Sho Ohno

A bHLH transcription factor, DvIVS, is involved in regulation of anthocyanin synthesis in dahlia (Dahlia variabilis)

Dahlias (Dahlia variabilis) exhibit a wide range of flower colours because of accumulation of anthocyanin and other flavonoids in their ray florets. Two lateral mutants were used that spontaneously occurred in ‘Michael J’ (MJW) which has yellow ray florets with orange variegation. MJOr, a bud mutant producing completely orange ray florets, accumulates anthocyanins, flavones, and butein, and MJY, another mutant producing completely yellow ray florets, accumulates flavones and butein. Reverse transcription–PCR analysis showed that expression of chalcone synthase 1 (DvCHS1), flavanone 3-hydroxylase (DvF3H), dihydroflavonol 4-reductase (DvDFR), anthocyanidin synthase (DvANS), and DvIVS encoding a basic helix–loop–helix transcription factor were suppressed, whereas that of chalcone isomerase (DvCHI) and DvCHS2, another CHS with 69% nucleotide identity with DvCHS1, was not suppressed in the yellow ray florets of MJY. A 5.4 kb CACTA superfamily transposable element, transposable element of Dahlia variabilis 1 (Tdv1), was found in the fourth intron of the DvIVS gene of MJW and MJY, and footprints of Tdv1 were detected in the variegated flowers of MJW. It is shown that only one type of DvIVS gene was expressed in MJOr, whereas these plants are likely to have three types of the DvIVS gene. On the basis of these results, the mechanism regulating the formation of orange and yellow ray florets in dahlia is discussed.

Simultaneous post-transcriptional gene silencing of two different chalcone synthase genes resulting in pure white flowers in the octoploid dahlia

Garden dahlias (Dahlia variabilis) are autoallooctoploids with redundant genes producing wide color variations in flowers. There are no pure white dahlia cultivars, despite its long breeding history. However, the white areas of bicolor flower petals appear to be pure white. The objective of this experiment was to elucidate the mechanism by which the pure white color is expressed in the petals of some bicolor cultivars. A pigment analysis showed that no flavonoid derivatives were detected in the white areas of petals in a star-type cultivar ‘Yuino’ and the two seedling cultivars ‘OriW1’ and ‘OriW2’ borne from a red-white bicolor cultivar, ‘Orihime’, indicating that their white areas are pure white. Semi-quantitative RT-PCR showed that in the pure white areas, transcripts of two chalcone synthases (CHS), DvCHS1 and DvCHS2 which share 69% nucleotide similarity with each other, were barely detected. Premature mRNA of DvCHS1 and DvCHS2 were detected, indicating that these two CHS genes are silenced post-transcriptionally. RNA gel blot analysis revealed that small interfering RNAs (siRNAs) derived from CHSs were produced in these pure white areas. By high-throughput sequence analysis of small RNAs in the pure white areas with no mismatch acceptance, small RNAs were mapped to two alleles of DvCHS1 and two alleles of DvCHS2 expressed in ‘Yuino’ petals. Therefore, we concluded that simultaneous siRNA-mediated post-transcriptional gene silencing of redundant CHS genes results in the appearance of pure white color in dahlias.

Endogenous post-transcriptional gene silencing of flavone synthase resulting in high accumulation of anthocyanins in black dahlia cultivars

Black color in flowers is a highly attractive trait in the floricultural industry, but its underlying mechanisms are largely unknown. This study was performed to identify the bases of the high accumulation of anthocyanidins in black cultivars and to determine whether the high accumulation of total anthocyanidins alone leads to the black appearance. Our approach was to compare black dahlia (Dahlia variabilis) cultivars with purple cultivars and a purple flowering mutant of a black cultivar, using pigment and molecular analyses. Black cultivars characteristically exhibited low lightness, high petal accumulation of cyanidin and total anthocyanidins without flavones, and marked suppression of flavone synthase (DvFNS) expression. A comparative study using black and purple cultivars revealed that neither the absence of flavones nor high accumulation of total anthocyanidins is solely sufficient for black appearance, but that cyanidin content in petals is also an important factor in the phenotype. A study comparing the black cultivar ‘Kokucho’ and its purple mutant showed that suppression of DvFNS abolishes the competition between anthocyanidin and flavone synthesis and leads to accumulation of cyanidin and total anthocyanidins that produce a black appearance. Surprisingly, in black cultivars the suppression of DvFNS occurred in a post-transcriptional manner, as determined by small RNA mapping.

A basic helix-loop-helix transcription factor DvIVS determines flower color intensity in cyanic dahlia cultivars

The study was aimed to identify the factors that regulate the intensity of flower color in cyanic dahlia (Dahlia variabilis), using fifteen cultivars with different color intensities in their petals. The cultivars were classified into three groups based on their flavonoid composition: ivory white cultivars with flavones; purple and pink cultivars with flavones and anthocyanins; and red cultivars with flavones, anthocyanins, and chalcones. Among the purple, pink, and ivory white cultivars, an inverse relationship was detected between lightness, which was used as an indicator for color intensity and anthocyanin content. A positive correlation was detected between anthocyanin contents and the expression of some structural genes in the anthocyanin synthesis pathway that are regulated by DvIVS, a basic helix-loop-helix transcription factor. A positive correlation between anthocyanin content and expression of DvIVS was also found. The promoter region of DvIVS was classified into three types, with cultivars carrying Type 1 promoter exhibited deep coloring, those carrying Type 2 and/or Type 3 exhibited pale coloring, and those carrying Type 1 and Type 2 and/or Type 3 exhibited medium coloring. The transcripts of the genes from these promoters encoded full-length predicted proteins. These results suggested that the genotype of the promoter region in DvIVS is one of the key factors determining the flower color intensity.

Post-transcriptional silencing of chalcone synthase is involved in phenotypic lability in petals and leaves of bicolor dahlia ( Dahlia variabilis ) ‘Yuino’

AbstractMain conclusionPost-transcriptional gene silencing (PTGS) of a chalcone synthase (DvCHS2) occurred in the white part of bicolor petals and flavonoid-poor leaves; however, it did not in red petals and flavonoid-rich leaves. Petal color lability is a prominent feature of bicolor dahlia cultivars, and causes plants to produce not only original bicolor petals with colored bases and pure white tips, but also frequently single-colored petals without white tips. In this study, we analysed the molecular mechanisms that are associated with petal color lability using the red-white bicolor cultivar ‘Yuino’. Red single-colored petals lose their white tips as a result of recover of flavonoid biosynthesis. Among flavonoid biosynthetic genes including four chalcone synthase (CHS)-like genes (DvCHS1, DvCHS2, DvCHS3, and DvCHS4), DvCHS1 and DvCHS2 had significantly lower expression levels in the white part of bicolor petals than in red petals, while DvCHS3, DvCHS4, and other flavonoid biosynthetic genes had almost the same expression levels. Small RNAs from the white part of a bicolor petal were mapped onto DvCHS1 and DvCHS2, while small RNAs from a red single-colored petal were not mapped onto any of the four CHS genes. A relationship between petal color and leaf flavonoid accumulation has previously been demonstrated, whereby red petal-producing plants accumulate flavonoids in their leaves, while bicolor petal-producing plants tend not to. The expression level of DvCHS2 was down-regulated in flavonoid-poor leaves and small RNAs from flavonoid-poor leaves were mapped onto DvCHS2, suggesting that the down-regulation of DvCHS2 in flavonoid-poor leaves occurs post-transcriptionally. Genomic analysis also suggested that DvCHS2 is the key gene involved in bicolor formation. Together, these results suggest that post-transcriptional gene silencing of DvCHS2 plays a key role in phenotypic lability in this bicolor dahlia.