A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus

The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by the Madagascar periwinkle (Catharanthus roseus). Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003–0.01% yields. Metabolic engineering efforts to either improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. A VIGS method was developed herein to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro.     

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.     

Peroxidase and the biosynthesis of terpenoid indole alkaloids in the medicinal plant Catharanthus roseus (L.) G. Don

The leaves of Catharanthus roseus (L.) G. Don produce the first natural drugs used in cancer therapy – the dimeric terpenoid indole alkaloids vinblastine and vincristine. The study of C. roseus further revealed two other terpenoid indole alkaloids with important pharmacological activity: ajmalicine, used as an antihypertensive, and serpentine, used as sedative. The biosynthetic pathway of the medicinal alkaloids has been investigated in much detail and a number of steps are now well characterized at the enzyme and gene level and, recently, several regulatory genes have also been isolated and characterized. Since early studies of the biosynthesis of vinblastine, during the 1970s and 1980s, the dimerization reaction has attracted much attention due to its possible regulatory importance and potential application for the semi synthetic production of the dimeric alkaloids. After initial, inconclusive work suggesting the involvement of peroxidase-like enzymes, the search for the dimerization enzyme in leaf tissue detected a single dimerization activity credited to the single class III plant peroxidase present in the leaves of the plant – the basic isoenzyme CRPRX1. The enzyme was purified to homogeneity, the respective cDNA and genomic sequences were characterized, and a channeling mechanism was proposed for the peroxidase-mediated-vacuolar synthesis of the first dimeric alkaloid intermediate, α-3′,4′-anhydrovinblastine. On the other hand, the oxidation of ajmalicine into serpentine has been attributed to basic peroxidase isoenzymes localized in the vacuole of C. roseus cells. An overview of the work implying class III plant peroxidases in the biosynthesis of terpenoid indole alkaloids in C. roseus is presented here. Abbreviations: CRPRX1 –Catharanthus roseus peroxidase 1; DAB – diaminobenzidine; IEF – isoelectric focusing; UV – ultraviolet.     

Somatic embryogenesis and indole alkaloid production in Catharanthus roseus

ABSTRACT

A new protocol to obtain an embryogenic cell line from cultured seedling explants of Catharanthus roseus is described. In order to assess the relationship between tissue differentiation and secondary metabolite biosynthesis, the biosynthetic capabilities (alkaloid production) of an embryogenic cell line and two non-embryogenic C. roseus strains were comparatively examined. Faster cell growth rate was associated with higher alkaloid production in the embryogenic cell line. The kinetics of ajmalicine and serpentine production by the three cell lines is also reported.     

Implementation of functional genomics for gene discovery in alkaloid producing plants

Two centuries after the discovery of the first alkaloids, many enzymes involved in plant alkaloid biosynthesis have been identified. Nevertheless, the biosynthetic pathways for most of the plant alkaloids still remain incompletely characterised and understanding the regulatory mechanisms controlling the onset and flux of alkaloid biosynthesis is virtually inexistent. This information is however crucial to allow modelling of metabolic networks and predictive metabolic engineering. In the postgenomics era, new functional genomics tools, enabling comprehensive investigations of biological systems, are continuously emerging and are now gradually being implemented in the field of plant secondary metabolism as well. Here we discuss the advances these promising new technologies have already brought and may still bring with regard to the dissection of plant alkaloid biosynthesis. Encouraging results were obtained in alkaloid producing species such as Papaver somniferum, Catharanthus roseus and Nicotiana tabacum. Therefore we anticipate that functional genomics and the knowledge it brings along, will eventually allow a better exploitation of the plant biosynthetic machinery.     

Transient gene expression of recombinant terpenoid indole alkaloid enzymes in<Emphasis Type="Italic">Catharanthus roseus</Emphasis> leaves

We developed a transient expression assay for Madagascar periwinkle (Catharanthus roseus [L.] G. Don.) that is based on vacuum infiltration of intact leaves with recombinantAgrobacterium tumefaciens. This simple and rapid technique was used to overexpresstryptophan decarboxylase (tdc) andstrictosidine synthase (str1) genes, which encode 2 key enzymes of the terpenoid indole alkaloid (TIA) biosynthesis pathway. Immunoblot analysis of crude leaf extracts demonstrated that recombinant TDC and STR1 accumulated to detectable levels when targeted to their native subcellular compartments (i.e., the cytosol and vacuole, respectively) or to the chloroplast. In this article, we discuss possible applications of the transient assay in studies on the overexpression of enzymes of the TIA pathway in intactC. roseus leaves.     

Agrobacterium-mediated transformation of the terpenoid indole alkaloid-producing plant species Tabernaemontana pandacaqui

Plants of the Apocynaceae family produce a wide range of terpenoid indole alkaloids (TIAs) which have important pharmaceutical applications. Studies of the molecular mechanisms controlling TIA biosynthesis may eventually provide possibilities to improve product yield by genetic modification of plants or cell cultures. However, these studies suffer from the lack of transformation/regeneration protocols for Apocynaceae plants. We chose to study the feasibility of Agrobacterium tumefaciens-mediated transformation of Tabernaemontana pandacaqui, because of the availability of an efficient regeneration procedure for this member of the Apocynaceae family. A procedure to produce transgenic T. pandacaqui plants was established, albeit with low efficiency. Transgenic expression was demonstrated of an intron-containing β-glucuronidase reporter gene and of a gene coding for the TIA biosynthetic enzyme strictosidine synthase from Catharanthus roseus, another Apocynaceae species. Received: 16 June 1997 / Revision received: 12 July 1997 / Accepted: 13 July 1997     

Proteome analysis of the medicinal plant <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

A proteomic approach is undertaken aiming at the identification of novel proteins involved in the alkaloid biosynthesis of Catharanthus roseus. The C. roseus cell suspension culture A11 accumulates the terpenoid indole alkaloids strictosidine, ajmalicine and vindolinine. Cells were grown for 21 days, and alkaloid accumulation was monitored during this period. After a rapid increase between day 3 and day 6, the alkaloid content reached a maximum on day 16. Systematic analysis of the proteome was performed by two-dimensional polyacrylamide gel electrophoresis. After day 3, the proteome started to change with an increasing number of protein spots. On day 13, the proteome changed back to roughly the same as at the start of the growth cycle. 88 protein spots were selected for identification by mass spectrometry (MALDI-MS/MS). Of these, 58 were identified, including two isoforms of strictosidine synthase (EC 4.3.3.2), which catalyzes the formation of strictosidine in the alkaloid biosynthesis; tryptophan synthase (EC 4.1.1.28), which is needed for the supply of the alkaloid precursor tryptamine; 12-oxophytodienoate reductase, which is indirectly involved in the alkaloid biosynthesis as it catalyzes the last step in the biosynthesis of the regulator jasmonic acid. Unique sequences were found, which may also relate to unidentified biosynthetic proteins.     

Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus

Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway. Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the pathway. Two such lines accumulated over 200 mg · L⁻¹ of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity, but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and appeared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway will yield positive results only in the presence of a favorable epigenetic environment. Received: 12 June 1997 / Accepted: 24 October 1997     

病毒诱导基因沉默鉴定穿心莲内酯生物合成关键酶ApCPS功能

该研究采用病毒诱导基因沉默技术(VIGS),以生长到第8片真叶期的穿心莲植株为实验材料,沉默参与穿心莲内酯生物合成的ent-柯巴基焦磷酸合酶基因(ApCPS),用半定量和荧光定量PCR检测病毒诱导沉默后ApCPS及其上游基因的表达,用HPLC法检测ApCPS沉默后穿心莲内酯的积累变化,同时检测茉莉酸甲酯(MeJA)处理后ApCPS及上游基因的表达,以全面分析穿心莲内酯代谢以及ApCPS在穿心莲内酯生物合成中的作用机制,验证其在植物体内的功能。结果显示:(1)ApCPS基因被成功沉默,基因表达显著下调,进而引起上游牻牛儿基牻牛儿基焦磷酸合成酶基因(GGPS)的表达下调,而3-羟-3-甲基戊二酰辅酶A还原酶基因(HMGR)和1-脱氧木酮糖-5-磷酸合成酶基因(DXS)的表达未受影响。(2)ApCPS基因沉默15d后穿心莲内酯积累量显著下降,表明ApCPS是穿心莲内酯生物合成关键酶基因,且能够负反馈影响上游基因表达。(3)茉莉酸甲酯(MeJA)显著诱导ApCPS及上游基因HMGR、DXS和GGPS的表达,表明穿心莲内酯生物合成基因受到MeJA的广泛调控。该研究首次使用VIGS证明ApCPS参与到穿心莲内酯生物合成,为利用该技术鉴定穿心莲内酯生物合成途径中其他基因功能奠定了基础。     

Nuclear factors GT-1 and 3AF1 interact with multiple sequences within the promoter of the Tdc gene from Madagascar periwinkle: GT-1 is involved in UV light-induced expression

Plant secondary metabolites of the terpenoid indole alkaloid (TIA) class comprise several compounds with pharmaceutical applications. A key step in the TIA biosynthetic pathway is catalysed by the enzyme tryptophan decarboxylase (TDC), which channels the primary metabolite tryptophan into TIA metabolism. In Catharanthus roseus (Madagascar periwinkle), the Tdc gene is expressed throughout plant development. Moreover, Tdc gene expression is induced by external stress signals, such as fungal elicitor and UV light. In a previous study of Tdc promoter architecture in transgenic tobacco it was shown that the ?538 to ?112 region is a quantitative determinant for the expression level in different plant organs. Within this sequence one particular region (?160 to ?99) was identified as the major contributor to basal expression and another region (?99 to ?37) was shown to be required for induction by fungal elicitor. Here, the in vitro binding of nuclear factors to the ?572 to ?37 region is described. In extracts from tobacco and C. roseus, two binding activities were detected that could be identified as the previously described nuclear factors GT-1 and 3AF1, based on their mobility and binding characteristics. Both factors appeared to interact with multiple regions in the Tdc promoter. Mutagenesis of GT-1 binding sites in the Tdc promoter did not affect the basal or elicitor-induced expression levels. However, induction of the Tdc promoter constructs by UV light was significantly lower, thereby demonstrating a functional role for GT-1 in the induction of Tdc expression by UV light.     

Indole alkaloid accumulation and tryptophan decarboxylase activity in Catharanthus roseus cells cultured in three different media

Catharanthus roseus cells were cultured in three types of media. These media were: a low sucrose subculture medium and two high sucrose media, each of which differed in their mineral and hormonal contents. The kinetics of tryptophan decarboxylase activity and the accumulations of tryptophan, tryptamine, ajmalicine and serpentine were different in each series but no correlation between maximum enzyme activity and alkaloid contents was observed. Ajmalicine and serpentine productions were unaffected by addition of Trp to the media, whereas addition of secologanin enhanced alkaloid production. The results seem to imply that the terpenoid pathway is the limiting factor in alkaloid production in C. roseus cells.