ABSTRACT
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.
Subject(s)
Ascorbate Peroxidases/genetics , Oryza/enzymology , Reactive Oxygen Species/metabolism , Signal Transduction , Alcohol Oxidoreductases/metabolism , Ascorbate Peroxidases/metabolism , Catalase/metabolism , Cellular Senescence , Chloroplasts/metabolism , Gene Knockdown Techniques , Hydrogen Peroxide/metabolism , Oryza/genetics , Oryza/physiology , Peroxisomes/enzymology , Plant Leaves/enzymology , Plant Leaves/genetics , Plant Leaves/physiology , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Roots/enzymology , Plant Roots/genetics , Plant Roots/physiology , Plants, Genetically Modified , Stress, PhysiologicalABSTRACT
The maintenance of H2O2 homeostasis and signaling mechanisms in plant subcellular compartments is greatly dependent on cytosolic ascorbate peroxidases (APX1 and APX2) and peroxisomal catalase (CAT) activities. APX1/2 knockdown plants were utilized in this study to clarify the role of increased cytosolic H2O2 levels as a signal to trigger the antioxidant defense system against oxidative stress generated in peroxisomes after 3-aminotriazole-inhibited catalase (CAT). Before supplying 3-AT, silenced APX1/2 plants showed marked changes in their oxidative and antioxidant profiles in comparison to NT plants. After supplying 3-AT, APX1/2 plants triggered up-expression of genes belonging to APX (OsAPX7 and OsAPX8) and GPX families (OsGPX1, OsGPX2, OsGPX3 and OsGPX5), but to a lower extent than in NT plants. In addition, APX1/2 exhibited lower glycolate oxidase (GO) activity, higher CO2 assimilation, higher cellular integrity and higher oxidation of GSH, whereas the H2O2 and lipid peroxidation levels remained unchanged. This evidence indicates that redox pre-acclimation displayed by silenced rice contributed to coping with oxidative stress generated by 3-AT. We suggest that APX1/2 plants were able to trigger alternative oxidative and antioxidant mechanisms involving signaling by H2O2, allowing these plants to display effective physiological responses for protection against oxidative damage generated by 3-AT, compared to non-transformed plants.
Subject(s)
Acclimatization/drug effects , Amitrole/toxicity , Ascorbate Peroxidases/metabolism , Catalase/antagonists & inhibitors , Cytosol/enzymology , Gene Silencing/drug effects , Oryza/enzymology , Oxidative Stress/drug effects , Antioxidants/metabolism , Ascorbic Acid/metabolism , Catalase/metabolism , Cell Respiration/drug effects , Cytosol/drug effects , Gene Expression Regulation, Plant/drug effects , Gene Knockdown Techniques , Genes, Plant , Glutathione/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Models, Biological , Oryza/drug effects , Oryza/genetics , Oryza/physiology , Oxidation-Reduction/drug effects , Photosynthesis/drug effects , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified , RNA, Messenger/genetics , RNA, Messenger/metabolism , Time FactorsABSTRACT
The biochemical mechanisms underlying the involvement of cytosolic ascorbate peroxidases (cAPXs) in photosynthesis are still unknown. In this study, rice plants doubly silenced in these genes (APX1/2) were exposed to moderate light (ML) and high light (HL) to assess the role of cAPXs in photosynthetic efficiency. APX1/2 mutants that were exposed to ML overexpressed seven and five proteins involved in photochemical activity and photorespiration, respectively. These plants also increased the pheophytin and chlorophyll levels, but the amount of five proteins that are important for Calvin cycle did not change. These responses in mutants were associated with Rubisco carboxylation rate, photosystem II (PSII) activity and potential photosynthesis, which were similar to non-transformed plants. The upregulation of photochemical proteins may be part of a compensatory mechanism for APX1/2 deficiency but apparently the finer-control for photosynthesis efficiency is dependent on Calvin cycle proteins. Conversely, under HL the mutants employed a different strategy, triggering downregulation of proteins related to photochemical activity, Calvin cycle and decreasing the levels of photosynthetic pigments. These changes were associated to strong impairment in PSII activity and Rubisco carboxylation. The upregulation of some photorespiratory proteins was maintained under that stressful condition and this response may have contributed to photoprotection in rice plants deficient in cAPXs. The data reveal that the two cAPXs are not essential for photosynthesis in rice or, alternatively, the deficient plants are able to trigger compensatory mechanisms to photosynthetic acclimation under ML and HL conditions. These mechanisms involve differential regulation in protein expression related to photochemistry, Calvin cycle and photorespiration.
Subject(s)
Ascorbate Peroxidases/metabolism , Oryza/physiology , Oxygen Consumption/physiology , Photosynthesis/physiology , Plant Proteins/metabolism , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Ascorbate Peroxidases/genetics , Blotting, Western , Catalase/genetics , Catalase/metabolism , Cytosol/enzymology , Dose-Response Relationship, Radiation , Gene Expression Regulation, Plant/radiation effects , Glutamate-Ammonia Ligase/genetics , Glutamate-Ammonia Ligase/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Light , Light-Harvesting Protein Complexes/metabolism , Mutation , Oryza/genetics , Oryza/metabolism , Oxygen Consumption/genetics , Oxygen Consumption/radiation effects , Pheophytins/metabolism , Photosynthesis/genetics , Photosynthesis/radiation effects , Photosystem II Protein Complex/metabolism , Plant Proteins/genetics , Plants, Genetically Modified , Reverse Transcriptase Polymerase Chain Reaction , Ribulose-Bisphosphate Carboxylase/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolismABSTRACT
Current studies, particularly in Arabidopsis, have demonstrated that mutants deficient in cytosolic ascorbate peroxidases (APXs) are susceptible to the oxidative damage induced by abiotic stress. In contrast, we demonstrate here that rice mutants double silenced for cytosolic APXs (APx1/2s) up-regulated other peroxidases, making the mutants able to cope with abiotic stress, such as salt, heat, high light and methyl viologen, similar to non-transformed (NT) plants. The APx1/2s mutants exhibited an altered redox homeostasis, as indicated by increased levels of H2O2 and ascorbate and glutathione redox states. Both mutant and NT plants exhibited similar photosynthesis (CO2) assimilation and photochemical efficiency) under both normal and stress conditions. Overall, the antioxidative compensatory mechanism displayed by the mutants was associated with increased expression of OsGpx genes, which resulted in higher glutathione peroxidase (GPX) activity in the cytosolic and chloroplastic fractions. The transcript levels of OsCatA and OsCatB and the activities of catalase (CAT) and guaiacol peroxidase (GPOD; type III peroxidases) were also up-regulated. None of the six studied isoforms of OsApx were up-regulated under normal growth conditions. Therefore, the deficiency in cytosolic APXs was effectively compensated for by up-regulation of other peroxidases. We propose that signalling mechanisms triggered in rice mutants could be distinct from those proposed for Arabidopsis.
Subject(s)
Ascorbate Peroxidases/metabolism , Catalase/metabolism , Glutathione Peroxidase/metabolism , Oryza/enzymology , Ascorbate Peroxidases/genetics , Carbon Dioxide/metabolism , Catalase/genetics , Chlorophyll/metabolism , Chloroplasts/metabolism , Cytosol/enzymology , Cytosol/metabolism , Gene Expression Regulation, Plant , Glutathione/analysis , Glutathione Peroxidase/genetics , Homeostasis , Hydrogen Peroxide/analysis , Lipid Peroxidation , Oryza/genetics , Oryza/physiology , Oxidation-Reduction , Oxidative Stress , Phenotype , Photosynthesis , Plant Leaves/enzymology , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified , Sequence Deletion , Signal Transduction , Stress, Physiological , Up-RegulationABSTRACT
Hydrolysis of DNA is of increasing importance in biotechnology and medicine. In this Letter, we present the DNA-cleavage potential of metal-free hydroxylamines and oximes as new members of nucleic acid cleavage agents.
Subject(s)
Chemistry/methods , DNA/chemistry , Deoxyribonucleases/chemistry , Deoxyribonucleases/chemical synthesis , Hydroxylamines/chemistry , Metals/chemistry , Oximes/chemistry , Distamycins/pharmacology , Drug Design , Glycerol/chemistry , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Nucleic Acid Conformation , Nucleic Acids/chemistry , SolventsABSTRACT
Transgenic plants have been used widely as expression systems of recombinant proteins in recent years. This process can be an efficient alternative for the large-scale production of proteins. In this work, we present the establishment of transgenic sugarcane expressing a His-tagged canecystatin under the control of the maize ubiquitin promoter. A number of studies have demonstrated that cystatins, which are natural inhibitors of cysteine proteinases, can be used for protection against insect attacks. A transformed sugarcane plant that presented high levels of (HIS)CaneCPI-1 expression, was selected for the purification of this protein through affinity chromatography in a nickel column. This purified (HIS)CaneCPI-1 was immunodetected using a polyclonal antibody, which was also able to detect the (HIS)CaneCPI-1 in a crude extract from transgenic plant leaves. Assays of inhibitory activity performed with the purified (HIS)CaneCPI-1 revealed its ability to inhibit the catalytic activity of midgut cysteine proteinase partially purified from the sugarcane weevil Sphenophorus levis and human cathepsin L in nanomolar order. These studies demonstrate that sugarcane is a viable expression system for recombinant protein production.
