Transcript Environmental factors that induce oxidative stress

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.
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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
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Basics of Redox Chemistry
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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
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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
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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.
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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
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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
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• Ground-state oxygen has 2-unpaired electrons
: :
: :
. O:O .
• The unpaired electrons have parallel spins
• Oxygen molecule is minimally reactive
due to spin restrictions
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 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.
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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)
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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
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“Longevity” of reactive species
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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
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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
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Radical-mediated reactions
Addition
R.
+
H2C=CH2
R-CH2-CH2.
Hydrogen abstraction
R.
+
LH
RH
+
L.
Electron abstraction
R.
+
ArNH2
R-
+
ArNH2.+
Termination
R.
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+
Y.
Disproportionation
CH3CH2. + CH3CH2.
R-Y
CH3CH3 + CH2=CH2
Fenton reaction (1894)
Cu1+

Cu2+
Haber and Weiss extension (1934)
Oxidizing molec
Reducung molec
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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
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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.-
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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.
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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
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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 ,
High Light + Chilling
Effects
Sulfur Dioxide
Paraquat
High Light + Chilling
Sulfur Dioxide
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ROS subcellular
sites unclear
,
The electron transport system in the thylakoid membrane
showing 3 possible sites of activated oxygen production
auto-oxidizable
Mehler reaction
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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
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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
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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
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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
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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
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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
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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.
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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
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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.
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64
Oxidative Burst in the Plasma Membrane
apoplastic peroxidase
NADPH oxidase
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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
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Activation of NADPH Oxidase Occurs within
Intracellular Compartments
animals
Molec. Cell 11, 35-47
(2003)
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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 ???
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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)
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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
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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)
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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
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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
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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
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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):
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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.
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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
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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