Thèse Biosynthèse des Strigolactones chez la Mousse Physcomitrium Patens. H/F - Doctorat.Gouv.Fr

Établissement : Université Paris-Saclay GS Biosphera - Biologie, Société, Ecologie & Environnement, Ressources, Agriculture & Alimentation
École doctorale : Sciences du Végétal : du gène à l'écosystème
Laboratoire de recherche : IJPB - Institut Jean-Pierre Bourgin-Sciences du Végétal
Direction de la thèse : Sandrine BONHOMME ORCID 0000000249002221
Début de la thèse : 2026-10-01
Date limite de candidature : 2026-04-30T23:59:59Les strigolactones (SL), buténolides dérivés des caroténoïdes, sont synthétisées par toutes les plantes terrestres, vasculaires comme non vasculaires (bryophytes). Les SL sont à la fois des signaux allélochimiques exsudés dans le sol, et des phytohormones qui régulent le développement des plantes et leur réponse à différents stress. Alors que les multiples rôles et les structures chimiques de ces composés sont de mieux en mieux décrits, les étapes finales de leur biosynthèse par les plantes restent à découvrir pour un grand nombre de SL. Chez la mousse Physcomitrium patens, nous avons montré que les SL agissent en tant qu'hormones et en tant que signaux dans les interactions plante-plante, mais la voie de biosynthèse de ces molécules en aval du précurseur commun à toutes les SL, la carlactone (CL), demeure inconnue. De manière frappante, l'homologue du cytochrome P450 (CYP) CYP711A qui catalyse la transformation de la CL chez la plupart des plantes terrestres n'est pas retrouvé chez P. patens. Le projet de thèse vise à identifier les enzymes de la voie de biosynthèse des SL en aval de la CL chez P. patens, à partir d'une liste de gènes CYP candidats établie sur la base de nos résultats antérieurs. Le.la doctorant.e sélectionnera les meilleurs gènes candidats en fonction du phénotype de mutants simples pour ces gènes, obtenus par CRISPR-Cas9. Il caractérisera l'activité catalytique des protéines codées par ces CYP. Le rôle dans la voie de biosynthèse des SL des gènes CYP retenus sera confirmé par la caractérisation de mutants multiples. Enfin, les exsudats des mutants obtenus au cours de la thèse seront analysés, afin de préciser le rôle hormonal et/ou allélopathique des produits (SL) des enzymes CYP caractérisées.

Strigolactones (SLs) are specialized metabolites, derived from carotenoids, which are produced by all land plants. Exuded in the soil, SLs act as allelochemical signals, by promoting the symbiosis with arbuscular mycorrhizal fungi (AMF)[1], but also by inducing the germination of root parasitic plant seeds [2]. In addition, endogenous SLs act as phytohormones, regulating plant architecture and tolerance to abiotic stress [3,4,5]. More than 40 natural SLs are known to date [6], but their precise role, as allelochemical or as hormone, is often still elusive. Each plant synthesizes a unique cocktail of SLs, derived from a common precursor called carlactone (CL), which is produced through the action of Carotenoid Cleavage Deoxygenase (CCD) enzymes [7]. CL is modified into carlactonoic acid (CLA) by cytochrome P450 (CYP) enzymes [8] of the CYP711A family, among which the MORE AXILLARY GROWTH1 (MAX1) from Arabidopsis . MAX1 homologs show functional diversity and lead to various SLs, in a species-specific manner [9,10]. The model P. patens can synthesize CL, but lacks a true homologue of CYP711A, and the SL biosynthetic pathway downstream of CL remains unresolved to date in this moss [11]. SL biosynthesis genes have been shown to be regulated by SL levels in many species [9,13]. Our transcriptomic data following SL application, and data from P. patens transcriptomic atlas [12], led us to identify a list of 11 CYP candidate genes (see also below), which role in SL biosynthesis will be tested.

The objective of the thesis is to elucidate the strigolactone biosynthesis pathway in P. patens. In particular, the steps downstream the production of carlactone by the PpCCD8 enzyme will be investigated, focusing on the possible role of Cytochrome P450 enzymes in this biosynthesis. Eventually, the project will lead the PhD student to explore the diversity of SL compounds synthesized by P. patens, and consider their role in moss development and/or plant-plant interactions.

Among the 70 predicted CYP genes in P. patens, we have selected those possibly involved in SL synthesis based on the following criteria:
(1)The transcriptional regulation by SLs: Evidence for a negative feedback regulation of SL synthesis on both CCD7 and CCD8 genes has been reported in many species, including P. patens [13]. However, this negative feedback does not systematically affect CYP genes, which happen to be either down or up-regulated by SL [15]. We previously obtained transcriptomic data, following application of a synthetic SL [(±)-GR24] or exudates from WT moss, versus application of mock or exudates from the Ppccd8 mutant. These data led us to identify 25 CYP genes down-regulated or up-regulated by SLs and/or by moss WT exudates.
(2)The tissue-specific expression profile of P. patens CYP genes: our rationale is that genes involved in SL synthesis downstream of PpCCD7 and PpCCD8 should have similar expression patterns, with notably higher expression at the base of gametophores and in protonema filaments [13]. Through in silico analysis of the expression of these genes [12], 11 out of the 25 above-mentioned CYP genes show such an expression pattern.
The expression of all 11 CYP genes is now being checked by RT-qPCR, following application of (+)-GR24, a better mimic of natural SLs than the aforementioned racemic (±)-GR24. Thus, the PhD student will start the project with a short list of maximum 11 (probably less) CYP genes.

I Selecting a set of candidate genes potentially involved in CL modification in P. patens

The objective of this first part is to reduce the number of CYP enzymes potentially involved in SL biosynthesis.
I-2- Homology modelling to predict 3D structures of the 11 CYPs using prediction softwares (e. g. Alphafold) will be done early in the project. Structures will be compared to those of CYP proteins known to be involved in SL biosynthesis, in other plant species.
I-3- Single Ppcyp KO mutants will be obtained, through CRISPR-Cas9 [16], for all CYP genes showing a confirmed expression profile (SL regulated). In P. patens, obtaining these mutants is rapid. Their phenotypic analysis will include plant moss size comparison to that of Ppccd8, and complementation assays by SL mimic ((+)-GR24) application. Mutants affected in SL biosynthesis are expected to show an extended size, that can be reverted by adding (+)-GR24 to the medium.

II Functional analysis of selected CYP proteins

II-1-Recombinant CYP proteins (selected from I) will be produced and enriched in yeast microsomal fractions. These preparations will be used in in vitro enzyme assays, with a range of substrates, including CL, 19-hydroxy-CL, and other more stable CL derivatives. Substrate metabolization will be monitored by UHPLC-MS, either through the appearance of a product compound or consumption of the substrate. This part will be done in collaboration with Hugues Renault (CNRS, University of Strasbourg), which group is expert in CYP functional analysis. The PhD student will learn these assays in Strasbourg. François-Didier Boyer (ICSN Gif sur Yvette), with whom our team collaborates for many years, will furnish the substrates.
II-2-For the CYP candidate(s) with a validated substrate, docking experiments will be performed, to further explore the catalytic determinants at atomic resolution, as described recently by the group of H. Renault [17].

III Characterization of SLs produced by Ppcyp mutants and determination of their role as hormones or allelochemicals

III-1-Based on the results from I and II, and to confirm the role of CYP proteins, more Ppcyp mutants will be obtained, in various backgrounds: Ppccd8 (= CL deficient) to address the possibility of multiple pathways, and OE-PpCCD8, to test whether CL is accumulated. For the case genetic redundancy would be suspected, multiple mutants affecting 2 or more CYP genes will be obtained. Similar phenotypic characterization as in I-3 will be done.
III-2- The exudates from selected mutants (combining results obtained in I, II and III-1) will be analyzed, to confirm the activity of selected CYP proteins in vivo. The production of SLs will be evaluated by assays on parasitic seeds (Collaboration US2B Nantes) and analysis in LC-MS/MS (Collaboration IJPB OV-Chemistry). In addition, the transcript levels of routinely used SL-response marker genes [18] will be monitored, following application of exudates from WT versus Ppccd8 and Ppcyp mutant.
III-3-Comparing the phenotypes of WT, Ppccd8 and selected Ppcyp mutants will enable to conclude whether the selected CYP genes catalyze the biosynthesis of a SL hormone. In parallel, the use of WT and the various mutant lines in intraspecific allelopathic assays (plant-plant interaction) will assess a role as allelochemicals.

-Formation en biologie moléculaire et cellulaire et en biochimie
-Intérêt pour les modèles de plantes non vasculaires