EPR Spin-Trapping for Monitoring Temporal Dynamics of Singlet Oxygen during Photoprotection in Photosynthesis
Collin J. Steen, Jens Niklas, Oleg G. Poluektov, Richard D. Schaller, Graham R. Fleming, Lisa M. Utschig
Abstract
High Resolution Image Download MS PowerPoint Slide A central goal of photoprotective energy dissipation processes is the regulation of singlet oxygen ( 1 O 2 *) and reactive oxygen species in the photosynthetic apparatus. Despite the involvement of 1 O 2 * in photodamage and cell signaling, few studies directly correlate 1 O 2 * 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 1 O 2 * during photoprotection in plant thylakoid membranes. The EPR spin-trapping method for detection of 1 O 2 * was first optimized for photosensitization in dye-based chemical systems and then used to establish methods for monitoring the temporal dynamics of 1 O 2 * in chlorophyll-containing photosynthetic membranes. We find that the apparent 1 O 2 * concentration in membranes changes throughout a 1 h period of continuous illumination. During an initial response to high light intensity, the concentration of 1 O 2 * 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 1 O 2 * during high light. Upon saturation of NPQ, the concentration of 1 O 2 * 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).