Thèse Régulation du Complexe Mbw par le Stress Haute Température au Cours du Développement des Graines d'Arabidopsis et son Impact sur le Métabolisme des Flavonoïdes H/F - 75

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 : Isabelle DEBEAUJON ORCID 0000000283935340
Début de la thèse : 2026-10-01
Date limite de candidature : 2026-05-06T23:59:59

Il est établi que les gènes codant pour les flavonoïdes s'expriment lors du développement de tissus spécifiques ou sont induits en réponse à divers stress. Certains régulateurs transcriptionnels MYB et bHLH orchestrent ces régulations. Nous avons identifié ces régulateurs dans les graines d'Arabidopsis comme étant les protéines TT2 (MYB) et TT8 (bHLH), qui interagissent avec une troisième protéine, TTG1 (une protéine WDR), et parfois également TTG2 (une protéine WRKY), pour former les complexes régulateurs « MBW ». Ces complexes agissent lors du développement des téguments de la graine d'Arabidopsis afin de déclencher l'accumulation de proanthocyanidines (tanins condensés). Cependant, les connaissances sur les mécanismes qui régissent la régulation de ce complexe en réponse aux stress environnementaux restent très limitées. L'objectif de cette thèse de doctorat est d'approfondir la compréhension des mécanismes impliqués dans le contrôle environnemental des gènes MBW en étudiant l'impact des stress thermiques (stress thermique modéré et constant et choc thermique) au cours du développement de la graine. Des plantes transgéniques porteuses des gènes rapporteurs TT2, TT8, TTG1 et TTG2 seront utilisées pour caractériser l'expression de ces gènes MBW et de leurs cibles par des approches génétiques et moléculaires. De plus, une attention particulière sera portée à l'impact de l'épigénétique et de l'état redox modulé par le stress thermique sur la fonction des complex MBW et la biosynthèse des tanins.

Flavonoids are one of the largest groups of plant specialized metabolites, well known for the colours they can confer to plant vegetative or reproductive tissues. They play important roles throughout the plant life cycle and for plant fitness, contributing to plant responses to a wide range of abiotic and biotic stresses (Corso et al., 2020). Consistent with these functions the biosynthetic genes involved are tightly controlled at the transcriptional level by developmental, physiological and environmental signals, mainly through MYB and bHLH transcriptional regulators and often by specific MBW (MYB-bHLH-WDR) complexes (Xu et al., 2015 ; Bulgakov, 2024). For instance, in Arabidopsis, the developmental regulation of tannin metabolism exclusively in the seed testa is known to be orchestrated by a complex of three master regulatory factors, namely TRANSPARENT TESTA 2 (TT2)/AtMYB123, TT8/bHLH042 and TT GLABRA 1 (TTG1)/WD40 that cooperatively activate BANYULS (BAN) encoding an anthocyanidin reductase (ANR) (Lepiniec et al., 2006). TTG1 interacts with the TTG2/WRKY44 protein to perform this function (Gonzalez et al., 2016).
Albeit condensed tannins (proanthocyanidins) are powerful antioxidants with protective functions towards multiple stresses, the regulation of their biosynthetic pathway under environmental stresses are still unclear. Our team has recently characterized a massive induction of the specialized metabolome including flavonoids in dry seeds of A. thaliana Col-0 plants subjected to a mild and constant heat stress (HS) of 27°C versus 21°C from flowering onwards (Barreda et al., 2025). In this study, HS caused a decrease in flavan-3-ol (tannin monomer) accumulation in developing seeds.
Reactive oxygen species (ROS) are generated in plants submitted to various stresses including HS and are hypothesized to be involved in stress signaling notably through post-translational modification of transcription factors (Foyer and Noctor, 2016 ; Hendrix et al., 2023). The R2R3 MYB transcription factor Pericarp Color1 (P1) from maize regulates the biosynthesis of phlobaphene, a flavonoid end-product similar to tannins. P1 requires reducing conditions for DNA binding : under non-reducing conditions, conserved cysteines Cys-49 and Cys-53 located in the MYB DNA binding domain form a disulfide bond that prevents P1 from binding DNA (Heine et al., 2004). Whether this regulatory mechanisms is also at play for AtTT2 MYB requires further investigations.

Recently, Jacob et al. (2021) have observed that overexpression (OE) of the TT2 gene triggered upregulation of HEAT SHOCK FACTOR A2 (HSFA2) thus conferring thermotolerance to in-vitro-cultured 6-day-old seedlings exposed to a heat stress (HS) of 44°C during 80 min. We are keen to establish whether TT2 can also activate HSFA2 in seeds upon HS and whether the expression of its direct target BAN and tannin accumulation are impacted by HS.

The main objective of the project is to elucidate the regulation of the MBW transcriptional regulatory complex by environmental controls (i.e. heat stress, HS), using molecular and genetic approaches.

1. Heat stress regulation of the MBW complex
1a. Heat stress regulation of MBW gene expression
Transgenic plants carrying proTT:TTgene-GFP constructs are available for TT2, TT8, TTG1 and TTG2. The correct activity of the reporter genes has been demonstrated by the complementation of corresponding mutants. These plants will be grown in a growth chamber under standard environmental conditions (21C°D/N) until the flowering stage and will be submitted to heat stresses. Flowers will be tagged before HS for gene expression analysis. Gene expression and protein localization will be studied in seeds (4-day-old corresponding to the peak of tannin biosynthesis) of the transgenic plants by confocal microscopy, qRT-PCR, Western blot, and immunolocalization with available anti-GFP antibodies.

1b. Heat stress regulation of flavonoid genes and tannin accumulation
The expression of the known MBW target genes will be quantified by qRT-PCR in developing seeds (4 dap). To determine whether tannin biosynthesis is affected by HS, a targeted metabolomic analysis (UPLC-MS) will be carried out on mature seeds. Measurement of the antioxidant activity will be realized on the same seed lots using the DPPH assay according to a protocol routinely used at IJPB.

2. Mechanisms involved in the regulation of MBW genes by heat stresses
2a. Epigenetics
Potential epigenetic regulations will be investigated by studying DNA accessibility and chromatin modifications (e.g. H3K27me3) at MBW loci using ATAC-qPCR and ChIP-qPCR, respectively during seed development.

2b. Redox state
The student will investigate the structure/function regulation of the MBW complex both in vitro and in vivo. The putative role of two cysteines present in the TT2 binding site will be investigated through site-directed mutagenesis. The TT2 proteins will be produced in E. coli or insect cells and tested by EMSA with target promoters (e.g. BAN). Moreover, the activity of the proteins will be tested in vivo by both transient expression in moss protoplast and by expression in tt2 mutants under the control of the TT2 promoter or the strong constitutive UBIQUITIN promoter that will allow to test TT2 in seed testa and in vegetative organs (seedlings), respectively.

2c. Promoter dissection
If time available, functional dissection of promoters of interest will be carried out and used for looking for new regulatory genes by 1H screening in yeast (a transcription factor library is available for 1H in yeast).