Abstract
Artemisia annua is a medicinal plant rich in terpenes and flavonoids with useful biological activities such as antioxidant, anticancer, and antimalarial activities. The transcriptional regulation of flavonoid biosynthesis in A. annua has not been well-studied. In this study, we identified a YABBY family transcription factor, AaYABBY5, as a positive regulator of anthocyanin and total flavonoid contents in A. annua. AaYABBY5 was selected based on its similar expression pattern to the phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavonol synthase (FLS) genes. A transient dual-luciferase assay in Nicotiana bethamiana with the AaYABBY5 effector showed a significant increase in the activity of the downstream LUC gene, with reporters AaPAL, AaCHS, AaCHI, and AaUFGT. The yeast one-hybrid system further confirmed the direct activation of these promoters by AaYABBY5. Gene expression analysis of stably transformed AaYABBY5 overexpression, AaYABBY5 antisense, and control plants revealed a significant increase in the expression of AaPAL, AaCHS, AaCHI, AaFLS, AaFSII, AaLDOX, and AaUFGT in AaYABBY5 overexpression plants. Moreover, their total flavonoid content and anthocyanin content were also found to increase. AaYABBY5 antisense plants showed a significant decrease in the expression of flavonoid biosynthetic genes, as well as a decrease in anthocyanin and total flavonoid contents. In addition, phenotypic analysis revealed deep purple-pigmented stems, an increase in the leaf lamina size, and higher trichome densities in AaYABBY5 overexpression plants. Together, these data proved that AaYABBY5 is a positive regulator of flavonoid biosynthesis in A. annua. Our study provides candidate transcription factors for the improvement of flavonoid concentrations in A. annua and can be further extended to elucidate its mechanism of regulating trichome development.
Highlights
Artemisia annua is a renowned plant rich in terpenes and flavonoids
Flavonoid biosynthetic genes contain YABBY-binding motifs The cloned promoter sequences of flavonoid biosynthetic genes used in the present study, AaPAL, AaCHS, AaCHI, AaFLS, AaFSII, and genes regulating anthocyanins AaDFR, AaLDOX, and AaUFGT, were analyzed for putative YABBY-binding sequences[37] using PlantPAN 3.0 (PlantPAN; http://PlantPAN.itps.ncku.edu.tw)
YABBY-binding motifs are represented by AT-rich sites in Arabidopsis with consensus binding sequences defined as AATNATAA and AATNATTA
Summary
Artemisia annua is a renowned plant rich in terpenes and flavonoids. Flavonoids are polyphenolic plant secondary metabolites. Anthocyanins, flavonols, flavanols, and proanthocyanidins (PAs) or condensed tannins are the major classes of flavonoids. These compounds show differential expression patterns according to plant growth or developmental stages and in a species-specific manner[1]. Flavonoids perform various functions in plants, including antioxidant activity, protection against UV light, Increasing evidence recommends using the dried leaves of A. annua, which contain both artemisinin and flavonoids, to cure malaria more effectively than using only artemisinin[3]. The mechanism for increased bioavailability of artemisinin when using dried leaves that supply artemisinin and flavonoids synergistically was shown[5]. Flavonoids of A. annua exhibit efficacious antioxidant effects[6], strong antiinflammatory properties[7], and potent anticancer characteristics[8].
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