Determination of Oxygen Emission and Uptake in Leaves by Pulsed, Time Resolved Photoacoustics

Abstract

Pulsed, time resolved photoacoustics has sufficient sensitivity to determine oxygen emission and uptake by single tumover flashes to leaves. The advantage over previous methodologies is that when combined with single turnover flashes the kinetics of the thermal and the gas signals can be resolved to 0.1 millisecond and separated. The S-state oscillations of oxygen formation are readily observed. The gas signal from common spongy leaves such as spinach (Spinacia sp.), Japanese andromeda (Pieris japonica), mock orange (Philadelphus coronarius) and viburnum (Viburnum tomentosum), after correction for instrumental rise time, show a lag of only 1 millisecond and a rise time of 5 milliseconds in the formation of oxygen. Thus a recent proposal that the formation of oxygen requires over 100 milliseconds cannot be true for choroplasts in vivo. The rapid emission is correlated with structure of the leaf. At low light flash energies a rapid gas uptake is observed. The uptake has slightly slower kinetics than oxygen evolution, and its magnitude increases with damage to the leaf. The pulse methodology shows that the uptake begins with the very first flash after dark adaption, and allows the detection of a positive signal (oxygen) on the third flash. These observations, the long wavelength of excitation (695 nanometers) and the magnitude of the signal support the contention that the gas uptake is oxygen reduction by electrons from photosystem 1. These results show that important physiological aspects of a leaf can be studied by pulsed, time resolved photoacoustics.The photoacoustic effect is caused by the thermal expansion of a fluid when the energy of absorbed photons is degraded to heat (17 ever, the inherent complexity of photosynthetic systems coupled with the limited data obtained in the usual photoacoustic methodology using modulated light has made progress slow. A complete analysis would require a dense set of both amplitude and phase measurements over a range of at least three orders of magnitude in frequency. Since the measurements are made one frequency at a time, it is not surprising that such data assets are rare (23). Further, the analysis of such a data set with multiple relaxation times having both positive and negative amplitudes is itself very difficult and may lead to ambiguous results. By contrast, the analysis of the photoacoustic response to a single short pulse of light is relatively straightforward. The signal to noise ratio is typically sufficient to obtain the complete kinetic trace of the signal in a single measurement over the accessible time range of 30 ,us to 100 ms. In addition to this enormous advantage of obtaining all of the accessible time data (i.e. reciprocal frequency) in one measurement, the use of classical single turnover flashes (so short the system can only turnover once [8]) provides the further advantage of identifying and distinguishing processes related to oxygen formation. The slower oxygen pressure signal is easily resolved from the faster photothermal signal because mol...

Energy Storage of Linear and Cyclic Electron Flows in Photosynthesis

“…The technique of photoacoustics has been applied to photosynthetic systems for some time (10,26,30) with a variety of useful results, including the direct measurement of 02 formation in intact leaves (8,11,28 …”

“…By selection of excitation wavelength, we have determined the TE for linear electron flow through PSII to PSI and that for cyclic electron flow in PSI and possibly in PSII. The energy storage in PSII and in PSI were determined by varying flash intervals and by use of inhibitors.The technique of photoacoustics has been applied to photosynthetic systems for some time (10,26,30) with a variety of useful results, including the direct measurement of 02 formation in intact leaves (8,11,28 …”

SO2 Effect on Photosynthetic Activities of Intact Sugar Maple Leaves as Detected by Photoacoustic Spectroscopy

“…Recent studies have demonstrated that the PA 02 signal is influenced by a PSI-mediated 02 uptake process and possibly by other 02-consuming processes of the cell (9,14). If S02 has any inhibitory effect on these processes, it could cause an increase in 02 signal amplitude.…”

Enthalpy, Entropy, and Volume Changes of Electron Transfer Reactions in Photosynthetic Proteins

“…A gas-coupled microphone in a closed chamber is used as a detector on the ms time scale. This method allows measurements of the photosynthetic thermal efficiency, or energy storage, and of the optical cross-section of the light harvesting systems (Canaani et al, 1984;Mauzerall, 1990;Braslavsky & Heibel, 1992). However, the time resolution is low, typically about 30 μs, and inappropriate for obtain thermodynamic parameters on the microsecond and nanosecond time scales.…”

“…Measuring the energetics of photobiological reactions in vivo is of more interest because the local environment of photosystems may exert a prevailing consequence on their kinetics and thermodynamics. Pulsed photoacoustics via a microphone detector revealed the oxygen evolution and oxygen uptake in vivo on the millisecond time scale (Mauzerall, 1990). Using a piezo film detector, the in vivo enthalpy and volume changes of photosystem I and photosystem II of Synechocysits sp.…”