Iridoid Production Research Articles

Integrated Transcriptomics and Metabolomics Reveal Key Insights into Iridoid Biosynthesis in Gentiana crassicaulis Seeds during Germination.

Background:Gentiana crassicaulis Duthie ex Burk., a key species used in traditional Chinese medicine for treating rheumatic pain and stroke, contains iridoids as its primary active component. However, the biosynthetic mechanisms underlying iridoid production are not fully understood. Methods: This study focused on iridoid biosynthesis during the germination of G. crassicaulis seeds, integrating metabolomic and transcriptomic analyses to uncover the underlying pathways and key candidate genes. Results: 196,132 unigenes and 10 iridoid compounds were identified through RNA-seq and ultra performance liquid chromatography-quadrupole time of flight-mass spectrometer (UPLC-Q-TOF-MS), respectively. The intersection of results from Pearson correlation analysis and weighted gene co-expression network analysis (WGCNA) revealed a significant correlation between 26 genes and iridoid levels, suggesting their potential role in the iridoid metabolism. Notably, six highly expressed candidate genes (DL7H, SLS, CYP76, CYP72A2, CYP84A1, and 13-LOX3) and five iridoids (loganic acid, sweroside, swertiamarin, gentiopicroside, and 6'-O-β-D-glucosyl-gentiopicroside) responded to methyl jasmonate stimulation in G. crassicaulis seedlings. Conclusions: by combining the known functions of candidate gene families, It is hypothesized that the CYP716 and LOX families exert indirect influences on iridoid metabolism, while the CYP71, CYP81, CYP72, CYP76, CYP710 families, 2OG-FeII family, and the glucosyltransferase family are likely to play direct roles in the biosynthetic transformations of the five iridoids. This study provides a theoretical basis for further functional gene validation and metabolic engineering aimed at enhancing iridoid production. The insights gained could lead to improved iridoid production efficiency in medicinal plants, ultimately benefiting the quality and efficacy of medicinal materials.

A fused hybrid enzyme of 8-hydroxygeraniol oxidoreductase (8HGO) from Gardenia jasminoides and iridoid synthase (ISY) from Catharanthus roseus significantly enhances nepetalactol and iridoid production.

The operation of 8HGO-ISY fusion enzymes can increase nepetalactol flux to iridoid biosynthesis, and the Gj8HGO-CrISY expression in Gardenia jasminoides indicates that seco-iridoids and closed-ring iridoids share a nepetalactol pool. Nepetalactol is a common precursor of (seco)iridoids and their derivatives, which are a group of noncanonical monoterpenes. Functional characterization of an 8HGO (8-hydroxygeraniol oxidoreductase) from Catharanthus roseus, a seco-iridoids producing plant, has been reported; however, the 8HGO from G. jasminoides with plenty of closed-ring iridoids remains uninvestigated. In this work, a Gj8HGO was cloned and biochemically characterized. In addition, the relatively low production of nepetalactol in plants and engineered microbial host is likely to be attributed to the fact that Cr8HGO and CrISY (iridoid synthase) are substrate-promiscuous enzymes catalyzing unexpected substrates to the undesired products. Herein, a bifunctional enzyme consisting of an 8HGO fused to an ISY was designed for the proximity to the substrate and recycling of NADP+ and NADPH cofactor to reduce the undesired intermediate in the synthesis of nepetalactol. Of four fusion enzymes (i.e., Gj8HGO-GjISY, Gj8HGO-GjISY2, Gj8HGO-GjISY4, and Gj8HGO-CrISY), interestingly, only the last one can enable cascade reaction to form cis-trans-nepetalactol. Furthermore, we establish a reliable Agrobacterium-mediated transformation system. The expression of Gj8HGO-CrISY in G. jasminoides led to a significant enhancement of nepetalactol production, about 19-fold higher than that in wild-type plants, which further resulted in the twofold to fivefold increase of total iridoids and representative iridoid such as geniposide, indicating that seco-iridoids in C. roseus and closed-ring iridoids in G. jasminoides share a nepetalactol pool. All results suggest that 8HGO and ISY can be manipulated to maximize metabolic flux for nepetalactol and iridoid production.

Biosynthesis of iridoid sex pheromones in aphids

Iridoid monoterpenes, widely distributed in plants and insects, have many ecological functions. While the biosynthesis of iridoids has been extensively studied in plants, little is known about how insects synthesize these natural products. Here, we elucidated the biosynthesis of the iridoids cis-trans-nepetalactol and cis-trans-nepetalactone in the pea aphid Acyrthosiphon pisum (Harris), where they act as sex pheromones. The exclusive production of iridoids in hind legs of sexual female aphids allowed us to identify iridoid genes by searching for genes specifically expressed in this tissue. Biochemical characterization of candidate enzymes revealed that the iridoid pathway in aphids proceeds through the same sequence of intermediates as described for plants. The six identified aphid enzymes are unrelated to their counterparts in plants, conclusively demonstrating an independent evolution of the entire iridoid pathway in plants and insects. In contrast to the plant pathway, at least three of the aphid iridoid enzymes are likely membrane bound. We demonstrated that a lipid environment facilitates the cyclization of a reactive enol intermediate to the iridoid cyclopentanoid-pyran scaffold invitro, suggesting that membranes are an essential component of the aphid iridoid pathway. Altogether, our discovery of this complex insect metabolic pathway establishes the genetic and biochemical basis for the formation of iridoid sex pheromones in aphids, and this discovery also serves as a foundation for understanding the convergent evolution of complex metabolic pathways between kingdoms.

Spatial and temporal patterns of secoiridoid and xanthone biosynthetic pathways during early development of Centaurium erythraea Rafn, as altered by ploidy level

Centaurium erythraea, used as medical plant from the earliest times, is an immense depot of quite rare bioactive compounds. Secoiridoids (sweroside, swertiamarin, and gentiopicrin) and xanthones (methylbellidifolin and decussatin) are predominant bioactive compounds in C. erythraea. The present study aims at providing new insights into how the content of these bioactive principles can be related with plant ploidy level by characterizing possible differences in their biosynthesis and accumulation between diploid and tetraploid genotypes from both spatial and temporal aspects. In general, shoots are determined as the major site of secoiridoids’ and xanthones’ accumulation, whose ratio vary during the development. Genes involved in iridoid and xanthone metabolic pathways were found to be coordinately regulated at the transcriptional level both during the development and among organs. Biosynthetic gene expression levels were found highly correlated with the content of major compounds from these two classes. Diversification in chemical profiles between tetraploid and diploid genotypes may result from the expression difference between homologous loci correspondent to several key biosynthetic genes, which trigger changes in the two metabolic routes. Thus, enhanced expression of genes coding for geraniol synthase (GES), 8-hydroxygeraniol oxidoreductase (8HGO), and 7-deoxyloganic acid hydrolase (7DLH2) is strongly associated with intensive production of iridoids. Interestingly, transcript levels of beta-glucosidase (CebGLU), a candidate to catalyze the first step in the secoiridoid catabolism, is significantly positively correlated with the content of major secoiridoids. Elevated expression of genes coding for benzophenone synthase (BS) and 3-hydroxybenzoate:CoA ligase (3HBL) appear to account for enhanced production of hexa-substituted xanthones. Regarding content of iridoids and xanthones, a diploid genotype appeared to be more productive than a tetraploid genotype under controlled in vitro conditions, therewithal displaying significantly higher biomass.

Domestic cat damage to plant leaves containing iridoids enhances chemical repellency to pests

SummaryCatnip (Nepeta cataria) and silver vine (Actinidia polygama) produce iridoids with arthropod-repellent effects. Cats rub and roll against these plants, transferring iridoids to their fur that repels mosquitoes. Cats also lick and chew plant leaves during this response, although the benefit of this additional behavior has remained unknown. Here, we show that feline leaf damage substantially increases iridoid emission from both plants while also diversifying iridoids in silver vine. Cats show an equivalent duration of response to the complex cocktail of iridoids in damaged silver vine and to the much higher level of a single iridoid produced by damaged catnip. The more complex iridoid cocktail produced when silver vine is licked and chewed by cats increases mosquito repellency at low concentration. In conclusion, feline leaf damage contributes by releasing more mosquito-repellent iridoids. Feline olfactory and behavioral sensitivity is fine-tuned to plant-specific iridoid production for maximizing the mosquito repellency gained.

Aspergillus oryzae Biosynthetic Platform for de Novo Iridoid Production.

The iridoids and their derivatives monoterpene indole alkaloids (MIAs) are two broad classes of plant-derived natural products with valuable pharmaceutical properties. However, the poor source limited their application. Nepetalactol, a common iridoid scaffold of MIAs, was heterologously produced in Saccharomyces cerevisiae. Although the optimization of nepetalactol production in S. cerevisiae was achieved by metabolic engineering, the inherent metabolic constraints impose a restriction on the production. Herein, we developed a high nepetalactol-producing Aspergillus oryzae platform strain. First, the co-expression of 5 nepetalactol biosynthetic genes, in a high isopentenyl pyrophosphate (IPP)-producing strain A. oryzae AK2, succeeded in the biosynthesis of nepetalactol. Second, the improvement of the IPP supply and the suppression of the byproduct citronellol formation were simultaneously achieved. Finally, the highest titer of nepetalactol of 7.2 mg/L was obtained with the engineered strain, after the optimization of the carbon source. To the best of our knowledge, this is the highest reported titer of nepetalactol in microbial cells. The developed A. oryzae strain represents an attractive biosynthetic platform host for the de novo production of iridoids and MIAs.

Anti-Inflammation and Antimicrobial Constituents from the Roots and Their Production in Callus Cultures of Valeriana jatamansi Jones

Background: Valeriana jatamansi Jones (Syn. V. wallichii DC.; Fam. – Valerianaceae) is a medicinal plant species, endemic to the Himalayan regions of India and rich in presence of iridoids. This plant species possessed antimicrobial, antioxidant and anti-inflammatory properties. Methods: The shade-dried roots were powdered, percolated with 95% ethanol for 36 h at room temperature (3-times) and filtrate used for isolation of iridoids. The isolated compounds identified based on physical and spectral data analysis. For the enhancement of production of iridoids, the callus cultures established on Murashige and Skoog (MS) culture medium with variable concentrations of growth hormones. The isolated iridoids estimated by using High Performance Liquid Chromatography (HPLCSPD- M10A photodiode array detector) and Gas Chromatography–Mass Spectrometry (GC-MS) analysis. The anti-inflammatory activity of iridoids assessed by using carrageenan and Complete Freund's Adjuvant (CFA-induced adjuvant) models in experimental rats. The total eight bacterial and five fungal strains used for determination of antimicrobial activity. The activity assessed by using microdilution method. Results: Total seven iridoids were isolated from ethyl acetate fraction and their production estimated in cell cultures. The maximum accumulation (69.39±0.45 mg/g) of jatamanvaltrate S was observed in 2, 4- D (4.0 mg/l) and kinetin (1.0 mg/l) supplemented MS culture medium. Maximum anti-inflammatory activity demonstrated by jatamanvaltrate R (46.8%) at the dose of 20 mg/kg body weight (bw) at 8 h after carrageenan injection. Similarly, the jatamanvaltrate R also displayed maximum inhibitory activity (49.9%) to CFA-induced adjuvant arthritis in rats on day 8. The strongest antibacterial activity was exhibited by jatamanvaltrate S (21 μg/ml) against Staphylococcus aureus while maximum antifungal efficacy displayed by jatamanin B (30 μg/ml) against Penicillium chrysogenum. Conclusion: In this study, all the isolated iridoids found as bioactive molecules and exhibited promising anti-inflammatory and antimicrobial activities.

Phylogenomic Mining of the Mints Reveals Multiple Mechanisms Contributing to the Evolution of Chemical Diversity in Lamiaceae

The evolution of chemical complexity has been a major driver of plant diversification, with novel compounds serving as key innovations. The species-rich mint family (Lamiaceae) produces an enormous variety of compounds that act as attractants and defense molecules in nature and are used widely by humans as flavor additives, fragrances, and anti-herbivory agents. To elucidate the mechanisms by which such diversity evolved, we combined leaf transcriptome data from 48 Lamiaceae species and four outgroups with a robust phylogeny and chemical analyses of three terpenoid classes (monoterpenes, sesquiterpenes, and iridoids) that share and compete for precursors. Our integrated chemical–genomic–phylogenetic approach revealed that: (1) gene family expansion rather than increased enzyme promiscuity of terpene synthases is correlated with mono- and sesquiterpene diversity; (2) differential expression of core genes within the iridoid biosynthetic pathway is associated with iridoid presence/absence; (3) generally, production of iridoids and canonical monoterpenes appears to be inversely correlated; and (4) iridoid biosynthesis is significantly associated with expression of geraniol synthase, which diverts metabolic flux away from canonical monoterpenes, suggesting that competition for common precursors can be a central control point in specialized metabolism. These results suggest that multiple mechanisms contributed to the evolution of chemodiversity in this economically important family.

Character evolution and missing (morphological) data across Asteridae.

Our current understanding of flowering plant phylogeny provides an excellent framework for exploring various aspects of character evolution through comparative analyses. However, attempts to synthesize this phylogenetic framework with extensive morphological data sets have been surprisingly rare. Here, we explore character evolution in Asteridae (asterids), a major angiosperm clade, using an extensive morphological data set and a well-resolved phylogeny. We scored 15 phenotypic characters (spanning chemistry, vegetative anatomy, and floral, fruit, and seed features) across 248 species for ancestral state reconstruction using a phylogenetic framework based on 73 plastid genes and the same 248 species. Iridoid production, unitegmic ovules, and cellular endosperm were all reconstructed as synapomorphic for Asteridae. Sympetaly, long associated with asterids, shows complex patterns of evolution, suggesting it arose several times independently within the clade. Stamens equal in number to the petals is likely a synapomorphy for Gentianidae, a major asterid subclade. Members of Lamianae, a major gentianid subclade, are potentially diagnosed by adnate stamens, unilacunar nodes, and simple perforation plates. The analyses presented here provide a greatly improved understanding of character evolution across Asteridae, highlighting multiple characters potentially synapomorphic for major clades. However, several important parts of the asterid tree are poorly known for several key phenotypic features (e.g., degree of petal fusion, integument number, nucellus type, endosperm type, iridoid production). Further morphological, anatomical, developmental, and chemical investigations of these poorly known asterids are critical for a more detailed understanding of early asterid evolution.

A two-season impact study on Globularia alypum: adaptive leaf structures and secondary metabolite variations

A detailed histo-anatomical and phytochemical study on Globularia alypum was carried out to investigate season-induced plant responses related to leaf morphology and content of secondary metabolites (SMs). Leaf tissue analysis by light and scanning electron microscopy showed neither morphological nor significant anatomical adaptions/variations to the coldest and the hottest seasons. Both summer and winter leaf tissue resulted amphistomatic with compact mesophyll and capitate glandular trichomes. Epidermal and mesophyll cells showed a significant accumulation of osmiophilic compounds, further characterized as phenolics by the histochemical screening. Beside the morpho-anatomical similarities in tissue arrangement, SMs production, within and among the different natural product classes, resulted seasonally dependent. In total, 24 SMs belonging to iridoids, flavonoids and phenylethanoid glycosides were tentatively assigned by UHPLC-HRMS analysis. Among iridoids, (E)-globularicisin showed to be the prevalent constituent, with content up to 70%. Total iridoid production was increased by 48.1% during summer. Similarly, flavonoid anabolism was more active during the same period, although with less remarkable seasonal variation reaching 25.6%. In contrast to the overall higher production of iridoids and flavonoids, a drastic fall (33.5%) of phenylethanoids was observed during summer.

Engineering the biocatalytic selectivity of iridoid production in Saccharomyces cerevisiae

Monoterpene indole alkaloids (MIAs) represent a structurally diverse, medicinally essential class of plant derived natural products. The universal MIA building block strictosidine was recently produced in the yeast Saccharomyces cerevisiae, setting the stage for optimization of microbial production. However, the irreversible reduction of pathway intermediates by yeast enzymes results in a non-recoverable loss of carbon, which has a strong negative impact on metabolic flux. In this study, we identified and engineered the determinants of biocatalytic selectivity which control flux towards the iridoid scaffold from which all MIAs are derived. Development of a bioconversion based production platform enabled analysis of the metabolic flux and interference around two critical steps in generating the iridoid scaffold: oxidation of 8-hydroxygeraniol to the dialdehyde 8-oxogeranial followed by reductive cyclization to form nepetalactol. In vitro reconstitution of previously uncharacterized shunt pathways enabled the identification of two distinct routes to a reduced shunt product including endogenous ‘ene’-reduction and non-productive reduction by iridoid synthase when interfaced with endogenous alcohol dehydrogenases. Deletion of five genes involved in α,β-unsaturated carbonyl metabolism resulted in a 5.2-fold increase in biocatalytic selectivity of the desired iridoid over reduced shunt product. We anticipate that our engineering strategies will play an important role in the development of S. cerevisiae for sustainable production of iridoids and MIAs.

Differential iridoid production as revealed by a diversity panel of 84 cultivated and wild blueberry species.

Cultivated blueberry (Vaccinium corymbosum, Vaccinium angustifolium, Vaccinium darrowii, and Vaccinium virgatum) is an economically important fruit crop native to North America and a member of the Ericaceae family. Several species in the Ericaceae family including cranberry, lignonberry, bilberry, and neotropical blueberry species have been shown to produce iridoids, a class of pharmacologically important compounds present in over 15 plant families demonstrated to have a wide range of biological activities in humans including anti-cancer, anti-bacterial, and anti-inflammatory. While the antioxidant capacity of cultivated blueberry has been well studied, surveys of iridoid production in blueberry have been restricted to fruit of a very limited number of accessions of V. corymbosum, V. angustifolium and V. virgatum; none of these analyses have detected iridoids. To provide a broader survey of iridoid biosynthesis in cultivated blueberry, we constructed a panel of 84 accessions representing a wide range of cultivated market classes, as well as wild blueberry species, and surveyed these for the presence of iridoids. We identified the iridoid glycoside monotropein in fruits and leaves of all 13 wild Vaccinium species, yet only five of the 71 cultivars. Monotropein positive cultivars all had recent introgressions from wild species, suggesting that iridoid production can be targeted through breeding efforts that incorporate wild germplasm. A series of diverse developmental tissues was also surveyed in the diversity panel, demonstrating a wide range in iridoid content across tissues. Taken together, this data provides the foundation to dissect the molecular and genetic basis of iridoid production in blueberry.

The Influence of Methyl Jasmonate and Salicylic Acid on Secondary Metabolite Production in Rehmannia Glutinosa Libosch. Hairy Root Culture

Abstract Rehmannia glutinosa hairy roots were used to evaluate the effect of methyl jasmonate (MeJa) and salicylic acid (SA) on increase of root biomass and production of iridoids (catalpol, harpagide) and phenylethanoids (verbascoside and isoverbascoside). The elicitors were added to 23-day-old culture separately at concentrations between 50 and 200 μM or in combinations at concentrations of 50 and 100 μM. Roots were harvested 72 h and 120 h after elicitation. The type of elicitor, its concentration and exposure time were found to strongly affect the content of each analyzed compound. A 72-hour treatment with 200 μM MeJa was the most effective in increase of verbascoside content (60.07 mg·DW−1 equivalent to 845.45 mg·L−1) and isoverbascoside (1.77 mg·DW−1 equivalent to 24.94 mg·L−1): these respective amounts were roughly 10- and 6.4-fold higher than the control values (unelicited roots). Exposure to 150 μM MeJa provided optimal harpagide content after 72 hours (0.136 mg·DW−1; 7.5-fold increase compared to the control), and catalpol content after 120 hours (up to 2.145 mg·DW−1). The combination of MeJa and SA also resulted in higher levels of secondary metabolites compared to the control culture, although these levels were lower than those observed for MeJa alone at the optimal concentration and exposure time. SA alone was less efficient in enhancing metabolite production than MeJa.

Isolation and purification of six iridoid glycosides from gardenia jasminoides fruit by medium-pressure liquid chromatography combined with macroporous resin chromatography.

Gardeniae fructus is one of the most frequently used herbs in traditional Chinese medicine. In the present study, a process for the enrichment of six iridoid glycosides from Gardeniae fructus was developed using medium-pressure liquid chromatography combined with macroporous resin and reversed-phase chromatography. The purities of different fractions from Gardeniae fructus were assessed using quantitative high-performance liquid chromatography. After fractionation using HPD-100 column chromatography, a 30% ethanol fraction was selected based on high-performance liquid chromatography and liquid chromatography with mass spectrometry qualitative analysis to separate and purify. Based on the orientation analysis results, six compounds-deacetyl asperulosidic acid methyl ester, gardenoside, ixoroside, scandoside methyl ester, genipin-1-O-β-d-gentiobioside, and geniposide-were successfully isolated and purified in three to four combined steps from Gardeniae fructus. The purities of these compounds were found by high-performance liquid chromatography analysis to be 97.9, 98.1, 95.5, 96.3, 97.1, and 98.7%, respectively. Moreover, their structures were elucidated by NMR spectroscopy and liquid chromatography with tandem mass spectrometry. The separation process was highly efficient, rapid, and accurate, making it a potential approach for the large-scale production of iridoids in the laboratory and providing several marker compounds for quality control. This procedure may be meaningful for the purification of other natural products used in traditional Chinese medicine.

Glandular β-glucosidases in juvenile Chrysomelina leaf beetles support the evolution of a host-plant-dependent chemical defense

Plant-feeding insects are spread across the entire plant kingdom. Because they chew externally on leaves, leaf beetle of the subtribe Chrysomelina sensu stricto are constantly exposed to life-threatening predators and parasitoids. To counter these pressures, the juveniles repel their enemies by displaying glandular secretions that contain defensive compounds. These repellents can be produced either de novo (iridoids) or by using plant-derived precursors. The autonomous production of iridoids pre-dates the evolution of phytochemical-based defense strategies. Both strategies include hydrolysis of the secreted non-toxic glycosides in the defensive exudates. By combining in vitro as well as in vivo experiments, we show that iridoid de novo producing as well as sequestering species rely on secreted β-glucosidases to cleave the pre-toxins. Our phylogenetic analyses support a common origin of chrysomeline β-glucosidases. The kinetic parameters of these β-glucosidases demonstrated substrate selectivity which reflects the adaptation of Chrysomelina sensu stricto to the chemistry of their hosts during the course of evolution. However, the functional studies also showed that the broad substrate selectivity allows building a chemical defense, which is dependent on the host plant, but does not lead to an “evolutionary dead end”.

Independently recruited oxidases from the glucose-methanol-choline oxidoreductase family enabled chemical defences in leaf beetle larvae (subtribe Chrysomelina) to evolve.

Larvae of the leaf beetle subtribe Chrysomelina sensu stricto repel their enemies by displaying glandular secretions that contain defensive compounds. These repellents can be produced either de novo (iridoids) or by using plant-derived precursors (e.g. salicylaldehyde). The autonomous production of iridoids, as in Phaedon cochleariae, is the ancestral chrysomeline chemical defence and predates the evolution of salicylaldehyde-based defence. Both biosynthesis strategies include an oxidative step of an alcohol intermediate. In salicylaldehyde-producing species, this step is catalysed by salicyl alcohol oxidases (SAOs) of the glucose-methanol-choline (GMC) oxidoreductase superfamily, but the enzyme oxidizing the iridoid precursor is unknown. Here, we show by in vitro as well as in vivo experiments that P. cochleariae also uses an oxidase from the GMC superfamily for defensive purposes. However, our phylogenetic analysis of chrysomeline GMC oxidoreductases revealed that the oxidase of the iridoid pathway originated from a GMC clade different from that of the SAOs. Thus, the evolution of a host-independent chemical defence followed by a shift to a host-dependent chemical defence in chrysomeline beetles coincided with the utilization of genes from different GMC subfamilies. These findings illustrate the importance of the GMC multi-gene family for adaptive processes in plant-insect interactions.

Shoot proliferation and rooting treatments influence secondary metabolite production and antioxidant activity in tissue culture-derived Aloe arborescens grown ex vitro

Aloe species are valuable plants with great ornamental and medicinal value. Although micropropagation protocols have been developed to meet the increasing global demand, the effects of the series of events during micropropagation on the phytochemical and pharmacological efficacy of ex-vitro plants remains poorly understood. Thus, we evaluated the effects of cytokinin and rooting compounds used during the shoot regeneration and rooting phases respectively, on secondary metabolite production in greenhouse-grown in vitro-derived Aloe arborescens. Shoots derived from meta-methoxytopolin (MemT)-containing medium and rooted with either smoke–water (SW) or indole butyric acid (IBA) had higher levels of total phenolics and flavonoids than those rooted on plant growth regulator (PGR)-free medium. Iridoid content was significantly reduced in cytokinin-regenerated shoots rooted with IBA in comparison to PGR-free regenerated shoots rooted with IBA. Conversely, the use of SW for rooting in cytokinin-regenerated shoots significantly increased iridoid content when compared to PGR-free regenerated shoots rooted with SW. These findings suggest an antagonistic interaction between cytokinins used in this study and IBA as well as a possible synergistic or additive interaction of the cytokinins with SW on iridoid production. Significantly higher antioxidant activity was recorded in shoots regenerated from meta-topolin riboside (mTR) and MemT and rooted with IBA or SW when compared to those rooted without PGR. Overall, the type of cytokinin and rooting treatments individually and interactively had a significant carry-over effect on secondary metabolite production and antioxidant potential of tissue culture-derived A. arborescens. Therefore, when micropropagating plants for medicinal uses, it is prudent to select the right cytokinin and rooting compound for optimal production of secondary metabolites and ultimately the pharmacological efficacy of acclimatized plants.

An alternative route to cyclic terpenes by reductive cyclization in iridoid biosynthesis

The iridoids comprise a large family of distinctive bicyclic monoterpenes that possess a wide range of pharmacological activities, including anticancer, anti-inflammatory, antifungal and antibacterial activities. Additionally, certain iridoids are used as sex pheromones in agriculturally important species of aphids, a fact that has underpinned innovative and integrated pest management strategies. To harness the biotechnological potential of this natural product class, the enzymes involved in the biosynthetic pathway must be elucidated. Here we report the discovery of iridoid synthase, a plant-derived enzyme that generates the iridoid ring scaffold, as evidenced by biochemical assays, gene silencing, co-expression analysis and localization studies. In contrast to all known monoterpene cyclases, which use geranyl diphosphate as substrate and invoke a cationic intermediate, iridoid synthase uses the linear monoterpene 10-oxogeranial as substrate and probably couples an initial NAD(P)H-dependent reduction step with a subsequent cyclization step via a Diels-Alder cycloaddition or a Michael addition. Our results illustrate how a short-chain reductase was recruited as cyclase for the production of iridoids in medicinal plants. Furthermore, we highlight the prospects of using unrelated reductases to generate artificial cyclic scaffolds. Beyond the recognition of an alternative biochemical mechanism for the biosynthesis of cyclic terpenes, we anticipate that our work will enable the large-scale heterologous production of iridoids in plants and microorganisms for agricultural and pharmaceutical applications.

Habitat climate-dependent gardenieae iridoid productivity revealed through callus induction

Here we report the callus induction of Gardenia, Genipa, and Tarenna plants of the tribe Gardenieae growing throughout the Pacificrim area and South Africa to reveal the habitat climate-linked diversity of the production of iridoids comprising germination and growth-inhibitory GE

Production of iridoid glucosides in cell suspension cultures of Penstemon serrulatus: effects of nutritional factors

The cell growth and iridoid production were studied in suspension cultures derived from anther-callus of Penstemon serrulatus. Penstemide and serrulatoloside were major iridoids produced by the cell cultures. Cells (14 days old) produced up to 7.8% of dry weight of penstemide and 5.1% of serrulatoloside. However, formation of both iridoids gradually decreased during consecutive growth cycles. It was possible to improve the quantity of penstemide and serrulatoloside in cell cultures by altering the concentrations of some nutritional factors in the medium. The best results were obtained by increasing the concentration of sucrose (to 5% or 7%); the contents of penstemide and serrulatoloside were 4-fold higher than those obtained in the presence of 3% sucrose. Sucrose concentration from 5% to 7% improved not only the content of iridoids, but also cell growth. Further stimulation of iridoid production was achieved by the selection of high iridoid-producing cell line and transferring the cells to optimized medium.