Molecular understanding of anthocyanin biosynthesis activated by PAP1 in engineered redArtemisia annuacells and regulation of 2, 4-dichlorophenoxyacetic acid

Abstract

AbstractArtemisia annuais an effective antimalarial medicinal crop. We have established anthocyanin-producing red cell cultures from this plant with the overexpression ofProduction of Anthocyanin Pigment 1(PAP1) encoding a R2R3MYB transcription factor. To understand the molecular mechanism by which PAP1 activated the entire anthocyanin pathway, we mined the genomic sequences ofA. annuaand obtained eight promoters of the anthocyanin pathway genes. Sequence analysis identified four types of AC cis-elements from six promoters, the MYB response elements (MRE) bound by PAP1. In addition, six promoters were determined to have at least one G-Box cis-element. Eight promoters were cloned for activity analysis. Duel luciferase assays showed that PAP1 significantly enhanced the promoting activity of seven promoters, indicating that PAP1 turned on the biosynthesis of anthocyanins via the activation of these pathway gene expression. To understand how 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin, regulates the PAP1-activated anthocyanin biosynthesis, five different concentrations (0, 0.05, 0.5, 2.5, and 5 μM) were tested to characterize anthocyanin production and profiles. The resulting data showed that the concentrations tested decreased the fresh weight of callus growth, anthocyanin levels, and the production of anthocyanins per petri dish. HPLC-qTOF-MS/MS based profiling showed that these concentrations did not alter anthocyanin profiles. Real time RT-PCR was completed to characterize the expressionPAP1and four representative pathway genes. The results showed that the five concentrations reduced the expression levels of the constitutivePAP1transgene and three pathway genes significantly and eliminated chalcone synthase gene in expression either significantly or slightly. These data indicate that the constitutivePAP1expression depends on gradients added in the medium. Based on these findings, the regulation of 2,4-D is discussed for anthocyanin engineering in red cells ofA. annua.ConclusionPromoters of eight anthocyanin pathway genes were cloned. Four types of AC cis-elements were identified from six promoters and G-Box elements were also determined from six promoters. PAP1 enhanced the activity of eight promoters. 2,4-D downregulated the expression of the constitutive PAP1 transgene leading to the decrease the biosynthesis of anthocyanins.