Carotenoid Cleavage Dioxygenase (CmCCD4a) Contributes to White Color Formation in Chrysanthemum Petals

The white petals of chrysanthemum ( Chrysanthemum morifolium Ramat.) are believed to contain a factor that inhibits the accumulation of carotenoids. To find this factor, we performed PCR-Select subtraction screening and obtained a clone expressed differentially in white and yellow petals. The deduced amino acid sequence of the protein (designated CmCCD4a) encoded by the clone was highly homologous to the sequence of carotenoid cleavage dioxygenase. All the white-flowered chrysanthemum cultivars tested showed high levels of CmCCD4a transcript in their petals, whereas most of the yellow-flowered cultivars showed extremely low levels. Expression of CmCCD4a was strictly limited to flower petals and was not detected in other organs, such as the root, stem, or leaf. White petals turned yellow after the RNAi construct of CmCCD4a was introduced. These results indicate that in white petals of chrysanthemums, carotenoids are synthesized but are subsequently degraded into colorless compounds, which results in the white color.


INTRODUCTION
difference between the expression levels of carotenoid biosynthetic genes in white and yellow petals during the course of development. In addition, the carotenoid content in immature white petals is almost equal to that in yellow petals, and the carotenoid content decreases to undetectable levels as the white petals mature. These results indicate that the formation of white color is caused neither by down-regulation nor by disruption of the carotenoid biosynthetic pathway.
To find a factor that controls carotenoid content in chrysanthemum petals, we performed PCR-Select subtraction screening to search for cDNAs that were differentially expressed in white and yellow petals. In this paper, we show that a gene encoding a carotenoid cleavage dioxygenase (designated as CmCCD4a) is expressed specifically in white petals and that this enzyme contributes to the formation of white color in chrysanthemum petals.

Cloning of CmCCD4a by PCR-Select Subtraction Screening
We performed subtraction of messages expressed in the white petals of chrysanthemum cv. Paragon from those in the yellow petals of cv. Yellow Paragon, a bud sport arising from Paragon. We then screened for cDNAs that were differentially expressed in the white and yellow petals and obtained 31 cDNA clones whose expression was higher in Paragon, and 71 clones whose expression was higher in Yellow Paragon. On the basis of signal-to-noise ratios, we selected 15 clones for further analysis. Quantitative real-time RT-PCR analysis showed that only one of the clones was expressed specifically in the petals of white cultivars. We obtained full-length cDNA of the clone by the rapid amplification of cDNA ends (RACE) method, and we determined its nucleotide sequence. The nucleotide sequence of the clone contained an open reading frame of 1797 bp, predicting a 599-amino acid polypeptide, and had an estimated molecular mass of 67 kD. Comparison of the deduced amino acid sequence of the protein encoded by the clone with available databases revealed the sequence to be highly homologous with that of carotenoid cleavage dioxygenase (CCD) (Fig. 1A). The protein showed the highest homology with AtCCD4, a CCD homolog found in Arabidopsis (Fig. 1B); we therefore designated the clone as CmCCD4a. The amino acid sequence of CmCCD4a showed 61% homology with the sequence of AtCCD4, and 51% homology with that of OsCCD4a from rice.

Cloning of CCD and NCED Homologs and Expression in Petals
Homologs of CCD and NCED were screened from cDNAs of petals and leaves of chrysanthemums using degenerate primers of CCD and NCED, respectively. In addition to CmCCD4a, we obtained one CCD homolog and two NCED homologs from leaf cDNA.
The amino acid sequence of the protein encoded by the CCD homolog showed the highest homology to AtCCD4, and the proteins encoded by the NCED homologs showed the highest homology to AtNCED3 among the Arabidopsis CCD family. Therefore, we designated these proteins as CmCCD4b, CmNCED3a, and CmNCED3b, respectively (Fig.   1B). We performed genomic PCR using degenerate primers to search for additional homologs, but we did not find any. On the basis of sequence comparison of these homologs, we designed primers specific to each homolog for genomic PCR and real-time RT-PCR analyses. Figure 2A shows the levels of transcripts of CCD and NCED homologs in the white and yellow petals of various chrysanthemum cultivars. All the white petals tested showed high levels of expression of CmCCD4a, whereas transcripts in the yellow petals were not detected, except in Yellow Paragon, which had approximately half the amount of CmCCD4a transcripts as Paragon. The levels of CmCCD4b, CmNCED3a, and CmNCED3b transcripts in petals were extremely low compared to the level of CmCCD4a transcripts. In addition, there was no significant difference between the expression levels of these homologs in white and yellow petals.
Genomic PCR analysis showed that the bands that corresponded to CmCCD4a was not amplified in yellow-flowered cultivars with extremely low expression of CmCCD4a ( Fig.   2A). In contrast, the bands corresponding to CmCCD4b, CmNCED3a, and CmNCED3b were amplified in all the cultivars we tested. Genomic CmCCD4a has two BamHI sites: one at −464 bp and another at 1565 bp from the start codon; BamHI digestion produced a 2.0-kbp fragment. In Southern blot analysis probed with the CCD fragment, the band corresponding to CmCCD4a was not detected in these cultivars (Fig. 2B). The band at 7 kbp existed in all the cultivars tested. Because the CmCCD4b gene existed in all the cultivars, the band may correspond to CmCCD4b. The band pattern of Paragon coincided with that of Yellow Paragon, a bud sport of Paragon. On the other hand, Florida Marble, a bud sport of White Marble, had three bands in common with White Marble but lacked a band corresponding to CmCCD4a. When the same filter was reprobed with the NCED fragment, all cultivars showed two to three bands of different sizes. Different band patterns were observed in membranes hybridized with CCD and NCED probes, which indicates that these probes are not cross-hybridized. Figure 3 shows the organ-specific expression pattern of CCD and NCED homologs.

Organ-and Stage-Specific Expression of CCD and NCED Homologs
Expression of CmCCD4a was strictly limited to flower petals and was not detected in other organs, such as the root, stem, or leaf. The chrysanthemum flower is often dimorphic: in the center of the capitulum is the small disc floret; in the marginal ray floret, the corolla is conspicuous as a long "petal." White-flowered cultivars usually have yellow disc florets.
The level of CmCCD4a transcripts in the yellow disc floret of Paragon was extremely low compared with the level in the white petals of the ray floret. Yellow disc florets of the other white-flowered cultivars, such as Sei-Marine, Fiducia, and White Marble, also showed extremely low levels of CmCCD4a transcripts (data not shown). The abundance of transcripts of CmCCD4b, CmNCED3a, and CmNCED3b showed different organ-specificity from that of CmCCD4a. These transcripts were extremely low in petals, disc florets, and roots. The levels of CmCCD4b and CmNCED3a transcripts were high in leaves. The expression of CmCCD4b was extremely high in stems; this high level of expression may have been caused partly by the normalization against actin levels, if stem tissue had a substantially lower level of actin transcripts than other tissues. Expression of CmNCED3b was low in all the tissues we tested.
White petals of Paragon contained 47 μg/gfw of carotenoids at the very early stage of flower development (Fig. 4). Carotenoid content decreased as the petals matured, and carotenoids were not detected in the fully opened petals. In contrast, the level of CmCCD4a transcripts increased drastically during the course of petal development.

Suppression of CmCCD4a Expression by RNAi
To determine the role of CmCCD4a gene products in the formation of petal color, we produced transgenic chrysanthemum plants with reduced expression of CmCCD4a. We

CmCCD4a in Wild Chrysanthemum Species
Genomic PCR analysis was also performed in white-and yellow-flowered wild species of chrysanthemum (Fig. 6). The bands that corresponded to CmCCD4a were observed in all the white-flowered species but not in the yellow-flowered species. In contrast, the bands that corresponded to CmCCD4b were observed in both white-and yellow-flowered species.

Light Microscope Observation of Transverse Sections of Petals
Among yellow-flowered cultivars, only Yellow Paragon expressed CmCCD4a in petals.
It is possible that petals of Yellow Paragon are periclinal chimera, and either the L1 or the L2 layer may behave genetically in a manner identical to that of the white progenitor Paragon. Periclinal structures were determined by microscope examination of transverse sections of petals (Fig. 7). In the sections of Yellow Paragon, yellow pigmentation was localized in the adaxial epidermis (L1 layer), and the underlying mesophyll (L2 layer) appeared to be white. In contrast, both the L1 and L2 layers were yellow in petals of Super Yellow.

DISCUSSION
By means of PCR-Select subtraction screening, we obtained a clone that encoded a carotenoid cleavage dioxygenase and was differentially expressed in white and yellow petals of chrysanthemums. Several types of CCDs were recently reported in various plant species. Arabidopsis contains nine homologs of the CCD family (Tan et al., 2003). Five of the homologs have been designated as 9-cis-epoxycarotenoid dioxygenases (AtNCED2, AtNCED3, AtNCED5, AtNCED6, and AtNCED9), which catalyze cleavage of the 11,12 double bond of 9-cis-neoxanthin and 9-cis-violaxanthin to form xanthoxin, an ABA precursor. The rest of these CCDs (designated AtCCD1, AtCCD4, AtCCD7, and AtCCD8) cleave carotenoids into apocarotenoids at different double bond positions. AtCCD1 symmetrically cleaves the 9,10 (9′,10′) double bonds of multiple carotenoid substrates in vitro (Schwartz et al., 2001). Homologs of this enzyme have recently been identified in various plant species; some of the homologs contribute to the formation of flavor volatiles, such as β-ionone, pseudoionone, and geranylacetone (Simkin et al., 2004a,b). AtCCD7 and AtCCD8 sequentially cleave β-carotene into 13-apo-β-carotenone (C18), a signaling molecule that is necessary for the regulation of lateral branching (Schwartz et al., 2004).
The biochemical and enzymatic activities of AtCCD4 have not been characterized. The deduced amino acid sequence of CmCCD4a showed the highest homology with that of AtCCD4 among members of Arabidopsis CCD family, showing 61% homology. CmCCD4a also shares a common feature with AtCCD4 in that both contain a plastid-targeting transit peptide at the N-terminus and four highly conserved histidine residues that may be involved in coordinating a non-heme iron that is required for enzymatic activity (Schwartz et al., 1997, Tan et al., 1997. Sequencing of a genomic clone encoding CmCCD4a revealed the presence of a 105-bp intron at 1442 bp from the start codon (data not shown); this intron was not present in AtCCD4.

Quantitative real-time RT-PCR analysis showed a clear relationship between
CmCCD4a mRNA abundance and carotenoid content in various chrysanthemum tissues: when the level of CmCCD4a transcripts was high, the carotenoid content was low.
Kishimoto and Ohmiya (2006) compared expression of genes encoding isoprenoid and carotenoid biosynthetic enzymes in both white-and yellow-flowered cultivars. The difference they observed in the levels of transcripts, however, could not account for the differences between the carotenoid content values for yellow and white petals. In addition, white petals of chrysanthemums accumulate carotenoids during the early stage of petal development at levels similar to the levels in the yellow petals, which shows that the carotenoid biosynthetic pathway is not blocked in white petals. It is possible that white petals can synthesize carotenoids even when the carotenoid content decreases to undetectable levels. We therefore assumed that in white petals, carotenoid degradation by CmCCD4a causes the white color, even though carotenoid biosynthesis continues to take place.
We therefore performed an RNAi experiment to see whether the suppression of CmCCD4a expression affected the petal color of chrysanthemums. In RNAi lines of white-flowered cultivars, in which CmCCD4a expression was reduced to 2-4%, petal color became yellow. This result clearly indicates that CmCCD4a contributed to the white color formation in chrysanthemum petals by cleaving carotenoids into colorless compounds.
During petal development of yellow-flowered chrysanthemum cultivars, carotenoid concentration increases approximately 10-fold, owing to the accumulation of lutein and its derivatives (Kishimoto and Ohmiya, 2006). In transgenic lines introduced the RNAi construct of CmCCD4a, carotenoids decreased as petals matured, and carotenoid levels in late-stage petals were lower than those in yellow-flowered cultivars. This phenomenon is probably because of the enzymatic activity of CmCCD4a that existed in transgenic lines, although the level was low. It may be possible to increase the carotenoid content in petals to make a vivid yellow petal color by completely knocking out the expression of CmCCD4a.
We obtained one CCD homolog (CmCCD4b) and two NCED homologs (CmNCED3a and CmNCED3b). The expression of these homologs in petals was lower than the expression of CmCCD4a. In addition, significant differences between the expression levels of these homologs in white and yellow petals were not observed. We therefore assumed that among the currently available CCD and NCED homologs of chrysanthemums, only CmCCD4a was involved in white color formation in petals. Expression of CmCCD4a was detected only in white petals, which indicates that the function of CmCCD4a was limited to white color formation in petals. Kishimoto and Ohmiya (2006) reported that at the very early stage of petal development, Paragon contains carotenoids such as lutein and β-carotene but that the levels of these compounds are drastically decreased during petal development. Their results suggest that CmCCD4a cleaves these carotenoids into colorless However, the precise mechanism remains to be elucidated.
Sporting, a well-known phenomenon in chrysanthemums, is a process by which new cultivars arise vegetatively from the parental cultivar. Generally, variants that arise from radiation breeding or bud sports are involved in genomic deletions. Dowrick and Bayoumi (1966) showed that changes in chromosome number and chromosome fragmentation are usually responsible for color changes. About one-third of commercial chrysanthemum cultivars are thought to have arisen in this way (Wasscher, 1956). In the case of chrysanthemums, yellow-flowered bud sports arise from white-flowered cultivars, but the mutation for the reverse orientation has never been observed. A single gene was postulated by Hattori (1991): the dominant allele gives white cultivars, and the recessive gives yellow cultivars. He speculated that such a gene might act as a suppressor of carotenoid formation, although the precise mechanism remains to be elucidated. We propose herein that CmCCD4a is this so-called suppressor of carotenoid formation; however, our results suggest that rather than suppressing carotenoid formation it catalyzes carotenoid breakdown.
It is assumed that yellow-flowered bud sports may lose the CmCCD4a gene during somatic 5′-TAYCAYYMRTTYGAYGGNGAYGGNATG-3′,

CCD
reverse, 5′-TANCGNGGNAARNACNCCNAAYCT-3′. We screened cDNAs of petals and leaves of chrysanthemums and obtained one CCD homolog (designated as CmCCD4b) and two NCED homologs (designated as CmNCED3a and CmNCED3b) from leaf cDNAs.
Full-length cDNAs for these homologs were obtained by rapid amplification of cDNA ends.
By avoiding homologous parts among these homologs, we designed specific primers for  μmol s -1 m -2 ). Paromomycin-resistant shoots were then transferred to soil.

Measurement of Carotenoid Concentration
Tissues (0.5 g) were ground in acetone, then were partitioned between diethyl ether and aqueous NaCl.