The assembly of (+)‐vincadifformine‐ and (−)‐tabersonine‐derived monoterpenoid indole alkaloids in Catharanthus roseus involves separate branch pathways

The biological activity of monoterpenoid indole alkaloids (MIAs) has led to their use in cancer treatment and other medical applications. Their biosynthesis has involved the formation of reactive intermediates by responsible enzymes to elaborate several different chemical scaffolds. Modification of scaffolds through different substitution reactions has produced chemically diverse MIAs and related biological activities. The present study characterizes the three‐step pathway involved in the formation of (+)‐echitovenine, the major O‐acetylated MIA of Catharanthus roseus roots, and differentiates it from a parallel pathway involved in the formation of hörhammericine. Separate hydrolases convert a common reactive MIA intermediate to aspidosperma skeletons of opposite specific rotations, that is (+)‐vincadifformine and (?)‐tabersonine, respectively. The formation of (+) minovincinine from (+) vincadifformine 19‐hydroxylase (V19H) is catalyzed by a root‐specific cytochrome P450 with high amino acid sequence similarity to the leaf‐specific tabersonine‐3‐hydroxylase involved in vindoline biosynthesis. Similarly, O‐acetylation of (+)‐minovincinine to form (+) echitovenine involves minovincinine‐O‐acetytransferase. The substrate specificity of V19H and MAT for their respective (+)‐enantiomers defines the separate enantiomer‐specific pathway involved in (+)‐echitovenine biosynthesis and differentiates it from a parallel (?)‐enantiomer‐specific pathway involved in the formation of hörhammericine from (?)‐tabersonine.     

Genome‐guided investigation of plant natural product biosynthesis

The medicinal plant Madagascar periwinkle, Catharanthus roseus (L.) G. Don, produces hundreds of biologically active monoterpene‐derived indole alkaloid (MIA) metabolites and is the sole source of the potent, expensive anti‐cancer compounds vinblastine and vincristine. Access to a genome sequence would enable insights into the biochemistry, control, and evolution of genes responsible for MIA biosynthesis. However, generation of a near‐complete, scaffolded genome is prohibitive to small research communities due to the expense, time, and expertise required. In this study, we generated a genome assembly for C. roseus that provides a near‐comprehensive representation of the genic space that revealed the genomic context of key points within the MIA biosynthetic pathway including physically clustered genes, tandem gene duplication, expression sub‐functionalization, and putative neo‐functionalization. The genome sequence also facilitated high resolution co‐expression analyses that revealed three distinct clusters of co‐expression within the components of the MIA pathway. Coordinated biosynthesis of precursors and intermediates throughout the pathway appear to be a feature of vinblastine/vincristine biosynthesis. The C. roseus genome also revealed localization of enzyme‐rich genic regions and transporters near known biosynthetic enzymes, highlighting how even a draft genome sequence can empower the study of high‐value specialized metabolites.     

Expression of deacetylvindoline-4-O-acetyltransferase in Catharanthus roseus hairy roots

Madagascar periwinkle [Catharanthus roseus (L.) G Don] is a pantropical plant of horticultural value that produces the powerful anticancer drugs vinblastine and vincristine that are derived from the dimerization of the monoterpenoid indole alkaloids (MIAs), vindoline and catharanthine. The present study describes the genetic engineering and expression of the terminal step of vindoline biosynthesis, deacetylvindoline-4-O-acetyltransferase (DAT) in Catharanthus roseus hairy root cultures. Biochemical analyses showed that several hairy root lines expressed high levels of DAT enzyme activity compared to control hairy root cultures expressing β-gulucuronidase activity (GUS) activity. Metabolite analysis using high performance liquid chromotagraphy established that hairy root extracts had an altered alkaloid profile with respect to hörhammericine accumulation in DAT expressing lines in comparison to control lines. Further analyses of one hairy root culture expressing high DAT activity suggested that DAT expression and accumulation of hörhammericine (9) were related. It is concluded that expression of DAT in hairy roots altered their MIA profile and suggests that further expression of vindoline pathway genes could lead to significant changes in alkaloid profiles. Evidence is provided that hörhammericine (9) accumulates via a DAT interaction with the root specific minovincinine-19-O-acetyltransferase (MAT) that inhibits the MAT mediated conversion of hörhammericine (9) into 19-O-acetyl-hörhammericine (12).     

Strictosidine activation in Apocynaceae: towards a "nuclear time bomb"?

Background  

The first two enzymatic steps of monoterpene indole alkaloid (MIA) biosynthetic pathway are catalysed by strictosidine synthase (STR) that condensates tryptamine and secologanin to form strictosidine and by strictosidine β-D-glucosidase (SGD) that subsequently hydrolyses the glucose moiety of strictosidine. The resulting unstable aglycon is rapidly converted into a highly reactive dialdehyde, from which more than 2,000 MIAs are derived. Many studies were conducted to elucidate the biosynthesis and regulation of pharmacologically valuable MIAs such as vinblastine and vincristine in Catharanthus roseus or ajmaline in Rauvolfia serpentina. However, very few reports focused on the MIA physiological functions.     

Virus‐induced gene silencing in Catharanthus roseus by biolistic inoculation of tobacco rattle virus vectors

Catharanthus roseus constitutes the unique source of several valuable monoterpenoid indole alkaloids, including the antineoplastics vinblastine and vincristine. These alkaloids result from a complex biosynthetic pathway encompassing between 30 and 50 enzymatic steps whose characterisation is still underway. The most recent identifications of genes from this pathway relied on a tobacco rattle virus‐based virus‐induced gene silencing (VIGS) approach, involving an Agrobacterium‐mediated inoculation of plasmids encoding the two genomic components of the virus. As an alternative, we developed a biolistic‐mediated approach of inoculation of virus‐encoding plasmids that can be easily performed by a simple bombardment of young C. roseus plants. After optimisation of the transformation conditions, we showed that this approach efficiently silenced the phytoene desaturase gene, leading to strong and reproducible photobleaching of leaves. This biolistic transformation was also used to silence a previously characterised gene from the alkaloid biosynthetic pathway, encoding iridoid oxidase. Plant bombardment caused down‐regulation of the targeted gene (70%), accompanied by a correlated decreased in MIA biosynthesis (45–90%), similar to results obtained via agro‐transformation. Thus, the biolistic‐based VIGS approach developed for C. roseus appears suitable for gene function elucidation and can readily be used instead of the Agrobacterium‐based approach, e.g. when difficulties arise with agro‐inoculations or when Agrobacterium‐free procedures are required to avoid plant defence responses.     

Epidermis is a pivotal site of at least four secondary metabolic pathways in Catharanthus roseus aerial organs

Catharanthus roseus produces a wide range of secondary metabolites, some of which present high therapeutic values such as antitumoral monoterpenoid indole alkaloids (MIAs), vinblastine and vincristine, and the hypotensive MIA, ajmalicine. We have recently shown that a complex multicellular organisation of the MIA biosynthetic pathway occurred in C. roseus aerial organs. In particular, the final steps of both the secoiridoid–monoterpene and indole pathways specifically occurred in the epidermis of leaves and petals. Chorismate is the common precursor of indole and phenylpropanoid pathways. In an attempt to better map the spatio-temporal organisation of diverse secondary metabolisms in Catharanthus roseus aerial organs, we studied the expression pattern of genes encoding enzymes of the phenylpropanoid pathway (phenylalanine ammonia-lyase [PAL, E.C. 4.3.1.5], cinnamate 4-hydroxylase [C4H, E.C. 1.14.13.11] and chalcone synthase [CHS, E.C. 2.3.1.74]). In situ hybridisation experiments revealed that CrPAL and CrC4H were specifically localised to lignifying xylem, whereas CrPAL, CrC4H and CrCHS were specifically expressed in the flavonoid-rich upper epidermis. Interestingly, these three genes were co-expressed in the epidermis (at least the upper, adaxial one) together with three MIA-related genes, indicating that single epidermis cells were capable of concomitantly producing a wide range of diverse secondary metabolites (e.g. flavonoïds, indoles, secoiridoid–monoterpenes and MIAs). These results, and data showing co-accumulation of flavonoids and alkaloids in single cells of C. roseus cell lines, indicated the spatio-temporal feasibility of putative common regulation mechanisms for the expression of these genes involved in at least four distinct secondary metabolisms.     

Catharanthus roseus mitogen-activated protein kinase 3 confers UV and heat tolerance to Saccharomyces cerevisiae

Catharanthus roseus is an important source of pharmaceutically important Monoterpenoid Indole Alkaloids (MIAs). Accumulation of many of the MIAs is induced in response to abiotic stresses such as wound, ultra violet (UV) irradiations, etc. Recently, we have demonstrated a possible role of CrMPK3, a C. roseus mitogen-activated protein kinase in stress-induced accumulation of a few MIAs. Here, we extend our findings using Saccharomyces cerevisiae to investigate the role of CrMPK3 in giving tolerance to abiotic stresses. Yeast cells transformed with CrMPK3 was found to show enhanced tolerance to UV and heat stress. Comparison of CrMPK3 and SLT2, a MAPK from yeast shows high-sequence identity particularly at conserved domains. Additionally, heat stress is also shown to activate a 43 kDa MAP kinase, possibly CrMPK3 in C. roseus leaves. These findings indicate the role of CrMPK3 in stress-induced MIA accumulation as well as in stress tolerance.     

Quantification of Anti‐Addictive Alkaloids Ibogaine and Voacangine in In Vivo‐ and In Vitro‐Grown Plants of Two Mexican Tabernaemontana Species

Tabernaemontana alba and Tabernaemontana arborea are Apocynaceae species used in Mexican traditional medicine for which little phytochemical information exists. In this study, preliminary gas chromatography/mass spectrometry analyses of different organs obtained from wild plants of both species identified a total of 10 monoterpenoid indole alkaloids (MIAs) and one simple indole alkaloid, nine of which were reported for the first time in these species. Furthermore, callus cultures were established from T. alba leaf explants and regeneration of whole plants was accomplished via somatic embryogenesis. The anti‐addictive MIAs ibogaine and voacangine were then quantified by gas chromatography with flame ionization detection in wild plants of both species, as well as greenhouse‐grown plants, in vitro‐grown plantlets and embryogenic callus of T. alba. Ibogaine and voacangine were present in most samples taken from the whole plants of both species, with stem and root barks showing the highest concentrations. No alkaloids were detected in callus samples. It was concluded that T. alba and T. arborea are potentially viable sources of ibogaine and voacangine, and that these MIAs can be produced through somatic embryogenesis and whole plant regeneration of T. alba. Approaches to increase MIA yields in whole plants and to achieve alkaloid production directly in cell cultures are discussed.     

Oxidative stress and production of bioactive monoterpene indole alkaloids: biotechnological implications

Monoterpene indole alkaloids (MIAs) encompass plant natural products with important pharmacological relevance. They include the anti-tumoral MIAs found in Catharanthus roseus and Camptotheca acuminata. The often low yields of bioactive alkaloids in plants has prompted research to identify the factors regulating MIA production. Oxidative stress is a general response associated with biotic and abiotic stresses leading to several secondary responses, including elicitation of MIA production. These changes in secondary metabolism may take place directly or via second messengers, such as Ca²⁺ and reactive oxygen species (ROS). H₂O₂ is the main ROS that participates in MIA biosynthesis. This review analyzes the links between oxidative stress, elicitation of bioactive MIA production and their potential roles in antioxidant defense, as well as exploring the implications to developing biotechnological strategies relevant for alkaloid supply.     

Gas composition strategies for the successful scale-up of Catharanthus roseus cell cultures for the production of ajmalicine

Ajmalicine, serpentine, catharanthine, and vindoline are monoterpenoid indole alkaloids (MIAs) of commercial interest which are produced by the Catharanthus roseus plant. Cultures of C. roseus have been investigated as a potential source of these pharmaceutically important compounds since the early 1960s. In addition, their production from C. roseus cultures has served as a model system for investigating secondary metabolism and for evaluating production-enhancing strategies. Initially, this review will survey (1) the MIAs of interest for large-scale production from plant cell cultures and (2) the volumetric productivities of a specific MIA, ajmalicine, achieved and projected using plant cell cultures. To meet the need for these valuable compounds, the production of these MIAs from plant cell cultures must be successfully reproduced in large-scale aerated and agitated reactors. While the large-scale cultivation of plant cell cultures is currently feasible, initial attempts at scale-up may yield results that differ from that optimized in flasks. To bridge the jump between production in flasks and production in large-scale bioreactors, changes introduced with scale-up such as gas composition must be identified and rationally manipulated to reproduce or even improve growth and secondary metabolite production. Hence, this review will (1) identify the effects of gas composition (i.e., O₂, CO₂, ethylene, or other endogenous volatile compounds) on growth and secondary metabolism and (2) draw operating strategies for optimizing the gas composition for growth of C. roseus cultures and the production of ajmalicine.     

Optimization of the transient transformation of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> cells by particle bombardment and its application to the subcellular localization of hydroxymethylbutenyl 4-diphosphate synthase and geraniol 10-hydroxylase

The monoterpene indole alkaloids (MIA) synthesized in Catharanthus roseus are highly valuable metabolites due to their pharmacological properties. In planta, the MIA biosynthetic pathway exhibits a complex compartmentation at the cellular level, whereas subcellular data are sparse. To gain insight into this level of organization, we have developed a high efficiency green fluorescent protein (GFP) imaging approach to systematically localize MIA biosynthetic enzymes within C. roseus cells following a biolistic-mediated transient transformation. The biolistic transformation protocol has been first optimized to obtain a high number of transiently transformed cells with a ~12-fold increase compared to previous protocols and thus to clearly and easily identify the fusion GFP expression patterns in numerous cells. On the basis of this protocol, the subcellular localization of hydroxymethylbutenyl 4-diphosphate synthase (HDS), a methyl erythritol phosphate pathway enzyme and geraniol 10-hydroxylase (G10H), a monoterpene-secoiridoid pathway enzyme has been next characterized. Besides showing the accumulation of HDS within plastids of C. roseus cells, we also provide evidences of the presence of HDS in long stroma-filled thylakoid-free extensions budding from plastids, i.e. stromules that are in close association with other organelles such as endoplasmic reticulum (ER) or mitochondria in agreement with their proposed function in enhancing interorganelle metabolite exchanges. Furthermore, we also demonstrated that G10H is an ER-anchored protein, consistent with the presence of a transmembrane helix at the G10H N-terminal end, which is both necessary and sufficient to drive the ER anchoring. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Involvement of LeMRP,an ATP‐binding cassette transporter,in shikonin transport and biosynthesis in Lithospermum erythrorhizon

• Shikonin and its derivatives are important medicinal secondary metabolites accumulating in roots of Lithospermum erythrorhizon. Although some membrane proteins have been identified as transporters of secondary metabolites, the mechanisms underlying shikonin transport and accumulation in L. erythrorhizon cells still remain largely unknown.
• In this study, we isolated a cDNA encoding LeMRP, an ATP‐binding cassette transporter from L. erythrorhizon, and further investigated its functions in the transport and biosynthesis of shikonin using the yeast transformation and transgenic hairy root methods, respectively. Real‐time PCR was applied for expression analyses of LeMRP and shikonin biosynthetic enzyme genes.
• Functional analysis of LeMRP using the heterologous yeast cell expression system showed that LeMRP could be involved in shikonin transport. Transgenic hairy roots of L. erythrorhizon demonstrated that LeMRP overexpressing hairy roots produced more shikonin than the empty vector (EV) control. Real‐time PCR results revealed that the enhanced shikonin biosynthesis in the overexpression lines was mainly caused by highly up‐regulated expression of genes coding key enzymes (LePAL, HMGR, Le4CL and LePGT) involved in shikonin biosynthesis. Conversely, LeMRP RNAi decreased the accumulation of shikonin and effectively down‐regulated expression level of the above genes. Typical inhibitors of ABC proteins, such as azide and buthionine sulphoximine, dramatically inhibited accumulation of shikonin in hairy roots.
• Our findings provide evidence for the important direct or indirect role of LeMRP in transmembrane transport and biosynthesis of shikonin.