Atropa belladonna L. is one of the most important herbal plants that produces hyoscyamine or atropine, and it also produces anisodamine and scopolamine. However, the in planta hyoscyamine content is very low, and it is difficult and expensive to independently separate hyoscyamine from the tropane alkaloids in A. belladonna. Therefore, it is vital to develop A. belladonna plants with high yields of hyoscyamine, and without anisodamine and scopolamine. In this study, we generated A. belladonna plants without anisodamine and scopolamine, via the CRISPR/Cas9-based disruption of hyoscyamine 6β-hydroxylase (AbH6H), for the first time. Hyoscyamine production was significantly elevated, while neither anisodamine nor scopolamine were produced, in the A. belladonna plants with homozygous mutations in AbH6H. In summary, new varieties of A. belladonna with high yields of hyoscyamine and without anisodamine and scopolamine have great potential applicability in producing hyoscyamine at a low cost.

Catharanthus roseus exhibits vibrant petals and displays robust resistance to disease and drought, making it highly valuable for ornamental and gardening applications. While the application of C. roseus as a source of anticancer drugs has gained considerable attention in recent years, there has been limited investigation into the regulatory mechanism underlying anthocyanin accumulation in the petals of C. roseus. This study comprehensively analyzed the metabolome and transcriptome of three distinct C. roseus varieties exhibiting different petal colors. Out of the 39 identified flavonoids, 10 anthocyanins exhibited significant variations in accumulation, directly contributing to the diverse coloration of C. roseus petals. Among them, malvidin 3-O-glucoside and petunidin 3-O-glucoside were identified as primary contributors to the purple petal phenotype, while peonidin 3-O-glucoside and delphinidin 3-O-glucoside exhibited the highest contribution rates to the red petals. Additionally, the variation content of cyanidin 3-O-rutinoside, delphinidin 3-O-glucoside, and petunidin 3-O-rutinoside also influenced the color transformation of C. roseus petals. RNA sequencing identified a total of 4173 differentially expressed genes (DEGs), including 1003 overlapping DEGs. A combined transcriptome and metabolome analysis showed that the coordinately regulated anthocyanin biosynthetic genes including chalcone isomerase (CHS), flavonoid 3′-hydroxylase (F3′H), and dihydroflavonol 4-reductase (DFR) played critical roles in the formation of the anthocyanins. MYB and bHLH transcription factors were also found to be significantly correlated with differences in flower color. These results serve as a foundation for future investigations into anthocyanin biosynthesis and regulatory mechanisms in C. roseus.

Hyoscyamine and scopolamine, belonging to medicinal tropane alkaloids (MTAs), are potent anticholinergic drugs. Their industrial production relies on medicinal plants, but the levels of the two alkaloids are very low in planta. Engineering the MTA’s production is an everlasting hot topic for pharmaceutical industry. With understanding the MTA’s biosynthesis, biotechnological approaches are established to produce hyoscyamine and scopolamine in an efficient manner. Great advances have been obtained in engineering MTA’s production in planta. In this review, we summarize the advances on the biosynthesis of MTAs and engineering the MTA’s production in hairy root cultures, as well in plants. The problems and perspectives on engineering the MTA’s production are also discussed.

Anthocyanins are water-soluble flavonoid pigments that play a crucial role in plant growth and metabolism. They serve as attractants for animals by providing plants with red, blue, and purple pigments, facilitating pollination and seed dispersal. The fruits of solanaceous plants, tomato (Solanum lycopersicum) and eggplant (Solanum melongena), primarily accumulate anthocyanins in the fruit peels, while the ripe fruits of Atropa belladonna (Ab) have a dark purple flesh due to anthocyanin accumulation. In this study, an R2R3-MYB transcription factor (TF), AbMYB1, was identified through association analysis of gene expression and anthocyanin accumulation in different tissues of A. belladonna. Its role in regulating anthocyanin biosynthesis was investigated through gene overexpression and RNA interference (RNAi). Overexpression of AbMYB1 significantly enhanced the expression of anthocyanin biosynthesis genes, such as AbF3H, AbF3′5′H, AbDFR, AbANS, and Ab3GT, leading to increased anthocyanin production. Conversely, RNAi-mediated suppression of AbMYB1 resulted in decreased expression of most anthocyanin biosynthesis genes, as well as reduced anthocyanin contents in A. belladonna. Overall, AbMYB1 was identified as a fruit-expressed R2R3-MYB TF that positively regulated anthocyanin biosynthesis in A. belladonna. This study provides valuable insights into the regulation of anthocyanin biosynthesis in Solanaceae plants, laying the foundation for understanding anthocyanin accumulation especially in the whole fruits of solanaceous plants.

Monoterpenoids serve as essential components of plant essential oils and play significant roles in plant growth, development, and insect resistance. Artemisia annua, an important medicinal plant, produces abundant terpenoids. While previous research on A. annua has predominantly focused on artemisinin biosynthesis and its regulation, studies on other terpenoids in this plant have significantly lagged behind. To comprehensively investigate monoterpene biosynthesis in A. annua, we analyzed monoterpenes across its different tissues using optimized extraction and chromatographic conditions developed to enhance sensitivity and resolution in our GC-MS-based analytical method. In A. annua, 31 monoterpenoid compounds were identified. Subsequently, eight candidate monoterpene synthases (mTPS) were characterized in Escherichia coli, confirming their catalytic activity in converting geranyl pyrophosphate (GPP) into distinct monoterpene products. Subcellular localization revealed these TPSs in chloroplasts, consistent with the widely reported chloroplast localization of TPS enzymes. These enzymes were functionally defined as monoterpenoid synthases, collectively responsible for synthesizing 18 monoterpenoid metabolites. Notably, AaTPS13, AaTPS19, and AaTPS20 exhibited substantial product promiscuity. Critically, the AaTPS19 was identified as the first known terpene synthase producing 2-pinanol. These findings systematically elucidate the biosynthesis of monoterpenoids in A. annua and provide key enzymatic elements for metabolic engineering and synthetic biology applications in monoterpenoid production.

Artemisinin, a crucial antimalarial compound synthesized in Artemisia annua, is tightly controlled by various transcription factors. Despite significant progress in understanding this regulatory network, further exploration of additional factors is needed to improve artemisinin biosynthesis. Here, we identified a novel R2-MYB-like transcription factor, AaMYB121, which responds to methyl jasmonate (MeJA). Overexpression of AaMYB121 in transgenic A. annua plants resulted in dihydroartemisinic acid (DHAA) content being elevated 2~3 times compared to the control, while artemisinin levels increased to 1.4~2 times, significantly boosting artemisinin biosynthesis. Dual-Luciferase (Dual-LUC) assay and yeast one-hybrid (Y1H) analysis demonstrated that AaMYB121 directly binds to the promoter motifs of DBR2 (−1146~−1103) and ALDH1 (−1558~−1504), thereby triggering the transcriptional initiation of these genes. Notably, AaMYB121 features an elongated DNA-binding domain that specifically recognizes AT-rich cis-elements in the promoters of key artemisinin biosynthetic genes. These findings established AaMYB121 as a novel MYB-like transcription factor with strong potential to enhance the regulation of artemisinin production through targeted modulation, offering a valuable approach to improve artemisinin yields for therapeutic purposes.