Photosynthesis Research 2,3: 313-318, 1990. © 1990 Kluwer Academic Publishers. Printed in the Netherlands.

Regular paper

On the nature of the photosynthetic energy storage monitored by photoacoustic spectroscopy Robert Carpentier, Roger M. Leblanc & Murielle Mimeault Centre de Recherche en Photobiophysique, Universit~ de, Quebec ~ Trois-Rivibres, 3351, Boul. des Forges, C.P. 500, Trois-Rivibres (Qukbec), G9A 5H7, Canada Received 10 March 1989; accepted 17 September 1989

Key words: thylakoid membranes, electron transfer, photoacoustic spectroscopy, energy storage, photosynthesis, plastoquinone

Abstract The photosynthetic energy storage yield of uncoupled thylakoid membranes was monitored by photoacoustic spectroscopy at various measuring beam intensities. The energy storage rate as evaluated by the half-saturation measuring beam intensity 05o) was inhibited by 3-(3,4-dichlorophenyl)-1,1 dimethylurea, by heat inactivation or by artificial electron acceptors specific for photosystem I or photosystem II; and was activated by electron donors to photosystem I. The reactions involving both photosystems were all characterized by a similar maximal energy storage yield of 16 + 2 percent. The data could be interpreted if we assumed that the energy storage elicited by the photosystems at 35Hz is detected at the level of the plastoquinone pool.

Abbreviations; PS - photosystem, Tes - N-Tris [hydroxymethl] methyl-2-aminoethanesulfonic acid, D C M U - 3-(3,4-dichlorophenyl)-l,l-dimethylurea, DCIP - 2,6-dichlorophenolindophenol, FeCN - potassium ferricyanide, DCBQ - 2,5-dichlorobenzoquinone, TMPD - N,N,N'-tetramethyl-p-phenilenediamine

Introduction There is an emerging interest for the application of photoacoustic spectroscopy in studies of photosynthetic electron transport. This technique is used to evaluate the conversion efficiency of absorbed modulated light energy into heat. Energy storage during photosynthetic reactions decreases the thermal emission yield. Therefore, the comparison of photochemically active and inactive samples allows the determination of the photosynthetic energy storage yield (Malkin and Cahen 1979, Leblanc et al. 1983). Energy storage mediated by PSII (Carpentier et al. 1984, 1985) and/or PSI (Carpentier et al. 1984, 1986 Lasser-Ross et al. 1980) was reported in intact algal cells (Carpentier et al. 1984, Yamagishi and Katoh 1984, O'Hara et al. 1983, Cannani 1986) as well as in isolated thy-

lakoid membranes (Lasser-Ross 1980, Carpentier et al. 1988) or submembrane fractions enriched in photosystem II (Carpentier et al. 1985). Recently, we have shown that measurements of energy storage as a function of the measuring beam intensity could be analysed similarly to enzyme kinetics (Carpentier et al. 1985, 1988). This treatment provides the determination of the maximal energy storage yield in absence of light saturation and the half-saturation measuring beam intensity which was shown to be proportional to the electron transfer rate (Carpentier et al. 1988). An important parameter that has not been clarified, however, is the nature of the component(s) responsible for the energy storage monitored during photoacoustic experiments. Because of the modulation frequency usually involved during the measurements (30-100Hz), the events monitored

314 are occurring in the millisecond range of time. Therefore, a relevant starting hypothesis would be that the energy storage is detected at the level of intermediates of the electron transport chain. In order to verify this assumption, energy storage by PSI and PS II was studied in native and inhibited thylakoid membranes with the use of various electron acceptors and donors.

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Materials and methods

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Fig. 1. Acousticsignal from thylakoidmembranesaspirated on Thylakoid membranes were isolated from spinach leaves homogenized in 20mM Tes-NaOH pH 7.5, 5 mM MgCI2 and 350mM sorbitol. After filtration through eight layers of cheesecloth, the mixture was centrifuged for 1 min at 2000 x g and resuspended in a ten-fold dilution of the homogenization buffer. The membranes were collected by centrifugation (1 min at 4000 x g) and resuspended at a Chl concentration of 250 #g ml-1 in 50 mM TesNaOH pH7.5, 2mM MgCI2, l m M NH4CI and 330 mM sorbitol. For photoacoustic measurements, 1 ml of the thylakoid membrane preparation was aspirated onto a nitrocellulose filter (Carpentier et al. 1988). The filter was cut into a disk of appropriate dimensions (1.5mm, diameter) and introduced into the cell of a home-made apparatus previously described (Carpentier et al. 1984). The measuring beam (680nm) was modulated in intensity at a frequency of 35 Hz. To monitor the photochemical energy storage yield, the non-modulated white background light of saturating intensity (100 W m-2) from a quartz-halogen lamp (250 W) was directed into the cell through a fiber optic guide.

Results

The photoacoustic signal obtained from thylakoid membranes aspirated onto a nitrocellulose filter is shown in Fig. 1. The signal intensity (Qm) obtained with a continuous strong background illumination which saturates photosynthesis represents the maximal conversion of the absorbed modulated light intensity of the measuring beam into thermal emission. However, in the absence of this side illumination, a weaker signal (Qc) is monitored because

a nitrocellulosefilter, t , measuring beam (2.25Wm-2) on; $, J,, backgroundillumination on and off,respectively.~ was 12.5 percent. some of the absorbed energy is stored in photochemical intermediates during modulated photosynthesis (electron transfer) and therefore not released as heat. The ratio (Qm - Qc/Qm) x 100% thus represents the percentage of energy stored by the photochemical process and will be refered to as the photosynthetic energy storage yield (~b~) (Carpentier et al. 1988). The value of ~b~varies with the modulated measuring beam intensity. This is due to partial saturation of photosynthesis by the measuring beam itself (Carpentier et al. 1988). The reciprocal of tk', versus the measuring beam intensity (I) provides a straight relationship which intercepts the ordinate at the value of ~b~for I = O (~b'r0) according to the following relation (Carpentier et al. 1988):

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This true energy storage yield is then related to the quantum yield of photochemistry (~) and to the ratio of stored over absorbed energy through the following equation (Malkin and Cahen 1979, Leblanc et al. 1983):

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(2)

where N is the Avogadro number, h is the Planck constant, c is the light velocity, 2 is the measuring beam wavelength and AEp is the amount of thermal energy per mole stored by the photochemical reaction. On the other hand, an extrapolation of the relation described by Eq. (1) to the absciss provides the modulated light intensity producing

315 A

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Fig. 2. The reciprocalof ~b~as a functionof the measuringbeam

intensity for thylakoid with and without additives.The values for i50(Wm-2) are indicated in brackets, ~b~0was 16 ___2 percent. A: zx, control thylakoids[6.6]; o, 0.5 mM methylviologen and 0.8raM NaN3 [3.8]; e, 5 × 10-SM DCMU [0.9]; A, 5 x 10-SM DCMU, 0.5raM methylviologenand 0.8raM NaN3[0.9]. B: zx,controlthylakoids[6.3]; e, heat treated sample (20 min, 70°C)[1.0]; *, heat treatedsamplewith0.5 nM, methylviologenand 0.SmM NaN3[1.0]. Experimentsin A and B were carried out with two differentthylakoidpreparations. half-saturation of photosynthesis 050) (Carpentier et al. 1988). Experiments similar to the one reported in Fig. 1 were performed at various measuring beam intensities and the data was treated according to Eq. (1) to obtain ~o and i50 with native and inhibited preparations in the presence of electron donors and acceptors. In these experiments, only the first cycle of actinic beam on and off was used because prolonged exposure to the background light induces photoinhibition of electron transport [11]. About these measurements, we could speculate that an increase of gas pressure following oxygen evolution in the photoacoustic cell could influence the value of the above parameters. However, in the type of preparation used, the participation of oxygen to the pressure wave at the phase angle of the thermal signal probably represents less than 10 percent of the photoacoustic signal (Carpentier et al. 1984, 1985, Cannani 1986).

The reciprocal of ~b~versus the modulated beam intensity is given for thylakoid membranes without additives in Fig. 2A (control). The value found for ~b~o was 16 + 2 percent and was similar in all the experiments reported in this work. On the other hand, i5o in the control samples varied between 5.5 to 7 . 0 W m -2 from one preparation to another depending on their photosynthetic activity. For the preparation used in Fig. 2A, it was evaluated to be 6.6 W m -2. After the addition of the PSI acceptor methylviologen, ~0 remained unaffected. However, the slope of the straight line represented by Eq. (1) increased. The above resulted in a decreased is0 (from 6.6 to 3.8 W m -2) which is explained by the leakage of electrons towards methylviologen, which reduces energy storage in the electron transfer chain (Popovic et al. 1987). The data in Fig. 2A represents typical results obtained from one of three separate experiments. In the presence of D C M U (Fig. 2A), inhibition of the plastoquinone pool reduction resulted in an even lower i50 (0.9Wm-2). This parameter remained at the same value when methylviologen was added to D C M U treated samples. In Fig. 2B, control and methylviologen treated samples are compared with thylakoids inhibited by heat treatment (70°C for 20min). After this treatment, oxygen evolution monitored by a Clark electrode was fully inhibited, but oxygen uptake by photosystem I with DCIP as donor and methylviologen as acceptor was still detected at a rate of 225 #mol 02 (mg Chl h)- t. In the preparation used in Fig. 2B, the addition of methylviologen to the control samples caused a drop in is0 (from 6.3 to 4 . 0 W m -2) similar to the experiment reported in Fig. 2A (not shown). As for DCMU, heat treatment also produced a large decline in i50 (to 1 W m -2) which could not be affected by further addition of methylviologen. We must note that even when oxygen evolution was fully inhibited by 5 x 10-SM D C M U or by heat treatment, a non negligible energy storage remained (Fig. 2). Because no effect was produced upon the onset of the background light when using non-photoactive samples (i.e. carbon black) under the same illumination conditions (results not shown), we believe this remaining energy storage to originate from photochemical activity whose nature will be discussed later on. In Fig. 2, it is shown that the PSI electron

316 A

strated by the occurrence of an increased is0 when using these donors. This energy storage is further confirmed by the effect of methlviologen. It was shown in Fig. 2 that methylviologen has no measurable effect in D C M U or heat-treated samples. In Fig. 4, methylviologen induced a decrease of is0 in such samples where DCIP or TMPD were added as donor.

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z (win "2) Fig. 3. The effect of electron acceptors on the reciprocal of~b; as a function of the measuring beam intensity. The values for is0 (Wm -2) are indicated in brackets, q~0 was 16 _ 2 percent. A: o, control thylakoids [5.5]; /,, 5mM FeCN [2.3]; - , 5mM FeCN and 5 x 10-SM DCMU [1.0]. B: o, control thylakoids [7.0]; O, 0.63raM DCBQ [2.8]; I , 0.6mM DCBQ and 5 × 10-SM DCMU [0.9].

acceptor methylviologen decreases the energy storage yield at partially saturating light intensities when PS II is active. A similar effect is shown in Fig. 3A for the acceptor FeCN, which can accept electrons from both PS I and PS II in this type of material (Izawa et al. 1980), and in Fig. 3B for the PSII acceptor DCBQ. Both acceptors caused nearly a 60 percent decrease in is0 at the concentration usually saturating for electron transfer measurements. The addition of the inhibitor D C M U to the thylakoid membranes in the presence of acceptors produced a much stronger decrease in is0 than with the acceptors alone. It is clear, from the results presented in Figs. 2 and 3, that electron acceptors from both PS II and PSI can affect the value of is0 when water is used as electron donor. In Fig. 4, we show an experiment more specific for PS I where DCIP (Fig. 4A and B) and TMPD (Fig. 4C and D) are used as donors in samples previously inhibited by D C M U or heat treatment. Energy storage by PSI alone is thus demon-

From the data presented above, it is clear that artificial electron acceptors affect energy storage in thylakoid membranes in a similar way as do inhibitors. Both PS II and PSI acceptors could decrease the half-saturation light intensity for energy storage, is0. The latter was shown to be proportional to the electron transfer rate measured by oxygen evolution (Carpentier et al. 1988). Since oxygen evolution is stimulated by the addition of acceptors although energy storage is inhibited, it should be mentioned that is0 is proportional to the electon transfer rate only when it is monitored in the absence of acceptors. The value of is0 therefore represents the rate of energy storage in the form of a reduced intermediate of the electron transport chain, the reduction of which is prevented to some extent in the presence of acceptors. In a previous study, we have found that a PS II submembrane fraction could retain large energy storage ability (Carpentier et al. 1988). Because the last electron transfer intermediate present in these fractions is plastoquinone (Lavorel et al. 1984), and also because the events monitored by photoacoustic spectroscopy at the frequencies commonly used (30-100Hz) occur in a time period in the millisecond range (Carpentier et al. 1985), we have hypothetized that energy storage could, in fact, represent the reduction of plastoquinone molecules. The present data is largely confirmatory to this hypothesis if we give the following interpretations to the energy storage measured in uncoupled thylakoid membranes: (i) DCMU and heat treatment reduce the rate of energy storage 050) by preventing plastoquinone reduction; (ii) when present, acceptors can partially prevent the reduction of plastoquinone molecule or reoxidize them as they are reduced;

317

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Fig. 4 The effect of electron donors specific for PSI on the reciprocal of ~ as a function of the measuring beam intensity. The values for i50 (Wm -2) are given in brackets, ~b;0was 16 + 2 percent. A, samples inhibited with 5 x 10-SM DCMU: e, no additive [0.9]; 0, 0.8 mM DCIP and 3 mM ascorbate [1.5]; A, 0.8 mM DCIP, 3 mM ascorbate, 0.5 mM methylviologen and 0.8 mM NaN 3 [1.2]. B, heat treated samples (20 min, 70°C); e, no additive [0.8]; o, 0.8 mM DCIP and 3 mM ascorbate [1.5]; A, 0.8 mM DCIP, 3 mM ascorbate, 0.5mM methylviologen and 0.8mM NaN 3 [1.2]. C, samples inhibited with 5 x 10-SM DCMU: e, no additive [0.9]: o, 0.5mM TMPD and 3 mM ascorbate [1.8]; A, 0.5 mM methylviologen and 0.8 mM NaN3 [1.3]. D, heat treated samples: e, no additive [0.8]; o, 0.5 mM TMPD and 3 mM ascorbate [1.8]; A, 0.8 mM DCIP, 0.5 mM ascorbate, 0.5 mM methylviologen and 0.8 mM NaN 3 [1.3].

(iii)storage by photosystem I with DCIP or TMPD as donor in the absence of acceptors is due to reduction of plastoquinone through cyclic electron transfer. This interpretation could also explain the energy storage in PSI of cyanobacterial heterocysts where NADH and NADPH were used as donors (Carpentier et al. 1986); (iv)a common energy storage site in PSI and in PS II (i.e. the plastoquinone pool) results in the similar value of (~0 (16 percent) obtained in both reactions. If we suppose, as usually accepted, that the quantum yield for charge separation in both photosystems is nearly one, ~b~0will vary with AEp [see Eq. (2) above]. A similar ~b;0 for PS II and PSI therefore implies that the energy is stored with a similar value of the ratio AEp/(NhC/2) by both photosystems. Finally, another point of discussion to be raised is the incomplete inhibition of energy storage after treatements (5 x 10 5MDCMU, heat treatment) which fully inhibit oxygen evolution. The lack of effect of methylviologen on this remaining energy storage activity when added to DCMU or heattreated thylakoids (Fig. 2) indicates that this activity is not due to cyclic PS I as previously proposed

(Carpentier et al. 1984). In fact, methylviologen would be able to partly exaust the electrons implicated in this cycle, as is the case when DCIP or TMPD are used as donors (Fig. 4). Another possibility could be that the energy is stored in primary acceptors of PSI and/or PS II which are still connected to active reaction centers. Energy storage in pheophytine as been reported in PS II core complexes (Fragata et al. 1987), and storage in primary acceptors of both photosystems was detected by time-resolved photoacoustic measurements (Nitsch et al. 1988). Further investigations will be necessary to clarify the nature of this energy storage which also occurs with a ~b~0of 16 percent.

References 1. Cannani 0 (1986) Photoacoustic detection of oxygen evolution and state 1-state 2 transitions in cyanobacteria. Biochem Biophys Acta 852:74-80 2. Carpentier R, Larue B and Leblanc RM (1984) Photoacoustic spectroscopy of Anacystis nidulans III. Detection of photosynthetic activities. Arch Biochem Biophys 228: 534-543 3. Carpentier R, Nakatani HY and Leblanc RM (1985) Pho-

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5.

6.

7.

8. 9.

10.

toacoustic detection of energy conversion in a photosystem II submembrane preparation from spinach. Biochim Biophys Acta 808:470-473 Carpentier R, Matthijs HCP, Leblanc RM and Hind G (1986) Monitoring energy conversion in photosystem I of cyanobacterial heterocysts by photoacoustic spectroscopy. Can J Phys 64:1136-1138 Carpentier R, Leblanc RM and Mimeault M (1987) Photoinhibition and chlorophyll photobleaching in immobilized thylakoid membrane. Enzyme Microb Technol 9:489-493 Carpentier R, Leblanc RM and Mimeault M (1988) Monitoring electron transfer by photoacoustic spectroscopy in native and immobilized thylakoid membranes. Biotechnol Bioeng 32:64-67 Fragata M, Popovic R, Camm EL and Leblanc RM (1987) Pheophytin-mediated energy storage of photosystem II particles detected by photoacoustic spectroscopy. Photosynth Res 14:71-80 Izawa S (1980) Acceptors and donors for chloroplast electron transport. Methods Enzymol 69:413-434 Lasser-Ross N, Malkin S and Cahen D (1980) Photoacoustic detection of photosynthetic activities in isolated broken chloroplasts. Biochim Biophys Acta 593:330-341 Lavorel J, Seibert M, Maison-Peteri B and Briantais J-M (1984) Evaluation of the plastoquinone pool size in

11.

12.

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oxygen-evolvingphotosystem II (OES) preparations. Adv Photosyn Res 1:199-202 Leblanc RM, Carpentier R and Larue B (1983) Action spectra for thermal deactivation and photosynthesis in Anacystis nidulans. In: C Sybesma (ed) Advances in Photosynthesis Research, pp 681-684. Dordrecht: Martinus Nijhoff Malkin S and Cahen D (1979) Photoacoustic spectroscopy and radiant energy conversion: theory of the effect with special emphasis on photosynthesis. Photochem Photobiol 29:803-813 Nitsch C, Braslavsky SE and Schatz GH (1988) Laserinduced optoacoustic calorimetry of primary processes in isolated photosystem I and photosystem II particles. Biochim Biophys Acta 934:210-212 O'Hara EP, Tom RD and Moore TA (1983) Determination of the in vivo absorption and photosynthetic properties of the lichen Acarospora schleicheri using photoacoustic spectroscopy. Photochem Photobiol 38:709-715 Popovic R, Beauregard M and Leblanc RM (1987) Study of energy storage processes in bundle sheath cells of Zea mays. Plant Physiol 84:1437-1441 Yamagishi A and Katoh S (1984) Photoacoustic measurements of the energy-conversion efficiency of photosynthesis in thalli of the green alga Bryopsis maxima. Biochim Biophys Acta 766:215-221

On the nature of the photosynthetic energy storage monitored by photoacoustic spectroscopy.

The photosynthetic energy storage yield of uncoupled thylakoid membranes was monitored by photoacoustic spectroscopy at various measuring beam intensi...
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