Penthorum chinense Pursh has been used for centuries as an herbal medicine and food in East Asia. The main active substances in P. chinense are galloylated macrocyclic polyphenolic compounds, which have excellent medicinal properties. Galloylation and glycosylation are key steps in the formation of polyphenolic compounds, as the glycosylation of flavonoids is required for the acylation of flavonoid glycosides, and the glycosylation of gallic acid is necessary for its role as an acyl donor. Therefore, glycosylation to generate the acyl donor or acceptor is a core step in the biosynthesis of polyphenolic compounds. However, how this glycosylation occurs in P. chinense is unknown. In this study, we determined that the UDP-glucose transferase PcUGT84A82 mediates the glycosylation of gallic acid and pinocembrin to produce 1-O-Galloyl-β-D-glucose and pinocembroside, respectively. Metabolic profiling of polyphenolic compounds using UHPLC-ESI–Q-TOF/MS revealed high levels of polyphenols in flowers, leaves, and roots, and low levels in stems of P. chinense. We performed isoform-sequencing (Iso-seq) to assemble a full-length transcriptome of P. chinense, from which we identified 58 UGT family members. PcUGT84A82 is highly similar to functional UGTs in other plant species, and PcUGT84A82 transcript levels were positively correlated with the levels of various polyphenolic compounds. We validated the function of PcUGT84A82 via in vitro enzyme assays and transient expression in Nicotiana benthamiana leaves. Subcellular localization tests showed that PcUGT84A82 localizes to the nucleus and cytoplasm. In summary, PcUGT84A82 catalyzes the conversion of gallic acid to 1-O-Galloyl-β-D-glucose as the acyl donor and pinocembrin to pinocembroside as the acyl acceptor, mediating the biosynthesis of galloylated macrocyclic polyphenolic compounds in P. chinense. These findings lay the foundation for elucidating the entire biosynthetic pathway of active polyphenols in this important herbal plant species.

Artemisinin is primarily synthesized and stored in the subepidermal space of the glandular trichomes of Artemisia annua. The augmentation of trichome density has been demonstrated to enhance artemisinin yield. However, existing literature lacks insights into the correlation between the stratum corneum and trichomes. This study aims to unravel the involvement of TrichomeLess Regulator 3 (TLR3), which encodes the transcription factor, in artemisinin biosynthesis and its potential association with the stratum corneum. TLR3 was identified as a candidate gene through transcriptome analysis. The role of TLR3 in trichome development and morphology was investigated using yeast two-hybrid, pull-down analysis, and RNA electrophoresis mobility assay. Our research revealed that TLR3 negatively regulates trichome development. It modulates the morphology of Arabidopsis thaliana trichomes by inhibiting branching and inducing the formation of abnormal trichomes in Artemisia annua. Overexpression of the TLR3 gene disrupts the arrangement of the stratum corneum and reduces artemisinin content. Simultaneously, TLR3 possesses the capacity to regulate stratum corneum development and trichome follicle morphology by interacting with TRICHOME AND ARTEMISININ REGULATOR 1, and CycTL. Consequently, our findings underscore the pivotal role of TLR3 in the development of glandular trichomes and stratum corneum biosynthesis, thereby influencing the morphology of Artemisia annua trichomes.