In vascular plants, the final photosynthetic electron transfer from ferredoxin (Fd) to NADP+ is catalyzed by the flavoenzyme ferredoxin:NADP+ oxidoreductase (FNR). FNR is recruited to thylakoid membranes via an integral membrane protein TROL (thylakoid rhodanese-like protein) and the membrane associated protein Tic62. We have previously demonstrated that the absence of TROL triggers a very efficient superoxide (O2•-) removal mechanism. The dynamic TROL-FNR interaction has been shown to be an apparently overlooked mechanism that maintains linear electron flow before alternative pathway(s) is(are) activated. In this work, we aimed to further test our hypothesis that the FNR-TROL pair could be the source element that triggers various downstream networks of chloroplast ROS scavenging. Tandem affinity purification followed by the MS analysis confirmed the TROL-FNR interaction and revealed possible interaction of TROL with the thylakoid form of the enzyme ascorbate peroxidase (tAPX), which catalyzes the H2O2-dependent oxidation of ascorbate and is, therefore, the crucial component of the redox homeostasis system in plants. Further, EPR analyses using superoxide spin trap DMPO showed that, in comparison with the wild type, plants overexpressing TROL (TROL OX) propagate more O2•- when exposed to high light stress. This indicates an increased sensitivity to oxidative stress in conditions when there is an excess of membrane-bound FNR and less free FNR is found in the stroma. Finally, immunohistochemical analyses of glutathione in different Arabidopsis leaf cell compartments showed highly elevated glutathione levels in TROL OX, indicating an increased demand for this ROS scavenger in these plants, likely needed to prevent the damage of important cellular components caused by reactive oxygen species.
Keywords: EPR; ROS; co-immunoprecipitation; glutathione; redox homeostasis; stress response; superoxide; tAPX.