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We investigated the effects of graphene on the model herb Artemisia annua, which is renowned for producing artemisinin, a widely used pharmacological compound. Seedling growth and biomass were promoted when A. annua was cultivated with low concentrations of graphene, an effect which was attributed to a 1.4-fold increase in nitrogen uptake, a 15%-22% increase in chlorophyll fluorescence, and greater abundance of carbon cycling-related bacteria. Exposure to 10 or 20 mg/L graphene resulted in a ∼60% increase in H 2 O 2 , and graphene could act as a catalyst accelerator, leading to a 9-fold increase in catalase (CAT) activity in vitro and thereby maintaining reactive oxygen species (ROS) homeostasis. Importantly, graphene exposure led to an 80% increase in the density of glandular secreting trichomes (GSTs), in which artemisinin is biosynthesized and stored. This contributed to a 5% increase in artemisinin content in mature leaves. Interestingly, expression of miR828 was reduced by both graphene and H 2 O 2 treatments, resulting in induction of its target gene AaMYB17, a positive regulator of GST initiation. Subsequent molecular and genetic assays showed that graphene-induced H 2 O 2 inhibits micro-RNA (miRNA) biogenesis through Dicers and regulates the miR828-AaMYB17 module, thus affecting GST density. Our results suggest that graphene may contribute to yield improvement in A. annua via dynamic physiological processes together with miRNA regulation, and it may thus represent a new cultivation strategy for increasing yield capacity through nanobiotechnology., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Numerous green-synthesized nanoparticles are being evaluated for their potential application in soil, yet understanding their influence on plant performance and their rhizocompartments associated microbes is a gap that needs to be addressed. In this study, we investigated how applying iron oxide nanoparticles (control 0, low 25 and high 50 mg/kg) impacted the rhizocompartments associated microbial communities and plant secondary metabolites grown in natural soil microcosm. The transcriptional analysis showed that the majority of artemisinin and flavonoid biosynthetic pathway genes were significantly upregulated in plants with the application of low concentration of green-synthesized iron oxide nanoparticles (FeO.NPs) when compared with control plants, suggesting a fine collaboration between these metabolites' pathways, as indicated by a remarkable increase in artemisinin content (2-fold), ferulic acid (7.6-fold), luteolin (4.3-fold) and syringic acid (6.2-fold). The response of rhizocompartments microbial communities and associated soil enzymes was investigated using high-throughput sequencing and profiler test kits, respectively. The soil microbial community structure was altered upon FeO.NPs exposure in both low and high treated soils as indicated by multivariate analysis. For instance, bacterial phyla Bacteroidetes and Gemmatimonadetes and fungal phyla Ascomycota demonstrated a significant rise in relative abundance in both the rhizosphere and rhizoplane in low and high treatments relative to the control. Moreover, genera level analysis revealed that Cupriavidus , Vibrionimonas , and Burkholderia displayed higher relative abundance (p < 0.05) in low FeO.NPs concentration while Giaella and Nonomuraea didn't show any noticeable response to FeO.NPs. In terms of fungal genera, Oidiodendron and Cryptococcus showed high relative abundance under low treatment. The altered microbiome profile is possibly associated with soil enzymes, for instance, urease and catalase activities were increased by 1.76 and 1.71-fold, respectively in low FeO.NPs. Thus, this study demonstrates the effects of FeO.NPs gradients on plant secondary metabolites and soil enzyme activities, while contributing new insights into their specific impacts on the microbial community composition in the rhizocompartments of plant root system. [Display omitted] • Biogenic FeO.NPs were synthesized from a medicinal plant Catharanthus roseus. • FeO.NPs were characterized by standard material characterization techniques. • FeO.NPs enhanced secondary metabolites, particularly under low concentration. • FeO.NPs altered rhizocompartments' microbial community structure. [ABSTRACT FROM AUTHOR]

Artemisinin is a sesquiterpene lactone synthesized by the Chinese medicinal plant Artemisia annua L., and is the principal component used for the treatment of malaria. Recent studies have demonstrated the potential role of B-box (BBX) transcription factors in regulating plant secondary metabolism. However, the roles and functions of BBX members in the biosynthesis of artemisinin in A. annua have not been reported yet. In this study, a total of 27 BBX members (AaBBXs) were identified and classified into five sub-groups (I-V). The AaBBXs in each sub-group shared similar gene structures, conserved domains and motifs. Additionally, multiple cis -acting elements which are responsible for light, hormones, and stress signals were found in the promoter regions of AaBBX members. Based on transcriptome data, 27 AaBBXs showed constitutive expression in different organs/tissues, while several BBX genes exhibited higher expression levels in glandular secretory trichomes (GSTs), the site of artemisinin biosynthesis. To explore the roles of AaBBX members in regulating the artemisinin biosynthesis, 7 AaBBX s (AaBBX5/6/8/15/22/23/24) were selected for further investigation. The expressions of all 7 selected AaBBXs were strongly induced by MeJA and ABA, and they were all found to be nuclear localized. Additionally, all seven proteins were found to have the ability to physically interact with AaJAZ8 in vitro. Dual-luciferase assays revealed that AaBBX5/6/8/15/22/23 can activate the promoter activity of the artemisinin biosynthetic pathway genes such as AaADS , AaCYP71AV1 , AaDBR2 and AaALDH1 at different degrees. AaBBX22 showed highest expression level in A. annua GSTs, and can significantly activate the promoters of all four artemisinin biosynthetic pathway genes. Furthermore, transgenic assay results indicated that AaBBX22 acted as a positive regulator for the biosynthesis of artemisinin in A. annua. Taken together, this study provided a comprehensive genome-wide characterization of the BBX family and identified several significant BBX genes, including AaBBX22 , which paves the way for further investigation into the regulatory role of the BBX gene family in artemisinin biosynthesis. [Display omitted] • 27 AaBBX members in Artemisia annua L. were identified using genome-wide analysis. • AaBBX s highly expressing in glandular secretory trichomes were selected. • AaBBX5 / 6 / 8 / 15 / 22 / 23 / 24 were induced by jasmonic acid and abscisic acid treatment. • AaBBX5/6/8/15/22/23 activated the genes in artemisinin biosynthesis pathway. • AaBBX22 upregulated artemisinin biosynthesis in Artemisia annua L. [ABSTRACT FROM AUTHOR]