Ebook Anoxia and Oxidative Stress: Lipid Peroxidation, Antioxidant Status and Mitochondrial Functions in Plants
Research on the formation of reactive oxygen species (ROS) and the consequences in the cell under anoxia is of great importance in the elucidation of essential questions in stress physiology. Oxygen deprivation stress, and particularly transient hypoxia, has been suggested recently as a convenient model for the investigation of O2/ROS sensing. Hence, it is of importance to show direct ROS formation under oxygen deprivation in plant tissues with respect to anoxia tolerance. Another problem, which is of great practical importance, includes physiological processes underlying anoxia tolerance. In the present study emphasis has been placed on the differences between anoxia tolerant (Iris pseudacorus, Oryza sativa or Ovena sativa) and anoxia-intolerant (Iris germanica, Triticum aestivum) plant species in ROS production, development of lipid peroxidation (LP) during propagation and termination phases and antioxidant status of the cells under anoxic stress. In addition, the consequences of re-admission of oxygen (reoxygenation injury) have been studied. The above mentioned parameters can be affected by the metabolic changes brought about by anoxic stress: a decrease in adenylate energy charge, acidification of cytoplasm, elevation of cytosolic Ca2+ concentration, changes in the redox state and alterations in membrane structure and functions. Possible correlations between the parameters representative of oxidative stress and anoxia induced metabolic changes are discussed.
As further evidence for ROS formation, anoxia and especially post-anoxic reoxygenation caused cell wall and plasma membrane associated H2O2 accumulation, visualised by CeCl3 detection and transmission electron microscopy. The results suggest that anoxic stress together with other stresses shares a common mechanism of induction, i.e. generation of ROS. In addition, the peroxidation of lipids was more intensive in anoxia-intolerant plants (Triticum aestivum and Iris germanica), as measured by conjugated diene and triene formation during the propagation phase. The same tendency was observed on the termination stage of LP, characterised by thiobarbituric acid reactive substances (TBARS) accumulation (with the exception of the extremely anoxia-tolerant I. pseudacorus).
Different responses of antioxidant systems in anoxia-tolerant and intolerant plants suggest that there is no universal mechanism incorporating all the antioxidants and leading to ROS detoxification. The plants studied here differed significantly in initial antioxidant content, which did not correlate with anoxia tolerance. The most important characteristic of anoxia tolerance was the ability to maintain a high ratio of reduced to oxidised forms of antioxidants, rather than their absolute levels. However, prolonged anoxia and subsequent reoxygenation led to a decrease in all antioxidants studied (ascorbate, glutathione, tocopherol, superoxide dismutase) indicating oxidative stress and revealing a decrease in the redox state of the cell.
An important feature determining stress tolerance is the ability to preserve energy resources and/or to efficiently terminate ROS formation. Mitochondria are responsible not only for energy conservation, but also for the regulation of Ca2+ fluxes. They produce ROS and are an essential component in the signalling pathway leading to programmed cell death. A tighly regulated inner membrane channel a permeability transition pore (PTP) is induced in animal mitochondria by high matrix Ca2+, dissipation of the inner membrane potential, redox changes, oxidation of GSH and an elevation in ROS level. Consideration of the role of PTP in animal tissues and metabolic changes under anoxia in plant cells imply the possibility of PTP induction in plant mitochondria under stress conditions, although the phenomenon of permeability transition has not been described in plants. In the present study mitochondrial functions under anoxia were studied with respect to PTP induction. High amplitude mitochondrial swelling indicative of the PTP opening (in mammalian mitochondria) was observed in wheat root mitochondria after Ca2+ uptake and energisation by a respiratory substrate. However, this process was insensitive to cyclosporin A, a specific inhibitor of the permeability transition in mammalian mitochondria and, hence, the results are not conclusive on the presence of the PTP in plant mitochondria and require further investigation.
In general, the formation of ROS under oxygen deprivation stress represents a common mechanism of stress response initiation. It was shown that low amounts of oxygen present in the system were sufficient for H2O2 accumulation. Restoration of normoxic conditions caused secondary oxidative stress and led to an increase in LP, membrane damage and exhaustion of antioxidant resources. Lower intensity of oxidative damage in anoxia-tolerant plants demonstrated the higher stability of their membranes. The mechanisms responsible for such stability probably incorporate structural properties of the membranes as well as the antioxidative capacity and the ability to control metabolic functions for a longer time under stress conditions.
CONTENTS
Abbreviations
Original publications
Summary
Preface
1. Introduction
1.1. Physiology of anoxic stress
1.2. Structural adaptations to anoxia
1.3. Anoxia induced metabolic changes
1.4. Anoxia and gene expression
1.5. Membrane function and structure under anoxia
1.6. Role of ROS in the stress response
1.7. Chemistry of reactive oxygen species: Types of ROS
1.8. Lipid peroxidation
1.9. Antioxidant system
- 1.9.1. Superoxide dismutase (SOD)
1.9.2. Catalase and peroxidases
1.9.3. Phospholipid hydroperoxide glutathione peroxidase
1.9.4. Enzymes regenerating active forms of ascorbate and glutathione
1.9.5. Redox active compounds: ascorbate and glutathione
1.9.6. Phenolic compounds
1.9.7. Tocopherols
1.10. Antioxidative network
1.11. Antioxidant status of the cell under stress conditions
1.12. Role of mitochondria in stress response
1.13. Aims of the present study
2. Materials and methods
2.1. Experimental design
2.2. Plant material
2.3. Growth conditions
2.4. Anoxic stress treatment
2.5. Cytochemical visualisation of hydrogen peroxide
2.6. Extraction of lipids
2.7. Detection of lipid-conjugated dienes and trienes
2.8. Second derivative spectrophotometry of conjugated dienes
2.9. Thiobarbituric acid reactive substances (TBARS) assay
2.10. Superoxide dismutase activity determination
2.11. Extraction and analysis of tocopherols by HPLC and massspectrometry
2.12. Ascorbic acid assay
2.13. Determination of reduced and oxidised forms of glutathione
2.14. Isolation of mitochondria
2.15. Measurement of oxygen consumption
2.16. Observation of the swelling of mitochondria
2.17. Measurement of Ca2+ transport across inner mitochondrial membrane
2.18. Determination of membrane potential with Safranine O
3. Results
3.1. Formation of reactive oxygen species (H2O2) and lipid peroxidation
- 3.1.1. Ultrastructural changes caused by anoxic stress
3.1.2. Visualisation of H2O2 under anoxia and reaeration
3.1.3. Formation of conjugated dienes and trienes
3.1.4. Accumulation of TBARS
3.2. Antioxidant status under oxygen deprivation
- 3.2.1. Superoxide dismutase activity
3.2.2. Ascorbic and dehydroascorbic acid content
3.2.3. Changes in glutathione concentration
3.2.4. Tocopherols under anoxia and aeration
3.3. Characterisation of mitochondrial functions
- 3.3.1. Ca2+ uptake by plant mitochondria
3.3.2. Swelling of mitochondria
3.3.3. Inner membrane potential
4. Discussion
4.1. Correlation between ROS formation, lipid peroxidation and anoxia tolerance
- 4.1.1. Anatomical and ultrastructural features
4.1.2. H2O2 formation
4.1.3. Anoxia-induced lipid peroxidation: CD, CT and TBARS
4.2. Antioxidant status under anoxia and reoxygenation
- 4.2.1. Superoxide dismutase
4.2.2. Ascorbate and glutathione pools under anoxia and reoxygenation
4.2.3. Changes in the tocopherol content
4.3. Characterisation of mitochondrial functions and PTP
5. Conclusions and future prospects
6. References
Posted in :
