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 SCFTIR¹. Binding of Aux/IAA proteins leads to degradation via the 26S proteasome, but evidence for SCFTIR¹-mediated poly-ubiquitination of Aux/IAA proteins is lacking. Here we used an Arabidopsis cell suspension-based protoplast system to find evidence for SCFTIR¹-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 SCFTIR¹-dependent poly-ubiquitination of Aux/IAA proteins marks these proteins for degradation by the 26S proteasome, leading to activation of auxin-responsive gene expression.
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.
The (seco)iridoids and their derivatives, the monoterpenoid indole alkaloids (MIAs), form two large families of plant-derived bioactive compounds with a wide spectrum of high-value pharmacological ...and insect-repellent activities. Vinblastine and vincristine, MIAs used as anticancer drugs, are produced by Catharanthus roseus in extremely low levels, leading to high market prices and poor availability. Their biotechnological production is hampered by the fragmentary knowledge of their biosynthesis. Here we report the discovery of the last four missing steps of the (seco)iridoid biosynthesis pathway. Expression of the eight genes encoding this pathway, together with two genes boosting precursor formation and two downstream alkaloid biosynthesis genes, in an alternative plant host, allows the heterologous production of the complex MIA strictosidine. This confirms the functionality of all enzymes of the pathway and highlights their utility for synthetic biology programmes towards a sustainable biotechnological production of valuable (seco)iridoids and alkaloids with pharmaceutical and agricultural applications.
Summary
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.
Significance Statement
Madagascar periwinkle produces the valuable anticancer therapeutic compounds vinblastine and vincristine.The biosynthesis of these monoterpenoid indole alkaloids is strictly controlled by developmental and environmental cues. Here we describe the identification of a transcriptional regulator invovled in the jasmonate‐dependent activation and amplification loop governing one specific branch of the monoterpenoid indole alkaloid pathway.
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 β-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.
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 helixâloopâhelix (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.
Significance Terpenoids are the largest group of plant-specialized metabolites and include many valuable bioactive compounds, such as the blockbuster anticancer drugs vincristine and vinblastine, that are monoterpenoid indole alkaloids from the medicinal plant Catharanthus roseus (Madagascar periwinkle). A master regulator was discovered that activates the biosynthesis of the iridoids, the monoterpenoid precursors of vinblastine and vincristine, and the rate-limiting branch in their biosynthetic pathway. This master regulator can be used to boost production of iridoids and monoterpenoid indole alkaloids in C. roseus cell cultures and thus represents an interesting tool for the metabolic engineering of the sustainable production of these high-value compounds in cultures of the endogenous plant species.
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.
Plant defense against pathogens depends on the action of several endogenously produced hormones, including jasmonic acid (JA) and ethylene. In certain defense responses, JA and ethylene signaling ...pathways synergize to activate a specific set of defense genes. Here, we describe the role of the Arabidopsis (Arabidopsis thaliana) APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) domain transcription factor ORA59 in JA and ethylene signaling and in defense. JA- and ethylene-responsive expression of several defense genes, including PLANT DEFENSIN1.2 (PDF1.2), depended on ORA59. As a result, overexpression of ORA59 caused increased resistance against the fungus Botrytis cinerea, whereas ORA59-silenced plants were more susceptible. Several AP2/ERF domain transcription factors have been suggested to be positive regulators of PDF1.2 gene expression based on overexpression in stably transformed plants. Using two different transient overexpression approaches, we found that only ORA59 and ERF1 were able to activate PDF1.2 gene expression, in contrast to the related proteins AtERF1 and AtERF2. Our results demonstrate that ORA59 is an essential integrator of the JA and ethylene signal transduction pathways and thereby provide new insight into the nature of the molecular components involved in the cross talk between these two hormones.
Biosynthesis of many classes of secondary metabolites in plants is induced by the stress hormone jasmonate. The gene for ORCA3, a jasmonate-responsive APETALA2 (AP2)-domain transcription factor from ...Catharanthus roseus, was isolated by transferred DNA activation tagging. Orca3 overexpression resulted in enhanced expression of several metabolite biosynthetic genes and, consequently, in increased accumulation of terpenoid indole alkaloids. Regulation of metabolite biosynthetic genes by jasmonate-responsive AP2-domain transcription factors may link plant stress responses to changes in metabolism.
This review presents an overview of promoter sequences and transcription factors involved in jasmonate-responsive gene expression.
Plants possess inducible defense systems to oppose attack by ...pathogens and herbivores. Jasmonates are important signaling molecules produced by plants which regulate in positive or negative crosstalk with ethylene subsets of genes involved in defense against necrotrophic microorganisms or herbivorous insects, respectively. This review presents an overview of promoter sequences and transcription factors involved in jasmonate-responsive gene expression with the most important components summarized here. Frequently occurring jasmonate-responsive promoter sequences are the GCC motif, which is commonly found in promoters activated synergistically by jasmonate and ethylene, and the G-box, which is commonly found in promoters activated by jasmonates and repressed by ethylene. Important transcription factors conferring jasmonate-responsive gene expression in Arabidopsis are ORA59 and AtMYC2. ORA59 interacts with the GCC motif and controls the expression of genes that are synergistically induced by jasmonates and ethylene, whereas AtMYC2 interacts with the G-box and related sequences, and controls genes activated by jasmonate alone. AtMYC2 can interact with JAZ proteins, which are hypothesized to act as repressors. The bioactive jasmonate (+)-7-
iso-JA-
l-Ile promotes the interaction between the ubiquitin ligase complex SCF
COI1 and JAZ proteins, resulting in their degradation by the 26S proteasome, thereby liberating AtMYC2 from repression according to the prevailing model. Literature up to 1 June 2009 was used for this review.
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