Diversity and evolution of cytochrome P450s of Jacobaea vulgaris and Jacobaea aquatica.

BACKGROUND: Collectively, plants produce a huge variety of secondary metabolites (SMs) which are involved in the adaptation of plants to biotic and abiotic stresses. The most characteristic feature of SMs is their striking inter- and intraspecific chemical diversity. Cytochrome P450 monooxygenases (CYPs) often play an important role in the biosynthesis of SMs and thus in the evolution of chemical diversity. Here we studied the diversity and evolution of CYPs of two Jacobaea species which contain a characteristic group of SMs namely the pyrrolizidine alkaloids (PAs). RESULTS: We retrieved CYPs from RNA-seq data of J. vulgaris and J. aquatica, resulting in 221 and 157 full-length CYP genes, respectively. The analyses of conserved motifs confirmed that Jacobaea CYP proteins share conserved motifs including the heme-binding signature, the PERF motif, the K-helix and the I-helix. KEGG annotation revealed that the CYPs assigned as being SM metabolic pathway genes were all from the CYP71 clan but no CYPs were assigned as being involved in alkaloid pathways. Phylogenetic analyses of full-length CYPs were conducted for the six largest CYP families of Jacobaea (CYP71, CYP76, CYP706, CYP82, CYP93 and CYP72) and were compared with CYPs of two other members of the Asteraceae, Helianthus annuus and Lactuca sativa, and with Arabidopsis thaliana. The phylogenetic trees showed strong lineage specific diversification of CYPs, implying that the evolution of CYPs has been very fast even within the Asteraceae family. Only in the closely related species J. vulgaris and J. aquatica, CYPs were found often in pairs, confirming a close relationship in the evolutionary history. CONCLUSIONS: This study discovered 378 full-length CYPs in Jacobaea species, which can be used for future exploration of their functions, including possible involvement in PA biosynthesis and PA diversity.

An E. coli biosensor for screening of cDNA libraries for isochorismate pyruvate lyase-encoding cDNAs.

Salicylic acid (SA) is an essential hormone for development and induced defense against biotrophic pathogens in plants. The formation of SA mainly derives from chorismate via demonstrated isochorismate synthase (ICS) and presumed isochorismate pyruvate lyase (IPL)-mediated steps in Arabidopsis thaliana, but so far no plant enzyme displaying IPL activity has been identified. Here, we developed an E. coli SA biosensor to screen for IPL activity based on the SalR regulator/salA promoter combination from Acinetobacter sp ADP1, to control the expression of the reporter luxCDABE. The biosensor was responsive to micromolar concentrations of exogenous SA, and to endogenous SA produced after transformation with a plasmid permitting IPTG-inducible expression of bacterial IPL in this biosensor strain. After screening a cDNA library constructed from turnip crinkle virus (TCV)-infected Arabidopsis ecotype Di-17, we identified an enzyme, PRXR1, as a putative IPL that converts isochorismate into SA. Our results provide a new experimental approach to identify IPL and new insights into the SA biosynthesis pathway in Arabidopsis.

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.

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.

Salicylic acid suppresses jasmonic acid signaling downstream of SCFCOI1-JAZ by targeting GCC promoter motifs via transcription factor ORA59.

Antagonism between the defense hormones salicylic acid (SA) and jasmonic acid (JA) plays a central role in the modulation of the plant immune signaling network, but the molecular mechanisms underlying this phenomenon are largely unknown. Here, we demonstrate that suppression of the JA pathway by SA functions downstream of the E3 ubiquitin-ligase Skip-Cullin-F-box complex SCF(COI1), which targets JASMONATE ZIM-domain transcriptional repressor proteins (JAZs) for proteasome-mediated degradation. In addition, neither the stability nor the JA-induced degradation of JAZs was affected by SA. In silico promoter analysis of the SA/JA crosstalk transcriptome revealed that the 1-kb promoter regions of JA-responsive genes that are suppressed by SA are significantly enriched in the JA-responsive GCC-box motifs. Using GCC:GUS lines carrying four copies of the GCC-box fused to the ß-glucuronidase reporter gene, we showed that the GCC-box motif is sufficient for SA-mediated suppression of JA-responsive gene expression. Using plants overexpressing the GCC-box binding APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors ERF1 or ORA59, we found that SA strongly reduces the accumulation of ORA59 but not that of ERF1. Collectively, these data indicate that the SA pathway inhibits JA signaling downstream of the SCF(COI1)-JAZ complex by targeting GCC-box motifs in JA-responsive promoters via a negative effect on the transcriptional activator ORA59.

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.

Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization.

Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and sub-cellular localization of VoGES and LdGES were characterized in stable transformed tobacco and using transient expression in Nicotiana benthamiana. Transgenic tobacco expressing VoGES or LdGES accumulate geraniol, oxidized geraniol compounds like geranial, geranic acid and hexose conjugates of these compounds to similar levels. Geraniol emission of leaves was lower than that of flowers, which could be related to higher levels of competing geraniol-conjugating activities in leaves. GFP-fusions of the two GES proteins show that VoGES resides (as expected) predominantly in the plastids, while LdGES import into to the plastid is clearly impaired compared to that of VoGES, resulting in both cytosolic and plastidic localization. Geraniol production by VoGES and LdGES in N. benthamiana was nonetheless very similar. Expression of a truncated version of VoGES or LdGES (cytosolic targeting) resulted in the accumulation of 30% less geraniol glycosides than with the plastid targeted VoGES and LdGES, suggesting that the substrate geranyl diphosphate is readily available, both in the plastids as well as in the cytosol. The potential role of GES in the engineering of the TIA pathway in heterologous hosts is discussed.

Geraniol hydroxylase and hydroxygeraniol oxidase activities of the CYP76 family of cytochrome P450 enzymes and potential for engineering the early steps of the (seco)iridoid pathway.

The geraniol-derived (seco)iridoid skeleton is a precursor for a large group of bioactive compounds with diverse therapeutic applications, including the widely used anticancer molecule vinblastine. Despite of this economic prospect, the pathway leading to iridoid biosynthesis from geraniol is still unclear. The first geraniol hydroxylation step has been reported to be catalyzed by cytochrome P450 enzymes such as CYP76B6 from Catharanthus roseus and CYP76C1 from Arabidopsis thaliana. In the present study, an extended functional analysis of CYP76 family members was carried-out to identify the most effective enzyme to be used for pathway reconstruction. This disproved CYP76C1 activity and led to the characterization of CYP76C4 from A. thaliana as a geraniol 9- or 8-hydroxylase. CYP76B6 emerged as a highly specialized multifunctional enzyme catalyzing two sequential oxidation steps leading to the formation of 8-oxogeraniol from geraniol. This dual function was confirmed in planta using a leaf-disc assay. The first step, geraniol hydroxylation, was very efficient and fast enough to outcompete geraniol conjugation in plant tissues. When the enzyme was expressed in leaf tissues, 8-oxogeraniol was converted into further oxidized and/or reduced compounds in the absence of the next enzyme of the iridoid pathway.

The basic helix-loop-helix transcription factor CrMYC2 controls the jasmonate-responsive expression of the ORCA genes that regulate alkaloid biosynthesis in Catharanthus roseus.

Jasmonates are plant signalling molecules that play key roles in defence against insects and certain pathogens, among others by controlling the biosynthesis of protective secondary metabolites. In Catharanthus roseus, the AP2/ERF-domain transcription factor ORCA3 controls the jasmonate-responsive expression of several genes encoding enzymes involved in terpenoid indole alkaloid biosynthesis. ORCA3 gene expression is itself induced by jasmonate. The ORCA3 promoter contains an autonomous jasmonate-responsive element (JRE) composed of a quantitative sequence responsible for the high level of expression and a qualitative sequence that acts as an on/off switch in response to methyl-jasmonate (MeJA). Here, we identify the basic helix-loop-helix (bHLH) transcription factor CrMYC2 as the major activator of MeJA-responsive ORCA3 gene expression. The CrMYC2 gene is an immediate-early jasmonate-responsive gene. CrMYC2 binds to the qualitative sequence in the ORCA3 JRE in vitro, and transactivates reporter gene expression via this sequence in transient assays. Knock-down of the CrMYC2 expression level via RNA interference caused a strong reduction in the level of MeJA-responsive ORCA3 mRNA accumulation. In addition, MeJA-responsive expression of the related transcription factor gene ORCA2 was significantly reduced. Our results show that MeJA-responsive expression of alkaloid biosynthesis genes in C. roseus is controlled by a transcription factor cascade consisting of the bHLH protein CrMYC2 regulating ORCA gene expression, and the AP2/ERF-domain transcription factors ORCA2 and ORCA3, which in turn regulate a subset of alkaloid biosynthesis genes.

Two GCC boxes and AP2/ERF-domain transcription factor ORA59 in jasmonate/ethylene-mediated activation of the PDF1.2 promoter in Arabidopsis.

Plant defense against microbial pathogens depends on the action of several endogenously produced hormones, including jasmonic acid (JA) and ethylene (ET). In defense against necrotrophic pathogens, the JA and ET signaling pathways synergize to activate a specific set of defense genes including PLANT DEFENSIN1.2 (PDF1.2). The APETALA2/Ethylene Response Factor (AP2/ERF)-domain transcription factor ORA59 acts as the integrator of the JA and ET signaling pathways and is the key regulator of JA- and ET-responsive PDF1.2 expression. The present study was aimed at the identification of elements in the PDF1.2 promoter conferring the synergistic response to JA/ET and interacting with ORA59. We show that the PDF1.2 promoter was activated synergistically by JA and the ET-releasing agent ethephon due to the activity of two GCC boxes. ORA59 bound in vitro to these GCC boxes and trans-activated the PDF1.2 promoter in transient assays via these two boxes. Using the chromatin immunoprecipitation technique we were able to show that ORA59 bound the PDF1.2 promoter in vivo. Finally, we show that a tetramer of a single GCC box conferred JA/ethephon-responsive expression, demonstrating that the JA and ET signaling pathways converge to a single type of GCC box. Therefore ORA59 and two functionally equivalent GCC box binding sites form the module that enables the PDF1.2 gene to respond synergistically to simultaneous activation of the JA and ET signaling pathways.

The jasmonate-responsive element from the ORCA3 promoter from Catharanthus roseus is active in Arabidopsis and is controlled by the transcription factor AtMYC2.

Jasmonates are plant signaling molecules that play key roles in protection against certain pathogens and against insects by switching on the expression of genes encoding defense proteins including enzymes involved in the biosynthesis of toxic secondary metabolites. In Catharanthus roseus, the ethylene response factor (ERF) transcription factor ORCA3 controls the jasmonate-responsive activation of terpenoid indole alkaloid biosynthetic genes. ORCA3 gene expression is itself induced by jasmonate. Its promoter contains an autonomous jasmonate-responsive element (JRE). Here we describe the jasmonate-responsive activity of the JRE from the ORCA3 promoter in Arabidopsis thaliana. We found that it interacts in vitro and in vivo with the basic helix-loop-helix transcription factor AtMYC2. Analysis of JRE-mediated reporter gene expression in an atmyc2-1 mutant background showed that the activity was strictly dependent on AtMYC2.

Auxin-induced, SCF(TIR1)-mediated poly-ubiquitination marks AUX/IAA proteins for degradation.

The plant hormone auxin (indole-3-acetic acid or IAA) regulates plant development by inducing rapid cellular responses and changes in gene expression. Auxin promotes the degradation of Aux/IAA transcriptional repressors, thereby allowing auxin response factors (ARFs) to activate the transcription of auxin-responsive genes. Auxin enhances the binding of Aux/IAA proteins to the receptor TIR1, which is an F-box protein that is part of the E3 ubiquitin ligase complex SCF(TIR1). Binding of Aux/IAA proteins leads to degradation via the 26S proteasome, but evidence for SCF(TIR1)-mediated poly-ubiquitination of Aux/IAA proteins is lacking. Here we used an Arabidopsis cell suspension-based protoplast system to find evidence for SCF(TIR1)-mediated ubiquitination of the Aux/IAA proteins SHY2/IAA3 and BDL/IAA12. Each of these proteins showed a distinct abundance and repressor activity when expressed in this cell system. Moreover, the amount of endogenous TIR1 protein appeared to be rate-limiting for a proper auxin response measured by the co-transfected DR5::GUS reporter construct. Co-transfection with 35S::TIR1 led to auxin-dependent degradation, and excess of 35S::TIR1 even led to degradation of Aux/IAAs in the absence of auxin treatment. Expression of the mutant tir1-1 protein or the related F-box protein COI1, which is involved in jasmonate signaling, had no effect on Aux/IAA degradation. Our results show that SHY2/IAA3 and BDL/IAA12 are poly-ubiquitinated and degraded in response to increased auxin or TIR1 levels. In conclusion, our data provide experimental support for the model that SCF(TIR1)-dependent poly-ubiquitination of Aux/IAA proteins marks these proteins for degradation by the 26S proteasome, leading to activation of auxin-responsive gene expression.

Ethylene signaling renders the jasmonate response of Arabidopsis insensitive to future suppression by salicylic Acid.

Cross-talk between jasmonate (JA), ethylene (ET), and Salicylic acid (SA) signaling is thought to operate as a mechanism to fine-tune induced defenses that are activated in response to multiple attackers. Here, 43 Arabidopsis genotypes impaired in hormone signaling or defense-related processes were screened for their ability to express SA-mediated suppression of JA-responsive gene expression. Mutant cev1, which displays constitutive expression of JA and ET responses, appeared to be insensitive to SA-mediated suppression of the JA-responsive marker genes PDF1.2 and VSP2. Accordingly, strong activation of JA and ET responses by the necrotrophic pathogens Botrytis cinerea and Alternaria brassicicola prior to SA treatment counteracted the ability of SA to suppress the JA response. Pharmacological assays, mutant analysis, and studies with the ET-signaling inhibitor 1-methylcyclopropene revealed that ET signaling renders the JA response insensitive to subsequent suppression by SA. The APETALA2/ETHYLENE RESPONSE FACTOR transcription factor ORA59, which regulates JA/ET-responsive genes such as PDF1.2, emerged as a potential mediator in this process. Collectively, our results point to a model in which simultaneous induction of the JA and ET pathway renders the plant insensitive to future SA-mediated suppression of JA-dependent defenses, which may prioritize the JA/ET pathway over the SA pathway during multi-attacker interactions.

The use of genetics to dissect plant secondary pathways.

Plant secondary metabolism comprises an enormous diversity in compounds and enzymes, and wide spectra of mechanisms of gene regulation and of transport of metabolites and enzymes. Genetic approaches using the model plant Arabidopsis thaliana have contributed importantly to recent progress in understanding glucosinolate biosynthesis and its intricate linkage with auxin homeostasis. Arabidopsis genetics have also caused revolutionary changes in the existing views on the metabolic intermediates and enzyme activities that are involved in phenylpropanoid biosynthesis. Some progress has been achieved in understanding the transcriptional regulation of the flavonoid pathway. Transcriptional regulators have also been identified for glucosinolate and terpenoid indole alkaloid biosynthesis.

Tailoring the plant metabolome without a loose stitch.

Metabolic engineering holds great promise as a technique for improving crop plants. However, introducing new metabolic steps can disturb normal metabolism and gene expression, affecting phenotype and quality in undesired ways. Recently, Charlotte Kristensen et al. reported that introducing the sorghum pathway for biosynthesis of the cyanogenic glucoside dhurrin into Arabidopsis plants resulted in high dhurrin levels and only marginal side effects on the metabolome and the transcriptome.

Jasmonate-responsive transcription factors regulating plant secondary metabolism.

Plants produce a large variety of secondary metabolites including alkaloids, glucosinolates, terpenoids and phenylpropanoids. These compounds play key roles in plant-environment interactions and many of them have pharmacological activity in humans. Jasmonates (JAs) are plant hormones which induce biosynthesis of many secondary metabolites. JAs-responsive transcription factors (TFs) that regulate the JAs-induced accumulation of secondary metabolites belong to different families including AP2/ERF, bHLH, MYB and WRKY. Here, we give an overview of the types and functions of TFs that have been identified in JAs-induced secondary metabolite biosynthesis, and highlight their similarities and differences in regulating various biosynthetic pathways. We review major recent developments regarding JAs-responsive TFs mediating secondary metabolite biosynthesis, and provide suggestions for further studies.

Transcription factors: tools to engineer the production of pharmacologically active plant metabolites.

Plants produce a variety of secondary metabolites, some of which are used as pharmaceuticals or are health promoting as food components. Recent genetic studies on the flavonoid biosynthetic pathway show that transcription factors are efficient new molecular tools for plant metabolic engineering to increase the production of valuable compounds. The use of specific transcription factors would avoid the time-consuming step of acquiring knowledge about all enzymatic steps of a poorly characterized biosynthetic pathway. Although genetic approaches are difficult for most plant species, promoter studies of single-pathway genes and T-DNA activation tagging are feasible alternative approaches for isolating transcription factors, as illustrated for terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

Iridoid synthase activity is common among the plant progesterone 5ß-reductase family.

Catharanthus roseus, the Madagascar periwinkle, synthesizes bioactive monoterpenoid indole alkaloids, including the anti-cancer drugs vinblastine and vincristine. The monoterpenoid branch of the alkaloid pathway leads to the secoiridoid secologanin and involves the enzyme iridoid synthase (IS), a member of the progesterone 5ß-reductase (P5ßR) family. IS reduces 8-oxogeranial to iridodial. Through transcriptome mining, we show that IS belongs to a family of six C. roseus P5ßR genes. Characterization of recombinant CrP5ßR proteins demonstrates that all but CrP5ßR3 can reduce progesterone and thus can be classified as P5ßRs. Three of them, namely CrP5ßR1, CrP5ßR2, and CrP5ßR4, can also reduce 8-oxogeranial, pointing to a possible redundancy with IS (corresponding to CrP5ßR5) in secoiridoid synthesis. In-depth functional analysis by subcellular protein localization, gene expression analysis, in situ hybridization, and virus-induced gene silencing indicate that besides IS, CrP5ßR4 may also participate in secoiridoid biosynthesis. We cloned a set of P5ßR genes from angiosperm plant species not known to produce iridoids and demonstrate that the corresponding recombinant proteins are also capable of using 8-oxogeranial as a substrate. This suggests that IS activity is intrinsic to angiosperm P5ßR proteins and has evolved early during evolution.

Transcription factors controlling plant secondary metabolism: what regulates the regulators?

Plants produce secondary metabolites, among others, to protect themselves against microbial and herbivore attack or UV irradiation. Certain metabolite classes also function in beneficial interactions with other organisms. For example, anthocyanin pigments and terpenoid essential oils have key roles in attraction of flower pollinators. Secondary metabolites also have direct uses for man. Flavonoids and terpenoids for example have health-promoting activities as food ingredients, and several alkaloids have pharmacological activities. Controlled transcription of biosynthetic genes is one major mechanism regulating secondary metabolite production in plant cells. Several transcription factors involved in the regulation of metabolic pathway genes have been isolated and studied. There are indications that transcription factor activity itself is regulated by internal or external signals leading to controlled responses. The aim of this review is to discuss the regulation of transcription factors involved in secondary metabolism in plants at gene and protein levels, using phenylpropanoid and terpenoid indole alkaloid pathways as two well-studied examples.

T-DNA activation tagging.

T-DNA activation tagging is a method to generate dominant mutations in plants or plant cells by random insertion of a T-DNA carrying constitutive enhancer elements, which can cause transcriptional activation of flanking plant genes. The method consists of generating a large number of transformed plants or plant cells using a specialized T-DNA construct, followed by selection for the desired phenotype. Subsequently, the activated plant gene is rescued from selected mutant transformants for further functional analysis. Since the exact procedure depends on the plant material and the selected phenotype, this chapter describes one specific example of T-DNA activation tagging of suspension-cultured cells, including, where possible, cross-references to more general applications of the technique.