Analysis of anthocyanins and the expression patterns of genes involved in biosynthesis in two Vanda hybrids

Pigment accumulation, anthocyanin structure and the expression of floral anthocyanin genes were evaluated in anthocyanin-based colored florets of a pale-mauve Vanda hybrid (V. teres x V. hookeriana) and compared to those of a white Vanda hybrid (V. teres var. alba x V. Cooperi var. White Wing). In the pale-mauve Vanda hybrid, anthocyanins gradually accumulated during all of the three developmental stages of the florets, whereas no anthocyanins detected in the florets of the white Vanda hybrid at any of the stages. The chlorophyll a content accumulated to higher levels in the white Vanda hybrid florets and slightly decreased from the unopened-floret stage (B) to the full-bloom floret stage (FB) in both hybrids. In the pale-mauve Vanda hybrid, the anthocyanin contents in each of the floral parts revealed a direct correlation to the a* values (R2=0.7276) and an inverse relation to the L* values (R2=0.9023). Based on HPLC-PDA and LC-ESI-MSn analyses, the anthocyanins in the pale-mauve hybrid were only derived from cyanidin molecules, which were diversely conjugated with some hexose sugars and organic acids, such as ferrulic acid, sinapic acid and malonic acid. In addition, the semiquantitative expression of four genes involved in the anthocyanin biosynthetic pathway, including phenylalanine ammonia-lyase (Va-PAL1), chalcone synthase (Va-CHS1), flavanone 3-hydroxylase (Va-F3H1) and dihydroflavonol 4-reductase (Va-DFR1), of the pale-mauve Vanda hybrid showed that all of the genes, except Va-CHS1, were highly expressed in all of the three stages of the florets, whereas Va-F3H1 was rarely expressed in the vegetative organs. When Va-PAL1 was expressed at high levels in the florets at all of the floral stages, Va-F3H1 expression was very low only in the developing florets of the white hybrid. Therefore, Va-F3H1 could play an important role in controlling the biosynthesis of anthocyanin in the Vanda hybrids.