Measurement of Chlorophyll Fluorescence by Synchronous Detection

Environ. Sci. Technol. 1998, 32, 2640-2645

Measurement of Chlorophyll Fluorescence by Synchronous Detection in Integrating Sphere: A Modified Analytical Approach for the Accurate Determination of Photosynthesis Parameters for Whole Plants RICHARD SALVETAT,† PHILIPPE JUNEAU, AND RADOVAN POPOVIC* Institut des Sciences de l’Environnement, Universite´ du Que´bec a` Montre´al, De´partement de Chimie/TOXEN, Case Postale 8888, Succursalle Centre-Ville, H3C 3P8 Montre´al, Que´bec, Canada

In photosynthesis research, pulse-amplified modulated (PAM) fluorescence is used to study the chlorophyll fluorescence induction of plants in order to investigate the mechanisms of the energy transfer in photosynthesis and to determine the plant physiological state. However, in many cases the lack of accuracy and reproducibility in measurement introduces some problems in the determination of fluorescence parameters and therefore in their interpretation concerning environmental research domain. In this paper, a new type of modulated fluorometer designed to resolve these problems is presented. An integrating sphere was used as a measuring chamber associated with a PAM fluorometer system that was originally built in our laboratory. To demonstrate the performance and the reliability of our measuring system, we presented results of fluorescence measurements of plants that have been exposed to different environmental conditions (ultraviolet-B radiation, low- and high-temperature stress). The advantages of measuring different fluorescence parameters with synchronous detection by this type of PAM system are discussed.

Introduction Measurement of oxygen evolution and CO2 exchanges are commonly used to evaluate the effects of pollutants or environmental stress on the physiological state of plants. These measurements, under certain conditions, are very slow and suffer from lack of precision (1). Measurement of chlorophyll a (Chl a) fluorescence in intact plants proved in the past to be an efficient tool for estimating photosynthesis values and therefore evaluating a plant physiological state (2-4). As is well-known, after a period of adaptation to darkness, illuminated plants show a time-dependent fluorescence induction that reflects mechanisms of photochemi* Corresponding author e-mail: [email protected]; fax: 514987-4054. † Present address: ENSIL, E Ä cole Nationale Supe´rieure d’Inge´nieurs de Limoges, Parc Ester, Technopole, 87068 Limoges Cedex, France. 2640

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cal energy transfer and energy storage in photosynthesis (5). Since carbon metabolism and related processes (in plants) are directly dependent on the primary photosynthesis process, the kinetics of dissipated fluorescence by plants may give accurate information about their physiological states (6). To define instantaneously these states, fluorescence kinetics offer such worthwhile photosynthesis parameters as the “dead fluorescence” (Fo); the variable fluorescence (Fv); the quantum yield of photochemistry (Φ); the first complementary area, CA (6, 7); and the fluorescence nonphotochemical and photochemical quenching coefficients, qN and qP (8). The standard laboratory techniques for studying fluorescence kinetics in plants employ different types of fluorometers. Frequently used is a modulated fluorometer (9), which was founded on the principle of synchronous detection (10). The advantage of pulse-amplified modulated (PAM) fluorometer utilization is that only one measurement is sufficient to determine numerous important photosynthetic parameters. The fluorescence value Fo is estimated with a high accuracy, minimizing bias errors in the determination of the other photosynthetic parameters [(Fv)max, Φ, CA, qN, and qP]. In photosynthesis studies, CA, qN, and qP are related respectively to the photosystem II (PSII) working capacity, nonphotochemical energy dissipation, and photochemical energy dissipation. Similar studies currently use (Fv)max and Φ as indicators of maximum PSII photochemistry efficiency (7, 8). Therefore, all these parameters are very important for evaluation of the plant physiological state as affected by different environmental conditions (6). To evaluate these parameters, the modulated fluorometer demonstrated some advantages as compared to other fluorometric systems employed in measurements of fluorescence with algae or with fractions of plants (chloroplasts, thylakoids, or PSII submembrane fractions) (9). However, this instrument cannot give reliable information on the physiologic state of whole plants or even of intact leaves, since measurement by fiber optics cannot provide easily reproducible and representative signals for all plants. Fluorescence parameters obtained by these measurements are dependent on the measured leaf zone or on the part of the plant, and consequently, it may affect the data reproducibility. Earlier, it had been proposed (11) that the use of an integrating sphere in measurements of plant fluorescence has the following advantages: First, the detected signal represents the integrated value of fluorescence of the whole plant surface. Second, the reproducibility of measurements made using this method is very high since the signal of emitted fluorescence is independent of the exact position of the plant inside the sphere. However, the first integrated-sphere fluorometer, which used an incandescent lamp and a camera shutter as light source, did not permit accurate determination of the Fo fluorescence. Moreover, the determination of fluorescence quenching coefficients under saturating-light condition is difficult or even impossible (12). So, to study the effects of environmental factors on whole plants, the measure of fluorescence parameters in an integrating sphere became a necessary tool. In the past this type of analytical approach, useful for practical and fundamental research, was not possible since the light emitting diode (LED) used did not have sufficient luminosity to saturate the photosynthesis of whole plants (13). Also, there were technical problems with the properties of the design (11). On the other hand, a highly sensitive detector system was necessary to detect modulated fluorescence in a voluminous integrating sphere (14). S0013-936X(97)01039-0 CCC: $15.00

 1998 American Chemical Society Published on Web 07/17/1998

FIGURE 1. Schematic diagram of the integrating modulated fluorometer: (1) pulsed analytic light; (2) continuous actinic light; (3) pulsed saturating light; (4) integrating sphere; (5) reflector; (6) iris diaphragm; (7) detection system: (a) 730 nm interference filter (Ealing Electrooptics 35-6337), (b) PIN photodiode (Hamamatsu S1723-04), (c) current-to-voltage converter (105 V/A amplification); (8) high pass filter; (9) lock-in amplifier; (10) data acquisition system and microcomputer. The lines noted Trig.AL and Trig.SL are the trigger lines of, respectively, the actinic and the saturating light monitored by the microcomputer. In this paper, we present a new design for an integrating modulated fluorometer. We use a high-brightness LED as a light source to fully saturate the photosynthesis of plants in the condition of an integrating sphere. We also introduce our developed photodetection system that resolved the detection problem of low fluorescence intensity. With this new measuring system, we are able to determine the photochemistry quantum yield and other fluorescence parameters for whole intact plants. To demonstrate the performance of our fluorometric measuring system, we measure fluorescence induction in pea plants affected by ultraviolet-B (UV-B) radiation and tomato plants exposed to low- and high-temperature stress.

Materials Figure 1 shows a block diagram of the new measuring system. The analytic light source (1) is provided by four ultrabrightness LEDs (Ledtronic L200CWY3K; maximum peak wavelength λo ) 590 nm) modulated with a 25-kHz sinusoidal wave. The current pulse is adjustable to permit a variation of photosynthetic photon flux density (PPFD) in a range from 0 to 5 µmol m-2 s-1. A short-pass filter (Oriel 58856) removes wavelengths over 600 nm. The actinic light source (2) consists of 110 high-brightness solid-state lamps (Hewlett-Packard HLMT-CH00) made with the transparent substrate AlInGaP technology. To avoid the overlap with the chlorophyll fluorescence domain (680-750 nm), we use LED with maximum emission at λp ) 621 nm. The light intensity can be adjusted up to 600 µmol m-2 s-1. The saturating pulse light source (3) consists of the same 110 TS-AlInGaP reddish-orange LEDs as are used for the actinic light source. We succeed in obtaining in the integrating sphere a PPFD of over 5000 µmol m-2 s-1 by pulsing the LED with 1 µs width pulse at a frequency of 300 kHz (Figure 2a). The integrating sphere (4) has a diameter of 20 cm (Oriel model 70451). In this sphere we use spherical skullcap diffusers (5) behind each source in order to homogenize the illumination and to prevent the direct illumination of samples. All parts inside the sphere are covered with white, nonfluorescent reflectance coating (Kodak Chemical 6080). The plants are inserted into the sphere through an iris diaphragm (6).

The detector (7) is a PIN photodiode (7b) (Hamamatsu S1723-04) with 15 V negative bias (Figure 2b). The photocurrent is converted to voltage with a fast FET input operational amplifier (Analogue Devices AD795) mounted in a transconductance converter with a 105 V/A amplification (7c). This current-to-voltage converter is connected to a voltage amplifier. The photodetector is protected against reflected and scattered light by a 730-nm interference filter (7a) (Ealing Electrooptics 35-6337). The lock-in amplifier (9) is where the signal from the detector (7) is demodulated synchronously with a balanced modulator/demodulator (Analogue Devices AD630) by using in-phase information derived from the modulator (EXCAR, XR2206). The signal is filtered by using a two-poles, simple, low-pass filter that provides a gain of 100 to the output. Figure 2c shows the lock-in amplifier with the AD630 employed as a synchronous demodulator. The acquisition data system (10) consists of a 16-bit analog-digital converter connected by the ISA bus to an IBMPS2 microcomputer. The computer also controls the different light sources. The acquisition and digital signal processing software is written in C language (Turbo C version 2.0, Borland). Plant materials used for fluorescence measurements were pea seedlings and tomato plants. Pea plants were grown under visible light (400-700 nm) for 14 days at 25 °C, and tomato plants were bought from a local producer. Pea treated plants were exposed to UV-B radiation (280-320 nm) for the last 7 days of growing. The UV-B radiation was provided by fluorescent tubes (UVB-40, Philips). Plants were exposed to two different UV-B radiation levels: 2 and 4 µmol m-2 s-1. During the exposure to UV-B radiation, we used a cellulose acetate filter to eliminate the presence of UV-C light. For both control and treated plants, the photosynthetic photon flux density (PPFD) was 300 µmol m-2 s-1. To induce temperature stress on tomato plants, we exposed them in a controlled growing chamber to a temperature of 4 and 45 °C for 1.5 h before the fluorescence measurement.

Methods To measure modulated fluorescence kinetics related to the physiological state of plants, we adapted plants for 20 min to the dark to obtain the equilibrium state of the photosynthetic apparatus. After this dark-adaptation period, the VOL. 32, NO. 17, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Scheme of photodetection systems: (A) saturating light pulse compartment, (B) fluorescence photodetector organization, and (C) lock-in amplifier setup. See Materials for further details.

FIGURE 3. Dark-light induction curves of whole pea plants measuring in integrating sphere: (a) determination of the dead fluorescence (Fo); (b) determination of (Fv)max; (c) determination of fluorescence quenching components (qN and qP). See Methods for more details and terms definition. plant sample was illuminated during 10 s solely by the modulated analytical light, which had a very low intensity in order to avoid photochemical energy transfer by photosynthesis. This light source was pulsed by a high-frequency signal to provide a high signal-to-noise ratio at the output of the lock-in amplifier (15). Under this condition, a modulated fluorescence signal with constant amplitude was emitted by the plant (Figure 3a). The constant fluorescence, so-called dead fluorescence (Fo), represented the light emission by excited antennae of Chl a molecules before the excitation migrated to reaction centers of PSII and induced photochemical activity. When the continuous actinic light was turned on, fluorescence rose rapidly to a peak level, Fp. 2642

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The increase of chlorophyll fluorescence emission is caused by the photochemical reduction of primary quinone electron acceptor QA associated with the PSII reaction centers. Once the process of electron transfer was triggered by actinic light, the level of the fluorescence induced by the modulated analytical light is directly dependent on the QA redox state. The modulated signal measured through the lock-in amplifier is, therefore, proportional to the fluorescence kinetic induced by the actinic light (16, 17). At saturating light intensities of actinic light, when the electron acceptor QA pool is fully reduced, the yield of variable fluorescence reaches a maximal level at Fp (4). In this case (Figure 3b), the fluorescence peak (Fp) is called the saturation

TABLE 1. Values of Some Photosynthetic Parameters of Pea Seedling Measured in Four Different Positions in the Integrating Sphere positions parameters

1

2

3

4

av values

Fo (a.u.) FMAX (a.u.) Φ CA qN qP

0.456 2.56 0.822 2.56 0.348 0.813

0.460 2.61 0.824 2.60 0.342 0.818

0.450 2.55 0.824 2.20 0.354 0.824

0.448 2.40 0.813 2.22 0.361 0.800

0.454 (0.006) 2.53 (0.09) 0.821 (0.005) 2.40 (0.2) 0.351 (0.008) 0.814 (0.01)

a The values in parentheses are the standard deviation calculated for the four positions.

FIGURE 4. Kinetics of chlorophyll fluorescence induction on exposure of a dark-adapted pea leaf. Upon illumination, fluorescence (curve a) rises immediately to level O (so-called Fo), followed by a rapid rise to a peak level P, and a slower reduction to a steadystate level T. Curve b shows the first 4 s of kinetics. The different phases conventionally lettered O-I-D-P-S-M-T are involved in the calculation of the photosynthetic parameters. level of variable fluorescence, noted as FMAX. Therefore, if a dark-adapted plant is illuminated with a 500-ms saturating pulse light, the detected modulated signal represents the maximal chlorophyll fluorescence, FMAX, emitted when all QA were reduced. So, to determine FMAX accurately, we initially applied the saturating light pulse and then waited 30 s (the time required by the plant to reoxidize fully its primary electron acceptors QA and reobtain the equilibrium state of the photosynthetic apparatus) before turning on the actinic light in order to measure the variable fluorescence. The determination of the maximum saturation level of variable fluorescence FMAX permitted us to evaluate the quantum yield of photochemistry in intact plants by using the ratio: Φ ) (FMAX - Fo)/FMAX ) (Fv)max/FMAX (18). The fluorescence phase P was followed by a fluorescence transient via phases S, M, and the steady-state level T in pea plants that we used as experimental material (Figure 4). When a saturating light pulse was applied repetitively to monitor the maximum level of variable fluorescence during the P-SM-T phase, we were able to determine the chlorophyll fluorescence quenching components at any point of this transient (Figure 3c). The value of maximal fluorescence did not remain constant during the P-S-M-T transient, showing the complexity of the fluorescence quenching mechanism (19). This quenching effect had two components: the photochemical fluorescence quenching (qP) dependent on the electron-transport activity and the nonphotochemical fluorescence quenching (qN) related to the nonphotochemical reaction by which part of the absorbed light energy was dissipated. We demonstrate here that the superposition of actinic and saturating lights in an integrating sphere is the only technique that allows the continuous determination of qP and qN in intact whole plants at any time of illumination. The value of qN and qP were calculated, respectively, by these formulas: (Fv)max - (Fv)S and ((Fv)S Fv)/(Fv)S) (20). An integrated sphere requires a highly sensitive detection system since the modulated actinic light has a very low intensity (to avoid photochemical energy transfer) and consequently induces a very low yield of fluorescence. To obtain a lock-in amplifier output signal with a valuable signalto-noise ratio, the photodetection system should have a rapid rise time. Figure 2 presents the scheme of our photodetection system. A bias-negative voltage is applied to the large area PIN diode in order to reduce its rise time and to increase its

sensitivity. A fast FET-input operational amplifier (with 250 V/µs slew rate) was used as a current-to-voltage transducer. The scale factor of our circuit is 105 V/A. To prevent the lock-in amplifier from reaching saturation, a band-pass filter (flow ) 1.6 kHz; fhigh ) 50 kHz) suppressed the continuous component that was introduced by the fluorescence signal from the actinic light and also by the pulsed signal generated by the LEDs of the saturating light. We used different sampling frequencies to optimize the quantity of information and the size of the data file. Thus, during the 5 s after the actinic light had been turned on, the fluorescence signal was sampled at 2.5 kHz in order to analyze the fast phase O-I-D-P (Figure 4) and to calculate the complementary area (CA), which gave valuable information on the photochemistry capacity of PSII (6, 7). During the P-S-M-T transients, the sampling frequency was decreased to 25 Hz, except during the pulse of saturating light when a 250-Hz sampling frequency was used to determine accurately the maximum peak value. The kinetics related to the photochemical and nonphotochemical fluorescence quenching were determined by a cubic spline interpolation (21).

Results and Discussion To evaluate the effect of plant position in the sphere on the reproducibility of fluorescence measurements, we examined four different positions of an intact plant during the evaluation of fluorescence parameters (Table 1). At the beginning of the measurement, the first position of the plant in the sphere was taken arbitrarily. The three other plant positions were induced after a rotational motion of 90° around its vertical axis. The low value of the standard deviation for each photosynthetic parameter shows clearly that the fluorescence kinetic is independent of the plant position inside the sphere. It is evident that synchronous detection of modulated fluorescence signal allows us to distinguish accurately, for whole plants, the Fo, FMAX, Φ, CA, qN, and qP values. We have shown here that the use of TS-AlInGaP LEDs in pulsed mode gives, in the integrating sphere, a light intensity that saturates PSII activity in whole plants, since the obtained value of 0.821 for Φ was similar to the value determined for healthy plants (16). Under our experimental conditions we have been able, by synchronous detection of modulated fluorescence, to record the photochemical and nonphotochemical fluorescence quenching values for a whole plant. Therefore, the use of high-brightness LEDs in an integrating sphere provides an optimal probe for designing a measuring system for whole plants as an important and a necessary tool in environmental studies, since we obtained a low variability between our results and known values for all photosynthetic parameters. Table 2 represents values of fluorescence parameters for plants affected by UV-B radiation. As seen, UV-B radiation does not modify the Fo fluorescence. Since Fo fluorescence originates from the Chl a antennae associated with the PSII VOL. 32, NO. 17, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. Principal Photosynthetic Parameters Measured in Control and UV-B-Treated Pea Plantsa parameters

control

UV-B (2 µmol m-2 s-1)

variation (%)

UV-B-treated (4 µmol m-2 s-1)

variation (%)

Fo (a.u.) FMAX (a.u.) Φ CA qN qP

0.461 (0.007) 2.56 (0.06) 0.820 (0.009) 2.39 (0.2) 0.354 (0.01) 0.810 (0.02)

0.458 (0.009) 1.24 (0.05) 0.631 (0.01) 1.86 (0.1) 0.283 (0.02) 0.616 (0.02)

0.7 52 23 22 20 24

0.459 (0.01) 0.974 (0.06) 0.529 (0.01) 1.67 (0.15) 0.244 (0.02) 0.518 (0.01)

0.4 62 35 30 31 36

a

The values in parentheses indicate the standard deviation (n ) 4).

TABLE 3. Temperature Dependency of Some Photosynthetic Parameters for Tomato Planta

a

parameters

control

4 °C (1.5 h)

variation (%)

45 °C (1.5 h)

variation (%)

Fo (a.u.) FMAX (a.u.) Φ CA qN qP

0.131 (0.01) 0.916 (0.04) 0.857 (0.1) 3.35 (0.15) 0.210 (0.03) 0.906 (0.01)

0.130 (0.02) 0.929 (0.06) 0.860 (0.008) 5.50 (0.09) 0.487 (0.13) 0.751 (0.07)

0 1 0 64 132 17

0.134 (0.01) 0.730 (0.03) 0.816 (0.02) 4.54 (0.06) 0.400 (0.07) 0.897 (0.04)

2 20 5 36 90 1

The values in parentheses indicate the standard deviation (n ) 4).

light-harvesting complex (5), it is evident that this complex was not altered by UV-B radiation. This UV-B “immunity” can probably be explained by the well-sunk antennae of Chl a in the photosynthetic membranes and the protection given by the other pigments and proteins. No effect on Fo values was seen earlier for spinach and pea leaves (22, 23). However, it was obvious that FMAX as the maximal fluorescence yield was decreased in the plant treated with UV-B radiation. Therefore, the quantum yield of photochemistry (Φ) was approximately 20% lower for the UV-B-exposed plant than for the control plants. We also noticed a decrease of the CA for UV-B-exposed plants since PSII function was altered. Furthermore, we noticed a decrease in the nonphotochemical and the photochemical quenching values that correlated with the UV-B damage of many components of the photosynthetic membrane (22, 24, 25). We show in Table 3 the temperature dependency of some photosynthetic parameters for tomato plants treated at 4, 20, and 45 °C. Effects of temperature stress on photosynthetic parameters were different as compared to those obtained after UV-B irradiation of plants. This difference came as a consequence of their different action sites, since UV-B damages PSII protein complexes and other photosynthetic membrane components (22, 24, 25), while temperature stress primarily alters liquid enzymatic system in chloroplast (26, 27). However, Φ and qP were less indicative parameters for the change of the tomato physiological state induced by a temperature stress. On the other hand, we showed that CA and qN were highly indicative parameters that may indicate the change of physiological state of plant induced by lowand high-temperature stress. The change of the CA values indicates that the photochemical capacity of PSII in plants was altered by temperature effect on the PSII water splitting enzyme system and consequently reduced PSII electron transport (6). Temperature alteration on the oxygen evolving system was earlier reported for tomato and pea (26, 27). As seen by this effect, CA was increased by 64% and 36% in plants exposed to 4 and 45 °C, respectively. Tomato plants exposed to temperature stress showed also an important increase of qN value since the qN for low and high temperature were respectively 2.3 and 1.9 time higher than the qN of control plants. It appears that low temperature limits the photosynthetic electron transport and consequently results in a decrease of NADPH and ATP synthesis (28); therefore, qN became higher as compared to the control. The increase in the qN value for high-temperature treated plants 2644

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may be caused by the deactivation of the CO2 fixation, which restricts the ATP consumption and produced an increase of proton gradient in thylakoid membranes (29, 30). With shown results, it became obvious that any effect of environmental stress, inducing the change of plant physiological state, will be manifested by the changes of the photosynthetic activity. Therefore, some photosynthetic fluorescence parameters can be used as a powerful tool to evaluate the impact of environmental conditions. We are proposing a new system to detect the modulated fluorescence signal in an integrating sphere by implementing a large active-area PIN photodiode and using a high-speed FET-input operational amplifier as a current-to-voltage converter (Figure 2). We demonstrate that the measurement of chlorophyll fluorescence by synchronous detection in an integrating sphere permits the determination of the photosynthesis parameters for whole plants with very high precision and very good reproducibility. For these reasons, our instrumentation may be considered for use in studies of plants affected by different pollutants or environmental stresses.

Acknowledgments This work was supported by the Green Plan of Canada and the Natural Science and Engineering Council of Canada (NSERC) through Grant GP0093404 awarded to R.P. R.S. was supported by a Fondation UQAM postdoctoral fellowship. P.J. was supported by a FCAR fellowship.

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(21) Mathews, J. H. Numerical Methods for Computer Science, Engineers and Mathematics; Prentice Hall Inc: Englewood Cliffs, NJ, 1987; pp 238-249. (22) Melis, A.; Nemson, J. F.; Harrisson, M. A. Biochim. Biophys. Acta 1992, 1000, 312-320. (23) Day, T. A.; Volgelmann, T. C. Physiol. Plant. 1995, 94, 433-440. (24) Trebst, A.; Depka, B. Z. Naturforsch 1990, 45C, 765-771. (25) Nedunchezhian, N.; Kulandaivelu, G. Physiol. Plant. 1991, 81, 558-562. (26) Berry, J.; Bjo¨rkman, O. Annu. Rev. Plant Physiol. 1980, 31, 491543. (27) Martin, B.; Ort, D. R.; Boyer, J. S. Plant Physiol. 1981, 68, 329334. (28) Janssen, L. H. J.; Wams, H. W.; van Hasselt, P. R. J. Plant Physiol. 1992, 139, 549-554. (29) Weis, E. Planta 1981, 151, 33-39. (30) Georgieva, K.; Yordanov, I. J. Plant Physiol. 1994, 144, 754-759.

Received for review December 2, 1997. Revised manuscript received May 28, 1998. Accepted June 3, 1998. ES971039F

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