Cell Death and Differentiation (2005) 12, 1277–1284 & 2005 Nature Publishing Group All rights reserved 1350-9047/05 $30.00 www.nature.com/cdd Chloroplasts regulate leaf senescence: delayed senescence in transgenic ndhF-defective tobacco JM Zapata1,2, A Gue´ra1,2, A Esteban-Carrasco1, M Martı´n1 and mitochondria in animal PCD could be assumed by chloro- B Sabater*,1 plasts in leaves. This possibility seems feasible because, in leaves, chloroplasts are the main source of reactive oxygen 1 Departamento de Biologı´a Vegetal, Universidad de Alcala´ de Henares, Alcala´ species (ROS)3,7 that increase and are involved both in de Henares, 28871-Madrid, Spain animal PCD8,9 and leaf senescence.6,7,10 In fact, ROS 2 These authors contributed equally to this study. scavengers, such as superoxide dismutase (SOD), extend * Corresponding author: B Sabater, Departamento de Biologı´a Vegetal, the life span when overexpressed in Drosophila111 whereas Universidad de Alcala´ de Henares, Alcala´ de Henares, 28871-Madrid, Spain. 12 Tel: þ 34 91 8854934; Fax: þ 34 91 8855066; their induction is impaired during leaf senescence. The E-mail: [email protected] involvement of chloroplasts, instead of mitochondria, in the senescence of leaves would reveal a prominent role of ROS in Received 03.12.04; revised 16.2.05; accepted 30.3.05; published online 20.5.05 PCD and the emergence of different systems13 during Edited by M Piacentini evolution to trigger the production of ROS. Chloroplasts of higher plants contain the Ndh complex (one per 100–200 photosystem complexes), homologous to the Abstract NADH dehydrogenase or complex I (EC 1.6.5.3) of the Mitochondrial involvement has not been identified in the mitochondrial respiratory chain, which catalyzes the reduction 14–16 programmed cell death (PCD) of leaf senescence which of plastoquinone with NADH. In all, 11 genes (ndh)of 17–19 suggests that processes such as those involving reactive plastid DNA encode components of the Ndh complex oxygen species (ROS) are controlled by chloroplasts. We which participates in a chlororespiratory electron transport chain15,20,21 that regulates the redox state of transporters to report that transgenic tobacco (DndhF), with the plastid optimize22 the rate of cyclic electron transport. In chloro- ndhF gene knocked-out, shows low levels of the plastid Ndh respiration, the Ndh complex provides electrons to plastoqui- complex, homologous to mitochondrial complex I, and more none, and the Mehler reaction (producing superoxide anion than a 30-day-delay in leaf senescence with respect to wt. The À radical, Á O2 , which is converted to H2O2 by SOD) and comparison of activities and protein levels and analyses of peroxidase/oxidase reactions remove electrons from reduced genetic and phenotypic traits of wtxDndhF crosses indicate iron sulfur protein and reduced plastoquinone, respectively. that regulatory roles of mitochondria in animal PCD are The expression of ndh genes and the activity of the Ndh assumed by chloroplasts in leaf senescence. The Ndh complex increase during senescence.15,21,23–25 These in- complex would increase the reduction level of electron creases are accompanied by an increase of thylakoid 12,23 transporters and the generation of ROS. Chloroplastic control peroxidase but not of SOD, in contrast to the response of leaf senescence provides a nonclassical model of PCD and of young leaves in which the three chlororespiratory activities increase in response to different stress situations. By reveals an unexpected role of the plastid ndh genes that are increasing the level of the reduced forms of electron present in most higher plants. transporters, the increase of Ndh in senescent leaves must Cell Death and Differentiation (2005) 12, 1277–1284. À 6,7,10 increase the rate of the Mehler reaction producing Á O2 . doi:10.1038/sj.cdd.4401657; published online 20 May 2005 By comparison, the release of cytochrome c from mitochon- dria in PCD3,8 interrupts the respiratory electron transport, Keywords: chloroplast; leaf senescence; ndh genes; program- increasing the level of the reduced forms of electron med cell death; reactive oxygen species transporters and the production of ROS. Evidence for a role of the mitochondrial complex I during Abbreviations: MDA, malondialdehyde; PCD, programmed animal senescence26,27 suggests additional similarities be- cell death; ROS, reactive oxygen species; SOD, superoxide tween mitochondria-regulated PCD and chloroplast-regulated dismutase leaf senescence. However, the increase of ndh gene expression during senescence does not by itself demonstrate a regulatory role of the Ndh complex and chloroplasts in leaf senescence similar to that of mitochondria in animal PCD. In Introduction this work, we report a more definitive proof with a transgenic Leaf senescence is a regulated process of programmed cell tobacco (containing an insertion-inactivated plastid ndhF death (PCD)1,2 coordinated with the development of other gene), which shows delayed leaf senescence. plant organs such as seeds, more apical leaves or storage structures. Apoptotic PCD processes take place during the senescence of different types of plant cells in which mitochondria integrate diverse signals.3–5 However, evidence Results remains elusive for the involvement of mitochondria during The DndhF transgenic tobacco was described previously20 leaf senescence1 which suggests3,6 that the regulatory role of and was obtained from Nicotiana tabacum, cv. Petit Havana Chloroplast control of leaf senescence JM Zapata et al 1278 (wt) by inactivating the plastid ndhF gene by insertion of a show for successive time periods after germination. The cassette containing the spectinomycin resistance gene aadA delayed senescence in DndhF was also tested by the classical (Figure 1a). The DndhF and wt tobacco genotypes were assay of chlorophyll loss in detached leaves incubated in distinguishable because the amplification with F2/F4 primers water in the dark.28 Figure 3a shows that, after 24 and 48 h (Figure 1a) produced a 515 bp band with wt DNA and a incubations, wt leaf disks lost 15 and 25% chlorophyll, 1928 bp band with DndhF DNA.20 A faint 515 bp band was respectively, while leaf disks of DndhF barely lost 5% amplified, in addition to the stronger 1928 bp band, with chlorophyll after 48 h incubations. The level and the activity DndhF tobacco DNA (Figure 1b). Also faint bands of NDH-F, of the Ndh complex increased during the incubation of leaf J and K polypeptides and of Ndh activity were detected in disks of wt but not of DndhF tobacco (Figure 3b). The Ndh DndhF tobacco (Figure 1c and d) indicating that it was not complex is responsible for the increase in chlorophyll completely homoplastomic. Among the approximately 10 000 fluorescence (dependent on the level of reduced plastoqui- copies of plastid DNA estimated per cell, DndhF tobacco still none) after the interruption of actinic illumination.20 Accor- contains a few copies of wt nontransformed plastid DNA. In dingly, fluorescence rose in wt but not DndhF incubated leaf any case, the level of the Ndh complex was very low in DndhF disks (Figure 3c). This is another indication that the Ndh tobacco in comparison with wt. PCR amplification with complex remained at a low level during incubations of DndhF different primers and sequencing around cassette/ndhF joints but not of wt leaf disks. As a comparison, the activity of the (Figures S1 and S2 in Online Supplementary Information) chlororespiratory thylakoid peroxidase strongly increased confirmed that the spectinomycin cassette was inserted at the during the incubation of leaf disks of wt tobacco (Figure 3d) indicated positions of the plastid ndhF gene in DndhF tobacco. but to a lesser extent in DndhF. No significant difference was detected in the growth and One of the best characterized actions of ROS is the damage morphology of wt and DndhF before the reproductive stage of to membrane lipids, which may be estimated by the development (around 80 days after germination) (Figure 2). production of malondialdehyde (MDA).21 Indicative of the Usually, DndhF bloomed 7 days before wt tobacco. Similarly relative production of ROS, the levels of MDA were to many plants, the flowering in wt tobacco is accompanied by significantly lower in DndhF (black bars) than in wt (white the senescence (as judged by the yellowing that progresses bars) basal leaves of 72-day-old plants (Figure 4a). At this from basal to apical leaves) of the branch supporting the age, no leaf yellowing was yet apparent, although the first reproductive structures. However, in contrast to wt, the leaves flowers had appeared in DndhF whereas no flowering was of DndhF tobacco did not show senescence until the late detected in wt. For a comparison at similar physiological stages of fruit development, around 40 days after flowering stages, MDA was also determined in leaves of 79-day-old wt (Figure 2). The leaf senescence in DndhF is delayed by more (gray bars) that by then showed the first flowers. Again, the than 30 days with regard to wt tobacco as plates in Figure 2 MDA level was approximately 135 and 85% higher in the Figure 1 Insertion–inactivation of ndhF gene in tobacco. (a) Map showing the antiparallel insertion of the aadA cassette in the ndhF gene of DndhF tobacco and the primers used to identify the genotypes. (b) PCR amplification products using primers F2/F4 and DNA isolated from the fourth leaves of wt and DndhF 40 days after germination. (c) Western blot with antisera raised against NDH-F, NDH-J and NDH-K polypeptides of the Ndh complex after SDS-PAGE of 15 mg protein extracts prepared from the fourth leaves of wt and DndhF tobacco 40 days after germination. (d) Zymographic detection of the NADH dehydrogenase activity of the Ndh complex after native electrophoresis of 120 mg protein solubilized from thylakoid preparations of the fourth leaves of wt and DndhF tobacco 40 days after germination.