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.Carotenoids are 40-carbon isoprenoids with polyene chains that may contain up to 15 conjugated double bonds. More than 700 naturally occurring carotenoids have been identified (Britton et al., 2004). Carotenoids are essential for photosynthesis, and they furnish flowers and fruits with distinct colors designed to attract insects and other animals. Carotenoids also serve as precursors for the biosynthesis of the plant growth regulator abscisic acid .The chrysanthemum (Chrysanthemum morifolium Ramat.), which has been bred for more than 2,000 years, is one of the most important ornamental flowers in the world. The petal color of yellow-flowered cultivars originates mainly from carotenoids. Understanding the mechanism that controls carotenoid accumulation in petals will not only contribute greatly to the breeding of chrysanthemums and other flowering plants but also provide important information about the molecular evolutionary mechanisms responsible for different petal colors. Cultivated chrysanthemums are thought to have originated from hybrids between white-and yellow-flowered wild species. On the basis of an experiment in which white-and yellow-flowered chrysanthemums were crossed, Hattori (1991) observed that the white petal color is dominant over yellow and suggested that a single dominant gene that inhibits carotenoid accumulation may exist. The detailed function of such a gene, however, is still unknown. Kishimoto and Ohmiya (2006) demonstrated no significant 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 i...

Coexpression of two anthocyanin modification genes elicits blue flower coloration through interaction with colorless flavonoids.

No abstract

Chrysanthemums (Chrysanthemum morifolium Ramat.) have no purple-, violet- or blue-flowered cultivars because they lack delphinidin-based anthocyanins. This deficiency is due to the absence of the flavonoid 3',5'-hydroxylase gene (F3'5'H), which encodes the key enzyme for delphinidin biosynthesis. In F3'5'H-transformed chrysanthemums, unpredictable and unstable expression levels have hampered successful production of delphinidin and reduced desired changes in flower color. With the aim of achieving delphinidin production in chrysanthemum petals, we found that anthocyanin biosynthetic gene promoters combined with a translational enhancer increased expression of some F3'5'H genes and accompanying delphinidin-based anthocyanin accumulation in transgenic chrysanthemums. Dramatic accumulation of delphinidin (up to 95%) was achieved by simple overexpression of Campanula F3'5'H controlled by a petal-specific flavanone 3-hydroxylase promoter from chrysanthemum combined with the 5'-untranslated region of the alcohol dehydrogenase gene as a translational enhancer. The flower colors of transgenic lines producing delphinidin-based anthocyanins changed from a red-purple to a purple-violet hue in the Royal Horticultural Society Colour Charts. This result represents a promising step toward molecular breeding of blue chrysanthemums.

No abstract

No abstract

The most widely used gene editing technology-the CRISPR/Cas9 system-employs a bacterial monomeric DNA endonuclease known as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) and single-guide RNA (sgRNA) that directs Cas9 to a complementary target DNA. However, introducing mutations into higher polyploid plant species, especially for species without genome information, has been difficult. Chrysanthemum morifolium (chrysanthemum) is one of the most important ornamental plants, but it is a hexaploid with a large genome; moreover, it lacks whole-genome information. These characteristics hinder genome editing in chrysanthemum. In the present study, we attempted to perform gene editing using the CRISPR/Cas9 system to introduce mutations into chrysanthemum. We constructed transgenic chrysanthemum plants expressing the yellowish-green fluorescent protein gene from Chiridius poppei (CpYGFP) and targeted CpYGFP for gene editing. We compared the activity of a Cauliflower mosaic virus (CaMV) 35S promoter and parsley ubiquitin promoter in chrysanthemum calli and chose the parsley ubiquitin promoter to drive Cas9. We selected two sgRNAs to target different positions in the CpYGFP gene and obtained transgenic calli containing mutated CpYGFP genes (CRISPR-CpYGFP-chrysanthemum). A DNA sequencing analysis and fluorescence observations indicated that cells containing the mutated CpYGFP gene grew independently of cells containing the original CpYGFP gene in one callus. We finally obtained the CRISPR-CpYGFP-chrysanthemum shoot containing a mutation in the CpYGFP sequence. This is the first report of gene editing using the CRISPR/Cas9 system in chrysanthemum and sheds light on chrysanthemum genome editing.

No abstract