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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Artemisinin is biosynthesized in Artemisia annua and widely used for the treatment of malaria. Abscisic acid (ABA)‐responsive kinase 1 (AaAPK1), a member of the SnRK2 family, is involved in the regulation of artemisinin biosynthesis through the phosphorylation of AabZIP1, which directly transactivates genes involved in artemisinin biosynthesis. Through diverse assays – including yeast two‐hybrid and bimolecular fluorescence complementation assays – we report that the ABA‐responsive protein phosphatase AaPP2C1 physically interacts with AaAPK1. In addition, phos‐tag mobility shift assays indicate that AaPP2C1 dephosphorylates AaAPK1. Moreover, dual‐luciferase assays demonstrate that the presence of AaPP2C1 reduces the transactivation of artemisinin biosynthesis genes by AabZIP1. These results further refine the post‐translational regulatory network of artemisinin biosynthesis, showing that AaPP2C1 is negatively involved through dephosphorylation of AaAPK1. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Rhodiola crenulata is a Tibetan native herbal plant belonging to the family of Crassulaceae, which produces the pharmaceutical icariside D2 with the activities of inhibiting angiotensin-converting enzyme and killing leukemia cancer cells. In this study, we functionally characterized a novel UDP-glycosyltransferase (RcUGT1) that converted tyrosol to specifically produce icariside D2 from R. crenulata at molecular and biochemical levels. RcUGT1 was highly expressed in flowers and roots, while the icariside D2 content was much higher in stems than that in other organs, suggesting the potential translocation of icariside D2 from flowers and roots to stems. The high production of icariside D2 in stems provided a reasonable suggestion to farmers to harvest stems instead of roots for icariside D2 production. Enzymatic assays of recombinant RcUGT1 indicated that it converted tyrosol to specifically form icariside D2, with the values of Km 0.97±0.10 mM, Vmax 286±8.26 pKat/mg, Kcat 0.01552 s−1, and Kcat/Km 159.55 s−1 M−1. Functional identification of RcUGT1 facilitated the icariside D2 production through metabolic engineering in plants or synthetic biology in microbes. [ABSTRACT FROM AUTHOR]

In recent years, although several related genes had been cloned and characterized, the role of aldehyde dehydrogenase 1 ( ALDH1), the newly cloned gene involved in artemisinin biosynthesis pathway, is still not clear. In this study, a 2,100-bp ALDH1 promoter region fused with GUS reporter gene was stably transferred into Arabidopsis thaliana. Histochemical staining showed the methyl jasmonate (MeJA) and wounding treatment induced the GUS gene expression specifically in the trichomes of transgenic A. thaliana, consistent with the results that the expression level of ALDH1 gene was increased in the A. annua under MeJA and wounding treatments. Two RAA motifs (AP2/ERF binding site) but no W box (WRKY binding site) motif were identified in the ALDH1 promoter by the analysis through PLACE and plantCARE. Through the dual luciferase reporter assay, we revealed that both AaORA and AaERF2, rather than AaWRKY1, could activate the expression of ALDH1 promoter. Our study shed light on the in-depth understanding of the role of ALDH1 in artemisinin biosynthesis. [ABSTRACT FROM AUTHOR]

Artemisinic aldehyde Δ11 (13) reductase ( DBR2) is the checkpoint enzyme catalyzing artemisinic aldehyde to form dihydroartemisinic aldehyde directly involved in artemisinin biosynthetic pathway. In the present study, DBR2 was employed to engineer the biosynthetic pathway of artemisinin in transgenic plants of Artemisia annua L. Seven independent transgenic plants of A. annua with DBR2 overexpression driven by the cauliflower mosaic virus 35S promoter were obtained by Agrobacterium-mediated genetic transformation and confirmed by genomic PCR. The results of real-time q PCR analysis showed that the expression levels of DBR2 gene in all the seven transgenic lines were significantly higher than in nontransgenic control. The high-performance liquid chromatography analysis of artemisinin and its relative metabolites demonstrated that the contents of artemisinin and its direct precursor dihydroartemisinic acid were remarkably increased in the transgenic plants of A. annua with DBR2 overexpression. Interestingly, it was also found that the contents of arteannuin B and its direct precursor artemisinic acid in the branch pathway competing against artemisinin biosynthesis were also improved in DBR2-overexpressed A. annua plants. The transgenic results in the present study indicated that DBR2 is a useful structural gene in engineering the artemisinin biosynthetic pathway to develop genetically modified A. annua with the higher yield of artemisinin. [ABSTRACT FROM AUTHOR]

Tyrosine decarboxylase initializes salidroside biosynthesis. Metabolic characterization of tyrosine decarboxylase gene from Rhodiola crenulata (RcTYDC) revealed that it played an important role in salidroside biosynthesis. Recombinant 53 kDa RcTYDC converted tyrosine into tyramine. RcTYDC gene expression was induced coordinately with the expression of RcUDPGT (the last gene involved in salidroside biosynthesis) in SA/MeJA treatment; the expression of RcTYDC and RcUDPGT was dramatically upregulated by SA, respectively 49 folds and 36 folds compared with control. MeJA also significantly increased the expression of RcTYDC and RcUDPGT in hairy root cultures. The tissue profile of RcTYDC and RcUDPGT was highly similar: highest expression levels found in stems, higher expression levels in leaves than in flowers and roots. The gene expressing levels were consistent with the salidroside accumulation levels. This strongly suggested that RcTYDC played an important role in salidroside biosynthesis in R. crenulata. Finally, RcTYDC was used to engineering salidroside biosynthetic pathway in R. crenulata hairy roots via metabolic engineering strategy of overexpression. All the transgenic lines showed much higher expression levels of RcTYDC than non-transgenic one. The transgenic lines produced tyramine, tyrosol and salidroside at higher levels, which were respectively 3.21–6.84, 1.50–2.19 and 1.27–3.47 folds compared with the corresponding compound in non-transgenic lines. In conclusion, RcTYDC overexpression promoted tyramine biosynthesis that facilitated more metabolic flux flowing toward the downstream pathway and as a result, the intermediate tyrosol was accumulated more that led to the increased production of the end-product salidroside. [ABSTRACT FROM AUTHOR]

Putrescine, produced via the arginine decarboxylase (ADC)/ornithine decarboxylase (ODC)-mediated pathway, is an initial precursor for polyamines metabolism and the root-specific biosynthesis of medicinal tropane alkaloids (TAs). These alkaloids are widely used as muscarinic acetylcholine antagonists in clinics. Although the functions of ODC in biosynthesis of polyamines and TAs have been well investigated, the role of ADC is still poorly understood. In this study, enzyme inhibitor treatment showed that ADC was involved in the biosynthesis of putrescine-derived metabolites and root growth in Atropa belladonna. Further analysis found that there were six ADC unigenes in the A. belladonna transcriptome, with two of them, AbADC1 and AbADC2 , exhibiting high expression in the roots. To investigate their roles in TAs/polyamines metabolism and root growth, RNA interference (RNAi) was used to suppress either AbADC1 or AbADC2 expression in A. belladonna hairy roots. Suppression of the AbADC1 expression resulted in a significant reduction in the putrescine content and hairy root biomass. However, it had no noticeable effect on the levels of N -methylputrescine and the TAs hyoscyamine, anisodamine, and scopolamine. On the other hand, suppression of AbADC2 expression markedly reduced the levels of putrescine, N -methylputrescine, and TAs, but had no significant effect on hairy root biomass. According to β -glucuronidase (GUS) staining assays, AbADC1 was mainly expressed in the root elongation and division region while AbADC2 was mainly expressed in the cylinder of the root maturation region. These differences in expression led to functional divergence, with AbADC1 primarily regulating root growth and AbADC2 contributing to TA biosynthesis. • RNAi reduced putrescine biosynthesis by suppressing AbADC1 or AbADC2 in A. belladonna hairy roots. • Knocking down AbADC1 reduced hairy root biomass, while knocking down AbADC2 did not. • Knocking down AbADC2 suppressed the biosynthesis of N -methylputrescine and TAs. • AbADC1 exhibited high expression in the root elongation and division region. • AbADC2 was highly expressed in the cylinder of the root maturation region. [ABSTRACT FROM AUTHOR]

Abstract Ethnopharmacological relevance Plants of Epilobium angustifolium are popular in China to treatment of traumatic injury, subduing inflammation and menstrual disorders. In European, the preparations or extracts containing E. angustifolium are popular to treat prostate diseases. Recent research suggested that E. angustifolium showed therapeutic effects in early stage of BPH, inflammation of urethra and prostate, as well as micturition problems. And the related researches were focus on aqueous extract and its main constituent of oenothein B. Aim of the study This study aims to evaluate the therapeutic effect against BPH of the ethyl acetate extracts (EAE) and n -butanol extracts (BUE) from E. angustifolium and to chemical investigation of the active constituents. Materials and methods The in vitro anti-BPH activity was assessed by determining the benign prostatic hyperplasia epithelial-1 (BPH-1) cell viability using MTT assay as well as suppressing of prostate specific antigen (PSA) secretion in prostate epithelial cancer hormone-dependent (LNCaP) cells measured by ELISA method. The in vivo anti-BPH was evaluated by testosterone propionate induced BPH SD rats. After oral administration of BUE at 100, 200 and 400 mg/kg B.W. for 28 days, the prostate weight and index, plasma androgen level, histopathological alteration, oxidative and inflammatory-related factors in prostate were assessed. Phytochemical investigation on active extracts was carried by chromatographic and spectroscopic techniques. Anti-BPH activities of the isolates were evaluated in vitro. Results BUE and EAE from E. angustifolium exhibited significant anti-BPH effect in vitro. Further in vivo study demonstrated that BUE exhibited therapeutic effects against TP-induced BPH in SD rats via down-regulating of the androgen level, suppressing the expression of NF-κB and eventually alleviating the inflammatory responses and oxidative stress. Phytochemical research on BUE and EAE extracts led to the isolation and identification of 50 compounds. In vitro anti-BPH screening revealed that 26 compounds exhibited anti-proliferation in BHP-1 cell and 36 compounds showed PSA inhibition in LNCap cell, in which 7 compounds exhibited very significant anti-BPH activities in both two cell lines (P < 0.01), 5 compounds with extremely significant activities in one of the cell lines (P < 0.001), and compound 25 exhibited the most potent anti-BPH activity (P < 0.001). Conclusions E. angustifolium exhibited the therapeutic potential against BPH, and its active compounds may be used as candidate for treatment of BPH. Graphical abstract fx1 [ABSTRACT FROM AUTHOR]