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...

Changes in CuZn-SOD activity and content in isolated wheat chloroplasts under the light, and the involvement of protease(s) and/or active oxygen species in this process were studied. Both SOD activity and content decayed with exposure time to photooxidative stress. Ascorbate, a H2O2 scavenger, prevented photooxidation-associated inactivation of SOD, while benzoate, a .OH scavenger, prevented SOD degradation. Wheat chloroplasts incubated in the dark did not hydrolyze exogenous or endogenous SOD, either H2O2-pretreated or not. Protease inhibitors did not prevent SOD degradation under photooxidative treatment, suggesting that plastid protease(s) did not participate in this process. Purified chloroplast CuZn-SOD was exposed to H2O2 and O2- or .OH-generating systems. O2- had no effect on either SOD activity or stability (estimated by native PAGE). H2O2 up to 700 microM inhibited SOD in a dose-dependent manner and induced charge/mass changes as seen by native PAGE. .OH also reduced SOD activity by inducing its fragmentation. High levels of active oxygen, as can be generated under strong stress conditions, could directly inactivate and degrade chloroplastic SOD.

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.

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