Alkaloid Production Research Articles

Metabolic responses of Euglena gracilis under photoheterotrophic and heterotrophic conditions

The protist Euglena gracilis has various trophic modes including heterotrophy and photoheterotrophy. To investigate how cultivation mode influences metabolic regulation, the chemical composition of cellular metabolites of Euglena gracilis grown under heterotrophic and photoheterotrophic conditions was monitored from the early exponential phase to the mid-stationary phase using two different techniques, i.e, nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HRMS). The combined metabolomics approach allowed an in-depth understanding of the mechanism of photoheterotrophic and heterotrophic growth for biomolecule production. Heterotrophic conditions promoted the production of polar amino and oxygenated compounds such as proteins and polyphenol compounds, especially at the end of the exponential phase while photoheterotrophic cells enhanced the production of organoheterocyclic compounds, carbohydrates, and alkaloids.

Eye Diseases: When the Solution Comes from Plant Alkaloids.

Plants are an incredible source of metabolites showing a wide range of biological activities. Among these, there are the alkaloids, which have been exploited for medical purposes since ancient times. Nowadays, many plant-derived alkaloids are the main components of drugs used as therapy for different human diseases. This review deals with providing an overview of the alkaloids used to treat eye diseases, describing the historical outline, the plants from which they are extracted, and the clinical and molecular data supporting their therapeutic activity. Among the different alkaloids that have found application in medicine so far, atropine and pilocarpine are the most characterized ones. Conversely, caffeine and berberine have been proposed for the treatment of different eye disorders, but further studies are still necessary to fully understand their clinical value. Lastly, the alkaloid used for managing hypertension, reserpine, has been recently identified as a potential drug for ameliorating retinal disorders. Other important aspects discussed in this review are different solutions for alkaloid production. Given that the industrial production of many of the plant-derived alkaloids still relies on extraction from plants, and the chemical synthesis can be highly expensive and poorly efficient, alternative methods need to be found. Biotechnologies offer a multitude of possibilities to overcome these issues, spanning from genetic engineering to synthetic biology for microorganisms and bioreactors for plant cell cultures. However, further efforts are needed to completely satisfy the pharmaceutical demand.

Induction of Monoterpenoid Oxindole Alkaloids Production and Related Biosynthetic Gene Expression in Response to Signaling Molecules in Hamelia patens Plant Cultures.

Hamelia patens (Rubiaceae), known as firebush, is a source of bioactive monoterpenoid oxindole alkaloids (MOAs) derived from monoterpenoid indole alkaloids (MIAs). With the aim of understanding the regulation of the biosynthesis of these specialized metabolites, micropropagated plants were elicited with jasmonic acid (JA) and salicylic acid (SA). The MOA production and MIA biosynthetic-related gene expression were evaluated over time. The production of MOAs was increased compared to the control up to 2-fold (41.3 mg g DW-1) at 72 h in JA-elicited plants and 2.5-fold (42.4 mg g DW-1) at 120 h in plants elicited with SA. The increment concurs with the increase in the expression levels of the genes HpaLAMT, HpaTDC, HpaSTR, HpaNPF2.9, HpaTHAS1, and HpaTHAS2. Interestingly, it was found that HpaSGD was downregulated in both treatments after 24 h but in the SA treatment at 120 h only was upregulated to 8-fold compared to the control. In this work, we present the results of MOA production in H. patens and discuss how JA and SA might be regulating the central biosynthetic steps that involve HpaSGD and HpaTHAS genes.

Epichloë fungal endophyte strains and their Lolium hosts affect resistance to Listronotus bonariensis (Coleoptera: Curculionidae)

ABSTRACT Both adults and larvae of Listronotus bonariensis damage ryegrass (Lolium spp.), a dominant component of New Zealand pasture. Damage can be reduced by Epichloë festucae var. lolii, a biotrophic fungal symbiont of ryegrass, through the production of alkaloids with deterrent and/or toxic effects to insect herbivores including L. bonariensis. Here we report results from two pot trials and a field trial comparing endophyte strains with different alkaloid profiles that have been introduced to reduce or eliminate adverse effects on stock. Adult L. bonariensis feeding scars, number of eggs and the proportion of tillers with larval damage and the severity of that damage were recorded. Samples of pseudostem and leaf blades were analysed for alkaloids. AR1 endophyte reduced adult feeding, oviposition, and larval damage whereas AR37 had no effect on adults but was highly effective in reducing larval damage. Reduction in adult feeding and oviposition by different NEA strains was governed by concentrations of peramine and ergovaline. For plants infected with AR37, alkaloid concentrations that determined the extent of larval damage were influenced by host species and ploidy; tetraploid L. perenne was more severely damaged than its diploid equivalent; L. multiflorum cultivars were more susceptible than the L. hybridum and L. perenne cultivars.

The effects of green and chemically-synthesized copper oxide nanoparticles on the production and gene expression of morphinan alkaloids in Oriental poppy

Oriental poppy (Papaver orientale L.) belonging to the Papaveraceae family, has the capacity to synthesize a wide range of benzylisoquinoline alkaloids (BIAs). This experiment was conducted to investigate the effects of green and chemical copper oxide nanoparticles (CuO NPs) elicitors on oxidative stress and the BIAs biosynthesis pathway in the cell suspension culture of P. orientale. This research shows that both green and chemical CuO NPs at concentrations of 20 mg/L and 40 mg/L, induce oxidative stress in the cell suspension of P. orientale by increasing the production of H2O2 and the activity of antioxidant enzymes. The comparison of treatments revealed that utilizing a lower concentration of CuO NPs (20 mg/L) and extending the duration of cell suspension incubation (up to 48 h) play a more influential role in inducing the expression of the BIAs biosynthesis pathway genes (PsWRKY, TYDC, SalSyn, SalR, SalAT, T6ODM, COR and CODM) and increasing the production of morphinan alkaloids (thebaine, codeine, and morphine). The overarching results indicate that the concentration of CuO NPs and the duration of cell treatment have a more significant impact than the nature of CuO NPs in inducing oxidative stress and stimulating the expression of the BIAs pathway genes.

Development of Catharanthus callus cell lines for indole-derived alkaloids production

In this work, we evaluated the effect of 29 PGR combinations and photoperiods on callogenesis while analyzing the fate of calluses during a two-year period. Variation of PGR combinations in the culture medium strongly affected callogenesis in terms of growth and callus morphology. Although most PGR combinations resulted in callogenesis, only few ones supported cell survivor in long-term basis for the generation of cell lines as homogenous-looking calluses. Analysis by tandem liquid chromatography quadrupole time of flight mass spectrometry (LC-QTOF-MS/MS) revealed production of vincristine (9.25 mg/kg), vinblastine (0.17 mg/kg), and vindoline (1.02 mg/kg) in cell lines when proper combinations of PGR are employed. These callus cell lines are a promising platform to establish suspension cultures seeking production of alkaloids, particularly vincristine and vinblastine.

Phytochemical analysis and enhanced production of alkaloids in non-dormant corm-derived callus of Gloriosa superba (L.) using plant growth regulators and abiotic elicitors

Phytochemical analysis and enhanced production of alkaloids in non-dormant corm-derived callus of Gloriosa superba (L.) using plant growth regulators and abiotic elicitors

Differential Tolerance of Primary Metabolism of Annona emarginata (Schltdl.) H. Rainer to Water Stress Modulates Alkaloid Production

Annona emarginata produces alkaloids of ecological and pharmacological interest and is tolerant to water and biotic stress, so it is used as rootstock for other Annonaceae fruits. There are few reports in the literature on how contrasting water stress impacts the production of specialized metabolites in Annonaceae and how primary metabolism adjusts to support such production. The objective of this investigation was to evaluate how drought and flooding stress affect alkaloid concentration and the primary metabolism of young A. emarginata plants. Three water levels (flooding, field capacity, and drought) were studied at two moments (stress and recovery). Variables analyzed were gas exchange levels, chlorophyll a fluorescence, leaf sugars, total alkaloid content, alkaloid profile, and Liriodenine concentration. The photosynthetic metabolism of A. emarginata was affected by water stress, with plants having a greater ability to adapt to drought conditions than to flooding. During the drought, a reduction in photosynthetic efficiency with subsequent recovery, higher starch and trehalose concentrations in leaves, and total alkaloids in roots (480 µg.g−1) were observed. Under flooding, there was a reduction in photochemical efficiency during stress, indicating damage to the photosynthetic apparatus, without reversal during the recovery period, as well as a higher concentration of total sugars, reducing sugars, sucrose, glucose, and fructose in leaves, and Liriodenine in roots (100 µg.g−1), with a lower concentration of total alkaloids (90 µg.g−1). It could be concluded that there is differential tolerance of A. emarginata to water stress, inducing the modulation of alkaloid production, while drought promotes a higher concentration of total alkaloids and flooding leads to an increase in the Liriodenine concentration.

Seed priming with corona discharge plasma modified growth performance, improved metabolism, and elicited production of tropane alkaloids in Datura inoxia seedlings; plasma technology for application in plant in‐vitro cultures

AbstractThis study monitored the growth, biochemical, and developmental responses of Datura seedlings to the cold plasma. The effectiveness of different methods was explored to break seed dormancy. Germinating seeds were exposed to corona discharge plasma for different durations, including 0, 60, 120, 180, and 300 s. A procedure consisting of illumination with a red light (20 min), soaking in water (4°C for 48 h), and removing a part of Testa was the best method for breaking seed dormancy. The plasma treatments of 60, 120, and 180 s improved the plant growth performance, while this trait was declined in response to the plasma treatment of 300 s. The proline concentrations in both root and leaves displayed a linear significant upward trend in response to the plasma treatments. The plasma for 180 s was the most effective method to increase tropane alkaloids. With increasing the exposure time from 60 to 300 s, the leaf soluble phenols were linearly enhanced. The plasma application for 60, 120, and 180 s significantly augmented total protein concentration, while the exposure of seeds for 300 s significantly diminished it. The highest amounts of photosynthetic pigments (chlorophylls and carotenoids) were recorded in the seedlings treated with plasma for 120 and 180 s. The activities of enzymatic antioxidants (catalase and peroxidase) showed a similar upward trend to that of proline. The plasma priming also improved the phenylalanine ammonia‐lyase activity (a secondary metabolism index). Further investigations are needed to optimize plasma parameters and understand the involved mechanisms to maximize its benefits while minimizing potential risks.

Enhancement of in vitro production of tropane alkaloids and phenolic compounds in Hyoscyamus niger by culture types and elicitor treatments

This study aimed to determine the effects of 24-epibrasinolide (EBL) and methyl jasmonate (MJ) treatments on growth parameters and secondary metabolite synthesis in adventitious root and cell suspension cultures of Hyoscyamus niger. Therefore, different concentrations (0.5, 1 and 2 mg L−1) of EBL alone and combined with 224.3 mg L−1 (1 mM) MJ were applied to root and cell suspension cultures. 2 mg L−1 and 1 mg L−1 EBL were determined as the treatments in which the highest values were obtained in terms of growth criteria in root and cell cultures, respectively. In root cultures, the highest scopolamine accumulation (2.57 mg g−1) was obtained from the combination of 2 mg L−1 EBL and MJ, while the highest value (0.66 mg g−1) for hyoscyamine was observed in the roots treated with 1 mg L−1 EBL and MJ. In cell cultures, 2 mg L−1 EBL for scopolamine and 0.5 mg L−1 EBL for hyoscyamine were found to be the best applications and calculated as 0.51 µg g−1 and 0.28 µg g−1, respectively. EBL and MJ treatments also stimulated total phenolic content (TPC). The highest TPC in root cultures was detected as 18.01 mg g−1 with the combination of MJ while in cell cultures, maximum TPC was observed in cells applied with 2 mg L−1 EBL and MJ as 11.56 mg g−1. When EBL and MJ were applied to root and cell suspension cultures, significant changes occurred in the amount of phenolic compounds. Co-application of EBL and MJ significantly increased the amount of gallic acid, catechin, epicatechin, cinnamic acid and chlorogenic acid in root cultures. The application of 2 mg L−1 EBL was determined as the most suitable application for gallic acid, catechin, epicatechin, p-coumaric acid, and caffeic acid in cell cultures. It was also found that the metabolite production performance of adventitious roots was higher than that of cells. In conclusion, it was suggested that the use of MJ and EBL may be a promising strategy to enhance the accumulation of scopolamine, hyoscyamine and phenolics in root and cell cultures of H. niger.

Evolution and diversification of carboxylesterase-like [4+2] cyclases in aspidosperma and iboga alkaloid biosynthesis

Monoterpene indole alkaloids (MIAs) are a large and diverse class of plant natural products, and their biosynthetic construction has been a subject of intensive study for many years. The enzymatic basis for the production of aspidosperma and iboga alkaloids, which are produced exclusively by members of the Apocynaceae plant family, has recently been discovered. Three carboxylesterase (CXE)-like enzymes from Catharanthus roseus and Tabernanthe iboga catalyze regio- and enantiodivergent [4+2] cycloaddition reactions to generate the aspidosperma (tabersonine synthase, TS) and iboga (coronaridine synthase, CorS; catharanthine synthase, CS) scaffolds from a common biosynthetic intermediate. Here, we use a combined phylogenetic and biochemical approach to investigate the evolution and functional diversification of these cyclase enzymes. Through ancestral sequence reconstruction, we provide evidence for initial evolution of TS from an ancestral CXE followed by emergence of CorS in two separate lineages, leading in turn to CS exclusively in the Catharanthus genus. This progression from aspidosperma to iboga alkaloid biosynthesis is consistent with the chemotaxonomic distribution of these MIAs. We subsequently generate and test a panel of chimeras based on the ancestral cyclases to probe the molecular basis for differential cyclization activity. Finally, we show through partial heterologous reconstitution of tabersonine biosynthesis using non-pathway enzymes how aspidosperma alkaloids could have first appeared as "underground metabolites" via recruitment of promiscuous enzymes from common protein families. Our results provide insight into the evolution of biosynthetic enzymes and how new secondary metabolic pathways can emerge through small but important sequence changes following co-option of preexisting enzymatic functions.

Indole Alkaloid Production by the Halo Blight Bacterium Treated with the Phytoalexin Genistein.

When Pseudomonas savastanoi pv. phaseolicola, the bacterium that causes halo blight, induces hypersensitive immunity in common bean leaves, salicylic acid and phytoalexins accumulate at the site of infection. Both salicylic acid and the phytoalexin resveratrol exert antibiotic activities and toxicities in vitro, adversely disrupting the P. savastanoi pv. phaseolicola proteome and metabolism and stalling replication and motility. These efficacious properties likely contribute to the cessation of bacterial spread in beans. Genistein is an isoflavonoid phytoalexin that also accumulates during bean immunity, so we tested its antibiotic potential in vitro. Quantitative proteomics revealed that genistein did not induce proteomic changes in P. savastanoi pv. phaseolicola in the same way that salicylic acid or resveratrol did. Rather, a dioxygenase that could function to metabolize genistein was among the most highly induced enzymes. Indeed, high-throughput metabolomics provided direct evidence for genistein catabolism. Metabolomics also revealed that genistein induced the bacterium to produce indole compounds, several of which had structural similarity to auxin. Additional mass spectrometry analyses proved that the bacterium produced an isomer of the auxin indole-3-acetic acid but not indole-3-acetic acid proper. These results reveal that P. savastanoi pv. phaseolicola can tolerate bean genistein and that the bacterium likely responds to bean-produced genistein during infection, using it as a signal to increase pathogenicity, possibly by altering host cell physiology or metabolism through the production of potential auxin mimics.

Root-associated Streptomyces produce galbonolides to modulate plant immunity and promote rhizosphere colonization.

The rhizosphere, which serves as the primary interface between plant roots and the soil, constitutes an ecological niche for a huge diversity of microbial communities. Currently, there is little knowledge on the nature and the function of the different metabolites released by rhizospheric microbes to facilitate colonization of this highly competitive environment. Here, we demonstrate how the production of galbonolides, a group of polyene macrolides that inhibit plant and fungal inositol phosphorylceramide synthase (IPCS), empowers the rhizospheric Streptomyces strain AgN23, to thrive in the rhizosphere by triggering the plant's defence mechanisms. Metabolomic analysis of AgN23-inoculated Arabidopsis roots revealed a strong induction in the production of an indole alkaloid, camalexin, which is a major phytoalexin in Arabidopsis. By using a plant mutant compromised in camalexin synthesis, we show that camalexin production is necessary for the successful colonization of the rhizosphere by AgN23. Conversely, hindering galbonolides biosynthesis in AgN23 knock-out mutant resulted in loss of inhibition of IPCS, a deficiency in plant defence activation, notably the production of camalexin, and a strongly reduced development of the mutant bacteria in the rhizosphere. Together, our results identified galbonolides as important metabolites mediating rhizosphere colonization by Streptomyces.

Transcriptomic profiling of adding cobalt chloride to improve dendrobine-type total alkaloid production.

Trichoderma longibrachiatum UN32 is known for its efficient production of dendrobine-type total alkaloids (DTTAs). This study aimed to determine the optimal medium composition for the UN32 strain using response surface methodology. Key factors, including glucose, beef extract, and CoCl2, were selected through the Plackett-Burman design. Subsequently, a factorial optimization approach was employed using the steepest ascent design, and 17 trial sets were completed via the Box-Behnken design. The optimal medium composition was found to consist of 29.4g/L of glucose, 17.3g/L of beef extract, and 0.28mmol/L of CoCl2. This optimized medium resulted in an impressive 80.8% increase in mycelial dry weight (reaching 12.303g/L) and a substantial 76.4% boost in DTTA production (reaching 541.63 ± 46.95μg). Furthermore, the fermentation process was scaled up to a 5-L bioreactor, leading to a DTTA production approximately 1.95 times than the control. Transcriptome analysis of strain UN32 in response to CoCl2 supplementation revealed significant changes in the expression of critical genes associated with the TCA cycle and L-valine, L-leucine, and L-isoleucine biosynthesis changed. These alterations resulted in a heightened influx of acetyl-CoA into DTTA production. Additionally, the expression of genes related to antioxidant enzymes was modified to maintain homeostasis of reactive oxygen species (ROS). A potential mechanism for the accumulation of DTTAs based on ROS as a signal transduction was proposed. These findings provide valuable insights into the regulatory mechanisms of DTTA biosynthesis, potentially offering a method to enhance the production of secondary metabolites in the UN32 strain. KEY POINTS: • After the RSM optimization, there is a substantial increase of 80.8% in biomass production and a significant 76.4% rise in DTTA production. • Transcriptome analysis revealed that the inclusion of CoCl2 supplements resulted in an enhanced influx of acetyl-CoA. • Proposed a mechanism for the accumulation of DTTAs for the role of ROS as a signal transduction pathway.

Monocrotaline presence in the Crotalaria (Fabaceae) plant genus and its influence on arthropods in agroecosystems.

Crotalaria (Fabaceae) occurs abundantly in tropical and subtropical regions and has about 600 known species. These plants are widely used in agriculture, mainly as cover plants and green manures, in addition to their use in the management of phytonematodes. A striking feature of these species is the production of pyrrolizidine alkaloids (PAs), secondary allelochemicals involved in plant defense against herbivores. In Crotalaria species, monocrotaline is the predominant PA, which has many biological activities reported, including cytotoxicity, tumorigenicity, hepatotoxicity and neurotoxicity, with a wide range of ecological interactions. Thus, studies have sought to elucidate the effects of this compound to promote an increase in flora and fauna (mainly insects and nematodes) associated with agroecosystems, favoring the natural biological control. This review summarizes information about the monocrotaline, showing such effects in these environments, both above and below ground, and their potential use in pest management programs.

Isolation of Bacterial Endophytes Associated with Cinchona ledgeriana Moens. and Their Potential in Plant-growth Promotion, Antifungal and Quinoline Alkaloids Production.

For centuries, quinoline alkaloids from the tree bark of Cinchona ledgeriana (C. ledgeriana) have been used in the treatment of malaria. However, unsustainable harvesting and poor growth conditions greatly limit its use as raw materials. Since plant endophytes are known to contribute to the physiology of the host and its metabolism for survival, this study showed the potential of endophytes isolated from C. ledgeriana roots in promoting the germination of Catharathus roseus (C. roseus) seedlings and the biosynthesis of quinoline alkaloid. In this present study, we found that the Enterobacteriaceae family comprised the majority of the bacterial community, with Klebsiella pneumoniae being the most abundant species at the C. ledgeriana roots. Characterization of culturable bacterial endophytes from the C. ledgeriana roots showed that all the isolates displayed plant growth-promoting factors and antifungal activities. Interestingly, chromatographic analyses led to the identification of the quinoline alkaloids producing Achromobacter xylosoxidans (A. xylosoxidans) A1. Moreover, the co-cultures of A. xylosoxidans A1, Cytobacillus solani (C. solani) A3, and Klebsiella aerogenes A6 increased the fresh and dry weight of the C. roseus seedlings. These results suggest that these bacterial endophytes may enhance quinine and quinidine production as well as the growth of the plant host.

New Set of EST-STR Markers for Discrimination of Related Papaver somniferum L. Varieties.

Papaver somniferum L. is cultivated for its edible seeds and for the production of alkaloids. A serious problem in seed trade and processing is the intentional mixing of excellent food-quality seeds with non-food-grade-quality seeds. Tracking the correct or illegitimate handling of seeds requires an efficient method for discrimination and individualization of poppy varieties. As in human and animal forensics, DNA variable regions containing short tandem repeats (STRs) located either in non-coding DNA or in gene sequences (EST-STRs) are preferred markers for discrimination between genotypes. Primers designed for 10 poppy EST-STR loci not analyzed so far were tested for their discriminatory ability on a set of 23 related P. somniferum L. genotypes. Thirty-three EST-STR alleles were identified together. Their polymorphic information content (PIC) values were in the range of 0.175-0.649. The PI value varied in the range of 0.140-0.669, and the cumulative PI was 1.2 × 10-5. PIsibs values varied between 0.436 and 0.820 and the cumulative value was lower (5.0 × 10-3). All analyzed genotypes were distinguished mutually, each with its own unique EST-STR profile. These newly developed EST-STR markers more effectively discriminated P. somniferum L. genotypes, even those genotypes whose DNA profiles were previously identical.

Ergot alkaloids as pharmaceuticals: Status and prospects of commercial cultivation of ergot crop for natural alkaloids

Ergot is a plant disease symptom induced by a fungal pathogen of the genus Claviceps in grains of cereal crops, wherein the ergot alkaloids are produced in the cereal grains which are transformed into ergot sclerotia. The natural occurrence of ergot grains in cereal crops is dependent on the environmental factors conducive to ergot development. Under natural fungal infection, the ergot percent varies from 1.2 to 3.3 in the infected crops which hamper the yield or recovery of natural ergot alkaloids. Ergot alkaloids are specific pharmaceutical alkaloids used to treat certain ailments like migraines, induction of childbirth, and the control of postpartum bleeding in humans. Ergot sclerotia contain about 0.15% to 0.5% alkaloids, with medicinally useful compounds. Annual world production of ergot alkaloids has been estimated at 5,000-8,000 kg of all ergopeptines (peptide ergot alkaloids) and 10,000-15,000 kg of lysergic acid, the latter being mainly used in the manufacture of semisynthetic derivatives. The greater part of this production occurs as a result of fermentation (around 60%) while field cultivation of triticale (a hybrid of wheat and rye) accounts for the balance. This is because the artificial cultivation of ergot sclerotia on a host crop plant is lacking as a business module. Due to the low recovery of ergot grains and ergot alkaloids under natural crop production conditions, commercial cultivation for ergot grains is discussed in this paper.

Histochemical Localization of Alkaloids in the Bulbs of In Vitro-Regenerated Snake’s Head Fritillary (Fritillaria meleagris L.): The Effect of a Temperature Regime

The genus Fritillaria is characterized by the production of alkaloids, primarily of the isosteroidal type. The aim of this preliminary study was to perform a histolocalization screening aimed at revealing the presence of alkaloids in the bulbs of F. meleagris, cultured in vitro under different growth regimes and at different developmental stages, as a powerful tool to identify preferable in vitro plant material for alkaloid isolation. Histochemical localization of alkaloids was performed using Wagner’s and Dragendorff’s reagents in fresh sections of bulbs cultured at 24 °C or 7 °C for 4 weeks, as well as those cultured at 24 °C following the 4-week chilling treatment, which were sampled at the beginning of sprouting. A positive reaction was observed with both reagents and was particularly intense in cold-treated bulbs. Alkaloids were mainly distributed in the bulbs and partly in the sprouts of the in vitro grown F. meleagris. The most intense staining, indicative of high alkaloid content, was observed in the bulb scales of pre-chilled bulbs that sprouted at 24 °C, rendering them preferable as in vitro plant material for alkaloid isolation. The results suggest that alkaloid production in the bulbs of F. meleagris can be improved by manipulating growth in the microenvironment of in vitro cultures, in order to meet the increasing industrial demand for medicinally or commercially important metabolites used as traditional medicines and herbal remedies.

Genome-wide identification of NAC transcription factors and regulation of monoterpenoid indole alkaloid biosynthesis in Catharanthus roseus.

NAC transcription factors (TFs) are crucial to growth and defense responses in plants. Though NACs have been characterized for their role in several plants, comprehensive information regarding their role in Catharanthus roseus, a perennial ornamental plant, is lacking. Homology modelling was employed to identify and characterize NACs in C. roseus. In-vitro propagation of C. roseus plants was carried out using cell suspension and nodal culture and were elicited with two auxin-antagonists, 5-fluoro Indole Acetic Acid (5-F-IAA) and α-(phenyl ethyl-2-oxo)-Indole-Acetic-Acid (PEO-IAA) for the enhanced production of monoterpenoid indole alkaloids (MIAs) namely catharanthine, vindoline, and vinblastine. Analyses revealed the presence of 47 putative CrNAC genes in the C. roseus genome, primarily localized in the nucleus. Phylogenetic analysis categorized these CrNACs into eight clusters, demonstrating the highest synteny with corresponding genes in Camptotheca acuminata. Additionally, at least one defense or hormone-responsive cis-acting element was identified in the promoter region of all the putative CrNACs. Of the two elicitors, 5-F-IAA was effective at 200 µM to elicit a 3.07-fold increase in catharanthine, 2.76-fold in vindoline, and 2.4-fold in vinblastine production in nodal culture. While a relatively lower increase in MIAs was recorded in suspension culture. Validation of RNA-Seq by qRT-PCR showed upregulated expression of stress-related genes (CrNAC-07 and CrNAC-24), and downregulated expression of growth-related gene (CrNAC-25) in elicited nodal culture of C. roseus. Additionally, the expression of genes involved in the biosynthesis of MIAs was significantly upregulated upon elicitation. The current study provides the first report on the role of CrNACs in regulating the biosynthesis of MIAs.