RESUMO
Retrograde signalling pathways that are triggered by changes in cellular redox homeostasis remain poorly understood. Transformed rice plants that are deficient in peroxisomal ascorbate peroxidase APX4 (OsAPX4-RNAi) are known to exhibit more effective protection of photosynthesis against oxidative stress than controls when catalase (CAT) is inhibited, but the mechanisms involved have not been characterized. An in-depth physiological and proteomics analysis was therefore performed on OsAPX4-RNAi CAT-inhibited rice plants. Loss of APX4 function led to an increased abundance of several proteins that are involved in essential metabolic pathways, possibly as a result of increased tissue H2O2 levels. Higher photosynthetic activities observed in the OsAPX4-RNAi plants under CAT inhibition were accompanied by higher levels of Rubisco, higher maximum rates of Rubisco carboxylation, and increased photochemical efficiencies, together with large increases in photosynthesis-related proteins. Large increases were also observed in the levels of proteins involved in the ascorbate/glutathione cycle and in other antioxidant-related pathways, and these changes may be important in the protection of photosynthesis in the OsAPX4-RNAi plants. Large increases in the abundance of proteins localized in the nuclei and mitochondria were also observed, together with increased levels of proteins involved in important cellular pathways, particularly protein translation. Taken together, the results show that OsAPX4-RNAi plants exhibit significant metabolic reprogramming, which incorporates a more effective antioxidant response to protect photosynthesis under conditions of impaired CAT activity.
Assuntos
Ascorbato Peroxidases/metabolismo , Catalase/metabolismo , Oryza/metabolismo , Estresse Oxidativo , Fotossíntese , Interferência de RNARESUMO
The physiological role of plant mitochondrial glutathione peroxidases is scarcely known. This study attempted to elucidate the role of a rice mitochondrial isoform (GPX1) in photosynthesis under normal growth and salinity conditions. GPX1 knockdown rice lines (GPX1s) were tested in absence and presence of 100 mM NaCl for 6 d. Growth reduction of GPX1s line under non-stressful conditions, compared with non-transformed (NT) plants occurred in parallel to increased H2 O2 and decreased GSH contents. These changes occurred concurrently with photosynthesis impairment, particularly in Calvin cycle's reactions, since photochemical efficiency did not change. Thus, GPX1 silencing and downstream molecular/metabolic changes modulated photosynthesis differentially. In contrast, salinity induced reduction in both phases of photosynthesis, which were more impaired in silenced plants. These changes were associated with root morphology alterations but not shoot growth. Both studied lines displayed increased GPX activity but H2 O2 content did not change in response to salinity. Transformed plants exhibited lower photorespiration, water use efficiency and root growth, indicating that GPX1 could be important to salt tolerance. Growth reduction of GPX1s line might be related to photosynthesis impairment, which in turn could have involved a cross talk mechanism between mitochondria and chloroplast originated from redox changes due to GPX1 deficiency.
Assuntos
Inativação Gênica , Glutationa Peroxidase/metabolismo , Mitocôndrias/metabolismo , Oryza/fisiologia , Fotossíntese , Proteínas de Plantas/metabolismo , Salinidade , Biomassa , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/efeitos da radiação , Gases/metabolismo , Inativação Gênica/efeitos dos fármacos , Inativação Gênica/efeitos da radiação , Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Luz , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos da radiação , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/efeitos da radiação , Oryza/efeitos dos fármacos , Oryza/efeitos da radiação , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Fenótipo , Fotossíntese/efeitos dos fármacos , Fotossíntese/efeitos da radiação , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Raízes de Plantas/efeitos da radiação , Cloreto de Sódio/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/efeitos da radiaçãoRESUMO
The physiological role of peroxisomal ascorbate peroxidases (pAPX) is unknown; therefore, we utilized pAPX4 knockdown rice and catalase (CAT) inhibition to assess its role in CAT compensation under high photorespiration. pAPX4 knockdown induced co-suppression in the expression of pAPX3. The rice mutants exhibited metabolic changes such as lower CAT and glycolate oxidase (GO) activities and reduced glyoxylate content; however, APX activity was not altered. CAT inhibition triggered different changes in the expression of CAT, APX and glutathione peroxidase (GPX) isoforms between non-transformed (NT) and silenced plants. These responses were associated with alterations in APX, GPX and GO activities, suggesting redox homeostasis differences. The glutathione oxidation-reduction states were modulated differently in mutants, and the ascorbate redox state was greatly affected in both genotypes. The pAPX suffered less oxidative stress and photosystem II (PSII) damage and displayed higher photosynthesis than the NT plants. The improved acclimation exhibited by the pAPX plants was indicated by lower H2 O2 accumulation, which was associated with lower GO activity and glyoxylate content. The suppression of both pAPXs and/or its downstream metabolic and molecular effects may trigger favourable antioxidant and compensatory mechanisms to cope with CAT deficiency. This physiological acclimation may involve signalling by peroxisomal H2 O2 , which minimized the photorespiration.
Assuntos
Antioxidantes/metabolismo , Ascorbato Peroxidases/genética , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Oryza/fisiologia , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Ascorbato Peroxidases/metabolismo , Catalase/genética , Catalase/metabolismo , Respiração Celular , Técnicas de Silenciamento de Genes , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Oryza/enzimologia , Oryza/genética , Oryza/efeitos da radiação , Oxirredução , Estresse Oxidativo , Peroxissomos/enzimologia , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente ModificadasRESUMO
Photosynthesis involves the conversion of sunlight energy into stored chemical energy, which is achieved through electron transport along a series of redox reactions. Excess photosynthetic electron transport might be dangerous due to the risk of molecular oxygen reduction, generating reactive oxygen species (ROS) over-accumulation. Avoiding excess ROS production requires the rate of electron transport to be coordinated with the capacity of electron acceptors in the chloroplast stroma. Imbalance between the donor and acceptor sides of photosystem I (PSI) can lead to inactivation, which is called PSI photoinhibition. We used a light-inducible PSI photoinhibition system in Arabidopsis thaliana to resolve the time dynamics of inhibition and to investigate its impact on ROS production and turnover. The oxidation state of the PSI reaction center and rates of CO2 fixation both indicated strong and rapid PSI photoinhibition upon donor side/acceptor side imbalance, while the rate of inhibition eased during prolonged imbalance. PSI photoinhibition was not associated with any major changes in ROS accumulation or antioxidant activity; however, a lower level of lipid oxidation correlated with lower abundance of chloroplast lipoxygenase in PSI-inhibited leaves. The results of this study suggest that rapid activation of PSI photoinhibition under severe photosynthetic imbalance protects the chloroplast from over-reduction and excess ROS formation.
RESUMO
H2O2, which is continually produced by aerobic metabolism, is a cytotoxic molecule when in high levels. However, low levels can act as a signaling molecule able to regulate the expression of stress responses, senescence, programmed cell death, plant growth, and development. Ascorbate peroxidase (APX) enzyme plays an essential role in the control of intracellular H2O2 levels. Here, the function of a gene encoding a peroxisomal APX (OsAPX4) from rice (Oryza sativa L.) was studied. OsAPX4 gene expression can be detected in roots and panicles, but the highest expression level occurs in leaves. Silencing of OsAPX4 and OsAPX3 expression in RNAiOsAPX4 did not affect the growth of plants under growth chamber conditions, but aging transgenic plants interestingly displayed an early senescence phenotype. Leaf fragments from silenced plants were also more sensitive to induced senescence conditions. RNAiOsAPX4 plants did not present detectable changes in intracellular H2O2 levels, but biochemical analyses showed that transgenic plants displayed some decreased APX activity in the chloroplastic fraction. Also, the peroxisomal enzyme glycolate oxidase exhibited lower activity, whereas catalase activity was similar to non-transformed rice. The results imply that OsAPX4 gene has an important role in leaf senescence pathway mediated by ROS signaling.