Vismione H (VH) is a fluorescent prenylated anthranoid produced by plants from the Hypericaceae family, with antiprotozoal activities against malaria and leishmaniosis. Little is known about its biosynthesis and metabolism in plants or its mode of action against parasites. When VH is isolated from Psorospermum glaberrimum, it is rapidly converted into madagascine anthrone and anthraquinone, which are characterized by markedly different fluorescent properties. To locate the fluorescence of VH in living plant cells and discriminate it from that of the other metabolites, an original strategy combining spectral imaging (SImaging), confocal microscopy, and non-targeted metabolomics using mass spectrometry, was developed. Besides VH, structurally related molecules including madagascine (Mad), emodin (Emo), quinizarin (Qui), as well as lapachol (Lap) and fraxetin (Fra) were analyzed. This strategy readily allowed a spatiotemporal characterization and discrimination of spectral fingerprints from anthranoid-derived metabolites and related complexes with cations and proteins. In addition, our study validates the ability of plant cells to metabolize VH into madagascine anthrone, anthraquinones and unexpected metabolites. These results pave the way for new hypotheses on anthranoid metabolism in plants.

Type-I protein prenylation, the post-translational modification of CaaX motif-containing proteins, relies on two substrates: the target protein and a mevalonate-derived prenyl diphosphate co-substrate, either farnesyl diphosphate (FPP) or geranylgeranyl diphosphate (GGPP). Two enzymes, protein farnesyltransferase and type-I geranylgeranyltransferase, recognize and bind both co-substrates. Modifying potentially hundreds of distinct protein targets within a constrained timeframe poses a major regulatory challenge for the cell. However, the mechanisms controlling prenyltransferase activity, including substrate availability, enzyme specificity, and catalytic efficiency, remain poorly understood, particularly in plants. Plant prenylation systems exhibit distinctive features. The diversity of prenyl diphosphate donors is expanded by the plastidial methylerythritol phosphate pathway, which supplements the mevalonate pathway and may provide alternative prenyl groups beyond the canonical FPP and GGPP. Additionally, many CaaX-containing proteins are plant-specific, and post-transcriptional modifications generate multiple prenylatable isoforms, increasing substrate complexity. In this review, we examine the diversification of both prenyl diphosphate donors and protein substrates in plants, hypothesizing that such diversification may illuminate key mechanisms underlying the cellular regulation of protein prenylation.

In plants, the plastidial mevalonate (MVA)-independent pathway is required for the modification with geranylgeranyl groups of CaaL-motif proteins, which are substrates of protein geranylgeranyltransferase type-I (PGGT-I). As a consequence, fosmidomycin, a specific inhibitor of 1-deoxy-d-xylulose (DX)-5 phosphate reductoisomerase/DXR, the second enzyme in this so-called methylerythritol phosphate (MEP) pathway, also acts as an effective inhibitor of protein prenylation. This can be visualized in plant cells by confocal microscopy by expressing GFP-CaM-CVIL, a prenylation sensor protein. After treatment with fosmidomycin, the plasma membrane localization of this GFP-based sensor is altered, and a nuclear distribution of fluorescence is observed instead. In tobacco cells, a visual screen of conditions allowing membrane localization in the presence of fosmidomycin identified jasmonic acid methyl esther (MeJA) as a chemical capable of gradually overcoming inhibition. Using Arabidopsis protein prenyltransferase loss-of-function mutant lines expressing GFP-CaM-CVIL proteins, we demonstrated that in the presence of MeJA, protein farnesyltransferase (PFT) can modify the GFP-CaM-CVIL sensor, a substrate the enzyme does not recognize under standard conditions. Similar to MeJA, farnesol and MVA also alter the protein substrate specificity of PFT, whereas DX and geranylgeraniol have limited or no effect. Our data suggest that MeJA adjusts the protein substrate specificity of PFT by promoting a metabolic cross-talk directing the origin of the prenyl group used to modify the protein. MVA, or an MVA-derived metabolite, appears to be a key metabolic intermediate for this change in substrate specificity.

Vismione H (VH) is a fluorescent prenylated anthranoid produced by plants from the Hypericaceae family, with antiprotozoal activities against malaria and leishmaniosis. Little is known about its biosynthesis and metabolism in plants or its mode of action against parasites. When VH is isolated from Psorospermum glaberrimum , it is rapidly converted into madagascine anthrone and anthraquinone, which are characterized by markedly different fluorescent properties. To locate the fluorescence of VH in living plant cells and discriminate it from that of the other metabolites, an original strategy combining spectral imaging (SImaging), confocal microscopy and non-targeted metabolomics using mass spectrometry, was developed. Besides VH, structurally related molecules including madagascine, emodin, quinizarin as well as lapachol and fraxetin were analyzed. This strategy readily allowed a spatiotemporal characterization and discrimination of spectral fingerprints from anthranoids-derived metabolites and related complexes with cations and proteins. In addition, our study validates the capability of plant cells to metabolize VH into madagascine anthrone, anthraquinones and unexpected metabolites, leading to new hypotheses on the metabolism of anthranoids in plants.

The modification of GFP-CaaL sensor proteins by geranylgeranyl groups in plants requires an active plastidial mevalonate (MVA)-independent pathway. As a consequence, fosmidomycin, a specific inhibitor of 1-deoxy-D-xylulose (DX)-5 phosphate reductoisomerase/DXR, the second enzyme in this so-called methylerythritol phosphate (MEP) pathway, impedes protein prenylation, which results in GFP-labeling of nuclei instead of the plasma membrane. In tobacco cells, a visual screen of conditions that allowed membrane localization of GFP-CaaL proteins in the presence of fosmidomycin, pinpointed jasmonic acid methyl esther (Me-JA) as a chemical able to overcome the inhibition in a gradual manner. Using Arabidopsis protein prenyltransferase loss-of-function mutant lines expressing GFP-CaaL proteins, we demonstrated that Me-JA induces a modification in protein substrate recognition by protein farnesyltransferase (PFT). Similar to Me-JA, farnesol and MVA also alter the protein substrate specificity of PFT, whereas DX and geranylgeraniol have limited or no effect. Our data indicate that stimulation of the MVA pathway adjusts the protein substrate specificity of PFT, which is corroborated with metabolic cross-talk of the origin of the prenyl group used to modify the protein. To illustrate how activation of the MVA pathway impacting PFT substrate specificity benefits a plant under stress conditions, a model is proposed.

Dynamique de la flexibilité des activités des protéines prényltransférases: La prénylation des protéines est une modification post-traductionnelle essentielle pour le développement, la signalisation et l’adaptation à l’environnement des cellules eucaryotes. Bien que de nombreux inhibiteurs actifs et spécifiques des protéine-prenyltransférases aient été isolés à ce jour, la méconnaissance des bases biologiques contrôlant les modifications des protéines par des groupements isopréniques rend leur utilisation inefficace dans le traitement de cancer ou du paludisme. Pour caractériser la modification des protéines prénylées, nous avons utilisé un modèle cellulaire unique permettant de visualiser ces changements in vivo. De plus, des techniques de capture des protéines modifiées ont été développées afin d’analyser les mécanismes au niveau moléculaire par des approches protéomiques et métabolomiques. Dans un criblage d’inhibiteurs potentiels, une molécule efficace in vivo a été identifiée. Par ailleurs, le criblage a permis d’observer in vivo la fluorescence de vismiones (composés anthracéniques prénylé) extraites de plantes et décrites pour leurs propriétés antipaludiques. Une méthode ingénieuse associant l’imagerie spectrale et l’analyse métabolomique a été développée pour tracer et caractériser le métabolisme associé aux vismiones dans des cellules de plantes.