Unveiling the regulatory mechanisms of nodules development and quality formation in Panax notoginseng using multi-omics and MALDI-MSI.

INTRODUCTION: Renowned for its role in traditional Chinese medicine, Panax notoginseng exhibits healing properties including bidirectional regulatory effects on hematological system diseases. However, the presence of nodular structures near the top of the main root, known as nail heads, may impact the quality of the plant's valuable roots. OBJECTIVES: In this paper, we aim to systematically analyze nail heads to identify their potential correlation with P. notoginseng quality. Additionally, we will investigate the molecular mechanisms behind nail head development. METHODS: Morphological characteristics and anatomical features were analyzed to determine the biological properties of nail heads. Active component analysis and MALDI mass spectrometry imaging (MALDI-MSI) were performed to determine the correlation between nail heads and P. notoginseng quality. Phytohormone quantitation, MALDI-MSI, RNA-seq, and Arabidopsis transformation were conducted to elucidate the mechanisms of nail head formation. Finally, protein-nucleic acid and protein-protein interactions were investigated to construct a transcriptional regulatory network of nodule development and quality formation. RESULTS: Our analyses have revealed that nail heads originate from an undeveloped lateral root. The content of ginsenosides was found to be positively associated with the amount of nail heads. Ginsenoside Rb1 specifically accumulated in the cortex of nail heads, while IAA, tZR and JAs also showed highest accumulation in the nodule. RNA-seq analysis identified PnIAA14 and PnCYP735A1 as inhibitors of lateral root development. PnMYB31 and PnMYB78 were found to form binary complexes with PnbHLH31 to synergistically regulate the expression of PnIAA14, PnCYP735A1, PnSS, and PnFPS. CONCLUSION: Our study details the major biological properties of nodular structures in P. notoginseng and outlines their impact on the quality of the herb. It was also determined that PnMYB31- and PnMYB78-PnbHLH31 regulate phytohormones and ginsenosides accumulation, further affecting plant development and quality. This research provides insights for quality evaluation and clinical applications of P. notoginseng.

Metabolic Patterns of Flavonoid and Its Key Gene Expression Characteristics of Five Cultivars of Tulipa gesneriana during Flower Development.

Flower color is one of the most important ornamental traits of tulips (Tulipa gesneriana). Five typical tulip cultivars were selected to identify the flavonoid components and analyze their key gene expression in their tepals. Firstly, after preliminary determination of the pigment type, the flavonoids were identified by UPLC-Q-TOF-MS. A total of 17 anthoxanthins were detected in the five cultivars. The total anthoxanthin content in the white tulip and the red tulip showed a similar decreasing trend, while an increasing trend was observed in the black tulip. Similarly, a total of 13 anthocyanins were detected in five tulip cultivars. The black tulip contained the largest number of anthocyanins, mainly delphinidin derivatives (Dp) and cyanidin derivatives (Cy). The total anthocyanin content (TAC) in the orange, red, and black cultivars was higher than that in the white and yellow cultivars and presented an overall increase trend along with the flower development. TgCHS, TgFLS, TgF3H, TgF3'H, TgF3'5'H, and TgDFR, as key structural genes, were involved in the flavonoid synthesis pathway, and the expression patterns of these genes are basically consistent with the components and accumulation patterns of flavonoids mentioned above. Taken together, the flower color in tulips was closely related to the composition and content of anthocyanins and anthoxanthins, which were indeed regulated by certain key structural genes in the flavonoid pathway.

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.

AaABCG20 transporter involved in cutin and wax secretion affects the initiation and development of glandular trichomes in Artemisia annua.

Glandular trichomes are specialized structures found on the surface of plants to produce specific compounds, including terpenes, alkaloids, and other organic substances. Artemisia annua, commonly known as sweet wormwood, synthesizes and stores the antimalarial drug artemisinin in glandular trichomes. Previous research indicated that increasing the glandular trichome density could enhance artemisinin production, and the cuticle synthesis affected the initiation and development of glandular trichomes in A. annua. In this study, AaABCG12 and AaABCG20 were isolated from A. annua that exhibited similar expression patterns to artemisinin biosynthetic genes. Of the two, AaABCG20 acted as a specific transporter in glandular trichomes. Downregulating the expression of AaABCG20 resulted in a notable reduction in the density of glandular trichome, while overexpressing AaABCG20 resulted in an increase in glandular trichome density. GC-MS analysis demonstrated that AaABCG20 was responsible for the transport of cutin and wax in A. annua. These findings indicated that AaABCG20 influenced the initiation and development of glandular trichomes through transporting cutin and wax in A. annua. This glandular trichome specific half-size ABCG-type transporter is crucial in facilitating the transportation of cutin and wax components, ultimately contributing to the successful initiation and development of glandular trichomes.

Fabrication of highly dispersed Pd-Mn3O4 catalyst for efficient catalytic propane total oxidation.

Adjusting the interaction between dual active components for enhancing volatile organic compounds (VOCs) degradation is an effective but still challenging means of air pollution control. Herein, a limited pyrolysis oxidation strategy was adopted to prepare Pd-Mn3O4 spinel catalysts with uniform morphology and active component dispersion. Among these, 1.08Pd-Mn3O4 presented the highest catalytic efficiency with a T90 value of 240 °C, which was 94 °C lower than that of Mn3O4. Characterization and density functional theory (DFT) calculation results revealed that the strong metal-support interaction (SMSI) effect between Pd and Mn3O4 promoted the redistribution of surface charges, thus strengthening the oxidation-reduction ability of the active sites. Moreover, the SMSI effect led to a better migration of surface oxygen species, and boosted the generation of active surface oxygen species. Simultaneously, the Pd catalyst further reduced the energy barrier in the initial stage of the dehydrogenation of propane. Overall, this study provided a novel design strategy for dual active components catalysts with SMSI effect and extended the application of these catalysts in the important field of VOCs elimination.

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.

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.

Elevation of artemisinin content by co-transformation of artemisinin biosynthetic pathway genes and trichome-specific transcription factors in Artemisia annua.

Artemisinin, derived from Artemisia annua, is currently used as the first-line treatment for malaria. However, wild-type plants have a low artemisinin biosynthesis rate. Although yeast engineering and plant synthetic biology have shown promising results, plant genetic engineering is considered the most feasible strategy, but it is also constrained by the stability of progeny development. Here we constructed three independent unique overexpressing vectors harboring three mainstream artemisinin biosynthesis enzymes HMGR, FPS, and DBR2, as well as two trichomes-specific transcription factors AaHD1 and AaORA. The simultaneous co-transformation of these vectors by Agrobacterium resulted in the successful increase of the artemisinin content in T0 transgenic lines by up to 3.2-fold (2.72%) leaf dry weight compared to the control plants. We also investigated the stability of transformation in progeny T1 lines. The results indicated that the transgenic genes were successfully integrated, maintained, and overexpressed in some of the T1 progeny plants' genomes, potentially increasing the artemisinin content by up to 2.2-fold (2.51%) leaf dry weight. These results indicated that the co-overexpression of multiple enzymatic genes and transcription factors via the constructed vectors provided promising results, which could be used to achieve the ultimate goal of a steady supply of artemisinin at affordable prices around the world.

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.

Transcriptional regulatory network of high-value active ingredients in medicinal plants.

High-value active ingredients in medicinal plants have attracted research attention because of their benefits for human health, such as the antimalarial artemisinin, anticardiovascular disease tanshinones, and anticancer Taxol and vinblastine. Here, we review how hormones and environmental factors promote the accumulation of active ingredients, thereby providing a strategy to produce high-value drugs at a low cost. Focusing on major hormone signaling events and environmental factors, we review the transcriptional regulatory network mediating biosynthesis of representative active ingredients. In this network, many transcription factors (TFs) simultaneously control multiple synthase genes; thus, understanding the molecular mechanisms affecting transcriptional regulation of active ingredients will be crucial to developing new breeding possibilities.

AaWIN1, an AP2/ERF protein, positively regulates glandular secretory trichome initiation in Artemisia annua.

Exploring the genetic network of glandular trichomes and manipulating genes relevant to secondary metabolite biosynthesis are of great importance and value. Artemisinin, a key antimalarial drug ingredient, is synthesized and stored in glandular secretory trichomes (GSTs) in Artemisia annua. WIN/SHN proteins, a clade of AP2/ERF family, are known as regulators for cuticle biosynthesis. However, their function in glandular trichome development is less unknown. In this study, we identified a WIN/SHN gene from A. annua and named it as AaWIN1. AaWIN1 was predominantly expressed in buds, flowers and trichomes, and encoded a nuclear-localized protein. Overexpressing AaWIN1 in A. annua significantly increased the density of GST as well as the artemisinin content. Furthermore, AaGSW2 was reported to play an important role in promoting GST initiation, and the expression of AaGSW2 was induced in AaWIN1-overexpression lines. AaMIXTA1, a MYB protein positively regulating trichome initiation and cuticle biosynthesis, was confirmed to interact with AaWIN1. In addition, the ectopic expression of AaWIN1 resulted in slender and curled leaves, fewer trichomes, and rising expressions of cuticle biosynthesis genes in Arabidopsis thaliana. Taken together, based on phenotype observations, content measurements and gene expression detections, AaWIN1 was considered as a positive regulator for GST initiation in A. annua.

AaMYB108 is the core factor integrating light and jasmonic acid signaling to regulate artemisinin biosynthesis in Artemisia annua.

Artemisinin, a sesquiterpene compound synthesized and stored in the glandular trichome of Artemisia annua leaves, has been used to treat malaria. Previous studies have shown that both light and jasmonic acid (JA) can promote the biosynthesis of artemisinin, and the promotion of artemisinin by JA is dependent on light. However, the specific molecular mechanism remains unclear. Here, we report a MYB transcription factor, AaMYB108, identified from transcriptome analysis of light and JA treatment, as a positive regulator of artemisinin biosynthesis in A. annua. AaMYB108 promotes artemisinin biosynthesis by interacting with a previously characterized positive regulator of artemisinin, AaGSW1. Then, we found that AaMYB108 interacted with AaCOP1 and AaJAZ8, respectively. The function of AaMYB108 was influenced by AaCOP1 and AaJAZ8. Through the treatment of AaMYB108 transgenic plants with light and JA, it was found that the promotion of artemisinin by light and JA depends on the presence of AaMYB108. Taken together, our results reveal the molecular mechanism of JA regulating artemisinin biosynthesis depending on light in A. annua. This study provides new insights into the integration of light and phytohormone signaling to regulate terpene biosynthesis in plants.

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.

AaSPL9 affects glandular trichomes initiation by positively regulating expression of AaHD1 in Artemisia annua L.

Glandular trichomes can secrete and store a large number of secondary metabolites in plants, some of which are of high medicinal and commercial value. For example, artemisinin, isolated from Artemisia annua L. plants, and its derivatives have great high medicinal value. Previous research indicated that artemisinin was synthesized in the glandular trichomes on the leaves of A. annua. It is an important study direction to improve artemisinin yield by promoting the initiation and development of glandular trichome. In this study, SQUAMOSA promoter-binding protein-like 9 (AaSPL9) was identified. In AaSPL9 overexpression transgenic plants, the glandular trichomes density was increased by 45-60 %, and the content of artemisinin was increased by 33-60 %, indicating that AaSPL9 positively regulate the glandular trichomes initiation. Yeast one-hybrid(Y1H), Dual-luciferase (Dual-Luc), Electrophoretic Mobility Shift Assay (EMSA) demonstrated that AaSPL9 activated the expression of AaHD1 by combining directly the GTAC-box of the AaHD1 promoter. Taken together, we identified AaSPL9, a positive transcription factor, regulating the glandular trichome initiation in A. annua, and revealed a novel molecular mechanism by which a SPL protein to promote glandular trichome initiation.