RESUMEN

Carotenoids are photosynthetic pigments and antioxidants that contribute to different plant colors. However, the involvement of TOPLESS (TPL/TPR)-mediated histone deacetylation in the modulation of carotenoid biosynthesis through ethylene-responsive element-binding factor-associated amphiphilic repression (EAR)-containing transcription factors (TFs) in apple (Malus domestica Borkh.) is poorly understood. MdMYB44 is a transcriptional repressor that contains an EAR repression motif. In the present study, we used functional analyses and molecular assays to elucidate the molecular mechanisms through which MdMYB44-MdTPR1-mediated histone deacetylation influences carotenoid biosynthesis in apples. We identified two carotenoid biosynthetic genes, MdCCD4 and MdCYP97A3, that were confirmed to be involved in MdMYB44-mediated carotenoid biosynthesis. MdMYB44 enhanced ß-branch carotenoid biosynthesis by repressing MdCCD4 expression, whereas MdMYB44 suppressed lutein level by repressing MdCYP97A3 expression. Moreover, MdMYB44 partially influences carotenoid biosynthesis by interacting with the co-repressor TPR1 through the EAR motif to inhibit MdCCD4 and MdCYP97A3 expression via histone deacetylation. Our findings indicate that the MdTPR1-MdMYB44 repressive cascade regulates carotenoid biosynthesis, providing profound insights into the molecular basis of histone deacetylation-mediated carotenoid biosynthesis in plants. These results also provide evidence that the EAR-harboring TF/TPL repressive complex plays a universal role in histone deacetylation-mediated inhibition of gene expression in various plants.

Asunto(s)

RESUMEN

Morphological novelties, including formation of trait combinations, may result from de novo gene origination and/or co-option of existing genes into other developmental contexts. A variety of shape-color combinations of capitular florets occur in Chrysanthemum and its allies. We hypothesized that co-option of a carotenoid cleavage dioxygenase gene into the floral symmetry gene network would generate a white zygomorphic ray floret. We tested this hypothesis in an evolutionary context using species in Chrysanthemum sensu lato, a monophyletic group with diverse floral shape-color combinations, based on morphological investigation, interspecific crossing, molecular interaction and transgenic experiments. Our results showed that white color was significantly associated with floret zygomorphy. Specific expression of the carotenoid cleavage dioxygenase gene CCD4a in marginal florets resulted in white color. Crossing experiments between Chrysanthemum lavandulifolium and Ajania pacifica indicated that expression of CCD4a is trans-regulated. The floral symmetry regulator CYC2g can activate expression of CCD4a with a dependence on TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING (TCP) binding element 8 on the CCD4a promoter. Based on all experimental findings, we propose that gene co-option of carotenoid degradation into floral symmetry regulation, and the subsequent dysfunction or loss of either CYC2g or CCD4a, may have led to evolution of capitular shape-color patterning in Chrysanthemum sensu lato.

Asunto(s)

RESUMEN

Coloring is one of the most important characteristics in commercial flowers and fruits, generally due to the accumulation of carotenoid pigments. Enzymes of the CCD4 family in citrus intervene in the generation of ß-citraurin, an apocarotenoid responsible for the reddish-orange color of mandarins. Citrus CCD4s enzymes could be capable of interacting with the thylakoid membrane inside chloroplasts. However, to date, this interaction has not been studied in detail. In this work, we present three new complete models of the CCD4 family members (CCD4a, CCD4b, and CCD4c), modeled with a lipid membrane. To identify the preference for substrates, typical carotenoids were inserted in the active site of the receptors and the protein-ligand interaction energy was evaluated. The results show a clear preference of CCD4s for xanthophylls over aliphatic carotenes. Our findings indicate the ability to penetrate the membrane and maintain a stable interaction through the N-terminal α-helical domain, spanning a contact surface of 2250 to 3250 Å2. The orientation and depth of penetration at the membrane surface suggest that CCD4s have the ability to extract carotenoids directly from the membrane through a tunnel consisting mainly of hydrophobic residues that extends up to the catalytic center of the enzyme.

Asunto(s)

RESUMEN

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

RESUMEN

Carotenoids are important natural pigments that give bright colors to plants. The difference in the accumulation of carotenoids is one of the key factors in the formation of various colors in carrot taproots. Carotenoid cleavage dioxygenases (CCDs), including CCD and 9-cis epoxycarotenoid dioxygenase, are the main enzymes involved in the cleavage of carotenoids in plants. Seven CCD genes have been annotated from the carrot genome. In this study, through expression analysis, we found that the expression level of DcCCD4 was significantly higher in the taproot of white carrot (low carotenoid content) than orange carrot (high carotenoid content). The overexpression of DcCCD4 in orange carrots caused the taproot color to be pale yellow, and the contents of α- and ß-carotene decreased sharply. Mutant carrot with loss of DcCCD4 function exhibited yellow color (the taproot of the control carrot was white). The accumulation of ß-carotene was also detected in taproot. Functional analysis of the DcCCD4 enzyme in vitro showed that it was able to cleave α- and ß-carotene at the 9, 10 (9', 10') double bonds. In addition, the number of colored chromoplasts in the taproot cells of transgenic carrots overexpressing DcCCD4 was significantly reduced compared with that in normal orange carrots. Results showed that DcCCD4 affects the accumulation of carotenoids through cleavage of α- and ß-carotene in carrot taproot.

Asunto(s)

RESUMEN

Apocarotenoids contribute to fruit color and aroma, which are critical quality and marketability attributes. Previously, we reported that the red peels of citrus fruits, which are characterized by higher expression levels of a carotenoid cleavage dioxygenase 4b (CitCCD4b) gene, accumulate higher levels of ß-citraurin and ß-citraurinene than yellow peels. Here, we identified and quantified 12 apocarotenoids, either volatile or nonvolatile, in citrus peel using liquid chromatography-mass spectrometry (LC-MS). Our results show that red peels contain also dramatically higher amounts of ß-apo-8'-carotenal, crocetin dialdehyde known from saffron, ß-citraurol, ß-cyclocitral, and 3-OH-ß-cyclocitral and up to about 17-fold higher levels of 3-OH-ß-cyclocitral glucoside (picrocrocin isomer). The content of these apocarotenoids was also significantly increased in different CitCCD4b-overexpressing transgenic callus lines, compared with corresponding controls. Transient expression of CitCCD4b in Nicotiana benthamiana leaves resulted in a striking increase in the 3-OH-ß-cyclocitral level and the accumulation of picrocrocin. Thus, our work reinforces the specific function of CitCCD4b in producing C10 apocarotenoid volatiles and C30 pigments in citrus peel and uncovers its involvement in the biosynthesis of picrocrocin, C20 dialdehyde, and C30 alcohol apocarotenoids, suggesting the potential of this enzyme in metabolic engineering of apocarotenoids and their derivatives.

Asunto(s)

RESUMEN

The carotenoid-derived volatile ß-ionone plays an important role in the formation of green and black tea flavors due to its low odor threshold, but its formation and the gene(s) involved in its biosynthesis during the tea withering process is(are) still unknown. In this study, we found that the content of ß-ionone increased during the tea withering process catalyzed by an unknown enzyme(s). Correlation analysis of expression patterns of Camellia sinensis carotenoid cleavage dioxygenase genes (CsCCDs) and the ß-ionone content during the withering period revealed CsCCD4 as the most promising candidate. The full-length CsCCD4 gene was amplified from C. sinensis, and the biochemical function of the recombinant CsCCD4 protein was studied after coexpression in Escherichia coli strains engineered to accumulate ß-carotene. The recombinant protein was able to cleave a variety of carotenoids at the 9-10 and 9'-10' double bonds. Volatile ß-ionone was detected as the main product by gas and liquid chromatography-mass spectrometry. The accumulation of ß-ionone was consistent with the expression levels of CsCCD4 in different tissues and during the withering process. The CsCCD4 expression was induced by low temperature and mechanical damage stress but not by dehydration stress. The results demonstrate that CsCCD4 catalyzes the production of ß-ionone in the tea plant and provide insight into its formation mechanism during the withering process.

Asunto(s)

RESUMEN

Red-peeled huyou has a distinct red peel color due mainly to the presence of red apocarotenoid ß-citraurin as well as the increase in amount of total carotenoids. The expression level of carotenoid cleavage dioxygenase 4b1 (CCD4b1) accounted for 99.0% of total transcript abundance of CCD4s in red-peeled huyou peel and was nearly 100 times higher than that in ordinary huyou. ß-Citraurin accumulation and peel coloration was mostly favored at 15 °C but strongly inhibited at moderately high temperatures 20 °C and 25 °C. Exogenous ethylene application for 3 d had no obvious effect on ß-citraurin accumulation in red-peeled huyou but holding fruit at moderately higher temperatures (20 °C and 25 °C) for 3 d had a significant adverse effect on ß-citraurin accumulation. The expression of phytoene synthase 1 (PSY1) and CCD4b1 was higher at 10 °C and 15 °C and significantly lower at 20 °C and 25 °C. The mechanisms governing the accumulation of ß-citraurin are discussed.

Asunto(s)

RESUMEN

The coloration of Citrus fruits is related with the concentration of carotenoids, isoprenoid pigments of 40 carbon atoms (C40). Rodrigo et al. and Ma et al. reported a CCD4-type citrus dioxygenase responsible for the generation of C30 apocarotenoids providing a reddish-orange pigmentation to the peel of many mandarins and oranges. Among them, CCD4b was the first case described of a dioxygenase that cleaves carotenoids C40 in the double bond 7', 8' or 7, 8, generating ß-citraurin or 8-ß-apocarotenal. Here we report the three-dimensional structures of CCD4a and CCD4b, modeled by sequence homology (2BIW) and validated by molecular dynamics (MD). Docking calculations were performed in CCD4a and CCD4b structures with thousands of rotated initial carotenoid conformations and all the possible poses in the active site were found. The interaction energy was measured by means of ASE scoring, Amber99 refinement and London ΔG rescoring. For the case of CCD4a model, the results showed London ΔG score of -19, -17 and -15 kcal/mol for zeaxanthin, ß-cryptoxanthin and ß-carotene, respectively. The same sequence in the estimated interaction strength for the three ligands was obtained using MD. The interaction energy of CCD4b indicated that, in agreement with experimental data, zeaxanthin and ß-cryptoxanthin could be cleaved by the enzyme, ß- and α-carotene have chances to be oxidized and lycopene has not good interaction energy to be predicted as substrate. These findings will be discussed considering the potential in vivo substrates and products, and the physiological role in Citrus fruits. Communicated by Ramaswamy H. Sarma.

Asunto(s)

RESUMEN

Narcissus pseudonarcissus is an important bulbous plant with white or yellow perianths and light yellow to orange-red coronas, but little is known regarding the biochemical and molecular basis related to flower color polymorphisms. To investigate the mechanism of color formation, RNA-Seq of flower of two widely cultured cultivars ('Slim Whitman' and 'Pinza') with different flower color was performed. A total of 84,463 unigenes were generated from the perianths and coronas. By parallel metabolomic and transcriptomic analyses, we provide an overview of carotenoid biosynthesis, degradation, and accumulation in N. pseudonarcissus. The results showed that the content of carotenoids in the corona was higher than that in the perianth in both cultivars. Accordingly, phytoene synthase (PSY) transcripts have a higher abundance in the coronas than that in perianths. While the expression levels of carotenoid biosynthetic genes, like GGPPS, PSY, and LCY-e, were not significantly different between two cultivars. In contrast, the carotenoid degradation gene NpCCD4 was highly expressed in white-perianth cultivars, but was hardly detected in yellow-perianth cultivars. Silencing of NpCCD4 resulted in a significant increase in carotenoid accumulation, especially in all-trans-ß-carotene. Therefore, we presume that NpCCD4 is a crucial factor that causes the low carotenoid content and color fading phenomenon of 'Slim Whitman' by mediating carotenoid turnover. Our findings provide mass RNA-seq data and new insights into carotenoid metabolism in N. pseudonarcissus.

Asunto(s)

RESUMEN

Gardenia jasminoides is used in traditional Chinese medicine and has drawn attention as a rich source of crocin, a compound with reported activity against various cancers, depression and cardiovascular disease. However, genetic information on the crocin biosynthetic pathway of G. jasminoides is scarce. In this study, we performed a transcriptome analysis of the leaves, green fruits, and red fruits of G. jasminoides to identify and predict the genes that encode key enzymes responsible for crocin production, compared with Crocus sativus. Twenty-seven putative pathway genes were specifically expressed in the fruits, consistent with the distribution of crocin in G. jasminoides. Twenty-four of these genes were reported for the first time, and a novel CCD4a gene was predicted that encodes carotenoid cleavage dioxygenase leading to crocin synthesis, in contrast to CCD2 of C. sativus. In addition, 6 other candidate genes (ALDH12, ALDH14, UGT94U1, UGT86D1, UGT71H4, and UGT85K18) were predicted to be involved in crocin biosynthesis following phylogenetic analysis and different gene expression profiles. Identifying the genes that encode key enzymes should help elucidate the crocin biosynthesis pathway.

RESUMEN

Carotenoids play an essential role in light harvesting and protection from excess light. During chloroplast senescence carotenoids are released from their binding proteins and are eventually metabolized. Carotenoid cleavage dioxygenase 4 (CCD4) is involved in carotenoid breakdown in senescing leaf and desiccating seed, and is part of the proteome of plastoglobules (PG), which are thylakoid-associated lipid droplets. Here, we demonstrate that CCD4 is functionally active in PG. Leaves of Arabidopsis thaliana ccd4 mutants constitutively expressing CCD4 fused to yellow fluorescent protein showed strong fluorescence in PG and reduced carotenoid levels upon dark-induced senescence. Lipidome-wide analysis indicated that ß-carotene, lutein, and violaxanthin were the principle substrates of CCD4 in vivo and were cleaved in senescing chloroplasts. Moreover, carotenoids were shown to accumulate in PG of ccd4 mutant plants during senescence, indicating translocation of carotenoids to PG prior to degradation.

RESUMEN

The Arabidopsis carotenoid cleavage dioxygenase 4 (AtCCD4) is a negative regulator of the carotenoid content of seeds and has recently been suggested as a candidate for the generation of retrograde signals that are thought to derive from the cleavage of poly-cis-configured carotene desaturation intermediates. In this work, we investigated the activity of AtCCD4 in vitro and used dynamic modeling to determine its substrate preference. Our results document strict regional specificity for cleavage at the C9-C10 double bond in carotenoids and apocarotenoids, with preference for carotenoid substrates and an obstructing effect on hydroxyl functions, and demonstrate the specificity for all-trans-configured carotenes and xanthophylls. AtCCD4 cleaved substrates with at least one ionone ring and did not convert acyclic carotene desaturation intermediates, independent of their isomeric states. These results do not support a direct involvement of AtCCD4 in generating the supposed regulatory metabolites. In contrast, the strigolactone biosynthetic enzyme AtCCD7 converted 9-cis-configured acyclic carotenes, such as 9-cis-ζ-carotene, 9'-cis-neurosporene, and 9-cis-lycopene, yielding 9-cis-configured products and indicating that AtCCD7, rather than AtCCD4, is the candidate for forming acyclic retrograde signals.

Asunto(s)

RESUMEN

Carotenoids are metabolized to apocarotenoids through the pathway catalysed by carotenoid cleavage oxygenases (CCOs). The apocarotenoids are economically important as it is known to have therapeutic as well as industrial applications. For instance, bixin from Bixa orellana and crocin from Crocus sativus are commercially used as a food colourant and cosmetics since prehistoric time. In our present study, CCD4a gene has been identified and isolated from leaves of B. orellana for the first time and named as BoCCD4a; phylogenetic analysis was carried out using CLUSTAL W. From sequence analysis, BoCCD4a contains two exons and one intron, which was compared with the selected AtCCD4, RdCCD4, GmCCD4 and CmCCD4a gene. Further, the BoCCD4a gene was cloned into pCAMBIA 1301, transformed into Agrobacterium tumefaciens EHA105 strain and subsequently transferred into hypocotyledons and callus of B. orellana by agro-infection. Selection of stable transformation was screened on the basis of PCR detection by using GUS and hptII specific primer, which was followed by histochemical characterization. The percent transient GUS expression in hypocotyledons and callus was 84.4 and 80 %, respectively. The expression of BoCCD4a gene in B. orellana was confirmed through RT-PCR analysis. From our results, the sequence analysis of BoCCD4a gene of B. orellana was closely related to the CsCCD4 gene of C. sativus, which suggests this gene may have a role in various processes such as fragrance, insect attractant and pollination.

Asunto(s)

RESUMEN

Carotenoids comprise one of the major groups of pigments in flowers. Because carotenoids are physiologically indispensable pigments for all photosynthetic plants, their catabolism must be discretely regulated in photosynthetic organs and non-photosynthetic organs such as petals or fruits. In the chrysanthemum, carotenoid cleavage dioxygenase 4a (CmCCD4a), which is dominantly expressed in petals, cleaves carotenoid, leading to a white flower. CmCCD4a-5 was recently identified as a new member of the CmCCD4a family, but its detailed expression profile in plant tissues has not yet been established. In this study, we sequenced a 1094-bp region upstream of CmCCD4a-5 and assessed its petal-specific promoter activity. To evaluate the activity of this gene, we constructed two types of transgenic Arabidopsis thaliana that possessed, respectively, a fusion gene of a 1090-bp or 505-bp segment of the upstream region plus the ß-d-glucuronidase (GUS) gene (1090bUR::GUS and 505bUR::GUS). GUS activity in the 505bUR::GUS strain was observed mainly in the anthers/pollen in flower buds, whereas GUS activity of the 1090bUR::GUS strain was observed in immature petals of the flower buds. Among the cis-acting elements located between positions -505 and -1090, no elements that have previously been reported to enhance the expression in petals or to suppress it in anthers/pollen were detected by PLACE analysis, indicating the existence of unknown cis-element(s). A semiquantitative reverse transcription-polymerase chain reaction analysis revealed that CmCCD4a-5 transcription was prominent in petals but was undetectable in roots, stems and leaves.

Asunto(s)