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UV-B radiation is a pivotal photomorphogenic signal and positively regulates plant growth and metabolite biosynthesis. In order to elucidate the transcriptional regulation mechanism underlying UV-B-induced artemisinin and flavonoid biosynthesis in Artemisia annua, the transcriptional responses of A. annua L. leaves to UV-B radiation were analyzed using the Illumina transcriptome sequencing. A total of 10705 differentially expressed genes (DEGs) including 533 transcription factors (TFs), were identified. Based on the expression trends of the differentially expressed TFs as well as artemisinin and flavonoid biosynthesis genes, we speculated that TFs belonging to 6 clusters were most likely to be involved in the regulation of artemisinin and/or flavonoid biosynthesis. The regulatory relationship between TFs and artemisinin/flavonoid biosynthetic genes was further studied. Dual-LUC assays results showed that AaMYB6 is a positive regulator of AaLDOX which belongs to flavonoid biosynthesis pathway. In addition, we identified an R2R3 MYB TF, AaMYB4 which potentially mediated both artemisinin and flavonoid biosynthesis pathways by activating the expression of AaADS and AaDBR2 in artemisinin biosynthesis pathway and AaUFGT in flavonoid biosynthesis pathway. Overall, our findings would provide an insight into the elucidation of the parallel transcriptional regulation of artemisinin and flavonoid biosynthesis in A. annua L. under UV-B radiation.

Summary Artemisinin, a sesquiterpene lactone widely used in malaria treatment, was discovered in the medicinal plant Artemisia annua. The biosynthesis of artemisinin is efficiently regulated by jasmonate (JA) and abscisic acid (ABA) via regulatory factors. However, the mechanisms linking JA and ABA signalling with artemisinin biosynthesis through an associated regulatory network of downstream transcription factors (TFs) remain enigmatic. Here we report AaTCP15, a JA and ABA dual‐responsive teosinte branched1/cycloidea/proliferating (TCP) TF, which is essential for JA and ABA‐induced artemisinin biosynthesis by directly binding to and activating the promoters of DBR2 and ALDH1, two genes encoding enzymes for artemisinin biosynthesis. Furthermore, AaORA, another positive regulator of artemisinin biosynthesis responds to JA and ABA, interacts with and enhances the transactivation activity of AaTCP15 and simultaneously activates AaTCP15 transcripts. Hence, they form an AaORA‐AaTCP15 module to synergistically activate DBR2, a crucial gene for artemisinin biosynthesis. More importantly, AaTCP15 expression is activated by the multiple reported JA and ABA‐responsive TFs that promote artemisinin biosynthesis. Among them, AaGSW1 acts at the nexus of JA and ABA signalling to activate the artemisinin biosynthetic pathway and directly binds to and activates the AaTCP15 promoter apart from the AaORA promoter, which further facilitates formation of the AaGSW1‐AaTCP15/AaORA regulatory module to integrate JA and ABA‐mediated artemisinin biosynthesis. Our results establish a multilayer regulatory network of the AaGSW1‐AaTCP15/AaORA module to regulate artemisinin biosynthesis through JA and ABA signalling, and provide an interesting avenue for future research exploring the special transcriptional regulation module of TCP genes associated with specialized metabolites in plants.

Artemisia annua is a medicinal plant rich in terpenes and flavonoids with useful biological activities such as antioxidant, anticancer, and antimalarial activities. The transcriptional regulation of flavonoid biosynthesis in A. annua has not been well-studied. In this study, we identified a YABBY family transcription factor, AaYABBY5, as a positive regulator of anthocyanin and total flavonoid contents in A. annua. AaYABBY5 was selected based on its similar expression pattern to the phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavonol synthase (FLS) genes. A transient dual-luciferase assay in Nicotiana bethamiana with the AaYABBY5 effector showed a significant increase in the activity of the downstream LUC gene, with reporters AaPAL, AaCHS, AaCHI, and AaUFGT. The yeast one-hybrid system further confirmed the direct activation of these promoters by AaYABBY5. Gene expression analysis of stably transformed AaYABBY5 overexpression, AaYABBY5 antisense, and control plants revealed a significant increase in the expression of AaPAL, AaCHS, AaCHI, AaFLS, AaFSII, AaLDOX, and AaUFGT in AaYABBY5 overexpression plants. Moreover, their total flavonoid content and anthocyanin content were also found to increase. AaYABBY5 antisense plants showed a significant decrease in the expression of flavonoid biosynthetic genes, as well as a decrease in anthocyanin and total flavonoid contents. In addition, phenotypic analysis revealed deep purple-pigmented stems, an increase in the leaf lamina size, and higher trichome densities in AaYABBY5 overexpression plants. Together, these data proved that AaYABBY5 is a positive regulator of flavonoid biosynthesis in A. annua. Our study provides candidate transcription factors for the improvement of flavonoid concentrations in A. annua and can be further extended to elucidate its mechanism of regulating trichome development.

Background The Agrobacterium-mediated transient transformation, which proved effective in diverse plant species, has been widely applied for high-throughput gene function studies due to its simplicity, rapidity, and high efficiency. Despite the efforts have made on Artemisia annua transient expression, achieving high-throughput gene functional characterization basing on a fast and easy-manipulated transient transformation system in A. annua remains challenging. Results The first pair of true leaves of A. annua is an ideal candidate for Agrobacterium injection. EHA105 was the optimal strain that can be used for the development of the transient expression system. The supplementation of Triton X-100 at a concentration of 0.005% greatly improved the transient expression frequency. According to the histochemical β-Glucuronidase (GUS) staining assay, high transient expression level of the reporter gene (GUS) maintained at least a week. Dual-luciferase (Dual-LUC) transient assays showed that the activity of cauliflower mosaic virus 35S (CaMV35S) promoter and its derivates varied between A. annua and tobacco. In A. annua, the CaMV35S promoter had comparable activity with double CaMV35S promoter, while in tobacco, CaMV35S exhibited approximately 50% activity of double CaMV35S promoter. Otherwise, despite the CaMV35S promoter and double CaMV35S promoter from GoldenBraid Kit 2.0 displayed high activity strength in tobacco, they demonstrated a very low activity in transiently expressed A. annua. The activity of UBQ10 promoter and endogenous UBQb promoter was investigated as well. Additionally, using our transient expression system, the transactivation of AaGSW1 and AaORA on AaCYP71AV1 promoter was confirmed. Dual-LUC assays demonstrated that AaHD8 activated the expression of two glandular secreting trichomes-specific lipid transfer protein genes AaLTP1 and AaLTP2, indicating that AaLTP1 and AaLTP2 might serve as downstream components of AaHD8-involved glandular trichome initiation and cuticle formation, as well as artemisinin secretion in A. annua. Conclusions A simple, rapid, good-reproducibility, high-efficiency and low-cost transient transformation system in A. annua was developed. Our method offered a new way for gene functional characterization studies such as gene subcellular localization, promoter activity and transcription activation assays in A. annua, avoiding the aberrant phenotypes resulting from gene expression in a heterologous system.

Due to overlapping and diverse morphological characters, Berberis is among the most taxonomically complex genera. Palynology is one of the taxonomic tools for delimitation and identification of complex species. In this study, pollens of 10 Berberis species were analyzed through light and scanning electron microscopy. Qualitative as well as quantitative features (pollen shape, size, presence or absence of colpi, colpi length and width, exine thickness, ornamentation, pollen class, aperture, and polar-equatorial ratio) were measured. Five species were observed to have colpate (pantocolpate) with elongated ends, radially symmetrical, isopolar, monads, and psilate-regulate pollens. In polar view, six pollen were spheroidal, two were ovoid, one spherical, and one oblate. Similarly, variation in pollen length was prominent and the largest pollen on polar view was recorded for B. psodoumbellata 60-65 μm (62.4 ± 0.9), while the smallest one was observed for B. lycium 29-35 μm (32.2 ± 1). The observed variation in both quantitative and qualitative features were important in taxonomic identification. This shows that palynological characters are helpful in identification of Berberis genus at the species level.

Artemisinins, as a class of bioactive molecules, are mainly derived from the extracts of Artemisia annua L. and are the mainstay for malaria treatment, including severe malaria, uncomplicated malaria and multi-drug resistant malaria. They are well-known for their good tolerability, safety and rapid onset of action. Their efficacy is not only limited to malaria but also extends to a variety of human diseases such as cancer, tuberculosis, viral diseases (e.g. Human cytomegalovirus), immune diseases and parasitic infections like schistosomiasis. Being a cheap and safe drug class, which saves millions of lives at risk from malaria around the globe, can also have significant potential in oncology as they have shown anti-cancer properties in both cell lines and animal models. Active derivatives (e.g. artesunate, artemether and arteether etc.) have also been synthesized which can be used for oral, rectal, intramuscular and intravenous administration. A comprehensive update on the non-malarial use of artemisinins and/or their derivatives and artemisinin-based drug development beyond anti-malarial is discussed in this review. With the collaborative efforts in the clinical pharmacology of artemisinins and novel synthesis of artemisinin analogues, it is likely that artemisinin-based drugs will become an important armamentarium impeding a number of diseases beyond malaria.

Aim of the current study was to investigate the effect of exogenously inoculated root endophytic fungus, Piriformospora indica, on molecular, biochemical, morphological and physiological parameters of Artemisia annua L. treated with different concentrations (0, 50, 100 and 150 μmol/L) of arsenic (As) stress. As was significantly accumulated in the roots than shoots of P. indica-inoculated plants. As accumulation and immobilization in the roots is directly associated with the successful fungal colonization that restricts most of As as compared to the aerial parts. A total of 4.1, 11.2 and 25.6 mg/kg dry weight of As was accumulated in the roots of inoculated plants supplemented with 50, 100 and 150 μmol/L of As, respectively as shown by atomic absorption spectroscopy. P. indica showed significant tolerance in vitro to As toxicity even at high concentration. Furthermore, flavonoids, artemisinin and overall biomass were significantly increased in inoculated-stressed plants. Superoxide dismutase and peroxidase activities were increased 1.6 and 1.2 fold, respectively under 150 μmol/L stress in P. indica-colonized plants. Similar trend was followed by ascorbate peroxidase, catalase and glutathione reductase. Like that, phenolic acid and phenolic compounds showed a significant increase in colonized plants as compared to their respective control/un-colonize stressed plants. The real-time PCR revealed that transcriptional levels of artemisinin biosynthesis genes, isoprenoids, terpenes, flavonoids biosynthetic pathway genes and signal molecules were prominently enhanced in inoculated stressed plants than un-inoculated stressed plants., Highlights • The level of artemisinin and flavonoids was increased by P. indica under arsenic stress condition. • P. indica accumulated and restricted arsenic in the roots. • P. indica showed higher tolerance to arsenic in vitro. • Antioxidant defense system and enzymatic activities were enhanced by P. indica.

Artemisinin is an effective antimalarial sesquiterpene lactone synthesized in Artemisia annua. Various transcription factors have been previously reported that can influence the biosynthesis of artemisinin; however, the effect of YABBY family transcription factors on artemisinin biosynthesis was unknown. In the present study, we cloned and characterized AaYABBY5: a homolog of MsYABBY5 in Mentha spicata which is involved in modulating the monoterpenes, as a positive regulator of artemisinin biosynthesis in A. annua. AaYABBY5 was found localized to the nucleus, and its expression was found to be induced by exogenous methyl jasmonic acid (MeJA) treatment. In the dual-luciferase reporter assay, it was found that AaYABBY5 significantly increased the activities of promoters of amorpha-4,11-diene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1), double-bond reductase 2 (DBR2), and aldehyde dehydrogenase 1 (ALDH1) genes. Yeast one hybrid assay showed that AaYABBY5 directly bonds to the promoters of CYP71AV1 and DBR2 genes. Quantitative real-time polymerase chain reaction (qPCR) of AaYABBY5 overexpression and AaYABBY5 antisense plants revealed a significant increase in the expression of ADS, CYP71AV1, DBR2, and ALDH1 in AaYABBY5 overexpression plants and a significant decrease in the expression of these genes in AaYABBY5 antisense A. annua, respectively. Furthermore, the results of high-performance liquid chromatography (HPLC) showed that the artemisinin and its precursor dihydroartemisinic acid were significantly increased in the AaYABBY5 overexpression plants while AaYABBY5 downregulation resulted in a significant decrease in the concentration of artemisinin. Taken together, these results explicitly represent that AaYABBY5 is a positive regulator of artemisinin biosynthesis in A. annua.

Taxonomy of the genus Berberis is quite complex, due to overlapping morphological characters, making it very difficult to differentiate the species within the genus. In order to resolve this taxonomic complexity, the foliar anatomy of 10 Berberis L. species was carried out, for the first time from Pakistan, using light microscopy (LM). Significant variation in terms of epidermal cells shape, size, cell wall pattern, and stomata type was observed. B. baluchistanica has the largest epidermal cells, Adaxial: length = 45-(53.9 ± 3.6)-62.5 μm; and width = 22.5-(26.3 ± 1.3)-30 μm; Abaxial: length = 37.5-(43.25 ± 2.5)-50 μm; and width = 20-(22.6 ± 0.8)-25. The highest number of stomata was observed in B. glaucocarpa as 62 on the abaxial surface while the lowest number of stomata was recorded in B. baluchistanica as 8 on the adaxial surface. Of 10 investigated species, 6 possess anomocytic type stomata, while 2 species that is, B. aitchisonii and B. parkeriana have both anomocytic and anisocytic stomata while B. baluchistanica and B. calliobotrys have only paracytic type stomata. The highest number of cells per unit area was present on the adaxial surface of B. calliobotrys ranging from 245-(252.4)-260 followed by B. parkeriana with 209-(227.8)-250 on the abaxial surface. Stomatal index (SI) also varied considerably and was the lowest (2.6) percentage in B. baluchistanica and highest (31.9) percentage in B. kunawurensis. A taxonomic key based on micro-morphological characters is provided for species identification.

Biological activity of human interleukin-6 (IL-6) is associated with a vast number of diseases such as rheumatoid arthritis, sepsis, and severe inflammatory diseases. In this study, human IL-6 cDNA was isolated from a cDNA library that was constructed with mRNA derived from human placental tissues. Subsequently, the complete human IL-6 cDNA was cloned and expressed in BL21DE3 cells. The recombinant human IL-6 (rhIL-6) protein was expressed in a form of an insoluble inclusion body. Inclusion bodies were solubilized under denaturing conditions and purified by immobilized metal affinity chromatography with gradual on-column refolding by the gradient elution method (from 6 to 0 M urea). The protein was purified to apparent homogeneity of about 99% with a yield of 50 mg/L. The purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), size exclusion high-performance liquid chromatography, and Western blotting analysis. The bioactivity was assessed by proliferation assay of TF-1 cells in a dose-dependent manner. The present study confirms the expression of the placenta-derived IL-6 gene in a prokaryotic expression system and matrix-assisted on-column refolding and purification of rhIL-6 by immobilized metal affinity chromatography.

The aim of present study was to enhance topical permeation of clotrimazole gel preparation by using various permeability enhancers such as coconut oil, pistachio oil and sodium lauryl sulphate (SLS). Clotrimazole gel preparations were prepared and optimized by using three factor, five level central composite design. A second-order polynomial equation was generated in order to estimate the effect of independent variables i.e. coconut oil (X1), pistachio oil (X2) and sodium lauryl sulphate (X3) at various dependent variables i.e. flux (Y1), lag time (Y2), diffusion coefficient (Y3), permeability coefficient (Y4), and input rate (Y5) of clotrimazole gel formulations. Ex vivo skin permeation study was performed through rat skin by using modified Franz diffusion cell system. Optimized formulation F8 exhibited highest flux 2.17 µg/cm2/min, permeability coefficient 0.0019 cm/min and input rate 1.543 µg/cm2/min, along with moderate lag time 77.27 min and diffusion coefficient 0.063 cm2/min, which is further supported by anti-fungal activity that exhibited more prominent zone of inhibition against Candida albicans, Aspergillus niger and Mucor. Thus, it can be concluded that permeation of clotrimazole gel was enhanced by various combination of coconut oil, pistachio oil and sodium lauryl sulphate but optimized formulation F8 containing 0.4 ml pistachio oil, 0.8 ml coconut oil and 0.04 g of SLS exhibited more pronounced and promising effect through rat skin.