Thèse le Rôle de la Ferrédoxine en Tant que Centre de Distribution d'Électrons dans le Chloroplaste des Plantes 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 : I2BC - Institut de Biologie Intégrative de la Cellule
Direction de la thèse : Anja KRIEGER-LISZKAY ORCID 0000000171414129
Début de la thèse : 2026-10-01
Date limite de candidature : 2026-04-30T23:59:59Le changement climatique et la croissance démographique mondiale font de la sécurité alimentaire une préoccupation majeure pour les pouvoirs publics. Une stratégie prometteuse pour relever ce défi consiste à améliorer l'efficacité de la photosynthèse. La photosynthèse est le processus par lequel les plantes utilisent l'énergie solaire pour synthétiser de la biomasse à partir du dioxyde de carbone et de l'eau. Plus précisément, la conversion de l'énergie solaire en énergie chimique se produit grâce au fonctionnement de la chaîne de transport d'électrons photosynthétique (PETC) dans les membranes thylakoïdes des chloroplastes. Les complexes pigments/protéines impliqués dans le transport linéaire des électrons photosynthétiques, produisant de l'ATP et du pouvoir réducteur (NADPH), sont désormais bien caractérisés. En revanche, on en sait beaucoup moins sur la compétition entre les différentes voies électroniques au-delà du photosystème I (PSI), qui contrôlent la photosynthèse et le métabolisme chloroplastique. Toutes partagent un donneur d'électrons protéique commun : la ferrédoxine (Fd), une protéine stromale soluble. Le mécanisme par lequel la Fd choisit entre ses différents accepteurs d'électrons reste difficile à cerner. Il est particulièrement intéressant de comprendre la régulation du transport cyclique des électrons (CET) et du transport pseudocyclique des électrons (PCET), qui sont des voies alternatives de transport des électrons qui stimulent la production d'ATP tout en protégeant les plantes contre les facteurs de stress environnementaux (par exemple, la sécheresse, la lumière intense et les températures extrêmes). Cependant, malgré leur importance dans l'acclimatation des plantes à l'environnement, ce processus de régulation reste obscur.
Dans le cadre de ce projet, nous visons (1) à caractériser par spectroscopie la préférence des différentes voies métaboliques pour l'oxydation du Fd, et (2) à étudier sur le plan biochimique et fonctionnel la régulation redox et l'activité de la CET et de la PCET chez les plantes exposées au stress. Ces informations seront utiles pour élaborer des stratégies visant à repenser les voies électroniques et à améliorer les réponses des plantes au stress dans le cadre d'études futures.

Ferredoxins are the major electron donors to various electron acceptors in chloroplasts. Mutants of different Fd-dependent enzymes have been previously studied, like for example the Ferredoxin-NADP+ Reductase (FNR), the Malate Dehydrogenase (NADP-MDH) and the Ferredoxin-Thioredoxin Reductase (FTR). For most of them, Km-values, Vmax and the midpoint redox potentials of the isolated proteins are known. However, how Fd chooses and distributes electrons to these different enzymes in vivo is still an open question. This depends not only on midpoint redox potentials but on the size of the Fd and NADP+ pool, and the concentration and cellular localisation of the electron accepting enzymes (Rodriguez-Heredia et al., 2022). Within this context, particular interest lies in understanding the regulation of alternative electron pathways such as cyclic electron transport (CET), which also depends on Fd as electron donor.
Absorption changes in the near-infrared allow monitoring Ferredoxin (Fd) reduction and oxidation kinetics in leaves (Schreiber, 2017; Sétif et al., 2019), which are a reliable indicator of CET activity (Ohnishi et al., 2023; Maekawa et al., 2024). Using a KLAS-NIR-PAM system, our preliminary data show that Fd reoxidation rates are decreased in an Arabidopsis thaliana mutant lacking FTRA2, one of the two structural isoforms of FTR. In contrast, we have seen that Fd reoxidation rates are faster in a Fd2 knockout mutant, the major Fd isoform in Arabidopsis (90 %). We have established recently a protocol allowing to follow Fd reduction and reoxidation kinetics in the micro- and millisecond time range via absorption changes in the near-infrared.Using this methodology, we plan to assess how different electron routes compete for Fd- using Arabidopsis mutants affected in CET and in different alternative electron routes beyond PSI.

Aims of the thesis are to demonstrate the role of CET via Fe reoxidation under different light conditions (Task 1), and to investigate the hierarchy and competition of Fe oxidation by FNR and FTR in vivo (Task 2).

Task 1: Characterisation of Fd-dependence of CET
Background: CET is difficult to measure, because it is a minor electron transport pathway. Up to now, the most reliable method to monitor CET is via the electrochromic shift (ECS) technique. We have first evidence that Fd reoxidation kinetics allow to follow CET. The Arabidopsis mutant fd2 show faster reoxidation kinetics in the microsecond range than the wild type, indicating that Fd1 is the electron donor for CET. Single turnover flashes will be used to reduce Fd (Sétif et al., 2019). Known mutants of CET (pgrl1ab (FQR-dependent pathway), crr2-2 (NDH-dependent pathway) will be analysed. In addition, several physiological conditions favouring CET will be studied: low light, transition to high light, drought and extreme temperatures (Johnson, 2011).

Material: Arabidopsis mutant fd2, fd1 antisense lines, mutants of cyclic electron flow pgrl1ab (FQR-dependent pathway, crr2-2 (NDH-dependent pathway). All mutants are available in the lab, except of the fd1 antisense lines, which will be generated.
All lines will be grown in control (short day, normal growth light, 20°C/18°C) and stress conditions (high light, drought, 28°C).

Methods: RNAi silencing in Arabidopsis thaliana will be used to generate viable fd1 mutants. Photosynthesis will be evaluated by gas exchange, chlorophyll fluorescence and absorption spectroscopy in the near-infrared. ECS and Fd reoxidation kinetics will be measured spectroscopically and their kinetics will be analysed.
Fd1 and Fd2 expression levels will be measured by RT-qPCR in the different mutants and under the different physiological conditions (low light, transition to high light, drought and extreme temperatures), and their protein levels will be estimated by immunoblots.

Task 2: Characterisation of Fd-dependent electron transport pathways beyond photosystem I
Background: In addition to CET, Fd can mediate electron transfer to other metabolic pathways central to chloroplast metabolism and plant acclimation to the environment (i.e., FNR, glutamate synthase -GOGAT-, nitrite reductase -NiR-, sulfite reductase -SiR-). Moreover, Fd can transfer electrons to thioredoxins (Trxs) via the FTR. Once reduced, Trxs can reduce disulfide bonds in different target proteins, regulating their activity. Trxs activate amongst others carbon fixation reactions and the NADP+-MDH. In addition, MDH also relies on on NADPH as electron donor. MDH is an important enzyme controlling the reduction state of the chloroplast (NADP+/NADPH ratio) via the malate valve. The mechanisms by which Fd differentially distributes electrons towards FNR and FTR, FTR to Trx remain poorly understood. An integrative approach addressing the preference for each electron route under different environmental conditions has, to the best of our knowledge, never been developed. We will study the oxidation and reduction kinetics of Fd in Arabidopsis thaliana wt plants and ftra1 and ftra2 (mutants of the variable subunit of FTR, Keryer et al., 2004), trxm4 and 2cpab (2-Cys Peroxiredoxin). trxm4 and 2cpab have been shown to regulate CET and PETC, respectively (Courteille et al. 2013; Hani et al. 2024). We will follow the Fd reoxidation kinetics after short (10s) preillumination with different light intensities. Furthermore, we will determine NADPH generation biochemically or via changes in NAD(P)H fluorescence.

Material: Arabidopsis mutant ftra, mdh, trxm4, 2cpab (several mutant lines will be provided by E. Issakidis-Bourguet, IPS2), fnr mutants (provided by G. Hanke, QMUL, London).

Methods: Photosynthesis will be measured by gas exchange, chlorophyll fluorescence and absorption spectroscopy in the near-infrared. Fd reoxidation kinetics will be measured spectroscopically and their kinetics will be analysed. RT-qPCR will be used to detect difference in expression levels of FTR, FNR, Fd in the different mutants.