Bartolomé Sabater

Chlororespiration and Poising of Cyclic Electron Transport PLASTOQUINONE AS ELECTRON TRANSPORTER BETWEEN THYLAKOID NADH DEHYDROGENASE AND PEROXIDASE

Polypeptides encoded by plastid ndhgenes form a complex (Ndh) which could reduce plastoquinone with NADH. Through a terminal oxidase, reduced plastoquinone would be oxidized in chlororespiration. However, isolated Ndh complex has low activity with plastoquinone and no terminal oxidase has been found in chloroplasts, thus the function of Ndh complex is unknown. Alternatively, thylakoid hydroquinone peroxidase could oxidize reduced plastoquinone with H2O2. By immunoaffinity chromatography, we have purified the plastid Ndh complex of barley (Hordeum vulgareL.) to investigate the electron donor and acceptor specificity. A detergent-containing system was reconstructed with thylakoid Ndh complex and peroxidase which oxidized NADH with H2O2 in a plastoquinone-dependent process. This system and the increases of thylakoid Ndh complex and peroxidase activities under photooxidative stress suggest that the chlororespiratory process consists of the sequence of reactions catalyzed by Ndh complex, peroxidase (acting on reduced plastoquinone), superoxide dismutase, and the non-enzymic one-electron transfer from reduced iron-sulfur protein (FeSP) to O2. When FeSP is a component of cytochrome b 6·fcomplex or of the same Ndh complex, O2 may be reduced with NADH, without requirement of light. Chlororespiration consumes reactive species of oxygen and, eventually, may decrease their production by lowering O2 concentration in chloroplasts. The common plastoquinone pool with photosynthetic electron transport suggests that chlororespiratory reactions may poise reduced and oxidized forms of the intermediates of cyclic electron transport under highly fluctuating light intensities.

Chloroplasts regulate leaf senescence: delayed senescence in transgenic ndhF -defective tobacco

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 (ΔndhF), 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 wtxΔndhF 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.

Sensitivity of Superoxide Dismutase Transcript Levels and Activities to Oxidative Stress Is Lower in Mature-Senescent Than in Young Barley Leaves

Antioxidant enzyme activities are inducible by oxidative stress and decrease during senescence. To determine if the age-dependent decrease of superoxide dismutase (SOD) activities is due to decreased sensitivity to oxidative stress, we have investigated the changes in steady-state levels of transcripts and activities of mitochondrial Mn-SOD (SOD1), chloroplastic Fe-SOD (SOD2), and cytoplasmic Cu-Zn-SOD (SOD3) in young and mature-senescent detached barley (Hordeum vulgare L.) leaves in response to incubation in darkness, growth light (20 W m-2), and photooxidative stress conditions (100 W m-2 with 21 or 100% O2). For a comparison, changes in the mRNA for ribulose bisphosphate carboxylase were also measured. After leaf detachment, the abundance of all three SOD mRNAs increased, then decreased and eventually stabilized after 6 h of incubation. After 20 h of incubation under darkness SOD transcripts decreased in both young and mature-senescent leaves. While under strong photooxidative stress the levels of the three SOD transcripts significantly increased in young leaves; in mature-senescent leaves SOD2 and, to lesser extent, SOD1 and SOD3 transcripts decreased. Generally, SOD activity changes were similar to those of mRNAs. It is proposed that oxidative damage during senescence could be favored by the inability of senescing leaves to modulate the steady-state level of SOD mRNA, and probably those of other antioxidant enzymes, concomitant with the rate of oxyradical formation.

Plastid ndh genes in plant evolution

The plastid ndh genes encode components of the thylakoid Ndh complex which purportedly acts as an electron feeding valve to adjust the redox level of the cyclic photosynthetic electron transporters. During the process of evolution from endosymbiosis to modern chloroplast, most cyanobacterial genes were lost or transferred to nucleus. Eleven ndh genes are among the 150-200 genes remaining in higher plant chloroplast DNA, out of some 3000 genes in the original prokaryotic Cyanobacteria in which homologues to ndh genes encode components of the respiratory Complex I and probably other complexes. The ndh genes are absent in all sequenced plastid DNAs of algae except for the Charophyceae and some Prasinophyceae. With the possible exclusion of some Conifers and Gnetales, the plastid DNA of all photosynthetic land plants contains the ndh genes, whereas they are absent in epiphytic plants that have also lost genes for the photosynthetic machinery. Therefore, the functional role of the ndh genes seems closely related to the land adaptation of photosynthesis. Transcripts of several plastid genes require C to U editing. The ndh genes concentrate about 50% of the editing sites of angiosperm plastid transcripts. Editing sites may be remnants from an ancestor in which a number of T to C inactivating mutations took place in the ndh genes which, during evolution, are being corrected back to T. The comparison of homologous editing sites in the mRNAs of angiosperm ndh genes provides a tool to investigate selective and permissive environmental conditions of past evolutionary events.

The Activity of the Chloroplastic Ndh Complex Is Regulated by Phosphorylation of the NDH-F Subunit

Hydrogen peroxide (H2O2) 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, H2O2, dimethylthiourea (a scavenger of H2O2), and/or EGTA (a Ca2+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 H2O2 in the modulation of both the protein level and activity of the Ndh complex and the participation of Ca2+ 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 H2O2- and Ca2+-mediated action.

Leaf age- and paraquat concentration-dependent effects on the levels of enzymes protecting against photooxidative stress

Abstract Antioxidant protective enzymes are usually induced in leaves under conditions of increased active oxygen generation, such as high light intensity, low CO2 fixation rate or in the presence of paraquat, which transports electrons from photosynthetic machinery to oxygen to form O2 −. However, at high photooxidative stress, even protective enzymes can be destroyed and leaf cells become dead. The protective role of several chloroplastic activities was evaluated at increasing photooxidative stress in barley leaves of different ages. We investigated the effects of different paraquat concentrations (combined with low and high light intensities) in expanding and aged-senescent leaves on the activity of plastid peroxidase and on the activity and protein levels of plastid superoxide dismutase (SOD), glutathione reductase (GR) and NADH dehydrogenase of the complex including polypeptides encoded by plastid ndh genes. The chloroplastic GR was the most sensitive to inactivation when photooxidative stress increased. SOD was preferentially induced in young-expanding leaves while NADH dehydrogenase and peroxidase were preferentially induced in adult-senescent leaves. The results suggest a limited role of GR in the protection against photooxidative stress and a close relation between the actions of Ndh complex and peroxidase.

Editing of the chloroplast ndhB encoded transcript shows divergence between closely related members of the grass family (Poaceae).

The ndhB-encoded transcript from barley chloroplasts deviates from the genomic ndhB sequence by nine C-to-U transitions, which is the maximum number of editing events for a chloroplast mRNA reported so far. Comparison with ndhB transcripts from other chloroplast species shows that six of the nine editing sites observed in barley are structurally and functionally conserved in maize, rice and tobacco. The remaining three sites, however, show divergent patterns of conservation even within the three members of the grass family. The conservation of two of these sites in tobacco but not in the closely related graminean species suggests that divergence of the ndhB editing sites is caused by the loss of preexisting editing sites rather than by gain of new sites.

Plastid DNA of five ecotypes of Arabidopsis thaliana: sequence of ndhG gene and maternal inheritance☆

Southern blots of BamHI, HindIII and EcoRI restriction fragments with 21 heterologous and homologous probes indicated that restriction and gene maps of plastid DNA of Arabidopsis thaliana (L.) cv. Landsberg (Ler-0) is similar to that known of Columbia (Col-0) ecotype. Restriction analysis and specific Southern blots of plastid DNA of Ler-0, Cape Verdi Islands (Cvi-0), Col-0, Leiden (Le-0) and Stockholm (St-0) ecotypes revealed that, with respect to other ecotypes, only Cvi-0 lacks a BamHI site and gains a new HindIII site in the small single copy (SSC) region of plastid DNA. As in most plants, Northern blots for ndh transcripts revealed that SSC is expressed in Arabidopsis. Sequencing of a 1519b fragment around ndhG gene DNA confirmed that plastid DNA of Cvi-0 lacks a BamHI site (present in other ecotypes) at positions 46–51 down the initiation codon (ATG) of ndhG. BamHI digestions of the corresponding fragments amplified from plastid DNA of Ler-0, Cvi-0 and Ler-0 × Cvi-0 first 38 offsprings always showed maternal inheritance of plastid DNA.

Identification of a thylakoid peroxidase of barley which oxidizes hydroquinone

Abstract The oxidation of hydroquinone by two peroxidases (EC 1.11.1.7) fractions obtained from barley leaves has been studied. The results indicated that thylakoid contains hydroquinone peroxidase which may be distinguished from soluble peroxidase by apparent K M s , influence of pH on activity and zymographic assays. Appropriate controls indicated that thylakoid peroxidase was not a contamination from other cellular fractions. Thylakoid peroxidase may be solubilized by Triton-containing buffers but not by buffers without detergent and can also be distinguished of other guaiacol peroxidases (as cell wall peroxidase) and of thylakoid ascorbate peroxidase (EC 1.11.1.11) by zymographic assays and stability and kinetic properties. The reduced form of quinone electron carrier of thylakoids may be substrates of HQ peroxidase.

Multiple phosphorylation sites in the β subunit of thylakoid ATP synthase

Proteomic analyses of the β subunit of the plastid ATP synthase of barley (Hordeum vulgare L.) revealed that mature protein was not carboxy terminus processed and suggested the correction of the 274 codon (GAT to AAT) in the data bank that was confirmed by DNA sequencing. Six isoforms of the ATP synthase β subunit with pI ranging from 4.95 to 5.14 were resolved by two-dimensional electrophoresis (2-DE). Mass spectrometry analyses indicated that the six isoforms differ in their phosphorylation degree, which was confirmed by the disappearance of more acidic forms after incubation with the protein phosphatase calcineurin. Six Ser and/or Thr were detected as phosphorylated, among them the conserved Thr-179 that is also phosphorylated in the β subunit of human mitochondria. The results are discussed in relation with the proposed regulation of the ATP synthase by phosphorylation and 14-3-3 proteins.