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The peanut plant is one of the most economically important crops around the world. Abiotic stress, such as drought, causes over five hundred million dollars in losses in peanut production per year. Peanuts are known to produce prenylated stilbenoids to counteract biotic stress. However, their role in abiotic stress tolerance has not been elucidated. To address this issue, hairy roots with the capacity to produce prenylated stilbenoids were established. An RNA-interference (RNAi) molecular construct targeting the stilbenoid-specific prenyltransferase AhR4DT-1 was designed and expressed via Agrobacterium rhizogenes-mediated transformation in hairy roots of peanut cultivar Georgia Green. Two transgenic hairy roots with the RNAi molecular construct were established, and the downregulation of AhR4DT-1 was validated using reverse transcriptase quantitative PCR. To determine the efficacy of the RNAi-approach in modifying the levels of prenylated stilbenoids, the hairy roots were co-treated with methyl jasmonate, hydrogen peroxide, cyclodextrin, and magnesium chloride to induce the production of stilbenoids and then the stilbenoids were analyzed in extracts of the culture medium. Highly reduced levels of prenylated stilbenoids were observed in the RNAi hairy roots. Furthermore, the hairy roots were evaluated in a polyethylene glycol (PEG) assay to assess the role of prenylated stilbenoids on water-deficit stress. Upon PEG treatment, stilbenoids were induced and secreted into the culture medium of RNAi and wild-type hairy roots. Additionally, the biomass of the RNAi hairy roots decreased by a higher amount as compared to the wild-type hairy roots suggesting that prenylated stilbenoids might play a role against water-deficit stress., 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 © 2024 Elsevier Masson SAS. All rights reserved.)

Prenylated stilbenoids are phenolic compounds produced in a small number of plants such as peanut ( Arachis hypogaea ) to counteract biotic and abiotic stresses. In addition to their role in plant defense, they exhibit biological activities with potential application in human health. Whereas non-prenylated stilbenoids such as resveratrol are commercially available, the availability of prenylated stilbenoids is limited. To this end, hairy root cultures of peanut were developed as an elicitor-controlled bioproduction platform for prenylated stilbenoids. An orthogonal array design approach led to the elucidation of an optimized elicitation procedure consisting of co-treatment of the hairy root cultures with 18 g/L methyl-β-cyclodextrin, 125 µM methyl jasmonate, 3 mM hydrogen peroxide (H 2 O 2 ) and medium supplementation with additional 1 mM magnesium chloride. After 168-h of elicitor treatment, the combined yield of the prenylated stilbenoids arachidin-1, arachidin-2, arachidin-3 and arachidin-5 reached approximately 750 mg/L (equivalent to 107 mg/g DW). Moreover, hairy root cultures from the wild Arachis species A. duranensis and A. ipaensis were developed and shown to produce prenylated stilbenoids upon elicitor treatment. These wild Arachis hairy root lines may provide a platform to elucidate the biosynthetic origin of prenylated stilbenoids in peanut., Competing Interests: The authors declare no conflict of interest.

Peanut (Arachis hypogaea) produces stilbenoids upon exposure to abiotic and biotic stresses. Among these compounds, the prenylated stilbenoids arachidin-1 and arachidin-3 have shown diverse biological activities with potential applications in human health. These compounds exhibit higher or novel biological activities in vitro when compared to their nonprenylated analogues piceatannol and resveratrol, respectively. However, assessment of these bioactivities in vivo has been challenging because of their limited availability. In this study, hairy root cultures of peanut were induced to produce stilbenoids upon treatment with elicitors. Co-treatment with 100 μM methyl jasmonate (MeJA) and 9 g/L methyl-β-cyclodextrin (CD) led to sustained high levels of resveratrol, piceatannol, arachidin-1, and arachidin-3 in the culture medium when compared to other elicitor treatments. The average yields of arachidin-1 and arachidin-3 were 56 and 148 mg/L, respectively, after co-treatment with MeJA and CD. Furthermore, MeJA and CD had a synergistic effect on resveratrol synthase gene expression, which could explain the higher yield of resveratrol when compared to treatment with either MeJA or CD alone. Peanut hairy root cultures were shown to be a controlled and sustainable axenic system for the production of the diverse types of biologically active stilbenoids.

Over 216 million malaria cases are reported annually worldwide and about a third of these cases, primarily children under the age of five years old, will not survive the infection. Despite this significant world health impact, only a limited number of therapeutic agents are currently available. The lack of scaffold diversity poses a threat in the event that multi-drug-resistant strains emerge. Terrestrial natural products have provided a major source of chemical diversity for starting materials in many FDA approved drugs over the past century. Bixa orellana L. is a popular plant used in South America for the treatment of malaria. In search of new potential therapeutic agents, the chemical constituents of a selected hairy root culture line of Bixa orellana L. were characterized utilizing NMR and mass spectrometry methods, followed by its biological evaluation against malaria strains 3D7 and K1. The crude extract and its isolated compounds demonstrated EC50 values in the micromolar range. Herein, we report our findings on the chemical constituents of Bixa orellana L. from hairy roots responsible for the observed antimalarial activity.

Stilbenoids are polyphenolic phytoalexins that exhibit potential health applications in humans. Hairy root cultures of muscadine grape (Vitis rotundifolia Michx.) were used to study the biochemical and molecular regulation of stilbenoid biosynthesis upon treatment with 100 μM methyl jasmonate (MeJA) or 10 mM hydrogen peroxide (H2O2) over a 96-h period. Resveratrol, piceid, and ε-viniferin were identified in higher concentrations in the tissue whereas resveratrol was the most abundant stilbenoid in the medium under either treatment. An earlier increase in resveratrol accumulation was observed for the MeJA-treated group showing a maximum at 12 h in the tissue and 18 h in the medium. Furthermore, the antioxidant capacity of extracts from the tissue and medium was determined by the 2,2'-azinobis[3-ethylbenzthiazoline sulfonic acid] (ABTS) and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays showing correlation with the stilbenoid content. Fourteen candidate reference genes for qPCR were tested under the described experimental conditions and resulted in the selection of 5 reference genes. Quantitative analyses of transcripts for phenylalanine ammonia-lyase (PAL), resveratrol synthase (RS), and two stilbene synthases (STS and STS2) showed the highest RNA level induction at 3 h for both treatments with a higher induction for the MeJA treatment. In contrast, the flavonoid-related chalcone synthase (CHS) transcripts showed induction and a decrease in expression for MeJA and H2O2 treatments, respectively. The observed responses could be related to an oxidative burst triggered by the exposure to abiotic stressor compounds with signaling function such as MeJA and H2O2 which have been previously related to the synthesis of secondary metabolites., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Stilbenoids are polyphenolic phytoalexins with health-related properties in humans. Muscadine grape ( Vitis rotundifolia ) hairy root cultures were established via Agrobacterium rhizogenes -mediated transformation, and the effects of growth regulators (3-indolebutyric acid and 6-benzylaminopurine) and methyl jasmonate (MeJA) on stilbenoid production were studied. Twenty-one-day-old hairy root cultures were treated with 100 μM MeJA for 24 h, and then the stilbenoids were extracted from the medium and tissue with ethyl acetate and analyzed by HPLC. Resveratrol, piceid, and ε-viniferin were observed preferentially in tissue, whereas piceatannol was observed only in medium. Growth regulators did not affect the yield of stilbenoids, whereas higher levels were found upon treatment with MeJA. Stilbenoids identified in the hairy root cultures were analyzed for their radical scavenging capacity showing piceatannol and ε-viniferin as the strongest antioxidants. Muscadine grape hairy root cultures were demonstrated to be amenable systems to study stilbenoid biosynthesis and a sustainable source of these bioactive compounds.

In order to identify a promoter system for high-level expression of transgenes in hairy roots, we characterized the chimeric super-promoter fused to the translational enhancer from tobacco etch virus (TEV). Transgenic tobacco plants and hairy roots were generated with the super-promoter:TEV sequence and a modified green fluorescence protein (mGFP5) as a reporter gene. To exploit the utility of hairy root cultures as a secretion-based expression system, the signal peptide of patatin was fused to mGFP5 to direct its secretion into the culture medium. Levels of mGFP5 RNA were on average sixfold higher in hairy roots than leaves. Likewise, GFP protein levels per gram of fresh weight were at least tenfold higher in hairy roots than leaves. Furthermore, more than 10% of the recombinant protein produced in the hairy root culture system was found in the medium. Immunoblotting with anti-GFP antibodies showed two products of 27.1 and 29.9 kDa in all leaf and hairy root tissue extracts, whereas a single 27.1-kDa product was detected in the medium. Inducibility of the promoter was studied with mature leaves and 14-day (midlog phase) hairy roots. A twofold increase in mRNA levels was found immediately after wounding in both mature leaves and hairy roots, with a corresponding increase in mGFP5 protein after 24 h. Our studies demonstrate the utility of the super-promoter:TEV system for high-level expression of recombinant proteins in hairy root bioreactors.

Antioxidant stilbenoids, such as resveratrol, arachidin-1, and arachidin-3, have demonstrated beneficial effects on human health. Although resveratrol is commercially available, arachidin-1 and arachidin-3 are not, resulting in an opportunity to explore purification methods and to confirm biological activity. Recently, Arachis hypogaea hairy root cultures (produced via Agrobacterium rhizogenes-mediated transformation) were reported to secrete stilbenoids into liquid growth media upon elicitation in quantities sufficient for commercial production. The purpose of this study was to purify substantial quantities of resveratrol, arachidin-1, and arachidin-3 from A. hypogaea hairy root cultures using centrifugal partition chromatography (CPC), determine the antioxidant activity of these compounds using the thiobarbituric acid reactive substances (TBARS) assay, and determine the cytotoxicity of the compounds using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In a single run of CPC, resveratrol, arachidin-1, and arachidin-3 were separated to a purity of 97.1%, 97.0%, and 91.8%, respectively. Lipid oxidation was inhibited by a 27 and 7 μM dose for reference standards of resveratrol and arachidin-1, respectively, while oxidation was not inhibited up to a 27 μM dose for reference standard of arachidin-3. Oxidation was inhibited at a 14, 7, and 14 μM doses for CPC-purified resveratrol, arachidin-1, and arachidin-3, respectively. Arachidin-1 and arachidin-3 demonstrated cytotoxicity at 27 and 55 μM in RAW 264.7 and HeLa cell lines, respectively; while resveratrol exhibited no cytotoxicity to either cell line. These results demonstrate the integration of a production and purification system for the manufacturing of A. hypogaea-derived stilbenoids., (© 2010 American Institute of Chemical Engineers)

Previously, we have shown that hairy root cultures of peanut provide a controlled, sustainable and scalable production system that can be induced to produce stilbenoids. However to leverage peanut hairy roots to study the biosynthesis of this polyphenolic biosynthetic pathway, growing conditions and elicitation kinetics of these tissue cultures must be defined and understood. To this end, a new peanut cv. Hull hairy root (line 3) that produces resveratrol and its prenylated analogues arachidin-1 and arachidin-3 upon sodium acetate-mediated elicitation was established. Two culture media were compared for impact on root growth and stilbenoid biosynthesis/secretion. The levels of ammonium, nitrate, phosphate and residual sugars were monitored along growth and elicitation period. A modified MS (MSV) medium resulted in higher root biomass when compared to B5 medium. The stilbenoid profile after elicitation varied depending on the age of the culture (6, 9, 12, and 15-day old). After elicitation at day 9 (exponential growth in MSV medium), over 90% of the total resveratrol, arachidin-1 and arachidin-3 accumulated in the medium. Our studies demonstrate the benefits of the hairy root culture system to study the biosynthesis of stilbenoids including valuable prenylated polyphenolic compounds., (2010 Elsevier Masson SAS. All rights reserved.)

Resveratrol and its derivatives are natural stilbenes associated with many health benefits that include those conferred by their antioxidant and anticancer properties. While stilbenes can be recovered as an extract from a selected number of plants, these products are not suitable for many applications in the food/pharmaceutical sectors due to high levels of impurities as well as the overall low concentration of resveratrol and its derivatives in the extract. To deliver a highly defined and enriched resveratrol product, hairy root cultures of peanut (Arachis hypogaea) were established and tested as a bioproduction system for resveratrol and associated derivatives. Analyses by HPTLC and GC-MS of ethyl acetate extracts showed that a single 24 h sodium acetate elicitation resulted in a 60-fold induction and secretion of trans-resveratrol into the medium of peanut hairy root cultures. trans-Resveratrol accumulated to levels of 98 microg/mg of the dried extract from the medium representing 99% of the total resveratrol produced. Other stilbenes, including trans-pterostilbene, were also detected in the medium. Our results demonstrate the capacity of hairy root cultures as an effective bioprocessing system for valued nutraceuticals like resveratrol and resveratrol derivatives. In being able to effectively induce and recover high levels of resveratrol and associated derivatives from the media fraction, hairy roots may offer a scalable and continuous product recovery platform for naturally-derived, high quality, enriched nutraceuticals.

Ricin B (RTB), the non-toxic lectin subunit of ricin, is a promising mucosal adjuvant and carrier for use in humans. RTB fusion proteins have been expressed in tobacco hairy root cultures, but the secreted RTB component of these proteins was vulnerable to protease degradation in the medium. Moreover, castor bean purified RTB spiked into tobacco hairy root culture media showed significant degradation after 24 h and complete loss of product after 72 h. Aqueous two-phase extraction (ATPE) was tested for fast recovery of RTB not only to partially purify the protein but also to improve its stability. Two different polyethylene glycol (PEG)/salt/water systems including PEG/potassium phosphate and PEG/sodium sulfate, were studied. RTB was shown to be favorably recovered in PEG/sodium sulfate systems. Statistical analysis indicated that the ionic strength of the system and the sodium sulfate concentration were important in optimizing the partition coefficient of RTB. A selectivity of almost three could be achieved for RTB in optimized systems, and RTB partitioned in the PEG-rich phase exhibited extended stability. Therefore, ATPE was shown to be effective in initial recovery/purification and stabilization of RTB and may hold promise for other unstable secreted proteins from hairy root culture.

Camptothecin (CPT) is an anticancer and antiviral alkaloid produced by the Chinese tree Camptotheca acuminata (Nyssaceae) and some other species belonging to the families Apocynaceae, Olacaceae, and Rubiaceae. Bark and seeds are currently used as sources for the drug. Several attempts have been made to produce CPT from cell suspensions; however, the low yields obtained limit this approach. Cultures of differentiated cell types may be an alternative source of alkaloid production. Hairy root cultures of C. acuminata were established from tissue transformed with Agrobacterium rhizogenes strains ATCC 15834 and R-1000. Integration of the genes responsible for the hairy-root phenotype ( rol genes) into the plant genome was verified by DNA gel blot analysis. The hairy roots produce and secrete CPT as well as the more potent and less toxic natural derivative, 10-hydroxycamptothecin (HCPT), into the medium. Remarkably, the cultures were able to synthesize the alkaloids at levels equal to, and sometimes greater than, the roots in planta, i.e., 1.0 and 0.15 mg/g dry weight for CPT and the HCPT, respectively.