The bHLH transcription factors play important roles in the regulation of plant growth, development, and secondary metabolism. β-Caryophyllene, epi-cedrol, and β-farnesene, three kinds of sesquiterpenes mainly found in plants, are widely used as spice in the food industry and biological pesticides in agricultural production. Furthermore, they also have a significant value in the pharmaceutical industry. However, there is currently a lack of knowledge on the function of bHLH family TFs in β-caryophyllene, epi-cedrol, and β-farnesene biosynthesis. Here, we found that AabHLH112 transcription factor had a novel function to positively regulate β-carophyllene, epi-cedrol, and β-farnesene biosynthesis in Artemisia annua. Exogenous MeJA enhanced the expression of AabHLH112 and genes of β-caryophyllene synthase (CPS), epi-cedrol synthase (ECS), and β-farnesene synthase (BFS), as well as sesquiterpenes content. Dual-LUC assay showed the activation of AaCPS, AaECS, and AaBFS promoters were enhanced by AabHLH112. Yeast one-hybrid assay showed AabHLH112 could bind to the G-box (CANNTG) cis-element in promoters of both AaCPS and AaECS. In addition, overexpression of AabHLH112 in A. annua significantly elevated the expression levels of AaCPS, AaECS, and AaBFS as well as the contents of β-caryophyllene, epi-cedrol, and β-farnesene, while suppressing AabHLH112 expression by RNAi reduced the expression of the three genes and the contents of the three sesquiterpenes. These results suggested that AabHLH112 is a positive regulator of β-caryophyllene, epi-cedrol, and β-farnesene biosynthesis in A. annua.

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.

Tropane alkaloids (TAs) are large secondary metabolite alkaloids that find extensive applications in the synthesis of antidotes, anesthetics, antiemetics, motion sickness drugs, and antispasmodics. The current production method primarily depends on extraction from medicinal plants of the Solanaceae family. Elicitation, as a highly effective biotechnological approach, offers significant advantages in augmenting the synthesis of secondary metabolites. The advantages include its simplicity of operation, low cost, and reduced risk of contamination. This review focuses on the impact of elicitation on the biosynthesis of TAs from three aspects: single-elicitor treatment, multiple-elicitor treatment, and the combination of elicitation strategy with other strategies. Some potential reasons are also proposed. Plant hormones and growth regulators, such as jasmonic acid (JA), salicylic acid (SA), and their derivatives, have been extensively employed in the separate elicitation processes. In recent years, novel elicitors represented by magnetic nanoparticles have emerged as significant factors in the investigation of yield enhancement in TAs. This approach shows promising potential for further development. The current utilization of multi-elicitor treatment is constrained, primarily relying on the combination of only two elicitors for induction. Some of these combinations have been found to exhibit synergistic amplification effects. However, the underlying molecular mechanism responsible for this phenomenon remains largely unknown. The literature concerning the integration of elicitation strategy with other strategies is limited, and several research gaps require further investigation. In conclusion, the impact of various elicitors on the accumulation of TAs is well-documented. However, further research is necessary to effectively implement elicitation strategies in commercial production. This includes the development of stable bioreactors, the elucidation of regulatory mechanisms, and the identification of more potent elicitors.

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.

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.

Isopentenyl diphosphate isomerase (IPPI) is a key enzyme catalyzing the interconversion between isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), two isoprenyl C5 units that are precursors for terpenoid biosynthesis. Previous study has identified two IPPIs, namely AaIPPI1 and AaIPPI2 from Artemisia annua, and revealed the positive role of AaIPPI1 in regulating the biosynthesis of terpenoids. However, the role of AaIPPI2 in regulating terpenoid biosynthesis remains to be clarified. In this study, we explored the contribution of AaIPPI2 to terpenoid biosynthesis in Artemisia annua. Overexpression of AaIPPI2 in A. annua significantly enhanced the contents of diverse monoterpenes and sesquiterpenes. Suppression of AaIPPI2 by RNA interference (RNAi) in A. annua markedly reduced the biosynthesis of these sesquiterpenes, but only caused a modest decrease in several monoterpenes’ levels with no change in other monoterpenes’ levels, suggesting that AaIPPI2 plays a lesser part in the regulation of monoterpenes biosynthesis in plastids compared to AaIPPI1. Taken together, these results demonstrate that AaIPPI2 plays a positive role in regulating the biosynthesis of terpenoids. Our findings enhanced the understanding of the distinction between the roles of the two IPPIs in the regulation of terpenoid biosynthesis in A. annua and provided candidate genes for optimizing terpene production in plants.