JA-Regulated AaGSW1-AaYABBY5/AaWRKY9 Complex Regulates Artemisinin Biosynthesis in Artemisia annua.

Artemisinin, a sesquiterpene lactone obtained from Artemisia annua, is an essential therapeutic against malaria. YABBY family transcription factor AaYABBY5 is an activator of AaCYP71AV1 (cytochrome P450-dependent hydroxylase) and AaDBR2 (double-bond reductase 2); however, the protein-protein interactions of AaYABBY5, as well as the mechanism of its regulation, have not yet been elucidated. AaWRKY9 protein is a positive regulator of artemisinin biosynthesis that activates AaGSW1 (glandular trichome-specific WRKY1) and AaDBR2 (double-bond reductase 2). In this study, YABBY-WRKY interactions are revealed to indirectly regulate artemisinin production. AaYABBY5 significantly increased the activity of the luciferase (LUC) gene fused to the promoter of AaGSW1. Toward the molecular basis of this regulation, AaYABBY5 interaction with AaWRKY9 protein was found. The combined effectors AaYABBY5 + AaWRKY9 showed synergistic effects toward the activities of AaGSW1 and AaDBR2 promoters, respectively. In AaYABBY5 overexpression plants, the expression of GSW1 was found to be significantly increased when compared to that of AaYABBY5 antisense or control plants. In addition, AaGSW1 was identified as an upstream activator of AaYABBY5. Further, it was found that AaJAZ8, a transcriptional repressor of jasmonate signaling, interacted with AaYABBY5 and attenuated its activity. Co-expression of AaYABBY5 and anti-AaJAZ8 in A. annua increased the activity of AaYABBY5 toward artemisinin biosynthesis. This current study provides the first indication of the molecular basis of regulation of artemisinin biosynthesis through YABBY-WRKY interactions, which are regulated through AaJAZ8. This knowledge presents AaYABBY5 overexpression plants as a powerful genetic resource for artemisinin biosynthesis.

Molecular approaches to enhance astaxanthin biosynthesis; future outlook: engineering of transcription factors in Haematococcus pluvialis.

Microalgae are the preferred species for producing astaxanthin because they pose a low toxicity risk than chemical synthesis. Astaxanthin has multiple health benefits and is being used in: medicines, nutraceuticals, cosmetics, and functional foods. Haematococcus pluvialis is a model microalga for astaxanthin biosynthesis; however, its natural astaxanthin content is low. Therefore, it is necessary to develop methods to improve the biosynthesis of astaxanthin to meet industrial demands, making its commercialization cost-effective. Several strategies related to cultivation conditions are employed to enhance the biosynthesis of astaxanthin in H. pluvialis. However, the mechanism of its regulation by transcription factors is unknown. For the first time, this study critically reviewed the studies on identifying transcription factors, progress in H. pluvialis genetic transformation, and use of phytohormones that increase the gene expression related to astaxanthin biosynthesis. In addition, we propose future approaches, including (i) Cloning and characterization of transcription factors, (ii) Transcriptional engineering through overexpression of positive regulators or downregulation/silencing of negative regulators, (iii) Gene editing for enrichment or deletion of transcription factors binding sites, (iv) Hormonal modulation of transcription factors. This review provides considerable knowledge about the molecular regulation of astaxanthin biosynthesis and the existing research gap. Besides, it provides the basis for transcription factors mediated metabolic engineering of astaxanthin biosynthesis in H. pluvialis.

Jasmonate- and abscisic acid-activated AaGSW1-AaTCP15/AaORA transcriptional cascade promotes artemisinin biosynthesis in Artemisia annua.

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.

The ameliorative effects of exogenous inoculation of Piriformospora indica on molecular, biochemical and physiological parameters of Artemisia annua L. under arsenic stress condition.

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.

Piriformospora indica alter root-associated microbiome structure to enhance Artemisia annua L. tolerance to arsenic.

Microorganisms in the rhizosphere are crucial allies for plant stress tolerance. Recent research suggests that by interacting with the rhizosphere microbiome, microorganisms can aid in the revegetation of soils contaminated with heavy metal(loid)s (HMs). However, it is unknown that how Piriformospora indica influences the rhizosphere microbiome to mitigate arsenic-toxicity in arsenic-enriched environments. Artemisia annua plants were grown in the presence or absence of P. indica and spiked with low (50) and high (150 µmol/L) concentrations of arsenic (As). After inoculation with P. indica, fresh weight increased by 37.7% and 10% in control and high concentration treated plants, respectively. Transmission electron microscopy showed that cellular organelles were severely damaged by As and even disappeared under high concentration. Furthermore, As was mostly accumulated by 5.9 and 18.1 mg/kg dry weight in the roots of inoculated plants treated with low and high concentrations of As, respectively. Additionally, 16 S and ITS rRNA gene sequencing were applied to analyze the rhizosphere microbial community structure of A. annua under different treatments. A significant difference was observed in microbial community structure under different treatments as revealed by non-metric multidimensional scaling ordination. The bacterial and fungal richness and diversity in the rhizosphere of inoculated plants were actively balanced and regulated by P. indica co-cultivation. Lysobacter and Steroidobacter were found to be the As-resistant bacterial genera. We conclude that P. indica inoculation could alter rhizosphere microecology, thereby mitigating As-toxicity without harming the environment.

Basic Helix-Loop-Helix Transcription Factors AabHLH2 and AabHLH3 Function Antagonistically With AaMYC2 and Are Negative Regulators in Artemisinin Biosynthesis.

Plants have evolved sophisticated systems for regulating the biosynthesis of specialized phytochemicals. Artemisinin, which is a sesquiterpene lactone widely used in anti-malaria treatment, is produced by the Artemisia annua L. plant. However, the artemisinin content in A. annua is low and difficult to meet market demands. Studies have shown that artemisinin biosynthesis in A. annua has complex temporal and spatial specificity and is under tightly transcriptional regulation. However, the mechanism of transcriptional regulation of artemisinin biosynthesis remains unclear. In this study, we identified two MYC-type bHLH transcription factors (AabHLH2 and AabHLH3) as novel regulators of artemisinin biosynthesis. These bHLH TFs act as transcription repressors and function redundantly to negatively regulate artemisinin biosynthesis. Furthermore, AabHLH2 and AabHLH3 are nuclear proteins that bind to DNA elements with similar specificity to that of AaMYC2, but lack the conserved activation domain, suggesting that repression is achieved by competition for the same cis-regulatory elements. Together, our findings reveal a novel artemisinin biosynthesis regulatory network, provide new insight into how specialized metabolites are modulated in plants, and propose a model in which different bHLH TFs coordinated in regulating artemisinin production in the plant. Finally, this study provides some useful target genes for metabolic engineering of artemisinin production via CRISPR/Cas9 gene editing.

Transcriptional regulation of flavonoid biosynthesis in Artemisia annua by AaYABBY5.

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.

A high-efficiency Agrobacterium-mediated transient expression system in the leaves of Artemisia annua L.

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 ß-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.

Transcriptomic analysis reveals the parallel transcriptional regulation of UV-B-induced artemisinin and flavonoid accumulation in Artemisia annua L.

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.

Light microscopy of Pakistani Berberis leaf cuticles and its taxonomic implications.

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.

Optimization of cell suspension culture of transformed and untransformed lettuce for the enhanced production of secondary metabolites and their pharmaceutical evaluation.

In vitro suspension culture techniques are cost effective for large-scale production of secondary metabolites. In the present study, firstly, suspension cultures of untransformed Lactuca sativa were prepared using different hormonal combinations and were subjected to different pH, temperature and salt concentrations. Maximum biomass was obtained for suspensions supplemented with 1.5 mg/L BAP and 0.1 mg/L NAA, at pH 5.8, temperature 28 °C and 0 mM NaCl concentration. Using these parameters, suspensions were produced for rol ABC- and rol C-transformed lines of L. sativa. All the transgenic lines showed prominent increase in fresh weight (FW) and dry weight (DW) with maximum values for rol ABC2 line producing 169.8 mg/mL FW and 25.3 mg/mL DW. The exudates of transformed and untransformed plants were tested for the antioxidant activity and in vivo assays on rats. Maximum phenolic content (261 µg/mL) and flavonoid content (637.6 µg/mL) were obtained for rol C1 transgenic line. Total antioxidant capacity was found maximum (1451.7 µg/mL) for untransformed lettuce, whereas rol C1 showed maximum total reducing power activity (637.6 µg/mL). In DPPH assay, maximum activity (104.7 µg/mL) was shown by rol ABC3 line. In rats analgesic assay, maximum activity (74.9%) was shown by rol C2. Line rol C1 showed maximum anti-inflammatory activity (69.2%) and maximum antidepressant activity (minimum immobility time of 55 s). Maximum anticoagulant activity was observed for rol ABC2 with maximum clotting time of 130 s. The present study could help in using lettuce suspension culture as platform for the enhanced production of important metabolites.

Pollen morphological variation of Berberis L. from Pakistan and its systematic importance.

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.

The YABBY Family Transcription Factor AaYABBY5 Directly Targets Cytochrome P450 Monooxygenase (CYP71AV1) and Double-Bond Reductase 2 (DBR2) Involved in Artemisinin Biosynthesis in Artemisia Annua.

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.