Repression of anthocyanin biosynthesis by R3-MYB transcription factors in lily (Lilium spp.).

KEY MESSAGE: Lily R3-MYB transcription factors are involved in negative regulation to limit anthocyanin accumulation in lily flowers and leaves and create notable color patterns on ectopically expressed petunia flowers. In eudicots, both positive and negative regulators act to precisely regulate the level of anthocyanin accumulation. The R3-MYB transcription factor is among the main factors repressing anthocyanin biosynthesis. Although, in monocots, the positive regulators have been well characterized, the negative regulators have not been examined. Two R3-MYBs, LhR3MYB1 and LhR3MYB2, which were identified in lily transcriptomes, were characterized in this study to understand the regulatory mechanisms of anthocyanin biosynthesis. LhR3MYB1 and LhR3MYB2 had a C2 suppressor motif downstream of a single MYB repeat; the similar amino acid motif appears only in AtMYBL2 among the eudicot R3-MYB proteins. Stable and transient overexpression of LhR3MYB1 and LhR3MYB2 in tobacco plants showed suppression of anthocyanin biosynthesis by both; however, suppression by LhR3MYB2 was stronger than that by LhR3MYB1. In the lily plant, the LhR3MYB2 transcript was detected in leaves with light stimulus-induced anthocyanin accumulation and in pink tepals. Although LhR3MYB1 was expressed in some, but not all tepals, its expression was not linked to anthocyanin accumulation. In addition, LhR3MYB1 expression levels in the leaves remained unchanged by the light stimulus, and LhR3MYB1 transcripts predominantly accumulated in the ovaries, which did not accumulate anthocyanins. Thus, although LhR3MYB1 and LhR3MYB2 have an ability to repress anthocyanin accumulation, LhR3MYB2 is more strongly involved in the negative regulation to limit the accumulation than that by LhR3MYB1. In addition, the overexpression of LhR3MYB2 generated notable color patterns in petunia flowers; thus, the usefulness of the LhR3MYB genes for creating unique color patterns by genetic engineering is discussed.