Silencing amorpha-4,11-diene synthase genes in Artemisia annua leads to FPP accumulation.

Artemisia annua is established as an efficient crop for the production of the anti-malarial compound artemisinin, a sesquiterpene lactone synthesized and stored in Glandular Secretory Trichomes (GSTs) located on the leaves and inflorescences. Amorpha-4,11-diene synthase (AMS) catalyzes the conversion of farnesyl pyrophosphate (FPP) to amorpha-4,11-diene and diphosphate, which is the first committed step in the synthesis of artemisinin. FPP is the precursor for sesquiterpene and sterol biosynthesis in the plant. This work aimed to investigate the effect of blocking the synthesis of artemisinin in the GSTs of a high artemisinin yielding line, Artemis, by down regulating AMS. We determined that there are up to 12 AMS gene copies in Artemis, all expressed in GSTs. We used sequence homology to design an RNAi construct under the control of a GST specific promoter that was predicted to be effective against all 12 of these genes. Stable transformation of Artemis with this construct resulted in over 95% reduction in the content of artemisinin and related products, and a significant increase in the FPP pool. The Artemis AMS silenced lines showed no morphological alterations, and metabolomic and gene expression analysis did not detect any changes in the levels of other major sesquiterpene compounds or sesquiterpene synthase genes in leaf material. FPP also acts as a precursor for squalene and sterol biosynthesis but levels of these compounds were also not altered in the AMS silenced lines. Four unknown oxygenated sesquiterpenes were produced in these lines, but at extremely low levels compared to Artemis non-transformed controls (NTC). This study finds that engineering A. annua GSTs in an Artemis background results in endogenous terpenes related to artemisinin being depleted with the precursor FPP actually accumulating rather than being utilized by other endogenous enzymes. The challenge now is to establish if this precursor pool can act as substrate for production of alternative sesquiterpenes in A. annua

Implication of two isoprenoid biosynthesis pathways in the specificity and regulation of protein prenylation in plants

La prénylation de type I des protéines correspond à une modification post-traductionnelle faisant intervenir une liaison thioéther entre une cystéine localisée dans un motif CaaX en position C terminale et un groupement prényle en C 15 (farnésyle) ou C 20 (géranylgéranyle). Ces réactions sont catalysées par des protéine prényltransférases (PPTs) appartenant à la même famille fonctionnelle et comprenant la protéine farnésyltransférase (PFT) et géranylgéranyltransférase de type I (PGGT-I). Les plantes se distinguent par une double origine des substrats prényle (farnésyle diphosphate et géranylgéranyle diphosphate) utilisés comme précurseurs pour la biosynthèse des isoprénoïdes. Ces derniers sont biosynthétisés par l'intermédiaire de deux voies métaboliques distinctes, la voie cytosolique du mévalonate (MVA) et la voie plastidiale du méthylérythritol phosphate (MEP). Il est maintenant clair que la géranylgéranylation des protéines végétales dépend de la voie du MEP. Durant ce travail de thèse doctorale une étude comparative des spécificités de substrat a été réalisée. Elle a permis de montrer que la PFT est spécifique de son substrat protéique alors que la PGGT-I est spécifique de son substrat prényle. Ces spécificités peuvent néanmoins être modifiées in vivo, par exemple lors d’une augmentation de la concentration en MVA, suggérant que cette flexibilité des propriétés enzymatiques a un rôle régulateur dans certaines conditions physiologiques. Pour cette raison, nous avons entrepris une caractérisation de la prénylation des protéines dans des plantes de tabac élicitées, qui induisent la synthèse de MVA pour produire le capsidiol, une phytoalexine sesquiterpénique. La biosynthèse de ce métabolite secondaire capsidiol dérivant de la voie du MVA, est dépendante de la prénylation des protéines, notamment de protéines géranylgéranylées d’origine plastidiale. Le monoterpène S-carvone a été identifié comme un inhibiteur de la biosynthèse de capsidiol en interférant avec l’activité des PPTs in vivo. Les travaux ont également permis d’envisager l’existence d’un nouveau mode de prénylation des protéines spécifique aux feuilles.Type-I protein prenylation is a post-translational modification of a protein bearing a CaaX motif with a prenyl moiety, this by a thioether linkage. The enzymes catalyzing those reactions are called protein prenyltransferase (PPTs). Two enzymes are involved, the protein farnesyltransferase (PFT) and the protein geranylgeranyltransferase type I (PGGT-I). They respectively use farnesyl diphosphate and geranylgeranyl diphosphate as substrate. Those precursors are synthetized in plants by two differentbiosynthetic pathways: the cytosolic mevalonate (MVA) and the plastidial methylerythritol phosphate (MEP) pathways. Protein geranylgeranylation is dependent of the MEP pathway. Those specificities can be modified A comparative analysis of PPTs specificity was done during this PhD thesis, revealing that PFT is specific for its protein substrate, while PGGT-I is specific for its prenyl substrate. But those specificities can be modulated in vivo, for instance by increasing the concentration of MVA. This suggests that the regulation of protein prenylation specificities can become functionally important during physiological processes. For that reason we characterized protein prenylation in elicited tobacco plants, which produce the sesquiterpene phytoalexin capsidiol. This metabolite is synthesized via the MVA pathway, and this process depends of protein prenylation, in particular geranylgeranylation, with the substrate coming from plastids. S-Carvone, a monoterpene, was identified as an inhibitor of PPTS, resulting in a lack of capsidiol production. This work also suggests that a new mechanism of prenylation might exist, specifically in leaves

Implication of two isoprenoid biosynthesis pathways in the specificity and regulation of protein prenylation in plants

La prénylation de type I des protéines correspond à une modification post-traductionnelle faisant intervenir une liaison thioéther entre une cystéine localisée dans un motif CaaX en position C terminale et un groupement prényle en C 15 (farnésyle) ou C 20 (géranylgéranyle). Ces réactions sont catalysées par des protéine prényltransférases (PPTs) appartenant à la même famille fonctionnelle et comprenant la protéine farnésyltransférase (PFT) et géranylgéranyltransférase de type I (PGGT-I). Les plantes se distinguent par une double origine des substrats prényle (farnésyle diphosphate et géranylgéranyle diphosphate) utilisés comme précurseurs pour la biosynthèse des isoprénoïdes. Ces derniers sont biosynthétisés par l'intermédiaire de deux voies métaboliques distinctes, la voie cytosolique du mévalonate (MVA) et la voie plastidiale du méthylérythritol phosphate (MEP). Il est maintenant clair que la géranylgéranylation des protéines végétales dépend de la voie du MEP. Durant ce travail de thèse doctorale une étude comparative des spécificités de substrat a été réalisée. Elle a permis de montrer que la PFT est spécifique de son substrat protéique alors que la PGGT-I est spécifique de son substrat prényle. Ces spécificités peuvent néanmoins être modifiées in vivo, par exemple lors d’une augmentation de la concentration en MVA, suggérant que cette flexibilité des propriétés enzymatiques a un rôle régulateur dans certaines conditions physiologiques. Pour cette raison, nous avons entrepris une caractérisation de la prénylation des protéines dans des plantes de tabac élicitées, qui induisent la synthèse de MVA pour produire le capsidiol, une phytoalexine sesquiterpénique. La biosynthèse de ce métabolite secondaire capsidiol dérivant de la voie du MVA, est dépendante de la prénylation des protéines, notamment de protéines géranylgéranylées d’origine plastidiale. Le monoterpène S-carvone a été identifié comme un inhibiteur de la biosynthèse de capsidiol en interférant avec l’activité des PPTs in vivo. Les travaux ont également permis d’envisager l’existence d’un nouveau mode de prénylation des protéines spécifique aux feuilles.Type-I protein prenylation is a post-translational modification of a protein bearing a CaaX motif with a prenyl moiety, this by a thioether linkage. The enzymes catalyzing those reactions are called protein prenyltransferase (PPTs). Two enzymes are involved, the protein farnesyltransferase (PFT) and the protein geranylgeranyltransferase type I (PGGT-I). They respectively use farnesyl diphosphate and geranylgeranyl diphosphate as substrate. Those precursors are synthetized in plants by two differentbiosynthetic pathways: the cytosolic mevalonate (MVA) and the plastidial methylerythritol phosphate (MEP) pathways. Protein geranylgeranylation is dependent of the MEP pathway. Those specificities can be modified A comparative analysis of PPTs specificity was done during this PhD thesis, revealing that PFT is specific for its protein substrate, while PGGT-I is specific for its prenyl substrate. But those specificities can be modulated in vivo, for instance by increasing the concentration of MVA. This suggests that the regulation of protein prenylation specificities can become functionally important during physiological processes. For that reason we characterized protein prenylation in elicited tobacco plants, which produce the sesquiterpene phytoalexin capsidiol. This metabolite is synthesized via the MVA pathway, and this process depends of protein prenylation, in particular geranylgeranylation, with the substrate coming from plastids. S-Carvone, a monoterpene, was identified as an inhibitor of PPTS, resulting in a lack of capsidiol production. This work also suggests that a new mechanism of prenylation might exist, specifically in leaves

Plant Glandular Trichomes: Natural Cell Factories of High Biotechnological Interest.

Multicellular glandular trichomes are epidermal outgrowths characterized by the presence of a head made of cells that have the ability to secrete or store large quantities of specialized metabolites. Our understanding of the transcriptional control of glandular trichome initiation and development is still in its infancy. This review points to some central questions that need to be addressed to better understand how such specialized cell structures arise from the plant protodermis. A key and unique feature of glandular trichomes is their ability to synthesize and secrete large amounts, relative to their size, of a limited number of metabolites. As such, they qualify as true cell factories, making them interesting targets for metabolic engineering. In this review, recent advances regarding terpene metabolic engineering are highlighted, with a special focus on tobacco (Nicotiana tabacum). In particular, the choice of transcriptional promoters to drive transgene expression and the best ways to sink existing pools of terpene precursors are discussed. The bioavailability of existing pools of natural precursor molecules is a key parameter and is controlled by so-called cross talk between different biosynthetic pathways. As highlighted in this review, the exact nature and extent of such cross talk are only partially understood at present. In the future, awareness of, and detailed knowledge on, the biology of plant glandular trichome development and metabolism will generate new leads to tap the largely unexploited potential of glandular trichomes in plant resistance to pests and lead to the improved production of specialized metabolites with high industrial or pharmacological value

S-Carvone suppresses cellulase-induced capsidiol production in Nicotiana tabacum by interfering with protein isoprenylation

International audienceS-Carvone has been described as a negative regulator of mevalonic acid (MVA) production by interfering with 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) activity, a key player in isoprenoid biosynthesis. The impact of this monoterpene on the production of capsidiol in Nicotiana tabacum, an assumed MVA-derived sesquiterpenoid phytoalexin produced in response to elicitation by cellulase, was investigated. As expected, capsidiol production, as well as early stages of elicitation such as hydrogen peroxide production or stimulation of 5-epi-aristolochene synthase activity, were repressed. Despite the lack of capsidiol synthesis, apparentHMGRactivity was boosted. Feeding experiments using (1-C-13) Glc followed by analysis of labeling patterns by C-13-NMR, confirmed an MVA-dependent biosynthesis; however, treatments with fosmidomycin, an inhibitor of the MVA-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) isoprenoid pathway, unexpectedly down-regulated the biosynthesis of this sesquiterpene as well. We postulated that S-carvone does not directly inhibit the production of MVA by inactivating HMGR, but possibly targets an MEP-derived isoprenoid involved in the early steps of the elicitation process. A new model is proposed in which the monoterpene blocks an MEP pathway-dependent protein geranylgeranylation necessary for the signaling cascade. The production of capsidiol was inhibited when plants were treated with some inhibitors of protein prenylation or by further monoterpenes. Moreover, S-carvone hindered isoprenylation of a prenylable GFP indicator protein expressed in N. tabacum cell lines, which can be chemically complemented with geranylgeraniol. The model was further validated using N. tabacum cell extracts or recombinant N. tabacum protein prenyltransferases expressed in Escherichia coli. Our study endorsed a reevaluation of the effect of S-carvone on plant isoprenoid metabolism