Low concentrations of tetracycline enhance the photophosphorylation and P/O ratio of chloroplasts

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1 Photosynthesis Research 79: , Kluwer Academic Publishers. Printed in the Netherlands. 201 Regular paper Low concentrations of tetracycline enhance the photophosphorylation and P/O ratio of chloroplasts Hui Dong & Jia-Mian Wei Shanghai Institute of Plant Physiology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, 300 Fenglin Road, Shanghai , China; Author for correspondence ( fax: ) Received 15 August 2003; accepted in revised form 17 November 2003 Key words: CF 1, chloroplast, electron transport, photophosphorylation, P/O ratio Abstract This study investigated the effects of tetracycline on photophosphorylation, electron transport and P/O ratio of spinach chloroplasts. When chloroplast preparations were treated with low concentrations of tetracycline, non-cyclic and cyclic photophosphorylation activities increased, electron transport rates and P/O ratios improved, chloroplast ms-dle also improved, and the Mg 2+ -ATPase activity of CF 1 increased in comparison to the control. These results indicate that spinach chloroplasts are sensitive to tetracycline. Next, we used the fluorescence emission spectra of CF 1 to examine the possible binding sites for tetracycline. The fluorescence emission spectra of CF 1 treated with glutaraldehyde, NEM and TNBS, which interact with CF 1 across its whole structure, at the γ subunit and at the β subunit, respectively, were compared with that of control CF 1. The peak sites of the various fluorescence emission spectra were the same, but the peak values for CF 1 treated with glutaraldehyde, NEM and TNBS were lower than that of control CF 1. The peak value of CF 1 treated with 50 µm tetracycline was very similar to that of CF 1 treated with NEM. The above results indicate that the acting site of tetracycline may be at or near the γ subunit of CF 1,and allows the creation of a model in which tetracycline binding strengthens the subunit interactions of ATP synthase, enlarges the proton motive force across the thylakoid membrane, and allows the excess proton motive force to increase ATP formation and improve the P/O ratio. Abbreviations: CF 1 coupling factor one; FeCY potassium ferricyanide; ms-dle millisecond delayed light emission; MV methylviologen; NEM N-ethylmalcimide; PMS phenazine methosulfate; TNBS 2,4,6- Trinitrobenzene Introduction The F-type ATP synthase is found in mitochondria, bacteria and chloroplasts, and is capable of catalyzing ATP formation from ADP and P i through oxidative phosphorylation or photophosphorylation. Chloroplast ATP synthase is composed of an integral membrane component called CF o, which consists of subunits I, II, III and IV and is responsible for mediating proton transport and providing specific sites for the attachment of CF 1. Membrane-bound CF 1 catalyzes both ATP synthesis and ATP hydrolysis in reactions that are coupled to proton translocation through the CF o.cf 1 contains five different subunits in the stoichiometric ratio of α 3 β 3 γδε. Extensive research has indicated that the catalytic site is located at the β subunit, or at the interface between the α and β subunits. The α subunit may play a role in the catalysis, regulation, or binding of CF 1 to CF o.theγ subunit regulates the catalytic activity of the ATPase and couples proton transport to this reaction. The δ subunit is required for photophosphorylation to achieve its maximal rate, and is capable of blocking the proton channel that extends through CF o.theε subunit is a potent inhibitor of the ATPase in both its soluble and bound forms and is also necessary for proton gate (McCarty and

2 202 Nalin 1986; Steinemann et al. 1994). The γ and ε subunits all rotate around its long axis inside the center of cavity formed by α 3 β 3 relatively (Aggeler et al. 1997; Schulenberg et al. 1997), inducing a conformational change of CF 1 that can lead to the synthesis or hydrolysis of ATP (Boyer 1993). A previous study in our laboratory showed that many reagents, such as aureomycin, polymyxin, polybasic acid and the cytokinin-like substance 6-benzylaminopurine could induce the conformational change of CF 1, resulting in increased photophosphorylation activity and a larger P/O ratio. Here,wehaveexaminedtetracyclineinasimilar manner. Tetracyclines are broad-spectrum agents that are active against a wide range of gram-positive and gram-negative bacteria, atypical organisms such as chlamydiae, mycoplasmas, and rickettsiae, and protozoan parasites (Chopra and Roberts 2001). Tetracycline is produced by catalytic hydrogenolysis of aureomycin, which consists of a partially saturated naftacene ring. Here, we show that tetracycline enhances spinach chloroplast photophosphorylation and P/O ratios, and provide evidence that tetracycline binds to CF 1 at or near the γ subunit. Materials and methods Spinach (Spinacia oleracea L.) leaves were fieldgrown at our institute for use in preparation of chloroplasts, and market-purchased spinach leaves were used for isolation of CF 1. Chloroplasts were isolated from fresh, prechilled spinach leaves with STN solution (0.4 M sucrose, 10 mm NaCl and 20 mm Tricine-NaOH, ph 7.4) at 4 C according to previously described methods (Wei et al. 1983), and the chlorophyll concentration was determined according to Arnon (1949). Isolation and purification of CF 1 was performed using Jagendorf s methods (1982). Chloroplast preparations containing 20 µg chlorophyll or 20 µg CF 1 were mixed with various concentrations of tetracycline or other reagents such as glutaraldehyde and NEM et al., incubated on ice for 10 min, and used for the following experiments. Photophosphorylation activity was measured according to Wei et al. (1998a). Chloroplast photophosphorylation reactions were carried out in 1 ml reaction mixtures containing 50 mm Tricine-NaOH (ph8.0), 5mMNaCl,5mMMgCl 2,10mMNa 2 HPO 4,1mM ADP, 1 mm FeCy (or alternatively, 0.1 mm MV for non-cyclic photophosphorylation or 0.05 mm PMS for cyclic photophosphorylation) and chloroplasts containing 20 µg chlorophyll. ATP content was measured by the luciferin/luciferase luminescence assay (Allnutt et al. 1991). Chloroplast electron transport rates were assayed with H 2 O as an electron donor and FeCY as an electron acceptor, according to Shen and Shen (1962), with the remainder of the reaction mixture being identical to the above photophosphorylation reaction. ATPase activities were determined according to Shi et al. (1998) in 1 ml reaction mixtures containing 50 mm Tris-HCl (ph8.8), 5 mm ATP, 2 mm MgCl 2, 20 mm NaCl, 33% methanol and chloroplasts containing 20 µg chlorophyll or 25% methanol and 20 µg CF 1. Inorganic phosphate content was determined according to Taussky and Shorr (1953). The millisecond delayed light emission (ms-dle) of the chloroplasts was measured according to Wei et al. (1998b). Room temperature reaction mixtures of 2.8 ml containing 50 mm Tricine-NaOH (ph 8.0), 2mMMgCl 2 and 0.1 mm MV were mixed with 0.2 ml of chloroplast preparation containing 40 µg chlorophyll, and samples were immediately put into a phosphoroscope for ms-dle measurement. Fluorescence emission spectra of isolated CF 1 were measured by a 970CRT fluorescence spectrophotometer (General Factory, Shanghai, China), with excitation at 277 nm and 295 nm separately. Scans ranged from 290 or 310 nm to 700 nm. CF 1 was Figure 1. Effects of tetracycline on photophosphorylation of spinach chloroplasts. The chloroplast preparations were treated with low concentrations (0 200 µm) of tetracycline. The photophosphorylation reactions were carried out at 25 C. The activity was measured by a luciferin/luciferase luminescence assay as described in Materials and methods.

3 203 Table 1. Effect of low concentrations of tetracycline on non-cycling photophosphorylation activity of spinach chloroplasts. ( MV as electron acceptor.) Five groups of data from five parallel experiments are shown in the table, numbered as 1, 2, 3, 4 and 5. The photophosphorylation activity was measured as described in Materials and methods with FeCy as an electron acceptor except group 4 with MV instead Photophosphorylation activity (µmol ATP mg 1 chl h 1 ) Exp. no: Control Tetracycline (50 µm) Table 2. Effect of low concentrations of tetracycline on non-cycling and cycling photophosphorylation activity of spinach chloroplasts. The photophosphorylation activity was measured as described in Materials and methods. FeCy was used as an electron acceptor for non-cyclic photophosphorylation while PMS was for cyclic photophosphorylation. Numbers 1, 2 and 3 represent 3 parallel experiments, respectively Photophosphorylation activity (µmol ATP mg 1 chl h 1 ) FeCy PMS Exp. no: Control Tetracycline (50 µm) treated with 0.05% glutaraldehyde, 50 µm tetracycline, 150 µm NEM or 0.005% TNBS, respectively. Results The effects of tetracycline on chloroplast photophosphorylation activity and electron transport rate The photophosphorylation activity of spinach chloroplasts was increased by treatment with low concentrations of tetracycline. Figure 1 shows that when chloroplast preparations were treated with <150 µm of tetracycline, non-cycling photophosphorylation activities increased. Indeed, low concentrations of tetracycline increased both non-cyclic photophosphorylation activity using FeCY or MV as an electron acceptor, and cyclic photophosphorylation activity using PMS as a cofactor (Tables 1 and 2). In contrast, when chloroplast preparations were treated with >150 µm of tetracycline, photophosphorylation activities were markedly inhibited. The degree to which the various high concentrations of tetracycline inhibited these activities was dependent upon the physiological conditions of the chloroplasts isolated from spinach leaves. It is well known that photophosphorylation is coupled to electron transport. Therefore, it is not surprising that tetracycline treatment affected the electron transport rate of the tested chloroplast preparations. Electron transport rates increased with increasing tetracycline concentrations, with low increases at Figure 2. Effect of tetracycline on coupling electron transport activity in chloroplasts. Experimental conditions were identical with those of the photophosphorylation reactions; electron transport activity was measured according to Shen and Shen (1962).

4 204 tetracycline concentrations below 100 µm, and rapid increases at higher concentrations (Figure 2). The efficiency (P/O ratio) of non-cyclic photophosphorylation and electron transport is an important issue related to the mechanism and efficiency both photophosphorylation and photosynthesis. There are 3 opinions about the P/O ratio, that is, 1, 1.33 and 2. In this experiment, when low concentrations of tetracycline were used to treat the chloroplast preparations, there was not only a gain in photophosphorylation activity, but also an increase in P/O ratio (Figure 3). The effects of tetracycline on the ms-dle of chloroplasts It is generally thought that the ms-dle originates from the back reaction of Photosystem II. The proton motive force across the thylakoid membrane may provide energy for the recombination of the reduced election acceptor with an oxidized electron donor to overcome the activation barrier, and therefore enhances the ms-dle. Therefore, the intensities of ms- DLE also reflect the extent of the proton motive force. The fast phase is the increase of ms-dle within 0.1 s at the beginning of illumination with flashing light; the slow phase follows this and reaches a plateau within Figure 3. Effects of tetracycline on photophosphorylation, electron transport and P/O ratio. The chloroplast preparations were treated with low concentrations of tetracycline (0 200 µm). The photophosphorylation activity and electron transport activity were measured as described in Materials and methods respectively. : Photophosphorylation (psp); : electron transport; : P/O ratio. Figure 5. Effects of tetracycline on the Mg 2+ -ATPase activity of spinach chloroplasts. The Mg 2+ -ATPase activity of chloroplasts was activated by 33% methanol at 37 C. Chloroplasts were preincubated in various concentrations of tetracycline from 0 to 400 µm at 0 C for 10 min. Figure 4. Comparison of ms-dle in untreated or tetracycline-treated chloroplasts. The ms-dle was measured at room temperature. a: control; b: chloroplasts treated with 50 µm tetracycline. Figure 6. Effects of tetracycline on the Mg 2+ -ATPase activity of CF 1. The Mg 2+ -ATPase activity of chloroplasts was activated by 25% methanol. Purified CF 1 was preincubated in various concentrations of tetracycline from 0 to 800 µm at0 C for 10 min.

5 205 Figure 7. Comparison of fluorescence emission spectra of dissociated CF 1 following various treatments and under various conditions at 20 C (A) Fluorescence emission spectra excited at 277 nm. a: control; b: CF 1 treated with glutaraldehyde; c: CF 1 treated with tetracycline; d: CF 1 treated with NEM; e: CF 1 treated with TNBS. (B) Fluorescence emission spectra excited at 295 nm. a: control; b: CF 1 treated with glutaraldehyde; c: CF 1 treated with 50 µm tetracycline; d: CF 1 treated with NEM; e: CF 1 treated with TNBS. a few seconds. Following treatment with 50 µm tetracycline, the fast phase (<0.1s) of the chloroplast ms-dle was obviously enhanced, and the slow phase (>0.1 s) showed a slight increase (Figure 4). The effects of tetracycline on Mg 2+ -ATPase activity We used activation by methanol to examine the Mg 2+ - ATPase activity of chloroplasts and isolated CF 1 in the presence and absence of tetracycline (Figures 5 and 6). The Mg 2+ -ATPase activity of chloroplasts activated by 33% methanol was increased 5 10% by treatment with µm tetracycline. Similarly, the Mg 2+ -ATPase activity of CF 1 activated by 25% methanol was increased more than 30% by treatment with 100 µm. The measurement of fluorescence spectra of CF 1 To determine the acting site of tetracycline on CF 1, the fluorescence emission spectra of CF 1 at 277 and

6 206 Table 3. Effect of low concentrations of tetracycline on photophosphorylation activity, electron transport rate and P/O ratio of spinach chloroplasts Photophosphorylation Electron transport P/O ratio (µmol ATP mg 1 chl h 1 ) (µmol FeCY mg 1 chl h 1 ) Exp. no: Control Tetracycline (50 µm) nm were measured before and after treatment with 50 µm tetracycline, and compared with the spectra of CF 1 treated with glutaraldehyde, NEM and TNBS, which act on the whole CF 1, and on the γ and β subunits, respectively (Figure 7). The peak site of fluorescence emission spectra was the same among the various test compounds, though lower than that of the untreated control, and the peak value of CF 1 treated with 50µM tetracycline was very similar to that of CF 1 treated with NEM, suggesting that the action site of tetracycline might be at or near the γ subunit. Discussion As shown in Figure 1 and Tables 1 and 2, low concentrations of tetracycline increased both cyclic photophosphorylation, which was only concerned with Photosystem I, and non-cyclic photophosphorylation, which was concerned with both Photosystem I and Photosystem II (Avron 1977). The electron transport rate was also elevated by tetracycline (Figure 2). In addition, the efficiency of ATP synthesis coupled to electron transport, which is commonly referred to as the P/O ratio, increased at low concentrations of tetracycline and decreased at higher concentrations (Figure 3 and Table 3). Within the current literature, 3 values for the P/O ratio are commonly cited (1.0, 1.33, 2.0) based partially on theoretical considerations and partially on experimental measurements (Hall 1976; Bendall and Manasse 1995). However, chemiosmotic stoichiometries are notoriously difficult to establish with any certainty (Bendall and Manasse 1995). One problem is that the detailed pathways of electron transfer through the cytochrome b/f complex remain controversial, and some form of the Q cycle is suggested to provide an explanation for electrogenic proton pumping. In addition, it has proved difficult to match theoretical and experimental calculations (Hope 1993; Bendall and Manasse 1995). Two factors might lead to overestimation of the measured values. When experiments are performed with an oxygen electrode, there is often an underestimation of the Hill reaction due to loss of oxygen from the aqueous medium into the air space, which becomes supersaturated during photosynthetic electron transport (Shen and Shen 1962). Additionally, cyclic or pseudocyclic photophosphorylation may be superimposed over non-cyclic photophosphorylation in the presence of higher concentrations of ferredoxin. In this case, the reduced ferredoxin may not only donate an electron to NADP + but may also carry out cyclic or pseudocyclic electron transport (Shen 1987). However, there exists yet another factor that could lower the measured P/O ratio. If chloroplasts are isolated from leaves stored in darkness, the P/O ratio measured will likely be lower than that obtained with chloroplasts isolated from illuminated leaves (Wei et al. 1987). However, our results (Figure 3 and Table 3) once more testified that coupling of non-cyclic photophosphorylation and electron transport in vivo is generally incomplete, and that the P/O ratio was variable and able to be determined experimentally. Overall, as the tetracycline concentration increased past about 150 µm, we observed corresponding decreases in the improved photophosphorylation activity, to the point where the activity decreased below that of the untreated control. In contrast, the electron transport rate increased with increasing tetracycline concentration in a dosedependent manner. This suggests that at high concentrations, tetracycline acted as an uncoupler (Figure 3), in that it separated photophosphorylation activity from electron transport. The chloroplast ms-dle is closely related to proton motive force in chloroplasts. Low concentrations (50 µm) of tetracycline significantly enhanced the fast phase and increased the slow phase to a lesser degree

7 207 (Figure 4), indicating that the proton motive force across the thylakoid membrane increased following treatment with 50 µm tetracycline. These results further confirmed the increase of photophosphorylation shown above. We measured the effect of tetracycline on the Mg 2+ -ATPase activity of spinach chloroplasts with and without tetracycline treatment, and found that tetracycline improve the chloroplasts Mg 2+ -ATPase activity with the concentration ranged from 0 to 400 µm. (Figure 5). Next, we sought to the more precise acting site of the tetracycline on the spinach chloroplasts. We focused specifically on the effect of tetracycline on the CF 1.TheMg 2+ -ATPase activity of CF 1 increased in response to tetracycline treatment in a manner similar to that of the chloroplasts (Figure 6), but to a greater degree. This suggested that at least one acting site of action of tetracycline is located in the CF 1. Both tryptophan and tyrosine are prevalent in CF 1 ; when tryptophan is excited at 277 nm, its emission peak is at 308 nm, and when excited at 295 nm, tryptophan and tyrosine show emission peaks at 332 and 350 nm, respectively (Beliveau et al. 1982). Thus, these peaks may be used to follow binding to various portions of the CF 1, by comparing emission spectra derived from treatments with various known binding agents to that of tetracycline. Glutaraldehyde is known to interact with the entirety of CF 1, TNBS acts on the β subunit, and NEM acts on the γ subunit (McCarty and Fagan 1973; Oliver and Jagendorf 1976; Boardman and Thorne 1977). Tetracycline could decrease the intensity of the peak of the endogenous fluorescence of CF 1, but had almost no effect on the peak value (Figure 7). Indeed, the fluorescence emission spectrum of tetracyclinetreated CF 1 was similar to that of CF 1 treated with NEM, suggesting that the action site of tetracycline might be at or near the γ subunit of CF 1. Thus, it is possible that low doses of tetracycline affect the subunit interactions of CF 1, and then cause the increase in the proton motive force across the thylakoid membranes, which results in more ATP formations through CF 1. In contrast, higher concentrations of tetracycline may disrupt the interaction between CF 1 and CF o, resulting in a loss of energy transduction and a concomitant decrease in ATP synthesis and effective uncoupling of the system. Although future study will be needed to confirm this possible model, our work herein provides a necessary next step in furthering our understanding of the effect of tetracycline on chloroplast ATPases. References Aggeler R, Ogilivie I and Capaldi RA (1997) ATP synthase complex. 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