Lycopene ε-cyclase mediated transition of α-carotene and ß-carotene metabolic flow in carrot fleshy root.

The accumulation of carotenoids, such as xanthophylls, lycopene, and carotenes, is responsible for the color of carrot (Daucus carota subsp. sativus) fleshy roots. The potential role of DcLCYE, encoding a lycopene ε-cyclase associated with carrot root color, was investigated using cultivars with orange and red roots. The expression of DcLCYE in red carrot varieties was significantly lower than that in orange carrots at the mature stage. Furthermore, red carrots accumulated larger amounts of lycopene and lower levels of α-carotene. Sequence comparison and prokaryotic expression analysis revealed that amino acid differences in red carrots did not affect the cyclization function of DcLCYE. Analysis of the catalytic activity of DcLCYE revealed that it mainly formed ε-carotene, while a side activity on α-carotene and γ-carotene was also observed. Comparative analysis of the promoter region sequences indicated that differences in the promoter region may affect the transcription of DcLCYE. DcLCYE was overexpressed in the red carrot 'Benhongjinshi' under the control of the CaMV35S promoter. Lycopene in transgenic carrot roots was cyclized, resulting in the accumulation of higher levels of α-carotene and xanthophylls, while the ß-carotene content was significantly decreased. The expression levels of other genes in the carotenoid pathway were simultaneously upregulated. Knockout of DcLCYE in the orange carrot 'Kurodagosun' by CRISPR/Cas9 technology resulted in a decrease in the α-carotene and xanthophyll contents. The relative expression levels of DcPSY1, DcPSY2, and DcCHXE were sharply increased in DcLCYE knockout mutants. The results of this study provide insights into the function of DcLCYE in carrots, which could serve as a basis for creating colorful carrot germplasms.

NinaB and BCO Collaboratively Participate in the ß-Carotene Catabolism in Crustaceans: A Case Study on Chinese Mitten Crab Eriocheir sinensis.

Carotenoid cleavage oxygenases can cleave carotenoids into a range of biologically important products. Carotenoid isomerooxygenase (NinaB) and ß, ß-carotene 15, 15'-monooxygenase (BCO1) are two important oxygenases. In order to understand the roles that both oxygenases exert in crustaceans, we first investigated NinaB-like (EsNinaBl) and BCO1-like (EsBCO1l) within the genome of Chinese mitten crab (Eriocheir sinensis). Their functions were then deciphered through an analysis of their expression patterns, an in vitro ß-carotene degradation assay, and RNA interference. The results showed that both EsNinaBl and EsBCO1l contain an RPE65 domain and exhibit high levels of expression in the hepatopancreas. During the molting stage, EsNinaBl exhibited significant upregulation in stage C, whereas EsBCO1l showed significantly higher expression levels at stage AB. Moreover, dietary supplementation with ß-carotene resulted in a notable increase in the expression of EsNinaBl and EsBCO1l in the hepatopancreas. Further functional assays showed that the EsNinaBl expressed in E. coli underwent significant changes in its color, from orange to light; in addition, its ß-carotene cleavage was higher than that of EsBCO1l. After the knockdown of EsNinaBl or EsBCO1l in juvenile E. sinensis, the expression levels of both genes were significantly decreased in the hepatopancreas, accompanied by a notable increase in the redness (a*) values. Furthermore, a significant increase in the ß-carotene content was observed in the hepatopancreas when EsNinaBl-mRNA was suppressed, which suggests that EsNinaBl plays an important role in carotenoid cleavage, specifically ß-carotene. In conclusion, our findings suggest that EsNinaBl and EsBCO1l may exhibit functional co-expression and play a crucial role in carotenoid cleavage in crabs.

Circadian Regulation of Expression of Carotenoid Metabolism Genes (PSY2, LCYE, CrtRB1, and NCED1) in Leaves of Tomato Solanum lycopersicum L.

The circadian dynamics of the expression of key genes of carotenoid metabolism (PSY2, LCYE, CrtRB1, and NCED1) in the photosynthetic tissue of tomato Solanum lycopersicum L. (cultivar Korneevsky) plants was characterized. An in silico analysis of the gene expression pattern was carried out and a high level of their transcripts was detected in the leaf tissue. qRT-PCR analysis of gene expression was performed at six time points during the day and showed the highest levels of PSY2, LCYE, and NCED1 transcripts in the second half of the light phase and CrtRB1 at the end of the dark phase. The content and composition of carotenoids in leaf tissue in the middle of the day was determined; it was shown that the leaf accumulates 1.5 times more compounds of the ɛ/ß-branch of carotenoid biosynthesis pathway than compounds of the ß/ß-branch.

Comparative transcriptome analysis reveals that chlorophyll metabolism contributes to leaf color changes in wucai (Brassica campestris L.) in response to cold.

BACKGROUND: Chlorophyll (Chl) is a vital photosynthetic pigment involved in capturing light energy and energy conversion. In this study, the color conversion of inner-leaves from green to yellow in the new wucai (Brassica campestris L.) cultivar W7-2 was detected under low temperature. The W7-2 displayed a normal green leaf phenotype at the seedling stage, but the inner leaves gradually turned yellow when the temperature was decreased to 10 °C/2 °C (day/night), This study facilitates us to understand the physiological and molecular mechanisms underlying leaf color changes in response to low temperature. RESULTS: A comparative leaf transcriptome analysis of W7-2 under low temperature treatment was performed on three stages (before, during and after leaf color change) with leaves that did not change color under normal temperature at the same period as a control. A total of 67,826 differentially expressed genes (DEGs) were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analysis revealed that the DEGs were mainly enriched in porphyrin and Chl metabolism, carotenoids metabolism, photosynthesis, and circadian rhythm. In the porphyrin and chlorophyll metabolic pathways, the expression of several genes was reduced [i.e. magnesium chelatase subunit H (CHLH)] under low temperature. Almost all genes [i.e. phytoene synthase (PSY)] in the carotenoids (Car) biosynthesis pathway were downregulated under low temperature. The genes associated with photosynthesis [i.e. photosystem II oxygen-evolving enhancer protein 1 (PsbO)] were also downregulated under LT. Our study also showed that elongated hypocotyl5 (HY5), which participates in circadian rhythm, and the metabolism of Chl and Car, is responsible for the regulation of leaf color change and cold tolerance in W7-2. CONCLUSIONS: The color of inner-leaves was changed from green to yellow under low temperature in temperature-sensitive mutant W7-2. Physiological, biochemical and transcriptomic studies showed that HY5 transcription factor and the downstream genes such as CHLH and PSY, which regulate the accumulation of different pigments, are required for the modulation of leaf color change in wucai under low temperature.

Red light-induced kumquat fruit coloration is attributable to increased carotenoid metabolism regulated by FcrNAC22.

Carotenoids play vital roles in the coloration of plant tissues and organs, particularly fruits; however, the regulation of carotenoid metabolism in fruits during ripening is largely unknown. Here, we show that red light promotes fruit coloration by inducing accelerated degreening and carotenoid accumulation in kumquat fruits. Transcriptome profiling revealed that a NAC (NAM/ATAF/CUC2) family transcription factor, FcrNAC22, is specifically induced in red light-irradiated fruits. FcrNAC22 localizes to the nucleus, and its gene expression is up-regulated as fruits change color. Results from dual luciferase, yeast one-hybrid assays and electrophoretic mobility shift assays indicate that FcrNAC22 directly binds to, and activates the promoters of three genes encoding key enzymes in the carotenoid metabolic pathway. Moreover, FcrNAC22 overexpression in citrus and tomato fruits as well as in citrus callus enhances expression of most carotenoid biosynthetic genes, accelerates plastid conversion into chromoplasts, and promotes color change. Knock down of FcrNAC22 expression in transiently transformed citrus fruits attenuates fruit coloration induced by red light. Taken together, our results demonstrate that FcrNAC22 is an important transcription factor that mediates red light-induced fruit coloration via up-regulation of carotenoid metabolism.

Uptake and metabolism of ß-apo-8'-carotenal, ß-apo-10'-carotenal, and ß-apo-13-carotenone in Caco-2 cells.

ß-Apocarotenoids are eccentric cleavage products of carotenoids formed by chemical and enzymatic oxidations. They occur in foods containing carotenoids and thus might be directly absorbed from the diet. However, there is limited information about their intestinal absorption. The present research examined the kinetics of uptake and metabolism of ß-apocarotenoids. Caco-2 cells were grown on 6-well plastic plates until a differentiated cell monolayer was achieved. ß-Apocarotenoids were prepared in Tween 40 micelles, delivered to differentiated cells in serum-free medium, and incubated at 37°C for up to 8 h. There was rapid uptake of ß-apo-8'-carotenal into cells, and ß-apo-8'-carotenal was largely converted to ß-apo-8'-carotenoic acid and a minor metabolite that we identified as 5,6-epoxy-ß-apo-8'-carotenol. There was also rapid uptake of ß-apo-10'-carotenal into cells, and ß-apo-10'-carotenal was converted into a major metabolite identified as 5,6-epoxy-ß-apo-10'-carotenol and a minor metabolite that is likely a dihydro-ß-apo-10'-carotenol. Finally, there was rapid cellular uptake of ß-apo-13-carotenone, and this compound was extensively degraded. These results suggest that dietary ß-apocarotenals are extensively metabolized in intestinal cells via pathways similar to the metabolism of retinal. Thus, they are likely not absorbed directly from the diet.

Carotenoid cleavage in chromoplasts of white and yellow-fleshed peach varieties.

BACKGROUND: In peach fruit, carotenoid accumulation in the mesocarp causes the difference between yellow and white genotypes. The latter are generally characterized by a peculiar and more intense aroma, because of higher release of volatiles deriving from dioxygenase-catalysed breakdown of the tetraterpene skeleton. The rate of carotenoid oxidation was investigated in peach (Prunus persica L.) fruits harvested at various stages of development. Two couples of white and yellow-fleshed isogenic varieties and an ancestral white-fleshed genotype were analysed, which had previously shown to differ in Carotenoid Cleavage Dioxygenase 4 allelic composition resulting in various combinations of putatively active/inactive proteins. RESULTS: Carotenoid bleaching activity was localized in the insoluble fraction of fruit flesh chromoplasts. Higher rates of trans-ß-apo-8'-carotenal than ß-carotene bleaching suggest that the first cleavage reaction is the rate-limiting step. Consistently, HPLC analysis did not show the appearance of coloured intermediates in reaction mixtures. High levels of substrate breakdown were found during the initial phases of fruit development in all genotypes examined, whereas significant differences were evident during the second exponential growth phase and ripening onset. Also, the ratio of carotene versus carotenale utilization varied significantly. CONCLUSION: Pattern comparison among activity levels measured in vitro on chromoplast enriched fractions suggests that cleavage enzyme(s) other than Carotenoid Cleavage Dioxygenase 4 play a significant role in carotenoid breakdown during fruit development and ripening. © 2018 Society of Chemical Industry.

Expression of a carotenoid-modifying gene and evolution of red coloration in weaverbirds (Ploceidae).

Red carotenoid colours in birds are widely assumed to be sexually selected quality indicators, but this rests on a very incomplete understanding of genetic mechanisms and honesty-mediating costs. Recent progress was made by the implication of the gene CYP2J19 as an avian carotenoid ketolase, catalysing the synthesis of red C4-ketocarotenoids from yellow dietary precursors, and potentially a major mechanism behind red coloration in birds. Here, we investigate the role of CYP2J19 in the spectacular colour diversification of African weaverbirds (Ploceidae), represented by five genera and 16 species: eight red, seven yellow and one without carotenoid coloration. All species had a single copy of CYP2J19, unlike the duplication found in the zebra finch, with high expression in the retina, confirming its function in colouring red oil droplets. Expression was weak or undetected in skin and follicles of pigment-depositing feather buds, as well as in beaks and tarsi, including those of the red-billed quelea. In contrast, the hepatic (liver) expression of CYP2J19 was consistently higher (>14-fold) in seven species with C4-ketocarotenoid coloration than in species without (including one red species), an association strongly supported by a phylogenetic comparative analysis. The results suggest a critical role of the candidate ketolase, CYP2J19, in the evolution of red C4-ketocarotenoid colour variation in ploceids. As ancestral state reconstruction suggests that ketocarotenoid coloration has evolved twice in this group (once in Euplectes and once in the Quelea/Foudia clade), we argue that while CYP2J19 has retained its ancestral role in the retina, it has likely been co-opted for red coloration independently in the two lineages, via increased hepatic expression.

An R2R3-MYB transcription factor represses the transformation of α- and ß-branch carotenoids by negatively regulating expression of CrBCH2 and CrNCED5 in flavedo of Citrus reticulate.

Although the functions of carotenogenic genes are well documented, little is known about the mechanisms that regulate their expression, especially those genes involved in α - and ß-branch carotenoid metabolism. In this study, an R2R3-MYB transcriptional factor (CrMYB68) that directly regulates the transformation of α- and ß-branch carotenoids was identified using Green Ougan (MT), a stay-green mutant of Citrus reticulata cv Suavissima. A comprehensive analysis of developing and harvested fruits indicated that reduced expression of ß-carotene hydroxylases 2 (CrBCH2) and 9-cis-epoxycarotenoid dioxygenase 5 (CrNCED5) was responsible for the delay in the transformation of α- and ß-carotene and the biosynthesis of ABA. Additionally, the expression of these genes was negatively correlated with the expression of CrMYB68 in MT. Further, electrophoretic mobility shift assays (EMSAs) and dual luciferase assays indicated that CrMYB68 can directly and negatively regulate CrBCH2 and CrNCED5. Moreover, transient overexpression experiments using leaves of Nicotiana benthamiana indicated that CrMYB68 can also negatively regulate NbBCH2 and NbNCED5. To overcome the difficulty of transgenic validation, we quantified the concentrations of carotenoids and ABA, and gene expression in a revertant of MT. The results of these experiments provide more evidence that CrMYB68 is an important regulator of carotenoid metabolism.

Retinal accumulation of zeaxanthin, lutein, and ß-carotene in mice deficient in carotenoid cleavage enzymes.

Carotenoid supplementation can prevent and reduce the risk of age-related macular degeneration (AMD) and other ocular disease, but until now, there has been no validated and well-characterized mouse model which can be employed to investigate the protective mechanism and relevant metabolism of retinal carotenoids. ß-Carotene oxygenases 1 and 2 (BCO1 and BCO2) are the only two carotenoid cleavage enzymes found in animals. Mutations of the bco2 gene may cause accumulation of xanthophyll carotenoids in animal tissues, and BCO1 is involved in regulation of the intestinal absorption of carotenoids. To determine whether or not mice deficient in BCO1 and/or BCO2 can serve as a macular pigment mouse model, we investigated the retinal accumulation of carotenoids in these mice when fed with zeaxanthin, lutein, or ß-carotene using an optimized carotenoid feeding method. HPLC analysis revealed that all three carotenoids were detected in sera, livers, retinal pigment epithelium (RPE)/choroids, and retinas of all of the mice, except that no carotenoid was detectable in the retinas of wild type (WT) mice. Significantly higher amounts of zeaxanthin and lutein accumulated in the retinas of BCO2 knockout (bco2-/-) mice and BCO1/BCO2 double knockout (bco1-/-/bco2-/-) mice relative to BCO1 knockout (bco1-/-) mice, while bco1-/- mice preferred to take up ß-carotene. The levels of zeaxanthin and lutein were higher than ß-carotene levels in the bco1-/-/bco2-/- retina, consistent with preferential uptake of xanthophyll carotenoids by retina. Oxidative metabolites were detected in mice fed with lutein or zeaxanthin but not in mice fed with ß-carotene. These results indicate that bco2-/- and bco1-/-/bco2-/- mice could serve as reasonable non-primate models for macular pigment function in the vertebrate eye, while bco1-/- mice may be more useful for studies related to ß-carotene.

Beyond topology: coevolution of structure and flux in metabolic networks.

Interactions between the structure of a metabolic network and its functional properties underlie its evolutionary diversification, but the mechanism by which such interactions arise remains elusive. Particularly unclear is whether metabolic fluxes that determine the concentrations of compounds produced by a metabolic network, are causally linked to a network's structure or emerge independently of it. A direct empirical study of populations where both structural and functional properties vary among individuals' metabolic networks is required to establish whether changes in structure affect the distribution of metabolic flux. In a population of house finches (Haemorhous mexicanus), we reconstructed full carotenoid metabolic networks for 442 individuals and uncovered 11 structural variants of this network with different compounds and reactions. We examined the consequences of this structural diversity for the concentrations of plumage-bound carotenoids produced by flux in these networks. We found that concentrations of metabolically derived, but not dietary carotenoids, depended on network structure. Flux was partitioned similarly among compounds in individuals of the same network structure: within each network, compound concentrations were closely correlated. The highest among-individual variation in flux occurred in networks with the strongest among-compound correlations, suggesting that changes in the magnitude, but not the distribution of flux, underlie individual differences in compound concentrations on a static network structure. These findings indicate that the distribution of flux in carotenoid metabolism closely follows network structure. Thus, evolutionary diversification and local adaptations in carotenoid metabolism may depend more on the gain or loss of enzymatic reactions than on changes in flux within a network structure.

Diversity, physiology, and evolution of avian plumage carotenoids and the role of carotenoid-protein interactions in plumage color appearance.

The diversity of vibrant plumage colors in birds has evolved as a direct result of social and environmental pressures. To fully understand these underlying pressures it is necessary to elucidate the mechanisms for the creation of novel plumage colors which include the metabolic transformations of dietary carotenoids and spectral tuning of the molecules within the feather protein environment. Recent advances in this field have greatly expanded the number and breadth of avian species for which plumage pigmentation has been characterized, making it possible to reconstruct the phylogenetic history of carotenoid usage in plumage. Resonance Raman and classical Raman spectroscopic techniques have been employed with great effect in the study of carotenoids in situ. The application of these methods have two benefits: to identify carotenoids in feathers that are unavailable for destructive sampling; and to study the spectral tuning resulting from the interaction between the carotenoids and the proteins to which they are bound. This review presents a summary of recent advances in the understanding of the molecular factors controlling the coloration of avian carotenoid plumage obtained through the application of both bioanalytical and spectroscopic methodologies.

Genetic variation predicts serum lycopene concentrations in a multiethnic population of postmenopausal women.

BACKGROUND: The consumption and blood concentrations of lycopene are both positively and inversely associated with the risk of several chronic diseases. The inconsistences in lycopene disease association studies may stem from a lack of knowledge about the genetic variation in the synthesis, metabolism, and deposition of transport and binding proteins, which potentially influence serum lycopene concentrations. OBJECTIVE: We examined the association between variation across the genome and serum concentrations of lycopene in a multiethnic population. METHODS: Participants included African (n = 914), Hispanic (n = 464), and European (n = 1203) American postmenopausal women from the Women's Health Initiative. We analyzed ∼7 million single nucleotide polymorphisms (SNPs). Linear regression models were used to assess associations between each SNP and serum concentrations (log transformed, continuous) of lycopene; we adjusted for age, body mass index, and population substructure. Models were run separately by ethnicity, and results were combined in a transethnic fixed-effects meta-analysis. RESULTS: In the meta-analysis, the scavenger receptor class B, member 1 (SCARB1) gene, which encodes for a cholesterol membrane transporter, was significantly associated with lycopene concentrations (rs1672879; P < 2.68 × 10(-9)). Each additional G allele resulted in a 12% decrease in lycopene concentrations for African Americans, 20% decrease for Hispanic Americans, and 9% decrease for European Americans. In addition, 2 regions were significantly associated with serum lycopene concentrations in African Americans: the slit homolog 3 gene (SLIT3), which serves as a molecular guidance cue in cellular migration, and the dehydrogenase/reductase (SDR family) member 2 (DHRS2) gene, which codes for an oxidoreductase that mitigates the breakdown of steroids. CONCLUSIONS: We found 3 novel loci associated with serum lycopene concentrations, 2 of which were specific to African Americans. Future functional studies looking at these specific genes may provide insight into the metabolism and underlying function of lycopene in humans, which may further elucidate lycopene's influence on disease risk and health. This trial was registered at clinicaltrials.gov as NCT00000611.

Lycopene and apo-10'-lycopenoic acid have differential mechanisms of protection against hepatic steatosis in ß-carotene-9',10'-oxygenase knockout male mice.

BACKGROUND: Nonalcoholic fatty liver disease is positively associated with obesity and cardiovascular disease risk. Apo-10'-lycopenoic acid (APO10LA), a potential oxidation product of apo-10'-lycopenal that is generated endogenously by ß-carotene-9',10'-oxygenase (BCO2) cleavage of lycopene, inhibited hepatic steatosis in BCO2-expressing mice. OBJECTIVE: The present study evaluated lycopene and APO10LA effects on hepatic steatosis in mice without BCO2 expression. METHODS: Male and female BCO2-knockout (BCO2-KO) mice were fed a high saturated fat diet (HSFD) with or without APO10LA (10 mg/kg diet) or lycopene (100 mg/kg diet) for 12 wk. RESULTS: Lycopene or APO10LA supplementation reduced hepatic steatosis incidence (78% and 72%, respectively) and severity in BCO2-KO male mice. Female mice did not develop steatosis, had greater hepatic total cholesterol (3.06 vs. 2.31 mg/g tissue) and cholesteryl ester (1.58 vs. 0.86 mg/g tissue), but had lower plasma triglyceride (TG) (229 vs. 282 mg/dL) and cholesterol (97.1 vs. 119 mg/dL) than male mice. APO10LA-mitigated steatosis in males was associated with reduced hepatic total cholesterol (18%) and activated sirtuin 1 signaling, which resulted in reduced fatty acids (FAs) and TG synthesis markers [stearoyl-coenzyme A (CoA) desaturase protein, 71%; acetyl-CoA carboxylase phosphorylation, 79%; AMP-activated protein kinase phosphorylation, 67%], and elevated cholesterol efflux genes (cytochrome P450 family 7A1, 65%; ATP-binding cassette transporter G5/8, 11%). These APO10LA-mediated effects were not mimicked by lycopene supplementation. Intriguingly, steatosis inhibition by lycopene induced peroxisome proliferator-activated receptor (PPAR)α- and PPARγ-related genes in mesenteric adipose tissue (MAT) that increases mitochondrial uncoupling [cell death-inducing DNA fragmentation factor, α subunit-like effector a, 55%; PR domain-containing 16, 47%; uncoupling protein 3 (Ucp3), 55%], FA ß-oxidation (PPARα, 53%; very long chain acyl-CoA dehydrogenase, 38%), and uptake (FA transport protein 4, 29%; lipoprotein lipase 43%). Expressions of 10 MAT PPAR-related genes were inversely correlated with steatosis score, suggesting that lycopene reduced steatosis by increasing MAT FA utilization. CONCLUSIONS: Our data suggest that lycopene and APO10LA inhibit HSFD-induced steatosis in BCO2-KO male mice through differential mechanisms. Sex disparity of BCO2-KO mice was observed in the outcomes of HSFD-induced liver steatosis and plasma lipids.

An LC/MS/MS method for stable isotope dilution studies of ß-carotene bioavailability, bioconversion, and vitamin A status in humans.

Isotope dilution is currently the most accurate technique in humans to determine vitamin A status and bioavailability/bioconversion of provitamin A carotenoids such as ß-carotene. However, limits of MS detection, coupled with extensive isolation procedures, have hindered investigations of physiologically-relevant doses of stable isotopes in large intervention trials. Here, a sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) analytical method was developed to study the plasma response from coadministered oral doses of 2 mg [(13)C10]ß-carotene and 1 mg [(13)C10]retinyl acetate in human subjects over a 2 week period. A reverse phase C18 column and binary mobile phase solvent system separated ß-carotene, retinol, retinyl acetate, retinyl linoleate, retinyl palmitate/retinyl oleate, and retinyl stearate within a 7 min run time. Selected reaction monitoring of analytes was performed under atmospheric pressure chemical ionization in positive mode at m/z 537→321 and m/z 269→93 for respective [(12)C]ß-carotene and [(12)C] retinoids; m/z 547→330 and m/z 274→98 for [(13)C10]ß-carotene and [(13)C5] cleavage products; and m/z 279→100 for metabolites of [(13)C10]retinyl acetate. A single one-phase solvent extraction, with no saponification or purification steps, left retinyl esters intact for determination of intestinally-derived retinol in chylomicrons versus retinol from the liver bound to retinol binding protein. Coadministration of [(13)C10]retinyl acetate with [(13)C10]ß-carotene not only acts as a reference dose for inter-individual variations in absorption and chylomicron clearance rates, but also allows for simultaneous determination of an individual's vitamin A status.

The P450-type carotene hydroxylase PuCHY1 from Porphyra suggests the evolution of carotenoid metabolism in red algae.

Carotene hydroxylases catalyze the hydroxylation of α- and ß-carotene hydrocarbons into xanthophylls. In red algae, ß-carotene is a ubiquitously distributed carotenoid, and hydroxylated carotenoids such as zeaxanthin and lutein are also found. However, no enzyme with carotene hydroxylase activity had been previously identified in red algae. Here, we report the isolation of a gene encoding a cytochrome P450-type carotene hydroxylase (PuCHY1) from Porphyra umbilicalis, a red alga with an ancient origin. Sequence comparisons found PuCHY1 belongs to the CYP97B subfamily, which has members from different photosynthetic organisms ranging from red algae to land plants. Functional complementation in Escherichia coli suggested that PuCHY1 catalyzed the conversion from ß-carotene to zeaxanthin. When we overexpressed PuCHY1 in the Arabidopsis thaliana chy2 mutant, pigment analysis showed a significant accumulation of hydroxylated carotenoids, including neoxanthin, violaxanthin, and lutein in the leaves of transgenic plants. These results confirmed a ß-hydroxylation activity of PuCHY1, and also suggested a possible ϵ-hydroxylation function. The pigment profile and gene expression analyses of the algal thallus under high-light stress suggested that P. umbilicalis is unlikely to operate a partial xanthophyll cycle for photoprotection.

An abscisic acid-responsive transcriptional regulatory module CsERF110-CsERF53 orchestrates citrus fruit coloration.

Carotenoid biosynthesis is closely associated with abscisic acid (ABA) during the ripening process of non-climacteric fruits, but the regulatory mechanism between ABA signaling and carotenoid metabolism remains largely unclear. Here, we identified two master regulators of ABA-mediated citrus fruit coloration, CsERF110 and CsERF53, which activated the expression of carotenoid metabolism genes (CsGGPPS, CsPSY, CsPDS, CsCRTISO, CsLCYB2, CsLCYE, CsHYD, CsZEP, and CsNCED2) to facilitate carotenoid accumulation. Further investigations showed that CsERF110 not only activated the expression of CsERF53 by binding to its promoter, but also interacted with CsERF53 to form a transcriptional regulatory module CsERF110-CsERF53. Furthermore, we discovered a positive feedback regulation loop between the ABA signal and carotenoid metabolism regulated by the transcriptional regulatory module CsERF110-CsERF53. Our results reveal that the transcriptional regulatory module CsERF110-CsERF53 responded to ABA signaling, thereby orchestrating citrus fruit coloration. Considering the importance of carotenoid content for citrus and many other carotenoid-rich crops, the revelation of molecular mechanisms underlying ABA-mediated carotenoid biosynthesis in plants will facilitate transgenic/gene editing approach development, further contributing to improving the quality of citrus and other carotenoid-rich crops.

Melatonin treatment delays postharvest senescence of broccoli with regulation of carotenoid metabolism.

The effect of melatonin treatment on the carotenoid metabolism in broccoli florets during storage was explored. The results indicated that 100 µmol/L of melatonin maintained the sensory quality of broccoli florets, which retarded the increase of the L* value and the decrease of the H value. Melatonin treatment increased the activities of tryptophan decarboxylase (TDC), tryptamine 5-hydroxylase (T5H), serotonin N-acetyltransferase (SNAT) and N-acetylserotonin methyltransferase (ASMT), leading to the enrichment of endogenous melatonin content in broccoli florets. Meanwhile, the treatment inhibited the concentrations of ß-carotene, ß-cryptoxanthin, zeaxanthin and lutein, which was beneficial in delaying the yellowing of broccoli. In addition, a series of carotenoid biosynthetic genes such as BoPSY, BoPDS, BoZDS, BoLCYß and BoZEP was also suppressed by melatonin. Further analysis revealed that the lower carotenoid content and the down-regulated BoNCED expression in treated broccoli resulted in less accumulation of abscisic acid precursors, inhibiting abscisic acid production during the yellowing process.

Magnesium accelerates changes in the fruit ripening and carotenoid accumulation in Satsuma Mandarin pulp.

This study aims to further examine the effect of Magnesium (Mg) application on fruit quality and carotenoid metabolism in Satsuma mandarin pulp. For this, a field experiment was using 20-year-old Satsuma mandarin (C. unshiu Marc.) for two treatment; (1) CK treatment (without Mg), (2) Mg fertilizer treatment (200 g MgO plant-1). Compared with CK, Mg treatment substantially raised the Mg content in pulp at 90 to 150 DAF (the fruit expansion period), increasing by 15.69%-21.74%. Mg treatment also increased fruit TSS content by 15.84% and 9.88%, decreased fruit TA content in by 34.25% and 33.26% at 195 DAF and 210 DAF (the fruit ripening period). Moreover, at 120 to 195 DAF, Mg treatment significantly increased the levels of lutein, ß-cryptoxanthin, zeaxanthin and violaxanthin in the pulp. This can be explained by the increased expression of important biosynthetic genes, including CitPSY, CitPDS, CitLCYb1, CitLCYb2, CitLCYe, CitHYb, and CitZEP, that played a role in altering the carotenoid composition. The findings of this research offer a novel approach for augmenting both the economic and nutritional worth of citrus fruits.