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.

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.

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.

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.

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.

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.

ROP GTPases with a geranylgeranylation motif modulate alkaloid biosynthesis in Catharanthus roseus.

Rho of Plant (ROP) GTPases function as molecular switches that control signaling processes essential for growth, development, and defense. However, their role in specialized metabolism is poorly understood. Previously, we demonstrated that inhibition of protein geranylgeranyl transferase (PGGT-I) negatively impacts the biosynthesis of monoterpene indole alkaloids (MIA) in Madagascar periwinkle (Catharanthus roseus), indicating the involvement of prenylated proteins in signaling. Here, we show through biochemical, molecular, and in planta approaches that specific geranylgeranylated ROPs modulate C. roseus MIA biosynthesis. Among the six C. roseus ROP GTPases (CrROPs), only CrROP3 and CrROP5, having a C-terminal CSIL motif, were specifically prenylated by PGGT-I. Additionally, their transcripts showed higher expression in most parts than other CrROPs. Protein-protein interaction studies revealed that CrROP3 and CrROP5, but not ΔCrROP3, ΔCrROP5, and CrROP2 lacking the CSIL motif, interacted with CrPGGT-I. Further, CrROP3 and CrROP5 exhibited nuclear localization, whereas CrROP2 was localized to the plasma membrane. In planta functional studies revealed that silencing of CrROP3 and CrROP5 negatively affected MIA biosynthesis, while their overexpression upregulated MIA formation. In contrast, silencing and overexpression of CrROP2 had no effect on MIA biosynthesis. Moreover, overexpression of ΔCrROP3 and ΔCrROP5 mutants devoid of sequence coding for the CSIL motif failed to enhance MIA biosynthesis. These results implicate that CrROP3 and CrROP5 have a positive regulatory role on MIA biosynthesis and thus shed light on how geranylgeranylated ROP GTPases mediate the modulation of specialized metabolism in C. roseus.

Integrated transcriptomic and proteomic analyses uncover the early response mechanisms of Catharanthus roseus under ultraviolet-B radiation.

Catharanthus roseus produces a large array of terpenoid indole alkaloids (TIAs) that are important natural source for many drugs. Ultraviolet B (UVB) radiation have been proved to have regulatory effect towards biosynthesis of TIAs, which were meaningful for boost of TIA production. To decipher more comprehensive molecular characteristics in C. roseus under UVB radiation, integrated analysis of the nuclear proteome together with the transcriptome data under UVB radiation were performed. Expression of genes related to transmembrane transporters gradually increased during the prolonged exposure to UVB radiation. Some of known TIA transporters were affected by UVB. Abundance of proteins associated with spliceosome and nucleocytoplasmic transport increased. Homologs belonging to ORCA and CrWRKY transcription factors family increased at both transcriptomic and proteomic levels. At the same time, the numbers of differential alternative splicing events between UVB-radiated and white-light-treated plants continuously increased. These results suggest that the nucleus participated in early response of C. roseus under UVB radiation, where alternative splicing events occurred and might regulate multiple pathways. Furthermore, integrative omics analysis indicates that expression of enzymes at the terminal stages of seco-iridoid pathway decreased with the prolonged radiation exposure, potentially inhibiting further rise of TIA synthesis under extended UVB exposure.

A fused hybrid enzyme of 8-hydroxygeraniol oxidoreductase (8HGO) from Gardenia jasminoides and iridoid synthase (ISY) from Catharanthus roseus significantly enhances nepetalactol and iridoid production.

MAIN CONCLUSION: The operation of 8HGO-ISY fusion enzymes can increase nepetalactol flux to iridoid biosynthesis, and the Gj8HGO-CrISY expression in Gardenia jasminoides indicates that seco-iridoids and closed-ring iridoids share a nepetalactol pool. Nepetalactol is a common precursor of (seco)iridoids and their derivatives, which are a group of noncanonical monoterpenes. Functional characterization of an 8HGO (8-hydroxygeraniol oxidoreductase) from Catharanthus roseus, a seco-iridoids producing plant, has been reported; however, the 8HGO from G. jasminoides with plenty of closed-ring iridoids remains uninvestigated. In this work, a Gj8HGO was cloned and biochemically characterized. In addition, the relatively low production of nepetalactol in plants and engineered microbial host is likely to be attributed to the fact that Cr8HGO and CrISY (iridoid synthase) are substrate-promiscuous enzymes catalyzing unexpected substrates to the undesired products. Herein, a bifunctional enzyme consisting of an 8HGO fused to an ISY was designed for the proximity to the substrate and recycling of NADP+ and NADPH cofactor to reduce the undesired intermediate in the synthesis of nepetalactol. Of four fusion enzymes (i.e., Gj8HGO-GjISY, Gj8HGO-GjISY2, Gj8HGO-GjISY4, and Gj8HGO-CrISY), interestingly, only the last one can enable cascade reaction to form cis-trans-nepetalactol. Furthermore, we establish a reliable Agrobacterium-mediated transformation system. The expression of Gj8HGO-CrISY in G. jasminoides led to a significant enhancement of nepetalactol production, about 19-fold higher than that in wild-type plants, which further resulted in the twofold to fivefold increase of total iridoids and representative iridoid such as geniposide, indicating that seco-iridoids in C. roseus and closed-ring iridoids in G. jasminoides share a nepetalactol pool. All results suggest that 8HGO and ISY can be manipulated to maximize metabolic flux for nepetalactol and iridoid production.

The leaf idioblastome of the medicinal plant Catharanthus roseus is associated with stress resistance and alkaloid metabolism.

Catharanthus roseus leaves produce a range of monoterpenoid indole alkaloids (MIAs) that include low levels of the anticancer drugs vinblastine and vincristine. The MIA pathway displays a complex architecture spanning different subcellular and cell type localizations, and is under complex regulation. As a result, the development of strategies to increase the levels of the anticancer MIAs has remained elusive. The pathway involves mesophyll specialized idioblasts where the late unsolved biosynthetic steps are thought to occur. Here, protoplasts of C. roseus leaf idioblasts were isolated by fluorescence-activated cell sorting, and their differential alkaloid and transcriptomic profiles were characterized. This involved the assembly of an improved C. roseus transcriptome from short- and long-read data, IDIO+. It was observed that C. roseus mesophyll idioblasts possess a distinctive transcriptomic profile associated with protection against biotic and abiotic stresses, and indicative that this cell type is a carbon sink, in contrast to surrounding mesophyll cells. Moreover, it is shown that idioblasts are a hotspot of alkaloid accumulation, suggesting that their transcriptome may hold the key to the in-depth understanding of the MIA pathway and the success of strategies leading to higher levels of the anticancer drugs.

Genome-wide identification and characterization of wall-associated kinases, molecular docking and polysaccharide elicitation of monoterpenoid indole alkaloids in micro-propagated Catharanthus roseus.

Wall-associated kinases (WAKs) are a unique family of proteins that are predominantly localized on the plasma membrane and simultaneously bound to the cell wall. WAKs play a pivotal role in signal transduction to regulate growth, defense, and response to environmental stimuli in plants. These kinases have been identified and characterized in various plant species, however, similar information for Catharanthus roseus is scarce. C. roseus is an evergreen ornamental plant that produces a repertoire of biologically active compounds. The plant is best characterized for the production of antineoplastic monoterpenoid indole alkaloids (MIAs) namely vinblastine and vincristine. Owing to the diverse composition of phytochemicals, C. roseus is known as a "model non-model" plant for secondary metabolite research. Genome analyses showed 37 putative CrWAK genes present in C. roseus, largely localized on the plasma membrane. Phylogenetic analysis revealed six clusters of CrWAKs. Diverse cis-acting elements, including those involved in defense responses, were identified on the promotor regions of CrWAK genes. The highest binding affinity (- 12.6 kcal/mol) was noted for CrWAK-22 against tri-galacturonic acid. Tri-galacturonic acid stimulated 2.5-fold higher production of vinblastine, sixfold upregulation of the expression of ORCA3 transcription factor, and 6.14-fold upregulation of CrWAK-22 expression. Based on these results it was concluded that the expression of CrWAK genes induced by biotic elicitors may have an important role in the production of MIAs. The current findings may serve as a basis for functional characterization and mechanistic explanation of the role of CrWAK genes in the biosynthesis of MIAs upon elicitation.

Regulation of T16H subcellular localization for promoting its catalytic efficiency in yeast cells.

To investigate the effect of subcellular localization on the transformation efficiency of heterologous expressed functional P450s in yeast. Microbial biotransformation offers a promising substitute for the direct extraction of natural products, but its viability in industrial applications depends on achieving high transformation efficiencies. To investigate the influence of subcellular microenvironments on the activity of heterologously expressed P450s, Catharanthus roseus tabersonine 16-hydroxylase (T16H) was chosen, and its subcellular localization was regulated by fusing organelle-localization signals. Interestingly, this manipulation had no effect on the gene expression levels of T16H, but resulted in varying conversion rates from tabersonine to 16-hydroxy tabersonine. Notably, the highest transformation efficiency was observed in yeast cells expressing peroxisome-localized T16H. Given the alkaline pH optimum for P450s, the alkaline peroxisomal lumen could be a suitable compartment for P450s reactions to achieve high transformation efficiency using yeast cells. Different organelle-localization of T16H in yeast cells resulted in varying conversion rates, suggesting that compartmentalizing the expression of target enzymes could be a viable approach to increase transformation efficiency in yeast.

Temperature-induced lipocalin-mediated membrane integrity: Possible implications for vindoline accumulation in Catharanthus roseus leaves.

Plant lipocalins perform diverse functions. Recently, allene oxide cyclase, a lipocalin family member, has been shown to co-express with vindoline pathway genes in Catharanthus roseus under various biotic/abiotic stresses. This brought focus to another family member, a temperature-induced lipocalin (CrTIL), which was selected for full-length cloning, tissue-specific expression profiling, in silico characterization, and upstream genomic region analysis for cis-regulatory elements. Stress-mediated variations in CrTIL expression were reflected as disturbances in cell membrane integrity, assayed through measurement of electrolyte leakage and lipid peroxidation product, MDA, which implicated the role of CrTIL in maintaining cell membrane integrity. For ascertaining the function of CrTIL in maintaining membrane stability and elucidating the relationship between CrTIL expression and vindoline content, if any, a direct approach was adopted, whereby CrTIL was transiently silenced and overexpressed in C. roseus. CrTIL silencing and overexpression confirmed its role in the maintenance of membrane integrity and indicated an inverse relationship of its expression with vindoline content. GFP fusion-based subcellular localization indicated membrane localization of CrTIL, which was in agreement with its role in maintaining membrane integrity. Altogether, the role of CrTIL in maintaining membrane structure has possible implications for the intracellular sequestration, storage, and viability of vindoline.

A near-complete genome assembly of Catharanthus roseus and insights into its vinblastine biosynthesis and high susceptibility to the Huanglongbing pathogen.

This study reports the assembly of a near-complete genome of Catharanthus roseus, consisting of 561.7 Mb scaffolded into 8 pseudochromosomes with a contig N50 of 24.7 Mb and a scaffold N50 of 71.1 Mb. The assembly enables the construction of a gene regulatory network of the vinblastine biosynthetic pathway and provides insights into the high susceptibility of C. roseus to the Huanglongbing pathogen.

Small RNA Profiling of Aster Yellows Phytoplasma-Infected Catharanthus roseus Plants Showing Different Symptoms.

Micropropagated Catharantus roseus plants infected with 'Candidatus Phytoplasma asteris' showed virescence symptoms, witches' broom symptoms, or became asymptomatic after their planting in pots. Nine plants were grouped into three categories according to these symptoms, which were then employed for investigation. The phytoplasma concentration, as determined by qPCR, correlated well with the severity of symptoms. To reveal the changes in the small RNA profiles in these plants, small RNA high-throughput sequencing (HTS) was carried out. The bioinformatics comparison of the micro (mi) RNA and small interfering (si) RNA profiles of the symptomatic and asymptomatic plants showed changes, which could be correlated to some of the observed symptoms. These results complement previous studies on phytoplasmas and serve as a starting point for small RNA-omic studies in phytoplasma research.

Single-cell multi-omics in the medicinal plant Catharanthus roseus.

Advances in omics technologies now permit the generation of highly contiguous genome assemblies, detection of transcripts and metabolites at the level of single cells and high-resolution determination of gene regulatory features. Here, using a complementary, multi-omics approach, we interrogated the monoterpene indole alkaloid (MIA) biosynthetic pathway in Catharanthus roseus, a source of leading anticancer drugs. We identified clusters of genes involved in MIA biosynthesis on the eight C. roseus chromosomes and extensive gene duplication of MIA pathway genes. Clustering was not limited to the linear genome, and through chromatin interaction data, MIA pathway genes were present within the same topologically associated domain, permitting the identification of a secologanin transporter. Single-cell RNA-sequencing revealed sequential cell-type-specific partitioning of the leaf MIA biosynthetic pathway that, when coupled with a single-cell metabolomics approach, permitted the identification of a reductase that yields the bis-indole alkaloid anhydrovinblastine. We also revealed cell-type-specific expression in the root MIA pathway.

Establishment of recombinant Catharanthus roseus stem cells stably overexpressing ORCA4 for terpenoid indole alkaloids biosynthesis.

Catharanthus roseus is a perennial herb of the Apocynaceae family, from which about 200 kinds of alkaloids have been characterized. Most alkaloids from C. roseus are terpenoid indole alkaloids (TIAs), such as vinblastine and vincristine, which are widely used in the clinic for their good antitumor activity. However, they were only biosynthesized in C. roseus, and their content in C. roseus is extremely low. The access to these valuable compounds is by plant extraction or chemical semisynthesis from their precursors catharanthine and vindoline. Since catharanthine and vindoline are also obtained from C. roseus, the supply of vinblastine and vincristine makes it difficult to meet market demands. Therefore, how to improve the yield of TIAs is an attractive issue. In this study, we compared the regulatory effect of two critical transcription factors, octadecanoid-derivative responsive Catharanthus AP2-domain protein 3 (ORCA3) and octadecanoid-derivative responsive Catharanthus AP2-domain protein 4 (ORCA4), on the biosynthesis of TIAs in C. roseus. The results showed that overexpressing both two transcription factors could increase the accumulation of TIAs. The effect was more significant when ORCA4 was overexpressed. To acquire C. roseus TIAs on a continuous and consistent basis, we then created and acquired C. roseus stem cells stably overexpressing ORCA4. This is the first time a recombinant C. roseus stem cell system with stable ORCA4 overexpression has been developed, which not only provides new ideas for future research in this area but also breaches new life into the industrial application of using plant cell culture to obtain natural products.

Engineering the Biosynthesis of Late-Stage Vinblastine Precursors Precondylocarpine Acetate, Catharanthine, Tabersonine in Nicotiana benthamiana.

Vinblastine is a chemotherapy agent produced by the plant Catharanthus roseus in small quantities. Currently, vinblastine is sourced by isolation or semisynthesis. Nicotiana benthamiana is a plant heterologous host that can be used for reconstitution of biosynthetic pathways as an alternative natural product sourcing strategy. Recently, the biosynthesis of the late-stage vinblastine precursors precondylocarpine acetate, catharanthine, and tabersonine have been fully elucidated. However, the large number of enzymes involved in the pathway and the unstable nature of intermediates make the reconstitution of late-stage vinblastine precursor biosynthesis challenging. We used the N. benthamiana chassis and a state-of-art modular vector assembly to optimize the six biosynthetic steps leading to production of precondylocarpine acetate from the central intermediate strictosidine (∼2.7 mg per 1 g frozen tissue). After selecting the optimal regulatory element combination, we constructed four transcriptional unit assemblies and tested their efficiency. Finally, we successfully reconstituted the biosynthetic steps leading to production of catharanthine and tabersonine.