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Antioxidant enzymes
1.
2.
3. CONTENTS
Introduction
Oxidative stress & reactive oxygen species
Antioxidants
Classification of antioxidants
Site of production of enzymatic antioxidants
Protective role of enzymatic antioxidants
Case studies.
4.
5. What are free radicals?
Any molecule containing one or
more unpaired electrons.
Highly reactive
Very short half-life
Generate new radicals by chain
reaction
Cause damage to biomolecules,
cells and tissues.
Types of free radical
7. SOURCES OF ROS IN PLANT CELLS AND THEIR REACTIVITY IN
VARIOUS CELLULAR COMPONENTS
1. Flavoenzyme ERO1
2. Lipoxygenases
3. Cyclo oxygenases
4. Flavin dependent demethylase
5. Oxidases for polyamines and amino acids
9. ROS : nature and origin
Reactive Oxygen Species (ROS)
The
formed
chemical
upon
species
incomplete
of molecularreduction
oxygen
Reduction
Accumulation of ROS leads
to cell
disturbances
injury
in
and
seed
development or germination
processes
Reduction Reduction Reduction
Figure 1. Formation of ROS through energy- and electron-transfer reactions
Krumova and Cosa, 2016
Non radical
derivatives
Radical
derivatives
12. Autoxidation
Oxidation of lipid in presence of oxygen around the
unsaturated fatty acid resulting in formation of peroxides
and hydroperoxides.
Lipid peroxidation
The oxidative degradation of lipids
The process in which free radicles steal electrons from
the lipids in cell membranes, resulting in cell damage.
It is stimulated by Lipoxygenases enzyme
Occurs in all seeds, more in oil seeds
ROS : cause of seed deterioration
15. An antioxidant is a molecule capable
of inhibiting the oxidation of other
molecules. Oxidation is a chemical
reaction that transfers electrons or
hydrogen from a substance to an
oxidizing agent.
Oxidation reactions can produce free
radicals. In turn, these radicals can
start chain reactions.
16. What are Antioxidants ?
An antioxidant defence differ from species to species.
The presence of antioxidant defence is universal.
Enzymatic antioxidants work by breaking down and
removing free radicals.
Non-enzymatic antioxidants work by interrupting free
radical chain reactions.
It fill electron needs of free radicals that are ferociously
searching for their missing electrons, without becoming
free radicals themselves
Antioxidants are the substances that are present in plants or in
seeds at lower concentration compared to that of oxidizable
substrates, significantly delays or prevent oxidation of
substrates.
17. Antioxidants and Plants
SOD, GP,CAT
Se, Cu, Mn, Zn
Vitamin A, C, E (α-Tocopherol)
Enzymes Minerals &
Vitamins
weight
compounds
High molecular Low molecular
weight
compounds
Why plant produce antioxidants?
To protect themselves from the ultraviolet
light from the sun and the reactive
oxygen species generated during
photosynthesis that would cause
irreparable damage in plants
1958:- Kaloyereast introduced the (FRTA- Free-
Radical Theory of Ageing) into seed science by
arguing that lipid oxidation might underlie loss of
viability in seeds
Non enzymatic
antioxidants
Lipid soluble antioxidant
Water soluble antioxidant
Proteins:-
albumins,
transferin,
Late 20th century - role of antioxidants in biology
focused on their use in preventing the oxidation of
unsaturated fats, which is the cause of rancidity
4
Gupta and Sharma, 2006
20. First line defence
SOD - quenching superoxide radical
CAT - catalysing decomposition of
hydrogen peroxide to water
GPX- Se containing enzyme, catalyse
reduction of H2O2
GR- efficient scavenging of peroxides
from cytosol and cell membrane
Role of Antioxidants in plants- seeds ?
21. Second line defence
Glutathione (GSH)- good scavenger of free
radiclas like O2
-, hydroxyl, and various lipid
hyperperoxides.
Vitamin E (α-tocopherol) - interacts directly with
free radicals like O2
- , hydroxyl, scavenge peroxyl
radical intermediates in lipid peroxidation, also
protects LDL
Carotenoid (β- carotene)- excellent scavenger of
singlet oxygen
Flavinoid- phenolic compound that inhibit lipid
peroxidation and lipoxygenase activity
Role of Antioxidants in plants- seeds ?
22. Role of Antioxidants in plants- seeds ?
Third line defence
Complex group of enzymes repair damaged DNA,
damaged proteins, oxidized lipids and peroxides and
also stop chain propagation of peroxyl lipid radical
Enzymes repair the damage to biomolecules and
reconstitute the damaged cell membrane
DNA repair enzymes, transferase, methionine
sulphoxide reductase
23. Action of enzymatic antioxidants in seed
Ascorbate (ASA)
Ascorbate peroxidase (APX)
H2O H2O2 H2O
O2
-
OH +O2
CAT
SOD
APX
ASA M DHA
Catalase (CAT)
Dehydroascorbate reductase (DHAR)
Glutathion reductase (GR)
Monodehydroascorbate reductase
(MDAR) Oxidized glutathione
(GSSG)
Reduced glutathione (GSH )
MDHAR
DHA
DHAR
GSSG GSH
GR
Superoxide dismutase (SOD)
NADPH NADP
electrontransport 12
A metabolic pathway that detoxifies
hydrogen peroxide (H2O2- a ROS and
a waste product in plant metabolism).
A metabolic pathway
Abbrevations
Fover-Halliwell- Asada cycle, 1976
OR
Ascorbate-Glutathione cycle
25. SOD
Important protective mechanism against oxidative damage
It works in collaboration with POD and CAT which act to remove O2
-
and Hydrogen peroxide
Removal of ROS produced under various stress conditions thus
avoiding oxidative damage
POD and CAT keeps H2O2 balance in seeds
In oil seeds it removes H2O2 produced during β –oxidation of fatty acid
Associated with essential metabolic processes:- Cell elongation,
lignification, phenolic oxidation, pathogen defence and defence against
stress
Important role in seed germination, growth, morphogenesis, senescence
and death
Seed and enzymatic antioxidants: Functions
CAT
POD
26. List of all the enzymatic and non-enzymatic antioxidants along with
their functions and cellular localization
27. Orthodox seeds
Maximum effective antioxidant system before maturation
drying of orthodox seeds, and again when seeds take up
water upon imbibition.
Seeds are devoid of ascorbate peroxidase (AP) at end of
desiccation period
They are desiccation tolerant
Recalcitrant seeds
Maximum effective antioxidant system when shed from the
mother plant at a high moisture content and very active
metabolism. This declines in case of stress
Seeds contain high ascorbate peroxidase (AP) activity to
eliminate Toxic H2O2 as they never reach the quiescent
phase
Desiccation sensitive
Variation in antioxidants among different seeds
Berjak and Pammenter, 2002
28.
29. Changes in total protein content, SOD, POD and PAL activities
during seed maturation
Hea and Gao, 2008
20
Changes of antioxidant enzyme and phenylalanine ammonia-lyase
activities during Chimonanthus praecox seed maturation
Developmental stage
weeks after flowering
Protein
content
(mg/g fresh
weight)
SOD
activity
(U/g fresh
weight)
POD
activity
(U/g fresh
weight)
PAL
activity
(U/g fresh
weight)
8 21.4 87 234 336
10 23.7 112 345 242
12 63.3 180 475 229
15 52.1 175 348 211
30. Fig. 1. PAGE showing isoenzyme pattern of a. superoxide dismutase
(SOD) and b. peroxidase (POD) during seed maturation.
Hea and Gao, 2008
20
Changes of antioxidant enzyme and phenylalanine ammonia-lyase
activities during Chimonanthus praecox seed maturation
31. Lipid Peroxidation and Antioxidant Responses during Seed
Germination of Jatropha curcas
Is considered sensitive marker used for assessing membrane lipid peroxidation
An increase in free radicals causes over production of MDA
Increased MDA contents in endosperms and cotyledons suggest an increase in lipid
peroxidation during seed germination Cai etal.,2011
Changes of malondialdehyde (MDA) contents in
endosperms and cotyledons during seed germination.
32. Lipid Peroxidation and Antioxidant Responses during Seed
Germination of Jatropha curcas
Cai etal.,2011
Fig. 5: Native PAGE for SOD isoenzymes
in endosperms and cotyledons during
germination. A: endosperms; B:
cotyledons.
Fig. 2: Changes in SOD contents in
endosperms and cotyledons during
seed germination.
33. Lipid Peroxidation and Antioxidant Responses during Seed
Germination of Jatropha curcas
Cai etal.,2011
Fig. 4: Changes in peroxidase (POD)
contents in endosperms and
cotyledons during seed germination.
Fig. 5: Native PAGE for POD isoenzymes
in endosperms and cotyledons during
germination. A: endosperms; B:
cotyledons.
34. Lipid Peroxidation and Antioxidant Responses during Seed
Germination of Jatropha curcas
Cai etal.,2011
Fig. 6: Changes in Catalase (CAT)
contents in endosperms and
cotyledons during seed germination.
Fig. 7: Native PAGE for CAT isoenzymes
in endosperms and cotyledons during
germination. A: endosperms; B:
cotyledons.
35. Improvement of quality, membrane integrity and antioxidant systems in
sweet pepper (Capsicum annuum Linn.) seeds affected by osmopriming
Changes in the total peroxide (A) and MDA content (B) of aged and primed sweet pepper seeds.
Siri et al., 2013
36. Improvement of quality, membrane integrity and antioxidant systems in
sweet pepper (Capsicum annuum Linn.) seeds affected by osmopriming
Changes in the total TAA (C) and Toatal ascorbate content (D) of aged and primed sweet pepper
seeds. Siri et al., 2013
37. Improvement of quality, membrane integrity and antioxidant systems in sweet
pepper (Capsicum annuum Linn.) seeds affected by osmopriming
Siri et al., 2013
38. Seeds were desiccated at 15% relative humidity at 15⁰C temperature at five different
intervals of time 0, 20, 40, 60 and 80 hours
Effect of desiccation on antioxidant enzymes activity of
recalcitrant tea (Camellia sinensis L.) seeds
Jamalomodi et al., 2013
39. Dormancy and enzymatic activity of rice cultivars seeds
stored in different environments
Figure 1. Specific activity of the catalase enzyme (CAT) (mmol of H2O2 min -1μg of prot-1) in rice cultivar seeds during storage
in different environments. Marques et al., 2014
40. Dormancy and enzymatic activity of rice cultivars seeds
stored in different environments
Specific activity of the enzyme ascorbate peroxidase (APX) (μmol de asc min-1 μg of prot-1)
in seeds of rice cultivars during storage in different environments. Marques et al., 2014
41. b.
Figure 1 a. Lipoxygenase (nm/g.f.w) activity, b. Catalase (μ mol g-1 dw/min) activity on
zero day, three days and five days of ageing respectively in different varieties (SL 525, PK 262,
PK 416 and PK 327) of soybean Chandelet et al., 2016
a.
Changes in enzyme activity during accelerated ageing in
soybean (Glycine max L.)
42. Figure 2 a. Peroxidase (μmol g-1 dw/min) activity, b. Superoxide dismutase (unit/mgdw)
activity on zero day, three days and five days of ageing respectively in different varieties (SL
525, PK 262, PK 416 and PK 327) of soybean Chandelet et al., 2016
a.
Changes in enzyme activity during acclerared ageing in
soybean (Glycine max L.)
43. Isoenzyme patterns of sunflower seeds, (BRS 122) subjected to different storage durations (0, 4, 8, and 12 months) and conditions
(P10°C, 25°C, and 30°C, V 10°C, 25°C, and 30°C) revealed for enzymes: (A) superoxide dismutase (SOD), (B) catalase (CAT),
(C) dehydrogenase alcohol (ADH), and (D) malate dismutase (MDH). C=control, P=paper, V=vacuum.
Physiological, enzymatic, and microstructural analyses of sunflower
seeds during storage
Lins et al., 2014
44. Activity of antioxidant enzymes and proline accumulation in Erythrina velutina
Willd. seeds subjected to abiotic stresses during germination
Ribeiro et al., 2014
45. Activity of antioxidant enzymes and proline accumulation in Erythrina velutina
Willd. seeds subjected to abiotic stresses during germination
Ribeiro et al., 2014
46. Activity of antioxidant enzymes and proline accumulation in Erythrina velutina
Willd. seeds subjected to abiotic stresses during germination
Ribeiro et al., 2014
47. Antioxidant enzyme activity and germination characteristics of
different maize hybrid seeds during ageing
Mansouri et al., 2014
SC704
SC700
SC647
SC500
SC370
SC260
48. Changes in activities of antioxidant enzymes
during Chenopodium murale seed germination
Bogdanovic et al., 2008
49. Antioxidant response and related gene
expression in aged oat seed
Effect of storage duration on germination and O2
.-
production rate and H2O2 content of oat seed with
different MC at 4◦C.
Kong et al., 2015
50. Antioxidant response and related gene
expression in aged oat seed
Effect of storage duration on antioxidant enzymes and gene expression of oat seed
Kong et al., 2015
51. Antioxidant response and related gene
expression in aged oat seed
Effect of storage duration on proline
content and gene expression of P5CS1 and
PDH1 of oat seed with different MC at 4◦C.
Kong et al., 2015
52. Impact of ultra-dry storage on vigor capacity and antioxidant
enzyme activities in seed of Ammopiptanthus mongolica
Li et al., 2010
53. Impact of ultra-dry storage on vigor capacity and antioxidant
enzyme activities in seed of Ammopiptanthus mongolica
Li et al., 2010
54. Gene expression of antioxidant enzymes
and coffee seed quality during pre- and post-physiological maturity
Santos et al., 2014
55. Gene expression of antioxidant enzymes
and coffee seed quality during pre- and post-physiological maturity
Santos et al., 2014
56. Enzymatic antioxidants and seed storability
CROP SCIENTIST SUMMARY OF WORK DONE
Erythrina Shen and oden, 1998 There exist relationship between seed
deterioration and the enzymes involved in
lipid peroxidation, free radical removal,
and seed respiratory metabolism
Radish Scialaba et al., 2002 Peroxidase activity decreased in aged seeds
as compare to fresh seeds
Sunflower Pallavi et al., 2003
Abreu et al., 2013
Sharp decline in peroxidase enzyme
during ageing
The oil content in seeds of sunflower
decreases along time
Chenapodium Metrovic et al., 2005 Peroxidase and catalase activities were
found higher in younger seeds
Arabidopsis Loycrajjou et al., 2008 Ageing induced deterioration increase
the extent of protein oxidation thus
inducing loss of functional properties of
proteins and enzymes
Soybean Chandel et al., 2016 Activities of CAT, POD, SOD and APX
enzymes decreased during seed storage
thus play an important role longevity
57. Activity of enzymatic antioxidants SOD, CAT,
APX and SOD are up regulated as an
antioxidant defence system against
endogenous oxidant radicals generated during
seed germination, desiccation, maturation,
storage and ageing
Enzymatic antioxidants are an important class
of phytochemicals that plays a very crucial
role in maintaining seed quality by
counteracting the oxidative stress
Conclusion
58. Future line of work
Quantification of antioxidants presentin seeds of wild and cultivable
species
Identification of gene responsible for the production of enzymatic
antioxidants and transfer to the cultivable species
Seed quality enhancement through treatment with chemicals that
improve the activity of enzymatic antioxidants
Priming duration, combinations of antioxidant substances and their
effects on seed longevity.
Prediction of seed longevity based on antioxidant content of seed in
storage