Localized Repression of Two bHLH Genes is Involved in the Formation of White Margins and White Abaxial Surfaces in Carnation Petals by Inducing the Absence of Anthocyanin Synthesis

Flower color patterns are attractive traits of floricultural plants. However, the mechanisms underlying these traits remain mostly unknown. Carnation (Dianthus caryophyllus L.) and interspecific hybrids thereof exhibit many flower color patterns involving white margins on reddish petals, as observed in the cultivar 'Minerva'. Flowers with white margins also have white abaxial surfaces. We studied the factors regulating the formation of white margins and a white abaxial surface in flowers through analyses of pigments and related gene expression. HPLC analysis revealed an absence of anthocyanins in white margins, although the accumulation of other flavonoid pigments (flavonols) was almost identical between the dark-red central and white marginal regions of petals. RNA-seq analyses of the dark red central regions and white marginal regions of 'Minerva' petals resulted in the extraction of 18 genes related to anthocyanin biosynthesis and transportation, including some transcription factors, as candidate regulatory genes for the formation of white margins. Further analysis of the expression of these genes by real-time RT-PCR and a comparison of two white-margin-flowered cultivars and three red-unicolor-flowered cultivars indicated that the expressions of two bHLH transcription factor genes and seven structural genes were positively correlated with anthocyanin accumulation. Although DcbHLH1, which is a homolog of JAF13 in Petunia xhybrida, was expressed in both flower color groups, DcbHLH2, a homolog of AN1, was expressed only in white-marginal-flowered cultivars. Moreover, in the petals of 'Minerva', the expression of those nine genes was repressed in the abaxial epidermal layer without red pigmentation conferred by anthocyanins. Therefore, we could postulate that the localized repression of both bHLH genes is involved in the formation of white margins in carnation petals by inducing the absence of anthocyanin synthesis and that the white abaxial surface of the petals may result from similar regulatory mechanisms. In particular, DcbHLH2 could act as a key gene because of its restricted expression only in cultivars with white-margined flowers.