranylgeranyl pyrophosphate to phytoene and has been shown to be rate limiting for the synthesis of carotenoids. To elucidate if the capacity to produce phytoene is limiting also in the seed of Arabidopsis (Wassilewskija), a gene coding for an endogenous phytoene synthase was cloned and coupled to a seed-specific promoter, and the effects of the overexpression were examined. The resulting transgenic plants produced darker seeds, and extracts from the seed of five overexpressing plants had a 43-fold average increase of beta-carotene and a total average amount of beta-carotene of approximately 260 microg g-1 fresh weight. Lutein, violaxanthin, and chlorophyll were significantly increased, whereas the levels of zeaxanthin only increased by a factor 1.1. In addition, substantial levels of lycopene and alpha-carotene were produced in the seeds, whereas only trace amounts were found in the control plants. Seeds from the transgenic plants exhibited delayed germination, and the degree of delay was positively correlated with the increased levels of carotenoids. The abscisic acid levels followed the increase of the carotenoids, and plants having the highest carotenoid levels also had the highest abscisic acid content. Addition of gibberellic acid to the growth medium only partly restored germination of the transgenic seeds.     

The effect of enhanced carotenoid content of transgenic maize grain on fungal colonization and mycotoxin content

Novel strategies that address vitamin A deficiency have been developed, such as high-carotenoid maize, a biofortified transgenic maize line rich in carotenoids generated by genetic transformation. The South African white maize inbred (M37W), which is devoid of carotenoids, was engineered to accumulate high levels of β-carotene (provitamin A), lutein, and zeaxanthin. Maize seeds contaminated with fumonisins and other mycotoxins pose a serious threat to both humans and livestock. During three consecutive harvests, the fungal incidence and the fumonisin and aflatoxin content of maize seeds grown in an experimental field in Catalonia (Northeastern Spain) were evaluated. Fungal infection was similar in high-carotenoid maize and its isogenic line, with Fusarium verticillioides being the most prevalent fungus in all the harvests. Neither Aspergillus spp. nor aflatoxin contamination was found. Fumonisin levels were lower in high carotenoid than in its isogenic line, but this reduction was statistically significant in only 2 of the 3 years of study. Our results suggest that high carotenoid content reduces fumonisin levels in maize grains.     

Effects of light and nitrogen starvation on the content and composition of carotenoids of the green microalga <Emphasis Type="Italic">Parietochloris incisa</Emphasis>

The changes in pigment content and composition of the unicellular alga Parietochloris incisa comb. nov (Trebouxiophyceae, Chlorophyta) were studied. This alga is unique in its ability to accumulate high amounts of arachidonic acid in the cell during cultivation under different irradiances and nitrogen availability in the medium. Under low irradiance of 35 μE/(m₂ s) photosynthetically active radiation the P. incisa cultures possessed slow growth and a relatively low carotenoid-to-chlorophyll ratio. At higher irradiances (200 and 400 μE/(m₂ s)) on complete medium, the alga displayed higher growth rate and an increase in the carotenoid content, especially that of β-carotene and lutein. Both on nitrogen-free (regardless of illumination intensity) and nitrogen-replete medium (under high light), a considerable increase in the ratio of carotenoid and chlorophyll contents was recorded. Predominant accumulation of xanthophylls took place in thylakoid membranes, whereas β-carotene deposition occurred mainly in the cytoplasmic lipid globules (oil bodies); lower amounts of carotenoids were accumulated in the absence of nitrogen. Under high light and nitrogen-deficiency conditions, an increase in violaxanthin de-epoxidation and nonphotochemical quenching was recorded together with a decline in variable chlorophyll fluorescence (F _v/F _m) level. A possible photoprotective role of carotenoids in adaptation of P. incisa to high light under nitrogen starvation conditions is discussed.     

Role of chlorophylls and carotenoids in seed tolerance to abiotic stressors

It is found that chlorophylls are not fully destructed during seed ripening and can be detected in appreciable quantities in physiologically mature seeds. The elevated content of residual chlorophylls reduces seed tolerance to abiotic stresses. The seed carotenoids were represented mainly by lutein and, in much smaller quantities, by β-carotene. Carotenoids were found to accumulate in seeds during accelerated aging and during seed germination at high temperatures. The ratio of carotenoid to chlorophyll content (Car/Chl) is proposed to be a measure of seed tolerance to stress factors. The seeds with elevated Car/Chl ratio were characterized by higher tolerance to stress treatments. It is supposed that the presence of chlorophylls in seeds enhances oxidative stress induced by abiotic stress factors. Carotenoids are considered as antioxidants protecting the seeds against oxidative stress.     

Variations in the carotenoid content of Chlamydomonas reinhardii throughout the cell cycle.

Synchronous cultures of Chlamydomonas reinhardii have been examined for the total amounts of carotenoid and chlorophyll present throughout a 12 hrs light -- 4 hrs dark life cycle. Variations in the carotenoid distribution at different points within the cell cycle have been found. During the greater part of the light period all major carotenoids increased at a proportionally similar rate. However, the increases in lutein and violaxanthin preceded those in beta-carotene and neoxanthin by some 2 hrs and that in loroxanthin, and algal xanthophyll, by abour 3 hrs. A marked drop in total carotenoid accumulation, corresponding to similar temporary falling away in the accumulation of beta-carotene, lutein and violaxanthin occurred at 9 hrs. The correspondence of this with the established drop in RNA accumulation and the break-up of the nucleolus was pointed out. Considerable redistribution among the carotenoids occurred during the dark period, notably the amount of beta-carotene increased relative to the total xanthophylls. The full significance of these results can not be estimated in the absence of comparative data on related organisms.     

Carotenoid content in Lota lota (L.) individuals in various biological activity periods

The authors investigated the carotenoid content in the particular parts of Lota lota in summer, autumn, and winter, i.e. when burbots exhibit the lowest and highest activity. By means of columnar and thin-layer chromatography, the following carotenoids were found to be present: alpha-carotene, beta-carotene, e-carotene, beta-cryptoxanthin, neothxanthin, lutein, 3'-epilutein, zeaxanthin, tunaxanthin, antheraxanthin, lutein epoxide, echinenone, 3'-hydroxyechinenone, idoxanthin, canthaxanthin, alpha-doradexanthin, beta-doradexanthin, astaxanthin, diatoxanthin, parasiloxanthin, monadoxanthin, 7,8-dihydroparasiloxanthin, mutatoxanthin and rhodoxanthin. In the Lota lota individuals examined, the content of carotenoids was found to differ in winter and summer. The total carotenoid content ranged from 0.067 (gonads of males) of to 6.095 micrograms g-1 wet weight (fins of males from December).     

Mapping of the genomic regions controlling seed storability in soybean (Glycine max L.)

Seed storability is especially important in the tropics due to high temperature and relative humidity of storage environment that cause rapid deterioration of seeds in storage. The objective of this study was to use SSR markers to identify genomic regions associated with quantitative trait loci (QTLs) controlling seed storability based on relative germination rate in the F_2:3 population derived from a cross between vegetable soybean line (MJ0004-6) with poor longevity and landrace cultivar from Myanmar (R18500) with good longevity. The F_2:4 seeds harvested in 2011 and 2012 were used to investigate seed storability. The F₂ population was genotyped with 148 markers and the genetic map consisted of 128 SSR loci which converged into 38 linkage groups covering 1664.3 cM of soybean genome. Single marker analysis revealed that 13 markers from six linkage groups (C1, D2, E, F, J and L) were associated with seed storability. Composite interval mapping identified a total of three QTLs on linkage groups C1, F and L with phenotypic variance explained ranging from 8.79 to 13.43%. The R18500 alleles increased seed storability at all of the detected QTLs. No common QTLs were found for storability of seeds harvested in 2011 and 2012. This study agreed with previous reports in other crops that genotype by environment interaction plays an important role in expression of seed storability.     

Comparative Study of the Carotenoid Composition of the Seeds of Ripening Momordica charantia and Tomatoes

The total carotenoid concentration of the seeds of Momordica charantia rose about 100-fold from the immature to the ripe stage. The massive increase was almost exclusively attributable to lycopene, which accounted for 96% of the carotenoids of the ripe seeds. The carotenoid pattern of the seed was found to be drastically different from that of the pericarp. The seed, which contained fewer carotenoids, had a total concentration 12 times greater than that in the pericarp at the ripe stage. The acyclic lycopene selectively accumulated in the seed, whereas the cyclic carotenoids, cryptoxanthin, zeaxanthin and β-carotene, were the principal pigments of the ripe pericarp. The seed of ripe tomatoes reflected the qualitative pattern of the whole fruit. The total carotenoid concentration was, however, much lower and the lycopene content was particularly low. β-Carotene, having a comparatively high concentration, emerged as the major pigment of the seed.