Terpenoid Indole Alkaloid Pathway Research Articles

The role of the Golden2-like (GLK) transcription factor in regulating terpenoid indole alkaloid biosynthesis in Catharanthus roseus

Key messageA GLK homologue was identified and functionally characterized in Catharanthus roseus. Silencing CrGLK with VIGS or the chloroplast retrograde signaling inducer lincomycin increased terpenoid indole alkaloid biosynthesis.Catharanthus roseus is the sole source of the chemotherapeutic terpenoid indole alkaloids (TIAs) vinblastine and vincristine. TIA pathway genes, particularly genes in the vindoline pathway, are expressed at higher levels in immature versus mature leaves, but the molecular mechanisms responsible for this developmental regulation are unknown. We investigated the role of GOLDEN2-LIKE (GLK) transcription factors in contributing to this ontogenetic regulation since GLKs are active in seedlings upon light exposure and in the leaf’s early development, but their activity is repressed as leaves age and senesce.We identified a GLK homologue in C. roseus and functionally characterized its role in regulating TIA biosynthesis, with a focus on the vindoline pathway, by transiently reducing its expression through two separate methods: virus-induced gene silencing (VIGS) and application of chloroplast retrograde signaling inducers, norflurazon and lincomycin. Reducing CrGLK levels with each method reduced chlorophyll accumulation and the expression of the light harvesting complex subunit (LHCB2.2), confirming its functional homology with GLKs in other plant species. In contrast, reducing CrGLK via VIGS or lincomycin increased TIA accumulation and TIA pathway gene expression, suggesting that CrGLK may repress TIA biosynthesis. However, norflurazon had no effect on TIA gene expression, indicating that reducing CrGLK alone is not sufficient to induce TIA biosynthesis. Future work is needed to clarify the specific molecular mechanisms leading to increased TIA biosynthesis with CrGLK silencing. This is the first identification and characterization of GLK in C. roseus and the first investigation of how chloroplast retrograde signaling might regulate TIA biosynthesis.

Elicitation with sodium nitroprusside and Trichoderma improves vincristine and vinblastine yield in Catharanthus roseus cell suspension culture by modulating terpenoid indole alkaloid pathway genes

Elicitation with sodium nitroprusside and Trichoderma improves vincristine and vinblastine yield in Catharanthus roseus cell suspension culture by modulating terpenoid indole alkaloid pathway genes

Genome-wide identification of Uncaria rhynchophylla bHLH transcription factors and in-vitro validation of UrbHLH1 through interaction with terpenoid indole alkaloid synthesis pathway members

Uncaria rhynchophylla (Gouteng), as an evergreen woody vine belong to Rubiaceae family, is a traditional medicinal herb in China. Its terpenoid indole alkaloids (TIAs), which have good antidepressant and combined therapeutic effects on Alzheimer's disease, have attracted widespread attention. However, the content of TIAs is relatively low in U.rhynchophylla, which is unable to meet the growing market demand. The basic helix loop-helix (bHLH) transcription factor family exists in all three eukaryotic kingdoms and can participate in regulating secondary metabolite pathways. So far, there has been no comprehensive analysis of the bHLH gene in U. rhynchophylla, and their role in TIAs is almost unknown. In this study, a total of 171 UrbHLH genes (UrbHLHs) were unevenly distributed on 22 chromosomes and divided into 23 subfamilies. In addition, the physicochemical properties of UrbHLHs were analyzed. Most UrbHLHs in each subgroup had similar gene structures and conserved motifs. Intraspecific collinearity analysis showed that UrbHLH1 may be related to the biosynthesis of TIAs. Subcellular localization experiments revealed that UrbHLH1 is located in the nucleus; Dual luciferase reporter gene analysis (Dual-LUC) showed that UrbHLH1 could activate the expression of UrG10H and Ur10HGO in the TIAs synthesis pathway of U. rhynchophylla. Finally, using yeast one hybrid (Y1H) it was found that the promoter regions of these two genes both have E-box binding elements, which can be bound by UrbHLH1 and produced strong interactions. Therefore, UrbHLH1 may participate in the synthesis of TIAs pathway. In conclusion, this study provides foundation data on the role of UrbHLH transcription factors in regulating TIAs of U. rhynchophylla.

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

BackgroundVirus-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.ResultsWe 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.ConclusionsThe 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.

Modulation of terpenoid indole alkaloid pathway via elicitation with phytosynthesized silver nanoparticles for the enhancement of ajmalicine, a pharmaceutically important alkaloid.

The use of silver nanoparticles as elicitors in cell cultures of Rauwolfia serpentina resulted in increased levels of ajmalicine, upregulated structural and regulatory genes, elevated MDA content, and reduced activity of antioxidant enzymes. These findings hold potential for developing a cost-effective method for commercial ajmalicine production. Plants possess an intrinsic ability to detect various stress signals, prompting the activation of defense mechanisms through the reprogramming of metabolites to counter adverse conditions. The current study aims to propose an optimized bioprocess for enhancing the content of ajmalicine in Rauwolfia serpentina callus through elicitation with phytosynthesized silver nanoparticles. Initially, callus lines exhibiting elevated ajmalicine content were established. Following this, a protocol for the phytosynthesis of silver nanoparticles using seed extract from Rauwolfia serpentina was successfully standardized. The physicochemical attributes of the silver nanoparticles were identified, including their spherical shape, size ranging from 6.7 to 28.8nm in diameter, and the presence of reducing-capping groups such as amino, carbonyl, and amide. Further, the findings indicated that the presence of 2.5mg L-1 phytosynthesized silver nanoparticles in the culture medium increased the ajmalicine content. Concurrently, structural genes (TDC, SLS, STR, SGD, G10H) and regulatory gene (ORCA3) associated with the ajmalicine biosynthetic pathway were observed to be upregulated. A notable increase in MDA content and a decrease in the activities of antioxidant enzymes were observed. A notable increase in MDA content and a decrease in the activities of antioxidant enzymes were also observed. Our results strongly recommend the augmentation of ajmalicine content in the callus culture of R. serpentina through supplementation with silver nanoparticles, a potential avenue for developing a cost-effective process for the commercial production of ajmalicine.

De Novo transcriptome assembly and differential expression analysis of catharanthus roseus in response to salicylic acid

The anti-cancer vinblastine and vincristine alkaloids can only be naturally found in periwinkle (Catharanthus roseus). Both of these alkaloids' accumulations are known to be influenced by salicylic acid (SA). The transcriptome data to reveal the induction effect (s) of SA, however, seem restricted at this time. In this study, the de novo approach of transcriptome assembly was performed on the RNA-Sequencing (RNA-Seq) data in C. roseus. The outcome demonstrated that SA treatment boosted the expression of all the genes in the Terpenoid Indole Alkaloids (TIAs) pathway that produces the vinblastine and vincristine alkaloids. These outcomes supported the time-course measurements of vincristine alkaloid, the end product of the TIAs pathway, and demonstrated that SA spray had a positive impact on transcription and alkaloid synthesis. Additionally, the abundance of transcription factor families including bHLH, C3H, C2H2, MYB, MYB-related, AP2/ ERF, NAC, bZIP, and WRKY suggests a role for a variety of transcription families in response to the SA stimuli. Di-nucleotide and tri-nucleotide SSRs were the most prevalent SSR markers in microsatellite analyses, making up 39% and 34% of all SSR markers, respectively, out of the 77,192 total SSRs discovered.

Hairy roots: An untapped potential for production of plant products.

While plants are an abundant source of valuable natural products, it is often challenging to produce those products for commercial application. Often organic synthesis is too expensive for a viable commercial product and the biosynthetic pathways are often so complex that transferring them to a microorganism is not trivial or feasible. For plants not suited to agricultural production of natural products, hairy root cultures offer an attractive option for a production platform which offers genetic and biochemical stability, fast growth, and a hormone free culture media. Advances in metabolic engineering and synthetic biology tools to engineer hairy roots along with bioreactor technology is to a point where commercial application of the technology will soon be realized. We discuss different applications of hairy roots. We also use a case study of the advancements in understanding of the terpenoid indole alkaloid pathway in Catharanthus roseus hairy roots to illustrate the advancements and challenges in pathway discovery and in pathway engineering.

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.

Biosynthesis and Modulation of Terpenoid Indole Alkaloids in Catharanthus roseus: A Review of Targeting Genes and Secondary Metabolites

The medicinal plant C. roseus synthesizes biologically active alkaloids via the terpenoid indole alkaloid (TIAs) biosynthetic pathway. Most of these alkaloids have high therapeutic value, such as vinblastine and vincristine. Plant signaling components, plant hormones, precursors, growth hormones, prenylated proteins, and transcriptomic factors regulate the complex networks of TIA biosynthesis. For many years, researchers have been evaluating the scientific value of the TIA biosynthetic pathway and its potential in commercial applications for market opportunities. Metabolic engineering has revealed the major blocks in metabolic pathways regulated at the molecular level, unknown structures, metabolites, genes, enzyme expression, and regulatory genes. Conceptually, this information is necessary to create transgenic plants and microorganisms for the commercial production of high-value dimer alkaloids, such as vinca alkaloids, vinblastine, and vincristine In this review, we present current knowledge of the regulatory mechanisms of these components in the C. roseus TIA pathway, from genes to metabolites.

Repression of ZCT1, ZCT2 and ZCT3 affects expression of terpenoid indole alkaloid biosynthetic and regulatory genes.

Terpenoid indole alkaloids (TIAs) include several valuable pharmaceuticals. As Catharanthus roseus remains the primary source of these TIA pharmaceuticals, several research groups have devoted substantial efforts to increase production of these compounds by C. roseus. Efforts to increase TIA production by overexpressing positive regulators of TIA biosynthetic genes have met with limited success. This limited success might be due to the fact that overexpression of several positive TIA regulators turns on expression of negative regulators of TIA biosynthetic genes. Consequently, a more effective approach for increasing expression of TIA biosynthetic genes might be to decrease expression of negative regulators of TIA biosynthetic genes. Towards this end, an RNAi construct was generated that expresses a hairpin RNA carrying nucleotide fragments from three negative transcriptional regulators of TIA genes, ZCT1, ZCT2 and ZCT3, under the control of a beta-estradiol inducible promoter. Transgenic C. roseus hairy root lines carrying this ZCT RNAi construct exhibit significant reductions in transcript levels of all three ZCT genes. Surprisingly, out of eight TIA biosynthetic genes analyzed, seven (CPR, LAMT, TDC, STR, 16OMT, D4H and DAT) exhibited decreased rather than increased transcript levels in response to reductions in ZCT transcript levels. The lone exception was T19H, which exhibited the expected negative correlation in transcript levels with transcript levels of all three ZCT genes. A possible explanation for the T19H expression pattern being the opposite of the expression patterns of the other TIA biosynthetic genes tested is that T19H shunts metabolites away from vindoline production whereas the products of the other genes tested shunt metabolites towards vindoline metabolism. Consequently, both increased expression of T19H and decreased expression of one or more of the other seven genes tested would be expected to have similar effects on flux through the TIA pathway. As T19H expression is lower in the ZCT RNAi hairy root lines than in the control hairy root line, the ZCTs could act directly to inhibit expression of T19H. In contrast, ZCT regulation of the other seven TIA biosynthetic genes tested is likely to occur indirectly, possibly by the ZCTs turning off expression of a negative transcriptional regulator of some TIA genes. In fact, transcript levels of a negative TIA transcriptional regulator, GBF1, exhibited a strong, and statistically significant, negative correlation with transcript levels of ZCT1, ZCT2 and ZCT3. Together, these findings suggest that the ZCTs repress expression of some TIA biosynthetic genes, but increase expression of other TIA biosynthetic genes, possibly by turning down expression of GBF1.

Improved virus-induced gene silencing allows discovery of a serpentine synthase gene in Catharanthus roseus.

Specialized metabolites are chemically complex small molecules with a myriad of biological functions. To investigate plant-specialized metabolite biosynthesis more effectively, we developed an improved method for virus-induced gene silencing (VIGS). We designed a plasmid that incorporates fragments of both the target gene and knockdown marker gene (phytoene desaturase, PDS), which identifies tissues that have been successfully silenced in planta. To demonstrate the utility of this method, we used the terpenoid indole alkaloid (TIA) pathway in Madagascar periwinkle (Catharanthus roseus) as a model system. Catharanthus roseus is a medicinal plant well known for producing many bioactive compounds, such as vinblastine and vincristine. Our VIGS method enabled the discovery of a previously unknown biosynthetic enzyme, serpentine synthase (SS). This enzyme is a cytochrome P450 (CYP) that produces the β-carboline alkaloids serpentine and alstonine, compounds with strong blue autofluorescence and potential pharmacological activity. The discovery of this enzyme highlights the complexity of TIA biosynthesis and demonstrates the utility of this improved VIGS method for discovering unidentified metabolic enzymes in plants.

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.

The expression of Terpenoid Indole Alkaloid (TIAs) pathway genes in Catharanthus roseus in response to salicylic acid treatment.

Vinblastine and vincristine are two important anti-cancer drugs that are synthesized by the Terpenoid Indole Alkaloids (TIAs) pathway in periwinkle (Catharanthus roseus). The major challenge in the pharmaceutical industry is the low production rate of these Alkaloids. TIA pathway is affected by elicitors, such as salicylic acid (SA). This study aimed to investigate the expression pattern of some key genes in TIAs pathway under SA treatment. Foliar application of SA (0.01 and 0.1mM) was used and leaves samples were taken at 0, 12, 18, 24 and 48h after the treatment. qRT-PCR was used to investigate the expression pattern of Chorismate mutase (Cm), tryptophan decarboxylase (Tdc), Geraniol-10-hydroxylase (G10h), Secologanin synthase (Sls), Strictosidine synthase (Str), Desacetoxyvindoline-4-hydroxylase (D4h) and Deacetylvindoline-4-O-acetyltransferase (Dat) genes, following the SA treatment. The results of this experiment showed that transcript levels of Tdc, G10h, Sls, Str, D4h and Dat genes were significantly up-regulated in both SA concentration treatments. Furthermore, the highest transcript levels of Dat was observed after 48h of the SA treatments. qRT-PCR results suggests that SA induces transcription of major genes involved in Alkaloids biosynthesis in Catharanthus roseus. It can be concluded that up-regulation of Tdc, G10h, Sls, Str, D4h and Dat genes can result in a higher production rate of Vinblastine and vincristine Alkaloids.

TIAs pathway genes and associated miRNA identification in Vinca minor: supporting aspidosperma and eburnamine alkaloids linkage via transcriptomic analysis.

V. minor contains monomeric eburnamine-type of indole alkaloids having utilization as a neuro-medicinal plant. The biosynthetic pathway studies using miRNAs has been the focal point for plant genomic research in recent years and this technique is utilized to get an insight into a possible pathway level study in V. minor as understanding of genes in this prized medicinal plant is meagrely understood. The de novo transcriptomic analysis using Illumina Next gen sequencing has been performed in glasshouse shifted plant and transformed roots to elucidate the possible non confirmed steps of terpenoid indole alkaloids (TIAs) pathway in V. minor. A putative TIA pathway is elucidated in the study including twelve possible TIAs biosynthetic genes. The specific miRNA associated with TIAs pathway were identified and their roles were discussed for the first time in V. minor. The comparative analysis of transcriptomic data of glasshouse shifted plant and transformed roots showed that the raw reads of transformed roots were higher (83,740,316) compared to glasshouse shifted plant (67,733,538). The EST-SSR prediction showed the maximum common repeats among glasshouse shifted plant and transformed roots, although small variation was found in trinucleotide repeats restricted to glasshouse shifted plant. The study reveals overall 37 miRNAs which were observed to be true and can have a role in pathway as they can regulate the growth and alkaloid production. The identification of putative pathway genes plays an important role in establishing linkage between Aspidosperma and Eburnamine alkaloids.

Metabolic Regulation Analysis of Ajmalicine Biosynthesis Pathway in Catharanthus roseus (L.) G. Don Suspension Culture Using Nanosensor

Ajmalicine is one of the most popular antihypertensive drugs obtained from the root barks of Cathranthus roseus (L.) G. Don and Rauvolfia serpentine (L.) Benth. ex Kurz. It has also potential antimicrobial, cytotoxic, central depressant and antioxidant activities. As the demand for the alkaloid is significantly high, metabolic engineering approaches are being tried to increase its production in both homologous and heterologous systems. The metabolic engineering approach requires knowledge of the metabolic regulation of the alkaloid. For understanding the metabolic regulation, fluxomic analysis is important as it helps in understanding the flux of the alkaloid through the complicated metabolic pathway. The present study was conducted to analyse the flux analysis of the ajmalicine biosynthesis, using a genetically encoded Fluorescent Resonance Energy Transfer FRET-based nanosensor for ajmalicine (FLIP-Ajn). Here, we have silenced six important genes of terpenoid indole alkaloid (TIA), namely G10H, 10HGO, TDC, SLS, STR and SDG, through RNA-mediated gene silencing in different batches of C. roseus suspension cells, generating six silenced cell lines. Monitoring of the ajmalicine level was carried out using FLIP-Ajn in these silenced cell lines, with high spatial and temporal resolution. The study offers the rapid, high throughput real-time measurement of ajmalicine flux in response to the silenced TIA genes, thereby identifying the regulatory gene controlling the alkaloid flux in C. roseus suspension cells. We have reported that the STR gene encoding strictosidine synthase of the TIA pathway could be the regulatory gene of the ajmalicine biosynthesis.

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.

Endophytes enhance the production of root alkaloids ajmalicine and serpentine by modulating the terpenoid indole alkaloid pathway in Catharanthus roseus roots.

The aim of this study was to identify and characterize promising endophytes capable of enhancing the content of root alkaloids ajmalicine and serpentine in low alkaloid yielding genotype of Catharanthus roseus cultivar Prabal and the possible mechanisms involved. Of the four strains isolated from alkaloid-rich genotype of C. roseus cultivar Dhawal, endophytic strains CATDLF5 (Curvularia sp.) and CATDLF6 (Choanephora infundibulifera) enhanced serpentine content by 211·7-337·6%, while CATDRF2 (Aspergillus japonicus) and CATDS5 (Pseudomonas sp.) increased the content of ajmalicine by 123·4-203·8% in cultivar Prabal. Upregulated expression of key genes, geraniol 10-hydroxylase, tryptophan decarboxylase and strictosidine synthase involved in terpenoid indole alkaloid (TIA) biosynthetic pathway was observed in endophyte inoculated plants. Upregulated Octadecanoid-derivative Responsive Catharanthus AP2/ERF domain transcription activators like ORCA3 while, and downregulation of transcriptional repressor, ZCTs (Cys2/His2-type zinc finger protein family) enhanced the expression of genes for secondary metabolite production in endophyte-inoculated plants. The present work concluded that the selected endophytes of C. roseus can enhance the ajmalicine and serpentine contents by modulating the expression of structural and regulatory genes of TIA biosynthetic pathway in root. Endophytes can play an important role to enhance in planta content of pharmaceutically important alkaloids in C. roseus and can therefore be useful in reducing the cost of production of important alkaloids.

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.

Effect of abiotic elicitation and pathway precursors feeding over terpenoid indole alkaloids production in multiple shoot and callus cultures of Catharanthus roseus

Catharanthus roseus is a well-known herb with great pharmaceutical value for being the only source of natural antineoplastic drugs vinblastine and vincristine. The terpenoid indole alkaloids (TIAs) biosynthetic pathway responsible for the production of these antineoplastic drug molecules is under a strict spatiotemporal regulation that requires five different cellular as well as at least four intracellular compartmentations for its completion. Therefore, several efforts were made to investigate the cell and tissue cultures of Catharanthus roseus to establish as an alternative source of TIAs production. Cell suspension, hairy roots and callus cultures of Catharanthus roseus do not provide the required cellular complexity for the completion of entire TIAs pathway, therefore, failed to produce vinblastine and vincristine. However, the multiple shoot cultures do provide the required cellular complexity to complete the entire TIAs pathway. Therefore, in the present study, the multiple shoot cultures along with callus cultures were subjected to the abiotic elicitors and TIAs pathway precursors feeding for the first time to chase the effect of these abiotic elicitors and pathway precursors on the production of major TIAs of Catharanthus roseus. The multiple shoot cultures treated with TIAs pathway precursor tryptamine 300 mg/L accumulated highest vinblastine content (0.0277% dry wt) followed by tryptophan feeding at 300 mg/L (0.0180% dry wt) and 500 mg/L (0.0175% dry wt) whereas, callus cultures failed to produce vinblastine.

Stepwise response of MeJA-induced genes and pathways in leaves of C. roseus

Catharanthus roseus is a perennial herb known for the production of important terpenoid indole alkaloids (TIAs) in addition to a variety of phenolic compounds. The goal of the present work was to detect the prolonged effects of MeJA (6 uM) treatment across time (up to 24 days) in order to detect the stepwise response of MeJA-induced genes and pathways in leaves of C. rouses. Prolonged exposure of plants to MeJA (6 uM) treatment for different time points (6, 12 and 24 days) indicated that genes in the indole alkaloid biosynthesis pathway and upstream pathways were triggered earlier (e.g., 6 days) than those in the anthocyanin biosynthesis pathway and its upstream pathways (e.g., 12 days). Three enzymes, e.g., T16H, OMT, and D4H, in the six-step vindoline biosynthesis and two enzymes, e.g., TDC and STR, acting consecutively in the conversion of tryptophan to strictosidine, were activated after 6 days of MeJA treatment. Two other key enzymes, e.g., TRP and CYP72A1, acting concurrently upstream of the TIA biosynthesis pathway were upregulated after 6 days. The genes encoding TDC and STR might concurrently act as a master switch of the TIA pathway towards the production of the indole alkaloids. On the other hand, we speculate that the gene encoding PAL enzyme also acts as the master switch of phenylpropanoid biosynthesis and the downstream flavonoid biosynthesis and anthocyanin biosynthesis pathways towards the production of several phenolic compounds. PAL and the downstream enzymes were activated 12 days after treatment. Cluster analysis confirmed the concordant activities of the flower- and silique-specific bHLH25 transcription factor and the key enzyme in the TIA biosynthesis pathway, e.g., STR. Due to the stepwise response of the two sets of pathways, we speculate that enzymes activated earlier likely make TIA biosynthesis pathway a more favourable target in C. roseus than anthocyanin biosynthesis pathway.