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Plastids of some groups of green algae, bryophytes and most of the vascular plants contain an NADH dehydrogenase (Ndh) complex homologous to the mitochondrial and eubacterial complex I (Friedrich et al., 1995; Friedrich & Weiss, 1997). Since its discovery (Matsubayashi et al., 1987), this complex has been associated to the so called ‘chlororespiration’ process (for a review see Peltier & Cournac, 2002). The Ndh complex catalyses the electron flow from NADH to plastoquinone (Endo et al., 1997; Feild et al., 1998), which is further oxidized by a peroxidase (Casano et al., 2000) or oxidase activity (Joët et al., 2002). The construction of several Ndh-inactivated plastid insertion mutants (Δndh) (Burrows et al., 1998; Kofer et al., 1998; Shikanai et al., 1998; Horváth et al., 2000; Martín et al., 2004) has made it possible to assess the function of the Ndh complex against (photo)oxidative stress (Endo et al., 1999; Martín et al., 2004; Quiles, 2006); water stress (Burrows et al., 1998); heat stress (Sazanov et al., 1998; Quiles, 2006); chilling stress (Li et al., 2004); and ozone-mediated stress (Guéra et al., 2005). According to a model proposed by Casano et al. (2000), the chlororespiratory process consists of an electron transfer from NADH to H2O2 involving the Ndh complex, a thylakoidal plastoquinol peroxidase (Zapata et al., 1998), superoxide dismutase (SOD) and the nonenzymatic electron transfer from reduced iron–sulphur protein to O2 (Mehler reaction). This model helps explain the role of the Ndh complex as an element of a relatively complex electron chain, which consumes the reactive oxygen species (ROS) produced in the chloroplast under most stress conditions, and eventually decreases their production by lowering the O2 concentration in chloroplasts. An alternative model presented by Joët et al. (2002) considered a simpler chain that includes the Ndh complex, plastoquinone, a plastid terminal oxidase and oxygen. This chain would also operate under dark conditions, consistent with a function of chlororespiration in etiolated or nonphotosynthetic tissues (Guéra et al., 2000; Carol & Kuntz, 2001; Bennoun, 2002; Guéra & Sabater, 2002; Peltier & Cournac, 2002; Lennon et al., 2003; Kuntz, 2004).
The Ndh complex also participates in cyclic electron transport around photosystem I (PSI) (Burrows et al., 1998; Endo et al., 1998; Shikanai et al., 1998; Joët et al., 2001, 2002; Munekage et al., 2004; Takabayashi et al., 2005). According to the model presented by Casano et al. (2000), the Ndh complex poises the redox state of the photosynthetic electron transport intermediates by optimizing cyclic electron flow. Suitable rates of electron transport around PSI would ensure the maintenance of: (i) the transthylakoidal proton gradient (ΔµΗ+) required for photophosphorylation; (ii) and the luminal acidification-dependent dissipation – related to the nonphotochemical quenching parameter (NPQ) of chlorophyll a (Chla) fluorescence – of the excess of incident energy on photosystem II (PSII), which is associated with many stress conditions. This model is supported by a great deal of experimental evidence (Joët et al., 2002; Li et al., 2004; Martín et al., 2004; Guéra et al., 2005). Otherwise, several reports have presented evidence supporting the presence of redundant routes of cyclic electron flow: an antimycin A-resistant Ndh-dependent pathway, and another antimycin A-sensitive ferredoxin : plastoquinone oxidoreductase-dependent flow (Munekage et al., 2002, 2004; Takabayashi et al., 2005).
Although the above-mentioned functions have been suggested for the Ndh complex, little or no phenotypic change was observed in most studies carried out with ndh-inactivated plants grown under glasshouse conditions (Burrows et al., 1998; Shikanai et al., 1998; Horváth et al., 2000). More recently, Zapata et al. (2005) have reported that leaf senescence was delayed 30 d with respect to the wild type in a ΔndhF tobacco plant. Leaf senescence is the last phase before leaf death and develops after leaves have reached their maximum length and photosynthetic capacity (Humbeck & Krupinska, 2003). The onset and progression of leaf senescence are controlled by an array of external and internal factors including age, levels of plant hormones, and reproductive growth (Leopold, 1980; Noodén et al., 1997; Jing et al., 2005; Wingler et al., 2005). Many environmental stresses, such as ozone fumigation, induce the expression of a set of senescence-related genes (Smart, 1994; Miller et al., 1999; Coupe et al., 2004; Guo & Gan, 2005), among them the ndh genes (Guéra et al., 2005). For a long time, leaf senescence has been seen as a programmed evolutionary selected process related to the filling of seeds (Noodén & Leopold, 1978; Leopold, 1980), but some debate still persists as to whether or not leaf senescence is a form of apoptosis or programmed cell death (Thomas et al., 2003; Wingler et al., 2005). According to Thomas et al. (2003), it can be distinguished from processes of programmed cell death by its reversibility.
In the present work, we perform a more detailed study of the consequences of Ndh complex suppression on the life cycle, accumulation of antioxidant activities and substances, growth, photosynthetic capacity and productivity of tobacco plants. Our results show that the Ndh-defective plants show subtle phenotypic changes and increased fitness compared with wild-type plants.
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Many articles on ndh-disrupted mutants report irrelevant phenotypic differences when the mutants are compared with wild-type plants (Burrows et al., 1998; Shikanai et al., 1998; Horváth et al., 2000; Takabayashi et al., 2002), but detailed data on the developmental cycle of these mutants are scarce. Recently, Zapata et al. (2005) described ndhF-disrupted tobacco plants showing a delay in leaf senescence. The results presented by Zapata et al. (2005) focused mainly on the last stages of leaf development. Now we have included observations at different stages of leaf and whole-plant development of Ndh-defective tobacco mutants. The ΔndhF plants had a higher PSII : PSI ratio than wild-type plants (Fig. 1). These results are complementary to those described by Baena-González et al., 2003) for psbA-deficient mutants in which the lack of the corresponding core PSII protein was accompanied by a 10-fold relative increase in the Ndh complex and the plastid terminal oxidase. These data indicate the high capacity for adaptation of the photosynthetic machinery of plants, able to compensate (at least partially) for the loss or dysfunction of some components. In this way, the lack of function of the Ndh complex, which has been associated with resistance to oxidative stress conditions, could also be compensated for in young mutant leaves by the higher ascorbate/dehydroascorbate ratio observed.
The ΔndhF mutation had no effect on foliar area, apical growth, sprouting of new leaves, blooming or germination (Fig. 4; Table 1). Otherwise, significant differences between wild-type and ΔndhF plants were found for ΦPSII, ΦNPQ and the onset of leaf senescence (Figs 2, 5, 6). Lower levels of NPQ are characteristic of ΔndhF mutants ( Martín et al., 2004; Guéra et al., 2005; Zapata et al., 2005). NPQ is a general photoprotective mechanism in plants with a main component (qe) dependent on thylakoid lumen acidification (Krause & Weis, 1991; Horton et al., 2005; Szabo et al., 2005). It is shown that the proportion of photons absorbed in the PSII antenna, the energy of which is dissipated by NPQ (ΦNPQ), was higher in wild-type than in ΔndhF plants (Figs 2, 7) under moderate light intensity. This fact permits higher levels of ΦPSII in the ΔndhF mutants, as long as ΦNO is not increased. Although no alterations in NPQ were found in several studies with Ndh-deficient plants (Shikanai et al., 1998; Joët et al., 2001; Munekage et al., 2004), Burrows et al. (1998) and Li et al. (2004) described ΔndhCKJ or ΔndhB tobacco mutants, respectively, as having a reduced ability to quench fluorescence nonphotochemically. Also, lower levels of NPQ have been described for sunflower nonmutant plants under conditions of low nonphotochemical reduction of the plastoquinone pool, apparently associated with low activity of the chlororespiratory chain (Feild et al., 1998). Similar observations have been made on the youngest leaves of wild-type barley, which present no or very low levels of the Ndh complex (Guéra et al., 2005). A plausible explanation for the lower levels of NPQ observed in ΔndhF mutants than in other Ndh-defective plants could be the possible involvement of the NdhF subunit in H+ pumping (Casano et al., 2004). Whatever the case, the NPQ levels maintained by the ΔndhF mutants appear to be sufficient to allow the complete development of plants under controlled conditions. One of the main functions of NPQ is to protect against sudden fluctuations of light intensity (Demmig-Adams & Adams, 1993), and the same has been proposed for the Ndh complex in experiments performed under laboratory conditions (Endo et al., 1999; Casano et al., 2000; Guéra et al., 2005). Here it is shown that abrupt light variations did not produce a differential pattern in the responses of the ΔndhF mutants compared with wild-type plants (Fig. 7). Therefore, although the Ndh complex can contribute to generating NPQ, this is probably only a complementary mechanism. Indeed, as illustrated in Fig. 2, at high light intensities the levels of NPQ became similar for ΔndhF and wild-type plants, indicating that the Ndh complex modulates, but is not an indispensable factor for the generation of, NPQ.
The basal leaves of wild-type plants developed senescence symptoms (lower levels of photosynthetic electron transport, increased membrane lipid peroxidation, chlorophyll loss and necrosis) before the leaves of ΔndhF plants (Zapata et al., 2005). We show that the levels of several antioxidants (ascorbate, β-carotene, peroxidase, the chloroplastic Cu/Zn SOD) diminished in both genotypes in an ageing-dependent way. Thus the delay in leaf senescence in ΔndhF mutants cannot, in principle, be attributed to the promotion of higher levels of antioxidants. In fact, Zapata et al. (2005) hypothesized that a possible decay of chloroplastic SOD during leaf ageing, together with high Ndh complex activity (which should lead to more reduced electron transporters and so higher levels of the Mehler reaction, thus increasing superoxide production) would contribute to the rise of steady-state levels of ROS, which could trigger programmed cell death processes. A role for ROS in leaf programmed cell death is usually accepted (Navabpour et al., 2003; Yoshida, 2003; Woo et al., 2004; Zimmermann & Zentgraf, 2005), both for their toxicity and for their function as signalling molecules at low concentrations. However, leaf senescence is a process that can be reverted (Zavaleta-Mancera et al., 1999a, 1999b) and therefore can be distinguished from programmed cell death (Thomas et al., 2003). We followed the evolution of induced senescence, a traditional way to study leaf senescence, by incubating leaf disks from both genotypes on distilled water in the dark. As for the observations made in whole plants, results with leaf disks revealed that the decrease in photosynthetic capacity is delayed in the ΔndhF mutants (Fig. 6). Therefore inactivation of the Ndh complex can delay the processes of both natural and induced dark-leaf senescence. The latter fact is important in looking for a function for the Ndh complex in senescence because, under dark conditions, the light-induced photosynthetic mechanisms for generating ROS evidently cannot be accomplished and the levels of the Mehler reaction and generation of superoxide radical in the dark must be very low.
It must be borne in mind that the main function attributed to the Ndh complex is to act as an intermediate in chlororespiration, reducing the plastoquinone pool, which in a further step will be oxidized by the quinol-oxidase or peroxidase activity described for thylakoid membranes (Zapata et al., 1998; Carol & Kuntz, 2001; Joët et al., 2002; Peltier & Cournac, 2002). During the onset of leaf senescence, enhancement of this route could lead to the generation of H2O2, which could, in turn, function as a signalling molecule triggering leaf senescence. This mechanism could be largely homologous to the plasma membrane NADPH-dependent oxidase that generates H2O2 in response to pathogen attack, which develops similar symptoms to those of leaf senescence (Lamb & Dixon, 1997; Apel & Hirt, 2004). It might also be of significance that, under induced senescence, the NPQ steady-state values were higher in ΔndhF than in the wild type (Fig. 7d), indicating a possible change in the functional role of the Ndh complex as an element implicated in one or more chains of electron transfer, and probably of energy dissipation.
Finally, the delay in leaf senescence might explain why ΔndhF plants present a very significant increase in seed production (Table 1), as has been described for other senescence-delayed mutants under nonlimiting nutrient supply conditions (Gan & Amasino, 1995; Spano et al., 2003).