Carotenoid Cleavage Dioxygenase Genes Research Articles

Cloning and functional characterization of carotenoid cleavage dioxygenase 4 genes

Although a number of plant carotenoid cleavage dioxygenase (CCD) genes have been functionally characterized in different plant species, little is known about the biochemical role and enzymatic activities of members of the subclass 4 (CCD4). To gain insight into their biological function, CCD4 genes were isolated from apple (Malus×domestica, MdCCD4), chrysanthemum (Chrysanthemum×morifolium, CmCCD4a), rose (Rosa×damascena, RdCCD4), and osmanthus (Osmanthus fragrans, OfCCD4), and were expressed, together with AtCCD4, in Escherichia coli. In vivo assays showed that CmCCD4a and MdCCD4 cleaved β-carotene well to yield β-ionone, while OfCCD4, RdCCD4, and AtCCD4 were almost inactive towards this substrate. No cleavage products were found for any of the five CCD4 genes when they were co-expressed in E. coli strains that accumulated cis-ζ-carotene and lycopene. In vitro assays, however, demonstrated the breakdown of 8′-apo-β-caroten-8′-al by AtCCD4 and RdCCD4 to β-ionone, while this apocarotenal was almost not degraded by OfCCD4, CmCCD4a, and MdCCD4. Sequence analysis of genomic clones of CCD4 genes revealed that RdCCD4, like AtCCD4, contains no intron, while MdCCD, OfCCD4, and CmCCD4a contain introns. These results indicate that plants produce at least two different forms of CCD4 proteins. Although CCD4 enzymes cleave their substrates at the same position (9,10 and 9′,10′), they might have different biochemical functions as they accept different (apo)-carotenoid substrates, show various expression patterns, and are genomically differently organized.

“Yellow Jimba”: Suppression of Carotenoid Cleavage Dioxygenase (CmCCD4a) Expression Turns White Chrysanthemum Petals Yellow

‘Jimba’ is the most popular white-flowered chrysanthemum cultivar in Japan. A yellow-flowered cultivar with the same growth properties as ‘Jimba’ will benefit growers because both forms could be produced under the same conditions. Many breeders have therefore tried to produce a “Yellow Jimba” by mutation breeding but have not yet succeeded. Previously, we showed that color mutation from white to yellow in the petals of ray florets is caused by the loss of a carotenoid cleavage dioxygenase gene, CmCCD4a. Here we introduced two separate CmCCD4a RNAi constructs into ‘Jimba’ by Agrobacterium-mediated transformation. The double-transformation effectively suppressed CmCCD4a expression in petals, which became yellow. The yellowest transformants contained 102 μg·g−1 FW carotenoids in the petals, and the expression level of CmCCD4a was 0.4% of the wild-type level. Although the transformed plants were significantly smaller than the wild type, flower size was unchanged. We consider that CmCCD4a could become a powerful tool for petal color manipulation from white to yellow.

Cytosolic and Plastoglobule-targeted Carotenoid Dioxygenases from Crocus sativus Are Both Involved in β-Ionone Release

Saffron, the processed stigma of Crocus sativus, is characterized by the presence of several apocarotenoids that contribute to the color, flavor, and aroma of the spice. However, little is known about the synthesis of aroma compounds during the development of the C. sativus stigma. The developing stigma is nearly odorless, but before and at anthesis, the aromatic compound beta-ionone becomes the principal norisoprenoid volatile in the stigma. In this study, four carotenoid cleavage dioxygenase (CCD) genes, CsCCD1a, CsCCD1b, CsCCD4a, and CsCCD4b, were isolated from C. sativus. Expression analysis showed that CsCCD1a was constitutively expressed, CsCCD1b was unique to the stigma tissue, but only CsCCD4a and -b had expression patterns consistent with the highest levels of beta-carotene and emission of beta-ionone derived during the stigma development. The CsCCD4 enzymes were localized in plastids and more specifically were present in the plastoglobules. The enzymatic activities of CsCCD1a, CsCCD1b, and CsCCD4 enzymes were determined by Escherichia coli expression, and subsequent analysis of the volatile products was generated by GC/MS. The four CCDs fell in two phylogenetically divergent dioxygenase classes, but all could cleave beta-carotene at the 9,10(9',10') positions to yield beta-ionone. The data obtained suggest that all four C. sativus CCD enzymes may contribute in different ways to the production of beta-ionone. In addition, the location and precise timing of beta-ionone synthesis, together with its known activity as a fragrance and insect attractant, suggest that this volatile may have a role in Crocus pollination.

Accumulation of Carotenoids and Expression of Carotenoid Biosynthetic Genes and Carotenoid Cleavage Dioxygenase Genes during Fruit Maturation in the Juice Sacs of ‘Tamami,’ ‘Kiyomi’ Tangor, and ‘Wilking’ Mandarin

To elucidate the mechanism of the high level of β-cryptoxanthin (β-cry) accumulation in ‘Tamami,’ the relationship between carotenoid accumulation and the expression of carotenoid-related genes during fruit maturation was investigated in the juice sacs of ‘Tamami,’ a hybrid between ‘Kiyomi’ tangor (Citrus unshiu Marc. × Citrus sinensis Osbeck) and ‘Wilking’ mandarin (Citrus nobilis Lour. × Citrus deliciosa Ten.), and its parental varieties, ‘Kiyomi’ tangor and ‘Wilking’ mandarin. In mature fruit, ‘Tamami’ predominantly accumulated a high level of β-cry. ‘Kiyomi’ tangor also accumulated β-cry. However, the level of β-cry in ‘Kiyomi’ tangor was lower than that in ‘Tamami.’ ‘Wilking’ mandarin accumulated a high level of 9-cis-violaxanthin predominantly. To analyze the expression of carotenoid-related genes, cDNA fragments for their genes were cloned from the three varieties, and the TaqMan MGB probe and a set of primers were designed for the respective genes on the basis of the common sequence of cDNA fragments among the three varieties. In ‘Tamami’ and ‘Wilking’ mandarin, the expression of genes that produce β,β-xanthophylls (CitPSY, CitPDS, CitZDS, CitLCYb, CitHYb, and CitZEP) increased simultaneously during the massive accumulation of β,β-xanthophylls (β-cry, zeaxanthin, all-trans-violaxanthin, and 9-cis-violaxanthin). In addition, higher levels of upstream synthesis genes (CitPSY, CitPDS, CitZDS, and CitLCYb) and lower levels of downstream synthesis genes (CitHYb and CitZEP) were observed in ‘Tamami’ than in ‘Wilking’ mandarin. In ‘Kiyomi’ tangor, simultaneous increases in the expression of genes that produce β,β-xanthophylls were observed in the early maturation stage. During the β,β-xanthophyll accumulation, the gene expression of carotenoid cleavage dioxygenases (CitCCD1, CitNCED2, and CitNCED3) increased in the three varieties. The results suggest that the simultaneous increases in gene expression under a balance of high expression of upstream synthesis genes and low expression of downstream synthesis genes led to a high level of β-cry accumulation in the juice sacs of ‘Tamami.’

Differential expression of putative 9- cis-epoxycarotenoid dioxygenases and abscisic acid accumulation in water stressed vegetative and reproductive tissues of citrus

The relationship between ABA accumulation and the expression of putative carotenoid cleavage dioxygenase ( CCD) genes in water-stressed vegetative and reproductive tissues from Clemenules mandarin ( Citrus clementina) scions grafted on Cleopatra mandarin ( Citrus reshni) rootstocks was investigated. Four full-length cDNAs ( CcNCED3, CcCCD4a, CcCCD4b, and CcNCED5) closely related to the CCD group and in particular to the 9- cis-epoxycarotenoid dioxygenase ( NCED) subfamily of Arabidopsis were cloned. In citrus seedlings drought-induced progressive ABA accumulation in roots, leaves and developing ovaries while re-hydration provoked sudden reduction of its levels. In these organs expression of CcCCD and CcNCED genes in normal conditions was low and similar. During the drought/re-watering cycles expression of CcNCED3, the major drought-induced gene, paralleled the pattern of ABA accumulation. In contrast, either minor or no up-regulation changes of CcNCED5 and CcCCD4a transcripts were observed. In green fruits, transcript levels of CcNCED3 and CcNCED5 in the flavedo (coloured fruit layer) were rather similar to those observed in developing ovaries. However, CcNCED5 expression dramatically increased at colour break and remained high in ripe fruit. Consistent with this observation, in the flavedo of detached ripe fruits a 37 °C dehydration treatment increased in parallel levels of ABA and CcNCED5 transcripts. The above results showed that in Clemenules mandarin water stress induced specific up-regulation of CcNCED3 in photosynthetically active tissues such as leaves and developing ovaries. On the other hand, CcNCED5 was preferentially induced in Clemenules mandarin ripening fruits.

Functional characterization of CmCCD1, a carotenoid cleavage dioxygenase from melon

Carotenoids are nutritionally important tetraterpenoid pigments that upon oxidative cleavage give rise to apocarotenoid (norisoprene) aroma volatiles. β-Carotene is the predominant pigment in orange-fleshed melon ( Cucumis melo L.) varieties, reaching levels of up to 50 μg/g FW. Pale green and white cultivars have much lower levels (0–10 μg/g FW). In parallel, β-ionone, the 9,10 cleavage product of β-carotene, is present (12–33 ng/g FW) in orange-fleshed melon varieties that accumulate β-carotene, and in much lower levels (0–5 ng/g FW) in pale green and white fleshed varieties. A search for a gene putatively responsible for the cleavage of β-carotene into β-ionone was carried out in annotated melon fruit EST databases yielding a sequence ( CmCCD1) highly similar (84%) to other plant carotenoid cleavage dioxygenase genes. To test its function, the clone was overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. We show here that the CmCCD1 gene product cleaves carotenoids at positions 9,10 and 9′,10′, generating geranylacetone from phytoene; pseudoionone from lycopene; β-ionone from β-carotene, as well as α-ionone and pseudoionone from δ-carotene. CmCCD1 gene expression is upregulated upon fruit development both in orange, pale-green and white melon varieties, despite the lack of apocarotenoid volatiles in the later. Thus, the accumulation of β-ionone in melon fruit is probably limited by the availability of carotenoid substrate.

A Carotenoid Cleavage Dioxygenase from Vitis vinifera L.: functional characterization and expression during grape berry development in relation to C13-norisoprenoid accumulation

A potential Carotenoid Cleavage Dioxygenase (CCD) gene was identified among a Vitis vinifera L. EST collection and a full-length cDNA (VvCCD1) was isolated. Recombinant expression of VvCCD1 confirmed that the gene encoded a functional CCD. Experimental evidence was obtained that VvCCD1 cleaves zeaxanthin symmetrically yielding 3-hydroxy-beta-ionone, a C(13)-norisoprenoidic compound, and a C(14)-dialdehyde. Expression of the gene was studied by real-time PCR at different developmental stages of grape berries from Muscat of Alexandria and Shiraz cultivars. A significant induction of the gene expression approaching véraison was observed in both cultivars. In parallel, the C(13)-norisoprenoid level increased from véraison to maturity in both cultivars.

キクの白色花弁で特異的に発現しているカロテノイド分解酵素遺伝子(CmCCD1)のクローニング

キクの白色花弁で特異的に発現しているカロテノイド分解酵素遺伝子(CmCCD1)のクローニング

Molecular characterization of the Arabidopsis 9-cis epoxycarotenoid dioxygenase gene family.

A key regulated step in abscisic acid (ABA) biosynthesis in plants is catalyzed by 9-cis epoxycarotenoid dioxygenase (NCED), which cleaves 9-cis xanthophylls to xanthoxin, a precursor of ABA. In Arabidopsis, ABA biosynthesis is controlled by a small family of NCED genes. Nine carotenoid cleavage dioxygenase (CCD) genes have been identified in the complete genome sequence. Of these, five AtNCEDs (2, 3, 5, 6, and 9) have been cloned and studied for expression and subcellular localization. Although all five AtNCEDs are targeted to plastids, they differ in binding activity of the thylakoid membrane. AtNCED2, AtNCED3, and AtNCED6 are found in both stroma and thylakoid membrane-bound compartments. AtNCED5 is exclusively bound to thylakoids, whereas AtNCED9 remains soluble in stroma. A quantitative real-time PCR analysis and histochemical staining of promoter::GUS activity in transgenic Arabidopsis revealed a complex pattern of localized NCED expression in well-watered plants during development. AtNCED2 and AtNCED3 account for the NCED activity in roots, with localized expression in root tips, pericycle, and cortex cells at the base of lateral roots. Localized AtNCED2 and AtNCED3 expression in pericycle cells is an early marker of lateral initiation sites. AtNCED5, AtNCED6, AtNCED3, and AtNCED2 are expressed in flowers with very high AtNCED6::GUS activity occurring in pollen. AtNCED5::GUS, and to lesser degrees, AtNCED2::GUS and AtNCED3::GUS are expressed in developing anthers. AtNCED5, AtNCED6, AtNCED9, and AtNCED3 contribute to expression in developing seeds with high levels of AtNCED6 present at an early stage. GUS analysis indicates that AtNCED3 expression is confined to the base of the seed, whereas AtNCED5 and AtNCED6 are expressed throughout the seed. Consistent with the studies conducted by Iuchi and his colleagues in 2001, AtNCED3 is the major stress-induced NCED in leaves. Our results indicate that developmental control of ABA synthesis involves localized patterns of AtNCED gene expression. In addition, differential membrane-binding capacity of AtNCEDs is a potential means of post-translational regulation of NCED activity.