Basic helix-loop-helix (bHLH) proteins are the second largest family of transcription factors (TFs) involved in developmental and physiological processes in plants. In this study, 205 putative bHLH TF genes were identified in the genome of Artemisia annua and expression of 122 of these was determined from transcriptomes used to construct the genetic map of A. annua. Analysis of gene expression association allowed division of the 122 bHLH TFs into five groups. Group V, containing 15 members, was tightly associated with artemisinin biosynthesis genes. Phylogenetic analysis indicated that two bHLH TFs, AabHLH106 and AabHLH112, were clustered with Arabidopsis ICE proteins. AabHLH112 was induced by low temperature, while AabHLH106 was not. We therefore chose AabHLH112 for further examination. AabHLH112 was highly expressed in glandular secretory trichomes, flower buds, and leaves. Dual-luciferase assays demonstrated that AabHLH112 enhanced the promoter activity of artemisinin biosynthesis genes and AaERF1, an AP2/ERF TF that directly and positively regulates artemisinin biosynthesis genes. Yeast one-hybrid assays indicated that AabHLH112 could bind to the AaERF1 promoter, but not to the promoters of artemisinin biosynthesis genes. Overexpression of AabHLH112 significantly up-regulated the expression levels of AaERF1 and artemisinin biosynthesis genes and consequently promoted artemisinin production.

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Przewalskia tangutica is a traditional medicinal plant from Tibet used for the analgesic effect from the tropane alkaloids (TAs) produced by the plant. Its roots have the highest yield of hyoscyamine in all plant species and so have been overharvested becoming an endangered medicinal plant species. Metabolic engineering is a good way to improve the yield of TAs in plants. In our study, two functionally distinct tropinone reductases genes, PtTRI and PtTRII, were cloned from P. tangutica and the functional divergence were characterized. The enzyme kinetics of PtTRI and PtTRII were investigated. The phylogenetic analysis classified them into different clades: PtTRI and PtTRII were in the clade of tropine-forming reductase and pseudotropine-forming reductase, respectively. We found PtTRI to be expressed in the roots but less in leaves, whereas PtTRII was expressed in the roots at higher levels than in the leaves. The kinetic parameters (K m , V max , and K cat ) were analyzed using purified recombinant enzymes at their optimum pH. Enzymatic analysis results showed that tropinone is a better substrate for PtTRII compared with PtTRI, suggesting that PtTRII might be a potential gene target for TA biosynthesis engineering. Compared with the reported TRIs, PtTRI exhibited a higher affinity for tropinone.