Carotenoid Cleavage Dioxygenase Genes Research Articles

Genome-wide analysis and identification of Carotenoid Cleavage Oxygenase (CCO) gene family in coffee (coffee arabica) under abiotic stress

The coffee industry holds importance, providing livelihoods for millions of farmers globally and playing a vital role in the economies of coffee-producing countries. Environmental conditions such as drought and temperature fluctuations can adversely affect the quality and yield of coffee crops.Carotenoid cleavage oxygenases (CCO) enzymes are essential for coffee plants as they help break down carotenoids contributing to growth and stress resistance. However, knowledge about the CCO gene family in Coffee arabica was limited. In this study identified 21 CCO genes in Coffee arabica (C. arabica) revealing two subfamilies carotenoid cleavage dioxygenases (CCDs) and 9-cis-epoxy carotenoid dioxygenases (NCED) through phylogenic analysis. These subfamilies exhibited distribution patterns in terms of gene structure, domains, and motifs. The 21 CaCCO genes, comprising 5 NCED and 16 CCD genes were found across chromosomes. Promoter sequencing analysis revealed cis-elements that likely interact with plant stress-responsive, growth-related, and phytohormones, like auxin and abscisic acid. A comprehensive genome-wide comparison, between C. arabica and A. thaliana was conducted to understand the characteristics of CCO genes. RTqPCR data indicated that CaNCED5, CaNCED6, CaNCED12, and CaNCED20 are target genes involved in the growth of drought coffee plants leading to increased crop yield, in a conditions, with limited water availability. This reveals the role of coffee CCOs in responding to abiotic stress and identifies potential genes useful for breeding stress-resistant coffee varieties.

Transcriptome analysis of Lantana camara flower petals reveals candidate anthocyanin biosynthesis genes mediating red flower color development.

Flower color plays a crucial role in the appeal and selection of ornamental plants, directly influencing breeding strategies and the broader horticulture industry. Lantana camara, a widely favored flowering shrub, presents a rich palette of flower colors. Yet, the intricate molecular mechanisms governing this color variation in the species have remained largely unidentified. With the aim of filling this gap, this study embarked on a comprehensive de novo transcriptome assembly and differential gene expression analysis across 3 distinct lantana accessions, each showcasing a unique flower color. By harnessing the capabilities of both PacBio and Illumina sequencing platforms, a robust transcriptome assembly, encompassing 123,492 gene clusters and boasting 94.2% BUSCO completeness, was developed. The differential expression analysis unveiled 72,862 unique gene clusters that exhibited varied expression across different flower stages. A pronounced upregulation of 8 candidate core anthocyanin biosynthesis genes in the red-flowered accession was uncovered. This was further complemented by an upregulation of candidate MYB75 (PAP1) and bHLH42 (TT8) transcription factors. A candidate carotenoid cleavage dioxygenase (CCD4a) gene cluster also manifested a marked upregulation in white flowers. The study unveils the molecular groundwork of lantana's flower color variation, offering insights for future research and potential applications in breeding ornamental plants with desired color traits.

Expression profile of the NCED/CCD genes in chickpea and lentil during abiotic stress reveals a positive correlation with increased plant tolerance

Carotenoid cleavage dioxygenase (CCD) gene family is organized in two subfamilies: (i) 9-cis epoxycarotenoid dioxygenase (NCED) genes and (ii) CCD genes. NCED genes are essential for catalyzing the first step of the abscisic-acid (ABA) biosynthesis, while CCD genes produce precursors of the strigolactones hormone. The functional characterization of these gene subfamilies has not been yet performed in chickpea and lentil. Herein, were identified and systematically characterized two NCED and five CCD genes in the chickpea and two NCED and six CCD genes in lentil. After in silico sequence analysis and phylogeny, the expression profile of the NCED/CCD genes was determined by meta-analysis and real-time PCR in plants under different stress conditions. Sequence data revealed that NCED/CCD genes are highly conserved between chickpea and lentil. This conservation was observed both at gene and protein sequence levels and phylogenetic relationships. Analysis of the promoter sequences revealed that all NCED/CCD genes have a considerable number of cis-regulatory elements responsive to biotic and abiotic stress. Protein sequence analysis evidenced that NCED/CCD genes share several conserved motifs and that they have a highly interconnected interaction network. Furthermore, the three-dimensional structure of these proteins was determined and indicated that some proteins have structures with considerable similarity. The meta-analysis revealed that NCED/CCD genes are dynamically modulated in different organs and under different stress conditions, but they have a positive correlation with plant tolerance. In accordance, real-time PCR data showed that both NCED and CCD genes are differentially modulated in plants under drought stress. In particular, CaNCED2, CaCCD5, LcNCED2, LcCCD1, and LcCCD2 genes have a positive correlation with improved plant tolerance to drought stress. Therefore, this study presented a detailed characterization of the chickpea and lentil NCED/CCD genes and provided new insights to improve abiotic stress tolerance in these two important crops.

Dynamic change of the carotenoid metabolic pathway profile during oolong tea processing with supplementary LED light

Carotenoid-derived volatiles are important contributors to tea aroma quality. However, the profile of the carotenoid pathway and carotenoid-derived volatiles (CDVs) artificial regulation in oolong tea processing has yet to be investigated. In the present work, the content and varieties of carotenoid-derived volatiles, the genome-wide identification of carotenoid cleavage dioxygenase (CsCCD) gene family, the expression level of CsCCD and other key genes in the carotenoid pathway, and the profile of carotenoid substances were analyzed by multi-omics and bioinformatics methods with innovative postharvest supplementary LED light during oolong tea processing. The results showed that during oolong tea processing, a total of 17 CDVs were identified. The content of β-ionone increased up to 26.07 times that of fresh leaves and its formation was significantly promoted with supplementary LED light from 0.54 μg/g to 0.83 μg/g in the third turning over treatment. A total of 11 CsCCD gene family members were identified and 119 light response cis-acting regulatory elements of CsCCD were found. However, the expression level of most genes in the carotenoid pathway including CsCCD were reduced due to mechanical stress. ‘Huangdan’ fresh tea leaves had a total of 1 430.46 μg/g 22 varieties of carotenoids, which mainly composed of lutein(78.10%), β-carotene(8.24%) and zeaxanthin(8.18%). With supplementary LED light, the content of antherxanthin and zeaxanthin in xanthophyll cycle was regulated and CDVs such as α-ionone, β-ionone, pseudoionone, damascenone, 6,10-dimethyl-5,9-undecadien-2-one, citral, geranyl acetate and α-farnesene were promoted significantly in different phases during oolong tea processing. Our results revealed the profile of the carotenoid metabolism pathway in oolong tea processing from the perspective of precursors, gene expression and products, and put forward an innovative way to improve CDVs by postharvest supplementary LED light.

Analysis of apocarotenoid volatiles from lettuce (Lactuca sativa) induced by insect herbivores and characterization of carotenoid cleavage dioxygenase gene.

The online version contains supplementary material available at 10.1007/s13205-023-03511-4.

Cloning and Prokaryotic Expression of Carotenoid Cleavage Dioxygenases from Mulberry (Morus notabilis).

Carotenoid cleavage dioxygenase (CCD) is the key enzyme for carotenoid cleavage, and the products of carotenoid cleavage regulate the ability of plants to stress. In this paper, six CCD genes were obtained from Morus notabilis (Mn) by reverse transcription-polymerase chain reaction (RT-PCR) and we classified them into three subgroups based on gene structures and phylogenetic analysis. The CDS (coding sequence) regions of the six MnCCD genes were 1617, 1620, 1635, 1713, 1746, and 1791 bp in full length, encoding 538, 539, 544, 570, 581, and 596 amino acids, respectively. Then, Pcold–TF-MnCCD plasmids were constructed and independently transferred into E. coli BL21 (DE3), and the MnCCD proteins were successfully expressed by prokaryotic expression with an expected molecular weight of recombinant proteins (∼120 kDa) and high solubility. These results will lay a foundation for the identification of mulberry carotenoid products.

Identification of the carotenoid cleavage dioxygenase genes and functional analysis reveal DoCCD1 is potentially involved in beta-ionone formation in Dendrobium officinale.

The carotenoids are the most widely distributed secondary metabolites in plants and can be degraded by carotenoid cleavage dioxygenase (CCD) to form apocarotenoids including an important C13 compound beta-ionone. Volatile beta-ionone can confer the violet and woody fragrance to plant essential oils, flowers, fruits, and vegetables, which therefore has been used in various industries. Dendrobium officinale is a traditional medicinal plant. However, there was limited information on the key enzymes involved in the biosynthesis of beta-ionone in D. officinale. In the present study, beta-ionone was detected in stems and leaves of D. officinale and genome-wide identification and expression profiles of CCD genes were subsequently carried out. There were nine DoCCD members in D. officinale. According to the phylogenetic relationship, DoCCD proteins were classified into six subfamilies including CCD1, CCD4, CCD7, CCD8, nine-cis-epoxycarotenoid dioxygenase (NCED) and zaxinone synthase (ZAS). DoCCD genes showed distinctive expression profiles and DoCCD1 gene was abundantly expressed in eight tissues. Induced expression of DoCCD1 gene resulted in discoloration of Escerichia coli strains that can accumulate carotenoids. Analysis of Gas Chromatography/Mass Spectrometer showed that DoCCD1 enzyme can cleave lycopene to produce 6-methyl-5-hepten-2-one and pseudoionone and also catalyze beta-carotene to form beta-ionone. Expression of DoCCD1 gene in Nicotiana benthamiana leaf resulted in production of abundant beta-ionone. Overall, the present study first provides valuable information on the CCD gene family in D. officinale, function of DoCCD1 gene as well as production of beta-ionone through genetic modification.

Metabolic Profiling and Transcriptional Analysis of Carotenoid Accumulation in a Red-Fleshed Mutant of Pummelo (Citrus grandis).

Citrus grandis ‘Tomentosa’, commonly known as ‘Huajuhong’ pummelo (HJH), is used in traditional Chinese medicine and can moisten the lungs, resolve phlegm, and relieve coughs. A spontaneous bud mutant, named R-HJH, had a visually attractive phenotype with red albedo tissue and red juice sacs. In this study, the content and composition of carotenoids were investigated and compared between R-HJH and wild-type HJH using HPLC–MS analysis. The total carotenoids in the albedo tissue and juice sacs of R-HJH were 4.03- and 2.89-fold greater than those in HJH, respectively. The massive accumulation of carotenoids, including lycopene, β-carotene and phytoene, led to the attractive red color of R-HJH. However, the contents of flavones, coumarins and most volatile components (mainly D-limonene and γ-terpinene) were clearly reduced in R-HJH compared with wild-type HJH. To identify the molecular basis of carotenoid accumulation in R-HJH, RNA-Seq transcriptome sequencing was performed. Among 3948 differentially expressed genes (DEGs), the increased upstream synthesis genes (phytoene synthase gene, PSY) and decreased downstream genes (β-carotene hydroxylase gene, CHYB and carotenoid cleavage dioxygenase gene, CCD7) might be the key factors that account for the high level of carotenoids in R-HJH. These results will be beneficial for determining the molecular mechanism of carotenoid accumulation and metabolism in pummelo.

Co-option of a carotenoid cleavage dioxygenase gene (CCD4a) into the floral symmetry gene regulatory network contributes to the polymorphic floral shape-color combinations in Chrysanthemum sensu lato.

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.

Integrative analyses of transcriptome and carotenoids profiling revealed molecular insight into variations in fruits color of Citrus Reticulata Blanco induced by transplantation.

Citrus fruits exhibit vivid color and are favored extensively. However, the biochemical and molecular mechanism of Citrus Reticulata Blanco fruits coloring, especially the effect of transplantation on fruits coloring, is unclear. Herein, RNA-Seq and carotenoids profiling were applied to investigate the effect of transplantation on Orah mandarin fruits coloring. Transplantation induces fruit color shallowing, Ca2+ and ACC level declining and IAA level increasing. Transplantation induced variation in fruit skin and pulp carotenoids, mainly β-citraurin as one of the important pigments of citrus peel. 2253 up-regulated genes, 1103 down-regulated genes in skin and 815 up-regulated genes, 534 down-regulated genes in pulp of transplanted tree fruits were identified by RNA-Seq. The DEGs involved hormone signal, carotenoids biosynthesis and TFs such as MYB and bHLH family TFs. The carotenoid cleavage dioxygenase gene (Ciclev10028113m.g) is positively correlated with β-citraurin and regulated directly and/or indirectly by MYB1R1, PIF4, ACC and IAA. Integrative analyses revealed potential molecular insights into Orah mandarin peel color variation during transplantation.

The Carotenoid Cleavage Dioxygenase Gene CCD7-B, at Large, Is Associated with Tillering in Common Wheat

Wheat, an important cereal crop, is responsible for the livelihoods of many people, and a component of national food security. Tillering, which determines plant architecture and spike number, is a critical agronomic trait of wheat. The carotenoid cleavage dioxygenase 7 (CCD7) has an important effect on the growth of tillers or lateral branches and lateral roots of plants. In order to study the relationship between CCD7 and tillering in wheat, CCD7-B was isolated from 10 Chinese wheat varieties with different tiller numbers. Subsequently, bioinformatics, allelic variation analysis, and field experiments were performed. Wheat CCD7-B belongs to the retinal pigment epithelial membrane receptor (RPE65) superfamily; it displays the greatest homology with monocot CCD7 proteins. Phylogenetic analysis of wheat CCD7-B proteins indicated division into dicotyledonous and monocotyledonous clades. Allelic variation analysis of CCD7-B via SrgAI enzyme digestion (a marker of cleaved amplified polymorphic sequences) suggested that 262 Chinese wheat micro-core collections and 121 Chinese wheat major cultivars from the Yellow and Huai River Valley winter wheat region can be divided into two groups: CCD7-B1 (C/T/T) and CCD7-B2 (G/C/A). CCD7-B1 showed better allelic variation than did CCD7-B2 for increasing the number of effective tillers of wheat varieties in China. This study provides reference data for the application of CCD7-B alleles to wheat breeding and supports further research regarding the mechanism of tillering in common wheat.

Genome-Wide Identification of CCD Gene Family in Six Cucurbitaceae Species and Its Expression Profiles in Melon.

The carotenoid cleavage dioxygenase (CCD) gene family in plants comprises two subfamilies: CCD and 9-cis-epoxycarotenoid dioxygenase (NCED). Genes in the NCED subfamily are mainly involved in plant responses to abiotic stresses such as salt, low temperature, and drought. Members of the NCED subfamily are the most important rate-limiting enzymes in the biosynthesis of abscisic acid (ABA). In the present study, genome-wide analysis was performed to identify CCD gene members in six Cucurbitaceae species, including watermelon (Citrullus lanatus), melon (Cucumis melo), cucumber (C.sativus), pumpkin (Cucurbita moschata), bottle gourd (Lagenaria siceraria), and wax gourd (Benincasa hispida). A total of 10, 9, 9, 13, 8, 8 CCD genes were identified in the six species, respectively, and these genes were unevenly distributed in different chromosomes. Phylogenetic analysis showed that CCD genes of the six species clustered into two subfamilies: CCD and NCED, with five and three independent clades, respectively. The number of exons ranged from 1 to 15, and the number of motifs were set to 15 at most. The cis-acting elements analysis showed that a lot of the cis-acting elements were implicated in stress and hormone response. Melon seedlings were treated with salt, low temperature, drought, and ABA, and then tissue-specific analysis of CCDs expression were performed on the root, stem, upper leaf, middle leaf, female flower, male flower, and tendril of melon. The results showed that genes in CCD family exhibited various expression patterns. Different CCD genes of melon showed different degrees of response to abiotic stress. This study presents a comprehensive analysis of CCD gene family in six species of Cucurbitaceae, providing a strong foundation for future studies on specific genes in this family.

Comprehensive Analysis of Carotenoid Cleavage Dioxygenases Gene Family and Its Expression in Response to Abiotic Stress in Poplar.

Carotenoid cleavage dioxygenases (CCDs) catalyzes the cleavage of various carotenoids into smaller apocarotenoids which are essential for plant growth and development and response to abiotic stresses. CCD family is divided into two subfamilies: 9-cis epoxycarotenoid dioxygenases (NCED) family and CCD family. A better knowledge of carotenoid biosynthesis and degradation could be useful for regulating carotenoid contents. Here, 23 CCD genes were identified from the Populus trichocarpa genome, and their characterizations and expression profiling were validated. The PtCCD members were divided into PtCCD and PtNCED subfamilies. The PtCCD family contained the PtCCD1, 4, 7, and 8 classes. The PtCCDs clustered in the same clade shared similar intron/exon structures and motif compositions and distributions. In addition, the tandem and segmental duplications resulted in the PtCCD gene expansion based on the collinearity analysis. An additional integrated collinearity analysis among poplar, Arabidopsis, rice, and willow revealed the gene pairs between poplar and willow more than that between poplar and rice. Identifying tissue-special expression patterns indicated that PtCCD genes display different expression patterns in leaves, stems, and roots. Abscisic acid (ABA) treatment and abiotic stress suggested that many PtCCD genes are responsive to osmotic stress regarding the comprehensive regulation networks. The genome-wide identification of PtCCD genes may provide the foundation for further exploring the putative regulation mechanism on osmotic stress and benefit poplar molecular breeding.

Elucidation of Phenomena Involving Cyanobacteria in Freshwater Ecosystem by Chemically Ecological Approach

Lakes Sagami and Tsukui are reservoirs constructed by connecting to the Sagami River. Because of eutrophication of the lakes, cyanobacteria have appeared every year. This review deals with phenomena related to occurrence of cyanobacteria that have been observed for 40 years since 1974 at the lakes. These 40 years of observations raised three interesting issues including the retention of cyanobacteria on their surfaces. These phenomena have been attributed to the usual factors, such as illuminance, nutrition and water temperature, but our research results suggested that they cannot be resolved without the introduction of another factor. We have attempted to elucidate various phenomena involving cyanobacteria in lake ecosystems by chemical ecological methods using volatile organic compounds (VOCs) produced by the cyanobacteria as indicators. One of the VOCs, β-cyclocitral, was significantly involved in the above phenomena, which was considered to be produced by the carotenoid cleavage dioxygenase (CCD) of the cyanobacteria. β-Cyclocitral was not produced in the two known CCDs, but two additional CCDs to Microcystis aeruginosa participated to produce the β-cyclocitral. These CCDs did not directly produce β-cyclocitral, but it was accumulated in cells as their precursors. The released β-cyclocitral underwent a Baeyer-Villiger-like oxidation. It was speculated that Microcystis activated the CCD genes through density stress and produced β-cyclocitral, which acted as an allelopathic substance. As a result, the number of cells of cyanobacteria decreased, and the resulting nitrogen and phosphorus were fed to the living cyanobacteria. It is postulated that this "quorum sensing" was functioning in the above-mentioned issues.

Cloning and Characterization of a Novel Carotenoid Cleavage Dioxygenase 1 from Helianthus annuus.

Natural β-ionone, a high-value flavoring agent, has been widely applied in the food, cosmetics, and perfume industry. However, attempts to overproduce β-ionone in microorganisms have been limited by the efficiency of carotenoid cleavage dioxygenases (CCDs), which catalyzes β-carotene in the biosynthesis pathway. In order to obtain CCD genes responsible for the specific cleavage of carotenoids generating β-ionone, a novel carotenoid cleavage dioxygenase 1 from Helianthus annuus was cloned and overexpressed in Escherichia coli BL21(DE3). The recombinant CCD was able to cleave a variety of carotenoids at the 9, 10 (9', 10') sites to produce C13 products in vitro, including β-ionone, pseudoionone, 3-hydroxy-4-oxo-β-ionone, 3-hydroxy-β-ionone, and 3-hydroxy-α-ionone, which vary depending on the carotenoid substrates. In comparison with lycopene and zeaxanthin, HaCCD1 also showed the high specificity for β-carotene to cleave the 9, 10 (9', 10') double bond to produce β-ionone in E. coli accumulating carotenoids. Finally, the expression of HaCCD1 in E. coli was optimized, and biochemical characterizations were further clarified. The optimal activity of HaCCD1 was at pH 8.8 and 50 °C. The Vmax for β-apo-8'-carotenal was 10.14 U/mg, while the Km was 0.32 mM. Collectively, our study provides a valuable enzyme for the synthesis of natural β-ionone by biotransformation and synthetic biology platform.

DcCCD4 catalyzes the degradation of α-carotene and β-carotene to affect carotenoid accumulation and taproot color in carrot.

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 β-carotene decreased sharply. Mutant carrot with loss of DcCCD4 function exhibited yellow color (the taproot of the control carrot was white). The accumulation of β-carotene was also detected in taproot. Functional analysis of the DcCCD4 enzyme in vitro showed that it was able to cleave α- and β-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 β-carotene in carrot taproot.

Genome-wide identification, characterization and expression profiles of the CCD gene family in Gossypium species.

The online version contains supplementary material available at 10.1007/s13205-021-02805-9.

Genome-wide identification and expression profiling of the carotenoid cleavage dioxygenase (CCD) gene family in Brassica napus L.

Carotenoid cleavage dioxygenase (CCD), a key enzyme in carotenoid metabolism, cleaves carotenoids to form apo-carotenoids, which play a major role in plant growth and stress responses. CCD genes had not previously been systematically characterized in Brassica napus (rapeseed), an important oil crop worldwide. In this study, we identified 30 BnCCD genes and classified them into nine subgroups based on a phylogenetic analysis. We identified the chromosomal locations, gene structures, and cis-promoter elements of each of these genes and performed a selection pressure analysis to identify residues under selection. Furthermore, we determined the subcellular localization, physicochemical properties, and conserved protein motifs of the encoded proteins. All the CCD proteins contained a retinal pigment epithelial membrane protein (RPE65) domain. qRT-PCR analysis of expression of 20 representative BnCCD genes in 16 tissues of the B. napus cultivar Zhong Shuang 11 (‘ZS11’) revealed that members of the BnCCD gene family possess a broad range of expression patterns. This work lays the foundation for functional studies of the BnCCD gene family.

Structural basis for carotenoid cleavage by an archaeal carotenoid dioxygenase

Apocarotenoids are important signaling molecules generated from carotenoids through the action of carotenoid cleavage dioxygenases (CCDs). These enzymes have a remarkable ability to cleave carotenoids at specific alkene bonds while leaving chemically similar sites within the polyene intact. Although several bacterial and eukaryotic CCDs have been characterized, the long-standing goal of experimentally visualizing a CCD-carotenoid complex at high resolution to explain this exquisite regioselectivity remains unfulfilled. CCD genes are also present in some archaeal genomes, but the encoded enzymes remain uninvestigated. Here, we address this knowledge gap through analysis of a metazoan-like archaeal CCD from Candidatus Nitrosotalea devanaterra (NdCCD). NdCCD was active toward β-apocarotenoids but did not cleave bicyclic carotenoids. It exhibited an unusual regiospecificity, cleaving apocarotenoids solely at the C14'-C13' alkene bond to produce β-apo-14'-carotenals. The structure of NdCCD revealed a tapered active site cavity markedly different from the broad active site observed for the retinal-forming Synechocystis apocarotenoid oxygenase (SynACO) but similar to the vertebrate retinoid isomerase RPE65. The structure of NdCCD in complex with its apocarotenoid product demonstrated that the site of cleavage is defined by interactions along the substrate binding cleft as well as selective stabilization of reaction intermediates at the scissile alkene. These data on the molecular basis of CCD catalysis shed light on the origins of the varied catalytic activities found in metazoan CCDs, opening the possibility of modifying their activity through rational chemical or genetic approaches.

Cloning and functional characterization of a carotenoid cleavage dioxygenase 2 gene in safranal and crocin biosynthesis from Freesia hybrida

Safranal and crocin, commonly derived from the oxidative cleavage reaction of zeaxanthin in plants, are two kinds of apocarotenoids with versatile functions, which were only found in limited number of plant species. In this study, both metabolites were detected and varied concomitantly with the expression of carotenoid cleavage dioxygenase (CCD) genes in Freesia hybrida, Red River® and Ambiance cultivars. The newly isolated CCD, denoted here as FhCCD2, was phylogenetically clustered with other reported saffron CCD2s. Besides, ten introns were also observed in the genomic DNA sequence of FhCCD2 and the presence of N-terminal transporter peptide suggested its plastidial sub-localization. Biochemical analysis showed that the FhCCD2 cleaved zeaxanthin at the 7, 8 and 7′, 8’ double bonds to generate intermediates prerequisite for the biosynthesis of safranal and crocin. Further, gene transient expression analysis showed that the promoter of FhCCD2 was functional in Ambiance as well as Red River® cultivars, even with slight variation in their promoter sequence. At present, CCD2 proteins have only been found in Freesia and Crocus genus of Iridaceae family. Phylogenetic and intron position analysis infer that CCD2 perhaps emerged after the intron loss during evolutionary process of CCD1 or their shared ancestry.