Some medicinal plants of the Solanaceae produce pharmaceutical tropane alkaloids (TAs), such as hyoscyamine and scopolamine. Littorine is a key biosynthetic intermediate in the hyoscyamine and scopolamine biosynthetic pathways. However, the mechanism underlying littorine formation from the precursors phenyllactate and tropine is not completely understood.Here, we report the elucidation of littorine biosynthesis through a functional genomics approach and functional identification of two novel biosynthesis genes that encode phenyllactate UDP-glycosyltransferase (UGT1) and littorine synthase (LS).UGT1 and LS are highly and specifically expressed in Atropa belladonna secondary roots. Suppression of either UGT1 or LS disrupted the biosynthesis of littorine and its TA derivatives (hyoscyamine and scopolamine). Purified His-tagged UGT1 catalysed phenyllactate glycosylation to form phenyllactylglucose. UGT1 and LS co-expression in tobacco leaves led to littorine synthesis if tropine and phenyllactate were added.This identification of UGT1 and LS provides the missing link in littorine biosynthesis. The results pave the way for producing hyoscyamine and scopolamine for medical use by metabolic engineering or synthetic biology.

An ABA-responsive SnRK2-type kinase, AaAPK1, is involved in positively regulating artemisinin biosynthesis in Artemisia annua through phosphorylation of AabZIP1.

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Artemisia annua produces artemisinin, an effective antimalarial drug. In recent decades, the later steps of artemisinin biosynthesis have been thoroughly investigated; however, little is known about the early steps of artemisinin biosynthesis. Comparative transcriptomics of glandular and filamentous trichomes and 13CO2 radioisotope study have shown that the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, rather than the mevalonate pathway, plays an important role in artemisinin biosynthesis. In this study, we have cloned three 1-deoxy-D-xylulose 5-phosphate synthase (DXS) genes from A. annua (AaDXS1, AaDXS2, and AaDXS3); the DXS enzyme catalyzes the first and rate-limiting enzyme of the MEP pathway. We analyzed the expression of these three genes in different tissues in response to multiple treatments. Phylogenetic analysis revealed that each of the three DXS genes belonged to a distinct clade. Subcellular localization analysis indicated that all three AaDXS proteins are targeted to chloroplasts, which is consistent with the presence of plastid transit peptides in their N-terminal regions. Expression analyses revealed that the expression pattern of AaDXS2 in specific tissues and in response to different treatments, including methyl jasmonate, light, and low temperature, was similar to that of artemisinin biosynthesis genes. To further investigate the tissue-specific expression pattern of AaDXS2, the promoter of AaDXS2 was cloned upstream of the β-glucuronidase gene and was introduced in arabidopsis. Histochemical staining assays demonstrated that AaDXS2 was mainly expressed in the trichomes of Arabidopsis leaves. Together, these results suggest that AaDXS2 might be the only member of the DXS family in A. annua that is involved in artemisinin biosynthesis.