Catalytic selectivity and evolution of cytochrome P450 enzymes involved in monoterpene indole alkaloids biosynthesis.

Cytochrome P450 enzyme (CYP)-catalyzed functional group transformations are pivotal in the biosynthesis of metabolic intermediates and products, as exemplified by the CYP-catalyzed C7-hydroxylation and the subsequent C7-C8 bond cleavage reaction responsible for the biosynthesis of the well-known antitumor monoterpene indole alkaloid (MIA) camptothecin. To determine the key amino acid residues responsible for the catalytic selectivity of the CYPs involved in MIA biosynthesis, we characterized the enzymes CYP72A728 and CYP72A729 as stereoselective 7-deoxyloganic acid 7-hydroxylases (7DLHs). We then conducted a comparative analysis of the amino acid sequences and the predicted structures of the CYP72A homologs involved in camptothecin biosynthesis, as well as those of the CYP72A homologs implicated in the pharmaceutically significant MIAs biosynthesis in Catharanthus roseus. The crucial amino acid residues for the catalytic selectivity of the CYP72A-catalyzed reactions were identified through fragmental and individual residue replacement, catalytic activity assays, molecular docking, and molecular dynamic simulations analysis. The fragments 1 and 3 of CYP72A565 were crucial for its C7-hydroxylation and C7-C8 bond cleavage activities. Mutating fragments 1 and 2 of CYP72A565 transformed the bifunctional CYP72A565 into a monofunctional 7DLH. Evolutionary analysis of the CYP72A homologs suggested that the bifunctional CYP72A in MIA-producing plants may have evolved into a monofunctional CYP72A. The gene pairs CYP72A728-CYP72A610 and CYP72A729-CYP72A565 may have originated from a whole genome duplication event. This study provides a molecular basis for the CYP72A-catalyzed hydroxylation and C-C bond cleavage activities of CYP72A565, as well as evolutionary insights of CYP72A homologs involved in MIAs biosynthesis.

Microbial production of 5-epi-jinkoheremol, a plant-derived antifungal sesquiterpene.

Synthetic biology using microbial chassis is emerging as a powerful tool for the production of natural chemicals. In the present study, we constructed a microbial platform for the high-level production of a sesquiterpene from Catharanthus roseus, 5-epi-jinkoheremol, which exhibits strong fungicidal activity. First, the mevalonate and sterol biosynthesis pathways were optimized in engineered yeast to increase the metabolic flux toward the biosynthesis of the precursor farnesyl pyrophosphate. Then, the transcription factor Hac1- and m6A writer Ime4-based metabolic engineering strategies were implemented in yeast to increase 5-epi-jinkoheremol production further. Next, protein engineering was performed to improve the catalytic activity and enhance the stability of the 5-epi-jinkoheremol synthase TPS18, resulting in the variant TPS18I21P/T414S, with the most improved properties. Finally, the titer of 5-epi-jinkoheremol was elevated to 875.25 mg/L in a carbon source-optimized medium in shake flask cultivation. To the best of our knowledge, this is the first study to construct an efficient microbial cell factory for the sustainable production of this antifungal sesquiterpene.IMPORTANCEBiofungicides represent a new and sustainable tool for the control of crop fungal diseases. However, hindered by the high cost of biofungicide production, their use is not as popular as expected. Synthetic biology using microbial chassis is emerging as a powerful tool for the production of natural chemicals. We previously identified a promising sesquiterpenoid biofungicide, 5-epi-jinkoheremol. Here, we constructed a microbial platform for the high-level production of this chemical. The metabolic engineering of the terpene biosynthetic pathway was firstly employed to increase the metabolic flux toward 5-epi-jinkoheremol production. However, the limited catalytic activity of the key enzyme, TPS18, restricted the further yield of 5-epi-jinkoheremol. By using protein engineering, we improved its catalytic efficiency, and combined with the optimization of regulation factors, the highest production of 5-epi-jinkoheremol was achieved. Our work was useful for the larger-scale efficient production of this antifungal sesquiterpene.

Genome-Wide Survey of the Potential Function of CrLBDs in Catharanthus roseus MIA Biosynthesis.

Catharanthus roseus (C. roseus) can produce over 150 types of monoterpenoid indole alkaloids (MIAs), including vinblastine and vincristine, which are currently the primary sources of these alkaloids. Exploring the complex regulatory mechanisms of C. roseus is significant for resolving MIA biosynthesis. The Lateral Organ Boundaries Domain (LBD) is a plant-specific transcription factor family that plays crucial roles in the physiological processes of plant growth, stress tolerance, and specialized metabolism. However, the LBD gene family has not been extensively characterized in C. roseus, and whether its members are involved in MIA biosynthesis is still being determined. A total of 34 C. roseus LBD (CrLBD) genes were identified. RNA-Seq data were investigated to examine the expression patterns of CrLBD genes in various tissues and methyl jasmonate (MeJA) treatments. The results revealed that the Class Ia member CrLBD4 is positively correlated with iridoid biosynthetic genes (p < 0.05, r ≥ 0.8); the Class IIb member CrLBD11 is negatively correlated with iridoid biosynthetic genes (p < 0.05, r ≤ -0.8). Further validation in leaves at different growth stages of C. roseus showed that CrLBD4 and CrLBD11 exhibited different potential expression trends with iridoid biosynthetic genes and the accumulation of vindoline and catharanthine. Yeast one-hybrid (Y1H) and subcellular localization assays demonstrated that CrLBD4 and CrLBD11 could bind to the "aattatTCCGGccgc" cis-element and localize to the nucleus. These findings suggest that CrLBD4 and CrLBD11 may be potential candidates for regulating MIA biosynthesis in C. roseus. In this study, we systematically analyzed the CrLBD gene family and provided insights into the roles of certain CrLBDs in the MIA biosynthesis of C. roseus.

[Bioactive secondary metabolites from an endophytic fungus Aspergillus sp. CCH-1E from Catharanthus roseus].

This study focused on the bioactive secondary metabolites of an endophytic fungus Aspergillus sp. CCH-1E from Catharanthus roseus. The secondary metabolites from Aspergillus sp. CCH-1E were isolated by using various chromatographic methods [such as normal-phase and reversed-phase chromatography and high-performance liquid chromatography(HPLC)], and their structures were identified by various spectroscopic methods [e.g., ultraviolet(UV) spectroscopy, infrared(IR) spectroscopy, nuclear magnetic resonance(NMR) spectroscopy, and high-resolution electrospray ionization mass spectrometry(HR-ESI-MS)]. Twelve compounds were yielded and identified from Aspergillus sp. CCH-1E, which are chermesinone H(1), chermesinone I(2), chermesinone B(3), 8,11-didehydrochermesinone B(4), chermesinone C(5), chermesinone A(6), chevalone B(7), barbacenic acid(8), 3,6,8-trihydroxy-3,5,7-trimethyl-3,4-dihydroisocoumarin(9), 5-hydroxy-2-methoxy-7-methyl-1,4-naphthoquinone(10), 1-hydroxy-6,8-dimethoxy-3-methylanthracene-9,10-dione(11), and 7-drimen-9α,11,12-triol(12). Among them, compounds 1 and 2 are new compounds. The growth inhibition effects of all compounds were evaluated against non-small cell lung cancer cell lines A549 and NCI-H1650, as well as human cervical cancer cell line HeLa by using methylthiazolyldiphenyl-tetrazolium bromide(MTT). Compound 7 significantly inhibited the growth of three tumor cells with the IC_(50) values of 1.22-2.43 µmol·L~(-1), respectively. Compounds 1-6 showed moderate cell growth inhibition with the IC_(50) values of 16.24-35.28 µmol·L~(-1).

Influence of biochar amendment on removal of heavy metal from soils using phytoremediation by Catharanthus roseus L. and Chrysopogon zizanioides L.

Advances in sustainable toxic heavy metal treatment technologies are crucial to meet our needs for safer land to develop an urban resilient future. The heavy metals bioaccumulate in the food chain due to their persistence in the soil, which poses a serious challenge to its removal and control. Utilisation of hyperaccumulators to reduce the mobility, accumulation and toxic impact of heavy metals is a promising and ecologically safe technique. Amendments such as biochar and chelates have been shown to enhance the phytoremediation efficiency. However, the potential soil improvement is influenced by the properties of the amendment, plant and metal heterogeneities. In this study, an organic sugarcane bagasse biochar amendment for the 60-day pot experiment using Catharanthus roseus L. (NT) and Chrysopogon zizanioides L. (VT) in a heavy metal-contaminated soil was applied. The influence of biochar on the phytoremediation of lead (Pb), zinc (Zn) and cadmium (Cd) from the soil was explored. The plant survival rate enhanced to 100% with biochar amendment, and the biomass increased from 5.83 to 15 g in Zn-contaminated samples. Nutrients such as potassium concentration are directly correlated to the amendment rates, whereas phosphate decreases beyond the 2% biochar amendment rate in both plants. High heavy metal accumulation capacities with improved growth with biochar indicate the sustainability of the process. The translocation factor (TF) > 1 for Zn in NT represents the phytoextraction efficiencies whereas VT indicates high BCF values in the range of 0.5-3.53 for the amended Zn-contaminated soils. The findings indicate that the amendment rate of 2% improves nutrient cycling, plant biomass and heavy metal removal efficiencies. The insights from this study establish that the synergy between biochar amendment and the selected medicinal plants improved the phytoremediation efficiency.

Characterization of the ZCTs, a subgroup of Cys2-His2 zinc finger transcription factors regulating alkaloid biosynthesis in Catharanthus roseus.

KEY MESSAGE: The C. roseus ZCTs are jasmonate-responsive, can be induced by CrMYC2a, and can act as significant regulators of the terpenoid indole alkaloid pathway when highly expressed. Catharanthus roseus is the sole known producer of the anti-cancer terpenoid indole alkaloids (TIAs), vinblastine and vincristine. While the enzymatic steps of the pathway have been elucidated, an understanding of its regulation is still emerging. The present study characterizes an important subgroup of Cys2-His2 zinc finger transcription factors known as Zinc finger Catharanthus Transcription factors (ZCTs). We identified three new ZCT members (named ZCT4, ZCT5, and ZCT6) that clustered with the putative repressors of the TIA pathway, ZCT1, ZCT2, and ZCT3. We characterized the role of these six ZCTs as potential redundant regulators of the TIA pathway, and their tissue-specific and jasmonate-responsive expression. These ZCTs share high sequence conservation in their two Cys2-His2 zinc finger domains but differ in the spacer length and sequence between these zinc fingers. The transient overexpression of ZCTs in seedlings significantly repressed the promoters of the terpenoid (pLAMT) and condensation branch (pSTR1) of the TIA pathway, consistent with that previously reported for ZCT1, ZCT2, and ZCT3. In addition, ZCTs significantly repressed and indirectly activated several promoters of the vindoline pathway (not previously studied). The ZCTs differed in their tissue-specific expression but similarly increased with jasmonate in a dosage-dependent manner (except for ZCT5). We showed significant activation of the pZCT1 and pZCT3 promoters by the de-repressed CrMYC2a, suggesting that the jasmonate-responsive expression of the ZCTs can be mediated by CrMYC2a. In summary, the C. roseus ZCTs are jasmonate-responsive, can be induced by CrMYC2a, and can act as significant regulators of the TIA pathway when highly expressed.

Characterization of a vacuolar importer of secologanin in Catharanthus roseus.

Monoterpenoid indole alkaloid (MIA) biosynthesis in Catharanthus roseus is a paragon of the spatiotemporal complexity achievable by plant specialized metabolism. Spanning a range of tissues, four cell types, and five cellular organelles, MIA metabolism is intricately regulated and organized. This high degree of metabolic differentiation requires inter-cellular and organellar transport, which remains understudied. Here, we have characterized a vacuolar importer of secologanin belonging to the multidrug and toxic compound extrusion (MATE) family, named CrMATE1. Phylogenetic analyses of MATEs suggested a role in alkaloid transport for CrMATE1, and in planta silencing in two varieties of C. roseus resulted in a shift in the secoiridoid and MIA profiles. Subcellular localization of CrMATE1 confirmed tonoplast localization. Biochemical characterization was conducted using the Xenopus laevis oocyte expression system to determine substrate range, directionality, and rate. We can confirm that CrMATE1 is a vacuolar importer of secologanin, translocating 1 mM of substrate within 25 min. The transporter displayed strict directionality and specificity for secologanin and did not accept other secoiridoid substrates. The unique substrate-specific activity of CrMATE1 showcases the utility of transporters as gatekeepers of pathway flux, mediating the balance between a defense arsenal and cellular homeostasis.

Quantitative Single-Cell Mass Spectrometry Provides a Highly Resolved Analysis of Natural Product Biosynthesis Partitioning in Plants.

Plants produce an extraordinary array of natural products (specialized metabolites). Notably, these structurally complex molecules are not evenly distributed throughout plant tissues but are instead synthesized and stored in specific cell types. Elucidating both the biosynthesis and function of natural products would be greatly facilitated by tracking the location of these metabolites at the cell-level resolution. However, detection, identification, and quantification of metabolites in single cells, particularly from plants, have remained challenging. Here, we show that we can definitively identify and quantify the concentrations of 16 molecules from four classes of natural products in individual cells of leaf, root, and petal of the medicinal plant Catharanthus roseus using a plate-based single-cell mass spectrometry method. We show that identical natural products show substantially different patterns of cell-type localization in different tissues. Moreover, we show that natural products are often found in a wide range of concentrations across a population of cells, with some natural products at concentrations of over 100 mM per cell. This single-cell mass spectrometry method provides a highly resolved picture of plant natural product biosynthesis partitioning at a cell-specific resolution.

Bioprocessing of camptothecin from Alternaria brassicicola, an endophyte of Catharanthus roseus, with a strong antiproliferative activity and inhibition to Topoisomerases.

Suppression of fungal camptothecin (CPT) biosynthesis with the preservation and successive subculturing is the challenge that impedes fungi from the industrial application, so, screening for a novel fungal isolate with a conceivable stable producing potency of CPT was the main objective of this work. Catharanthus roseus with diverse contents of bioactive metabolites could have a plethora of novel endophytes with unique metabolic properties. Among the endophytes of C. roseus, Alternaria brassicicola EFBL-NV OR131587.1 was the highest CPT producer (96.5 µg/L). The structural identity of the putative CPT was verified by HPLC, FTIR, HNMR and LC-MS/MS, with a molecular mass 349 m/z, and molecular fragmentation patterns that typically identical to the authentic one. The purified A. brassicicola CPT has a strong antiproliferative activity towards UO-31 (0.75 µM) and MCF7 (3.2 µM), with selectivity index 30.8, and 7.1, respectively, in addition to resilient activity to inhibit Topo II (IC50 value 0.26 nM) than Topo 1 (IC50 value 3.2 nM). The purified CPT combat the wound healing of UO-31 cells by ~ 52%, stops their matrix formation, cell migration and metastasis. The purified CPT arrest the cellular division of the UO-31 at the S-phase, and inducing their cellular apoptosis by ~ 20.4 folds, compared to the control cells. Upon bioprocessing with the surface response methodology, the CPT yield by A. brassicicola was improved by ~ 3.3 folds, compared to control. The metabolic potency of synthesis of CPT by A. brassicicola was attenuated with the fungal storage and subculturing, losing ~ 50% of their CPT productivity by the 6th month of storage and 6th generation. Practically, the CPT productivity of the attenuated A. brassicicola was restored by addition of 1% surface sterilized leaves of C. roseus, ensuring the eliciting of cryptic gene cluster of A. brassicicola CPT via the plant microbiome-A. brassicicola interactions. So, for the first time, a novel endophytic isolate A. brassicicola, from C. roseus, was explored to have a relatively stable CPT biosynthetic machinery, with an affordable feasibility to restore their CPT productivity using C. roseus microbiome, in addition to the unique affinity of the extracted CPT to inhibit Topoisomerase I and II.

Detection of vinblastine and related bioactive compounds from culture extracts of endophytic fungi of Catharanthus roseus.

This study investigates the synthesis of vinblastine by endophytic fungi isolated from leaf of C. roseus. A total of 10 endophytic fungi were selected for secretion of vinca alkaloids based on the initial screening by biochemical tests and thin-layer chromatography (TLC). Out of these ten, only four fungal extracts showed positive results for presence of vinblastine at same retention time (10 min.) compared to reference compound on HPLC analysis. The detected concentration of vinblastine was maximum (17 µg/ml) in isolate no. CRL 22 followed by CRL 52, CRL 17 and CRL 28. To validate the presence of vinblastine, ultra-high-performance liquid chromatography coupled with high-resolution accurate mass spectrometry (HRMS) was employed. This analysis confirmed the presence of anhydrovinblastine, a precursor of vinblastine through the detection of molecular ions at m/z 793.4185 in extract of CRL 17. In addition to anhydrovinblastine, the intermediate compounds essential to the biosynthetic pathway of vinblastine were also detected in the extract of CRL 17. These host-origin compounds strongly suggest the presence of a biosynthetic pathway within the endophytic fungus. Based on morphological observation and sequence analysis of the ITS region of rDNA, endophytic fungi were identified as Alternaria alternata (CRL 17), Curvularia lunata (CRL 28), Aspergillus terrus (CRL 52), and Aspergillus clavatonanicus (CRL 22).

Catharanthus roseus-mediated CuAl2O4 nanocomposites for evaluation of killing kinetics.

The present article portrayed on the killing kinetic of human pathogenic bacteria using bioinspired mesoporous CuAl2O4 nanocomposites (NCs). The NCs was fabricated using leaf extract of medicinal plant Catharanthus roseus (CR) as a green reducer and stabilizer. As bio-fabricated material was calcined at 800 °C and characterized by several analytical techniques like X-ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FT-IR), Ultraviolet-Visible Diffuse Reflectance Spectroscopy (UV-DRS), Energy Dispersive X-Ray Spectroscopy (EDS), X-Ray Photoelectron Spectroscopy (XPS), Raman, Brunauer-Emmett-Teller (BET), Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM) to authenticate its structure, phase, chemical bonding, chemical state, size and morphology behaviors. XRD and TEM revealed a reduced crystallite and nanoscale sizes of biosynthesized NCs. Moreover, XRD study exposed a cubic-structure of material, while transmission electron microscopy rendered an average particles size in range 10-15 nm. However, BET profile advocates a mesoporous nature of the particles. An effective biological molecular docking modulation assessed by substituting natural inhibitor by bioinspired NCs, while the protein PDB ID 4Z8D FabH as a receptor site for the present investigation. After assessment of molecular docking examination, the antibacterial activity of bioinspired NCs were performed against Staphylococcus aureus, Bacillus subtillis, Klebsiella pneumoniae and Escherichia coli using agar-well method. The broth culture method was employed on different pathogenic strains by kinetic growth assays and colony forming unit.

Crosstalk between melatonin and nitric oxide restrains Cadmium-induced oxidative stress and enhances vinblastine biosynthesis in Catharanthus roseus (L) G Don.

KEY MESSAGE: Nitric oxide functions downstream of the melatonin in adjusting Cd-induced osmotic and oxidative stresses, upregulating the transcription of D4H and DAT genes, and increasing total alkaloid and vincristine contents. A few studies have investigated the relationship between melatonin (MT) and nitric oxide (NO) in regulating defensive responses. However, it is still unclear how MT and NO interact to regulate the biosynthesis of alkaloids and vincristine in leaves of Catharanthus roseus (L.) G. Don under Cd stress. Therefore, this context was explored in the present study. Results showed that Cd toxicity (200 µM) induced oxidative stress, decreased biomass, Chl a, and Chl b content, and increased the content of total alkaloid and vinblastine in the leaves. Application of both MT (100 µM) and sodium nitroprusside (200 µM SNP, as NO donor) enhanced endogenous NO content and accordingly increased metal tolerance index, the content of total alkaloid and vinblastine. It also upregulated the transcription of two respective genes (D4H and DAT) under non-stress and Cd stress conditions. Moreover, the MT and SNP treatments reduced the content of H2O2 and malondialdehyde, increased the activities of superoxide dismutase and ascorbate peroxidase, enhanced proline accumulation, and improved relative water content in leaves of Cd-exposed plants. The scavenging NO by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy l-3-oxide (cPTIO) averted the effects of MT on the content of total alkaloid and vinblastine and antioxidative responses. Still, the effects conferred by NO on attributes mentioned above were not significantly impaired by p-chlorophenylalanine (p-CPA as an inhibitor of MT biosynthesis). These findings and multivariate analyses indicate that MT motivated terpenoid indole alkaloid biosynthesis and mitigated Cd-induced oxidative stress in the leaves of periwinkle in a NO-dependent manner.

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

KEY MESSAGE: A 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.

Structural insights into the molecular mechanism of phytoplasma immunodominant membrane protein.

Immunodominant membrane protein (IMP) is a prevalent membrane protein in phytoplasma and has been confirmed to be an F-actin-binding protein. However, the intricate molecular mechanisms that govern the function of IMP require further elucidation. In this study, the X-ray crystallographic structure of IMP was determined and insights into its interaction with plant actin are provided. A comparative analysis with other proteins demonstrates that IMP shares structural homology with talin rod domain-containing protein 1 (TLNRD1), which also functions as an F-actin-binding protein. Subsequent molecular-docking studies of IMP and F-actin reveal that they possess complementary surfaces, suggesting a stable interaction. The low potential energy and high confidence score of the IMP-F-actin binding model indicate stable binding. Additionally, by employing immunoprecipitation and mass spectrometry, it was discovered that IMP serves as an interaction partner for the phytoplasmal effector causing phyllody 1 (PHYL1). It was then shown that both IMP and PHYL1 are highly expressed in the S2 stage of peanut witches' broom phytoplasma-infected Catharanthus roseus. The association between IMP and PHYL1 is substantiated through in vivo immunoprecipitation, an in vitro cross-linking assay and molecular-docking analysis. Collectively, these findings expand the current understanding of IMP interactions and enhance the comprehension of the interaction of IMP with plant F-actin. They also unveil a novel interaction pathway that may influence phytoplasma pathogenicity and host plant responses related to PHYL1. This discovery could pave the way for the development of new strategies to overcome phytoplasma-related plant diseases.

Vincarostine A, a novel anti-malarial trimeric monoterpenoid indole alkaloid from Catharanthus roseus.

A novel trimeric monoterpenoid indole alkaloid, vincarostine A (1) consisting of an aspidosperma-iboga-aspidosperma type skeleton, was isolated from the whole plant of Catharanthus roseus. The structure including absolute stereochemistry was elucidated on the basis of 2D NMR data and CD spectrum. Vincarostine A (1) showed anti-malarial activity.

Catharanthus roseus intoxication mimicking acute cholangitis.

BACKGROUND: Catharanthus roseus, a Madagascar native flowering plant, is known for its glossy leaves and vibrant flowers, and its medicinal significance due to its alkaloid compounds. As a source of vinblastine and vincristine used in chemotherapy, Catharanthus roseus is also employed in traditional medicine with its flower and stalks in dried form. Its toxicity can lead to various adverse effects. We report a case of Catharanthus roseus juice toxicity presenting as acute cholangitis, emphasizing the importance of healthcare providers obtaining detailed herbal supplement histories. CASE PRESENTATION: A 65-year-old woman presented with abdominal pain, fever, anorexia, and lower limb numbness. Initial diagnosis of acute cholangitis was considered, but imaging excluded common bile duct stones. Further investigation revealed a history of ingesting Catharanthus roseus juice for neck pain. Laboratory findings showed leukocytosis, elevated liver enzymes, and hyperbilirubinemia. The patient developed gastric ulcers, possibly due to alkaloids in Catharanthus roseus. No bacterial growth was noted in blood cultures. The patient recovered after discontinuing the herbal extract. CONCLUSIONS: Catharanthus roseus toxicity can manifest as fever, hepatotoxicity with cholestatic jaundice, and gastric ulcers, mimicking acute cholangitis. Awareness of herbal supplement use and potential toxicities is crucial for healthcare providers to ensure prompt diagnosis and appropriate management. This case emphasizes the need for public awareness regarding the possible toxicity of therapeutic herbs and the importance of comprehensive patient histories in healthcare settings.

Discovery and Characterization of Two Highly Divergent Variants of a Novel Potyvirus Species Infecting Madagascar Periwinkle (Catharanthus roseus).

During the summer of 2022, a cluster of Madagascar periwinkle plants with white and mauve flowers were observed with foliar mild yellow mosaic symptoms on a private property in Harlingen, Cameron County, Texas. The symptoms were reproduced on mechanically inoculated periwinkle and Nicotiana benthamiana plants. Virions of 776 to 849 nm in length and 11.7 to 14.8 nm in width were observed in transmission electron microscopy of leaf dip preparations made from symptomatic periwinkle leaves. High-throughput sequencing (HTS) analysis of total RNA extracts from symptomatic leaves revealed the occurrence of two highly divergent variants of a novel Potyvirus species as the only virus-like sequences present in the sample. The complete genomes of both variants were independently amplified via reverse transcriptase PCR, cloned, and Sanger sequenced. The 5' and 3' of the genomes were acquired using random amplification of cDNA ends methodology. The assembled virus genomes were 9,936 and 9,944 nucleotides (nt) long, and they shared 99.9 to 100% identities with the respective HTS-derived genomes. Each genome encoded hypothetical polyprotein of 3,171 amino acids (aa) (362.6 kilodaltons [kDa]) and 3,173 aa (362.7 kDa), respectively, and they shared 77.3/84.4% nt/aa polyprotein identities, indicating that they represent highly divergent variants of the same Potyvirus species. Both genomes also shared below-species-threshold polyprotein identity levels with the most closely phylogenetically related known potyviruses, thus indicating that they belong to a novel species. The name periwinkle mild yellow mosaic virus (PwMYMV) is given to the potyvirus with complete genomes of 9,936 nt for variant 1 (PwMYMV-1) and 9,944 nt for variant 2 (PwMYMV-2). We propose that PwMYMV be assigned into the genus Potyvirus (family Potyviridae).

Characterization of a CrPME indicates its possible role in determining vindoline accumulation in Catharanthus roseus leaves.

The leaf-specific Catharanthus roseus alkaloid, vindoline, is the major bottleneck precursor in the production of scarce and costly anticancer bisindoles (vincristine and vinblastine). The final steps of its biosynthesis and storage occur in the laticifers. Earlier, we have shown that vindoline content is directly related to laticifer number. Pectin remodeling enzymes, like pectin methylesterase (PME), are known to be involved in laticifer development. A search in the croFGD yielded a leaf-abundant CrPME isoform that co-expressed with a few vindoline biosynthetic genes. Full-length cloning, tissue-specific expression profiling, and in silico analysis of CrPME were carried out. It was found to possess all the specific characteristics of a typical plant PME. Transient silencing (through VIGS) and overexpression of CrPME in C. roseus indicated a direct relationship between its expression and vindoline content. Comparative analysis of transcript abundance and enzyme activity in three familial C. roseus genotypes differing significantly in their vindoline content and laticifer count (CIM-Sushil > Dhawal > Nirmal) also corroborated the positive relationship of CrPME expression with vindoline content. This study highlights the possible role of CrPME, a cell wall remodeling enzyme, in modulating laticifer-associated secondary metabolism.

Functional annotation of proteins in Catharanthus roseus shoot cultures under biogenic zinc nanotreatment.

Nano-interactions are well known for their positive as well as negative impacts on the morphological and physiological systems of plants. Keeping in mind, the conformational changes in plant proteins as one of the key mechanisms for stress adaptation responses, the current project was designed to explore the effect of glutathione-capped and uncapped zinc nano-entities on Catharanthus roseus shoot cultures. Zinc nanotreatment (0.05 µg/mL) significantly induced ester production in C. roseus shoots as detected by Gas Chromatography-Mass spectrometry. These nanotreated shoots were further subjected to peptide-centric nano-LC-MS/MS analysis. Mass spectrometry followed by a Heat map revealed a significant effect of zinc nanoparticles on 59 distinct classes of proteins as compared to control. Proteins involved in regulating stress scavenging, transport, and secondary metabolite biosynthesis were robustly altered under capped zinc nanotreatment. UniProt database identified majority of the localization of the abundantly altered protein in cell membranes and chloroplasts. STRING and Cytoscape analysis assessed inter and intra coordination of triosephosphate isomerase with other identified proteins and highlighted its role in the regulation of protein abundance under applied stress. This study highlights the understanding of complex underlying mechanisms and regulatory networks involved in proteomic alterations and interactions within the plant system to cope with the nano-effect.

CrJAT1 Regulates Endogenous JA Signaling for Modulating Monoterpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus.

Many monoterpenoid indole alkaloids (MIAs) produced in Catharanthus roseus have demonstrated biological activities and clinical potential. However, their complex biosynthesis pathway in plants leads to low accumulation, limiting therapeutic applications. Efforts to elucidate the MIA biosynthetic regulatory mechanism have focused on improving accumulation levels. Previous studies revealed that jasmonic acid (JA), an important plant hormone, effectively promotes MIA accumulation by inducing the expression of MIA biosynthesis and transport genes. Nevertheless, excessive JA signaling can strongly inhibit plant growth, decreasing MIA productivity in C. roseus. Therefore, identifying key components balancing growth and MIA production in the JA signaling pathway is imperative for effective pharmaceutical production. Here, we identify a homolog of the jasmonate transporter 1, CrJAT1, through co-expression and phylogenetic analyses. Further investigation demonstrated that CrJAT1 can activate JA signaling to promote MIA accumulation without compromising growth. The potential role of CrJAT1 in redistributing intra/inter-cellular JA and JA-Ile may calibrate signaling to avoid inhibition, representing a promising molecular breeding target in C. roseus to optimize the balance between growth and specialized metabolism for improved MIA production.