A Cotyledon-based Virus-Induced Gene Silencing (Cotyledon-VIGS) approach to study specialized metabolism in medicinal plants.

BACKGROUND: Virus-induced gene silencing (VIGS) is widely used in plant functional genomics. However, the efficiency of VIGS in young plantlets varies across plant species. Additionally, VIGS is not optimized for many plant species, especially medicinal plants that produce valuable specialized metabolites. RESULTS: We evaluated the efficacy of five-day-old, etiolated seedlings of Catharanthus roseus (periwinkle) for VIGS. The seedlings were vacuum-infiltrated with Agrobacterium tumefaciens GV3101 cells carrying the tobacco rattle virus (TRV) vectors. The protoporphyrin IX magnesium chelatase subunit H (ChlH) gene, a key gene in chlorophyll biosynthesis, was used as the target for VIGS, and we observed yellow cotyledons 6 days after infiltration. As expected, the expression of CrChlH and the chlorophyll contents of the cotyledons were significantly decreased after VIGS. To validate the cotyledon based-VIGS method, we silenced the genes encoding several transcriptional regulators of the terpenoid indole alkaloid (TIA) biosynthesis in C. roseus, including two activators (CrGATA1 and CrMYC2) and two repressors (CrGBF1 and CrGBF2). Silencing CrGATA1 led to downregulation of the vindoline pathway genes (T3O, T3R, and DAT) and decreased vindoline contents in cotyledons. Silencing CrMYC2, followed by elicitation with methyl jasmonate (MeJA), resulted in the downregulation of ORCA2 and ORCA3. We also co-infiltrated C. roseus seedlings with TRV vectors that silence both CrGBF1 and CrGBF2 and overexpress CrMYC2, aiming to simultaneous silencing two repressors while overexpressing an activator. The simultaneous manipulation of repressors and activator resulted in significant upregulation of the TIA pathway genes. To demonstrate the broad application of the cotyledon-based VIGS method, we optimized the method for two other valuable medicinal plants, Glycyrrhiza inflata (licorice) and Artemisia annua (sweet wormwood). When TRV vectors carrying the fragments of the ChlH genes were infiltrated into the seedlings of these plants, we observed yellow cotyledons with decreased chlorophyll contents. CONCLUSIONS: The widely applicable cotyledon-based VIGS method is faster, more efficient, and easily accessible to additional treatments than the traditional VIGS method. It can be combined with transient gene overexpression to achieve simultaneous up- and down-regulation of desired genes in non-model plants. This method provides a powerful tool for functional genomics of medicinal plants, facilitating the discovery and production of valuable therapeutic compounds.

Light regulation of the biosynthesis of phenolics, terpenoids, and alkaloids in plants.

Biosynthesis of specialized metabolites (SM), including phenolics, terpenoids, and alkaloids, is stimulated by many environmental factors including light. In recent years, significant progress has been made in understanding the regulatory mechanisms involved in light-stimulated SM biosynthesis at the transcriptional, posttranscriptional, and posttranslational levels of regulation. While several excellent recent reviews have primarily focused on the impacts of general environmental factors, including light, on biosynthesis of an individual class of SM, here we highlight the regulation of three major SM biosynthesis pathways by light-responsive gene expression, microRNA regulation, and posttranslational modification of regulatory proteins. In addition, we present our future perspectives on this topic.

Partial desensitization of MYC2 transcription factor alters the interaction with jasmonate signaling components and affects specialized metabolism.

The activity of bHLH transcription factor MYC2, a key regulator in jasmonate signaling and plant specialized metabolism, is sensitive to repression by JASMONATE-ZIM-domain (JAZ) proteins and co-activation by the mediator subunit MED25. The substitution of a conserved aspartic acid (D) to asparagine (N) in the JAZ-interacting domain (JID) of Arabidopsis MYC2 affects interaction with JAZ, although the mechanism remained unclear. The effects of the conserved residue MYC2D128 on interaction with MED25 have not been investigated. Using tobacco as a model, we generated all possible substitutions of aspartic acid 128 (D128) in NtMYC2a. NtMYC2aD128N partially desensitized the repression by JAZ proteins, while strongly interacting with MED25, resulting in increased expression of nicotine pathway genes and nicotine accumulation in tobacco hairy roots overexpressing NtMYC2aD128N compared to those overexpressing NtMYC2a. The proline substitution, NtMYC2aD128P, negatively affected transactivation and abolished the interaction with JAZ proteins and MED25. Structural modeling and simulation suggest that the overall stability of the JID binding pocket is a predominant cause for the observed effects of substitutions at D128. The D128N substitution has an overall stabilizing effect on the binding pocket, which is destabilized by D128P. Our study offers an innovative tool to increase the production of plant natural products.

Metabolic engineering to enhance the accumulation of bioactive flavonoids licochalcone A and echinatin in Glycyrrhiza inflata (Licorice) hairy roots.

Echinatin and licochalcone A (LCA) are valuable chalcones preferentially accumulated in roots and rhizomes of licorice (Glycyrrhiza inflata). The licorice chalcones (licochalcones) are valued for their anti-inflammatory, antimicrobial, and antioxidant properties and have been widely used in cosmetic, pharmaceutical, and food industries. However, echinatin and LCA are accumulated in low quantities, and the biosynthesis and regulation of licochalcones have not been fully elucidated. In this study, we explored the potential of a R2R3-MYB transcription factor (TF) AtMYB12, a known regulator of flavonoid biosynthesis in Arabidopsis, for metabolic engineering of the bioactive flavonoids in G. inflata hairy roots. Overexpression of AtMYB12 in the hairy roots greatly enhanced the production of total flavonoids (threefold), echinatin (twofold), and LCA (fivefold). RNA-seq analysis of AtMYB12-overexpressing hairy roots revealed that expression of phenylpropanoid/flavonoid pathway genes, such as phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), and flavanone 3'-hydroxylase (F3'H), is significantly induced compared to the control. Transient promoter activity assay indicated that AtMYB12 activates the GiCHS1 promoter in plant cells, and mutation to the MYB-binding motif in the GiCHS1 promoter abolished activation. In addition, transcriptomic analysis revealed that AtMYB12 overexpression reprograms carbohydrate metabolism likely to increase carbon flux into flavonoid biosynthesis. Further, AtMYB12 activated the biotic defense pathways possibly by activating the salicylic acid and jasmonic acid signaling, as well as by upregulating WRKY TFs. The transcriptome of AtMYB12-overexpressing hairy roots serves as a valuable source in the identification of potential candidate genes involved in LCA biosynthesis. Taken together, our findings suggest that AtMYB12 is an effective gene for metabolic engineering of valuable bioactive flavonoids in plants.

Identification and Characterization of Transcription Factors Regulating Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus.

Biosynthesis of the therapeutically valuable terpenoid indole alkaloids (TIAs), in the medicinal plant Catharanthus roseus, is one of the most elaborate and complex metabolic processes. Although genomic and transcriptomic resources have significantly accelerated gene discovery in the TIA pathway, relatively few genes of transcription factors (TFs) have been identified and characterized thus far. Systematic identification of TFs and elucidation of their functions are crucial for understanding TIA pathway regulation. The successful discovery of TFs in the TIA pathway has relied mostly on three different approaches, (1) identification of cis-regulatory motifs (CRMs) present in the pathway gene promoters as they often provide clues on potential TFs that bind to the promoters, (2) co-expression analysis, based on the assumption that TFs regulating a metabolic or developmental pathway exhibit similar spatiotemporal expression as the pathway genes, and (3) isolation of homologs of TFs known to regulate structurally similar or diverse specialized metabolites in different plant species. TFs regulating TIA pathway have been isolated using either an individual or a combination of the three approaches. Here we describe transcriptome-based coexpression analysis and cis-element determination to identify TFs in C. roseus. In addition, we describe the protocols for generation of transgenic hairy roots, Agrobacterium infiltration of flowers, and electrophoretic mobility shift assay (EMSA). The methods described here are useful for the identification and characterization of potential TFs involved in the regulation of special metabolism in other medicinal plants.

Virus-Induced Gene Silencing as a Tool to Study Regulation of Alkaloid Biosynthesis in Medicinal Plants.

Advancements in genomics and transcriptomics have generated invaluable resources for the discovery of novel genes related to complex specialized metabolic pathways in plants. Virus-induced gene silencing (VIGS) has emerged as a powerful tool that is widely used for rapid functional characterization of genes in planta. VIGS has advantages over other reverse genetic approaches, such as RNAi-mediated suppression or T-DNA knockout, because it does not require the development of stable transgenic lines which is technically challenging and time consuming. Catharanthus roseus is an important medicinal plant that produces more than a hundred monoterpenoid indole alkaloids (MIAs), including the antineoplastic drugs vincristine and vinblastine. Biosynthesis of these alkaloids is strikingly complex, resulting in MIA accumulation in low quantities. Jasmonic acid (JA) is an elicitor of the MIA biosynthesis. Exogenous application of JA in C. roseus induces MIA pathway gene expression and increases MIA accumulation. The core JA signaling module comprises multiple components including the JA coreceptor Coronatine-Insensitive 1(COI1). COI1 plays a key role in JA-responsive gene expression in plants. Because generation of stable transgenic C. roseus plants is challenging, VIGS is being used for functional characterization of genes in the MIA pathway. Here we describe a detailed method for the VIGS-mediated suppression of C. roseus COI1(CrCOI1) expression to decipher the regulatory mechanism of JA-induced elicitation of MIA biosynthesis. When performing VIGS, gene silencing efficiency and the viral spread are monitored by the development of visible phenotype in the control plants. We use the C. roseus phytoene desaturase (CrPDS) and Protoporphyrin IX Mg-chelatase subunit H (CrChlH) as visual markers to access VIGS efficiency and viral spread. The protocol described here could be used for the functional characterization of genes involved in other metabolic pathways and in other medicinal plants.

Terpenoid indole alkaloid biosynthesis in Catharanthus roseus: effects and prospects of environmental factors in metabolic engineering.

Plants synthesize a vast array of specialized metabolites that primarily contribute to their defense and survival under adverse conditions. Many of the specialized metabolites have therapeutic values as drugs. Biosynthesis of specialized metabolites is affected by environmental factors including light, temperature, drought, salinity, and nutrients, as well as pathogens and insects. These environmental factors trigger a myriad of changes in gene expression at the transcriptional and posttranscriptional levels. The dynamic changes in gene expression are mediated by several regulatory proteins that perceive and transduce the signals, leading to up- or down-regulation of the metabolic pathways. Exploring the environmental effects and related signal cascades is a strategy in metabolic engineering to produce valuable specialized metabolites. However, mechanistic studies on environmental factors affecting specialized metabolism are limited. The medicinal plant Catharanthus roseus (Madagascar periwinkle) is an important source of bioactive terpenoid indole alkaloids (TIAs), including the anticancer therapeutics vinblastine and vincristine. The emerging picture shows that various environmental factors significantly alter TIA accumulation by affecting the expression of regulatory and enzyme-encoding genes in the pathway. Compared to our understanding of the TIA pathway in response to the phytohormone jasmonate, the impacts of environmental factors on TIA biosynthesis are insufficiently studied and discussed. This review thus focuses on these aspects and discusses possible strategies for metabolic engineering of TIA biosynthesis. PURPOSE OF WORK: Catharanthus roseus is a rich source of bioactive terpenoid indole alkaloids (TIAs). The objective of this work is to present a comprehensive account of the influence of various biotic and abiotic factors on TIA biosynthesis and to discuss possible strategies to enhance TIA production through metabolic engineering.

Reprogramming plant specialized metabolism by manipulating protein kinases.

Being sessile, plants have evolved sophisticated mechanisms to balance between growth and defense to survive in the harsh environment. The transition from growth to defense is commonly achieved by factors, such as protein kinases (PKs) and transcription factors, that initiate signal transduction and regulate specialized metabolism. Plants produce an array of lineage-specific specialized metabolites for chemical defense and stress tolerance. Some of these molecules are also used by humans as drugs. However, many of these defense-responsive metabolites are toxic to plant cells and inhibitory to growth and development. Plants have, thus, evolved complex regulatory networks to balance the accumulation of the toxic metabolites. Perception of external stimuli is a vital part of the regulatory network. Protein kinase-mediated signaling activates a series of defense responses by phosphorylating the target proteins and translating the stimulus into downstream cellular signaling. As biosynthesis of specialized metabolites is triggered when plants perceive stimuli, a possible connection between PKs and specialized metabolism is well recognized. However, the roles of PKs in plant specialized metabolism have not received much attention until recently. Here, we summarize the recent advances in understanding PKs in plant specialized metabolism. We aim to highlight how the stimulatory signals are transduced, leading to the biosynthesis of corresponding metabolites. We discuss the post-translational regulation of specialized metabolism and provide insights into the mechanisms by which plants respond to the external signals. In addition, we propose possible strategies to increase the production of plant specialized metabolites in biotechnological applications using PKs.

BHLH IRIDOID SYNTHESIS 3 is a member of a bHLH gene cluster regulating terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

Basic helix-loop-helix (bHLH) transcription factors (TFs) are key regulators of plant specialized metabolites, including terpenoid indole alkaloids (TIAs) in Catharanthus roseus. Two previously characterized subgroup-IVa bHLH TFs, BIS1 (bHLH Iridoid Synthesis 1) and BIS2 regulate iridoid biosynthesis in the TIA pathway. We reanalyzed the recently updated C. roseus genome sequence and discovered that BIS1 and BIS2 are clustered on the same genomic scaffold with a previously uncharacterized bHLH gene, designated as BIS3. Only a few bHLH gene clusters have been studied to date. Comparative analysis of 49 genome sequences from different plant lineages revealed the presence of analogous bHLH clusters in core angiosperms, including the medicinal plants Calotropis gigantea (giant milkweed) and Gelsemium sempervirens (yellow jessamine), but not in the analyzed basal angiosperm and lower plants. Similar to the iridoid pathway genes, BIS3 is highly expressed in roots and induced by methyl jasmonate. BIS3 activates the promoters of iridoid branch genes, geraniol synthase (GES), geraniol 10-hydroxylase (G10H), 8-hydroxygeraniol oxidoreductase (8HGO), iridoid synthase (IS), 7-deoxyloganetic acid glucosyl transferase (7-DLGT), and 7-deoxyloganic acid hydroxylase (7DLH), but not iridoid oxidase (IO). Transactivation of the promoters was abolished when BIS3 is converted to a dominant repressor by fusing with the ERF-associated amphiphilic repression (EAR) sequence. In addition, BIS3 acts synergistically with BIS1 and BIS2 to activate the G10H promoter in tobacco cells. Mutation of the known bHLH TF binding motif, G-box (CACGTG) in the G10H promoter significantly reduced but did not abolish the transactivation by BIS3. Promoter deletion analysis of G10H suggests that the sequences adjacent to the G-box are also involved in the regulation by BIS3. Overexpression of BIS3 in C. roseus flower petals significantly upregulated the expression of iridoid biosynthetic genes and increased loganic acid accumulation. BIS2 expression was significantly induced by BIS3 although BIS3 did not directly activate the BIS2 promoter. Our results advance our understanding of the regulation of plant specialized metabolites by bHLH TF clusters.

Protein phosphatase NtPP2C2b and MAP kinase NtMPK4 act in concert to modulate nicotine biosynthesis.

Protein phosphatases (PPs) and protein kinases (PKs) regulate numerous developmental, defense, and phytohormone signaling processes in plants. However, the underlying regulatory mechanism governing biosynthesis of specialized metabolites, such as alkaloids, by the combined effects of PPs and PKs, is insufficiently understood. Here, we report the characterization of a group B protein phosphatase type 2C, NtPP2C2b, that likely acts upstream of the NICOTINE2 locus APETALA 2/Ethylene Response Factors (AP2/ERFs), to regulate nicotine biosynthesis in tobacco. Similar to the nicotine pathway genes, NtPP2C2b is highly expressed in roots and induced by jasmonic acid (JA). Overexpression of NtPP2C2b in transgenic hairy roots or stable transgenic tobacco plants repressed nicotine pathway gene expression and reduced nicotine accumulation. Additionally, transient overexpression of NtPP2C2b, together with the NtERF221, repressed transactivation of the quinolinate phosphoribosyltransferase promoter in tobacco cells. We further demonstrate that the JA-responsive tobacco mitogen-activated protein kinase (MAPK) 4 interacts with NtPP2C2b in yeast and plant cells. Conditional overexpression of NtMPK4 in tobacco hairy roots up-regulated nicotine pathway gene expression and increased nicotine accumulation. Our findings suggest that a previously uncharacterized PP-PK module acts to modulate alkaloid biosynthesis, highlighting the importance of post-translational control in the biosynthesis of specialized plant metabolites.

Maleic hydrazide elicits global transcriptomic changes in chemically topped tobacco to influence shoot bud development.

MAIN CONCLUSION: Transcriptomic analysis revealed maleic hydrazide suppresses apical and axillary bud development by altering the expression of genes related to meristem development, cell division, DNA replication, DNA damage and recombination, and phytohormone signaling. Topping (removal of apical buds) is a common agricultural practice for some crop plants including cotton, cannabis, and tobacco. Maleic hydrazide (MH) is a systemic suckercide, a chemical that inhibits shoot bud growth, used to control the growth of apical (ApB) and axillary buds (AxB) following topping. However, the influence of MH on gene expression and the underlying molecular mechanism of controlling meristem development are not well studied. Our RNA sequencing analysis showed that MH significantly influences the transcriptomic landscape in ApB and AxB of chemically topped tobacco. Gene ontology (GO) enrichment analysis revealed that upregulated genes in ApB were enriched for phosphorelay signal transduction, and the regulation of transition timing from vegetative to reproductive phase, whereas downregulated genes were largely associated with meristem maintenance, cytokinin metabolism, cell wall synthesis, photosynthesis, and DNA metabolism. In MH-treated AxB, GO terms related to defense response and oxylipin metabolism were overrepresented in upregulated genes. GO terms associated with cell cycle, DNA metabolism, and cytokinin metabolism were enriched in downregulated genes. Expression of KNOX and MADS transcription factor (TF) family genes, known to be involved in meristem development, were affected in ApB and AxB by MH treatment. The promoters of MH-responsive genes are enriched for several known cis-acting elements, suggesting the involvement of a subset of TF families. Our findings suggest that MH affects shoot bud development in chemically topped tobacco by altering the expression of genes related to meristem development, DNA repair and recombination, cell division, and phytohormone signaling.

TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR4 Interacts with WRINKLED1 to Mediate Seed Oil Biosynthesis.

Cross-family transcription factor (TF) interactions play critical roles in the regulation of plant developmental and metabolic pathways. WRINKLED1 (WRI1) is a key TF governing oil biosynthesis in plants. However, little is known about WRI1-interacting factors and their roles in oil biosynthesis. We screened a TF library using Arabidopsis (Arabidopsis thaliana) WRI1 (AtWRI1) as bait in yeast two-hybrid assays and identified three TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) family TFs, namely TCP4, TCP10, and TCP24, as AtWRI1-interacting partners. The physical interaction between AtWRI1 and TCPs was further validated using bimolecular fluorescence complementation assays. TCPs play important roles in various plant developmental processes; however, their involvement in fatty acid biosynthesis was not previously known. Coexpression of TCP4, but not TCP10 or TCP24, with AtWRI1 reduced AtWRI1-mediated oil biosynthesis in Nicotiana benthamiana leaves. Transcriptomic analysis in transgenic Arabidopsis plants with enhanced TCP4 activity engineered by expressing rTCP4 (i.e. miR319-resistant TCP4) revealed that AtWRI1 target genes were significantly repressed. TCP4 expression is strongly correlated with AtWRI1 during embryo development. A tcp4 loss-of-function mutant, the jaw-D mutant with a strong reduction of TCP4 expression, and a tcp2 tcp4 tcp10 triple mutant accumulated more seed oil than wild-type Arabidopsis. In addition, TCP4 repressed the AtWRI1-mediated transactivation of the promoters of fatty acid biosynthetic genes. Collectively, our findings suggest that TCP4 represses fatty acid biosynthetic gene expression through interaction with AtWRI1, leading to a reduction of AtWRI1-mediated seed oil accumulation.

Revisiting the ORCA gene cluster that regulates terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

Transcription factor (TF) gene clusters in plants, such as tomato, potato, petunia, tobacco, and almond, have been characterized for their roles in the biosynthesis of diverse array of specialized metabolites. In Catharanthus roseus, three AP2/ERF TFs, ORCA3, ORCA4, and ORCA5, have been shown to be present on the same genomic scaffold, forming a cluster that regulates the biosynthesis of pharmaceutically important terpenoid indole alkaloids (TIAs). Our analysis of the recently updated C. roseus genome sequence revealed that the ORCA cluster comprises two additional AP2/ERFs, the previously characterized ORCA2 and a newly identified member designated as ORCA6. Transcriptomic analysis revealed that the ORCAs are highly expressed in stems, followed by leaves, roots and flowers. Expression of ORCAs was differentially induced in response to methyl-jasmonate and ethylene treatment. In addition, ORCA6 activated the strictosidine synthase (STR) promoter in tobacco cells. Activation of the STR promoter was significantly higher when ORCA2 or ORCA6 was coexpressed with the mitogen-activated protein kinase kinase, CrMPKK1. Furthermore, transient overexpression of ORCA6 in C. roseus flower petals activated TIA pathway gene expression and TIA accumulation. The results described here advance our understanding of regulation of TIA pathway by the ORCA gene cluster and the evolution for plant ERF gene clusters.

Mutually Regulated AP2/ERF Gene Clusters Modulate Biosynthesis of Specialized Metabolites in Plants.

APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) gene clusters regulate the biosynthesis of diverse specialized metabolites, including steroidal glycoalkaloids in tomato (Solanum lycopersicum) and potato (Solanum tuberosum), nicotine in tobacco (Nicotiana tabacum), and pharmaceutically valuable terpenoid indole alkaloids in Madagascar periwinkle (Catharanthus roseus). However, the regulatory relationships between individual AP2/ERF genes within the cluster remain unexplored. We uncovered intracluster regulation of the C. roseus AP2/ERF regulatory circuit, which consists of ORCA3, ORCA4, and ORCA5 ORCA3 and ORCA5 activate ORCA4 by directly binding to a GC-rich motif in the ORCA4 promoter. ORCA5 regulates its own expression through a positive autoregulatory loop and indirectly activates ORCA3 In determining the functional conservation of AP2/ERF clusters in other plant species, we found that GC-rich motifs are present in the promoters of analogous AP2/ERF clusters in tobacco, tomato, and potato. Intracluster regulation is evident within the tobacco NICOTINE2 (NIC2) ERF cluster. Moreover, overexpression of ORCA5 in tobacco and of NIC2 ERF189 in C. roseus hairy roots activates nicotine and terpenoid indole alkaloid pathway genes, respectively, suggesting that the AP2/ERFs are functionally equivalent and are likely to be interchangeable. Elucidation of the intracluster and mutual regulation of transcription factor gene clusters advances our understanding of the underlying molecular mechanism governing regulatory gene clusters in plants.

GATA and Phytochrome Interacting Factor Transcription Factors Regulate Light-Induced Vindoline Biosynthesis in Catharanthus roseus.

Catharanthus roseus is the exclusive source of an array of terpenoid indole alkaloids including the anticancer drugs vincristine and vinblastine, derived from the coupling of catharanthine and vindoline. Leaf-synthesized vindoline is regulated by light. A seven-step enzymatic process is involved in the sequential conversion of tabersonine to vindoline; however, the regulatory mechanism controlling the expression of genes encoding these enzymes has not been elucidated. Here, we identified CrGATA1, an Leu-Leu-Met domain GATA transcription factor that regulates light-induced vindoline biosynthesis in C. roseus seedlings. Expression of CrGATA1 and the vindoline pathway genes T16H2, T3O, T3R, D4H, and DAT was significantly induced by light. In addition, CrGATA1 activated the promoters of five light-responsive vindoline pathway genes in plant cells. Two GATC motifs in the D4H promoter were critical for CrGATA1-mediated transactivation. Transient overexpression of CrGATA1 in C. roseus seedlings resulted in up-regulation of vindoline pathway genes and increased vindoline accumulation. Conversely, virus-induced gene silencing of CrGATA1 in young C. roseus leaves significantly repressed key vindoline pathway genes and reduced vindoline accumulation. Furthermore, we showed that a C. roseus Phytochrome Interacting Factor, CrPIF1, is a repressor of CrGATA1 and vindoline biosynthesis. Transient overexpression or virus-induced gene silencing of CrPIF1 in C. roseus seedlings altered CrGATA1 and vindoline pathway gene expression in the dark. CrPIF1 repressed CrGATA1 and DAT promoter activity by binding to G/E-box/PBE elements. Our findings reveal a regulatory module involving Phytochrome Interacting Factor -GATA that governs light-mediated biosynthesis of specialized metabolites.

Cross-family transcription factor interaction between MYC2 and GBFs modulates terpenoid indole alkaloid biosynthesis.

Biosynthesis of medicinally valuable terpenoid indole alkaloids (TIAs) in Catharanthus roseus is regulated by transcriptional activators such as the basic helix-loop-helix factor CrMYC2. However, the transactivation effects are often buffered by repressors, such as the bZIP factors CrGBF1 and CrGBF2, possibly to fine-tune the accumulation of cytotoxic TIAs. Questions remain as to whether and how these factors interact to modulate TIA production. We demonstrated that overexpression of CrMYC2 induces CrGBF expression and results in reduced alkaloid accumulation in C. roseus hairy roots. We found that CrGBF1 and CrGBF2 form homo- and heterodimers to repress the transcriptional activities of key TIA pathway gene promoters. We showed that CrGBFs dimerize with CrMYC2, and CrGBF1 binds to the same cis-elements (T/G-box) as CrMYC2 in the target gene promoters. Our findings suggest that CrGBFs antagonize CrMYC2 transactivation possibly by competitive binding to the T/G-box in the target promoters and/or protein-protein interaction that forms a non-DNA binding complex that prevents CrMYC2 from binding to its target promoters. Homo- and heterodimer formation allows fine-tuning of the amplitude of TIA gene expression. Our findings reveal a previously undescribed regulatory mechanism that governs the TIA pathway genes to balance metabolic flux for TIA production in C. roseus.

Correction to: Genome-wide identification of hexokinase gene family in Brassica napus: structure, phylogenetic analysis, expression, and functional characterization.

In the original version of this article the name of the second author was misspelled. The correct name is: Xiaomin Wang.

Genome-wide identification of hexokinase gene family in Brassica napus: structure, phylogenetic analysis, expression, and functional characterization.

MAIN CONCLUSION: Genome-wide identification, expression analysis, and functional characterization of previously uncharacterized hexokinase family of oil crop, Brassica napus, underscore the importance of this gene family in plant growth and development. In plants, the multi-gene family of dual-function hexokinases (HXKs) plays important roles in sugar metabolism and sensing that affect growth and development. Rapeseed (Brassica napus L.) is an important oil crop; however, little is known about the B. napus HXK gene family. We identified 19 putative HXKs in B. napus genome. B. rapa and B. oleracea, the two diploid progenitors of B. napus, contributed almost equally to the BnHXK genes. Phylogenetic analysis divided the 19 BnHXKs into four groups. The exon-intron structures of BnHXKs share high similarity to those of HXKs in Arabidopsis and rice. The group III and IV BnHXKs are highly expressed in roots, whereas group I members preferentially express in leaves. Analysis of seed transcriptomes at different developmental stages showed that most of group I and IV HXKs are highly expressed 2-weeks after pollination (2WAP), compared to 4WAP for group III. BnHKXs are differentially expressed in susceptible and tolerant B. napus cultivars after fungal infection, suggesting the possible involvement in defense response. We generated rapeseed RNAi lines for BnHXK9, a member of relatively less characterized group IV, by pollen-mediated gene transformation. The seedlings of BnHXK9-RNAi lines showed delayed growth compared to the wild type. The RNAi plants were dwarf with curly leaves, suggesting the involvement of BnHXK9 in plant development. Collectively, our findings provides a comprehensive account of BnHXK gene family in an important crop and a starting point for further elucidation of their roles in sugar metabolism and sensing, as well as plant growth and development.

A network of jasmonate-responsive bHLH factors modulate monoterpenoid indole alkaloid biosynthesis in Catharanthus roseus.

The pharmaceutically valuable monoterpene indole alkaloids (MIAs) in Catharanthus roseus are derived from the indole and iridoid pathways that respond to jasmonate (JA) signaling. Two classes of JA-responsive bHLH transcription factor (TF), CrMYC2 and BIS1/BIS2, are known to regulate the indole and iridoid pathways, respectively. However, upregulation of either one of the TF genes does not lead to increased MIA accumulation. Moreover, little is known about the interconnection between the CrMYC2 and BIS transcriptional cascades and the hierarchical position of BIS1/BIS2 in JA signaling. Here, we report that a newly identified bHLH factor, Repressor of MYC2 Targets 1 (RMT1), is activated by CrMYC2 and BIS1, and acts as a repressor of the CrMYC2 targets. In addition, we isolated and functionally characterized the core C. roseus JA signaling components, including CORONATINE INSENSITIVE 1 (COI1) and JASMONATE ZIM domain (JAZ) proteins. CrMYC2 and BIS1 are repressed by the JAZ proteins in the absence of JA, but de-repressed by the SCFCOI1 complex on perception of JA. Our findings suggest that the repressors, JAZs and RMT1, mediate crosstalk between the CrMYC2 and BIS regulatory cascades to balance the metabolic flux in MIA biosynthesis.