EPR Spin-Trapping for Monitoring Temporal Dynamics of Singlet Oxygen during Photoprotection in Photosynthesis

A central goal of photoprotective energy dissipation processes is the regulation of singlet oxygen (¹O₂*) and reactive oxygen species in the photosynthetic apparatus. Despite the involvement of ¹O₂* in photodamage and cell signaling, few studies directly correlate ¹O₂* formation to nonphotochemical quenching (NPQ) or lack thereof. Here, we combine spin-trapping electron paramagnetic resonance (EPR) and time-resolved fluorescence spectroscopies to track in real time the involvement of ¹O₂* during photoprotection in plant thylakoid membranes. The EPR spin-trapping method for detection of ¹O₂* was first optimized for photosensitization in dye-based chemical systems and then used to establish methods for monitoring the temporal dynamics of ¹O₂* in chlorophyll-containing photosynthetic membranes. We find that the apparent ¹O₂* concentration in membranes changes throughout a 1 h period of continuous illumination. During an initial response to high light intensity, the concentration of ¹O₂* decreased in parallel with a decrease in the chlorophyll fluorescence lifetime via NPQ. Treatment of membranes with nigericin, an uncoupler of the transmembrane proton gradient, delayed the activation of NPQ and the associated quenching of ¹O₂* during high light. Upon saturation of NPQ, the concentration of ¹O₂* increased in both untreated and nigericin-treated membranes, reflecting the utility of excess energy dissipation in mitigating photooxidative stress in the short term (i.e., the initial ∼10 min of high light).