A Seed-Specific Regulator of Triterpene Saponin Biosynthesis in Medicago truncatula.

Plants produce a vast array of defense compounds to protect themselves from pathogen attack or herbivore predation. Saponins are a specific class of defense compounds comprising bioactive glycosides with a steroidal or triterpenoid aglycone backbone. The model legume Medicago truncatula synthesizes two types of saponins, hemolytic saponins and nonhemolytic soyasaponins, which accumulate as specific blends in different plant organs. Here, we report the identification of the seed-specific transcription factor TRITERPENE SAPONIN ACTIVATION REGULATOR3 (TSAR3), which controls hemolytic saponin biosynthesis in developing M. truncatula seeds. Analysis of genes that are coexpressed with TSAR3 in transcriptome data sets from developing M. truncatula seeds led to the identification of CYP88A13, a cytochrome P450 that catalyzes the C-16α hydroxylation of medicagenic acid toward zanhic acid, the final oxidation step of the hemolytic saponin biosynthesis branch in M. truncatula In addition, two uridine diphosphate glycosyltransferases, UGT73F18 and UGT73F19, which glucosylate hemolytic sapogenins at the C-3 position, were identified. The genes encoding the identified biosynthetic enzymes are present in clusters of duplicated genes in the M. truncatula genome. This appears to be a common theme among saponin biosynthesis genes, especially glycosyltransferases, and may be the driving force of the metabolic evolution of saponins.

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.

The MYB transcription factor Emission of Methyl Anthranilate 1 stimulates emission of methyl anthranilate from Medicago truncatula hairy roots.

Plants respond to herbivore or pathogen attacks by activating specific defense programs that include the production of bioactive specialized metabolites to eliminate or deter the attackers. Volatiles play an important role in the interaction of a plant with its environment. Through transcript profiling of jasmonate-elicited Medicago truncatula cells, we identified Emission of Methyl Anthranilate (EMA) 1, a MYB transcription factor that is involved in the emission of the volatile compound methyl anthranilate when expressed in M. truncatula hairy roots, giving them a fruity scent. RNA sequencing (RNA-Seq) analysis of the fragrant roots revealed the upregulation of a methyltransferase that was subsequently characterized to catalyze the O-methylation of anthranilic acid and was hence named M. truncatula anthranilic acid methyl transferase (MtAAMT) 1. Given that direct activation of the MtAAMT1 promoter by EMA1 could not be unambiguously demonstrated, we further probed the RNA-Seq data and identified the repressor protein M. truncatula plant AT-rich sequence and zinc-binding (MtPLATZ) 1. Emission of Methyl Anthranilate 1 binds a tandem repeat of the ACCTAAC motif in the MtPLATZ1 promoter to transactivate gene expression. Overexpression of MtPLATZ1 in transgenic M. truncatula hairy roots led to transcriptional silencing of EMA1, indicating that MtPLATZ1 may be part of a negative feedback loop to control the expression of EMA1. Finally, application of exogenous methyl anthranilate boosted EMA1 and MtAAMT1 expression dramatically, thus also revealing a positive amplification loop. Such positive and negative feedback loops seem to be the norm rather than the exception in the regulation of plant specialized metabolism.

FRS7 and FRS12 recruit NINJA to regulate expression of glucosinolate biosynthesis genes.

The sessile lifestyle of plants requires accurate physiology adjustments to be able to thrive in a changing environment. Plants integrate environmental timing signals to control developmental and stress responses. Here, we identified Far1 Related Sequence (FRS) 7 and FRS12, two transcriptional repressors that accumulate in short-day conditions, as regulators of Arabidopsis glucosinolate (GSL) biosynthesis. Loss of function of FRS7 and FRS12 results in plants with increased amplitudes of diurnal expression of GSL pathway genes. Protein interaction analyses revealed that FRS7 and FRS12 recruit the NOVEL INTERACTOR OF JAZ (NINJA) to assemble a transcriptional repressor complex. Genetic and molecular evidence demonstrated that FRS7, FRS12 and NINJA jointly regulate the expression of GSL biosynthetic genes, and thus constitute a molecular mechanism that modulates specialized metabolite accumulation.

The protein quality control system manages plant defence compound synthesis.

Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondary metabolites that serve in defence against attackers. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Here we show that, in the legume Medicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD) quality control system to manage the production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols. An ERAD-type RING membrane-anchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulation is prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues.

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.

A Repressor Protein Complex Regulates Leaf Growth in Arabidopsis.

Cell number is an important determinant of final organ size. In the leaf, a large proportion of cells are derived from the stomatal lineage. Meristemoids, which are stem cell-like precursor cells, undergo asymmetric divisions, generating several pavement cells adjacent to the two guard cells. However, the mechanism controlling the asymmetric divisions of these stem cells prior to differentiation is not well understood. Here, we characterized PEAPOD (PPD) proteins, the only transcriptional regulators known to negatively regulate meristemoid division. PPD proteins interact with KIX8 and KIX9, which act as adaptor proteins for the corepressor TOPLESS. D3-type cyclin encoding genes were identified among direct targets of PPD2, being negatively regulated by PPDs and KIX8/9. Accordingly, kix8 kix9 mutants phenocopied PPD loss-of-function producing larger leaves resulting from increased meristemoid amplifying divisions. The identified conserved complex might be specific for leaf growth in the second dimension, since it is not present in Poaceae (grasses), which also lack the developmental program it controls.

OSC2 and CYP716A14v2 catalyze the biosynthesis of triterpenoids for the cuticle of aerial organs of Artemisia annua.

Artemisia annua is widely studied for its ability to accumulate the antimalarial sesquiterpenoid artemisinin. In addition to producing a variety of sesquiterpenoids, A. annua also accumulates mono-, di-, and triterpenoids, the majority of which are produced in the glandular trichomes. A. annua also has filamentous trichomes on its aerial parts, but little is known of their biosynthesis potential. Here, through a comparative transcriptome analysis between glandular and filamentous trichomes, we identified two genes, OSC2 and CYP716A14v2, encoding enzymes involved in the biosynthesis of specialized triterpenoids in A. annua. By expressing these genes in Saccharomyces cerevisiae and Nicotiana benthamiana, we characterized the catalytic function of these proteins and could reconstitute the specialized triterpenoid spectrum of A. annua in these heterologous hosts. OSC2 is a multifunctional oxidosqualene cyclase that produces α-amyrin, ß-amyrin, and δ-amyrin. CYP716A14v2 is a P450 belonging to the functionally diverse CYP716 family and catalyzes the oxidation of pentacyclic triterpenes, leading to triterpenes with a carbonyl group at position C-3, thereby providing an alternative biosynthesis pathway to 3-oxo triterpenes. Together, these enzymes produce specialized triterpenoids that are constituents of the wax layer of the cuticle covering the aerial parts of A. annua and likely function in the protection of the plant against biotic and abiotic stress.

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.

The bHLH Transcription Factors TSAR1 and TSAR2 Regulate Triterpene Saponin Biosynthesis in Medicago truncatula.

Plants respond to stresses by producing a broad spectrum of bioactive specialized metabolites. Hormonal elicitors, such as jasmonates, trigger a complex signaling circuit leading to the concerted activation of specific metabolic pathways. However, for many specialized metabolic pathways, the transcription factors involved remain unknown. Here, we report on two homologous jasmonate-inducible transcription factors of the basic helix-loop-helix family, TRITERPENE SAPONIN BIOSYNTHESIS ACTIVATING REGULATOR1 (TSAR1) and TSAR2, which direct triterpene saponin biosynthesis in Medicago truncatula. TSAR1 and TSAR2 are coregulated with and transactivate the genes encoding 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A REDUCTASE1 (HMGR1) and MAKIBISHI1, the rate-limiting enzyme for triterpene biosynthesis and an E3 ubiquitin ligase that controls HMGR1 levels, respectively. Transactivation is mediated by direct binding of TSARs to the N-box in the promoter of HMGR1. In transient expression assays in tobacco (Nicotiana tabacum) protoplasts, TSAR1 and TSAR2 exhibit different patterns of transactivation of downstream triterpene saponin biosynthetic genes, hinting at distinct functionalities within the regulation of the pathway. Correspondingly, overexpression of TSAR1 or TSAR2 in M. truncatula hairy roots resulted in elevated transcript levels of known triterpene saponin biosynthetic genes and strongly increased the accumulation of triterpene saponins. TSAR2 overexpression specifically boosted hemolytic saponin biosynthesis, whereas TSAR1 overexpression primarily stimulated nonhemolytic soyasaponin biosynthesis. Both TSARs also activated all genes of the precursor mevalonate pathway but did not affect sterol biosynthetic genes, pointing to their specific role as regulators of specialized triterpene metabolism in M. truncatula.

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.

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 RING E3 Ligase KEEP ON GOING Modulates JASMONATE ZIM-DOMAIN12 Stability.

Jasmonate (JA) signaling in plants is mediated by the JASMONATE ZIM-DOMAIN (JAZ) proteins that repress the activity of several transcription factors regulating JA-inducible gene expression. The hormone JA-isoleucine triggers the interaction of JAZ repressor proteins with the F-box protein CORONATINE INSENSITIVE1 (COI1), part of an S-phase kinase-associated protein1/Cullin1/F-box protein COI1 (SCF(COI1)) E3 ubiquitin ligase complex, and their degradation by the 26S proteasome. In Arabidopsis (Arabidopsis thaliana), the JAZ family consists of 13 members. The level of redundancy or specificity among these members is currently not well understood. Here, we characterized JAZ12, encoded by a highly expressed JAZ gene. JAZ12 interacted with the transcription factors MYC2, MYC3, and MYC4 in vivo and repressed MYC2 activity. Using tandem affinity purification, we found JAZ12 to interact with SCF(COI1) components, matching with observed in vivo ubiquitination and with rapid degradation after treatment with JA. In contrast to the other JAZ proteins, JAZ12 also interacted directly with the E3 RING ligase KEEP ON GOING (KEG), a known repressor of the ABSCISIC ACID INSENSITIVE5 transcription factor in abscisic acid signaling. To study the functional role of this interaction, we circumvented the lethality of keg loss-of-function mutants by silencing KEG using an artificial microRNA approach. Abscisic acid treatment promoted JAZ12 degradation, and KEG knockdown led to a decrease in JAZ12 protein levels. Correspondingly, KEG overexpression was capable of partially inhibiting COI1-mediated JAZ12 degradation. Our results provide additional evidence for KEG as an important factor in plant hormone signaling and a positive regulator of JAZ12 stability.

Functional characterization of the Arabidopsis transcription factor bZIP29 reveals its role in leaf and root development.

Plant bZIP group I transcription factors have been reported mainly for their role during vascular development and osmosensory responses. Interestingly, bZIP29 has been identified in a cell cycle interactome, indicating additional functions of bZIP29 in plant development. Here, bZIP29 was functionally characterized to study its role during plant development. It is not present in vascular tissue but is specifically expressed in proliferative tissues. Genome-wide mapping of bZIP29 target genes confirmed its role in stress and osmosensory responses, but also identified specific binding to several core cell cycle genes and to genes involved in cell wall organization. bZIP29 protein complex analyses validated interaction with other bZIP group I members and provided insight into regulatory mechanisms acting on bZIP dimers. In agreement with bZIP29 expression in proliferative tissues and with its binding to promoters of cell cycle regulators, dominant-negative repression of bZIP29 altered the cell number in leaves and in the root meristem. A transcriptome analysis on the root meristem, however, indicated that bZIP29 might regulate cell number through control of cell wall organization. Finally, ectopic dominant-negative repression of bZIP29 and redundant factors led to a seedling-lethal phenotype, pointing to essential roles for bZIP group I factors early in plant development.

Change of a conserved amino acid in the MYC2 and MYC3 transcription factors leads to release of JAZ repression and increased activity.

The bHLH transcription factor MYC2, together with its paralogues MYC3 and MYC4, is a master regulator of the response to the jasmonate (JA) hormone in Arabidopsis (Arabidopsis thaliana). In the absence of JA, JASMONATE ZIM (JAZ) proteins interact with the MYC proteins to block their activity. Understanding of the mechanism and specificity of this interaction is key to unravel JA signalling. We generated mutant MYC proteins and assessed their activity and the specificity of their interaction with the 12 Arabidopsis JAZ proteins. We show that the D94N mutation present in the atr2D allele of MYC3 abolishes the interaction between MYC3 and most JAZ proteins. The same effect is observed when the corresponding conserved Asp (D105) was mutated in MYC2. Accordingly, MYC2(D105N) activated target genes in the presence of JAZ proteins, in contrast to wild-type MYC2. JAZ1 and JAZ10 were the only JAZ proteins still showing interaction with the mutant MYC proteins, due to a second MYC interaction domain, besides the classical Jas domain. Our results visualize the divergence among JAZ proteins in their interaction with MYC proteins. Ultimately, the transferability of the Asp-to-Asn amino acid change might facilitate the design of hyperactive transcription factors for plant engineering.

Transcription factor WRKY23 assists auxin distribution patterns during Arabidopsis root development through local control on flavonol biosynthesis.

Gradients of the plant hormone auxin, which depend on its active intercellular transport, are crucial for the maintenance of root meristematic activity. This directional transport is largely orchestrated by a complex interaction of specific influx and efflux carriers that mediate the auxin flow into and out of cells, respectively. Besides these transport proteins, plant-specific polyphenolic compounds known as flavonols have been shown to act as endogenous regulators of auxin transport. However, only limited information is available on how flavonol synthesis is developmentally regulated. Using reduction-of-function and overexpression approaches in parallel, we demonstrate that the WRKY23 transcription factor is needed for proper root growth and development by stimulating the local biosynthesis of flavonols. The expression of WRKY23 itself is controlled by auxin through the Auxin Response Factor 7 (ARF7) and ARF19 transcriptional response pathway. Our results suggest a model in which WRKY23 is part of a transcriptional feedback loop of auxin on its own transport through local regulation of flavonol biosynthesis.

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.

Ethylene Response Factor6 acts as a central regulator of leaf growth under water-limiting conditions in Arabidopsis.

Leaf growth is a complex developmental process that is continuously fine-tuned by the environment. Various abiotic stresses, including mild drought stress, have been shown to inhibit leaf growth in Arabidopsis (Arabidopsis thaliana), but the underlying mechanisms remain largely unknown. Here, we identify the redundant Arabidopsis transcription factors ETHYLENE RESPONSE FACTOR5 (ERF5) and ERF6 as master regulators that adapt leaf growth to environmental changes. ERF5 and ERF6 gene expression is induced very rapidly and specifically in actively growing leaves after sudden exposure to osmotic stress that mimics mild drought. Subsequently, enhanced ERF6 expression inhibits cell proliferation and leaf growth by a process involving gibberellin and DELLA signaling. Using an ERF6-inducible overexpression line, we demonstrate that the gibberellin-degrading enzyme GIBBERELLIN 2-OXIDASE6 is transcriptionally induced by ERF6 and that, consequently, DELLA proteins are stabilized. As a result, ERF6 gain-of-function lines are dwarfed and hypersensitive to osmotic stress, while the growth of erf5erf6 loss-of-function mutants is less affected by stress. Besides its role in plant growth under stress, ERF6 also activates the expression of a plethora of osmotic stress-responsive genes, including the well-known stress tolerance genes STZ, MYB51, and WRKY33. Interestingly, activation of the stress tolerance genes by ERF6 occurs independently from the ERF6-mediated growth inhibition. Together, these data fit into a leaf growth regulatory model in which ERF5 and ERF6 form a missing link between the previously observed stress-induced 1-aminocyclopropane-1-carboxylic acid accumulation and DELLA-mediated cell cycle exit and execute a dual role by regulating both stress tolerance and growth inhibition.

APETALA2/ETHYLENE RESPONSE FACTOR and basic helix-loop-helix tobacco transcription factors cooperatively mediate jasmonate-elicited nicotine biosynthesis.

Transcription factors of the plant-specific apetala2/ethylene response factor (AP2/ERF) family control plant secondary metabolism, often as part of signalling cascades induced by jasmonate (JA) or other elicitors. Here, we functionally characterized the JA-inducible tobacco (Nicotiana tabacum) AP2/ERF factor ORC1, one of the members of the NIC2-locus ERFs that control nicotine biosynthesis and a close homologue of ORCA3, a transcriptional activator of alkaloid biosynthesis in Catharanthus roseus. ORC1 positively regulated the transcription of several structural genes coding for the enzymes involved in nicotine biosynthesis. Accordingly, overexpression of ORC1 was sufficient to stimulate alkaloid biosynthesis in tobacco plants and tree tobacco (Nicotiana glauca) root cultures. In contrast to ORCA3 in C. roseus, which needs only the GCC motif in the promoters of the alkaloid synthesis genes to induce their expression, ORC1 required the presence of both GCC-motif and G-box elements in the promoters of the tobacco nicotine biosynthesis genes for maximum transactivation. Correspondingly, combined application with the JA-inducible Nicotiana basic helix-loop-helix (bHLH) factors that bind the G-box element in these promoters enhanced ORC1 action. Conversely, overaccumulation of JAZ repressor proteins that block bHLH activity reduced ORC1 functionality. Finally, the activity of both ORC1 and bHLH proteins was post-translationally upregulated by a JA-modulated phosphorylation cascade, in which a specific mitogen-activated protein kinase kinase, JA-factor stimulating MAPKK1 (JAM1), was identified. This study highlights the complexity of the molecular machinery involved in the regulation of tobacco alkaloid biosynthesis and provides mechanistic insights about its transcriptional regulators.