Confocal Laser Scanning Microscopy of Light-Independent ROS in Arabidopsis thaliana (L.) Heynh. TROL-FNR Mutants

Thylakoid rhodanese-like protein (TROL) serves as a thylakoid membrane hinge linking photosynthetic electron transport chain (PETC) complexes to nicotinamide adenine dinucleotide phosphate (NADPH) synthesis. TROL is the docking site for the flavoenzyme ferredoxin-NADP⁺ oxidoreductase (FNR). Our prior work indicates that the TROL-FNR complex maintains redox equilibrium in chloroplasts and systemically in plant cells. Improvement in the knowledge of redox regulation mechanisms is critical for engineering stress-tolerant plants in times of elevated global drought intensity. To further test this hypothesis and confirm our previous results, we monitored light-independent ROS propagation in the leaves of Arabidopsis wild type (WT), TROL knock-out (KO), and TROL ΔRHO (RHO-domain deletion mutant) mutant plants in situ by using confocal laser scanning microscopy with specific fluorescent probes for the three different ROS: O₂^·-, H₂O₂, and ¹O₂. Plants were grown under the conditions of normal substrate moisture and under drought stress conditions. Under the drought stress conditions, the TROL KO line showed ≈32% less O₂^·- while the TROL ΔRHO line showed ≈49% less H₂O₂ in comparison with the WT. This research confirms the role of dynamical TROL-FNR complex formation in redox equilibrium maintenance by redirecting electrons in alternative sinks under stress and also points it out as promising target for stress-tolerant plant engineering.