Leonardo M. Casano

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.

Antioxidant system response of different wheat cultivars under drought: field and in vitro studies

The participation of the antioxidant system in the drought tolerance of wheat cultivars (Triticum aestivum L.) was studied under field and in vitro conditions. Under field conditions, drought tolerance was evaluated by the capacity to maintain the grain yield under drought, which was higher in cvv. Elite and La Paz than in the sensitive cvv. Oasis and Cruz Alta. Tolerant cultivars showed lower relative water content (RWC) and lower above-ground vegetative biomass than sensitive cultivars. Field assays did not show a clear correlation between water-stress tolerance and antioxidant system behaviour. However, when leaves of cvv. with contrasting drought tolerance were subjected to osmotic stress in vitro, clear differences in the antioxidant system activity and oxidative damage between cvv. were observed. In the tolerant cultivar Elite, it was possible to observe an increase in ascorbate peroxidase (APX), superoxide dismutase (SOD) and glutathione reductase (GR) activities, a higher glutathione (GSH) and ascorbate content and less oxidative damage than in the sensitive cultivar Oasis, which showed no changes or only slight decreases in the enzyme activities. These results indicate that water stress tolerance is in part associated with the antioxidant system activity, and suggest that the behaviour of the antioxidant systemin vitro assays can be used as an early selection tool.

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.

Two Trebouxia algae with different physiological performances are ever‐present in lichen thalli of Ramalina farinacea. Coexistence versus Competition?

Ramalina farinacea is an epiphytic fruticose lichen that is relatively abundant in areas with Mediterranean, subtropical or temperate climates. Little is known about photobiont diversity in different lichen populations. The present study examines the phycobiont composition of several geographically distant populations of R. farinacea from the Iberian Peninsula, Canary Islands and California as well as the physiological performance of isolated phycobionts. Based on anatomical observations and molecular analyses, the coexistence of two different taxa of Trebouxia (working names, TR1 and TR9) was determined within each thallus of R. farinacea in all of the analysed populations. Examination of the effects of temperature and light on growth and photosynthesis indicated a superior performance of TR9 under relatively high temperatures and irradiances while TR1 thrived at moderate temperature and irradiance. Ramalina farinacea thalli apparently represent a specific and selective form of symbiotic association involving the same two Trebouxia phycobionts. Strict preservation of this pattern of algal coexistence is likely favoured by the different and probably complementary ecophysiological responses of each phycobiont, thus facilitating the proliferation of this lichen in a wide range of habitats and geographic areas.

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.

Oxidative stress induces distinct physiological responses in the two Trebouxia phycobionts of the lichen Ramalina farinacea

BACKGROUND AND AIMS Most lichens form associations with Trebouxia phycobionts and some of them simultaneously include genetically different algal lineages. In other symbiotic systems involving algae (e.g. reef corals), the relative abundances of different endosymbiotic algal clades may change over time. This process seems to provide a mechanism allowing the organism to respond to environmental stress. A similar mechanism may operate in lichens with more than one algal lineage, likewise protecting them against environmental stresses. Here, the physiological responses to oxidative stress of two distinct Trebouxia phycobionts (provisionally named TR1 and TR9) that coexist within the lichen Ramalina farinacea were analysed. METHODS Isolated phycobionts were exposed to oxidative stress through the reactive oxygen species propagator cumene hydroperoxide (CuHP). Photosynthetic pigments and proteins, photosynthesis (through modulated chlorophyll fluorescence), the antioxidant enzymes superoxide dismutase (SOD) and glutathione reductase (GR), and the stress-related protein HSP70 were analysed. KEY RESULTS Photosynthetic performance was severely impaired by CuHP in phycobionts, as indicated by decreases in the maximal PSII photochemical efficiency (F(v)/F(m)), the quantum efficiency of PSII (Φ(PSII)) and the non-photochemical dissipation of energy (NPQ). However, the CuHP-dependent decay in photosynthesis was significantly more severe in TR1, which also showed a lower NPQ and a reduced ability to preserve chlorophyll a, carotenoids and D1 protein. Additionally, differences were observed in the capacities of the two phycobionts to modulate antioxidant activities and HPS70 levels when exposed to oxidative stress. In TR1, CuHP significantly diminished HSP70 and GR but did not change SOD activities. In contrast, in TR9 the levels of both antioxidant enzymes and those of HSP70 increased in response to CuHP. CONCLUSIONS The better physiological performance of TR9 under oxidative conditions may reflect its greater capacity to undertake key metabolic adjustments, including increased non-photochemical quenching, higher antioxidant protection and the induction of repair mechanisms.

Balanced gene losses, duplications and intensive rearrangements led to an unusual regularly sized genome in Arbutus unedo chloroplasts.

Completely sequenced plastomes provide a valuable source of information about the duplication, loss, and transfer events of chloroplast genes and phylogenetic data for resolving relationships among major groups of plants. Moreover, they can also be useful for exploiting chloroplast genetic engineering technology. Ericales account for approximately six per cent of eudicot diversity with 11,545 species from which only three complete plastome sequences are currently available. With the aim of increasing the number of ericalean complete plastome sequences, and to open new perspectives in understanding Mediterranean plant adaptations, a genomic study on the basis of the complete chloroplast genome sequencing of Arbutus unedo and an updated phylogenomic analysis of Asteridae was implemented. The chloroplast genome of A. unedo shows extensive rearrangements but a medium size (150,897 nt) in comparison to most of angiosperms. A number of remarkable distinct features characterize the plastome of A. unedo: five-fold dismissing of the SSC region in relation to most angiosperms; complete loss or pseudogenization of a number of essential genes; duplication of the ndhH-D operon and its location within the two IRs; presence of large tandem repeats located near highly re-arranged regions and pseudogenes. All these features outline the primary evolutionary split between Ericaceae and other ericalean families. The newly sequenced plastome of A. unedo with the available asterid sequences allowed the resolution of some uncertainties in previous phylogenies of Asteridae.

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.

Different strategies to achieve Pb-tolerance by the two Trebouxia algae coexisting in the lichen Ramalina farinacea.

Lichen thalli are permeable to airborne substances, including heavy metals, which are harmful to cell metabolism. Ramalina farinacea shows a moderate tolerance to Pb. This lichen comprises two Trebouxia phycobionts, provisionally referred to as TR1 and TR9, with distinct physiological responses to acute oxidative stress. Thus, there is a more severe decay in photosynthesis and photosynthetic pigments in TR1 than in TR9. Similarly, under oxidative stress, antioxidant enzymes and HSP70 protein decrease in TR1 but increase in TR9. Since Pb toxicity is associated with increased ROS formation, we hypothesized greater Pb tolerance in this phycobiont. Accordingly, the aim of the present study was to characterize the physiological differences in the responses of TR1 and TR9 to Pb exposure. Liquid cultures of isolated phycobionts were incubated for 7 days in the presence of Pb(NO₃)₂. Thereafter, extracellular and intracellular Pb accumulation, photosynthetic pigments, and photosynthesis (as modulated chlorophyll fluorescence) were analyzed along with the antioxidant enzymes glutathione reductase (GR), superoxide dismutase (SOD), ascorbate peroxidase (APx), and catalase (CAT), and the stress-related protein HSP70. Pb uptake increased with the amount of supplied Pb in both algae. However, while significantly more metal was immobilized extracellularly by TR9, the amount of intracellular Pb accumulation was three times higher in TR1. In neither of the phycobionts were significant effects on photosynthetic pigments or photosynthetic electron transport observed. While under control conditions GR, SOD, and APx levels were significantly higher in TR1 than in TR9, only in the latter were these enzymes induced by Pb. This resulted in quantitatively similar antioxidant activities in the two algae when exposed to Pb. In conclusion, the phycobionts of R. farinacea make use of two different strategies against stress, in which the integration of distinct anatomical and physiological features affords similar levels of Pb tolerance.