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.

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 H₂O₂-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 andndhF transcripts in barley (Hordeum vulgare cv Hassan) leaves. Subapical leaf segments were incubated in growing light (GL), photooxidative light (PhL), GL and H₂O₂ (GL + H₂O₂), or PhL and 50 nm paraquat in the incubation medium. Treatments with H₂O₂ 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 H₂O₂-scavenger dimethyltiourea. NADH-DH and NDH-F polypeptide rapidly increased up to 16 h in PhL, GL+ H₂O₂, and, at higher rate, in PhL and paraquat. The observed increases of NADH-DH and NDH-F after 4 h in PhL and GL + H₂O₂ 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 ndhFtranscript levels. All these effects were prevented by dimethylthiourea. It is proposed that the induction of chloroplasticndh genes under photooxidative stress is mediated by H₂O₂ through mechanisms that involve a rapid translation of pre-existing transcripts and the increase of thendh 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 rβ-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.

Changes in CuZn-SOD actvity 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 700μM 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.

Lichens are symbiotic associations (holobionts) established between fungi (mycobionts) and certain groups of cyanobacteria or unicellular green algae (photobionts). This symbiotic association has been essential in the colonization of terrestrial dry habitats. Lichens possess key mechanisms involved in desiccation tolerance (DT) that are constitutively present such as high amounts of polyols, LEA proteins, HSPs, a powerful antioxidant system, thylakoidal oligogalactolipids, etc. This strategy allows them to be always ready to survive drastic changes in their water content. However, several studies indicate that at least some protective mechanisms require a minimal time to be induced, such as the induction of the antioxidant system, the activation of non-photochemical quenching including the de-epoxidation of violaxanthin to zeaxanthin, lipid membrane remodeling, changes in the proportions of polyols, ultrastructural changes, marked polysaccharide remodeling of the cell wall, etc. Although DT in lichens is achieved mainly through constitutive mechanisms, the induction of protection mechanisms might allow them to face desiccation stress in a better condition. The proportion and relevance of constitutive and inducible DT mechanisms seem to be related to the ecology at which lichens are adapted to.