Reactive Oxygen ROS Metabolism in Plants

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Reactive Oxygen (ROS) Metabolism in Plants

Ron Mittler, Department of Biochemistry,
University of Nevada (ronm_at_unr.edu)
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Reactive Oxygen Species (ROS)

• ROS are partially reduced or excited forms of
  atmospheric oxygen. They are generated in cells
  by the transfer of one, two, or three electrons
  to oxygen to produce, respectively, a superoxide
  radical (O2-), hydrogen peroxide (H2O2), or
  hydroxyl radical (HO. see Figure below), or by
  the excitation of oxygen to produce singlet
  oxygen (O21).

e-
e-
e-
e-
H2O
O2
O2
H2O2
HO.
-
H
H
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Some ROS exist as free radicals and some ROS
exist as excited forms of atmospheric oxygen
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The differences in orbital electrons between a
radical ROS and a non-radical ROS is shown below
for superoxide (radical) and hydrogen peroxide
(peroxide ion non radical).
Although atmospheric oxygen contains unpaired
electrons in its upper orbitals, due to their
parallel spins, oxygen is restricted from
interacting with most other cellular components.
This restriction no longer prevails when oxygen
is turned into a ROS.
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The table below shows some radical and
non-radical ROS
R donates an organic residue (e.g., a fatty acid)
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• ROS are capable of unrestricted oxidation of
  various cellular components and can lead to the
  oxidative destruction of the cell (see below for
  damage to DNA and the next two slides for damage
  to membranes and proteins) .

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The figure below shows the proposed reaction for
the oxidation of a lysine residue to form a
carbonyl. Similar reactions are possible for
several other amino acid residues. These will
inactivate proteins and result in protein
degradation.
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In the presence of heavy metals such as iron,
superoxide and peroxide can generate the highly
toxic hydroxyl radical (HO.) through the Fenton
or Haber- Weiss reactions. Because the reactivity
rate of HO. is equal to its diffusion rate there
are no real scavenging mechanism for HO. aside
from maintaining a low level of superoxide and
peroxide and restricting the level of free metals
in biological systems.
Fenton reaction
Fe3 O2- ? Fe2 O2 O2- O2- ? H2O2 O2
H2O2 Fe2 ? Fe3 OH? OH-
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Production of ROS in Plants

• Under normal conditions the rate of ROS
  production in cells is low. However, stress or
  disease can disrupt the cellular homeostasis of
  the cell and enhance the rate of ROS production.
  This process is usually countered in cells by the
  activation of anti-oxidative mechanisms that
  scavenge the enhanced levels of ROS.

Transient enhancement of ROS production in liver
cells
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Stresses that result in the accumulation of ROS
in plants include Drought, desiccation,
salinity, chilling, heat, heavy metals,
ultraviolet radiation, air pollutants,
nutritional deprivation, high light stress and
different combinations of the above.
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Main Sources of ROS in plant Cells

• Generally any organelle with a highly oxidizing
  metabolic activity or with and intense rate of
  electron flow can produce ROS
• Chloroplasts during photosynthesis
• Mitochondria during respiration
• Peroxisomes during photorespiration and fatty
  acid oxidation
• Membrane-bound NADPH oxidase
• Apoplastic amine oxidase and peroxidase
• Cell wall-bound peroxidase

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Electron-transfer chains within the mitochondria
(a) and chloroplast (b) are some of the main
sources of superoxide radical production in
cells.
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Excited chlorophyll molecule (3Chl) from the
photosynthetic antenna (left) can transfer their
high energy state to atmospheric oxygen 3O2 to
form singlet oxygen (1O2 a ROS see reaction
below).
A major defense mechanism of plants against
singlet oxygen is the presence of ß-carotene
(Car) molecules in close proximity to the antenna
chlorophylls. These will convert the excited
state of chlorophyll into heat (right).
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One of the main problems that plants encounter
during stress is the uncoupling of the light
reaction of photosynthesis from the fixation of
CO2. Inhibition of calvin cycle enzymes or
closure of stomata during stress prevents CO2
fixation. The electrons generated by the
photosynthetic apparatus during the light
reaction can then be turned into ROS instead of
being used for CO2 fixation.
Stress
ROS
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• ROS are not only bad for cells. In the next
  three slides the good aspect of ROS is shown
• ROS are used as signaling molecules to activate
  the defense response of plants against stress.
• ROS are used by cells to fight pathogens and
  activate the pathogen-response and programmed
  cell death-response of plants.
• ROS act as signal transduction molecules thet
  regulate cells growth and differentiation.

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• ROS accumulate during stress and may lead to cell
  injury and death. However, ROS are also used by
  cells as secondary messengers involved in the
  stress-response signal transduction pathway.

Stress
ROS
Signal
Defense response activation
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The infection of plants with pathogens, such as
bacteria, often results in the activation of a
programmed cell death response (see below for
lesions forming on an infected leaf). This
response prevents the spread of the pathogen to
uninfected parts of the plant. The recognition of
the invading pathogen results in the activation
of a membrane-bound NADPH oxidase (see right
model for animal enzyme). This enzyme produces
superoxide radicals that can dismutate into
hydrogen peroxide. NADPH oxidase is also
activated during abiotic stress to generate ROS
that are used as signal transduction molecules.
Bacteria
Lesion
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ROS are also used as important signaling
molecules involved in the regulation of growth,
differentiation and development in plants. The
image below shows ROS accumulation during root
hair growth in Arabidopsis (Foreman, J. et al.
(2003) Nature 422, 442-446 )
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Scavenging of ROS in cells

• Plants use two major strategies to control the
  level of ROS and prevent cellular damage
• Scavenge of ROS by anti-oxidants and
  anti-oxidative enzymes.
• Lower the rate of ROS production in cells by
  suppressing ROS-producing reactions and by
  different stress avoidance strategies.

The next few slides describe some of the major
antioxidants and anti-oxidative enzymes of plants
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Antioxidants
H2O2 or O2-
A sugar-based molecule that reacts with different
reactive oxygen species and detoxifies them. The
oxidized products of vitamin C reaction with ROS
can be reduced back using glutathione as a
reducing agent. Ascorbic acid can also function
in a cooperative manner with vitamin E to protect
membranes. Ascorbic acid is used as a substrate
for the hydrogen peroxide-scavenging enzyme
ascorbate peroxidase.
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Antioxidants
Reduced
Oxidized
A tripeptide molecule that contains an SH group
capable of reducing different ROS and
regenerating oxidized ascorbic acid. It is used a
a substrate for the protective enzyme
glutathione peroxidase for scavenging hydrogen
peroxide and lipid peroxides.
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Antioxidants

• A lipophilic molecule found mainly in membranes
  and plays a critical role in protecting the
  membrane from the effects of lipid peroxidation
  initiated by ROS. Oxidized vitamin E can be
  reduced by ascorbic acid.

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Antioxidants
ß-carotene is a lipophilic molecule mainly found
in association with the photosynthetic apparatus
where it is used to detoxify singlet oxygen.
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Superoxide Dismutase (SOD)
O2- O2- 2H ? H2O2 O2
A metal-containing enzyme (FeSOD in chloroplasts,
MnSOD in mitochondria and CuZnSOD in
chlotoplasts, cytosol, peroxisomes and apoplast)
with a very high KCAT. SODs are found in almost
all aerobic organisms and are key for ROS removal
reactions. They usually appear as dimers and are
relatively stable.
Structure of CuZnSOD
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Ascorbate Peroxidase (APX)
2 Asc H2O2 ? 2 MDA 2H2O
A heme-containing enzyme found in the chloroplast
(thylakoid-bound, stroma and lumen),
mitochondria, peroxisome (bound to the outer
surface) and apoplast. APX is found mainly in
plants and has high homology to yeast cytochrome
c-peroxidase. The chloroplast and mitochondrial
forms of APX are stable only in the presence of
ascorbic acid. ASC ascorbic acid MDA
Monodehydroascorbate
Structure of cytosolic APX monomer
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Catalase (CAT)
2H2O2 ? 2H2O O2
A tetrameric heme-containing enzyme
(25kD/subunit) found mainly in peroxisomes.
Catalase is key in the scavenging hydrogen
peroxide produced during photorespiration by
glycolate oxidase, but was also shown to scavenge
hydrogen peroxide that diffuses into the
peroxisome.
3D structure of catalase
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Glutathione Peroxidase (GPX)
H2O2 2 GSH ? 2H2O GSSG
A key enzyme for the removal of hydrogen peroxide
in animal cells. In plants GPX is found in the
chloroplast, cytosol and mitochondria. Some forms
of GPX bind selenium. At least one isozyme in
plants can detoxify lipid peroxides.
Structure of GPX
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Pathways for ROS detoxification (SOD, GPX, PrxR
and the Asada-Halliwell-Foyer pathway
The cascade of reactions used to scavenge
superoxide and hydrogen peroxide in the cytosol,
stroma and mitochondria of plants (some
components of these pathways are found in
peroxisomes). Superoxide is scavenged by SODs.
Hydrogen peroxide is scavenged by APX, GPX and
peroxiredoxin (PrxR). Oxidized ascorbic acid
(MDA) is reduced to ascorbic acid by MDAR
(monodehydroascorbate reductase), and DHR
(dehydroascrobate reductase) or GLR
(glutaredoxin). Oxidized glutathion (GSSG) is
reduced to GSH by glutathione reductase (GR). The
regenerating pathways require energy in the form
of NAD(P)H.
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Pathways for ROS detoxification (the water-water
pathway)
The water-water pathways is a thylakoid-specific
pathway that scavenges superoxide radicals
generated by the photosynthetic apparatus. An SOD
(CuZnSOD) and an APX (tylAPX) are used to
detoxify superoxide into water, and the oxidized
ascorbic acid (MDA) is reduced by electrons from
the photosynthetic apparatus through ferredoxin
(FD). A peroxiredoxin (PrxR) can also detoxify
hydrogen peroxide in this pathway. Because
electrons are taken from one water molecules (at
PSII) and are used to generate another water
molecule by the peroxidase reaction, the pathway
is called water-water pathway.
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Alternative Oxidase (AOX)
In both the mitochondrial electron-transport
chain (a) and the chloroplast electron-transport
chain (b), AOX diverts electrons that can be used
to reduce O2 into O2- and uses these electrons to
reduce O2 to H2O. AOX is indicated in yellow and
the different components of the
electron-transport chain are indicated in red,
green or gray. Abbreviations Cyt-b6f, cytochrome
b6f Cyt-c, cytochrome c Fd, ferredoxin PC,
plastocyanin PSI, PSII, photosystems I and II.
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Entire Cell
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• To prevent damage caused by ROS during abiotic
  stress plants may also use different avoidance
  mechanisms that prevent the production of ROS in
  cells
• Alterations in antenna protein structure and
  composition to reduce light-driven ROS
  production.
• Use of C4 and CAM mechanisms for CO2 fixation.
• Movement of chloroplasts within cells to avoid
  direct light.
• Anatomical adaptations such as leaf curling, hair
  cells and hidden stomata in special leaf
  structures.
• Dormancy and partial plant dormancy.
• Alternative oxidase.

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ROS
Summary of ROS producing, scavenging and avoiding
mechanisms in plants. References are after
Mittler 2002 Trends in Plant Sci 7, 405-410.
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In accordance with the important function of ROS
in plants, mutants with reduced activity of
different ROS-scavenging enzymes display reduced
growth and altered development (Apx1 cytosolic
APX, CSD2 chloroplastic CuZnSOD)
Pnueli et al., (2003) Plant J. 34, 187-203
Rizhsky et al., (2003) J. Biol. Chem. 278,
38921-38925.
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ROS during biotic and abiotic stresses, a
possible conflict?
ROS
ROS
ROS
ROS
ROS
ROS
A possible conflict in the steady-state level of
ROS may exist between biotic stress and abiotic
stress. Biotic stress (a) results in the
activation of NADPH oxidase and the suppression
of ascorbate peroxidase (APX) and catalase (CAT).
This leads to the over-accumulation of ROS and
the activation of defense mechanisms. Abiotic
stress (b) enhances ROS production by
chloroplasts and mitochondria. However, by
inducing ROS-scavenging enzymes such as APX and
CAT, it reduces ROS levels. The question mark
indicates that little is known about the
regulation of ROS metabolism during a combination
of biotic and abiotic stresses. Chloroplasts are
indicated in green, mitochondria in gray and the
steady-state levels of ROS in yellow.
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Because ROS are toxic but also play a key role in
signal transduction processes involved in plant
development, growth and defense, the level or ROS
in cells needs to be tightly regulated. This
control is achieved by a delicate balance between
the ROS-producing and the ROS-scavenging
mechanisms of the cell (The ROS network).
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• To balance between ROS production and ROS
  scavenging plant cells use a signal transduction
  pathway that detects the level of ROS and
  activates or suppress ROS production or ROS
  scavenging. In the next slide the putative ROS
  signal transduction pathway of plants is shown.
• ROS can be sensed by at least 3 different
  mechanisms
• Receptors such as two-component histidine kinase
  receptors.
• Redox-sensitive transcription factors such as HSF
  (heat shock transcription factor).
• Direct inhibition of phosphatases by ROS.

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See Figure legend in the next slide
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The Figure above shows a generalized model of the
reactive oxygen species (ROS) signal transduction
pathway. ROS can be detected by at least three
mechanisms (ROS receptors, redox-sensitive
transcription factors and phosphatases).
Detection of ROS by receptors results in the
generation of Ca2 signals and the activation of
a phospholipase C/D (PLC/PLD) activity that
generates phosphatidic acid (PA). PA and Ca2 are
thought to activate the protein kinase OXI1.
Activation of OXI1 results in the activation of a
mitogen-activated-protein kinase (MAPK) cascade
(MAPK3/6) and the induction or activation of
different transcription factors that regulate the
ROS-scavenging and ROS-producing pathways. The
activation or inhibition of redox-sensitive
transcription factors by ROS might also affect
the expression of OXI1 or other kinases and/or
the induction of ROS-specific transcription
factors. Inhibition of phosphatases by ROS might
result in the activation of kinases such as OXI1
or MAPK3/6. Two different loops are shown to be
involved in the ROS signal transduction pathway.
A localized or general defense response (a
negative feedback loop solid green line) can be
activated to suppress ROS, whereas a localized
amplification loop (positive feedback loop red
dashed line) can be activated to enhance ROS
signals via the activity of NADPH oxidases.
Salicylic acid (SA) and nitric oxide (NO) might
be involved in this amplification loop as
enhancers. Abbreviations HSF, heat shock factor
PDK, phosphoinositide-dependent kinase TF,
transcription factor.
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Summary and conclusions 1. ROS are toxic forms
of atmospheric oxygen produced in cells of
all aerobic organisms. 2. The production of ROS
is enhanced in cells during stress or disease. 3.
Cells contain many different pathways and
mechanisms to detoxify ROS. 4. ROS are important
signal transduction molecules involved in the
control of cell growth and differentiation and
the defense of cells against stress and
disease. 5. Because ROS are toxic, but also
participate in the control of many cellular
processes, the level of ROS in cells needs to be
tightly regulated. 6. A delicate balance between
the ROS-scavenging and ROS-producing mechanisms
of the cell determines the steady-state level of
ROS in cells, thereby determines the function of
ROS at any given time during the life cycle of a
plant.
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Review papers from the author of this
presentation describing ROS metabolism in plants.
Many excellent papers on ROS metabolism can be
found in the reference lists of these papers.
Mittler, R. (2002) Oxidative stress,
antioxidants, and stress tolerance. Ternds Plant
Sci. 7, 405-410 Mittler. R., Vanderauwera, S.,
Gollery, M., Van Breusegem, F. (2004) The
reactive oxygen gene network of plants. Trends
Plant Sci. 9, 490-498.
The author would like to thank the National
Science Foundation (NSF) for its generous support