Methyl Jasmonate Elicitation Research Articles

Response of Bioactive Metabolite and Biosynthesis Related Genes to Methyl Jasmonate Elicitation in Codonopsis pilosula.

Bioactive metabolites in Codonopsis pilosula are of particular interest as an immunostimulant. Methyl jasmonate (MeJA) plays an important role in the elicitation of metabolite biosynthesis. Here, we explored the response of metabolites to MeJA elicitation in C. pilosula adventitious roots and multiple shoots. The results showed that the biomass, polysaccharide, and lobetyolin content of adventitious roots exhibited the highest increases with 100 µmol·L−1 MeJA at the 16th day of subculture, whereas the atractylenolide III (a terpenoid) content increased extremely with 50 µmol·L−1 MeJA treatment at the 7th day of subculture. In addition, the biomass and lobetyolin content significantly increased at the 4th day after treatment. Similarly, the polysaccharide and lobetyolin content increased in multiple shoots. Further identification of different metabolites responding to MeJA by 1H-NMR showed an extremely significant increase of the lobetyolinin level, which coincided with lobetyolin. Accordingly, the precursor, fatty acids, showed a highly significant decrease in their levels. Furthermore, a significant increase in β-d-fructose-butanol glycoside was detected, which was accompanied by a decrease in the sucrose level. Accordingly, the enzyme genes responsible for terpenoid and carbohydrate biosynthesis, CpUGPase, and CpPMK, were up regulated. In conclusion, MeJA promoted culture growth and accelerated bioactive metabolite accumulation by regulating the expression of the metabolite biosynthesis related genes, CpUGPase and CpPMK in C. pilosula.

Identification and expression profiling of rosmarinic acid biosynthetic genes from Satureja khuzistanica under carbon nanotubes and methyl jasmonate elicitation

Satureja khuzistanica is a medicinal herb endemic to Iran which can serve as a source of rosmarinic acid (RA). In the present study, the effect of different concentrations of methyl jasmonate (MeJA) and multi-walled carbon nanotubes (MWCNTs) on the rosmarinic acid accumulation and the expression of genes involved in its biosynthetic pathway was evaluated in nodal segment cultures of S. khuzistanica. The concentration of RA varied in plant extracts derived from the field, plants obtained under in vitro solid media, calli, suspension cultures and nodal segment cultures in liquid medium, with the highest amount recorded in nodal segment cultures. Phenylalanine ammonia-lyase (PAL), tyrosine aminotransferase (TAT), 4-hydroxyphenylpyruvate reductase (HPPR) and RA synthase (RAS) cDNA clones as key biosynthetic genes of RA production were identified and expression patterns were assayed in response to MeJA and MWCNTs, exogenously applied at a range of 0, 50, 100, 250 mg L−1. The expression levels of HPPR, PAL and TAT were up-regulated at 100 mg L−1 MWCNTs, whereas down-regulated levels were observed at 250 mg L−1. RAS was up-regulated in all MWCNTs treatments. HPPR, PAL and TAT expression increased sharply at 250 mg L−1 MeJA. The highest levels of RAS transcripts were observed at 100 mg L−1 MeJA. The highest HPPR, PAL and TAT expression levels were observed at 250 mg L−1 MeJA. In accordance, HPLC analysis showed a high amount of RA under MeJA and MWCNT elicitors. Our results provide helpful information on the expression profiles of biosynthetic genes in connection with RA accumulation.

Use of a temporary immersion bioreactor system for the sustainable production of thapsigargin in shoot cultures of Thapsia garganica

BackgroundThapsigargin and nortrilobolide are sesquiterpene lactones found in the Mediterranean plant Thapsia garganica L. Thapsigargin is a potent inhibitor of the sarco/endoplasmic reticulum calcium ATPase pump, inducing apoptosis in mammalian cells. This mechanism has been used to develop a thapsigargin-based cancer drug first by GenSpera and later Inspyr Therapeutics (Westlake Village, California). However, a stable production of thapsigargin is not established.ResultsIn vitro regeneration from leaf explants, shoot multiplication and rooting of T. garganica was obtained along with the production of thapsigargins in temporary immersion bioreactors (TIBs). Thapsigargin production was enhanced using reduced nutrient supply in combination with methyl jasmonate elicitation treatments. Shoots grown in vitro were able to produce 0.34% and 2.1% dry weight of thapsigargin and nortrilobolide, respectively, while leaves and stems of wild T. garganica plants contain only between 0.1 and 0.5% of thapsigargin and below detectable levels of nortrilobolide. In addition, a real-time reverse transcription PCR (qRT-PCR) study was performed to study the regulatory role of the biosynthetic genes HMG-CoA reductase (HMGR), farnesyl diphosphate synthase (FPPS), epikunzeaol synthase (TgTPS2) and the cytochrome P450 (TgCYP76AE2) of stem, leaf and callus tissues. Nadi staining showed that the thapsigargins are located in secretory ducts within these tissues.ConclusionsShoot regeneration, rooting and biomass growth from leaf explants of T. garganica were achieved, together with a high yield in vitro production of thapsigargin in TIBs.

Proteomics (SWATH-MS) informed by transcriptomics approach of tropical herb Persicaria minor leaves upon methyl jasmonate elicitation.

BackgroundJasmonic acid (JA) and its derivative, methyl JA (MeJA) are hormonal cues released by plants that signal defense response to curb damages from biotic and abiotic stresses. To study such response, a tropical herbal plant, Persicaria minor, which possesses pungent smell and various bioactivities including antimicrobial and anticancer, was treated with MeJA. Such elicitation has been performed in hairy root cultures and plants such as Arabidopsis and rice, yet how MeJA influenced the proteome of an herbal species like P. minor is unknown.MethodIn this study, P. minor plants were exogenously elicited with MeJA and leaf samples were subjected to SWATH-MS proteomics analysis. A previously published translated transcriptome database was used as a reference proteome database for a comprehensive protein sequence catalogue and to compare their differential expression.ResultsFrom this proteomics informed by transcriptomics approach, we have successfully profiled 751 proteins of which 40 proteins were significantly different between control and MeJA-treated samples. Furthermore, a correlation analysis between both proteome and the transcriptome data sets suggests that significantly upregulated proteins were positively correlated with their cognate transcripts (Pearson’s r = 0.677) while a weak correlation was observed for downregulated proteins (r = 0.147).DiscussionMeJA treatment induced the upregulation of proteins involved in various biochemical pathways including stress response mechanism, lipid metabolism, secondary metabolite production, DNA degradation and cell wall degradation. Conversely, proteins involved in energy expensive reactions such as photosynthesis, protein synthesis and structure were significantly downregulated upon MeJA elicitation. Overall protein-transcript correlation was also weak (r = 0.341) suggesting the existence of post-transcriptional regulation during such stress. In conclusion, proteomics analysis using SWATH-MS analysis supplemented by the transcriptome database allows comprehensive protein profiling of this non-model herbal species upon MeJA treatment.

Transcriptome Analysis of JA Signal Transduction, Transcription Factors, and Monoterpene Biosynthesis Pathway in Response to Methyl Jasmonate Elicitation in Mentha canadensis L.

Mentha canadensis L. has important economic value for its abundance in essential oils. Menthol is the main component of M. canadensis essential oils, which is certainly the best-known monoterpene for its simple structure and wide applications. However, the regulation of menthol biosynthesis remains elusive in M. canadensis. In this study, transcriptome sequencing of M. canadensis with MeJA treatment was applied to illustrate the transcriptional regulation of plant secondary metabolites, especially menthol biosynthesis. Six sequencing libraries were constructed including three replicates for both control check (CK) and methyl jasmonate (MeJA) treatment and at least 8 Gb clean bases was produced for each library. After assembly, a total of 81,843 unigenes were obtained with an average length of 724 bp. Functional annotation indicated that 64.55% of unigenes could be annotated in at least one database. Additionally, 4430 differentially expressed genes (DEGs) with 2383 up-regulated and 2047 down-regulated transcripts were identified under MeJA treatment. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment indicated that “Monoterpenoid biosynthesis” was one of the most significantly enriched pathways in metabolism. Subsequently, DEGs involved in JA signal transduction, transcription factors, and monoterpene biosynthesis were analyzed. 9 orthologous genes involved in menthol biosynthesis were also identified. This is the first report of a transcriptome study of M. canadensis and will facilitate the studies of monoterpene biosynthesis in the genus Mentha.

Influence of methyl jasmonate on ginsenoside biosynthesis in suspension cultures of Panax quinquefolium L.

<p>Panax quinquefolium L., belonging to the Araliaceae family, along with P. ginseng is one of the well-known species of ginseng. Multidirectional pharmacological action of this plant is attributed to triterpene saponins called ginsenosides. Pharmacopoeial raw material are roots obtained from the field crops which are time-consuming and require expensive agrotechnical procedures. Therefore, the new sources of ginseng biomass are sought such as in vitro suspension cultures. P. quinquefolium L. cell cultures, treated with the elicitation of methyl jasmonate (MJ) in concentration 50 and 250 μmol L-1, synthesize more ginsenosides than control cultures. The highest increase (2.2-fold) of all examined compounds was noted using 250 μmol L-1 MJ. In this condition, the predominantly quantitative metabolite was Rb1 ginsenoside belonging to protopanaxadiol derivatives.</p>

Methyl jasmonate and salicylic acid elicitation increase content and yield of chlorogenic acid and its derivatives in Gardenia jasminoides cell suspension cultures

Chlorogenic acid and its derivatives (CADs) are the bioactive components in Gardenia jasminoides Ellis (GJE) fruits but occur at a low level. In the current paper, four types of calluses were induced from GJE leaf explants, and light yellow-green calluses with a fine appearance and texture were selected for the development of a cell suspension culture to produce CADs. Additionally, cultured cells were elicited by salicylic acid (SA) and methyl jasmonate (MeJA) to increase the CADs yield. Seven CADs compounds were identified via HPLC-TOF-MS/MS, namely, 3-caffeoylquinic acid, 4-caffeoylquinic acid, 5-caffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, 3,5-dicaffeoyl-4-o-(3-hydroxyl-3-methyl)-glutaroylquinic acid, and malonyl-4,5-o-dicaffeoylquinic acid. This is the first identification of malonyl-4,5-o-dicaffeoylquinic acid from GJE. Both SA and MeJA significantly promoted the CADs yield, but MeJA was more effective than SA. Cells elicited by 200 µM MeJA showed the maximum total CADs content and yield, up to 20.98 ± 1.1 mg g−1 and 232.32 ± 9.1 mg L−1, which were 19.51 times and 14.14 times those of the control, respectively. Furthermore, the total CADs extracted from cells elicited by 200 µM MeJA displayed a much higher antioxidant capacity than that of natural GJE fruits. Our research suggested that a GJE cell culture combined with MeJA elicitation has a promising application in natural CADs production.

Effects of a combination of elicitation and precursor feeding on grape amino acid composition through foliar applications to Garnacha vineyard

Vine foliar applications of phenylalanine (Phe) or methyl jasmonate (MeJ) could improve the synthesis of secondary metabolites. However, there are no reports focusing on the effects of elicitation supported by precursor feeding on must amino acid composition in grapevines. The aim of this research was to study the effect of the elicitation of methyl jasmonate (MeJ) supported by phenylalanine (Phe) as a precursor feeding (MeJ+Phe) and its application individually on must amino acid composition. Results showed that foliar Phe and MeJ treatments decreased the concentration of most of the studied amino acids with respect to the control (p≤0.05). MeJ+Phe treatment did not affect must nitrogen content. Musts obtained from MeJ+Phe showed higher concentration of several amino acids than samples from Phe and MeJ applications. Therefore, other sources of precursor feeding could support elicitation, to improve amino acid composition and be considered as a tool for viticulture.

Effects of Methyl Jasmonate and Chitosanon Shoot and Callus Growth of Iranian Hypericum perforatum L. in vitro Cultures

The present study was investigated the effects of two elicitors methyl jasmonate (0, 125, 250 and 500 µM) and chitosan (0, 50, 150 and 250 mg L-1) on shoot and callus growth of Iranian Hypericum perforatumL. in vitro cultures in separate experiments. The experiment was done in the base of completely randomized design with five replications and five explants in each replication. Methyl jasmonate significantly reduced the regeneration of cultured shoots and developed shoots necrosis as well as the growth of treated calluswith methyl jasmonate was ceased. In spite of methyl jasmonate, chitosanwas more effective in stimulating the growth of cultured explants. Chitosan significantly increase the regeneration of H. perforatum shoots and the highest regenerated shoots number (9.29 shoots per explant) and shoot weight (1.82 g) were obtained in 250 mg L-1chitosan. The growth of callus in response to chitosan was higher than control treatment and the treated calli with 250 mg L-1 chitosan showed the highest diameter and weight. The essential oil content and chemical compositions of in vitro proliferated Iranian H. perforatum shoots were also investigated. The essential oil content was 0.02% (w w-1). The characterization of essential oil by GC and GC/MS analyses, allowed the identification of 35 volatile constituents, accounting for 97.01% of the total oil composition and sesquiterpenes (51.72%) represented by γ–gurjunene (22.34%) were the main compounds.

Metabolite Production and Antibacterial Activities of Callus Cultures from Rosa damascena Mill. Petals

Rosa damascena Mill. is a very fragrant member of Rosaceae family, exhibiting strong antioxidant and antimicrobial activity. In this study, in vitro cell cultures from Rosa damascena Mill. flower petals were established and the effects of precursor L-phenylalanine, the elicitor methyl jasmonate and light/dark treatments on the accumulation of characteristic rose volatile compounds, tocopherols and β-carotene in addition to the antibacterial activity of petal callus extracts were investigated with Solid-Phase Microextraction Combined with Gas Chromatography–Mass Spectrometry (GC–MS). The amount of β-pinene increased by 236 times when 500 μM Phe was added to the culture medium or under light treatment. Strong antibacterial activity from petal callus extracts, up to 2.6- fold compared to amikacin as well as original petal tissues, was obtained under dark treatment for P. aeruginosa ATCC 27853 and S. aureus ATCC 25923. These results revealed that petal callus extracts are sources of natural antioxidants and antibacterial compounds.

Enhancement of anthraquinone production and release by combination of culture medium selection and methyl jasmonate elicitation in hairy root cultures of Rubia tinctorum

Anthraquinones (AQs) are secondary metabolites widely distributed in nature. Interesting applications of plant extracts containing AQs include the treatment of Hepatitis C and cancer. Plant in vitro culture is an alternative for producing plant-derived pharmaceuticals in controlled conditions and with low environmental impact. Moreover, it allows the application of different strategies for enhancing secondary metabolite production. Hairy root cultures, obtained after Agrobacterium rhizogenes infection, are able to produce high amounts of secondary metabolites at high growth rates. In this work, growth kinetics of hairy root cultures of Rubia tinctorum and AQ production were evaluated in two different culture media, Gamborg B5 with half of the saline strength (B51/2) and Lloyd & Mc Cown’s Woody Plant Medium (WPM). Although WPM allowed higher biomass production (58.6% higher) than B51/2, specific AQ production was higher in B51/2 (between 1.2 and 2.1 fold increases from day 21 to the end of the experiment). Moreover, AQ release to the culture medium was observed in B51/2 (∼10% of total AQs). The different performance of hairy roots in these culture media may be due to a limiting nutrient (other than carbon source) in B51/2. Elicitation in B51/2 with methyl jasmonate (100μM) resulted in a massive accumulation of intracellular (between 1.5 and 2.4-fold increases) and also extracellular AQs (up to 8.1 fold-increase compared with control at 4days post-elicitation), which could ease AQ purification. These results prove the usefulness of combining different approaches to enhance secondary metabolite accumulation in plant in vitro cultures, in order to develop an optimized productive process.

Capsicum spp in vitro liquid cell suspensions: A useful system for the production of capsaicinoids and polyphenols

Capsicum are among the most extensively cultivated and consumed plant species in the world, because of their unique pungency, aroma and colour. The typical burning sensation caused by chili peppers is due to the occurrence of a group of alkaloids named capsaicinoids. In the present study, the production of solid callus and cell suspensions from hypocotyl explants of three different chili pepper cultivars (Capsicum annuum L. cv. Mazzolino, Capsicum chinense Jacq. cv. Naga Morich and Pimenta de Neyde), was optimised. In addition, C. chinense cv. Naga Morich cell suspensions were supplemented with biotic elicitors (methyl-jasmonate and chitosan) and with precursors and intermediates of capsaicin biosynthesis (vanillin, phenylalanine and valine), and both cells and media were analysed for capsaicinoid, polyphenol, flavonoid contents and for antioxidant activity. This is the first report regarding capsaicinoid elicitation with pure chitosan and with a combination of precursors of both phenylpropanoid and valine pathways. Overall, the highest capsaicinoid levels were detected in cell extracts from cultures treated with 10 μM methyl-jasmonate and with a combination of phenylalanine and valine amino acids (100 μM each). The present results confirm the possibility of using hypocotyl chili pepper cell suspensions to produce high amounts of health beneficial metabolites.

How to induce defense responses in wild plant populations? Using bilberry (Vaccinium myrtillus) as example.

Inducible plant defense is a beneficial strategy for plants, which imply that plants should allocate resources from growth and reproduction to defense when herbivores attack. Plant ecologist has often studied defense responses in wild populations by biomass clipping experiments, whereas laboratory and greenhouse experiments in addition apply chemical elicitors to induce defense responses. To investigate whether field ecologists could benefit from methods used in laboratory and greenhouse studies, we established a randomized block‐design in a pine‐bilberry forest in Western Norway. We tested whether we could activate defense responses in bilberry (Vaccinium myrtillus) by nine different treatments using clipping (leaf tissue or branch removal) with or without chemical treatment by methyljasmonate (MeJA). We subsequently measured consequences of induced defenses through vegetative growth and insect herbivory during one growing season. Our results showed that only MeJA‐treated plants showed consistent defense responses through suppressed vegetative growth and reduced herbivory by leaf‐chewing insects, suggesting an allocation of resources from growth to defense. Leaf tissue removal reduced insect herbivory equal to the effect of the MeJa treatments, but had no negative impact on growth. Branch removal did not reduce insect herbivory or vegetative growth. MeJa treatment and clipping combined did not give an additional defense response. In this study, we investigated how to induce defense responses in wild plant populations under natural field conditions. Our results show that using the chemical elicitor MeJA, with or without biomass clipping, may be a better method to induce defense response in field experiments than clipping of leaves or branches that often has been used in ecological field studies.

Differential Gene Expression Analysis in Polygonum minus Leaf upon 24 h of Methyl Jasmonate Elicitation.

Polygonum minus is an herbal plant that grows in Southeast Asian countries and traditionally used as medicine. This plant produces diverse secondary metabolites such as phenolic compounds and their derivatives, which are known to have roles in plant abiotic and biotic stress responses. Methyl jasmonate (MeJA) is a plant signaling molecule that triggers transcriptional reprogramming in secondary metabolism and activation of defense responses against many biotic and abiotic stresses. However, the effect of MeJA elicitation on the genome-wide expression profile in the leaf tissue of P. minus has not been well-studied due to the limited genetic information. Hence, we performed Illumina paired-end RNA-seq for de novo reconstruction of P. minus leaf transcriptome to identify differentially expressed genes (DEGs) in response to MeJA elicitation. A total of 182,111 unique transcripts (UTs) were obtained by de novo assembly of 191.57 million paired-end clean reads using Trinity analysis pipeline. A total of 2374 UTs were identified to be significantly up-/down-regulated 24 h after MeJA treatment. These UTs comprising many genes related to plant secondary metabolite biosynthesis, defense and stress responses. To validate our sequencing results, we analyzed the expression of 21 selected DEGs by quantitative real-time PCR and found a good correlation between the two analyses. The single time-point analysis in this work not only provides a useful genomic resource for P. minus but also gives insights on molecular mechanisms of stress responses in P. minus.

Increasing the synthesis of bioactive abietane diterpenes in Salvia sclarea hairy roots by elicited transcriptional reprogramming.

Transcriptional activation of genes belonging to the plastidial MEP-derived isoprenoid pathway by elicitation with methyl jasmonate and coronatine enhanced the content of bioactive abietane diterpenes in Salvia sclarea hairy roots. We have shown that aethiopinone, an abietane diterpene synthesized in Salvia sclarea roots is cytotoxic and induces apoptosis in human melanoma cells. To develop a production platform for this compound and other abietane diterpenes, hairy root technology was combined with the elicitation of methyl jasmonate (MeJA) or the phytotoxin coronatine (Cor). Both MeJA and Cor induced a significant accumulation of aethiopinone, but prolonged exposure to MeJA irremediably caused inhibition of hairy root growth, which was unaffected by Cor treatment. Considering together the fold increase in aethiopinone content and the final hairy root biomass, the best combination was a Cor treatment for 28days, which allowed to obtain up to 105.34±2.30mg L-1 of this compound to be obtained, corresponding to a 24-fold increase above the basal content in untreated hairy roots. MeJA or Cor elicitation also enhanced the synthesis of other bioactive abietane-quinone diterpenes. The elicitor-dependent steering effect was due to a coordinated transcriptional activation of several biosynthetic genes belonging to the plastidial MEP-derived isoprenoid pathway. High correlations between aethiopinone content and MeJA or Cor-elicited level of gene transcripts were found for DXS2 (r 2=0.99), DXR (r 2=0.99), and GGPPS (r 2=0.98), encoding enzymes acting upstream of GGPP, the common precursor of diterpenes and other plastidial-derived terpenes, as well as CPPS (r 2=0.99), encoding the enzyme involved in the first cyclization steps leading to copalyl-diphosphate, the precursor of abietane-like diterpenes. These results point to these genes as possible targets of metabolic engineering approaches to establish a more efficient production platform for such promising anti-proliferative plant-derived compounds.

Transcriptional Responses and Gentiopicroside Biosynthesis in Methyl Jasmonate-Treated Gentiana macrophylla Seedlings.

Gentiana macrophylla, a medicinal plant with significant pharmacological properties, contains the bioactive compound gentiopicroside. Methyl jasmonate (MeJA) is an effective elicitor for enhancing the production of such compounds. However, little is known about MeJA-mediated biosynthesis of gentiopicroside. We investigated this phenomenon as well as gene expression profiles to determine the molecular mechanisms for MeJA-mediated gentiopicroside biosynthesis and regulation in G. macrophylla. Our HPLC results showed that Gentiana macrophylla seedlings exposed to MeJA had significantly higher concentrations of gentiopicroside when compared with control plants. We used RNA sequencing to compare transcriptional profiles in seedlings treated for 5 d with either 0 μmol L-1 MeJA (C) or 250 μmol L-1 MeJA (M5) and detected differentially expressed genes (DEGs). In total, 77,482 unique sequences were obtained from approximately 34 million reads. Of these, 48,466 (57.46%) sequences were annotated based on BLASTs performed against public databases. We identified 5,206 DEGs between the C and M5 samples, including genes related to the α-lenolenic acid degradation pathway, JA signaling pathway, and gentiopicroside biosynthesis. Expression of numerous enzyme genes in the glycolysis pathway was significantly up-regulated. Many genes encoding transcription factors (e.g. ERF, bHLH, MYB, and WRKY) also responded to MeJA elicitation. Rapid acceleration of the glycolysis pathway that supplies precursors for IPP biosynthesis and up-regulates the expression of enzyme genes in that IPP pathway are probably most responsible for MeJA stimulation of gentiopicroside synthesis. Our qRT-PCR results showed that the expression profiles of 12 gentiopicroside biosynthesis genes were consistent with the RNA-Seq data. These results increase our understanding about how the gentiopicroside biosynthesis pathway in G. macrophylla responds to MeJA.

Methyl jasmonate elicits the biotransformation of geraniol stored as its glucose conjugate into methyl geranate in Achyranthes bidentata plant

To investigate the biotransformation pathway of airborne geraniol by Achyranthes bidentata (A. bidentata), deuterium labeled geraniol was applied with or without methyl jasmonate (MeJA), and the biosynthesized metabolites were analyzed. In A. bidentata leaves, geraniol was conjugated with glucose. The conjugate was then metabolized to afford methyl geranate only under MeJA elicitation. MeJA elicits the biotransformation of geraniol into methyl geranate by inducing the conversion of the intermediate, glucose conjugate of geraniol.

Molecular characterization of an elicitor-responsive 3-hydroxy-3-methylglutaryl coenzyme A reductase gene involved in oleanolic acid production in cell cultures of Achyranthes bidentata

Achyranthes bidentata contains a rich source of important pharmaceutically active triterpene acids including oleanolic acid (OA) as a major one. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a key enzyme to provide mevalonate for biosynthesis of triterpene acids. In this study, in order to develop a sustainable source of OA, cell suspension cultures were established from shoot cultures of A. bidentata, and a full length cDNA encoding HMGR (designated as AbHMGR) was cloned and characterized. The cDNA contained 2078 nucleotides with a complete open reading frame of 1593 nucleotides, which was predicted to encode a peptide of 530 amino acids. Expression analysis by real-time PCR revealed that AbHMGR mRNA was abundant in A. bidentata roots, stems and leaves. When cultivated in Murashige and Skoog medium supplemented with 1.5 mg/L 1-naphthlcetic acid (NAA) and 1.5 mg/L 6-benzyladenine (6-BA), cells in suspension culture grew rapidly, yielding OA (100.9 mg/L) after 15 days. OA content in cell cultures was increased under the elicitation of methyl jasmonate (MeJA), yeast elicitor (YE) or cadmium chloride (CdCl2). The ultrahigh production of OA was achieved to 371.8 mg/L, a 5.4-fold of the control after 2-day treatment of 0.2 mM MeJA in the cell cultures. Quantitative real-time PCR analysis showed that AbHMGR was expressed at a higher level under the elicitation of MeJA or YE. Our results suggested that OA production may be the result of the up-regulated expression of AbHMGR under the treatment of various elicitors.

Metabolomics driven analysis of Erythrina lysistemon cell suspension culture in response to methyl jasmonate elicitation

An MS-based metabolomic approach was used to profile the secondary metabolite of the ornamental plant Erythrina lysistemon via ultra-performance liquid chromatography coupled to photodiode array detection and high resolution q-TOF mass spectrometry (UPLC-PDA-MS). Cultures maintained the capacity to produce E. lysistemon flavonoid subclasses with pterocarpans amounting for the most abundant ones suggesting that it could provide a resource of such flavonoid subclass. In contrast, alkaloids, major constituents of Erythrina genus, were detected at trace levels in suspension cultures. Methyl jasmonate (MeJA), phytohormone, was further supplied to culture with the aim of increasing secondary metabolites production and with metabolite profiles subjected to multivariate data analysis to evaluate its effect. Results revealed that triterpene i.e. oleanolic acid and fatty acid i.e. hydroxy-octadecadienoic acid were elicited in response to methyl jasmonate, whereas pterocarpans i.e., isoneorautenol showed a decline in response to elicitation suggesting for the induction of terpenoid biosynthetic pathway and concurrent with a down regulation of pterocarpans. In conclusion, a total of 53 secondary metabolites including 3 flavones, 12 isoflavones, 4 isoflavanones, 4 alkaloids, 11 pterocarpans, and 5 phenolic acids were identified.

Methyl jasmonate enhanced production of rosmarinic acid in cell cultures of Satureja khuzistanica in a bioreactor

The growing interest in rosmarinic acid (RA), an ester of caffeic acid and 3,4‐dihydroxyphenyl lactic acid, is due to its biological activities, which include cognitive‐enhancing effects, slowing the development of Alzheimer's disease, cancer chemoprotection, and anti‐inflammatory activity. Inspired by the challenge of meeting the growing demand for this plant secondary metabolite, we developed a biotechnological platform based on cell suspension cultures of Satureja khuzistanica. The high amounts of RA produced by this system accumulated mainly inside the cells. To further improve production, two elicitors, 100 μM methyl jasmonate (MeJA) and 40 mM cyclodextrin (CD), were tested, separately and together. MeJA increased RA productivity more than 3‐fold, the elicited cultures achieving an RA production of 3.9 g L−1 without affecting biomass productivity. CD did not have a clear effect on RA production, and under the combined treatment of MeJA + CD only a small amount of RA was released to the medium. When the cell culture was transferred from a shake flask to a wave‐mixed bioreactor, a maximum RA production of 3.1 g L−1 and biomass productivity of 18.7 g L−1 d−1 was achieved under MeJA elicitation, demonstrating the suitability of S. khuzistanica cell suspensions for the biotechnological production of this bioactive plant secondary metabolite.