Abiotic Stress

7/22/2019 Abiotic Stress

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Oxidative stress

Anju M PBCH 10 05 12


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One of the paradoxes of life on this planet is that

the molecule that sustains aerobic life, oxygen, is

not only fundamentally essential for energy

metabolism and respiration, but it has been

implicated in many diseases and degenerative

conditions

A common element in such diverse human

disorders as ageing, arthritis, cancer, Lou Gehrig's

disease and many others is the involvement ofpartially reduced forms of oxygen.


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In the sequential univalent process by which O2

undergoes reduction, several reactive intermediates

are formed, such as superoxide (O2), hydrogen

peroxide (H2O2), and the extremely reactive

hydroxyl radical (.OH): collectively termed as the

reactive oxygen species (ROS).

O2+e- O2+e- H2O2+e- .OH+e- H2O


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O2 can behave like a radical (a diradical) owing tothe presence of two unpaired electrons of parallel

spin, it does not exhibit extreme reactivity due to

quantum-mechanical restrictions.

Its electronic structure result in formation of water

by reduction with four electrons, i.e:

O2+4H +4e 2H O.


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Atm oxygen in its ground-state is a biradical, or it

has two unpaired electrons.

This makes oxygen paramagnetic; it also makes

oxygen very unlikely to participate in reactions with

organic molecules unless it is "activated".

The requirement for activation occurs because the

two unpaired electrons in oxygen have parallel

spins.

According to Pauli's exclusion principle, this

precludes reactions with a divalent reductant,

unless this reductant also has two unpaired

electrons with parallel spin opposite to that of the

oxygen, which is a very rare occurrence.


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Hence, oxygen is usually non-reactive to organic

molecules which have paired electrons with

opposite spins.

This spin restriction means that the most common

mechanisms of oxygen reduction in biochemical

reactions are those involving transfer of only a

single electron (monovalent reduction).

Activation of oxygen may occur by two differentmechanisms:

1. absorption of sufficient energy to reverse the spin on one

of the unpaired electrons, or monovalent reduction


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If triplet oxygen absorbs sufficient energy to reverse

the spin of one of its unpaired electrons, it will form

the singlet state, in which the two electrons have

opposite spins

This activation overcomes the spin restriction and

singlet oxygen can consequently participate in

reactions involving the simultaneous transfer of twoelectrons (divalent reduction).

Since paired electrons are common in organic

molecules, singlet oxygen is much more reactivetowards organic molecules than its triplet

counterpart.


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2. by the stepwise monovalent reduction of oxygen to

form superoxide ,hydrogen, hydroxyl radical and

finally water The first step in the reduction of oxygen forming

superoxide is endothermic but subsequent

reductions are exothermic


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REACTIONS THAT LEAD TO THE FORMATION OF SOME ROS

In the presence of trace amounts of iron, the reaction

of superoxide and hydrogen peroxide will form thedestructive hydroxyl radical and initiate the oxidation

of organic substrates


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What is oxidative stress?

our body constantly reacts with oxygen during

breathing and our cells produce energy. As a

consequence of this activity, highly reactive

molecules are produced known as free radicals.

Free radicals interact with other molecules within

cells. This can cause oxidative damage to proteins,

membranes and genes.

Oxidative damage has been implicated in the cause of

many diseases, such as cancer and Alzheimer's and

has an impact on the body's aging process.

External factors, such as pollution, sunlight and

smoking, also trigger the production of free radicals.


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BIOLOGICAL REACTIONS OF OXYGEN RADICALS

The reactions of activated oxygen in biologicalsystems there are even more complications due to

the surface properties of membranes, electrical

charges, binding properties of macromolecules, and

compartmentalisation of enzymes, substrates and

catalysts


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1. OXIDATIVE DAMAGE TO LIPIDS

The lipid bilayer membrane is composed of amixture of phospholipids and glycolipids that have

fatty acid chains

Initiation-the production of R/PUFA radical/ROO

by the interaction of PUFA with free radicals

generated by other means

R andROO are degraded to malon dialdehydean

indicator of fatty acid break down by free radicals


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Propagation-one free radical generates another free

radical in the neighbouring moleculeschain

reactiondestruction of fine architechture &

integrity of the membranes

Termination- reactions in membrane lipids are

terminated when the carbon or peroxy radicals

cross-link to form conjugated products that are notradicals


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Lipid peroxy radicals react with other lipids,

proteins, and nucleic acids; propagating thereby the

transfer of electrons and bringing about the

oxidation of substrates.

Cell membranes, which are structurally made up of

large amounts of PUFA, are highly susceptible to

oxidative attack and, consequently, changes in

membrane fluidity, permeability, and cellular

metabolic functions result.


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2.OXIDATIVE DAMAGE TO PROTEINS

Oxidative attack on proteins results in site-specific amino

acid modifications, fragmentation of the peptide chain,aggregation of cross-linked reaction products, altered

electrical charge and increased susceptibility to proteolysis

Sulphur containing amino acids, and thiol groupsspecifically, are very susceptible sites

the oxidation of iron-sulphur centres by superoxide

destroys enzymatic function

Thus it destroys the structure,functions of essential

proterins and enzymes and whole cell metabolism is

blocked


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In the process of cataractogenesis, oxidative

modification plays a significant role in cross-linking

of crystalline lens protein,leading to high-molecular-

weight aggregates, loss of solubility, and lens

opacity. Lipofuscinan aggregate of peroxidized

lipid andproteinsaccumulates in lysosomesofaged cells, Alzheimers disease brain cells, and iron

overloaded hepatocytes.

3 OXIDATIVE DAMAGE TO DNA


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3.OXIDATIVE DAMAGE TO DNA

Activated oxygen and agents that generate oxygenfree radicals, such as ionizing radiation, induce

numerous lesions in DNA that cause deletions,mutations and other lethal genetic effects

Degradation of the base will produce numerous

products, including 8-hydroxyguanine,hydroxymethyl urea, urea, thymine glycol, thymineand adenine ring-opened and -saturated products.

Characterizations of this damage to DNA has

indicated that both the sugar and the base moietiesare susceptible to oxidation, causing basedegradation, single strand breakage, and cross-linking to protein .


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Mutation arising from selective modification of G : C

sites specially indicates oxidative attack on DNA by

ROS.

The action of 8-oxodeoxy- guanosine as a

promutagen, as well as in altering the binding of

methylase to the oligomer so as to inhibit

methylation of adjacent cytosine has been reported

in cases of cancer development

ROS have also been shown to activate mutationsinhuman C-Ha-ras-1 protooncogene, and to induce

mutation in the p53 tumour-suppressor gene4

ROS i t f ith l ll i lli lti


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ROS may interfere with normal cell signalling, resultingthereby in alteration of the gene expression, anddevelopment of cancer by redox regulation of

transcriptional factors/activator and/or by oxidativelymodulating the protein kinase cascades.

ROS also induce various early response or stress-response genes like c-fos, c-jun, jun-B, jun-D, c-myc,erg-

1, and heme oxygenase-1. The oxidative damage of mitochondrial DNA also

involves base modification and strand breaks, whichleads to formation of abnormal componentsof the ETC

This results in the generation ofmore ROS throughincreased leakage of electrons, and cell damage.

Oxidative damage to mitochondrial DNA may promote

cancer and aging


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i h d i i i f d i f


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Mitochondria are a primary site of production of

free radicals. While more than 98% of the molecular

oxygen taken up by cells is fully utilized by

cytochrome c oxidase to form water, this enzymecan release partly reduced species. Other enzymes

of the respiratory chain, and in particular complexes

I and III, also produce partly reduced oxygen speciesincluding superoxide.


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V i id ti i l di id ti


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Various oxidative processes, including oxidation,hydroxylations, dealkylations, deaminations,dehalogenation and desaturation, occur on the SER.

Mixed function oxygenases that contain a hememoiety add an oxygen atom into an organicsubstrate using NAD(P)H as the electron donor

The generalised reaction catalysed by cytochromeP450

The best characterised cytochrome P450 in plants iscinnamate-4-hydroxylase which functions inflavonoid and lignin biosynthesis

Cytochrome P450 reacts first with its organic substrate RH The


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Cytochrome P450 reacts first with its organic substrate, RH. The

complex is reduced by a flavoprotein to form a radical intermediate

that can readily react with triplet oxygen because each has one

unpaired electron. This oxygenated complex may be reduced by

cytochrome b or occasionally the comples may decompose releasingsuperoxide


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The plant NAD(P)H oxidase have an analogous functionto the animal enzyme.

Leucocytes contain an NADH oxidase on the outermembrane surface which is activated in response to aforeign agent, generating superoxide that initiatesoxidative reactions that destroy the potential pathogen

In plants, fungal elicitors cause a similar formation ofsuperoxide that has been linked to the hypersensitiveresponse to some pathogenic fungi .

Wounding, heat shock and xenobiotics transientlyactivate this superoxide generating reaction, andconsequently, it has been proposed that thesesuperoxide generating reactions may serve as a signalin plant cells to elicit responses to biological, physical orchemical stress

R ti i d i


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Reactive oxygen species and aging

Eversince Harman104 first proposed the free-radical

theory of aging, as early as 1956, the molecular

basis of aging and the role of ROS

aging and age-related diseases result from ROS-

mediated oxidative damage of lipid, protein, and

nuclear and mitochondrial DNA molecules.

The concentration of oxidatively damaged proteins,

lipids, and DNA has been reported to increase with

age

The hydroxyl and peroxy radicals cause extensive

damage of proteins resulting in aging and age

related degenerative diseases


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mutation in mitochondrial DNA also leads to the

formation of defective respiratory enzymes which

not only result in decreased ATP synthesis but also

generate more ROS to cause further oxidative damage

This vicious cycle is mainly responsible for aging and

age-related disorders.

Melatonin, having antioxidant property, declines

significantly with the increase in age.

This decline in melatonin coincides with the

increased oxidative damage and pathogenesis.


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ROS have also been implicated in the regulation of

at least two well-defined transcription factors, AP-1

and NFkB

These transcription factors bind at the promoter

regions of a large variety of genes and play a very

significant role in the expression of various proteins

such as TNFa, interleukin-1 and -2, collagenase,

matrix metalloproteinase, etc. which are involved in

inflammatory responses and tissue injury Blocking the expression of these genes by suitable

antioxidants should be one of the approaches for

controlling the ROS-mediated pathogenesis


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Enhanced oxidative stress is now welldocumented to occur in a number ofdegenerative diseases includingParkinson s disease, Alzheimer s anddiabetes

DEFENCE MECHANISMS


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DEFENCE MECHANISMS

1.SUPEROXIDE DISMUTASE

was first isolated by Mann and Keilis and thought tobe a copper storage protein. SOD is now known to

catalyse the dismutation of superoxide to hydrogen

peroxide and oxygen

2 CATALASE


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2.CATALASE

Catalase is a heme-containing enzyme that catalysesthe dismutation of hydrogen peroxide into water and

oxygen. found in all aerobic eukaryotes and is important in the

removal of hydrogen peroxide generated inperoxisomes (microbodies) by oxidases involved in -

oxidation of fatty acids, the glyoxylate cycle and purinecatabolism

Careful examination of the structure of beef livercatalase has shown four NADPH binding sites percatalase tetramer , but these sites were not in closeassociation with the hydrogen peroxide reaction centre.Instead, NADPH functions in animal catalase to protectagainst inactivation by hydrogen peroxide

3 GLUTATHIONE


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3.GLUTATHIONE

Glutathione (GSH) is a tripeptide (Glu-Cys-Gly)

whose antioxidant function is facilitated by the

sulphydryl group of cysteine

is found in most tissues, cells and subcellular

compartments of higher plants

can react chemically with singlet oxygen,

superoxide and hydroxyl radicals and therefore

function directly as a free radical scavenger

stabilise membrane structure by removing acyl

peroxides formed by lipid peroxidation reactions


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The reduction of GSSG to GSH is catalysed by the

enzyme glutathione reductase (GR) in presence

NADPH(HMP)

GR is associated mainly with the chloroplast but

significant activity is also found in the cytosol and a

lesser amount in the mitochondria

GLUTATIONE PEROXIDASE


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GLUTATIONE PEROXIDASE

Glutathione peroxidase catalyses the reaction of

hydroperoxides with reduced glutathione (GSH) to

form glutathione disulphide (GSSG) and the

reduction product of the hydroperoxide

This enzyme is specific for its hydrogen donor, GSH,

andnonspecific for the hydroperoxides ranging from

H2O2 to organic hydroperoxides.

It is a seleno-enzyme; two-third of which (inliver) is

present in the cytosol and one-third in the

mitochondria2.


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Antioxidants and oxidative stress


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Antioxidants and oxidative stress

To counteract oxidative stress, the body produces an

armoury of antioxidants to defend itself. It's the job

of antioxidants to neutralise or 'mop up' free

radicals that can harm our cells.

our body's ability to produce antioxidants (its

metabolic process) is controlled by our genetic

makeup and influenced by our exposure to

environmental factors, such as diet and smoking.

wecan help your body to defend itself by increasingour dietary intake of antioxidants.

Ascorbic acid


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Ascorbic acid

L-ascorbic acid (vitamin C) is an important vitamin

in the human diet and is abundant in plant tissues.

Ascorbate functions as a reductant for many free

radicals, thereby minimising the damage caused by

oxidative stress

Structure of ascorbic acid and its metabolites

can directly scavenge oxygen free radicals with and


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can directly scavenge oxygen free radicals with andwithout enzyme catalysts and can indirectly scavenge them

by recycling tocopherol to the reduced form.

By reacting with activated oxygen more readily than anyother aqueous component, ascorbate protects critical

macromolecules from oxidative damage

Tocopherol


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Tocopherol

The tocopherol (vitamin E), have been studied

extensively in mammalian research as membrane

stabilisers and multifaceted antioxidants, that

scavenge oxygen free radicals, lipid peroxy radicals,

and singlet oxygen


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Carotenoids


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Carotenoids

Carotenoids are C40 isoprenoids and tetraterpenes

that are located in the plastids of both

photosynthetic and non-photosynthetic plant

tissues.

In chloroplasts, the carotenoids function act as

accessory pigments in light harvesting,

more important role is their ability to detoxify

various forms of activated oxygen and triplet

chlorophyll that are produced as a result ofexcitation of the photosynthetic complexes by light.


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-car + ROO -car + ROOH

-car + ROO inactive products

Structure of two common carotenoids found in plants, -carotene and

zeaxanthinin.

Foods and antioxidants


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Foods and antioxidants

Tomatoes

Tomatoes contain a pigment called lycopene that

is responsible for their red colour but is also a

powerful antioxidant.

Tomatoes in all their forms are a major source of

lycopene, including tomato products like canned

tomatoes, tomato soup, tomato juice

Lycopene is also highly concentrated in

watermelon


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Citrus fruits

Oranges, grapefruit, lemons and limes possess

many natural substances that appear to be

important in disease protection, such as

carotenoids, flavonoids, terpenes, limonoids and

coumarins.

It's always better to eat the fruit whole in its

natural form, because some of the potency is lost

when the juice is extracted

Tea


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Black tea, green tea and oolong teas have antioxidantproperties. All three varieties come from the plant

Camellia sinenis. Common brands of black tea do contain antioxidants,

but by far the most potent source is green tea(jasmine tea) which contains the antioxidant catechin.

Black tea has only 10 per cent as many antioxidants asgreen tea.

Oolong tea has 40 per cent as many antioxidants as

green tea. This because some of the catechins are destroyed

when green tea is processed (baked and fermented) tomake black tea.


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Carrots

Beta-carotene is an orange pigment that was

isolated from carrots 150 years ago.

It is found concentrated in deep orange and green

vegetables (the green chlorophyll covers up the

orange pigment).

Beta-carotene is an antioxidant that has been

much discussed in connection with lung cancer

rates.


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