Photosynthesis Research 25: 259-268, 1990. © 1990 Kluwer Academic Publishers. Printed in the Netherlands. Regular paper

Correlation between chlorophyll fluorescence and photoacoustic signal transients in spinach leaves Jan F.H. Snel, Martin Kooijman & Wim J. Vredenberg

Department of Plant Physiological Research, Agricultural University, Gen. Foulkesweg 72, 6703 B W Wageningen, The Netherlands Received 23 October i989; accepted in revised form 15 May 1990

Key words: fluorescence induction, heat emission, oxygen evolution, photoacoustics, quenching analysis, photosynthetic energy storage Abstract

Chlorophyll fluorescence and photoacoustic transients from dark adapted spinach leaves were measured and analyzed using the saturating pulse technique. Except for the first 30 s of photosynthetic induction, a good correlation was found between photoacoustically detected oxygen evolution at 35 Hz modulation frequency and electron flow calculated from the fluorescence quenching coefficients qe and qN. The induction kinetics of the photothermal signal, i.e., the photoacoustic signal at 370 Hz, reveal a fast (t r < 10 ms) and a slow (t,-~ 1 s) rise component. The fast component is suggested to be composed of the minimal thermal losses in photosynthesis and thermal losses from non-photosynthetic processes. The slow phase is attributed to variable thermal losses in photosynthesis. The variable thermal losses were normalized by measuring the minimal photothermal signal (H0) in the dark-adapted state and the maximal photothermal signal ( H a ) during a saturating light pulse. The kinetics of the normalized photochemical loss ( H - H 0 ) / ( H m - H 0 ) obtained from high-frequency PA measurements were found to correlate with the kinetics of oxygen evolution measured at low frequency.

Abbreviations: F m - m a x i m u m fluorescence; F 0 -initial fluorescence; F v -variable fluorescence; H photothermal signal; I - in-phase; LED - light emitting diode; PA - photoacoustic; PL - photochemical loss; Q - quadrature; qN -- non-photochemical quenching; qp - photochemical quenching; VCLS - voltage controlled light source

Introduction

In plant photosynthesis the primary reactions take place in the photosystems 1 and 2, where excitation energy is transferred from excited chlorophyll pigments to the reaction center. Here the energy is used to drive vectorial electron flow. These processes are accompanied by radiative (mainly chlorophyll a fluorescence associated with PS 2) and nonradiative (thermal) losses, which can be measured by chlorophyll fluorescence and photoacoustic spectroscopy, respec-

tively. Chlorophyll fluorescence originates mainly from chlorophyll a associated with photosystem 2. Two major types of fluorescence quenching are photochemical quenching related to deexcitation by photochemistry in photosystem 2, and non-photochemical quenching which has been suggested to result from enhanced nonradiative dissipation (Krause and Weiss 1984, Briantais et al. 1986). Schreiber et al. (1986) were able to measure photochemical (qp) and non-photochemical (qN) quenching in intact leaves with a pulse-amplitude-modulated fluoro-

260 meter by removing photochemical quenching with a short saturating light pulse (saturating pulse method). A good correlation between electron flow estimated from steady state carbon dioxide uptake and electron flow calculated on basis of qp and/or qN in steady state has been demonstrated (e.g., Schreiber and Bilger 1987, Weiss and Berry 1987). Photoacoustic (PA) spectroscopy has been successfully applied in the field of photosynthesis research (Buschmann et al. 1984, Braslavsky 1986). In addition to pressure changes associated with photothermal phenomena, pressure changes caused by the production of molecular oxygen during photosynthesis (photobaric component) have been observed in plants (Bults et al. 1982). Due to different mechanisms for the generation and propagation of the photobaric and photothermal signals, these components have a different delay (phase angle) with respect to the modulated light. The photobaric component can be determined by subtracting the photothermal contribution from the total signal. This photothermal component can be estimated from measurements at a high modulation frequency where the photobaric component is damped out and where only the photothermal component is left (Bults et al. 1982). Both the separation of photochemical and non-photochemical quenching of chlorophyll flu-

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orescence and the separation of the photothermal and the photobaric components of the PA signal require saturating light. The short, usually 500 ms, light pulse introduced with the saturating pulse method (Schreiber et al. 1986) has the advantage that (1) only photochemical quenching is removed and (2) the pulse can be given repetitively with a repetition rate of up to about 0.3 Hz. This allows the study of the kinetics of fluorescence quenching components, heat production and oxygen evolution (Bicanic et al. 1989, Buschmann and Kocsfinyi 1989, Snel et al. 1990). In this communication we compare the kinetics of electron flow derived from chlorophyll fluorescence and the kinetics of the photobaric and thermal transients measured using the saturating pulse method.

Materials and methods

Spinach plants (Spinacia oleracea cv. Amsterdams breedblad) were grown as described before (Snel 1985). The experimental setup is schematically depicted in Fig. 1 and consists of the following components: a photoacoustic cell plus 2-phase lock-in amplifier, a voltage controlled lightsource (VCLS) plus function generator, a pulsed chlorophyll fluorometer and a four-armed fiber-optic

L F

PAcell

103

PAM

101

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Fig. I. Schematic representation of the experimental setup. Components: }'AM 101: pulse-amplitude-modulation fluorometcr (basic unit); 101 ED: emitter~detector unit; 101F: 4-armed flexiblelightguide;FL 103: high-intensityhalogen |ightsourcc; P A M 103: timer module for FL 103; P M 5127: function generator; VCLS: voltage controlled lightsourcc;M: microphone; L: leaf;LA: 2-phase lock-in amplifier; R E C I. 2: digitaloscilloscope or pen-recorder.

261 lightguide for the fluorescence excitation pulse, for the fluorescence signal and for the saturating light pulse. The output signals of the lock-in amplifier and the PAM fluorometer were recorded on a Nicolet 2090 digital storage oscilloscope in the dual channel mode and plotted on a BBC XY-recorder resp. recorded on a penrecorder. A Tektronix 2601 Ramp/Pulse Generator delivered the gating voltage for the VCLS and the triggering pulses for the PAM 103 module and the Nicolet 2090 digital storage oscilloscope. Chlorophyll a fluorescence yield was measured using a PAM fluorometer (Heinz Walz, Messund Regeltechnik, Effeltrich, FRG) at 100 kHz modulation frequency. The configuration consisted of the basic module PAM-101, the emitter/ detector unit ED-101, the high intensity lightsource FL-103 with its control module PAM-103, and the optical fiber assembly 101-F. The fluorometer and its measuring principles have been described (Schreiber 1986, Schreiber et al. 1986). Sinusoidally modulated actinic light for the measurement of the PA signal was generated by the VCLS, of which a preliminary version has been described (Snel 1988). This version uses twelve high intensity LEDs (Stanley Electric Co. Ltd, type H-3k), driven by a single transconductance amplifier and coupled to a twelve-armed optical fiber (Schott, custom made). The emitted red light is centered around 660 nm with a spectral half-bandwidth of approx. 25nm. The sinusoidal input voltage for the VCLS was provided by a function generator (Philips, type PM5127). The light intensity of the VCLS was measured at the leaf surface using a United Detector Technology 80X Optometer. The signal from the integrated preamplifier in the microphone of the photoacoustic cell (Yeda Inc., Rehovot, Israel) was analysed by a lock-in amplifier ( E G & G / P A R model 5204). The quadrature channel ( Q ) of the lock-in amplifier was nulled during illumination with non-modulated saturating light by phase adjustment before dark adaptation. As the photobaric component is suppressed during illumination with non-modulated saturating light (Bults et al. 1982), the remaining PA-signal is only of photothermal origin. By adjusting the phase of the lock-in amplifier to the

phase of the photothermal signal during the saturating pulse, the Q-channel contains no photo-thermal component. The total photobaric signal J0, which is a measure of the rate of oxygen evolution, was calculated from the inphase signal (I) and the Q-signal by vector addition of the two signals (Poulet et al. 1989): Jo = ((I - / ~ ( 1 - PL)) 2 + Q2)O.5 with I, Q being the amplitude of the I respectively Q channel at the start of the saturating light pulse, I s is the amplitude inphase signal during the saturating pulse, and PL is the photochemical loss (see below). The photochemical loss is defined as the fraction of the maximal photothermal signal being stored in photosynthesis (e.g., Bults et al. 1982). The photochemical loss PL can be determined in a separate experiment at high modulation frequency by measuring the photothermal signal in the absence (H) and in the presence (Hm) of saturating non-modulated light: PL = (H m - H) / (Hm). The leaf was mounted in the PA-cell with its upper side facing the VCLS and its lower side directed towards the microphone and the 101-F fiberoptics for the fluorescence measurements. Only in the experiments shown in Figs. 2 and 3, the modulated light of the VCLS was transmitted to the sample via the remaining arm of the 101-F. All experiments were carried out on detached leaves at room temperature.

Results

Application of the saturating light pulse method to the photoacoustic technique requires that the PA signal can be detected with sufficient time resolution and signal-to-noise ratio. Figure 2 shows the effect of a 5 s pulse of saturating white light on the kinetics of chlorophyll fluorescence and the photobaric signal at 35 Hz of a spinach leaf in the steady state. The fluorescence response reaches a maximum after approx. 1 s and then declines until the end of the pulse. After the pulse we observe a fast decline followed by a slower decay to below the steady state, resulting in an undershoot and a slow relaxation to the

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Fig. 2. Chlorophyll fluorescence (A) and photoacoustic Q-channel (B) signal transients from a spinach leave upon application of a 5 s pulse of non-modulated saturating white background light to a spinach leaf adapted to the modulated actinic light. The photothermal component was suppressed as described in the methods. In this experiment the light output of the VCLS was connected to the fourth arm of the 101-F optical fiber. Average actinic light intensity: 6 W m 2; modulation frequency: 35 I4_z; time constant of the lock-in amplifier: 300 ms. The registrations are the average of 5 measurements on the same leaf with a repetition rate of 0.01 Hz.

steady state level. The Q-signal, which reflects modulated oxygen evolution, is suppressed within less than 1 s and recovers after the pulse with at least biphasic kinetics. At the light-dark transition a switching transient is often observed. This artefact, caused by the large change in PA signal due to the high intensity of the nonmodulated saturating white light was disregarded in further calculations. The PA signal contains only small non-photothermal contributions at high modulation frequency (Poulet et al. 1983). Figure 3 shows that under conditions that were similar to those in Fig. 2 except for the 20 s saturating light pulse, the PA signal at 470Hz rises with biphasic kinetics. The fast phase, which is approx. 85% of the maximum amplitude in this experiment, is accomplished within less than 1 s after application of saturating white light; the slower phase is not always observed. The data in Figs. 2 and 3 demonstrate that the photobaric signal can be suppressed and that the maximal photothermal signal can be determined during the saturating pulse. This means that the saturating pulse method can be applied to the PA technique with 1 s pulses at an average light intensity of 6 W m 2 (modulated light), albeit with a relatively poor signal-to-noise ratio. The signal-to-noise ratio of the PA-signal was improved by illuminating the leaf directly from

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the back side of the cell as shown in Fig. 1. This configuration eliminates the considerable optical losses in the fiberoptics resulting in a higher intensity (30 W m-2) of the modulated PA light. Figure 4 shows registrations of chlorophyl fluorescence (A) and photoacoustic signals from a dark adapted spinach leaf during illumination with modulated light at frequencies of 35 Hz (B, C) and 370Hz (D, E). The signals in Fig. 4A, B and C were recorded simultaneously, while the signals in D and E were recorded in separate experiments with the same leaf after another 45 min dark adaptation. The Q-channel (Fig. 4C) contains only a photobaric component while the/-channel (Fig. 4B) contains a mixture of the photothermal and the photobaric component. Figure 4C therefore depicts the kinetics of modulated photosynthetic oxygen production with a time resolution of 200 ms, which is much

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Fig. 4. Chlorophyll fluorescence yield (A) and photoacoustic signal transients at 35 Hz (B, C) resp. 370 Hz (D, E) from a detached, dark adapted spinach leaf. Before dark-adaptation the Q-channel was adjusted to zero duing the saturating pulse by adjustment of the phase of the lock-in amplifier (C) while in D and E the phase was adjusted to maximal signal amplitude. The photoacoustic induction transient at 370 Hz (E) was measured after a dark adaption of 45 min. This registration is the average of 8 measurements on the same leaf with a repetition rate of 0.08 Hz. Saturating light pulses of 800 ms duration were given every 6 s, starting at the beginning of illumination with modulated PA-light (A..D). Average intensity of modulated light: 30 W m -2. Timeconstants of the lock-in amplifier were 200 ms (B, C, D) and 10 ms (E).

faster than a conventional Clark-type of oxygen electrode.

equation: JE = Iqp(@po(1 - qN) + @PEqN)

Correlation between oxygen evolution and fluorescence quenching Weis and Berry (1987) have shown a linear relation between the quantum yield of electron flow in open PSII reaction centers (@p) and non-photochemical quenching during steady state photosynthesis. They proposed two quantum yield parameters (I)p0 and (~)PE which are the quantum yield of electron flow in open PS II reaction centers in the non-energized resp. energized state. These two parameters could be determined by extrapolation of (I)p to qr~ = 0 and qN = 1, respectively (Weis and Berry 1987). The rate of electron flow (JE) was then calculated from chlorophyll fluorescence using the following

(1)

with I being the light intensity (/zmole m -2 s -~) At low oxygen concentrations JE has been shown to be correlated with steady state electron flow calculated from gas exchange measurements (Weis and Berry 1987). Here we investigate this relation during induction. The kinetics of qp and qN (Fig. 5A), the total oxygen signal (Fig. 5B) and the photochemical loss (Fig. 5C) were calculated from the data in Fig. 4. Figure 6 shows the relation between the rate of oxygen evolution (J0) and the rate of electron flow (JE)" JE was calculated from the data in Fig. 5A using Eq. (1) with qbp0 = 1 and ~PE = 0 because these values have shown to give a good approximation after the first 30 s of induction in spinach leaves at the

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conditions used here (Snel et al. 1990). The relation between J0 and JE seems to be linear after the first 30 s of induction. The reason for the deviation in the first 30 s is not known; one complicating factor could be oxygen uptake by 50

photoreduction of oxygen (e.g., Robinson 1988). Oxygen uptake might cause a lowering of modulated oxygen evolution (Malkin 1987) which is not reflected in a decrease of photochemical fluorescence quenching as photoreduction of oxygen is driven by linear electron flow and therefore associated with photochemical quenching of chlorophyll fluorescence.

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Fig. 6. Kinetic relation between the rate of oxygen evolution (Jo) and the rate of the electron flow (JE) as estimated from qv and qN" The data for each point were taken from Fig. 4C (Jo) or calculated from Fig. 4A using Eq. (1) (JE), with consecutive data points being connected. The first point, which corresponds to the second saturating light pulse, is marked with a 'S'.

Heat is emitted in leaves during both photosynthetic and non-photosynthetic processes. Assuming that the yield of heat emission from excited non-photosynthetic pigments is not affected by light, the variable part will be solely the result of heat emission in photosynthesis. We have measured the induction of heat emission from the same dark-adapted spinach leaf disc by applying a 1 s pulse train of modulated (370 Hz) actinic light to the dark-adapted leaf every 120 s. Figure 4E shows the average of 8 registrations. This repetitive treatment hardly affected the

265 dark-level of chlorophyll fluorescence (not shown). The induction of the photothermal signal in Fig. 4E shows biphasic rise kinetics. The amplitude of the fast component (t r < 10 ms, instrument limited) is suggested to reflect the minimal heat emission from the leaf with the photosynthetic apparatus is in its most efficient state. The slow (t r ~ 1 s) component is attributed to closure of the photosystems as on this timescale there is no significant formation of qN" The normalized photochemical loss (i.e., photochemical energy storage) PL n at time t is then defined as follows: PE n = (H m -- H ) / ( H m -- Ho)

(2)

with H the actual heat emission at time t, H 0 the minimal heat emission from the dark-adapted leaf, and H m the maximal heat emission during the saturating light pulse. The advantage of this normalization is that the normalized photochemical loss gives the amount of energy stored in photosynthesis relative to the maximum amount as determined in the darkadapted state. This is illustrated in Fig. 7, where the timecourse of oxygen evolution correlates with the timecourse of the normalized photochemical loss PLn, although the relation appears to be nonlinear under the experimental conditions.

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Fig, 7. Relationship between the rate of oxygen evolution (J0) and the normalized photochemical loss (PL.). Each point corresponds to data taken from Fig. 4C (J0) or calculated from the data in Fig. 3 using Eq. (2) (PLn). Consecutive data points are connected by a line. The first point, which corresponds to the second saturating light pulse, is marked with a 'S'.

Discussion

The results show that simultaneous measurement of PA and chlorophyll a fluorescence transients in combination with the use of the saturating pulse technique provides (the kinetics of) a number of useful parameters related to energy conversion in photosynthesis: the chlorophyll fluorescence quenching parameters qe and qN, overall thermal loss, normalized photochemical loss and the relative rate of oxygen evolution. The saturating pulse method offers the additional advantage that the maximal photothermal signal is monitored during the pulse. Any change in the phase of the photothermal signal will be detected and can be accounted for in the calculation of the oxygen signal.

Correlation between fluorescence quenching and the photothermal signal The observation that the maximal photothermal signal, i.e., the amplitude of the PA signal at 370 Hz during the saturating pulses (Fig. 4D), does not increase during the induction phase while qN (Fig. 5A) does, demonstrates that a change of qN is not reflected in a significant change of overall heat production in the leaf (Bicanic et al. 1989, Buschmann and Kocs~inyi 1989). This suggests that the yield of modulated heat production by PS 2 during the saturating pulse is not significantly increased by qN- This is in agreement with the finding that the rate constant for nonradiative dissipation is much higher than the rate constant for fluorescence (e.g., Kitajima and Butler 1975). During application of the 20s saturating light pulse (Fig. 3) we observed a large decrease in fluorescence and after the pulse a fluorescence decrease to below the F0-1evel (data not shown). These observations suggest the existence of an extremely energized state during a prolonged saturating pulse. The small, slow increase of the photothermal signal during the 20 s saturating pulse (Fig. 3) might indicate that during extreme energization a small increase in the yield of heat emission occurs. This increase is unlikely to be solely attributable to PS II as PT00÷ is known to convert nearly all excitation energy in P S I into heat and as it has been shown that PT00 can be oxidized during

266 strong illumination (Weis et al. 1987, Schreiber et al. 1988, Weis and Lechtenberg 1988). A contribution of P S I to the photoacoustic signal has already been observed (Lasser-Ross et al. 1980, Kanstad et al. 1983, Buschmann and Kocsfinyi 1989). Therefore a PS I contribution to the photothermal signal during the saturating pulse in Fig. 3 is probable. After the pulse both the photothermal signal in Fig. 3 and the oxygen signal in Fig. 2B show biphastic relaxation kinetics. A similar slow phase is observed in the relaxation of chlorophyll fluorescence (Fig. 2A). The slow increase of both the chlorophyll fluorescence and the oxygen signal indicates that the yield of photochemistry in PS II is related to non-photochemical quenching of chlorophyll fluorescence at a timescale of a few seconds.

Correlation between oxygen evolution and fluorescence quenching parameters The signal in the Q-channel, which was adjusted to contain only a photobaric contribution, shows that the rate of oxygen evolution is zero or even negative during part of the induction transient (Fig. 4C). This is probably caused by a slight change of the phase of the photothermal component during the 45 rain dark adaptation. A negative amplitude is however not observed in the total oxygen signal (Fig. 5B). This is due to the contribution of the signal in the/-channel, which still contains a significant part of the photobaric component. The finding that Eq. (1) gives a qualitatively good description of the rate of oxygen evolution during a major part of photosynthetic induction (Fig. 6) is an important extension of the usefulness of this equation which was only validated for the steady state (Weis and Berry 1987). The values for the quantum yield parameters ~P0 = 1 and ~Pe = 0 (Snel et al. 1990) used in Fig. 6 are different from the values reported before (Weis and Berry 1987). This is because the parameters used here do not have the same meaning as the parameters introduced by Weis and Berry (1987). The first difference is that our parameters are relative since the photoacoustic technique does not yet provide a method for calibrating the rate of oxygen evolution. The second difference concerns the definition of ~P0, which

in our definition is the quantum yield of oxygen evolution in dark-adapted open PS II reaction centers. This definition is not necessarily identical to the quantum yield of electron flow in non-energized open PS II reaction centers during steady state photosynthesis as defined by Weis and Berry (1987). Extrapolation of the data in Fig. 6 to J0 = 0 indicates a non-zero value for JE" This might indicate underestimation of J0 due to photoreduction of oxygen (Robinson 1988) and/or overestimation of Jz" Photoreduction of oxygen might contain a modulated component (Malkin 1987, Havaux et al. 1988) resulting in a 'negative' photoacoustic oxygen signal. Photoreduction of oxygen has been shown to be associated with photochemical and non-photochemical quenching (Neubauer and Schreiber 1989). The overestimation of JE might be caused by the fact that the leaf is illuminated with modulated PA light from the backside, resulting in a more far-red enriched illumination at the side of the leaf which is probed by chlorophyll fluorescence. This could lead to a more oxidized PS 2 (higher qp) resulting in a higher value of JE at the surface facing the fluorescence detector. The photoacoustic signal emanates from layers adjacent to the surface of the leaf or adjacent to airspaces within the leaf (Bults et al. 1982). This means that the photoacoustic signal may result from cell layers experiencing a different light intensity/quality than the cell layers from which fluorescence is detected. This might also explain why the correlation in the first 30 s is poor: in the first 30s qp is the main factor determining the rate of electron flow JE (Fig. 5A). If the kinetics of photochemical quenching are not identical throughout the leaf, the correlation between the rate of oxygen evolution and the rate of electon flow calculated from Eq. (1) will break down.

Correlation between oxygen signal and photochemical loss Figure 4 shows that the photothermal signal at the end of the induction (Fig. 4D) is only slightly higher than the minimal photothermal signal in Fig. 4E. This indicates that steady state photosynthesis proceeds almost as efficient as in the

267 dark adapted state under non-energized conditions. This may be due to the relatively low actinic light intensity used in our experiments. The photochemical loss reflects the amount of energy stored in photosynthesis relative to the amount of energy lost as heat in the absence of photosynthesis (Malkin and Cahen 1979). In contrast, the normalized photochemical loss gives a measure of the energy stored in photosynthesis relative to the maximal amount of energy that can be stored in photosynthesis. Extrapolation of the data in Fig. 6 indicates that the photochemical loss is not zero in the absence of net oxygen evolution. This observation could be explained by (1) underestimation of the rate of electron flow as a result of modulated oxygen uptake (Malkin 1987) or (2) overestimation of the photochemical loss as a result of a PSI contribution. Linear electron flow in PSI and in PS II are not necessarily balanced during induction and therefore the PL of PSI may not correlate with oxygen evolution in PS II. Moreover cyclic electron flow in PSI could contribute to the overall PL (Lasser-Ross et al. 1980, Canaani et al. 1989). The accuracy of the calculation of the photothermal and the photobaric component (i.e., the rate of oxygen evolution) is limited by the fact that the oxygen signal and the photochemical loss had to be determined in two separate measurements. True simultaneous measurement at high and low frequency has been achieved using a second lock-in amplifier and modulating the light simultaneously at two frequencies (Snel and Geel, unpublished data).

Acknowledgements The authors wish to thank Drs Dane Bicanic (Dept. of Meteorology & Physics) and Ora Canaani (Dept. of Biochemistry, Weizmann Institute, Israel) for their kind advice and assistance with regard to the photoacoustic technique and Dr Olaf van Kooten for stimulating discussions and for reading the manuscript. We gratefully acknowledge the contribution of the 2phase lock-in amplifier by the Dept. of Molecular Physics.

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Correlation between chlorophyll fluorescence and photoacoustic signal transients in spinach leaves.

Chlorophyll fluorescence and photoacoustic transients from dark adapted spinach leaves were measured and analyzed using the saturating pulse technique...
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