A high-efficient protoplast transient system for screening gene editing elements in Salvia miltiorrhiza.

KEY MESSAGE: A high-efficiency protoplast transient system was devised to screen genome editing elements in Salvia miltiorrhiza. Medicinal plants with high-value pharmaceutical ingredients have attracted research attention due to their beneficial effects on human health. Cell wall-free protoplasts of plants can be used to evaluate the efficiency of genome editing mutagenesis. The capabilities of gene editing in medicinal plants remain to be fully explored owing to their complex genetic background and shortfall of suitable transformation. Here, we took the Salvia miltiorrhiza as a representative example for developing a method to screen favorable gene editing elements with high editing efficiency in medical plants by a PEG-mediated protoplast transformation. Results indicated that using the endogenous SmU6.1 of S. miltiorrhiza to drive sgRNA and the plant codon-optimized Cas9 driven by the promoter SlEF1α can enhance the efficiency of editing. In summary, we uncover an efficacious transient method for screening editing elements and shed new light on increasing gene editing efficiency in medicinal plants.

The Light- and Jasmonic Acid-Induced AaMYB108-like Positive Regulates the Initiation of Glandular Secretory Trichome in Artemisia annua L.

The plant Artemisia annua L. is famous for producing "artemisinin", which is an essential component in the treatment of malaria. The glandular secretory trichomes (GSTs) on the leaves of A. annua secrete and store artemisinin. Previous research has demonstrated that raising GST density can effectively raise artemisinin content. However, the molecular mechanism of GST initiation is not fully understood yet. In this study, we identified an MYB transcription factor, the AaMYB108-like, which is co-induced by light and jasmonic acid, and positively regulates glandular secretory trichome initiation in A. annua. Overexpression of the AaMYB108-like gene in A. annua increased GST density and enhanced the artemisinin content, whereas anti-sense of the AaMYB108-like gene resulted in the reduction in GST density and artemisinin content. Further experiments demonstrated that the AaMYB108-like gene could form a complex with AaHD8 to promote the expression of downstream AaHD1, resulting in the initiation of GST. Taken together, the AaMYB108-like gene is a positive regulator induced by light and jasmonic acid for GST initiation in A. annua.

AaSEPALLATA1 integrates jasmonate and light-regulated glandular secretory trichome initiation in Artemisia annua.

Glandular secretory trichomes (GSTs) can secrete and store a variety of specific metabolites. By increasing GST density, valuable metabolites can be enhanced in terms of productivity. However, the comprehensive and detailed regulatory network of GST initiation still needs further investigation. By screening a complementary DNA library derived from young leaves of Artemisia annua, we identified a MADS-box transcription factor, AaSEPALLATA1 (AaSEP1), that positively regulates GST initiation. Overexpression of AaSEP1 in A. annua substantially increased GST density and artemisinin content. The HOMEODOMAIN PROTEIN 1 (AaHD1)-AaMYB16 regulatory network regulates GST initiation via the jasmonate (JA) signaling pathway. In this study, AaSEP1 enhanced the function of AaHD1 activation on downstream GST initiation gene GLANDULAR TRICHOME-SPECIFIC WRKY 2 (AaGSW2) through interaction with AaMYB16. Moreover, AaSEP1 interacted with the JA ZIM-domain 8 (AaJAZ8) and served as an important factor in JA-mediated GST initiation. We also found that AaSEP1 interacted with CONSTITUTIVE PHOTOMORPHOGENIC 1 (AaCOP1), a major repressor of light signaling. In this study, we identified a MADS-box transcription factor that is induced by JA and light signaling and that promotes the initiation of GST in A. annua.

A high-efficiency trichome collection system by laser capture microdissection.

Trichomes, which are classified as glandular or non-glandular, are hair-like epidermal structures that are present on aerial parts of most plant species. Glandular secretory trichomes (GSTs) have the capacity to secrete and store specialized metabolites, which are widely used as natural pesticides, food additives, fragrance ingredients or pharmaceuticals. Isolating individual trichomes is an essential way for identifying trichome-specific gene functions and discovering novel metabolites. However, the isolation of trichomes is difficult and time-consuming. Here, we report a method to isolate the GSTs from leaf epidermis dispense with fixation using laser capture microdissection (LCM). In this study, 150 GSTs were captured efficiently from Artemisia annua leaves and enriched for artemisinin measurement. UPLC analysis of microdissected samples indicated specific accumulation of secondary metabolites could be detected from a small number of GSTs. In addition, qRT-PCR revealed that the GST-specific structural genes involved in artemisinin biosynthesis pathway were highly expressed in GSTs. Taken together, we developed an efficient method to collect comparatively pure GSTs from unfixed leaved, so that the metabolites were relatively obtained intact. This method can be implemented in metabolomics research of purely specific plant cell populations and has the potential to discover novel secondary metabolites.

MADS-box gene AaSEP4 promotes artemisinin biosynthesis in Artemisia annua.

The plant Artemisia annua is well known for its production of artemisinin, a sesquiterpene lactone that is an effective antimalarial compound. Although remarkable progress has been made toward understanding artemisinin biosynthesis, the effect of MADS-box family transcription factors on artemisinin biosynthesis is still poorly understood. In this study, we identified a MADS transcription factor, AaSEP4, that was predominantly expressed in trichome. AaSEP4 acts as a nuclear-localized transcriptional activator activating the expression of AaGSW1 (GLANDULAR TRICHOME-SPECIFIC WRKY1). Dual-luciferase and Yeast one-hybrid assays revealed that AaSEP4 directly bound to the CArG motif in the promoter region of AaGSW1. Overexpression of AaSEP4 in A. annua significantly induced the expression of AaGSW1 and four artemisinin biosynthesis genes, including amorpha-4,11-diene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1), double-bond reductase 2 (DBR2) and aldehyde dehydrogenase 1 (ALDH1). Furthermore, the results of high-performance liquid chromatography (HPLC) showed that the artemisinin content was significantly increased in the AaSEP4-overexpressed plants. In addition, RT-qPCR results showed that AaSEP4 was induced by methyl jasmonic acid (MeJA) treatment. Taken together, these results explicitly demonstrate that AaSEP4 is a positive regulator of artemisinin biosynthesis, which can be used in the development of high-artemisinin yielding A. annua varieties.

Engineering the expression of plant secondary metabolites-genistein and scutellarin through an efficient transient production platform in Nicotiana benthamiana L.

Plant natural products (PNPs) are active substances indispensable to human health with a wide range of medical and commercial applications. However, excessive population growth, overexploitation of natural resources, and expensive total chemical synthesis have led to recurrent supply shortages. Despite the fact that the microbial production platform solved these challenges, the platform still has drawbacks such as environmental pollution, high costs, and non-green production. In this study, an efficient platform for the production of PNPs based on the transient expression system of Nicotiana benthamiana L. combined with synthetic biology strategies was developed. Subsequently, the feasibility of the platform was verified by a simple "test unit." This platform was used to synthesize two high-value PNPs: genistein (5.51 nmol g-1 FW) and scutellarin (11.35 nmol g-1 FW). Importantly, this is the first report on the synthesis of scutellarin in heterologous plants. The platform presented here will possibly be adopted for the heterologous production of genistein and scutellarin in tobacco plants as a novel and sustainable production strategy.

The truncated AaActin1 promoter is a candidate tool for metabolic engineering of artemisinin biosynthesis in Artemisia annua L.

Malaria is a devastating parasitic disease with high levels of morbidity and mortality worldwide. Artemisinin, the active substance against malaria, is a sesquiterpenoid produced by Artemisia annua. To improve artemisinin content in the native A. annua plants, considerable efforts have been attempted, with genetic transformation serving as an effective strategy. Although, the most frequently-used cauliflower mosaic virus (CaMV) 35S (CaMV35S) promoter has proved to be efficient in A. annua transgenic studies, it appears to show weak activity in peltate glandular secretory trichomes (GSTs) of A. annua plants. Here, we characterized the 1727 bp fragment upstream from the translation start codon (ATG) of AaActin1, however, found it was inactive in tobacco. After removal of the 5' intron, the truncated AaActin1 promoter (tpACT) showed 69% and 50% activity of CaMV35S promoter in transiently transformed tobacco and stably transformed A. annua, respectively. ß-glucuronidase (GUS) staining analysis showed that the tpACT promoter was capable of directing the constant expression of a foreign gene in peltate GSTs of transgenic A. annua, representing higher activity than CaMV35S promoter. Collectively, our study provided a novel promoter available for metabolic engineering of artemisinin biosynthesis in A. annua.

Polymorphisms of Cytochromes P450 and Glutathione S-Transferases Synergistically Modulate Risk for Parkinson's Disease.

Background: Environmental substances such as pesticides are well-known in link with Parkinson's disease (PD) risk. Enzymes including cytochromes P450 (CYPs), esterases and glutathione S-transferases (GSTs) are responsible for the xenobiotic metabolism and may functionally compensate each other for subtypes in the same class. We hypothesize that the genetic effects of each class modulate PD risk stronger in a synergistic way than individually. Methods: We selected 14 polymorphic loci out of 13 genes which encode enzymes in the classes of CYP, esterase, and GST, and recruited a cohort of 1,026 PD and control subjects from eastern China. The genotypes were identified using improved multiplex ligation detection reaction and analyzed using multiple models. Results: A total of 13 polymorphisms remained after Hardy-Weinberg equilibrium analysis. None of the polymorphisms were independently associated with PD risk after Bonferroni correction either by logistic regression or genetic models. In contrast, interaction analyses detected increased resistance to PD risk in individuals carrying the rs12441817/CC (CYP1A1) and rs2070676/GG + GC (CYP2E1) genotypes (P = 0.002, OR = 0.393, 95% CI = 0.216-0.715), or carrying the GSTM1-present, GSTT1-null, rs156697/AG + GG (GSTO2) and rs1695/AA (GSTP1) genotypes (P = 0.003, OR = 0.348, 95% CI = 0.171-0.706). The synergistic effect of GSTs on PD was primarily present in females (P = 0.003). No synergistic effect was observed within genotypes of esterases. Conclusion: We demonstrate a presence of synergistic but not individual impact on PD susceptibility in polymorphisms of CYPs and GSTs. The results indicate that the genetic interplay leads the way to PD development for xenobiotic metabolizing enzymes.

AaWRKY17, a positive regulator of artemisinin biosynthesis, is involved in resistance to Pseudomonas syringae in Artemisia annua.

Artemisia annua, a traditional Chinese medicinal plant, remains the only plant source for artemisinin production, yet few genes have been identified to be involved in both the response to biotic stresses, such as pathogens, and artemisinin biosynthesis. Here, we isolated and identified the WRKY transcription factor (TF) AaWRKY17, which could significantly increase the artemisinin content and resistance to Pseudomonas syringae in A. annua. Yeast one-hybrid (Y1H), dual-luciferase (dual-LUC), and electrophoretic mobility shift assay (EMSA) results showed that AaWRKY17 directly bound to the W-box motifs in the promoter region of the artemisinin biosynthetic pathway gene amorpha-4,11-diene synthase (ADS) and promoted its expression. Real-time quantitative PCR (RT-qPCR) analysis revealed that the transcript levels of two defense marker genes, Pathogenesis-Related 5 (PR5) and NDR1/HIN1-LIKE 10 (NHL10), were greatly increased in AaWRKY17-overexpressing transgenic A. annua plants. Additionally, overexpression of AaWRKY17 in A. annua resulted in decreased susceptibility to P. syringae. These results indicated that AaWRKY17 acted as a positive regulator in response to P. syringae infection. Together, our findings demonstrated that the novel WRKY transcription factor AaWRKY17 could potentially be used in transgenic breeding to improve the content of artemisinin and pathogen tolerance in A. annua.

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.