Photoacoustic signals from intact leaves, produced upon excitation with single-turnover flashes, were shown to be dependent on their position in the flash sequence. Compared to the signal obtained from the first flash, all the others were time-shifted and had increased amplitudes. The signal from the third flash had the largest deviation, whereas that from the second flash deviated only minimally. The amplitude difference of the signals relative to that from the first flash was measured at a convenient time point (5 ms) and showed oscillations of period 4, similar to the O2-evolution pattern from algae. These oscillations were strongly damped, tending to a steady state from about the seventh flash on. The extra photoacoustic signal (relative to the first flash) was shown to be inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, heat treatment, or water infiltration. Its change with flash number, its saturation with increasing flash energy, and the above inhibition criteria indicate that it originates in pulsed O2 evolution. The sound wave produced by the first flash, however, arose by a photothermal mechanism only, as shown by its linear dependence on the flash intensity and insensitivity to the above treatments. The above flash pattern demonstrates that the photocycle of the S states (i.e., positive charge accumulation before two water molecules can be oxidized in a concerted way to produce molecular oxygen) occurs in intact leaves. It proves the applicability of the photoacoustic method for mechanistic studies of O2 evolution in leaves under physiological conditions. Water content of leaves is readily measureed by this method.
