Regulatory control of carotenoid accumulation in winter squash during storage

Main conclusion

Storage promotes carotenoid accumulation and converts amylochromoplasts into chromoplasts in winter squash. Such carotenoid enhancement is likely due to continuous biosynthesis along with reduced turnover and/or enhanced sequestration. Postharvest storage of fruits and vegetables is often required and frequently results in nutritional quality change. In this study, we investigated carotenoid storage plastids, carotenoid content, and its regulation during 3-month storage of winter squash butternut fruits. We showed that storage improved visual appearance of fruit flesh color from light to dark orange, and promoted continuous accumulation of carotenoids during the first 2-month storage. Such an increased carotenoid accumulation was found to be concomitant with starch breakdown, resulting in the conversion of amylochromoplasts into chromoplasts. The butternut fruits contained predominantly β-carotene, lutein, and violaxanthin. Increased ratios of β-carotene and violaxanthin to total carotenoids were noticed during the storage. Analysis of carotenoid metabolic gene expression and PSY protein level revealed a decreased expression of carotenogenic genes and PSY protein following the storage, indicating that the increased carotenoid level might not be due to increased biosynthesis. Instead, the increase likely resulted from a continuous biosynthesis with a possibly reduced turnover and/or enhanced sequestration, suggesting a complex regulation of carotenoid accumulation during fruit storage. This study provides important information to our understanding of carotenogenesis and its regulation during postharvest storage of fruits.     

Carotenoid Accumulation and Carotenogenic Genes Expression During Two Types of Persimmon Fruit (<Emphasis Type="Italic">Diospyros kaki</Emphasis> L.) Development

Persimmon (Diospyros kaki L.), belonging to the Ebenaceae family, has been used not only as a fresh fruit, but also for many medicinal uses. Carotenoids are the main pigment in persimmon fruit, which contribute significantly to fruit color and nutritional quality due to their composition and content. In this study, fruit quality indices, carotenoid contents and expression of carotenogenic genes were analyzed in two types of persimmon fruit. The results demonstrated that there was a positive correlation between fruit color and the contents of main composition and total carotenoids. Carotenoid accumulation in persimmon fruit resulted from the interaction of carotenogenic genes, but the molecular mechanisms responsible for accumulation of carotenoids in two types of persimmon fruit had a few differences. As a complete unit, the relatively low expression level of phytoene synthase gene (DkPSY) in “Niuxinshi” resulted in low carotenoid contents or even under the detection limit at the early fruit developmental stages; but low carotenoid contents in “Nishimurawase” were due to the relatively low expression level of carotenogenic genes other than DkPSY. At the late fruit developmental stages, increased expression levels of DkPSY, phytoene desaturase gene and beta-carotene hydroxylase gene (DkBCH) induced elevated carotenoid contents; because all carotenogenic genes strongly expressed in “Nishimurawase”, a large amount of carotenoids were accumulated. In addition, β-cryptoxanthin was the main composition whose content increased with the fruit maturity changes, which was mainly because of DkBCH which might lead more conversion of β-carotene to β-cryptoxanthin.     

Sucrose deficiency delays lycopene accumulation in tomato fruit pericarp discs

Tomato (Solanum lycopersicum) fruit ripening is characterized by a massive accumulation of carotenoids (mainly lycopene) as chloroplasts change to chromoplasts. To address the question of the role of sugars in controlling carotenoid accumulation, fruit pericarp discs (mature green fruits) were cultured in vitro in the presence of various sucrose concentrations. A significant difference in soluble sugar content was achieved depending on external sucrose availability. Sucrose limitation delayed and reduced lycopene and phytoene accumulation, with no significant effect on other carotenoids. Chlorophyll degradation and starch catabolism were not affected by variations of sucrose availability. The reduction of lycopene synthesis observed in sucrose-limited conditions was mediated through metabolic changes illustrated by reduced hexose accumulation levels. In addition, variations of sucrose availability modulated PSY1 gene expression. Taken together our results suggest that the modulation of carotenoid accumulation by sucrose availability occurs at the metabolic level and involves the differential regulation of genes involved in carotenoid biosynthesis.     

A Foundation for Provitamin A Biofortification of Maize: Genome-Wide Association and Genomic Prediction Models of Carotenoid Levels

Efforts are underway for development of crops with improved levels of provitamin A carotenoids to help combat dietary vitamin A deficiency. As a global staple crop with considerable variation in kernel carotenoid composition, maize (Zea mays L.) could have a widespread impact. We performed a genome-wide association study (GWAS) of quantified seed carotenoids across a panel of maize inbreds ranging from light yellow to dark orange in grain color to identify some of the key genes controlling maize grain carotenoid composition. Significant associations at the genome-wide level were detected within the coding regions of zep1 and lut1, carotenoid biosynthetic genes not previously shown to impact grain carotenoid composition in association studies, as well as within previously associated lcyE and crtRB1 genes. We leveraged existing biochemical and genomic information to identify 58 a priori candidate genes relevant to the biosynthesis and retention of carotenoids in maize to test in a pathway-level analysis. This revealed dxs2 and lut5, genes not previously associated with kernel carotenoids. In genomic prediction models, use of markers that targeted a small set of quantitative trait loci associated with carotenoid levels in prior linkage studies were as effective as genome-wide markers for predicting carotenoid traits. Based on GWAS, pathway-level analysis, and genomic prediction studies, we outline a flexible strategy involving use of a small number of genes that can be selected for rapid conversion of elite white grain germplasm, with minimal amounts of carotenoids, to orange grain versions containing high levels of provitamin A.     

Interspecific variation in dietary carotenoid assimilation in birds: links to phylogeny and color ornamentation

Many birds use carotenoid pigments to acquire rich red, orange, and yellow coloration in feathers and bare parts that is used as a signal of mate quality. Because carotenoids are derived from foods, much attention has been paid to the role of diet in generating color variation both within and among avian species. Less consideration has been given to physiological underpinnings of color variability, especially among species. Here, I surveyed published literature (e.g. captive feeding studies) on carotenoid assimilation in six bird species and completed additional controlled carotenoid-supplementation experiments in two others to consider the ability of different taxa to extract carotenoids from the diet in relation to phylogeny and coloration. I found that, for a given level of carotenoids in the diet, passerine birds (zebra finch, Taeniopygia guttata; house finch, Carpodacus mexicanus; American goldfinch, Carduelis tristis; society finch, Lonchura domestica) exhibit higher levels of carotenoids in circulation than non-passerines like gamebirds (domestic chicken, Gallus domesticus; red junglefowl, Gallus gallus; Japanese quail, Coturnix coturnix; red-legged partridge, Alectoris rufa). This difference in carotenoid accumulation is likely due to interspecific variation in micelle, chylomicron, or lipoprotein concentrations or affinities for xanthophyll carotenoids. Passerine birds more commonly develop carotenoid-based colors than do birds from ancient avian lineages such as Galliformes, and the physiological differences I uncover may explain why songbirds especially capitalize on carotenoid pigments for color production. Ultimately, because we can deconstruct color traits into component biochemical, physical, and physiological parts, avian color signals may serve as a valuable model for illuminating the proximate mechanisms behind interspecific variation in signal use in animals.     

Phenotypic, genetic, and environmental causes of variation in yellow skin pigmentation and serum carotenoids in Eurasian kestrel nestlings

In the context of sexual selection and parent-offspring communication, carotenoid-based coloration operates as a dynamic condition-dependent signal, as pigments stored in the skin and in the bill can be reallocated to other tissues in accordance with physiological needs. We studied the proximate factors affecting the carotenoid-dependent coloration of the Eurasian kestrel (Falco tinnunculus). Kestrel nestlings show carotenoid-based coloration at the integument level. Adult males and females share similar characteristics, but to a different extent. By cross-fostering nestlings, we evaluated the importance of the “nest of rearing” and the “nest of origin” to determine variation in skin color and blood carotenoids. The nest of rearing accounted for most of the observed variance in skin color, as well as serum carotenoids, while the nest of origin was not causal to the variability of carotenoids in young kestrels. The study indirectly shows that carotenoid-based color expressed by young kestrels is not affected by pre-laying conditions. Furthermore, we found that carotenoid coloration and blood carotenoid concentration were correlated at phenotypic and environmental levels, while the hereditary component of the carotenoid traits was too low to estimate their correlation at the genetic level.     

Effect of Dietary Carotenoid Supplementation on Food Intake and Immune Function in a Songbird with no Carotenoid Coloration

Studies of ornamental carotenoid coloration suggest that animals may have evolved specialized mechanisms for maximizing color expression and advertising their potential worth as a mate. For example, when given a choice of foods, many carotenoid‐pigmented fishes and birds select the more colorful, presumably carotenoid‐rich foods, and then accumulate these pigments at high levels in both the integument and systemically, in order to boost their immune system and hence directly advertise their health state with their colors. The majority of animals, however, do not exhibit sexually selected carotenoid coloration, which raises the question of whether they still optimize pigment intake and allocation in ways that boost endogenous accumulation and health. We tested the effect of carotenoid supplementation on food intake, carotenoid accumulation in blood, and both innate and adaptive immunity in male society finches (Lonchura domestica) – a non‐carotenoid‐colored estrildid finch relative of the zebra finch (Taeniopygia guttata; a species in which males do display sexually attractive and health‐revealing carotenoid color). Males fed a carotenoid‐rich diet for 2 wk did not consume more food than control males. Still, consumption of the carotenoid‐rich diet for 2 wk significantly elevated circulating levels of carotenoids in blood in male society finches, yielding the potential for immune enhancement. In fact, carotenoid‐enriched finches performed significantly better than control birds in our assay of constitutive innate immunity (bacterial‐killing activity of whole blood), although not in our test of inducible adaptive immunity (response to a mitogenic challenge with phytohemagglutinin). These results suggest that affinities for carotenoid‐rich foods may be particular to species with sexually selected carotenoid pigmentation, but that, as in humans and other mammals (e.g. mice, rats) without carotenoid color, the immune‐boosting action of carotenoids is conserved regardless of the strength of sexual selection on pigment use.     

Carotenoid synthesis in mustard seedlings as controlled by phytochrome and inhibitors

Summary Accumulation of carotenoids in the mustard seedling (Sinapis alba L.) is controlled by phytochrome (P_fr). Separation of the carotenoids shows that the control is quantitative rather than qualitative. Kinetic studies indicate that P_fr exerts a rapid and nearly reversible control over the rate of carotenoid accumulation. Whereas carotenoid accumulation between 36 and 60h after sowing is relatively insensitive towards Actinomycin D, the sensitivity towards cycloheximide and Puromycin is high. It is concluded that at least some of the enzymes required for carotenoid biosynthesis are made in the extraplastidal cytoplasm and it is suggested that P_fr acts at the level of carotenoid accumulation by providing a structural prerequisite for carotenoid accumulation in the plastid compartment. This latter suggestion is mainly based on the fact that carotenoid accumulation in light and dark is very sensitive towards chloramphenicol if the compound is applied at the time of sowing. If, however, the compound is applied 36 h after sowing, the effect of chloramphenicol is different in light and dark. In the dark there is no influence up to 200 g·ml^-1, whereas in the light there is a significant inhibition of carotenoid accumulation.Herrn Prof. Kurt Mothes mit guten Wünschen zum 70. Geburtstag.     

Multiple microscopic approaches demonstrate linkage between chromoplast architecture and carotenoid composition in diverse Capsicum annuum fruit

Increased accumulation of specific carotenoids in plastids through plant breeding or genetic engineering requires an understanding of the limitations that storage sites for these compounds may impose on that accumulation. Here, using Capsicum annuum L. fruit, we demonstrate directly the unique sub‐organellar accumulation sites of specific carotenoids using live cell hyperspectral confocal Raman microscopy. Further, we show that chromoplasts from specific cultivars vary in shape and size, and these structural variations are associated with carotenoid compositional differences. Live‐cell imaging utilizing laser scanning confocal (LSCM) and confocal Raman microscopy, as well as fixed tissue imaging by scanning and transmission electron microscopy (SEM and TEM), all demonstrated morphological differences with high concordance for the measurements across the multiple imaging modalities. These results reveal additional opportunities for genetic controls on fruit color and carotenoid‐based phenotypes.     

光照对柑橘果皮类胡萝卜素和色泽形成的影响

以“红柿柑”为试材,在柑橘果实膨大末期通过套袋遮光处理以抑制果皮光合作用,研究光照对果皮糖、叶绿素、类胡萝卜素及果实外观色泽的影响。结果表明,遮光后果皮叶绿素含量迅速下降引起果实转色提早,但各种类胡萝卜素含量及总量并未提高,而是显著下降;至果实成熟时由于遮光与光照处理的果皮叶绿素均消失,遮光果实类胡萝卜素含量低颜色变淡,与光照处理相比,遮光前期果皮糖含量下降不大,而后期下降明显;若在后期去袋照光,果皮糖含量上升,与此相应,类胡萝卜素,尤其是β-隐黄质的积累增加,颜色加深,表明光对果皮类胡萝卜素合成尤其是β-隐黄质的积累有促进作用,其原因是光以环境信号的方式影响果皮的类胡萝卜素形成。     

Carotenoid synthesis in a suspension culture of carrot cells

Biosynthesis of carotenoid in cultured carrot cells was studiedin relation to cell growth and acetate metabolism. Of the twostrains tested, one (GD-1) predominantly produces ß-caroteneand the other (GD-2) lycopene. In both strains, carotenoid wasproduced in parallel with cell growth. Incorporations of acetate-¹⁴Cinto carotenoids, organic acids and amino acids were acceleratedby increasing the concentration of 2,4-D in the medium. (Received November 17, 1970; )     

CHROMOPLAST ULTRASTRUCTURE AS AFFECTED BY GENES CONTROLLING GRANA RETENTION AND CAROTENOIDS IN FRUITS OF CAPSICUM ANNUUM

Plastids in the fruits of isogenic lines of pepper (Capsicum annuum) were examined by electron microscopy with reference to four genotypes determining the carotenoid composition and the colors red, yellow, brown, and green of the ripe fruit. One gene pair (y⁺/y) influences carotenoid content and the other pair (cl⁺/cl) controls the chlorophyll. The retention of the grana and chlorophyll in the ripe fruits of the brown and green phenotypes is correlated with the cl cl genotype. The y⁺ gene increases the total carotenoids and promotes the formation of red pigments. Giant grana were found in the yellow and green phenotypes, but during ripening these disappeared in the yellow. Unusual dichotomous and concentric grana were observed in the green. Globule-associated carotenoids forming fibrillar crystalloids were present in all color types, although to a lesser degree in the yellow fruit. Membrane-associated carotenoids occurred only in the yellow and green phenotypes.     

Select light spectra affect gonad color in the sea urchin Lytechinus variegatus

It is suggested that gonad color in sea urchins depends upon the in vivo accumulation and metabolism of red and yellow carotenoid pigments. We hypothesized that differential light exposure could affect carotenoid deposition and, hence, gonad color in sea urchins. We therefore performed two experiments to determine whether light spectra affect the gonad color of Lytechinus variegatus. In the first experiment, urchins were fed a formulated feed supplemented with or without β-carotene and held beneath three lighting regimes designed to emit differing wavelengths of the visible spectrum. After 12 weeks, urchins were dissected and gonad color (CIE L*a*b*) was measured with a Pantone Capsure RM200. Actinic light significantly increased the value of a* (red) in gonad color. Color in the orange and yellow spectra in the gonads increased in individuals fed the β-carotene supplemented diet. In the second experiment, we cultured urchins for nine weeks under lamps specialized to emit UV radiation. All urchins in this experiment received diets supplemented with β-carotene. There was no significant difference in harvested gonad color between these treatments. These data suggest that light quality and dietary carotenoids affect carotenoid deposition in the gonads.     

Pteridine, not carotenoid, pigments underlie the female-specific orange ornament of striped plateau lizards (Sceloporus virgatus)

Indicator models of sexual selection suggest that signal honesty is maintained via costs of ornament expression. Carotenoid-based visual signals are a well-studied example, as carotenoids may be environmentally limited and impact signaler health. However, not all bright yellow, orange and red ornaments found in vertebrates are carotenoid-based; pteridine pigments may also produce these colors. We examine the contribution of carotenoid and pteridine pigments to the orange reproductive color of female striped plateau lizards (Sceloporus virgatus). This color ornament reliably indicates female mate quality, yet costs maintaining signal honesty are currently unknown. Dietary carotenoid manipulations did not affect orange color, and orange skin differed from surrounding white skin in drosopterin, not carotenoid, content. Further, orange color positively correlated with drosopterin, not carotenoid, concentration. Drosopterin-based female ornaments avoid the direct trade-offs of using carotenoids for ornament production vs egg production, thus may relax counter-selection against color ornament exaggeration in females. Direct experimentation is needed to determine the actual costs of pteridine-based ornaments. Like carotenoids, pteridines influence important biological processes, including immune and antioxidant function; predation and social costs may also be relevant.     

Environmental Pollution Affects the Plumage Color of Great Tit Nestlings through Carotenoid Availability

Birds need to acquire carotenoids for their feather pigmentation from their diet, which means that their plumage color may change as a consequence of human impact on their environment. For example, the carotenoid-based plumage coloration of Great tit, Parus major, nestlings is associated with the degree of environmental pollution. Breast feathers of birds in territories exposed to heavy metals are less yellow than those in unpolluted environments. Here we tested two hypotheses that could explain the observed pattern: (I) deficiency of carotenoids in diet, and (II) pollution-related changes in transfer of carotenoids to feathers. We manipulated dietary carotenoid levels of nestlings and measured the responses in plumage color and tissue concentrations. Our carotenoid supplementation produced the same response in tissue carotenoid concentrations and plumage color in polluted and unpolluted environments. Variation in heavy metal levels did not explain the variation in tissue (yolk, plasma, and feathers) carotenoid concentrations and was not related to plumage coloration. Instead, the variation in plumage yellowness was associated with the availability of carotenoid-rich caterpillars in territories. Our results support the hypothesis that the primary reason for pollution-related variation in plumage color is carotenoid deficiency in the diet.     

Enhancement of carotenoid biosynthesis in the green microalga Dunaliella salina with light-emitting diodes and adaptive laboratory evolution

There is a particularly high interest to derive carotenoids such as β-carotene and lutein from higher plants and algae for the global market. It is well known that β-carotene can be overproduced in the green microalga Dunaliella salina in response to stressful light conditions. However, little is known about the effects of light quality on carotenoid metabolism, e.g., narrow spectrum red light. In this study, we present UPLC-UV-MS data from D. salina consistent with the pathway proposed for carotenoid metabolism in the green microalga Chlamydomonas reinhardtii. We have studied the effect of red light-emitting diode (LED) lighting on growth rate and biomass yield and identified the optimal photon flux for D. salina growth. We found that the major carotenoids changed in parallel to the chlorophyll b content and that red light photon stress alone at high level was not capable of upregulating carotenoid accumulation presumably due to serious photodamage. We have found that combining red LED (75 %) with blue LED (25 %) allowed growth at a higher total photon flux. Additional blue light instead of red light led to increased β-carotene and lutein accumulation, and the application of long-term iterative stress (adaptive laboratory evolution) yielded strains of D. salina with increased accumulation of carotenoids under combined blue and red light.     

Biosynthesis of Aryl Carotenoids: Studies on the Nature of the Precyclization Intermediate

The conversion of nicotine-induced pools of radioactive lycopene-¹⁴Cinto chlorobactene was studied in Chlorobium limicola. Removalof the nicotine and resuspension of cells in radioactivity-freemedium in the absence or presence of diphenylamine (to preventfurther carotenoid synthesis) indicates that.the lycopene ismetabolized to chlorobactene. Results suggest that some olderlycopene molecules, in the presence of nicotine, become inaccessibleto the carotenogenic enzymes, and are not further metabolizedupon removal of the inhibitor. Leucine is not effectively incorporatedinto carotenoids in C. limicola. ¹Present address: Division of Biology, California Instituteof Technology, Pasadena, California 91125, U.S.A. (Received November 20, 1981; Accepted May 5, 1981)     

CAROTENOID CLEAVAGE DIOXYGENASE4 Is a Negative Regulator of β-Carotene Content in Arabidopsis Seeds

Experimental approaches targeting carotenoid biosynthetic enzymes have successfully increased the seed β-carotene content of crops. However, linkage analysis of seed carotenoids in Arabidopsis thaliana recombinant inbred populations showed that only 21% of quantitative trait loci, including those for β-carotene, encode carotenoid biosynthetic enzymes in their intervals. Thus, numerous loci remain uncharacterized and underutilized in biofortification approaches. Linkage mapping and genome-wide association studies of Arabidopsis seed carotenoids identified CAROTENOID CLEAVAGE DIOXYGENASE4 (CCD4) as a major negative regulator of seed carotenoid content, especially β-carotene. Loss of CCD4 function did not affect carotenoid homeostasis during seed development but greatly reduced carotenoid degradation during seed desiccation, increasing β-carotene content 8.4-fold relative to the wild type. Allelic complementation of a ccd4 null mutant demonstrated that single-nucleotide polymorphisms and insertions and deletions at the locus affect dry seed carotenoid content, due at least partly to differences in CCD4 expression. CCD4 also plays a major role in carotenoid turnover during dark-induced leaf senescence, with β-carotene accumulation again most strongly affected in the ccd4 mutant. These results demonstrate that CCD4 plays a major role in β-carotene degradation in drying seeds and senescing leaves and suggest that CCD4 orthologs would be promising targets for stabilizing and increasing the level of provitamin A carotenoids in seeds of major food crops.