ABSTRACT
The inactivation of the chloroplast ascorbate peroxidases (chlAPXs) has been thought to limit the efficiency of the water-water cycle and photo-oxidative protection under stress conditions. In this study, we have generated double knockdown rice (Oryza sativa L.) plants in both OsAPX7 (sAPX) and OsAPX8 (tAPX) genes, which encode chloroplastic APXs (chlAPXs). By employing an integrated approach involving gene expression, proteomics, biochemical and physiological analyses of photosynthesis, we have assessed the role of chlAPXs in the regulation of the protection of the photosystem II (PSII) activity and CO2 assimilation in rice plants exposed to high light (HL) and methyl violagen (MV). The chlAPX knockdown plants were affected more severely than the non-transformed (NT) plants in the activity and structure of PSII and CO2 assimilation in the presence of MV. Although MV induced significant increases in pigment content in the knockdown plants, the increases were apparently not sufficient for protection. Treatment with HL also caused generalized damage in PSII in both types of plants. The knockdown and NT plants exhibited differences in photosynthetic parameters related to efficiency of utilization of light and CO2. The knockdown plants overexpressed other antioxidant enzymes in response to the stresses and increased the GPX activity in the chloroplast-enriched fraction. Our data suggest that a partial deficiency of chlAPX expression modulate the PSII activity and integrity, reflecting the overall photosynthesis when rice plants are subjected to acute oxidative stress. However, under normal growth conditions, the knockdown plants exhibit normal phenotype, biochemical and physiological performance.
Subject(s)
Ascorbate Peroxidases/genetics , Chloroplast Proteins/genetics , Oryza/genetics , Oxidative Stress/physiology , Photosynthesis/genetics , Plant Proteins/genetics , Ascorbate Peroxidases/metabolism , Chloroplast Proteins/metabolism , Electrophoresis, Gel, Two-Dimensional , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/radiation effects , Gene Expression Regulation, Plant/drug effects , Gene Expression Regulation, Plant/radiation effects , Herbicides/pharmacology , Isoenzymes/genetics , Isoenzymes/metabolism , Light , Oryza/drug effects , Oryza/radiation effects , Oxidative Stress/radiation effects , Paraquat/pharmacology , Photosynthesis/drug effects , Photosynthesis/radiation effects , Plant Proteins/metabolism , Plants, Genetically Modified , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, Mass, Electrospray IonizationABSTRACT
Ascorbate peroxidases (APX, EC 1.1.11.1) are class I heme-peroxidases, which catalyze the conversion of H(2)O(2) into H(2)O, using ascorbate as a specific electron donor. Previously, the presence of eight Apx genes was identified in the nuclear genome of rice (Oryza sativa), encoding isoforms that are located in different sub-cellular compartments. Herein, the generation of rice transgenic plants silenced for either both or each one of the cytosolic Apx1 and Apx2 genes was carried out in order to investigate the importance of cytosolic Apx isoforms on plant development and on plant stress responses. Transgenic double Apx1/2-silenced plants exhibited normal development, even though these plants showed a global reduction of Apx activity which strongly impacts the whole antioxidant system regulation. Apx1/2-silenced plants also showed increased H(2)O(2) accumulation under control and stress situations and presented higher tolerance to toxic concentration of aluminum when compared to wild type plants. On the other hand, silencing OsApx1 and OsApx2 genes individually resulted in strong effect on plant development producing semi-dwarf phenotype. These results suggested that the double silencing of cytosolic OsApx genes induced compensatory antioxidant mechanisms in rice while single knockdown of these genes did not, which resulted in the impairing of normal plant development.
Subject(s)
Adaptation, Physiological/genetics , Gene Silencing , Genes, Plant , Oryza/genetics , Oxidative Stress/genetics , Peroxidases/genetics , Plant Proteins/genetics , Aluminum/toxicity , Ascorbate Peroxidases , Cytosol , Hydrogen Peroxide/metabolism , Oryza/growth & development , Oryza/metabolism , Oxidation-Reduction , Peroxidases/metabolism , Plant Proteins/metabolism , Plants, Genetically ModifiedABSTRACT
Glutathione peroxidases (EC 1.11.1.9 and EC 1.11.1.12) catalyze the reduction of H(2)O(2) or organic hydroperoxides to water or corresponding alcohols using reduced glutathione. Some glutathione peroxidase isozymes have a selenium-dependent glutathione peroxidase activity and present a selenocysteine encoded by the opal TGA codon. In the present study, insights into the evolution of the whole glutathione peroxidase gene family were obtained after a comprehensive phylogenetic analysis using the improved number of glutathione peroxidase sequences recorded in the PeroxiBase database (http://peroxidase.isb-sib.ch/index.php). The identification of a common ancestral origin for the diverse glutathione peroxidase clusters was not possible. The complex relationships and evolutionary rates of this gene family suggest that basal glutathione peroxidase classes, present in all kingdoms, have originated from independent evolutionary events such as gene duplication, gene losses, lateral gene transfer among invertebrates and vertebrates or plants. In addition, the present study also emphasizes the possibility of some members being submitted to strong selective forces that probably dictated functional convergences of taxonomically distant groups.