Environmental factors that induce oxidative stress

1
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.

2
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

3
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

4

• 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 6CO2 36ATP
6H2O
5
There are just enough electrons to make the whole
atom electrically neutral
6
Basics of Redox Chemistry
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
7
Oxidation-reduction (redox) reactions comprisea
major class of biochemical reactions

• BioEnergetics, the reactions that lead to the
  generation of gt 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 --gt 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, NADHQ oxidoreductase

Good info source
http//www.plantstress.com/Articles/Oxidative20St
ress.htm
8
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.

9
Environmental factors that induce oxidative stress
Root growth
Good study source
http//cropsoil.psu.edu/Courses/AGRO518/Oxygen.htm

10
2 billion years of REDOX regulation

• ALL LIVING ORGANISMS are oxidationreduction
  (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.

11
Atomic and molecular oxygen
Molecular oxygen can accept a total of 4 electrons
atomic oxygen 1s22s22px22py12pz1
molecular oxygen s1s2 s1s2 s2s2 s2s2 s2pz2
p2px2 p2py2 p2px1 p2py1
12
Molecular oxygen is a di- or biradicalit has two
unpaired electrons and is paramagnetic
13
Superoxide
The addition of one electron to O2 gives the
electron configuration s1s2 s1s2 s2s2 s2s2
s2pz2 p2px2 p2py2 p2px2 p2py1 - superoxide, O2-
.
14
Peroxide (O-O2-)
And another gives the electron configuration s1s2
s1s2 s2s2 s2s2 s2pz2 p2px2 p2py2 p2px2 p2py2
- peroxide, O22-/H2O2
Bond order (10-8)/2 1 4 anti-bonding p
electrons, rapidly stabilised by accepting 2
protons ? H202
15
Hydroxyl radical and ion

HO
HO-
?
O2- (H2O) and O - (oxyl and/or hydroxyl
radical),
Bond order (10-9)/2 ½ Highly unstable
16
Oxygen-summary
17

• Ground-state oxygen has 2-unpaired electrons

• The unpaired electrons have parallel spins

• Oxygen molecule is minimally reactive due to spin
  restrictions

18

• 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.

19
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)

20
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

21
Longevity of reactive species
Reactive Species Half-life Hydrogen
peroxide Organic hydroperoxides
minutes Hypohalous acids Peroxyl radicals
seconds Nitric oxide Peroxynitrite
milliseconds Superoxide anion Singlet oxygen
microsecond Alcoxyl radicals Hydroxyl
radical nanosecond
22
Half-life of some reactive species
Reactive species
Half-life (s)
Physiol conc. (mol/l)
Hydroxyl radical (?OH) Alcoxyl radical
(RO?) Singlet oxygen (1O2) Peroxynitrite anion
(ONOO-) Peroxyl radical (ROO?) Nitric oxide
(?NO) Semiquinone radical Hydrogen peroxide
(H2O2) Superoxide anion (O2?-) Hypochlorous
acid (HOCl)
10-9 10-6 10-5 0.05 1.0 7 1 -
10 minutes/hours spontan. hours/days (accelerated
by enzymes) spontan. hours/days (by SOD accel. to
10-6) dep. on substrate
10-9
10-9 - 10-7
10-12 - 10-11
23
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. Xhalogen
24
Radical-mediated reactions
25
Fenton reaction (1894)
Cu1 ? Cu2
Haber and Weiss extension (1934)
Oxidizing molec
Reducung molec
26
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
27
Enzymatic sources of ROS

• Xanthine oxidase
• Hypoxanthine 2O2 --gt Xanthine
  O2.- H2O2
• NADPH oxidase
• NADPH O2 --gt NADP
  O2.-
• Amine oxidases
• R-CH2-NH2 H2O O2 --gt R-CHO NH3 H2O2
• Myeloperoxidase
  
• Hypohalous acid formation
• H2O2 X- H --gt HOX H2O
• NADH oxidase reaction
• Hb(Mb)-Fe3 ROOH --gt Compound I
  ROH
• Compound I NADPH --gt NAD
  Compound II
• Compound II NADH --gt NAD
  E-Fe3
• NAD O2 --gt
  NAD O2.-
• Aldehyde oxidase
• 2R-CHO 2O2 --gt 2R-COOH
  O2.-
• Dihydroorotate dehydrogenase
• Dihydroorotate NAD O2 --gt NADH O2.-
  Orotic acid

28
Nonenzymatic sources of ROS and autooxidation
reactions

• Fe2 O2 --gt Fe3 O2.-
• Hb(Mb)-Fe2 O2 --gt Hb(Mb)-Fe3
  O2.-
• Catecholamines O2 --gt Melanin O2.-
• Reduced flavin
• Leukoflavin O2 --gt Flavin
  semiquinone O2.-
• Coenzyme
• Q-hydroquinone O2 --gt Coenzyme Q
  (ubiquinone) O2 .-
• Tetrahydropterin 2 O2 --gt Dihydropterin
  2 O2.-

29
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.
30
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 acid forming a
lipid hydroperoxide (LOOH)
31
ROS Arise Throughout the Cell
Wounding
Chilling
Ozone
Pathogens
Cell Wall
Drought Salinity
Cytosol
Antioxidant genes
Nucleus
Gene
Expression
ROS subcellular sites unclear
32
The electron transport system in the thylakoid
membrane showing 3 possible sites of activated
oxygen production
auto-oxidizable
Mehler reaction
a) Singlet oxygen may be produced from triplet
chlorophyll in the light harvesting complex. b)
Superoxide and hydrogen peroxide may "leak" from
the oxidizing (water-splitting) 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).
33

• The waterwater cycle.
• The ascorbateglutathione cycle.
• The glutathione peroxidase (GPX) cycle.
• 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 (ac). 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.

34
The redox cycling of ascorbate in the chloroplast
often referred to as the Halliwell-Asada pathw
35
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, NADHQ 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
The more you eat the more mitochondria
respiration and more ROS you get Mol Cel Biol,
2000, p. 7311-7318, Vol. 20,
36
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
auto-oxidation 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
37
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 as a bypass to oxidize NADH and FADH2 under
ADP-limiting conditions under which the
cytochrome oxidase pathway is restricted
38
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
Annu. Rev. Plant Physiol. Plant Molec. Biol. 52,
561-591
39
 Extra- and intracellular sources of ROS in
plants. XOD, xanthine oxidase
40
Prooxidants
R3C. Carbon-centered R3N. Nitrogen-centered R-O. O
xygen-centered R-S. Sulfur-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

H2O2 Hydrogen peroxide HOCl- Hypochlorous
acid O3 Ozone 1O2 Singlet oxygen ONOO-
Peroxynitrite Men Transition metals

• Non-Radicals
• Species that have strong oxidizing potential
• Species that favor the formation of strong
  oxidants (e.g., transition metals)

41
Reactive Oxygen Species (ROS)
Radicals O2.- Superoxide .OH Hydroxyl RO2. Pero
xyl RO. Alkoxyl HO2. Hydroperoxyl
Non-Radicals H2O2 Hydrogen peroxide HOCl- Hypoc
hlorous acid O3 Ozone 1O2 Singlet
oxygen ONOO- Peroxynitrite
42
Oxidative Protection
Oxidative Stress
Oxidants
Antioxidants
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
43
ROS scavenging molecules
plant antioxidants Ascorbate Glutathione Polypheno
ls Flavonoids Lipoic acid
Ponce de León
Enzymes SOD Catalase Glutathione
peroxidase Ascorbate peroxidase Thioredoxins Gluta
redoxins
Nature 425, 132-133
44
Reactive Nitrogen Species (RNS)
Non-Radicals ONOO- Peroxynitrite ROONO Alkyl
peroxynitrites N2O3 Dinitrogen trioxide N2O4
Dinitrogen tetroxide HNO2 Nitrous
acid NO2 Nitronium anion NO- Nitroxyl
anion NO Nitrosyl cation NO2Cl Nitryl chloride
Radicals NO. Nitric Oxide NO2. Nitrogen dioxide
45
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.
• Used as neurotransmitter, bactericide.
• Environmental Pollutant
• First gas known to act as a biological messenger

46
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.

47
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
48
cGMP in NO-induced cell death
The effects of the caspase-1 inhibitor
Ac-YVAD-CMK on NO- and H2O2-induced cell death
Cells were pre-treated with ODQ (guanylate
cyclase inhibitor) and/or 8Br-cGMP prior to RBS.
49
NO and Cell Death
PBITU Psm (avrRpm 1)
NO H2O2 cause cell death
NO O2- react to form peroxynitrite
Cell Death
Peroxynitrite (ONOO -) does not cause cell death
Too much O2- mops up NO no death
Delladonne et al. (2001) PNAS 9813454
50
Endogenous sources of ROS and RNS (in animals)
51
PEROXISOME

• b-oxidation of fatty acids
• bile acid synthesis
• purine and polyamine catabolism
• amino acid catabolism
• oxygen metabolism

Fatty Acid Acyl-CoA Enoyl-CoA Hydroxyacyl-CoA
Ketoacyl-CoA Acetyl-CoA Acyl-CoA
shortened by two carbons
Fatty acyl-CoA synthetase Acyl-CoA
oxidase Enoyl-CoA hydrolase Hydroxyacyl-CoA dehy
drogenase Thiolase
H2O2
52
Oxidative Phosphorylation ROS
53
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 cofactors.
Porphyrins, chlorins, iron sulfur clusters,
flavins or quinones are common members of the
chains.
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.
54
Intracellular ROS abundance in WT and Aox1
transgenic cultured tobacco cells.
antisense
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

55
Oxygen consumption in oxidatively stressed
mitochondria.
56
ROS production in isolated mitochondria
B
A
0
.
5
0
.
4
mmol H2O2/µgProt/min
0
.
3
0
.
2
0
.
1
0
0
1
5
H2O2 (mM)
Control
G/GO
A) Mitochondria isolated from control or cells
treated for 3 h with G/GO and stained with
DHDR123
Amplification of the Oxidative Stress
control
H2O2 pretreated
57
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
citrate
cis-Aconitate
Isocitrate
Iron-sulphur clusters
58
(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
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.
60
Models for the release of cytochrome c from
mitochondria
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 ce, a large channel
forms in the outer membrane (via VDAC), allowing
cytochrome c release, but mitochondria are not
damaged
61
Integration of stress signals by Mitochondria
(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.
62
Mitochondria in Apoptosis
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
63
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.
64
(No Transcript)
65
Oxidative Burst in the Plasma Membrane
apoplastic peroxidase
NADPH oxidase
66
Activation of NADPH oxidase by pathogens
(elicitors)
Exogenous H2O2 rescues both Ca2 channel
activation and stomatal closing in atrbohD/F
placing it upstream of Ca2
rbohA
Arabidopsis
Rice
Human
EF hands Ca2-binding sites.
gp91phox
Resistance responses
67
Activation of NADPH Oxidase Occurs within
Intracellular Compartments
animals
plants
Molec. Cell 11, 35-47 (2003)
68
Oxidative Protection
Oxidative Stress
Oxidants
Antioxidants
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 ???

69
Oxidants
Oxidative Damage
Enzymatic Defences catalytically remove ROS
Antioxidants
(Repair Processes)
Other Protective Compounds e.g. HSPs
Metal Sequestration Proteins
Low MW Antioxidants
Amounts Variable - cell types
tissues Effectiveness Variable - (production
site, radical species)
70
ROS Detoxification
Catalytic Activity Mn3 O2?- ? (Mn3-O2?-) ?
Mn2 O2 Mn2 O2?- ? (Mn2-O2?-) 2H ? Mn3
H2O2
Catalases 2 H2O2 ---gt 2 H2O O2 Peroxidases
AH2 H2O2 ---gt A 2 H2O A is an electron
donor
71
Cellular localization of SODs
72
Halliwell-Asada pathway redox cycling of
ascorbate in the chloroplast
Antioxidant concentration in plant cells
ascorbate (10-100 mM), glutathione (1-10 mM)
73
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 (L-AA) and oxidized
(DHAA) ascorbate. (6 h and 48 h exposure to HL).
(B) Effect on reduced (GSH) and oxidized (GSSG)
glutathione
74
Flavonoids are Chemo-preventive Agents
75
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

76
Phenotypes associated with Bax expression in
transgenic plants
ROS Production in Plants Expressing Bax
PNAS 2001 vol. 98 12295 PNAS 1999 96
7956-7961.
77
Ascorbate reaction with superoxide can serve a
physiologically similar role to SOD 2 O 2 2H
ascorbate --gt 2H2O2 dehydroascorbate The
reaction with hydrogen peroxide is catalysed by
ascorbate peroxidase H2O2 2 ascorbate --gt
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) 2
GSH dehydroascorbate ? GSSG ascorbate
78
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. 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.
79
The role of Aquaporins and membrane damage in
chilling and hydrogen peroxide induced changes in
the hydraulic conductance of maize roots
Scheme summarizing the interpretation of the
results. Chilling causes an initial decrease of
Lo in both genotypes. After 3 d at 5C, 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.
80
Systemic Signaling and Acclimation in response to
excess light
H2O2 is a local and systemic signal involved in
the adaptation of leaves to high light
(the arrow indicates the apical region of the
rosette)
Photodamage APX2 induction Leaves grown in
LL (control) exposed to EL. (A) Chlorosis on
detached leaves after 2 hours in EL. (B) relative
luciferase activity
Systemic induction of APX2-LUC expression. Image
of luciferase activity. A part of the whole
rosette (as shown) was exposed to EL for 40 min
(arrow -gt the apical rosette region). A typical
primary (1) EL-exposed leaf and a secondary (2)
LL-exposed leaf are shown
catalase but not SOD diminished APX2 expr.
81
Systemic induction of H2O2 by wounding