A redundant transcription factor network steers spatiotemporal Arabidopsis triterpene synthesis.

Plant specialized metabolites modulate developmental and ecological functions and comprise many therapeutic and other high-value compounds. However, the mechanisms determining their cell-specific expression remain unknown. Here we describe the transcriptional regulatory network that underlies cell-specific biosynthesis of triterpenes in Arabidopsis thaliana root tips. Expression of thalianol and marneral biosynthesis pathway genes depends on the phytohormone jasmonate and is limited to outer tissues. We show that this is promoted by the activity of redundant bHLH-type transcription factors from two distinct clades and coactivated by homeodomain factors. Conversely, the DOF-type transcription factor DAG1 and other regulators prevent expression of the triterpene pathway genes in inner tissues. We thus show how precise expression of triterpene biosynthesis genes is determined by a robust network of transactivators, coactivators and counteracting repressors.

Touch signaling and thigmomorphogenesis are regulated by complementary CAMTA3- and JA-dependent pathways.

Plants respond to mechanical stimuli to direct their growth and counteract environmental threats. Mechanical stimulation triggers rapid gene expression changes and affects plant appearance (thigmomorphogenesis) and flowering. Previous studies reported the importance of jasmonic acid (JA) in touch signaling. Here, we used reverse genetics to further characterize the molecular mechanisms underlying touch signaling. We show that Piezo mechanosensitive ion channels have no major role in touch-induced gene expression and thigmomorphogenesis. In contrast, the receptor-like kinase Feronia acts as a strong negative regulator of the JA-dependent branch of touch signaling. Last, we show that calmodulin-binding transcriptional activators CAMTA1/2/3 are key regulators of JA-independent touch signaling. CAMTA1/2/3 cooperate to directly bind the promoters and activate gene expression of JA-independent touch marker genes like TCH2 and TCH4. In agreement, camta3 mutants show a near complete loss of thigmomorphogenesis and touch-induced delay of flowering. In conclusion, we have now identified key regulators of two independent touch-signaling pathways.

Evaluation of Antibiotic-Based Selection Methods for Camelina sativa Stable Transformants.

Camelina sativa (Camelina) is an oilseed crop that in recent years has gained importance due to its closeness to the plant model organism Arabidopsis thaliana (Arabidopsis), its low agronomical requirements, and the ability to grow under temperate conditions. To explore all the agronomical and biotechnological possibilities of this crop, it is important to evaluate the usability of the molecular procedures currently available for plants. One of the main tools for plant genetic modification and genetic studies is stable plant transformation. In the case of Arabidopsis, as well as Camelina, floral dipping is the easiest and most used method, which is followed by a selection for stable transformants. Commonly used selection methods for Camelina involve Discosoma sp. red protein (DsRed) fluorescence screening. However, many widely used plant transformation vector systems, for example those used in Arabidopsis and grasses, rely on antibiotic resistance selection. In this study, we evaluated the usability of different antibiotics including kanamycin (Kan), hygromycin (Hyg) and BASTA, and propose optimised protocols for selecting T1 and subsequent generation Camelina transformants, as well as crossing of Camelina lines expressing different transgenes. Finally, we also showed that overexpression of genes encoding enzymes from the seco-iridoid pathway of Catharanthus roseus using Hyg or BASTA-based expression constructs could be successfully achieved in Camelina, demonstrating the potential of these methods for metabolic engineering. Overall, in this study we show an efficient way to sterilize seeds, handle and perform selection of Camelina for use with transformation vectors designed for Arabidopsis thaliana. We also demonstrate a successful method to cross Camelina sativa and provide qRT-PCR results to prove its effectiveness.

A MYC2/MYC3/MYC4-dependent transcription factor network regulates water spray-responsive gene expression and jasmonate levels.

Mechanical stimuli, such as wind, rain, and touch affect plant development, growth, pest resistance, and ultimately reproductive success. Using water spray to simulate rain, we demonstrate that jasmonic acid (JA) signaling plays a key role in early gene-expression changes, well before it leads to developmental changes in flowering and plant architecture. The JA-activated transcription factors MYC2/MYC3/MYC4 modulate transiently induced expression of 266 genes, most of which peak within 30 min, and control 52% of genes induced >100-fold. Chromatin immunoprecipitation-sequencing analysis indicates that MYC2 dynamically binds >1,300 promoters and trans-activation assays show that MYC2 activates these promoters. By mining our multiomic datasets, we identified a core MYC2/MYC3/MYC4-dependent "regulon" of 82 genes containing many previously unknown MYC2 targets, including transcription factors bHLH19 and ERF109 bHLH19 can in turn directly activate the ORA47 promoter, indicating that MYC2/MYC3/MYC4 initiate a hierarchical network of downstream transcription factors. Finally, we also reveal that rapid water spray-induced accumulation of JA and JA-isoleucine is directly controlled by MYC2/MYC3/MYC4 through a positive amplification loop that regulates JA-biosynthesis genes.

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.

Identification of Iridoid Glucoside Transporters in Catharanthus roseus.

Monoterpenoid indole alkaloids (MIAs) are plant defense compounds and high-value pharmaceuticals. Biosynthesis of the universal MIA precursor, secologanin, is organized between internal phloem-associated parenchyma (IPAP) and epidermis cells. Transporters for intercellular transport of proposed mobile pathway intermediates have remained elusive. Screening of an Arabidopsis thaliana transporter library expressed in Xenopus oocytes identified AtNPF2.9 as a putative iridoid glucoside importer. Eight orthologs were identified in Catharanthus roseus, of which three, CrNPF2.4, CrNPF2.5 and CrNPF2.6, were capable of transporting the iridoid glucosides 7-deoxyloganic acid, loganic acid, loganin and secologanin into oocytes. Based on enzyme expression data and transporter specificity, we propose that several enzymes of the biosynthetic pathway are present in both IPAP and epidermis cells, and that the three transporters are responsible for transporting not only loganic acid, as previously proposed, but multiple intermediates. Identification of the iridoid glucoside-transporting CrNPFs is an important step toward understanding the complex orchestration of the seco-iridioid pathway.

Clade IVa Basic Helix-Loop-Helix Transcription Factors Form Part of a Conserved Jasmonate Signaling Circuit for the Regulation of Bioactive Plant Terpenoid Biosynthesis.

Plants produce many bioactive, specialized metabolites to defend themselves when facing various stress situations. Their biosynthesis is directed by a tightly controlled regulatory circuit that is elicited by phytohormones such as jasmonate (JA). The basic helix-loop-helix (bHLH) transcription factors (TFs) bHLH iridoid synthesis 1 (BIS1) and Triterpene Saponin Activating Regulator (TSAR) 1 and 2, from Catharanthus roseus and Medicago truncatula, respectively, all belong to clade IVa of the bHLH protein family and activate distinct terpenoid pathways, thereby mediating monoterpenoid indole alkaloid (MIA) and triterpene saponin (TS) accumulation, respectively, in these two species. In this study, we report that promoters of the genes encoding the enzymes involved in the specific terpenoid pathway of one of these species can be transactivated by the orthologous bHLH factor from the other species through recognition of the same cis-regulatory elements. Accordingly, ectopic expression of CrBIS1 in M. truncatula hairy roots up-regulated the expression of all genes required for soyasaponin production, resulting in strongly increased levels of soyasaponins in the transformed roots. Likewise, transient expression of MtTSAR1 and MtTSAR2 in C. roseus petals led to up-regulation of the genes involved in the iridoid branch of the MIA pathway. Together, our data illustrate the functional similarity of these JA-inducible TFs and indicate that recruitment of defined cis-regulatory elements constitutes an important aspect of the evolution of conserved regulatory modules for the activation of species-specific terpenoid biosynthesis pathways by common signals such as the JA phytohormones.

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.

CCoAOMT Down-Regulation Activates Anthocyanin Biosynthesis in Petunia.

Anthocyanins and volatile phenylpropenes (isoeugenol and eugenol) in petunia (Petunia hybrida) flowers have the precursor 4-coumaryl coenzyme A (CoA) in common. These phenolics are produced at different stages during flower development. Anthocyanins are synthesized during early stages of flower development and sequestered in vacuoles during the lifespan of the flowers. The production of isoeugenol and eugenol starts when flowers open and peaks after anthesis. To elucidate additional biochemical steps toward (iso)eugenol production, we cloned and characterized a caffeoyl-coenzyme A O-methyltransferase (PhCCoAOMT1) from the petals of the fragrant petunia 'Mitchell'. Recombinant PhCCoAOMT1 indeed catalyzed the methylation of caffeoyl-CoA to produce feruloyl CoA. Silencing of PhCCoAOMT1 resulted in a reduction of eugenol production but not of isoeugenol. Unexpectedly, the transgenic plants had purple-colored leaves and pink flowers, despite the fact that cv Mitchell lacks the functional R2R3-MYB master regulator ANTHOCYANIN2 and has normally white flowers. Our results indicate that down-regulation of PhCCoAOMT1 activated the anthocyanin pathway through the R2R3-MYBs PURPLE HAZE (PHZ) and DEEP PURPLE, with predominantly petunidin accumulating. Feeding cv Mitchell flowers with caffeic acid induced PHZ expression, suggesting that the metabolic perturbation of the phenylpropanoid pathway underlies the activation of the anthocyanin pathway. Our results demonstrate a role for PhCCoAOMT1 in phenylpropene production and reveal a link between PhCCoAOMT1 and anthocyanin production.

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.

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.

Iridoid Synthase Activity Is Common among the Plant Progesterone 5ß-Reductase Family.

Catharanthus roseus, the Madagascar periwinkle, synthesizes bioactive monoterpenoid indole alkaloids, among which 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. Characterisation 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, could also reduce 8-oxogeranial, pointing to a possible redundancy with IS (corresponding to CrP5ßR5) in secoiridoid synthesis. In depth functional analysis by subcellular protein localisation, gene expression analysis, in situ hybridisation and virus-induced gene silencing, indicates that besides IS, CrP5ßR4 may also participate in secoiridoid biosynthesis. Finally, 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.

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.

A peroxisomally localized acyl-activating enzyme is required for volatile benzenoid formation in a Petuniaxhybrida cv. 'Mitchell Diploid' flower.

Floral volatile benzenoid/phenylpropanoid (FVBP) biosynthesis is a complex and coordinate cellular process executed by petal limb cells of a Petunia×hybrida cv. 'Mitchell Diploid' (MD) plant. In MD flowers, the majority of benzenoid volatile compounds are derived from a core phenylpropanoid pathway intermediate by a coenzyme A (CoA) dependent, ß-oxidative scheme. Metabolic flux analysis, reverse genetics, and biochemical characterizations of key enzymes in this pathway have supported this putative concept. However, the theoretical first enzymatic reaction, which leads to the production of cinnamoyl-CoA, has only been physically demonstrated in a select number of bacteria like Streptomyces maritimus through mutagenesis and recombinant protein production. A transcript has been cloned and characterized from MD flowers that shares high homology with an Arabidopsis thaliana transcript ACYL-ACTIVATING ENZYME11 (AtAAE11) and the S. maritimus ACYL-COA:LIGASE (SmEncH). In MD, the PhAAE transcript accumulates in a very similar manner as bona fide FVBP network genes, i.e. high levels in an open flower petal and ethylene regulated. In planta, PhAAE is localized to the peroxisome. Upon reduction of PhAAE transcript through a stable RNAi approach, transgenic flowers emitted a reduced level of all benzenoid volatile compounds. Together, the data suggest that PhAAE may be responsible for the activation of t-cinnamic acid, which would be required for floral volatile benzenoid production in MD.

A model for combinatorial regulation of the petunia R2R3-MYB transcription factor ODORANT1.

The emission of floral volatiles requires coordinated expression of biosynthetic genes. In the regulatory network of the volatile benzenoid/phenylpropanoid pathway in Petunia hybrida two master regulators of the pathway have been identified. The R2R3-MYB transcription factor EMISSION OF BENZENOIDS II (EOBII) utilizes a specific MYB binding site to activate the expression of the R2R3-MYB ODORANT1 (ODO1). However, because EOBII is expressed early in flower development, when ODO1 is not, there must be other factors that play a role in regulating expression of ODO1. Through functional analyses of ODO1 promoter fragments from fragrant and non-fragrant flowers, we provide evidence for additional players and present a model for combinatorial regulation of ODO1 expression in Petunia.

Regulators of floral fragrance production and their target genes in petunia are not exclusively active in the epidermal cells of petals.

In which cells of the flower volatile biosynthesis takes place is unclear. In rose and snapdragon, some enzymes of the volatile phenylpropanoid/benzenoid pathway have been shown to be present in the epidermal cells of petals. It is therefore generally believed that the production of these compounds occurs in these cells. However, whether the entire pathway is active in these cells and whether it is exclusively active in these cells remains to be proven. Cell-specific transcription factors activating these genes will determine in which cells they are expressed. In petunia, the transcription factor EMISSION OF BENZENOIDS II (EOBII) activates the ODORANT1 (ODO1) promoter and the promoter of the biosynthetic gene isoeugenol synthase (IGS). The regulator ODO1 in turn activates the promoter of the shikimate gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Here the identification of a new target gene of ODO1, encoding an ABC transporter localized on the plasma membrane, PhABCG1, which is co-expressed with ODO1, is described. PhABCG1 expression is up-regulated in petals overexpressing ODO1 through activation of the PhABCG1 promoter. Interestingly, the ODO1, PhABCG1, and IGS promoters were active in petunia protoplasts originating from both epidermal and mesophyll cell layers of the petal, suggesting that the volatile phenylpropanoid/benzenoid pathway in petunia is active in these different cell types. Since volatile release occurs from epidermal cells, trafficking of (volatile) compounds between cell layers must be involved, but the exact function of PhABCG1 remains to be resolved.

The transcription factor EMISSION OF BENZENOIDS II activates the MYB ODORANT1 promoter at a MYB binding site specific for fragrant petunias.

Fragrance production in petunia flowers is highly regulated. Two transcription factors, ODORANT1 (ODO1) and EMISSION OF BENZENOIDS II (EOBII) have recently been identified as regulators of the volatile benzenoid/phenylpropanoid pathway in petals. Unlike the non-fragrant Petunia hybrida cultivar R27, the fragrant cultivar Mitchell highly expresses ODO1. Using stable reporter lines, we identified the 1.2-kbp ODO1 promoter from Mitchell that is sufficient for tissue-specific, developmental and rhythmic expression. This promoter fragment can be activated in non-fragrant R27 petals, indicating that the set of trans-acting factors driving ODO1 expression is conserved in these two petunias. Conversely, the 1.2-kbp ODO1 promoter of R27 is much less active in Mitchell petals. Transient transformation of 5' deletion and chimeric Mitchell and R27 ODO1 promoter reporter constructs in petunia petals identified an enhancer region, which is specific for the fragrant Mitchell cultivar and contains a putative MYB binding site (MBS). Mutations in the MBS of the Mitchell promoter decreased overall promoter activity by 50%, highlighting the importance of the enhancer region. We show that EOBII binds and activates the ODO1 promoter via this MBS, establishing a molecular link between these two regulators of floral fragrance biosynthesis in petunia.

A plant thiolase involved in benzoic acid biosynthesis and volatile benzenoid production.

The exact biosynthetic pathways leading to benzoic acid (BA) formation in plants are not known, but labeling experiments indicate the contribution of both beta-oxidative and non-beta-oxidative pathways. In Petunia hybrida BA is a key precursor for the production of volatile benzenoids by its flowers. Using functional genomics, we identified a 3-ketoacyl-CoA thiolase, PhKAT1, which is involved in the benzenoid biosynthetic pathway and the production of BA. PhKAT1 is localised in the peroxisomes, where it is important for the formation of benzoyl-CoA-related compounds. Silencing of PhKAT1 resulted in a major reduction in BA and benzenoid formation, leaving the production of other phenylpropanoid-related volatiles unaffected. During the night, when volatile benzenoid production is highest, it is largely the beta-oxidative pathway that contributes to the formation of BA and benzenoids. Our studies add the benzenoid biosynthetic pathway to the list of pathways in which 3-ketoacyl-CoA thiolases are involved in plants.