Implication du complexe Ndh dans le transfert cyclique des électrons autour du PS I

Implication du complexe Ndh dans le transfert cyclique des électrons autour du PS I

Increased Sensitivity of Photosynthesis to Antimycin A Induced by Inactivation of the Chloroplast ndhB Gene. Evidence for a Participation of the NADH-Dehydrogenase Complex to Cyclic Electron Flow around Photosystem I1

Tobacco (Nicotiana tabacum var Petit Havana) ndhB-inactivated mutants (ndhB2) obtained by plastid transformation (E.M. Horvath, S.O. Peter, T. Joe¨t, D. Rumeau, L. Cournac, G.V. Horvath, T.A. Kavanagh, C. Scha¨fer, G. Peltier, P. MedgyesyHorvath [2000] Plant Physiol 123: 1337–1350) were used to study the role of the NADH-dehydrogenase complex (NDH) during photosynthesis and particularly the involvement of this complex in cyclic electron flow around photosystem I (PSI). Photosynthetic activity was determined on leaf discs by measuring CO2 exchange and chlorophyll fluorescence quenchings during a dark-to-light transition. In the absence of treatment, both non-photochemical and photochemical fluorescence quenchings were similar in ndhB2 and wild type (WT). When leaf discs were treated with 5 mm antimycin A, an inhibitor of cyclic electron flow around PSI, both quenchings were strongly affected. At steady state, maximum photosynthetic electron transport activity was inhibited by 20% in WT and by 50% in ndhB2. Under non-photorespiratory conditions (2% O2, 2,500 mL L21 CO2), antimycin A had no effect on photosynthetic activity of WT, whereas a 30% inhibition was observed both on quantum yield of photosynthesis assayed by chlorophyll fluorescence and on CO2 assimilation in ndhB2. The effect of antimycin A on ndhB2 could not be mimicked by myxothiazol, an inhibitor of the mitochondrial cytochrome bc1 complex, therefore showing that it is not related to an inhibition of the mitochondrial electron transport chain but rather to an inhibition of cyclic electron flow around PSI. We conclude to the existence of two different pathways of cyclic electron flow operating around PSI in higher plant chloroplasts. One of these pathways, sensitive to antimycin A, probably involves ferredoxin plastoquinone reductase, whereas the other involves the NDH complex. The absence of visible phenotype in ndhB2 plants under normal conditions is explained by the complement of these two pathways in the supply of extra-ATP for photosynthesis. During oxygenic photosynthesis of C3 plants, both photosystem II (PSII) and photosystem I (PSI) cooperate to achieve NADP1 reduction using water as an electron donor and generate a trans-membrane proton gradient driving ATP synthesis. Although NADP1 reduction is recognized to be dependent on the activity of both photosystems through electron transport reactions of the “Z” scheme (Hill and Bendall, 1960; Redding et al., 1999), it has early been reported from studies on isolated thylakoids that ATP could be produced by the sole PSI through cyclic electron transfer reactions (Arnon, 1959). The cyclic electron flow around PSI has been extensively studied in thylakoids and/or chloroplasts of C3 plants (for review, see Fork and Herbert, 1993; Bendall and Manasse, 1995). This mechanism has been suggested to provide ATP for a variety of cellular processes, including stress adaptation (Havaux et al., 1991) and CO2 fixation (Furbank and Horton, 1987; Herbert et al., 1990). During photosynthetic CO2 fixation, both NADPH and ATP are used to regenerate ribulose-1,5-bisphosphate and allow functioning of the photosynthetic carbon reduction cycle (Calvin cycle). In the absence of Q cycle, when one NADPH is produced by linear electron transport reactions, four H1 are released in the lumen. If we consider that translocation of three H1 is required for the synthesis of one ATP (Hangarter and Good, 1982), the ATP to NADPH ratio produced during linear electron transport would be around 1.33. However in C3 plants, the ATP to NADPH ratio required for CO2 fixation has been reported to vary from 1.5 to 1.66, depending on the activity of photorespiration (Osmond, 1981). Insufficient ATP consequently would be synthesized for carbon reduction (Heber and Walker, 1992) and different mechanisms, including cyclic electron flow around PSI, have been proposed to fulfill this func1 This work was supported by the European Community Biotechnology program (grant no. BIO4–CT–97–2245). 2 Present address: Department of Genetics, Trinity College, University of Dublin, Dublin 2, Ireland. 3 Present address: Department of Biology, National University of Ireland, Maynooth, County Kildare, Ireland. * Corresponding author: e-mail gilles.peltier@cea.fr; fax 33–4– 42256265. Plant Physiology, April 2001, Vol. 125, pp. 1919–1929, www.plantphysiol.org © 2001 American Society of Plant Physiologists 1919 tion. A central question is the possible involvement of the Q-cycle, a cyclic electron flow inside the cytochrome (cyt) b6/f complex (Mitchell, 1975, 1977) able to translocate additional H1 and therefore provide extra ATP. However, the obligatory character or the flexibility of the Q-cycle during CO2 fixation remains a matter of debate (Davenport and McCarty, 1984; Ort, 1986; Heber and Walker, 1992; Cramer et al., 1996). Other mechanisms, like cooperation with mitochondrial respiration (Kro¨mer, 1995; Hoefnagel et al., 1998) and Mehler reactions (also known as waterwater cycle) (Schreiber and Neubauer, 1990) have also been suggested to re-equilibrate the chloroplastic ATP to NADPH ratio by generating extra-ATP, but their contribution during CO2 fixation remains to be established. Cyclic electron transfer reactions around PSI have been early reported to be inhibited by antimycin A (Tagawa et al., 1963). Most studies concluding to an involvement of cyclic electron flow during photosynthesis in C3 plants have been based on the effect of this compound on photosynthetic reactions such as photophosphorylation (Cleland and Bendall, 1992), rereduction of P7001 (Scheller, 1996), CO2-dependent O2 evolution (Furbank and Horton, 1987), 14CO2 fixation (Heber et al., 1978; Woo, 1983), or chlorophyll fluorescence (Ivanov et al., 1998). It was suggested that inhibition of photosynthetic reactions by antimycin A was related to the involvement of an antimycin A-sensitive ferredoxin plastoquinone reductase activity in cyclic reactions (Moss and Bendall, 1984; Cleland and Bendall, 1992). The actual efficiency of cyclic electron flow in vivo during photosynthesis of C3 plants is still unclear (Heber et al., 1995a). Photoacoustic measurements, which allow a direct and quantitative measurement of energy storage by cyclic electron flow around PSI in vivo, have been used to show the existence of cyclic electron transfer reactions in C4 plants, algae, and cyanobacteria (Herbert et al., 1990). However, until now, this technique failed to show significant cyclic activity in C3 plants (Herbert et al., 1990; Malkin et al., 1992). For the unicellular alga Chlamydomonas reinhardtii, Ravenel et al. (1994), by studying the effect of antimycin A and of different inhibitors on photoacoustic measurements, proposed that two pathways are operating in vivo around PSI. One pathway was shown to be sensitive to antimycin A, whereas the other would involve a NAD(P)H dehydrogenase activity (Ravenel et al., 1994). The existence of an antimycin A-insensitive cyclic electron pathways around PSI was also proposed in C3 plants from experiments performed in vitro (Hosler and Yocum, 1987; Scheller, 1996).

Involvement of the NDH Complex in Cyclic Electron Flow around PSI

We observed a sensitivity of photosynthesis to antimycin A in tobacco leaves (monitored either by chlorophyll fluorescence or gas exchange measurements), which was increased in ndhB2 mutants. Sensitivity of the electron transport to antimycin was highest at saturating light intensity and so was the difference between WT (20% inhibition) and mutant (50% inhibition). The additional inhibitory effect observed in ndhB2 mutants was not observed when using myxothiazol, thus showing that it is related to an inhibition of plastidial rather than mitochondrial reactions. In addition to its well-known effect on cyclic electron flow, antimycin A has been reported to affect qN (Oxborough and Horton, 1987; Ivanov et al., 1998). In our experiments, antimycin A induced a significant delay in the establishment of qN, but the steady-state level was virtually not affected and no differences were observed between WT and ndhB2 even at high intensities, when qN reaches its maximal values. In antimycin-treated leaves, Fs values at steady state remained higher in mutants than in WT, whereas the qN values were comparable. This higher Fs in mutants was then not related to variations in qN, but attributable to a more reduced state of the plastoquinone pool, indicating a less efficient functioning of electron acceptor reactions after PSI (Calvin cycle, etc.). We conclude that the simultaneous inhibition by antimycin A of cyclic electron flow around PSI and of NDH activity by gene inactivation leads to a reduced ability to use reducing power on the acceptor side of PSI. Previous studies, based on the disappearance of the transient postillumination rereduction of PQ in ndhinactivated mutants (Burrows et al., 1998; Cournac et al., 1998; Kofer et al., 1998; Shikanai et al., 1998) or on a decrease of the P7001 reduction rate in the dark (Burrows et al., 1998) already concluded to an involvement of the NDH complex in intersystem chain reduction and therefore to its potential implication during cyclic electron transport. It appears from our experiments that the NDH activity is involved in cyclic electron transport together with the antimycinsensitive pathway. Since photosynthesis is only slightly inhibited by antimycin A in WT, we conclude that the NDH-mediated pathway has a sufficient efficiency to compensate for the antimycin-sensitive pathway to a large extent. The existence of different cyclic electron pathways around PSI has previously been suggested in the literature. In spinach thylakoids, Hosler and Yocum (1985) reported the insensitivity to antimycin A of cyclic photophosphorylations measured in the presence of ferredoxin and NADP1. Based on photoacoustic measurements performed in vivo in C. reinhardtii cells, Ravenel et al. (1994) observed that antimycin A and N-ethyl-maleimide could inhibit PSI energy storage in vivo when added together, these compounds having no effect when added alone. More recently, based on P7001 rereduction measurements performed in barley thylakoids, Scheller (1996) proposed the existence of an antimycin-insensitive cyclic electron transport around PSI. The involvement of the NDH complex in cyclic electron flow in association with other pathways was shown in cyanobacteria (Mi et al., 1992; Yu et al., 1993) and recently suggested in higher plants from in vitro experiments performed on broken chloroplasts (Endo et al., 1998). Based on a differential sensitivity to antimycin A of PQ reduction in the WT and in a ndhB2 mutant, these authors concluded to the existence of two pathways, one of them involving the NDH complex. All of the evidences obtained in C3 plants are based on experiments performed on in vitro systems. Our study, performed on leaves, clearly shows the importance of cyclic pathways during photosynthesis in C3 plants in vivo