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1.
Virus‐induced gene silencing in Catharanthus roseus by biolistic inoculation of tobacco rattle virus vectors
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E. Marais A. Lanoue S. Besseau N. Papon M. Clastre T. Dugé de Bernonville G. Glévarec L. Atehortùa A. Oudin V. Courdavault 《Plant biology (Stuttgart, Germany)》2015,17(6):1242-1246
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. 相似文献
2.
Grégory Guirimand Vincent Courdavault Arnaud Lanoue Samira Mahroug Anthony Guihur Nathalie Blanc Nathalie Giglioli-Guivarc'h Benoit St-Pierre Vincent Burlat 《BMC plant biology》2010,10(1):182
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. 相似文献3.
Sarma Rajeev Kumar Avanish Rai Dikki Pedenla Bomzan Krishna Kumar Andra Hemmerlin Varun Dwivedi Rucha C. Godbole Vitthal Barvkar Karuna Shanker H. B. Shilpashree Ankita Bhattacharya Attibele Ramamurthy Smitha Namratha Hegde Dinesh A. Nagegowda 《The Plant journal : for cell and molecular biology》2020,103(1):248-265
4.
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. 相似文献
5.
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. 相似文献
6.
Inês Carqueijeiro Thomas Dugé de Bernonville Arnaud Lanoue Thu‐Thuy Dang Christiana N Teijaro Christian Paetz Kevin Billet Angela Mosquera Audrey Oudin Sébastien Besseau Nicolas Papon Gaëlle Glévarec Lucía Atehortùa Marc Clastre Nathalie Giglioli‐Guivarc'h Bernd Schneider Benoit St‐Pierre Rodrigo B. Andrade Sarah E. O'Connor Vincent Courdavault 《The Plant journal : for cell and molecular biology》2018,94(3):469-484
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Morteza Yousefzadi Mozafar Sharifi Mehrdad Behmanesh Elisabeth Moyano Mercedis Bonfill Rosa M. Cusido Javier Palazon 《Engineering in Life Science》2010,10(4):281-292
Many plant‐derived agents are being used to treat cancer, including taxol, vinblastine, vincristine, or camptothecin and podophyllotoxin derivatives, among others. Plant biotechnology can provide a new tool for the production of anticancer agents but in spite of considerable efforts to produce vinblastine and vincristine in cell cultures and knowledge of the biosynthetic pathway of Catharanthus roseus alkaloids, the biotechnological production of taxol has only been achieved at an industrial level by companies such as Phyton Biotech and Cytoclonal Pharmaceutics. Podophyllotoxin was isolated as the active antitumor agent from the roots of Podophyllum species and more recently from the genus Linum and others. Etoposide, teniposide, and etophos are semi‐synthetic derivatives of podophyllotoxin and are used in the treatment of cancer. Biotechnological approaches, including the use of cell cultures, biotransformation, or metabolic engineering techniques to manipulate the biosynthetic pathway, represent an alternative for the production of podophyllotoxin and are discussed in this review. 相似文献
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Pierre Poutrain Gr��gory Guirimand Ga?lle Gl��varec Vincent Courdavault Olivier Pichon 《Journal of Plant Growth Regulation》2011,30(2):235-241
In Catharanthus roseus cells, auxins are known to negatively regulate the biosynthesis of monoterpenoid indole alkaloids (MIA), a class of valuable
secondary metabolites. Despite extensive studies of this regulation, no protein of the auxin signaling pathway has been isolated
to date in this plant. We therefore decided to clone and characterize a C. roseus Aux/IAA protein that belongs to a family of gene expression repressors mediating auxin effects. Using PCR, a cDNA encoding
the first C. roseus Aux/IAA was cloned and named CrIAA1. The deduced amino acid sequence has four highly conserved domains that are typical of the Aux/IAA protein family and has
high homology to the Aux/IAA isoforms of Arabidopsis (>67%). The CrIAA1 gene expression, monitored by real-time PCR, was found to be dramatically induced by auxin treatment in C. roseus cells. Using GFP imagery and a bimolecular fluorescence complementation assay, we found that CrIAA1 can form oligomers in
the nucleus. We also found that CrIAA1 is quickly degraded following auxin treatments, suggesting that auxin regulates CrIAA1
availability via a feedback mechanism. These results should help to elucidate the molecular nature of the processes responsible
for the auxin-mediated regulation of MIA biosynthesis in C. roseus. 相似文献
11.
Danielle Williams Yang Qu Razvan Simionescu Vincenzo De Luca 《The Plant journal : for cell and molecular biology》2019,99(4):626-636
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. 相似文献
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14.
Thomas Dugé de Bernonville Emilien Foureau Claire Parage Arnaud Lanoue Marc Clastre Monica Arias Londono Audrey Oudin Benjamin Houillé Nicolas Papon Sébastien Besseau Ga?lle Glévarec Lucia Atehortùa Nathalie Giglioli-Guivarc’h Benoit St-Pierre Vincenzo De Luca Sarah E. O’Connor Vincent Courdavault 《BMC genomics》2015,16(1)
15.
Grégory Guirimand Vincent Burlat Audrey Oudin Arnaud Lanoue Benoit St-Pierre Vincent Courdavault 《Plant cell reports》2009,28(8):1215-1234
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. 相似文献
16.
Kotaro Yamamoto Dagny Grzech Konstantinos Koudounas Emily Amor Stander Lorenzo Caputi Tetsuro Mimura Vincent Courdavault Sarah E. OConnor 《Plant physiology》2021,187(2):846
Specialized metabolites are chemically complex small molecules with a myriad of biological functions. To investigate plant-specialized metabolite biosynthesis more effectively, we developed an improved method for virus-induced gene silencing (VIGS). We designed a plasmid that incorporates fragments of both the target gene and knockdown marker gene (phytoene desaturase, PDS), which identifies tissues that have been successfully silenced in planta. To demonstrate the utility of this method, we used the terpenoid indole alkaloid (TIA) pathway in Madagascar periwinkle (Catharanthus roseus) as a model system. Catharanthus roseus is a medicinal plant well known for producing many bioactive compounds, such as vinblastine and vincristine. Our VIGS method enabled the discovery of a previously unknown biosynthetic enzyme, serpentine synthase (SS). This enzyme is a cytochrome P450 (CYP) that produces the β-carboline alkaloids serpentine and alstonine, compounds with strong blue autofluorescence and potential pharmacological activity. The discovery of this enzyme highlights the complexity of TIA biosynthesis and demonstrates the utility of this improved VIGS method for discovering unidentified metabolic enzymes in plants.An improved virus-induced gene silencing approach led to the discovery of the alkaloid biosynthetic enzyme serpentine synthase. 相似文献
17.
Qifang Pan Yu Chen Quan Wang Fang Yuan Shihai Xing Yuesheng Tian Jingya Zhao Xiaofen Sun Kexuan Tang 《Plant Growth Regulation》2010,60(2):133-141
Catharanthuse roseus is a well-known medicinal plant for its two valuable anticancer compounds: vinblastine and vincristine, which belongs to
terpenoid indole alkaloids. Great efforts have been made to study the principles of its secondary metabolic pathways to regulate
the alkaloids biosynthesis. In this article, different plant growth regulators were shortly applied to Catharanthus roseus plants during the blooming period to study their effects on the biosynthesis of vinblastine, vindoline and catharanthine.
Salicylic acid and ethylene (ethephon) treatments resulted in a significant increase of vinblastine, vindoline and catharanthine
while abscisic acid and gibberellic acid had a strongly negative influence on the accumulation of the three important alkaloids.
Methyl jasmonate showed no great effect on the production of these valuable alkaloids. Chlormequat chloride highly enhanced
the accumulation of vinblastine but greatly decreased the contents of vindoline and catharanthine. 相似文献
18.
Guo X Geng P Bai F Bai G Sun T Li X Shi L Zhong Q 《Letters in applied microbiology》2012,55(2):162-169
Aims: The aims of this study are to obtain the draft genome sequence of Streptomyces coelicoflavus ZG0656, which produces novel acarviostatin family α‐amylase inhibitors, and then to reveal the putative acarviostatin‐related gene cluster and the biosynthetic pathway. Methods and Results: The draft genome sequence of S. coelicoflavus ZG0656 was generated using a shotgun approach employing a combination of 454 and Solexa sequencing technologies. Genome analysis revealed a putative gene cluster for acarviostatin biosynthesis, termed sct‐cluster. The cluster contains 13 acarviostatin synthetic genes, six transporter genes, four starch degrading or transglycosylation enzyme genes and two regulator genes. On the basis of bioinformatic analysis, we proposed a putative biosynthetic pathway of acarviostatins. The intracellular steps produce a structural core, acarviostatin I00‐7‐P, and the extracellular assemblies lead to diverse acarviostatin end products. Conclusions: The draft genome sequence of S. coelicoflavus ZG0656 revealed the putative biosynthetic gene cluster of acarviostatins and a putative pathway of acarviostatin production. Significance and Impact of the Study: To our knowledge, S. coelicoflavus ZG0656 is the first strain in this species for which a genome sequence has been reported. The analysis of sct‐cluster provided important insights into the biosynthesis of acarviostatins. This work will be a platform for producing novel variants and yield improvement. 相似文献
19.
Eucalyptus hairy roots,a fast,efficient and versatile tool to explore function and expression of genes involved in wood formation
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Anna Plasencia Marçal Soler Annabelle Dupas Nathalie Ladouce Guilherme Silva‐Martins Yves Martinez Catherine Lapierre Claudine Franche Isabelle Truchet Jacqueline Grima‐Pettenati 《Plant biotechnology journal》2016,14(6):1381-1393
20.
Qundan Lv Jie Qiu Jie Liu Zheng Li Wenting Zhang Qin Wang Jie Fang Junjie Pan Zhengdao Chen Wenliang Cheng Michael S. Barker Xuehui Huang Xin Wei Kejun Cheng 《The Plant journal : for cell and molecular biology》2020,103(5):1910-1923
Chimonanthus salicifolius, a member of the Calycanthaceae of magnoliids, is one of the most famous medicinal plants in Eastern China. Here, we report a chromosome‐level genome assembly of C. salicifolius, comprising 820.1 Mb of genomic sequence with a contig N50 of 2.3 Mb and containing 36 651 annotated protein‐coding genes. Phylogenetic analyses revealed that magnoliids were sister to the eudicots. Two rounds of ancient whole‐genome duplication were inferred in the C. salicifolious genome. One is shared by Calycanthaceae after its divergence with Lauraceae, and the other is in the ancestry of Magnoliales and Laurales. Notably, long genes with > 20 kb in length were much more prevalent in the magnoliid genomes compared with other angiosperms, which could be caused by the length expansion of introns inserted by transposon elements. Homologous genes within the flavonoid pathway for C. salicifolius were identified, and correlation of the gene expression and the contents of flavonoid metabolites revealed potential critical genes involved in flavonoids biosynthesis. This study not only provides an additional whole‐genome sequence from the magnoliids, but also opens the door to functional genomic research and molecular breeding of C. salicifolius. 相似文献