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.

Plant diterpenes such as ginkgolides are biosynthesized via the recently discovered 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The initial step of the MEP pathway is the formation of 1-deoxy-D-xylulose 5-phosphate (DXP) catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS, EC: 4.1.3.37), which may thus be considered the first committed step of the MEP pathway for ginkgolides biosynthesis. The full-length cDNA of DXS was isolated and characterized from the gymnosperm plant species, Ginkgo biloba. The full-length cDNA of GbDXS was 2795 bp containing a 2154 bp open reading frame (ORF) encoding 717 amino acids. Comparative and bioinformatic analyses revealed that GbDXS has extensive homology with DXSs from other plant species and, like these, contains a conserved transit peptide for plastid import, histidine residue, a putative thiamine diphosphate-binding site and a transketolase motif. Phylogenetic analysis indicates that GbDXS belongs to the plant DXS1 cluster and suggests it to be more ancient than other plant DXSs. GbDXS was found to be expressed in all tested tissues including roots, stems, leaves, pericarps and seeds. Expression profiling analyses revealed that GbDXS expression was induced by exogenous elicitors including methyl jasmonate, arachidonic acid, acetylsalicylic acid and ceric ammonium sulfate, and showed that the transcription levels were correlated with ginkgolide accumulation, suggesting that DXS might play a regulatory role in ginkgolide biosynthesis in cell culture of G. biloba at the transcriptional level.

Tropane alkaloids (TAs) are widely distributed in the Solanaceae, while some important medicinal tropane alkaloids (mTAs), such as hyoscyamine and scopolamine, are restricted to certain species/tribes in this family. Little is known about the genomic basis and evolution of TAs biosynthesis and specialization in the Solanaceae. Here, we present chromosome-level genomes of two representative mTAs-producing species:Atropa belladonnaandDatura stramonium. Our results reveal that the two species employ a conserved biosynthetic pathway to produce mTAs despite being distantly related within the nightshade family. A conserved gene cluster combined with gene duplication underlies the wide distribution of TAs in this family. We also provide evidence that branching genes leading to mTAs likely have evolved in early ancestral Solanaceae species but have been lost in most of the lineages, withA. belladonnaandD. stramoniumbeing exceptions. Furthermore, we identify a cytochrome P450 that modifies hyoscyamine into norhyoscyamine. Our results provide a genomic basis for evolutionary insights into the biosynthesis of TAs in the Solanaceae and will be useful for biotechnological production of mTAs via synthetic biology approaches.