No abstract

No abstract

Mitochondrial involvement has not been identified in the programmed cell death (PCD) of leaf senescence which suggests that processes such as those involving reactive oxygen species (ROS) are controlled by chloroplasts. We report that transgenic tobacco (DndhF), with the plastid ndhF gene knocked-out, shows low levels of the plastid Ndh complex, homologous to mitochondrial complex I, and more than a 30-day-delay in leaf senescence with respect to wt. The comparison of activities and protein levels and analyses of genetic and phenotypic traits of wtxDndhF crosses indicate that regulatory roles of mitochondria in animal PCD are assumed by chloroplasts in leaf senescence. The Ndh complex would increase the reduction level of electron transporters and the generation of ROS. Chloroplastic control of leaf senescence provides a nonclassical model of PCD and reveals an unexpected role of the plastid ndh genes that are present in most higher plants.

SummaryPost-transcriptional maturation of plastid-encoded mRNAs from land plants includes editing by making cytidine to uridine alterations at highly specific positions; this usually restores codon identities for conserved amino acids that are important for the proper function of the affected proteins. In contrast to the rather constant number of editing sites their location varies greatly, even between closely related taxa. Here, we experimentally determined the specific pattern of editing sites (the editotype) of the plastid genome of Arabidopsis thaliana ecotype Columbia (Col-0). Based on phylogenetic analyses of plastid open reading frames, we identified 28 editing sites. Two editing events in the genes matK and ndhB seem to have evolved late during the evolution of flowering plants. Strikingly, they are embedded in almost identical sequence elements and seem to be phylogenetically co-processed. This suggests that the two sites are recognized by the same trans-factor, which could help to explain the hitherto enigmatic gain of editing sites in evolution. In order to trace variations in editotype at the subspecies level we examined two other A. thaliana accessions, Cape Verde Islands (Cvi-0) and Wassilewskija (Ws-2), for the Col-0 editing sites. Both Cvi-0 and Ws-2 possess and process the whole set of editing sites as determined in Col-0, but the consequences of RNA editing differ at one position between the ecotypes.

Chloroplast-encoded NDH polypeptides (components of the plastid Ndh complex) and the NADH dehydrogenase activity of the Ndh complex (NADH-DH) increased under photooxidative stress. The possible involvement of H 2 O 2 -mediated signaling in the photooxidative induction of chloroplastic ndh genes was thoroughly studied. We have analyzed the changes in the NADH-DH and steady-state levels of NDH-F polypeptide and ndhB and ndhF transcripts in barley (Hordeum vulgare cv Hassan) leaves. Subapical leaf segments were incubated in growing light (GL), photooxidative light (PhL), GL and H 2 O 2 (GL ϩ H 2 O 2 ), or PhL and 50 nm paraquat in the incubation medium. Treatments with H 2 O 2 under GL mimicked the photooxidative stimulus, causing a dose-dependent increase of NADH-DH and NDH-F polypeptide. The kinetic of Ndh complex induction was further studied in leaves pre-incubated with or without the H 2 O 2 -scavenger dimethyltiourea. NADH-DH and NDH-F polypeptide rapidly increased up to 16 h in PhL, GLϩ H 2 O 2 , and, at higher rate, in PhL and paraquat. The observed increases of NADH-DH and NDH-F after 4 h in PhL and GL ϩ H 2 O 2 were not accompanied by significant changes in ndhB and ndhF transcripts. However, at 16-h incubations NADH-DH and NDH-F changes closely correlated with higher ndhB and ndhF transcript levels. All these effects were prevented by dimethylthiourea. It is proposed that the induction of chloroplastic ndh genes under photooxidative stress is mediated by H 2 O 2 through mechanisms that involve a rapid translation of pre-existing transcripts and the increase of the ndh transcript levels.

A 76 amino acid sequence of NDH-A (the protein encoded by plastid ndhA gene) from barley (Hordeum vulgare L.) was expressed as a fusion protein with beta-galactosidase in E. coli. The corresponding antibody generated in rabbits was used to investigate localization, expression and synthesis in vitro of NDH-A. NDH-A was identified as a 35 kDa polypeptide localized in thylakoid membrane. Western blots shows a large increase in NDH-A levels when barley leaves were incubated under photooxidative conditions, which was more pronounced in mature-senescent leaves than in young leaves. Immunoprecipitation of the [35S]methionine labelled proteins, synthesized in vitro by isolated chloroplasts, demonstrated the synthesis in chloroplasts of the NDH-A 35 kDa polypeptide when barley leaves had been incubated under photooxidative conditions. The results indicate that ndh genes may be involved in the protection of chloroplasts against photooxidative stress, particularly in mature-senescent leaves.

Hydrogen peroxide (H 2 O 2 ) induces increases, to different degrees, in transcripts, protein levels, and activity of the Ndh complex (EC 1.6.5.3). In the present work, we have compared the effects of relatively excess light, H 2 O 2 , dimethylthiourea (a scavenger of H 2 O 2 ), and/or EGTA (a Ca 2ϩ chelator) on the activity and protein levels of the Ndh complex of barley (Hordeum vulgare cv Hassan) leaf segments. The results show the involvement of H 2 O 2 in the modulation of both the protein level and activity of the Ndh complex and the participation of Ca 2ϩ mainly in the activity regulation of pre-existing protein. Changes in Ndh complex activity could not be explained only by changes in Ndh protein levels, suggesting posttranslational modifications. Hence, we investigate the possible phosphorylation of the Ndh complex both in thylakoids and in the immunopurified Ndh complex using monoclonal phosphoamino acid antibodies. We demonstrate that the Ndh complex is phosphorylated in vivo at threonine residue(s) of the NDH-F polypeptide and that the level of phosphorylation is closely correlated with the Ndh complex activity. The emerging picture is that full activity of the Ndh complex is reached by phosphorylation of its NDH-F subunit in a H 2 O 2 -and Ca 2ϩ -mediated action.A plastid Ndh complex, analogous to the NADH dehydrogenase (NADH-DH) or complex I (EC 1.6.5.3) of the mitochondrial respiratory chain, which catalyzes the transfer of electrons from NADH to plastoquinone, has been purified from pea (Pisum sativum; Sazanov et al., 1998) and barley (Hordeum vulgare cv Hassan; Casano et al., 2000). Eleven polypeptides of the Ndh complex (NDH polypeptides) are encoded by respective ndh genes of plastid DNA (Maier et al., 1995). Both the Ndh complex (providing electrons) and thylakoid plastoquinol peroxidase (Zapata et al., 1998) together with the Mehler reaction and superoxide dismutase (draining electrons) might poise the redox level of the photosynthetic electron carriers. This mechanism (chlororespiration) would most likely ensure the photosynthetic electron transport under a variety of environmental conditions, which include rapid changes of light intensity caused by sunflecks and leaf movements. In addition, chlororespiration may act as a scavenging system of reactive oxygen species generated under continuous photooxidative stress or by the successions of sunflecks and light gaps (Casano et al., 2000). In fact, NDH polypeptides and NADH-DH activity of the Ndh complex increase under photooxidative stress provoked by the herbicide paraquat (Martín et al., 1996; Catalá et al., 1997; Casano et al., 1999 Casano et al., , 2000 or bright light and chilling in field-grown barley (Teicher et al., 2000). In addition, ndh mutants show increased sensitivity to photooxidative stress (Endo et al., 1999; Horvath et al., 2000), which strongly suggests that the activity of the Ndh complex is involved in the protection against said stress.The increases of plastid-encoded NDH polypeptides and Ndh complex activity under ph...

An Ndh‐deficient mutant of tobacco (Nicotiana tabacum cv. Petit Havana) was prepared by disrupting the ndhF gene in a transplastomic approach. The mutant (ΔndhF) showed 10% of the Ndh complex activity (EC 1.6.5.3) and 8% of the NDH‐F polypeptide of that of non‐transformed plants. However, in ΔndhF, NDH‐A, another Ndh polypeptide, was still present at 50% of the level in non‐transformed plants. ΔndhF tobacco showed higher sensitivity than non‐transformed plants to photo‐oxidative stress (as judged by chlorophyll bleaching) caused by increased light intensity and paraquat applications. These photo‐oxidative treatments increased the amount and activity of the Ndh complex, thylakoid peroxidase, post‐illumination chlorophyll fluorescence and non‐photochemical quenching (NPQ) of chlorophyll fluorescence in non‐transformed but not in ΔndhF tobacco. Highly stressed non‐transformed plants showed a rapid post‐rise decline of chlorophyll fluorescence, probably indicating a re‐oxidation of reduced plastoquinone. The results indicate that, in normal plants, the Ndh complex and thylakoid peroxidase (EC 1.11.1.7) provide and remove electrons, respectively, to balance the redox level of the intermediates of cyclic electron transport. In this way, they optimize the generation of the transmembrane H+ gradient of thylakoids and, as a consequence, increase the NPQ and the protection against photo‐oxidative stress.