Observations on the in situ detection of free radicals in leaves using electron paramagnetic resonance spectrometry

Abstract

To follow in situ changes in free radicals in leaves subjected to changes in the composition of the surrounding air, a system has been developed for obtaining X-band (9 GHz) electron paramagnetic resonance (EPR) spectra of intact leaf tissue over extended periods. Studies with radish (Raphanus sativus), bluegrass (Poa pratensis), and ryegrass (Lolium perenne) leaves in darkness or illuminated with 650 nm, 710 nm, or white light in the spectrometer cavity readily reveal the photosynthetic signals I and II, and signals attributable to Mn2+ and Fe3+. Detached pieces of leaf cannot be used after about 1 h in the cavity because of the appearance of a large EPR signal resulting from excision. However, attached leaves of the grasses can be maintained in a fully functioning state in the cavity for several days, or can be withdrawn and replaced at will. Plants grown in high photon influx densities were found to reveal a large free radical signal, related to photoinhibition, that masks the typical photosynthetic signals but that can usually be eliminated by subjecting the tissue to 9 GHz microwave radiation. Signal changes in response to changes in the composition of the air stream can be directly tracked over extended periods, provided that no major changes occur in the underlying Mn2+ or Fe3+ signals. Preliminary studies with the air pollutants, sulphur dioxide, and ozone have demonstrated the potential of the system for revealing changes in the photosynthetic and other EPR signals resulting from the uptake of such phytotoxic gases. Key words: free radical, electron paramagnetic resonance spectrometry, intact leaf tissue, photosystem, air pollution, stress.