The photoacoustic frequency spectrum under steady-state conditions from Philodendron green leaves attached to the plant was measured in the 0.2â200 Hz ...
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Photosynthesis Research 52: 65–67, 1997. c 1997 Kluwer Academic Publishers. Printed in the Netherlands.
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Low frequency photoacoustics for monitoring the photobaric component in vivo of green leaves A. Frandas1 , H. Jalink2 & R. van der Schoor2 1
Institute of Isotopic and Molecular Technology, P.O.Box 700, 3400 Cluj-Napoca, Romania; 2 Centre for Plant Breeding and Reproduction Research, P.O.Box 16, 6700 AA Wageningen, the Netherlands
Received 16 August 1996; accepted in revised form 5 March 1997
Key words: oxygen evolution (uptake), photoacoustics
Abstract The photoacoustic frequency spectrum under steady-state conditions from Philodendron green leaves attached to the plant was measured in the 0.2–200 Hz frequency range. The PA amplitude spectrum showed a maximum at low frequency (around 1 Hz) which was attributed to an optimum frequency for oxygen evolution. The signal decreased at a lower frequency, where the oxygen or carbondioxide uptake starts to become important. Efficiency of the oxygen evolution as a function of excitation light intensity was determined for different levels of background light. Abbreviations: PA – photoacoustic; PS – photosystem; PD – photon density Photoacoustic and related photothermal methods have proven their capabilities in the study of photosynthesis (for review see Malkin and Canaani 1994). The mechanism of PA signal generation in green leaves originates from the appearance of sound waves due to the transformation of light energy to heat (photothermal component) and from the gas evolution due to photosynthetic activity (photobaric component). The photobaric component commonly involves O2 evolution, although O2 uptake through PS I may also be superimposed, as well as CO2 solubilization (Reising and Schreiber 1994). The PA method was used both in frequency (Havaux et al. 1987) and time domains (Kolbowski et al. 1990) mainly in a configuration using a gascoupled microphone arrangement. PA measurements were correlated with chlorophyll fluorescence quenching parameters (Snel et al. 1990) and cytochrome f spectroscopy (Havaux et al. 1986) showing that the PA technique is a valuable tool for obtaining complementary information on photosynthetic activity. Different types of stress, including pollutant effect on photochemical activity (Veeranjaneyulu et al. 1991)
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were investigated using PA spectroscopy. There are a few drawbacks to the PA technique: a low signal-tonoise ratio with a subsequent dispersion of results and a change of the gas composition during the measurement due to the closed PA cell. The latter can be partially overcome by using a gas permeable PA cell (Fork and Herbert 1991). Here we report the measurement of a high resolution PA frequency spectrum in the frequency domain with a particular emphasis on the extension to low frequencies (0.2–200Hz). The experimental setup is shown in Figure 1. The radiation source consisted of 16 LEDs (Hewlett Packard HLMP 8103, emission centered at 654 nm) mounted on a ring, thus ensuring an uniform illumination of the leaf. The LEDs were modulated electronically using the internal generator of the Bruel & Kjaer Spectrum Analyzer 2032 and a home-made amplifier. The amplitude of the modulation could be modified. The modulation function was a square wave and the modulation depth (modulation amplitude/medium light intensity) was 100%.
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Figure 1. The experimental setup.
The leaf was mounted in a PA cell, 50 mm diameter and 4 mm thickness, made from transparent plexiglass which minimized the residual PA signal due to light absorption in the PA cell itself. The cell was provided with one channel connecting the microphone to the cell through a silicone tube. Another channel allowed the measurement of leaves attached to a plant and a constant nutrition during measurement. The leaf stem was accommodated within the channel, the cell was covered by a plexiglass window, sealed by silicone grease and the ensemble was kept together by 3 string clamps. The PA signal was measured by a B&K microphone type 4182 and processed by the B&K Spectrum Analyzer which calculated the cross spectrum between the PA signal and the modulation signal from the internal generator of the Spectrum Analyzer. A leaf of a Philodendron plant (growing at 20 mol/m2 s 1 ) was accommodated in the PA cell. The leaf was illuminated by the 16 LEDs resulting an amplitude of photon density (PD) at the leaf of 60 mol/m2 s 1 . The measurement was performed in the dark after a dark adaptation period of 20 min. The PA spectrum was measured in the frequency range (0.2–200 Hz), using the experimental setup described above. The results are shown in Figure 2. Each point of the spectrum was measured as an average on at least 20 periods and was subsequently corrected for the frequency response of the B&K microphone (B&K Instruction Manual DK BE 1059-11). The frequency response of the cell itself was tested using a moistened filter paper as a sample. The PA spectrum was flat within the above frequency range. The frequency spectrum of the PA signal amplitude shows a maximum in the low frequency range
Figure 2. The PA frequency spectrum of a Philodendron leaf obtained in the dark, using an excitation of 60 mol/m2 s 1 photon density.
where the photobaric component is dominant. Therefore, this maximum can be associated with an optimum frequency for O2 evolution. At lower frequency the signal decreased, probably meaning that the O2 , and possibly CO2 uptake, starts to become important. This is consistent with the observation of Malkin (1987) while measuring PA transients, he showed that the first pulse of the PA signal was followed by a series of slower bursts of O2 uptake and evolution, reflecting the sequential activation of various parts of the photosynthetic apparatus. At higher frequencies, the PA signal decreases due to the dump out of the photobaric component. A relative minimum appears, followed by a constant level on the PA amplitude. The phase behaviour shows a jump in this region meaning a change in the dominant component of the PA signal, from photobaric to photothermal. The PA signal amplitude corresponding to the maximum in O2 evolution was measured at different LED intensities and different background light levels. The results are shown in Figure 3. Each curve was obtained at a specific background level: in the dark, in daylight (20 mol/m2 s 1 ) and superimposing the light from a fiber lamp (600 mol/m2 s 1 ). The points were normalized at the photon density of the measuring LED light. The line through the points was the best fit obtained. All functions were exponential. The transmission and reflection of the Philodendron leaf for different LED intensities were measured with a photodiode (data not shown). For the reflection
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67 All the disadvantages of the measuring system are inherent to the measurement of PA spectra of leaves. From the point of view of principle, using light as the excitation signal affects the plant, especially when measuring the photosynthetic activity which is a nonlinear process even at low light levels. The second problem comes from placing the leaf in a closed environment which distorts the results due to the change in gas composition. Further information about the mechanisms involved in the PA signal generation will be obtained by comparison to measurements of chlorophyll fluorescence.
Figure 3. The PA signal amplitude at 1.5 Hz normalized to the LED intensity, for different background lights (a) dark, (b) daylight (20 mol/m2 s 1 ), (c) fiber lamp (600 mol/m2 s 1 ).
measurements a gold mirror was used for calibration. The transmission varied between 8 and 4% by increasing the LED intensity, while the reflection was practically constant (about 2%). Therefore, the absorption by the leaf could be considered practically constant (90–94%). The curves fit were extrapolated to zero LED intensity, defining the efficiency of O2 evolution at an excitation intensity of zero for any background light. As expected, this efficiency is considerably higher for plants in the dark, where all the reaction centers are open. The O2 evolution efficiency for modulated light decreases with an increasing level of background light, since the reaction centers become saturated. This kind of curve can be measured for any additional background light. There are a number of advantages to our measuring system. The experiments were performed in vivo with the leaf still attached to the plant. The measurements were steady-state, all the measurements were performed after the plant adapted to the new measuring frequency. The PA frequency spectrum, extended for the first time to such low frequencies, allowed the observation of a maximum in the O2 evolution around 1 Hz and the monitoring of the O2 (and possible CO2 ) uptake at lower frequencies, close to dc. An advantage of a frequency domain measurement is the fact that processes occurring with different time constants can be separated. The large frequency domain investigated and the high signal to noise ratio of our measurements were possible by use of a high quality microphone.
Acknowledgments One of the authors (AF) acknowledges the receipt of a fellowship from The Netherlands Ministry of Agriculture, Nature Management and Fisheries which supported this work.
References Bruel & Kjaer Instruction Manual English DK BE 1059–11 Fork DC and Herbert SK (1991) A gas-permeable photoacoustic cell. Photosynth Res 27: 51–156 Havaux M, Canaani O and Malkin S (1986) Photosynthetic responses of leaves to water stress, expressed by photoacoustic and related methods. Plant Physiol 82: 834–839 Kolbowski J, Reising H and Schrieber U (1990) Computercontrolled pulse modulation system for analysis of photoacoustic signals in the time domain. Photosynth Res 25: 309–316 Malkin S (1987) Fast photoacoustic transients from dark adapted intact leaves: Oxygen evolution and uptake pulses during photosynthetic induction – a phenomenology record. Planta 171: 65–72 Malkin S and Canaani O (1994) The use and characteristics of the photoacoustic method in the study of photosynthesis. Annu Rev Plant Mol Biol 45: 493–526 Reising H and Schreiber U (1994) Inhibition by ethoxyzolamide of a photoacoustic uptake signal in leaves: Evidence for carbonic anydrase catalyzed CO2 -solubilization. Photosynth Res 42: 65– 73 Snel JFH, Kooiman M and Vrendenberg WJ (1990) Correlation between chlorophyll fluorescence and photoacoustic signal transients in spinach leaves. Photosynth Res 25: 259–268 Veeranjaneyulu K, N’soukpoe-Kossi CN and Leblanc RM (1991) SO2 effect on photosynthetic activities of intact sugar maple leaves as detected by photoacoustic spectroscopy. Plant Physiol 97: 50–54
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