Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
The Paradox of Aerobic Life • All life on earth is based on redox reactions (reduction; gain of ê, oxidation; loss of ê), using reductive processes to store energy and oxidative processes to release it. The unusual chemistry of O2 makes it possible to integrate highly reactive oxygen in life-giving redox metabolism. • Oxygen is essential, but toxic • Aerobic cells face constant danger from reactive oxygen species (ROS). • ROS can act as mutagens, cause lipid peroxidation and denature proteins. 1 The role of oxygen in plant growth and responses to environment Oxygen as the regulator of environmental responses We will talk about •What are ROS • ROS chemistry • ROS generation & decomposition (during Environmental stress) • ROS importance in plants • ROS signaling - ROS perception and signal transduction; - the downstream physiological effects of ROS •( ROS in plant disease) - induction of Programmed cell death (Apoptosis) - induction of defense reactions • The role of ROS in adaptation to stress(es) - the role of mitochondria and of intracellular repair systems - ROS in stress cross-talk 2 Free radicals a radical is any chemical species that has unpaired electrons, i.e. contains at least one electron that occupies an atomic or molecular orbital by itself. free radicals are capable of independent existence, while bound radicals are part of a larger molecular structure. Radicals can have positive, negative, or neutral charge • For example, O2- (superoxide anion radical) and OH- (hydroxyl ion) . are negatively charged radicals, while H (hydrogen radical) and OH (hydroxyl radical) are uncharged. • A) Ionization: H-O-H H+ + OH- • B) Radiolysis: H-O-H H + OH . . In A), 2ê are transferred to oxygen, with the resultant production of charged products; in B), 1 ê goes to oxygen and the other to hydrogen, with the consequence that the reaction products are uncharged . • The Earth was originally anoxic • Metabolism was anaerobic • O2 started appearing ~2.5 x 109 years ago Anaerobic metabolism-glycolysis Glucose + 2ADP + 2Pi Lactate + 2ATP + 2H2O O2 an electron acceptor in aerobic metabolism Glucose + 6O2 + 36ADP + 36Pi 4 6CO2 + 36ATP + 6H2O There are just enough electrons to make the whole atom electrically neutral 5 Basics of Redox Chemistry 6 Term Definition Oxidation Gain in oxygen Loss of electrons Reduction Loss of oxygen Gain of hydrogen Gain of electrons Oxidant Oxidizes another chemical by taking electrons, hydrogen, or by adding oxygen Reductant Reduces another chemical by supplying electrons, hydrogen, or by removing oxygen Oxidation-reduction (redox) reactions comprise a major class of biochemical reactions 1) BioEnergetics, the reactions that lead to the generation of > 95% of the energy utilized by aerobic organisms. 2) Chemical transformations e.g. alcohol dehydrogenase, fatty acid desaturase (introduces double bonds into fatty acids). 3) Detoxification-the conversion of the predominantly lipid-soluble toxic compounds present in our environment (e.g. DDT, many drugs) into water-soluble derivatives that can then be excreted. Electron transfers --> the oxidation of intermediary metabolites by O2 in the mitochondria . It often requires the successive transfer of H atoms or electrons, first to NAD+, then from NADH to an ubiquinone (Q), next from QH2 to ferricytochrome c and finally from ferrocytochrome c to O2. These reactions are catalysed, e.g., by an oxidoreductase using NAD+ or NADP+ as acceptor, NADH:Q oxidoreductase 7 Good info source: http://www.plantstress.com/Articles/Oxidative%20Stress.htm The Paradox of Aerobiosis • Oxygen is essential, but toxic. • Aerobic cells face constant danger from reactive oxygen species (ROS). • ROS can act as mutagens, they can cause lipid peroxidation and denature proteins. 8 Environmental factors that induce oxidative stress Root growth Good study source: http://cropsoil.psu.edu/Courses/AGRO518/Oxygen.htm 9 2 billion years of REDOX regulation • ALL LIVING ORGANISMS are oxidation– reduction (redox) systems. They use anabolic, reductive processes to store energy and catabolic, oxidative processes to release it. • Plants have perfected the art of redox control. Indeed, redox signals are key regulators of plant metabolism, morphology, and development. These signals exert control on nearly every aspect of plant biology from chemistry to development, growth, and eventual death. 10 Atomic and molecular oxygen Molecular oxygen can accept a total of 4 electrons atomic oxygen: 1s22s22px22py12pz1 molecular oxygen: s1s2 s*1s2 s2s2 s*2s2 s2pz2 p2px2 p2py2 p*2px1 p*2py1 11 Molecular oxygen is a di- or biradical it has two unpaired electrons and is paramagnetic Superoxide The addition of one electron to O2 gives the electron configuration s1s2 s*1s2 s2s2 s*2s2 s2pz2 p2px2 p2py2 p*2px2 p*2py1 - superoxide, O2- . Peroxide (O-O2-) And another gives the electron configuration s1s2 s*1s2 s2s2 s*2s2 s2pz2 p2px2 p2py2 p*2px2 p*2py2 - peroxide, O22-/H2O2 Bond order = (10-8)/2 = 1 4 anti-bonding p* electrons, rapidly stabilised by accepting 2 protons → H202 Hydroxyl radical and ion • HO• HO- Bond order = (10-9)/2 = ½; Highly unstable O2- (H2O) and O -· (oxyl and/or hydroxyl radical), Oxygen-summary 16 • Ground-state oxygen has 2-unpaired electrons : : : : . O:O . • The unpaired electrons have parallel spins • Oxygen molecule is minimally reactive due to spin restrictions 17 Free radicals have one or more unpaired electrons in their outer orbital, indicated in formulas as []. As a consequence they increased reactivity to other molecules. This reactivity is determined by the ease with which a species can accept or donate electrons. The prevalence of oxygen in biological systems means that oxygen centered radicals are the most common type found O2 is central to metabolism in aerobic life, as a terminal electron acceptor, being reduced to water. Transfer of electron to oxygen yields the reactive intermediates. 18 The beginnings 1775 - Priestley: discovery of O2 observation of toxic effect of O2 1900 – Moses Gomberg: discovery of triphenylmethyl radical Until 1950/60: minimal attention was given to biological actions of free radicals and reactive oxygen species (ROS) 19 Evidence on the existence of ROS 1954 - Gerschman et al. : Recognition of similarities between radiation and oxygen toxicity 1969 - McKord and Fridovich: Discovery of superoxide dismutase; suggested the existence of endogenous superoxide 1973 - Babior et al.: Recognition of the relationship between superoxide production and bactericidal activity of neutrophils 1981 - Granger et al.: recognition of the relationship between ROS production and ischemia/reperfusion induced gut injury 20 “Longevity” of reactive species 21 Reactive Species Half-life Hydrogen peroxide Organic hydroperoxides Hypohalous acids ~ minutes Peroxyl radicals Nitric oxide ~ seconds Peroxynitrite ~ milliseconds Superoxide anion Singlet oxygen Alcoxyl radicals ~ microsecond Hydroxyl radical ~ nanosecond Half-life of some reactive species Reactive species Half-life (s) Hydroxyl radical (OH) 10-9 Alcoxyl radical (RO) 10-6 Singlet oxygen (1O2) 10-5 Peroxynitrite anion (ONOO-) 0.05 – 1.0 Peroxyl radical (ROO) 7 Nitric oxide (NO) 1 - 10 Semiquinone radical minutes/hours Hydrogen peroxide (H2O2) spontan. hours/days Physiol conc. (mol/l) 10-9 10-9 - 10-7 (accelerated by enzymes) Superoxide anion (O2-) spontan. hours/days (by SOD accel. to 10-6) Hypochlorous acid (HOCl) dep. on substrate 22 10-12 - 10-11 Oxidation reactions Oxidation loss of H2 or gain of O, O2, or X2 Reduction gain of H2 or loss of O, O2, or X2 The loss or gain of H2O or HX are not considered oxidation-reduction reactions. X=halogen 23 Radical-mediated reactions Addition R. + H2C=CH2 R-CH2-CH2. Hydrogen abstraction R. + LH RH + L. Electron abstraction R. + ArNH2 R- + ArNH2.+ Termination R. 24 + Y. Disproportionation CH3CH2. + CH3CH2. R-Y CH3CH3 + CH2=CH2 Fenton reaction (1894) Cu1+ Cu2+ Haber and Weiss extension (1934) Oxidizing molec Reducung molec 25 Hydroxyl radical reactions addition of OH to the organic molecule Stable oxidised products abstraction reaction of the .OH radical: oxidation of organic substrates Chain reactions 26 Enzymatic sources of ROS Xanthine oxidase Hypoxanthine + 2O2 --> Xanthine + O2.- + H2O2 NADPH oxidase NADPH + O2 --> NADP+ + O2.- Amine oxidases R-CH2-NH2 + H2O + O2 --> R-CHO + NH3 + H2O2 Myeloperoxidase Hypohalous acid formation H2O2 + X- + H+ --> HOX + H2O NADH oxidase reaction Hb(Mb)-Fe3+ + ROOH --> Compound I + ROH Compound I + NADPH --> NAD· + Compound II Compound II + NADH --> NAD· + E-Fe3+ NAD· + O2 --> NAD+ + O2.- Aldehyde oxidase 2R-CHO + 2O2 --> 2R-COOH + O2.- Dihydroorotate dehydrogenase Dihydroorotate + NAD· + O2 --> NADH + O2.- + Orotic acid Nonenzymatic sources of ROS and autooxidation reactions Fe2+ + O2 --> Fe3++ O2.Hb(Mb)-Fe2+ + O2 --> Hb(Mb)-Fe3++ O2.Catecholamines + O2 --> Melanin + O2.- Reduced flavin Leukoflavin + O2 --> Flavin semiquinone + O2.- Coenzyme Q-hydroquinone + O2 --> Coenzyme Q (ubiquinone) + O2 .Tetrahydropterin + 2 O2 --> Dihydropterin + 2 O2.- 28 Lipid peroxidation 1.1 - Initiation Peroxidation sequence starts with the attack of a ROS (with sufficient reactivity) able to abstract a hydrogen atom from a methylene group (- CH2-), these hydrogen having very high mobility. This attack generates easily free radicals from polyunsaturated fatty acids. .OH is the most efficient ROS to do that attack, whereas O2.- is much less reactive Under aerobic conditions conjugated dienes are able to combine with O2 to give a peroxyl (or peroxy) radical, ROO .. peroxyl radical is able to abstract H from another lipid molecule (adjacent fatty acid), especially in the presence of Fe/Cu, causing a chain reaction. 29 The peroxidation of linoleic acid initiation, propagation and termination Peroxidation is initiated when a reactive oxygen species abstracts a methylene hydrogen from an unsaturated fatty acid found in the lipid membrane forming a lipid radical (L·). This lipid radical then reacts with molecular oxygen forming a lipid hydroperoxyl radical (LOO·) which can then react abstract a methylene hydrogen from a neighboring unsaturated fatty 30 acid forming a lipid hydroperoxide (LOOH) ROS Arise Throughout the Cell Wounding Pathogens Chilling Ozone Cell Wall Pathogens Wounding , Chilling Ozone Cell Wall Mitochondrion Mitochondrion Post-transcriptional Pos t-tra nscriptiona l Effects Effe cts Drought Salinity Drought , Salinity Cytosol Cytosol Antioxidant genes Antioxidant genes Nucleus (ROS su bce llul ar si tes un cle ar) Nucleus Gene Ex pression Chloroplast Gene Expression Chloroplast Pos t-tra nscriptiona l Effe cts Post-transcriptional Paraquat , Effects High Light + Chilling Sulfur Dioxide Paraquat High Light + Chilling Sulfur Dioxide 31 ROS subcellular sites unclear , The electron transport system in the thylakoid membrane showing 3 possible sites of activated oxygen production auto-oxidizable Mehler reaction 32 a) Singlet oxygen may be produced from triplet chlorophyll in the light harvesting complex. b) Superoxide and hydrogen peroxide may "leak" from the oxidizing (watersplitting) side of PSII. c) Triplet oxygen may be reduced to superoxide by ferredoxin on the reducing side of PSI, especially when NADP is limiting (NADPH oxidation by Calvin cycle low). (a) The water–water cycle. (b) The ascorbate–glutathione cycle. (c) The glutathione peroxidase (GPX) cycle. (d) CAT. SOD acts as the first line of defense converting O2− into H2O2. Ascorbate peroxidases (APX), GPX and CAT then detoxify H2O2. In contrast to CAT (d), APX and GPX require an ascorbate (AsA) and/or a glutathione (GSH) regenerating cycle (a–c). This cycle uses electrons directly from the photosynthetic apparatus (a) or NAD(P)H (b,c) as reducing power. ROIs are indicated in red, antioxidants in blue and ROI-scavenging enzymes in green. Abbreviations: DHA, dehydroascorbate; DHAR, DHA reductase; Fd, ferredoxin; GR, glutathione reductase; GSSG, oxidized glutathione; MDA, monodehydroascorbate; MDAR, MDA reductase; PSI, photosystem I; tAPX, thylakoid-bound APX. The redox cycling of ascorbate in the chloroplast often referred to as the Halliwell-Asada pathw 34 ROS production in Mitochondria Electron transfers oxidation of intermediary metabolites by O2 require the successive transfer of H+ or ê, first to NAD+, then from NADH to an ubiquinone (Q), next from QH2 to ferricytochrome c and finally from ferrocytochrome c to O2. These reactions are catalysed, e.g., by an oxidoreductase using NAD+ or NADP+ as acceptor, NADH:Q oxidoreductase ETC in the inner plant mitochondria membrane H+-pumping of CI, III, and IV. ROS production at the two main sites, CI and III. Since UQ• is bound to the inner and outer membranes in CIII, ROS can be formed on either side of the membrane. CI, NADH dehydrogenase; CII, succinate dehydrogenase; CIII, ubiquinol-cytochrome bc1 reductase; CIV, cytochrome c oxidase 35 The more you eat the more mitochondria respiration and more ROS you get Mol Cel Biol, 2000, p. 7311-7318, Vol. 20, Mitochondria as a source of ROS The source of mitochondrial ROS involves a non-heme Fe protein that transfers ê to O2. This occurs primarily at Complex I (NADH-coenzyme Q) and, to a lesser extent, following the autooxidation of coenzyme Q from the Complex II (succinate-coenzyme Q) and/or Complex III (coenzyme QH2-cytochrome c reductases) sites. The precise contribution of each site to total mitochondrial ROS production is probably determined by local conditions including chemical or physical damage to the mitochondria, oxygen availability and the presence of xenobiotics. Kehrer JP (2000) Toxicology 149: 43-50 36 Functions of the alternative oxidase Option for envir stress regulation In the electron-transport chains of mitochondrial (a) and chloroplast (b), AOX diverts electrons that can be used to reduce O2 into O2- and uses these electrons to reduce O2 to H2O. In addition, AOX reduces the overall level of O2, the substrate for ROI production, in the organelle. AOX is indicated in yellow and the different components of the electron-transport chain are indicated in red, green or gray. AOX may also work 37 as a bypass to oxidize NADH and FADH2 under ADP-limiting conditions under which the cytochrome oxidase pathway is restricted plant mitochondria in stress response In mammalian mitochondria, 1-5% of the oxygen consumed in vitro goes to ROS production. Antimycin, a complex III inhibitor that does not block O2.- formation, increased both O2.- generation and membrane damage (BBA1268,249) The major sites of ROS production are complex I and the ubisemiquinone in complex III. The latter activity is completely inhibited by the complex IV inhibitor KCN, which interrupts the Q cycle and prevents the formation of ubisemiquinone. KCN can thus be used to distinguish between complex I and III contributions to ROS 38 Annu. Rev. Plant Physiol. Plant Molec. Biol. 52, 561-591 Extra- and intracellular sources of ROS in plants. XOD, xanthine oxidase 39 Prooxidants R3C. Carbon-centered Free Radicals: Any species capable of independent existence that contains one or more unpaired electrons A molecule with an unpaired electron in an outer valence shell Non-Radicals: Species that have strong oxidizing potential Species that favor the formation of strong oxidants (e.g., transition metals) R3N. Nitrogen-centered R-O. Oxygen-centered R-S. Sulfur-centered H2O2 Hydrogen peroxide HOCl- Hypochlorous acid O3 Ozone 1O 2 Singlet oxygen ONOO- Peroxynitrite Men+ 40 Transition metals Reactive Oxygen Species (ROS) 41 Radicals: Non-Radicals: O2.- Superoxide H2O2 Hydrogen peroxide .OH Hydroxyl HOCl- Hypochlorous acid RO2. Peroxyl O3 Ozone RO. Alkoxyl 1O 2 Singlet oxygen HO2. Hydroperoxyl ONOO- Peroxynitrite Oxidative Protection Oxidative Stress Antioxidants Oxidants Oxidative Stress Oxidative Protection Oxidants: • Superoxide, Hydrogen peroxide, hydroxyl, nitric oxide, peroxynitrite • Auto-oxidation, Enzymes, Ischaemia-Reperfusion, Respiratory burst, organelles • Damage to lipids, protein, DNA • Consequences Repair, adaptation or death Antioxidants ??? Oxidative stress occurs when the ROS generation exceeds the ROS removal ROS scavenging molecules plant antioxidants Ascorbate Glutathione Polyphenols Flavonoids Lipoic acid Flavonoids Ponce de León Enzymes: SOD Catalase Glutathione peroxidase Ascorbate peroxidase Thioredoxins Glutaredoxins Nature 425, 132-133 Reactive Nitrogen Species (RNS) Radicals: NO. Nitric Oxide NO2. Nitrogen dioxide 44 Non-Radicals: ONOOPeroxynitrite ROONO Alkyl peroxynitrites N2O3 Dinitrogen trioxide N2O4 Dinitrogen tetroxide HNO2 Nitrous acid NO2+ Nitronium anion NONitroxyl anion NO+ Nitrosyl cation NO2Cl Nitryl chloride Nitric Oxide N O NO refers to nitrosyl radical (•NO) and its nitroxyl (NO–) and nitrosonium (NO+) ions Freely diffusible, gaseous free radical. First described in 1979 as a potent relaxant of peripheral vasculature. Used by the body as a signaling molecule. Nitric Oxide in plants Affects aspects of plant growth and development. Affects the responses to: light, gravity, oxidative stress, pathogens. Can be a maturation and senescence factor Has a concentration dependent cytotoxic or protective (antioxidant) effects. NO-induced cell death in Arabidopsis occurs independently of ROS Cells were treated with methyl viologen (MV) to generate O2 · , NO donor (RBS), and/or the peroxynitrite scavenger and SOD-mimetic MnTBAP cGMP in NO-induced cell death Cells were pre-treated with ODQ (guanylate cyclase inhibitor) and/or 8Br-cGMP prior to RBS. The effects of the caspase-1 inhibitor Ac-YVAD-CMK on NOand H2O2-induced cell death NO and Cell Death +PBITU NO + H2O2 cause cell death NO + O2- react to form peroxynitrite Peroxynitrite (ONOO -) does not cause cell death Too much O2- ‘mops up NO’ – no death 49 Delladonne et al. (2001) PNAS 98:13454 % Cell Death Psm (avrRpm 1) mM H O mM H O NO mM H O +NO Endogenous sources of ROS and RNS (in animals) Microsomal Oxidation, Flavoproteins, CYP enzymes Xanthine Oxidase, NOS isoforms Myeloperoxidase (phagocytes) Transition metals Endoplasmic Reticulum Cytoplasm Lysosomes Fe Cu Oxidases, Flavoproteins Peroxisomes Mitochondria Plasma Membrane 50 Lipoxygenases, Prostaglandin synthase NADPH oxidase Electron transport PEROXISOME • • • • • b-oxidation of fatty acids bile acid synthesis purine and polyamine catabolism amino acid catabolism oxygen metabolism Fatty Acid Fatty acyl-CoA synthetase Acyl-CoA H2O2 Acyl-CoA oxidase Enoyl-CoA Enoyl-CoA hydrolase Hydroxyacyl-CoA Hydroxyacyl-CoA dehydrogenase Ketoacyl-CoA Thiolase Acetyl-CoA 51 Acyl-CoA shortened by two carbons Oxidative Phosphorylation & ROS NADH + H+ e- Increasing Reducing Power FADH2 NAD+ -0.32 V O2 + e- => O2.- -0.45 V O2 + 2H+ + 2e- => H2O2 -0.11 V O2 + 4H+ + 4e- => H2O 0.82 V FAD -0.06 V e- bII bIII 0.04 V e- Cytochromes cII cIII 0.25 V aIII 0.29 V O2 0.82 V e- aII e- H2O 52 Many key oxidoreductases such as dehydrogenases, hydrogenases, nitrogenases, and the many oxygen enzymes of synthesis, drug detoxification, respiration photosynthesis, include a chain of single electron transferring redox Porphyrins, chlorins, iron sulfur clusters, flavins or quinones are common cofactors. members of the chains. 53 The chains, which can comprise 2 to 8 cofactors, serve to ferry single ê between one site of substrate oxidation/reduction and another, or to a place close to the surface of the enzyme where they are exchanged with other single ê transferring redox protein partners, such as cytochrome c or flavodoxin. The distance covered by these linear chains can be rather long. Intracellular ROS abundance in WT and Aox1 transgenic cultured tobacco cells. antisense • • 54 sense Plant Mitochondria also Contain an Uncoupling Protein Mammalian mitochondria do not contain the AOX. Instead they have an uncoupling protein that increases the proton permeability of the inner mitochondrial membrane and in that way dissipates the proton gradient. This is another mechanism for reducing the ATP production and increasing heat production. Surprisingly, plant mitochondria also contain a protein resembling the uncoupling protein Oxygen consumption in oxidatively stressed mitochondria. A C mal+glut ADP suc + ADP C rot KCN C G/GO 2 3 1 0 1 Time (min) ADP C H2O2 3 2 1 Time (min) 55 0 Time (min) mal+glut B G/GO 0 A) Arabidopsis cells were treated with G/GO. Electron transport was initiated by addition of complex I substrates, malate plus glutamate and NAD+. Coupling between the electron transport and ATP production was estimated by the addition of ADP. The role of complex I on oxygen consumption was examined by addition of rotenone. Numbers indicate the rate of oxygen consumption. B) Cells were spiked with 5 mM H2O2 and mitochondria were isolated 3 h later. Electron transport across complex I was measured as described in (A). C) Electron transport across complex III was measured with 10 mM succinate plus 100 mM ADP. The dependence of oxygen consumption on the cytochrome c pathway was examined by addition of 50 mM KCN ROS production in isolated mitochondria B A 0 .5 0 .4 0 .3 0 .2 0 .1 0 0 Control 5 H2O2 (mM) G/GO A) Mitochondria isolated from control or cells treated for 3 h with G/GO and stained with DHDR123 1 C 0.5 0.4 0.3 0.2 0.1 0 malate control 56 succinate H2O2 pretreated Mitochondrial Aconitase Is a Source of Hydroxyl Radical Aconitase (aconitate hydratase; EC 4.2.1.3) catalyses the stereospecific isomerisation of citrate to isocitrate via cisaconitate in the tricarboxylic acid cycle, a nonredox active process - H2O + H2O (1) + H2O citrate - H2O cis-Aconitate Isocitrate Iron-sulphur clusters 57 [Fe4S4](S Cys)3(H2O)n [Fe3S4](S Cys)3 (Because of the Aconitase role in cellular energy production, this enzyme function is well positioned as an important marker relative to biological decline) Recently it has been proposed that the reaction between mitochondrial aconitase and superoxide plays a major role in mitochondrial oxidative damage. During this reaction, the iron is released from m-aconitase as iron(II) with the concomitant generation of H2O2. This facilitates the formation of "free" hydroxyl radical in mitochondria. In the presence of intracellular reducing agents (e.g. glutathione, ascorbate, and NADPH), iron(II) is reincorporated into the inactive form of m-aconitase to regenerate the active form. According to this proposal, hydroxyl radical is continuously generated in mitochondria as a result of the reaction between superoxide and aconitase. J Biol Chem, Vol. 275, 14064-14069, 2000 58 59 The plant mitochondria may integrate stress signals for programmed cell death (PCD). There are many different situations that lead to cytochrome c release. These include oxidative stresses that induce permeability transition (PT) pore formation, stresses on electron transport and a rise in Ca2+ levels. It is proposed that when cells are unable to maintain metabolic homeostasis and the stresses overwhelm the cell, that mitochondria release cytochrome c triggering death. These stresses are normal components of PCD in plants. Models for the release of cytochrome c from mitochondria 60 In models a and b, the outer mitochondrial membrane ruptures as a result of swelling of the mitochondrial matrix, allowing cytochrome c to escape from mitochondria. Model a involves opening of the PTP whereas model b involves closure of the VDAC and hyperpolarization of the inner mitochondrial membrane as the causes of matrix swelling. In models c–e, a large channel forms in the outer membrane (via VDAC), allowing cytochrome c release, but mitochondria are not damaged Integration of stress signals by Mitochondria 61 (a) In all cases Cytochrome c release into the cytosol requires calcium flux at low cellular ATP levels. In the first (b), the permeability transition pore (PT pore) forms as a complex with the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT), cyclophilin D (not shown) and the benzodiazepine receptor (not shown). The PT pore permits water to move into the matrix; outer membrane rupturing occurs when the inner membrane swells. (c) Cytochrome c can also be released directly via the VDAC. Mitochondria in Apoptosis Bax 62 Increases in cytosolic Ca2+ due to activation of ion channel-linked receptors, can induce permeability transition (PT) of the mitochondrial membrane. PT constitutes the first rate-limiting event of the common pathway of apoptosis. Upon PT, apoptogenic factors leak into the cytoplasm from the mitochondrial intermembrane space. Two such factors, cytochrome c and apoptosis inducing factor (AIF), begin a cascade of proteolytic activity that ultimately leads to nuclear damage (DNA fragmentation) and cell death. Cytochrome c, a key protein in electron transport, appears to act by forming a multimeric complex with Apaf-1, a protease, which in turn activates procaspase 9, and begins a cascade of activation of downstream caspases. Smac/Diablo is released from the mitochondria and inhibits IAP (inhibitor of apoptosis) from interacting with caspase 9 leading to apoptosis. Bcl-2 and Bcl-X can prevent pore formation and block the release of cytochrome c from the mito Nitric oxide (NO) is a pleiotropic signalling molecule that binds to cytochrome c oxidase (complex IV) reversibly and in competition with oxygen. Endogenously generated NO disrupts the respiratory chain and causes changes in mitochondrial Ca2+ flux. 63 64 Oxidative Burst in the Plasma Membrane apoplastic peroxidase NADPH oxidase 65 Activation of NADPH oxidase by pathogens (elicitors) rbohA EF hands – Ca2+binding sites. gp91phox Arabidopsis Rice Human Exogenous H2O2 rescues both Ca2+ channel activation and stomatal closing in atrbohD/F placing it upstream of Ca2+ Resistance responses 66 Activation of NADPH Oxidase Occurs within Intracellular Compartments animals Molec. Cell 11, 35-47 (2003) 67 plants Oxidative Protection Oxidants Oxidative Stress Oxidative Stress Antioxidants Oxidative Protection Oxidants: • Superoxide, Hydrogen peroxide, hydroxyl, nitric oxide, peroxynitrite • Auto-oxidation, Enzymes, Ischaemia-Reperfusion, Respiratory burst, organelles • Damage to lipids, protein, DNA • Consequences Repair, adaptation or death Antioxidants ??? 68 Oxidants Oxidative Damage Enzymatic Defences – catalytically remove ROS Antioxidants Metal Sequestration Proteins Low MW Antioxidants (Repair Processes) Other Protective Compounds e.g. HSPs Amounts Variable - cell types & tissues Effectiveness Variable - (production site, radical species) 69 ROS Detoxification Catalytic Activity: Mn3+ + O2- (Mn3+-O2-) Mn2+ + O2 Mn2+ + O2- (Mn2+-O2-) + 2H+ Mn3+ + H2O2 Catalases: 2 H2O2 ---> 2 H2O + O2 Peroxidases: AH2 + H2O2 ---> A + 2 H2O 70 A is an electron donor Cellular localization of SODs 71 Halliwell-Asada pathway redox cycling of ascorbate in the chloroplast Antioxidant concentration in plant cells ascorbate (10-100 mM), glutathione (1-10 mM) 72 Light-induced necrosis in Cat1AS plants and protection by elevated CO2 Complementation by catalase Changes in ascorbate and glutathione contents in leaves of Cat1AS and wild-type tobacco during light stress (A) Effect of a shift from LL to HL on the levels of reduced (LAA) and oxidized (DHAA) ascorbate. (6 h and 48 h exposure to HL). (B) Effect on reduced (GSH) and oxidized (GSSG) glutathione 73 Flavonoids are Chemo-preventive Agents 74 Flavonoid Structure • 200-300 Related Polyphenols • Substitution on the C ring distinguishes the classes flavonoids • Substitution on the A and B rings distinguish structures within a class • Three potential metal binding sites exist 75 3' 2' 4' B 8 5' O 1 7 A C 5 4 6 2 6' 3 OH 1 OH HO O OH OH 3 O 2 Phenotypes associated with Bax expression in transgenic plants ROS Production in Plants Expressing Bax 76 PNAS | 2001 | vol. 98 | 12295 PNAS 1999; 96: 7956-7961. Ascorbate reaction with superoxide can serve a physiologically similar role to SOD: 2 O 2 + 2H+ + ascorbate --> 2H2O2 + dehydroascorbate The reaction with hydrogen peroxide is catalysed by ascorbate peroxidase : H2O2+ 2 ascorbate --> 2H2O + 2 monodehydroascorbate The indirect role of ascorbate as an antioxidant is to regenerate membrane-bound antioxidants, like a-tocopherol, that scavenge peroxyl radicals and singlet O2, respectively: tocopheroxyl radical + ascorbate tocopherol + monodehydroascorbate The above reactions indicate that there are two different products of ascorbate oxidation, monodehydroascorbate and dehydroascorbate, representing 1e and 2e transfers, respectively. The monodehydroascorbate can either spontaneously dismutate (below) or is reduced to ascorbate by NAD(P)H monodehydroascorbate reductase (below): 2 monodehydroascorbate ascorbate + dehydroascorbate monodehydroascorbate + NAD(P)H ascorbate + NAD(P) The dehydroascorbate is unstable above pH6, decomposing into tartrate and oxalate. To prevent this, dehydroascorbate is rapidly reduced to ascorbate by dehydroascorbate reductase using reducing equivalents from glutathione (GSH): 77 2 GSH + dehydroascorbate GSSG + ascorbate interactions that lead to recruitment of IP3 receptors during apoptosis The positive feedback between IP3 receptor-mediated Ca2+ release and mitochondria underlies the generation of Ca2+ signals that accelerate the rate of cell death. 78 The apoptosis-inducing cycle of Ca2+ between IP3 receptors and mitochondria can be initiated by a variety of mechanisms, including non-specific entry of Ca2+ following membrane damage. The role of Aquaporins and membrane damage in chilling and hydrogen peroxide induced changes in the hydraulic conductance of maize roots 79 Scheme summarizing the interpretation of the results. Chilling causes an initial decrease of Lo in both genotypes. After 3 d at 5°C, the tolerant genotype recovers its Lo thanks to the increase in aquaporin abundance and phosphorylation and to the maintenance of membrane integrity. On the contrary, the sensitive genotype does not recover its Lo because of membrane damage caused by oxidative stress. The tolerant genotype can cope with the oxidative stress, but the sensitive genotype cannot. Systemic Signaling and Acclimation in response to excess light H O is a local and systemic signal involved in the adaptation of leaves to high light 2 2 Photodamage & APX2 induction (the arrow indicates the apical region of the rosette) Leaves grown in LL (control) exposed to EL. (A) Chlorosis on detached leaves after 2 hours Systemic induction of APX2-LUC in EL. (B) relative luciferase activity expression. catalase but not SOD diminished APX2 expr. Image of luciferase activity. A part of the whole rosette (as shown) was exposed to EL for 40 min (arrow -> the apical rosette region). A typical primary (1°) EL-exposed leaf and a secondary (2°) LL-exposed leaf are shown Systemic induction of H2O2 by wounding