Interference between ER stress-related bZIP-type and jasmonate-inducible bHLH-type transcription factors in the regulation of triterpene saponin biosynthesis in Medicago truncatula.

Triterpene saponins (TS) are a structurally diverse group of metabolites that are widely distributed in plants. They primarily serve as defense compounds and their production is often triggered by biotic stresses through signaling cascades that are modulated by phytohormones such as the jasmonates (JA). Two JA-modulated basic helix-loop-helix (bHLH) transcription factors (TFs), triterpene saponin biosynthesis activating regulator 1 (TSAR1) and TSAR2, have previously been identified as direct activators of TS biosynthesis in the model legume Medicago truncatula. Here, we report on the involvement of the core endoplasmic reticulum (ER) stress-related basic leucine zipper (bZIP) TFs bZIP17 and bZIP60 in the regulation of TS biosynthesis. Expression and processing of M. truncatula bZIP17 and bZIP60 proteins were altered in roots with perturbed TS biosynthesis or treated with JA. Accordingly, such roots displayed an altered ER network structure. M. truncatula bZIP17 and bZIP60 proteins were shown to localize in the nucleus and appeared to be capable of interfering with the TSAR-mediated transactivation of TS biosynthesis genes. Furthermore, interference between ER stress-related bZIP and JA-modulated bHLH TFs in the regulation of JA-dependent terpene biosynthetic pathways may be widespread in the plant kingdom, as we demonstrate that it also occurs in the regulation of monoterpene indole alkaloid biosynthesis in the medicinal plant Catharanthus roseus.

Transient Gene Expression in Catharanthus roseus Flower Petals Using Agroinfiltration.

Transient transformation methods are frequently used to determine gene function. However, until recently only a few methods have been available in the model medicinal plant Catharanthus roseus. Here, we describe a rapid and highly reproducible protocol for the overexpression of genes of interest by agroinfiltration of C. roseus flower petals. This high throughput method is particularly suitable for screening purposes, for instance, target gene screening of transcription factor candidates, and complements other available methods.

Phytohormones: Multifunctional nutraceuticals against metabolic syndrome and comorbid diseases.

Metabolic syndrome is characterized by the co-occurrence of diverse symptoms initiating the development of type 2 diabetes, cardiovascular diseases, and a variety of comorbid diseases. The complex constellation of numerous comorbidities makes it difficult to develop common therapeutic approaches that ameliorate these pathological features simultaneously. The plant hormones abscisic acid, salicylic acid, auxin, and cytokinins, have shown promising anti-inflammatory and pro-metabolic effects that could mitigate several disorders relevant to metabolic syndrome. Intriguingly, besides plants, human cells and gut microbes also endogenously produce these molecules, indicating a role in the complex interplay between inflammatory responses associated with metabolic syndrome, the gut microbiome, and nutrition. Here, we introduce how bioactive phytohormones can be generated endogenously and through the gut microbiome. These molecules subsequently influence immune responses and metabolism. We also elaborate on how phytohormones can beneficially modulate metabolic syndrome comorbidities, and propose them as nutraceuticals.

CYP712K4 Catalyzes the C-29 Oxidation of Friedelin in the Maytenus ilicifolia Quinone Methide Triterpenoid Biosynthesis Pathway.

The native Brazilian plant Maytenus ilicifolia accumulates a set of quinone methide triterpenoids with important pharmacological properties, of which maytenin, pristimerin and celastrol accumulate exclusively in the root bark of this medicinal plant. The first committed step in the quinone methide triterpenoid biosynthesis is the cyclization of 2,3-oxidosqualene to friedelin, catalyzed by the oxidosqualene cyclase friedelin synthase (FRS). In this study, we produced heterologous friedelin by the expression of M. ilicifolia FRS in Nicotiana benthamiana leaves and in a Saccharomyces cerevisiae strain engineered using CRISPR/Cas9. Furthermore, friedelin-producing N. benthamiana leaves and S. cerevisiae cells were used for the characterization of CYP712K4, a cytochrome P450 from M. ilicifolia that catalyzes the oxidation of friedelin at the C-29 position, leading to maytenoic acid, an intermediate of the quinone methide triterpenoid biosynthesis pathway. Maytenoic acid produced in N. benthamiana leaves was purified and its structure was confirmed using high-resolution mass spectrometry and nuclear magnetic resonance analysis. The three-step oxidation of friedelin to maytenoic acid by CYP712K4 can be considered as the second step of the quinone methide triterpenoid biosynthesis pathway, and may form the basis for further discovery of the pathway and heterologous production of friedelanes and ultimately quinone methide triterpenoids.

Review: Endoplasmic Reticulum-Associated Degradation (ERAD)-Dependent Control of (Tri)terpenoid Metabolism in Plants.

Plants are sessile organisms. Therefore, they developed the capacity to quickly respond to biotic and abiotic environmental stresses, for instance by producing a broad spectrum of bioactive specialized metabolites. In this defense response, the jasmonate phytohormones can instigate a signaling cascade that leads to the specific elicitation and reprograming of numerous metabolic pathways. Recent research progress has provided several insights into the regulatory networks of many specialized metabolic pathways, mainly at the transcriptional level. Nonetheless, our view on the regulation of defense metabolism remains far from comprehensive. Here, we describe the recent advances obtained with regard to one aspect of the regulation of plant specialized metabolism, namely the posttranslational regulation of enzyme stability. We focus on terpenoid biosynthesis and in particular on the rate-limiting and well-investigated enzyme of the terpenoid precursor pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). There are clear similarities, as well as important mechanistic differences, among the components involved in the posttranslational regulation of terpenoid biosynthesis via HMGR in plants, yeasts, and mammals. Furthermore, in plants, several of these components evolved to respond to specific signaling cues. Indeed, the elements of the plant endoplasmic reticulum-associated degradation (ERAD) and ER stress-associated processes can be induced upon environmental stresses and during specific developmental processes, thereby allowing a unique posttranslational regulation of terpenoid biosynthesis pathways.

An engineered combinatorial module of transcription factors boosts production of monoterpenoid indole alkaloids in Catharanthus roseus.

To fend off microbial pathogens and herbivores, plants have evolved a wide range of defense strategies such as physical barriers, or the production of anti-digestive proteins or bioactive specialized metabolites. Accumulation of the latter compounds is often regulated by transcriptional activation of the biosynthesis pathway genes by the phytohormone jasmonate-isoleucine. Here, we used our recently developed flower petal transformation method in the medicinal plant Catharanthus roseus to shed light on the complex regulatory mechanisms steering the jasmonate-modulated biosynthesis of monoterpenoid indole alkaloids (MIAs), to which the anti-cancer compounds vinblastine and vincristine belong. By combinatorial overexpression of the transcriptional activators BIS1, ORCA3 and MYC2a, we provide an unprecedented insight into the modular transcriptional control of MIA biosynthesis. Furthermore, we show that the expression of an engineered de-repressed MYC2a triggers a tremendous reprogramming of the MIA pathway, finally leading to massively increased accumulation of at least 23 MIAs. The current study unveils an innovative approach for future metabolic engineering efforts for the production of valuable bioactive plant compounds in non-model plants.

Abscisic Acid as Pathogen Effector and Immune Regulator.

Abscisic acid (ABA) is a sesquiterpene signaling molecule produced in all kingdoms of life. To date, the best known functions of ABA are derived from its role as a major phytohormone in plant abiotic stress resistance. Different organisms have developed different biosynthesis and signal transduction pathways related to ABA. Despite this, there are also intriguing common themes where ABA often suppresses host immune responses and is utilized by pathogens as an effector molecule. ABA also seems to play an important role in compatible mutualistic interactions such as mycorrhiza and rhizosphere bacteria with plants, and possibly also the animal gut microbiome. The frequent use of ABA in inter-species communication could be a possible reason for the wide distribution and re-invention of ABA as a signaling molecule in different organisms. In humans and animal models, it has been shown that ABA treatment or nutrient-derived ABA is beneficial in inflammatory diseases like colitis and type 2 diabetes, which confer potential to ABA as an interesting nutraceutical or pharmacognostic drug. The anti-inflammatory activity, cellular metabolic reprogramming, and other beneficial physiological and psychological effects of ABA treatment in humans and animal models has sparked an interest in this molecule and its signaling pathway as a novel pharmacological target. In contrast to plants, however, very little is known about the ABA biosynthesis and signaling in other organisms. Genes, tools and knowledge about ABA from plant sciences and studies of phytopathogenic fungi might benefit biomedical studies on the physiological role of endogenously generated ABA in humans.

The ancient CYP716 family is a major contributor to the diversification of eudicot triterpenoid biosynthesis.

Triterpenoids are widespread bioactive plant defence compounds with potential use as pharmaceuticals, pesticides and other high-value products. Enzymes belonging to the cytochrome P450 family have an essential role in creating the immense structural diversity of triterpenoids across the plant kingdom. However, for many triterpenoid oxidation reactions, the corresponding enzyme remains unknown. Here we characterize CYP716 enzymes from different medicinal plant species by heterologous expression in engineered yeasts and report ten hitherto unreported triterpenoid oxidation activities, including a cyclization reaction, leading to a triterpenoid lactone. Kingdom-wide phylogenetic analysis of over 400 CYP716s from over 200 plant species reveals details of their evolution and suggests that in eudicots the CYP716s evolved specifically towards triterpenoid biosynthesis. Our findings underscore the great potential of CYP716s as a source for generating triterpenoid structural diversity and expand the toolbox available for synthetic biology programmes for sustainable production of bioactive plant triterpenoids.

Role and functioning of bHLH transcription factors in jasmonate signalling.

Plant growth, development and interaction with the environment involve the action of multiple phytohormones. Transcription factors (TFs) of diverse families play essential roles in the signalling cascades triggered by the perception of a particular hormone. TFs may act alone or in a combinatorial fashion with other TFs, and may act specifically in a single hormonal signalling cascade or as signalling hubs for multiple hormones. In the signalling cascades triggered by the phytohormone jasmonate (JA), which modulates a diverse, but specific, range of aspects of plant growth, development and defence, the TFs of the basic helix-loop-helix (bHLH) family play an essential and often conserved role in the plant kingdom. Here, we first discuss the bHLH TFs involved in all kinds of JA-modulated processes in the model plant Arabidopsis thaliana. Secondly, we elaborate on the identity and role of bHLH TFs in the conserved JA-mediated elicitation of specialized metabolism of medicinal and crop species. Finally, we discuss which directions future fundamental research on the functioning of bHLH TFs in JA signalling may head for and how this research can be translated from model plants into crop and medicinal plant species to engineer traits of agronomical and industrial interest.

The basic helix-loop-helix transcription factor BIS2 is essential for monoterpenoid indole alkaloid production in the medicinal plant Catharanthus roseus.

Monoterpenoid indole alkaloids (MIAs) are produced as plant defence compounds. In the medicinal plant Catharanthus roseus, they comprise the anticancer compounds vinblastine and vincristine. The iridoid (monoterpenoid) pathway forms one of the two branches that feed MIA biosynthesis and its activation is regulated by the transcription factor (TF) basic helix-loop-helix (bHLH) iridoid synthesis 1 (BIS1). Here, we describe the identification and characterisation of BIS2, a jasmonate (JA)-responsive bHLH TF expressed preferentially in internal phloem-associated parenchyma cells, which transactivates promoters of iridoid biosynthesis genes and can homodimerise or form heterodimers with BIS1. Stable overexpression of BIS2 in C. roseus suspension cells and transient ectopic expression of BIS2 in C. roseus petal limbs resulted in increased transcript accumulation of methylerythritol-4-phosphate and iridoid pathway genes, but not of other MIA genes or triterpenoid genes. Transcript profiling also indicated that BIS2 expression is part of an amplification loop, as it is induced by overexpression of either BIS1 or BIS2. Accordingly, silencing of BIS2 in C. roseus suspension cells completely abolished the JA-induced upregulation of the iridoid pathway genes and subsequent MIA accumulation, despite the presence of induced BIS1, indicating that BIS2 is essential for MIA production in C. roseus.

GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway.

Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules produced by solanaceous species. They contribute to pathogen defence but are toxic to humans and considered as anti-nutritional compounds. Here we show that GLYCOALKALOID METABOLISM 9 (GAME9), an APETALA2/Ethylene Response Factor, related to regulators of alkaloid production in tobacco and Catharanthus roseus, controls SGA biosynthesis. GAME9 knockdown and overexpression in tomato and potato alters expression of SGAs and upstream mevalonate pathway genes including the cholesterol biosynthesis gene STEROL SIDE CHAIN REDUCTASE 2 (SSR2). Levels of SGAs, C24-alkylsterols and the upstream mevalonate and cholesterol pathways intermediates are modified in these plants. Δ(7)-STEROL-C5(6)-DESATURASE (C5-SD) in the hitherto unresolved cholesterol pathway is a direct target of GAME9. Transactivation and promoter-binding assays show that GAME9 exerts its activity either directly or cooperatively with the SlMYC2 transcription factor as in the case of the C5-SD gene promoter. Our findings provide insight into the regulation of SGA biosynthesis and means for manipulating these metabolites in crops.

Transcript profiling of jasmonate-elicited Taxus cells reveals a ß-phenylalanine-CoA ligase.

Plant cell cultures constitute eco-friendly biotechnological platforms for the production of plant secondary metabolites with pharmacological activities, as well as a suitable system for extending our knowledge of secondary metabolism. Despite the high added value of taxol and the importance of taxanes as anticancer compounds, several aspects of their biosynthesis remain unknown. In this work, a genomewide expression analysis of jasmonate-elicited Taxus baccata cell cultures by complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) indicated a correlation between an extensive elicitor-induced genetic reprogramming and increased taxane production in the targeted cultures. Subsequent in silico analysis allowed us to identify 15 genes with a jasmonate-induced differential expression as putative candidates for genes encoding enzymes involved in five unknown steps of taxane biosynthesis. Among them, the TB768 gene showed a strong homology, including a very similar predicted 3D structure, with other genes previously reported to encode acyl-CoA ligases, thus suggesting a role in the formation of the taxol lateral chain. Functional analysis confirmed that the TB768 gene encodes an acyl-CoA ligase that localizes to the cytoplasm and is able to convert ß-phenylalanine, as well as coumaric acid, into their respective derivative CoA esters. ß-phenylalanyl-CoA is attached to baccatin III in one of the last steps of the taxol biosynthetic pathway. The identification of this gene will contribute to the establishment of sustainable taxol production systems through metabolic engineering or synthetic biology approaches.

The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus.

Plants make specialized bioactive metabolites to defend themselves against attackers. The conserved control mechanisms are based on transcriptional activation of the respective plant species-specific biosynthetic pathways by the phytohormone jasmonate. Knowledge of the transcription factors involved, particularly in terpenoid biosynthesis, remains fragmentary. By transcriptome analysis and functional screens in the medicinal plant Catharanthus roseus (Madagascar periwinkle), the unique source of the monoterpenoid indole alkaloid (MIA)-type anticancer drugs vincristine and vinblastine, we identified a jasmonate-regulated basic helix-loop-helix (bHLH) transcription factor from clade IVa inducing the monoterpenoid branch of the MIA pathway. The bHLH iridoid synthesis 1 (BIS1) transcription factor transactivated the expression of all of the genes encoding the enzymes that catalyze the sequential conversion of the ubiquitous terpenoid precursor geranyl diphosphate to the iridoid loganic acid. BIS1 acted in a complementary manner to the previously characterized ethylene response factor Octadecanoid derivative-Responsive Catharanthus APETALA2-domain 3 (ORCA3) that transactivates the expression of several genes encoding the enzymes catalyzing the conversion of loganic acid to the downstream MIAs. In contrast to ORCA3, overexpression of BIS1 was sufficient to boost production of high-value iridoids and MIAs in C. roseus suspension cell cultures. Hence, BIS1 might be a metabolic engineering tool to produce sustainably high-value MIAs in C. roseus plants or cultures.

Selection and validation of reference genes for transcript normalization in gene expression studies in Catharanthus roseus.

Quantitative Real-Time PCR (qPCR), a sensitive and commonly used technique for gene expression analysis, requires stably expressed reference genes for normalization of gene expression. Up to now, only one reference gene for qPCR analysis, corresponding to 40S Ribosomal protein S9 (RPS9), was available for the medicinal plant Catharanthus roseus, the only source of the commercial anticancer drugs vinblastine and vincristine. Here, we screened for additional reference genes for this plant species by mining C. roseus RNA-Seq data for orthologs of 22 genes known to be stably expressed in Arabidopsis thaliana and qualified as superior reference genes for this model plant species. Based on this, eight candidate C. roseus reference genes were identified and, together with RPS9, evaluated by performing qPCR on a series of different C. roseus explants and tissue cultures. NormFinder, geNorm and BestKeeper analyses of the resulting qPCR data revealed that the orthologs of At2g28390 (SAND family protein, SAND), At2g32170 (N2227-like family protein, N2227) and At4g26410 (Expressed protein, EXP) had the highest expression stability across the different C. roseus samples and are superior as reference genes as compared to the traditionally used RPS9. Analysis of publicly available C. roseus RNA-Seq data confirmed the expression stability of SAND and N2227, underscoring their value as reference genes for C. roseus qPCR analysis.

Taximin, a conserved plant-specific peptide is involved in the modulation of plant-specialized metabolism.

Small peptides play important roles in the signalling cascades that steer plant growth, development and defence, and often crosstalk with hormonal signalling. Thereby, they also modulate metabolism, including the production of bioactive molecules that are of high interest for human applications. Yew species (Taxus spp.) produce diterpenes such as the powerful anticancer agent paclitaxel, the biosynthesis of which can be stimulated by the hormone jasmonate, both in whole plants and cell suspension cultures. Here, we identified Taximin, as a gene encoding a hitherto unreported, plant-specific, small, cysteine-rich signalling peptide, through a transcriptome survey of jasmonate-elicited T. baccata suspension cells grown in two-media cultures. Taximin expression increased in a coordinated manner with that of paclitaxel biosynthesis genes. Tagged Taximin peptides were shown to enter the secretory system and localize to the plasma membrane. In agreement with this, the exogenous application of synthetic Taximin peptide variants could transiently modulate the biosynthesis of taxanes in T. baccata cell suspension cultures. Importantly, the Taximin peptide is widely conserved in the higher plant kingdom with a high degree of sequence conservation. Accordingly, Taximin overexpression could stimulate the production of nicotinic alkaloids in Nicotiana tabacum hairy root cultures in a synergistic manner with jasmonates. In contrast, no pronounced effects of Taximin overexpression on the specialized metabolism in Medicago truncatula roots were observed. This study increases our understanding of the regulation of Taxus diterpene biosynthesis in particular and plant metabolism in general. Ultimately, Taximin might increase the practical potential of metabolic engineering of medicinal plants.

Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase from Bupleurum falcatum.

The saikosaponins comprise oleanane- and ursane-type triterpene saponins that are abundantly present in the roots of the genus Bupleurum widely used in Asian traditional medicine. Here we identified a gene, designated CYP716Y1, encoding a cytochrome P450 monooxygenase from Bupleurum falcatum that catalyzes the C-16α hydroxylation of oleanane- and ursane-type triterpenes. Exploiting this hitherto unavailable enzymatic activity, we launched a combinatorial synthetic biology program in which we combined CYP716Y1 with oxidosqualene cyclase, P450, and glycosyltransferase genes available from other plant species and reconstituted the synthesis of monoglycosylated saponins in yeast. Additionally, we established a culturing strategy in which applying methylated ß-cyclodextrin to the culture medium allows the sequestration of heterologous nonvolatile hydrophobic terpenes, such as triterpene sapogenins, from engineered yeast cells into the growth medium, thereby greatly enhancing productivity. Together, our findings provide a sound base for the development of a synthetic biology platform for the production of bioactive triterpene sapo(ge)nins.

Differential transcriptome analysis of glandular and filamentous trichomes in Artemisia annua.

BACKGROUND: The medicinal plant Artemisia annua is covered with filamentous trichomes and glandular, artemisinin producing trichomes. A high artemisinin supply is needed at a reduced cost for treating malaria. Artemisinin production in bioreactors can be facilitated if a better insight is obtained in the biosynthesis of artemisinin and other metabolites. Therefore, metabolic activities of glandular and filamentous trichomes were investigated at the transcriptome level. RESULTS: By laser pressure catapulting, glandular and filamentous trichomes as well as apical and sub-apical cells from glandular trichomes were collected and their transcriptome was sequenced using Illumina RNA-Seq. A de novo transcriptome was assembled (Trinity) and studied with a differential expression analysis (edgeR).A comparison of the transcriptome from glandular and filamentous trichomes shows that MEP, MVA, most terpene and lipid biosynthesis pathways are significantly upregulated in glandular trichomes. Conversely, some transcripts coding for specific sesquiterpenoid and triterpenoid enzymes such as 8-epi-cedrol synthase and an uncharacterized oxidosqualene cyclase were significantly upregulated in filamentous trichomes. All known artemisinin biosynthesis genes are upregulated in glandular trichomes and were detected in both the apical and sub-apical cells of the glandular trichomes. No significant differential expression could be observed between the apical and sub-apical cells. CONCLUSIONS: Our results underscore the vast metabolic capacities of A. annua glandular trichomes but nonetheless point to the existence of specific terpene metabolic pathways in the filamentous trichomes. Candidate genes that might be involved in artemisinin biosynthesis are proposed based on their putative function and their differential expression level.

Metabolite profiling of plant tissues by liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry.

Plants accumulate an overwhelming variety of secondary metabolites that play important roles in defense and interaction of the plant with its environment. To investigate the dynamics of plant secondary metabolism, large-scale untargeted metabolite profiling (metabolomics) is mandatory. Here, we describe a detailed protocol for untargeted metabolite profiling in which methanol extracts of jasmonate-treated plant tissues are analyzed by reversed-phase liquid chromatography coupled to negative-ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (MS). By means of dedicated integration and alignment software, the relative abundance of thousands of mass peaks, corresponding to hundreds of compounds, is calculated, and mass peaks of which the area is significantly changed by jasmonate treatment are identified. Subsequently, the metabolites corresponding to the significantly changed peaks are tentatively annotated using the accurate mass prediction of the Fourier transform-MS and the generated MS/MS data. Via this method, compounds of medium polarity, such as glucosinolates, alkaloids, phenylpropanoids, flavonoids, polyamines, and saponins, can be analyzed.

CathaCyc, a metabolic pathway database built from Catharanthus roseus RNA-Seq data.

The medicinal plant Madagascar periwinkle (Catharanthus roseus) synthesizes numerous terpenoid indole alkaloids (TIAs), such as the anticancer drugs vinblastine and vincristine. The TIA pathway operates in a complex metabolic network that steers plant growth and survival. Pathway databases and metabolic networks reconstructed from 'omics' sequence data can help to discover missing enzymes, study metabolic pathway evolution and, ultimately, engineer metabolic pathways. To date, such databases have mainly been built for model plant species with sequenced genomes. Although genome sequence data are not available for most medicinal plant species, next-generation sequencing is now extensively employed to create comprehensive medicinal plant transcriptome sequence resources. Here we report on the construction of CathaCyc, a detailed metabolic pathway database, from C. roseus RNA-Seq data sets. CathaCyc (version 1.0) contains 390 pathways with 1,347 assigned enzymes and spans primary and secondary metabolism. Curation of the pathways linked with the synthesis of TIAs and triterpenoids, their primary metabolic precursors, and their elicitors, the jasmonate hormones, demonstrated that RNA-Seq resources are suitable for the construction of pathway databases. CathaCyc is accessible online (http://www.cathacyc.org) and offers a range of tools for the visualization and analysis of metabolic networks and 'omics' data. Overlay with expression data from publicly available RNA-Seq resources demonstrated that two well-characterized C. roseus terpenoid pathways, those of TIAs and triterpenoids, are subject to distinct regulation by both developmental and environmental cues. We anticipate that databases such as CathaCyc will become key to the study and exploitation of the metabolism of medicinal plants.