Title : Localized repression of two bHLH genes are involved in white margins in flowers of carnation by the absence of synthesis of anthocyanins
Flower colour patterns are attractive traits for floricultural plants. However, the mechanisms of such traits remain mostly unknown and obscure. Carnation (Dianthus caryophyllus L.) and its interspecific hybrids have many flower colour patterns, which involve white margins in reddish petals, represented by the cultivar ‘Minerva’. We studied the factors regulating the formation of white marginal flowers in given cultivars by the pigments and the related gene expression. HPLC analysis revealed the absence of anthocyanins in white margins, although the accumulation of other flavonoid pigments, namely flavonols, was almost identical between the central and marginal regions of petals. As the result of RNA-seq between the dark-red central regions and the white marginal ones of petals in ‘Minerva’, the expression of 18 genes related to biosynthesis and transportation of anthocyanins, including some transcription factors, were found to be different. Further analysis of the expression of these genes by real-time RT-PCR by comparison with two white-marginal-flowered cultivars and three red-unicolour-flowered ones indicated that the expressions of two bHLH transcription factor genes and seven structural genes, such as dehydroflavonol 4-reductase, anthocyanidin synthase, and glutathione S-transferase, were positively correlated with anthocyanin accumulation. Although DcbHLH1, which was a homolog of JAF13 in Petunia xhybrida, was expressed in both the flower colour groups, DcbHLH2, a homolog of AN1, was expressed only in white-marginal-flowered cultivars. Both bHLH genes may have a partially redundant function for the regulation of anthocyanin synthesis. Therefore, it can be considered that the localized repression of both bHLH genes was involved in the formation of white marginal flowers in carnation through the induction of the absence of anthocyanin accumulation in the marginal region of petals. Particularly, DcbHLH2 could act as a key gene because of its restricted expression only in cultivars with white marginal flowers.