Carotenoid cleavage dioxygenase (CmCCD4a) contributes to white color formation in chrysanthemum petals

Abstract

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 polymerase chain reaction-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.

Figure 1.

Sequence comparison of CCDs of various plant species. A, Alignment of amino acid sequence of CmCCD4a and CmCCD4b with related sequences. Identical amino acids are indicated with black backgrounds. Four conserved His residues are marked with asterisks. B, ClustalW tree analysis of chrysanthemum CCD and NCED homologs in various plant species. AtCCD1 (At3g63520), AtCCD4 (At4g19170), AtCCD7 (At2g44990), and AtCCD8 (At4g32810) are CCDs of Arabidopsis. AtNCED2 (At4g18350), AtNCED3 (At3g14440), AtNCED5 (At1g30100), AtNCED6 (At3g24220), and AtNCED9 (At1g78390) are NCEDs of Arabidopsis. Other CCDs include PhCCD1 of petunia (AY576003), LeCCD1A of tomato (Lycopersicon esculentum; AY576001), OsCCD4a of rice (AP007825), CsCCD (AJ132927) and CsZCD (AJ489276) of crocus, PaCCD1 (AF224670) of avocado (Persea americana), and PvCCD1 (AY029525) of kidney bean (Phaseolus vulgaris). Numbers at branch points indicate bootstrap values (1,000 replicates).

Figure 2.

Color-specific expression and genomic PCR analyses. Lanes 1 to 4, white-flowered cultivars; lanes 5 to 12, yellow-flowered cultivars: 1, Paragon; 2, White Marble; 3, Fiducia; 4, Sei-Marine; 5, Yellow Paragon; 6, Florida Marble; 7, Homaro; 8, Susie; 9, Morning Sun; 10, Statesman; 11, Super Yellow; and 12, Sunny Orange. A, Expression of chrysanthemum CCD and NCED homologs in petals of white- and yellow-flowered cultivars of chrysanthemum. Quantitative real-time RT-PCR analysis was performed in triplicate using specific primers for each homolog, and the expression levels were normalized against actin levels; mean values ±se are shown. Genomic PCR with homolog-specific primers of the same chrysanthemum cultivars analyzed in real-time RT-PCR are presented below. B, Genomic Southern-blot analysis of the chrysanthemum cultivars analyzed in A. Genomic DNAs (30 μg) were digested with BamHI and loaded onto each lane. Fragments were hybridized with DIG-labeled CmCCD4a cDNA. The filter was reprobed with DIG-labeled CmNCED3a cDNA. The arrowhead indicates the band that corresponds to CmCCD4a.

Figure 3.

Expression of chrysanthemum CCD and NCED homologs in various tissues of the white-flowered cultivar Paragon. Quantitative real-time RT-PCR analysis was performed in triplicate using primers specific to each homolog, and the expression levels were normalized against actin levels; mean values ± se are shown.

Figure 4.

Changes in carotenoid concentrations (black circles) and CmCCD4a transcript levels (bars) during flower petal development of Paragon. Quantitative real-time RT-PCR analysis was performed in triplicate using CmCCD4a-specific primers, and the expression levels were normalized against actin levels; mean values ± se are shown.

Figure 5.

Suppression of CmCCD4a expression by RNAi. A, Changes in carotenoid concentrations during petal development of wild-type (WT) and five independent CmCCD4a RNAi plants (1–5). Measurements were performed in triplicate, and mean values ± se are shown . E, Early; M, middle; L, late. B, Changes in CmCCD4a expression during petal development of wild-type and CmCCD4a RNAi plants. Quantitative real-time RT-PCR analyses were performed in triplicate using CmCCD4a-specific primers, and the expression levels were normalized against actin levels; mean values ± se are shown. C, Photographs of fully opened flowers of wild-type Sei-Marine and CmCCD4a RNAi plants (line 5).

Figure 6.

Genomic PCR of white- and yellow-flowered wild species of chrysanthemum with CmCCD4a and CmCCD4b primers: 1, Chrysanthemum boreale; 2, Chrysanthemum indicum; 3, Chrysanthemum makinoi; 4, Chrysanthemum japonese; 5, Chrysanthemum yezoense; 6, Chrysanthemum zawadskii; and 7, Chrysanthemum yoshinaganthum.

Figure 7.

Light microscope observation of transverse section through petals of yellow-flowered cultivars Yellow Paragon and Super Yellow. Bars = 100 μm.