Mechanism Of Oxidative Stress In Plants And Its Regulation
MATERIALS AND METHODS
3.1 TEST PLANTS:
Triticum aestivum L. (cv UP 2338) belongs to family poaceae. Wheat is the most
important food crop of the world and it is extensively cultivated in most parts of the
world. In India Wheat is grown as rabi crop. It is sown from early October to late
November. Wheat is adapted to a variety of climate. Wheat is an annual herb with 0.61.5m high culms, which are differentiated into nodes and internodes. The roots formed in
the seedling stage are ephemeral and are soon replaced by adventitious fibrous roots.
Grain is a dry, one-seeded, indehiscent fruit, known as caryopsis. The endosperm makes
up to about 82% of the grain by weight and is delimited by aleurone layer which is rich in
proteins and minerals.
Zea mays L. (cv 4212) belongs to family poaceae and is one of the oldest
cultivated plants. It is mainly grown in East India. The plant is an annual grass, tallest
amongst the cereals, attaining a height of about 3-15 feet. The plant bears two types of
infloresences, (A) uppermost tassel with male flower and (b) lower cob with pistillate
flowers. The spikelets flower are borne by a short branch know as shank. It grows best in
temperature range of 210C to 270C.In the Industry many produced of high value such as
corn starch and its derivatives, glucose or corn syrup, corn sugar, dextrins and Industrial
alcohol are prepared from corn.
Vigna mungo L. (cv PU 31) belongs to family papilionaceae and is one of the
most widely used pulses in India. It is herbaceous annual with procumbent branches.
Being thickly clothed with long brown hairs, stem and branches give a wooly appearance.
The leaves are trifoliate. The pods contain oblong and green or black seeds. It is
commonly grown as a mixed crop.
Pisum sativum L. (cv Azad) belongs to family fabaceae and is a native of
Southwest Asia. Plant is an annual plant, with a life cycle of one year. It is a cool season
crop grown in many parts of the world; planting can take place from winter to early
37
Mechanism Of Oxidative Stress In Plants And Its Regulation
summer depending on location. The average pea weighs between 0.1 and 0.36 grams.
The immature peas (and in snow peas the tender pod as well) are used as a vegetable,
fresh, frozen or canned; varieties of the species typically called field peas are grown to
produce dry peas like the split pea shelled from the matured pod.
3.2 DESIGN OF EXPERIMENTAL WORK DONE:

Experiments were conducted on four plants in form of replicates as described
above.

Experiments were divided into two group:
(a) Pot culture (b) Petridish culture
For pot culture controlled plants were grown in clay pots with total area of 310
cm-2 filled with soil and compost 5 kg in (3:1) ratio. Experiments were carried out
in soil as pot culture under wire house conditions. Test plants under separate
experiments were treated with different doses of light intensities, herbicide (2,4D) and heavy metal (cadmium).

To study the effect of different intensities of light plants were exposed to 343,
185, 78, 46 and 40μmolm-2s-1. Light intensity was controlled by shading plant pot
with 0, 1, 2, 3 and 4 layer of muslin cloth which were equivalent to 343, 185, 78,
46 and 40μmolm-2s-1 respectively.

For pot culture cadmium (2CdSO4.7H2O) doses supplied to test plants were the
(0.0, 0.1, 0.2, 0.4 and 1.0 mM) and at higher doses 0.4 and 1.0 mM, the test plants
were given 40ppm phosphorus and 10ppm copper respectively in combination to
Cd.

For petri-culture cadmium (3CdSO4.7H2O) doses supplied to test plants were 0.0,
0.1, 0.2, 0.4 and 1.0mM and plants subjected to higher doses like 0.4 and 1.0 mM
were rectified with 40ppm phosphorus and 10ppm copper.

For pot culture 2,4-D (2,4-dichloro phenoxyacetic acid) doses supplied to test
plants were 0.0,100, 500, 2000 and 4000ppm and higher doses 2000 and 4000
ppm in test plants were given 40 ppm phosphorus and 10 ppm copper
respectively.
38
Mechanism Of Oxidative Stress In Plants And Its Regulation
PHOTO PLATE-1
A
B
D
E
G
J
C
F
H
K
I
L
M
A.Wheat seedlings, B. Wheat grain spikes, C. Wheat seeds- D. Black gram plant,
DE- Black gram seeds, F- Black gram pods G- Maize plant, H- Maize spikes IMaize seeds J- Pea plant with flower, K- Pea plant with pods, L- Pea unripped
seeds, M- Pea ripped seeds
39
Mechanism Of Oxidative Stress In Plants And Its Regulation

Plants were observed daily for some abnormal changes in the form of chlorosis,
necrosis, browning of leaf tissues, stem curvature, etiolating, stunting growth and
other typical toxic symptoms resulted due to stress exposure.

At the end of experiments, plants were harvested and thoroughly washed with
distilled water and blotting the moisture for fresh weight and dry weight of plants.

In the leaf extract, stress effect of various oxidative stress causing agents like
cadmium, 2,4-D and light on morphological, physiological and biochemical
parameter, were measured.

The details of parameters undertaken in these studies were given below.
3.3 CHEMICALS:
Acetone, thiobarbituric acid (TBA), polyvinyl pyrophosphate (PVP), nitro blue
tetrazolium (NBT), nicotinamide adenine dinucleotide phosphate (NADPH), gluta thione
reduced (GSH), glutathione oxidized (GSSG), ninhydrin were purchased from merck
india pvt ltd, india. Salt for potassium phosphate buffer (K2HPO4 and KH2PO4), glacial
acetic acid (CH3COOH), nitric acid, ethanol, ethylene diamine tetraacetic acid disodium
salt (EDTA), potassium nitrate, calcium nitrate, magniseum sulphate, cadmium sulphate,
ferric chloride, boric acid, manganese sulphate, zinc sulphate, cupric salt, tricarboxylic
acid (TCA), sodium hydroxide (NaOH), perchloric acid, 2,4-dichlorophenoxyacetic acid
(2,4-D), sulphuric acid (H2SO4), hydrogen peroxide (H2O2), folin and ciocalteu’s phenol
regent hydrogen chloride, methionine, riboflavin, toluene, DTNB and other chemicals
and regents used throughout these investigations were purchased from Merck India Pvt.
Ltd., India.
3.4 INSTRUMENTS:
UV-Visible
Spectrophotometer
(UV5704SS),
double
atomic
absorption
spectrometer no AAS Elementas 4141, Centrifuge machine, autoclave, Oven, Electronic
balance, pH meter, Water bath, Lux meter,
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Mechanism Of Oxidative Stress In Plants And Its Regulation
3.5 NUTRIENT SOLUTION:
The basal nutrient solution was prepared by the method given by Hewitt (1966).
Macro and micronutrients solution were prepared by using AR (analytical regent) grade
chemical regents. Calcium nitrate was prepared by the method of Hewitt (1966) Iron
ethylene diamine tetra acetate (Fe-EDTA) was prepared by the method of Jacobson
(1941). First of all the stock solution of micronutrients including potassium nitrate,
magnesium sulphate and sodium hydrogen phosphate were prepared. For making basal
nutrient solution, Fe-EDTA and stock macronutrients were diluted 1000 times by adding
distilled water.
3.6 PREPARATION OF CADMIUM (Cd) TREATMENTS
The exposure of cadmium was given in the form of 3CdSO4.7H2O. A stock
solution of Cd 5.0 mM was prepared by dissolving 3.755g in1L double distilled water.
Following doses 0.0, 0.1, 0.2, 0.4 and 1.0 mM of cadmium were obtained by serial
dilution of stock solution with double distilled water. However two higher doses 0.4 and
1.0 mM test plants were given to 40 ppm phosphorus and 10 ppm of copper. 500 ml of
cadmium solution was supplied to the plant grown in pots at interval of 3 days for 15
days.
3.7 PREPARATION OF 2,4-D TREATMENTS:
The exposure of 2,4-D was given in the form of C8H6Cl2O3. A stock solution of
2,4-D 5000ppm was prepared by dissolving 5000mg in1L double distilled water.
Following doses 0.0,100, 500, 2000 and 4000ppm of 2,4-D were obtained by serial
dilution of stock solution using double distilled water. 2,4-D. Different doses of 2,4-D
were applied to leaf surface through separate sprayer.
3.8 TREATMENTS OF DIFFERENT INTENSITIES OF LIGHT:
Light intensity was controlled by shading plant pot with 0, 1, 2, 3 and 4 layer of
muslin cloth to achieve (343, 185, 78, 46 and 40 μmolm-2s-1). Illumination under shaded
pots was measured by using Lux meter and values were converted in photosythetically
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Mechanism Of Oxidative Stress In Plants And Its Regulation
active radiation (PAR) by using formula Lux value/54. For petridish culture, seedlings
were grown in different chambers illuminated with different intensities of light.
Nutrient elements
Form in which supplied Level of supply
1. Macronutrints
(in m. equiv/L)
Potassium(K)
KNO3
4
Calcium (Ca)
Ca(NO3)2
8
Nitrogen (N)
Nitrate of Ca&K
12
Phosphorus(P)
NaH2PO4.H2O
4
Magnesium(Mg)
MgSO4.&H2O
4
Sulphur(S)
Sulphate of magnesium
4
Sodium(Na)
NaH2PO4.2H2O
4
2. Micronutrients
Form in which supplied In ppm
Iron(Fe)
Fe-EDTA
5.600
Manganese(Mg)
MnSO4.H2O
0.550
Boron(B)
H3BO3
0.370
Copper(Cu)
CuSO4.5H2O
0.064
Zinc(Zn)
ZnSO4.2H2O
0.065
Molybdenum(Mo)
Na2Mo4.2H2O
0.020
Chloride(Cl)
NaCl
3.550
Cobalt(Co)
CoSO4.7H2O
0.006
Nickel(Ni)
NiSO4.7H2O
0.006
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Mechanism Of Oxidative Stress In Plants And Its Regulation
3.9 PETRIDISH CULTURE:
This culture based on short term experiments. The first step in these experiments
was to collect glass petridishes of equal shape and size. Now these petridishes were
washed with a common detergent like vim. After this petridishes were washed in tap
water followed by HCl-washing. Now these petridishes were finally washed with deionized and glass distilled water. After proper washing, these were kept in oven to make
it moisture free. Now these petridishes were lined with blotting seats to maintain proper
moisture for growth and development of plants. In case of controlled plants, these
blotting seats were soaked with only basal nutrient solution but in case of test plants,
respective doses of heavy metal solution were also provided along with basal nutrient
solution. The experiments were carried out in replicates in the temperature range of 1520±5ºC and 12 hours illumination 185 µmol m-2 s-1 every day. Experimental seeds were
soaked in basal nutrient solution for 12 hours and after that 30 such seeds were sown in
petridishes for their germination. For Cd experiments 8 days old normal growing plants
seedlings were treated with different levels of Cd up to 15 days. Nutrient solution was
changed regularly leaving enough nutrient solution to cover a height of about 2mm. At
the end of the exposure periods 8 days the control and treated plants were harvested and
assayed for the various undertaken parameters relating to toxicity/ and tolerance
evaluation.
3.10 POT CULTURE:
For conducting the pot culture experiments the soil was taken from university
field (no contaminated site), Badsahbag, Lucknow University Campus. Before collection
of soil it was ensured that the selected sites had not received any manure and fertilizer for
past 5 years. The surface soil up-to 35 to 50 cm was collected in bulk from an area having
soil of uniform texture. Cow dung was mixed in soil in 1:3 ratios. The experiment was
conducted in earthen pots (size 25x30 cm), filled with cow dung and soil (1:3) mixture.
Pot culture experiments were performed on Pea (Pisum sativam L.), Black gram (Vigna
mungo L. PU31) and Maize (Zea mays L. 4212) seeds. Seeds were grown in pots at
ambient temperature 20-30 0C. Twenty – twenty healthy seeds of pea, black gram and
43
Mechanism Of Oxidative Stress In Plants And Its Regulation
maize were soaked overnight at room temperature 20-25 0C in distilled water. For Cd
experiment following doses 0.0, 0.1, 0.2, 0.4 and 1.0 mM of cadmium were obtained by
serial dissolution of stock solution with double distilled water. 30 days old plants were
supplied to 500ml of cadmium solution at interval of 3 days for 15 days. For 2,4-D
experiments following doses 0.0,100, 500, 2000 and 4000ppm of 2,4-D were obtained by
serial dissolution of stock solution with double distilled water. 2,4-D. Different doses of
2,4-D applied to 30 days old plants leaf surface through separate spryer. For light
experiment light intensity was controlled by shading plant pot with 0, 1, 2, 3 and 4 layer
of muslin cloth to achieve (343, 185, 78, 46 and 40 μmolm-2s-1). Illumination under
shaded pots was measured by using Lux meter. And values were converted in
photosythetically active radiation (PAR) by using formula Lux value/54. Seeds of plants
were shown in pots illuminated with different intensity of light.
3.11 ESTIMATION OF METAL ACCUMULATION BY PLANT:
Irrespective of only Cd, the Triticum aestivum has been studied for the
accumulation of Fe and Zn also. At the end of experiment (15 days for petriculture and
45 days for pot culture) the control and test plants were harvested and assayed for the
heavy metal accumulation. The plants were washed in distilled water and dried for 48
hours at 65ºC in the oven. Metal were extracted from the dried plant tissues using the
method given by APHA et al. (2005). Plant tissues were wet digested with HNO3:HClO4
(3:1, v/v) by using hot plate. After cooling, the digested solution was filtered through
whatman No. 42 filters and final volume of filtrate make 10 ml. The contents of metals
(Cd, Fe and Zn) were determined with atomic absorption spectrometer (Elementa’S
4141).
3.12 FRESH AND DRY BIOMASS:
The determination of fresh and dry weight matter yield of seedlings and plants
were taken at 15th day in petridish culture and at final stage in pot culture i.e. 48 hours of
growth. For determining fresh matter yield, the fresh plant samples were collected,
thoroughly washed with running tap water and rinsed 2 to 3 times with distilled water.
After rinsing the plant samples were blotted gently to wipe off the absorbed water and
44
Mechanism Of Oxidative Stress In Plants And Its Regulation
weighed for fresh matter yield. Now these samples were kept in an oven at 65±5ºC for 48
hours and weighted on an electronic balance to determine the effect on dry biomass
production.
3.13 MOISTURE PERCENTAGE:
The moisture percentage was calculated as follows.
Moisture percentage (%) = Fresh Matter Yield-Dry Matter Yield X 100/Fresh Matter
Yield
3.14 PHOTOSYNTHETIC PIGMENTS:
Chlorophyll was estimated by the method of Arnon (1949). Leaves were plucked
and washed with distilled water and blotted. 100 mg leaves were taken and ground in 10
ml chilled acetone (85% v/v). Extract was centrifuged at 2000 rpm for 10 minutes. The
absorbance of supernatant was read at 663, 645, 510 and 480 nm using the double beam
UV-VIS spectrophotometer UV5704SS. For calculation the following formulas were
used and the content was expressed in mg g-1 fresh weight tissue.
Chl-a =
[12.7 (A663) -2.69 (A645)] X v/1000Xw
Chl-b =
[22.9 (A663) -4.78 (A645)] X v/1000Xw
Chl-T =
[20.2 (A645) -8.02 (A663)] X v//1000Xw
Carotenoids=
[7.60 (A663) -1.49 (A510)] X v/1000Xw
V= volume of initial samples in ml
W= weight of sample in mg
3.15 ANTIOXIDATIVE ENZYMES:
3.15.1 CATALASE:
Catalase activity was assayed by the method of Bisht (1972), a modified method
of Euler and Josephson 1927. The reaction mixture for catalase containing 0.01 mM
phosphate buffer (pH 7.0) and 0.5mM H2O2 in 10 ml was incubated with suitable aliquot
45
Mechanism Of Oxidative Stress In Plants And Its Regulation
from the extract. The reaction was run for 5 minutes at room temperature (25oC) and was
stopped by the addition of 5ml 2N H2SO4, Corresponding zero hour blanks with added
H2SO4 was also run. The mixture was titrated against 0.1 N KMnO4 and the activity of
catalase were expressed as µmol H2O2 decomposed / 100 mg fresh weight tissue.
3.15.2 PEROXIDASE:
The peroxidase activity was determined as per the method of Luck (1963). The
assay system for peroxidase contained 0.5 mM phosphate buffer (pH 6.0), 0.01% (v/v)
H2O2, 5 mg p-phenylenediamine and extract in 8 ml. The reaction was run at 250C for 5
minute and stopped with 2ml 5N H2SO4. Blanks with added H2SO4 were also taken. After
centrifugation, OD was measured at 485 nm on double beam UV-VIS spectrophotometer
UV5704SS.
3.15.3 SUPEROXIDE DISMUTASE:
The activity of SOD was assayed by the method of Beauchamp and Fridovich
(1971) by measuring its ability to inhibit the photochemical reduction of nitro blue
tetrazoliun (NBT). The reaction mixture (3ml) contained 40mM phosphate buffer (PH
7.8), 13mM methionine, 75µM NBT, 2µM riboflavin, 0.1mM EDTA and a suitable
aliquot of enzyme extract. Riboflavin was added in the end. The test tubes were shaken
and reaction was allowed to run for 20 min by keeping them in sun light. The reduction in
NBT was followed by reading the absorbance at 560nm using double beam UV-VIS
spectrophotometer UV5704SS.
Blanks were run in the same way but without
illumination.
3.15.4 GLUTATHIONE REDUCTASE:
The GR activity was assayed by the method of Smith et al.(1988). The reaction
mixture contained 1. ml of 0.2 M potassium phosphate buffer (pH 7.5) containing 1mM
EDTA, 0.5 ml 3 mM 5, 5’- dithiobis (2-nitrobenzoic acid) in 0.01M phosphate buffer (pH
7.5), 0.25 ml H2O, 0.1 ml 2 mM NADPH, 0.05 ml enzyme extract and 0.1 ml 20 mM
GSSG. The components were added in order of listed above directly to a cuvette and the
reaction was started by the addition of GSSG. The increase in absorbance was monitored
for 5 minute at 412 nm. The rate of enzymes activity was calculated using standard curve
46
Mechanism Of Oxidative Stress In Plants And Its Regulation
prepared by known amount of GR (Sigma, USA). Activity of enzyme was expressed as
µmoles of GSSG reduced min-1 g-1 fresh weight.
3.15.5 ASCORBATE PEROXIDASE:
The activity of APX was measured according to the Nakano and Asada (1981) by
estimating the rate of ascorbate oxidation (extinction coefficient 2.8 mM-1 cm-1). The 3
ml reaction mixture contained 50 mM phosphate buffer (pH 7.0), 0.1 mM H2O2, 0.5 mM
sodium ascorbate, 0.1 mM EDTA and a suitable aliquot of enzyme extract. The change in
absorbance was monitored at 290 nm and enzymes activity was expressed as µmol of
ascorbate oxidized min-1 g-1.
3.15.6 LIPID PEROXIDATION:
The level of lipid peroxidation in plant tissue was measured by the method of
Heath and Packer (1971) in terms of malondialdehyde content, a product of lipid
peroxidation determined by the thiobarbituric acid reaction. Fresh control and treated leaf
tissue (0.3g) were homogenized in 3ml of 20% (w/v) trichloroacetic acid (TCA). The
homogenate was centrifuged at 3,500 rpm for 20 min. To 1ml of aliquot of the
supernatant, 1ml of 20% TCA containing 0.5 %( w/v) TBA was added. The mixture was
heated at 950C for 30 min and then quickly cooled in ice bath. The contents were
centrifuged at 10,000 X g for 15 min and the absorbance was measured at 532 nm using
double beam UV-VIS spectrophotometer UV5704SS. Value for non – specific
absorbance at 600 nm was subtracted. The concentration of MDA was calculated using
an extinction coefficient of 155 Mm-1 cm-1
3.15.7 NON-PROTEIN THIOL:
Non protein thiol group was estimated by the method of Ellman (1959). Leaves
were plucked and washed with distilled water then blotted.700mg plant tissue grind in
3ml of 6.67% sulphosalicylic acid. Extract was centrifuged at 13,000 rpm for 10 minute
at 4oC. Reaction mixture contains 5mM of EDTA, 0.6 mM of DTNB and 120mM of
phosphate buffer. For the absorbance of supernatant was read at 412nm using double
beam UV-VIS spectrophotometer UV5704SS.
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Mechanism Of Oxidative Stress In Plants And Its Regulation
3.15.8 PROLINE:
Extraction and determination of proline was performed according to the method
of Bates et al. (1973) .500 mg plant tissue was homogenized in 5ml of 3% sulphosalicylic
acid . Extracts(2ml) were held for 1h in boiling water by adding 2 ml ninhydrin
regent(1.25g ninhydrin + 30ml glacial acetic acid + 20ml 6M phosphoric acid) and 2 ml
glacial acetic acid, after which cold toluene (4ml) was added. For the absorbance of
supernatant was read at 520nm using double beam UV-VIS spectrophotometer
UV5704SS and calculated as μmol g-1 FW against standard proline.
3.15.9 CYSTEINE:
Cysteine content in control and metal exposed plants was estimated following the
method of Gaitonde (1967). Plant material (500mg) was homogenized in 5% chilled
perchloric acid and centrifuged at 10,000×g for 10 min at 4 ͦ C. cysteine content was
measured in supernatant using acid- ninhydrin regent. For preparation of every 10 ml of
acid – ninhydrin reagent, 250mg of nihydrin was dissolved in 6 ml glacial acetic acid and
4 ml HCl. Reaction mixture (3ml) contained one ml each of supernatant, glacial acetic
acid and acid nin-hydrin reagent. Mixture was heated for 15min at 95 ͦ C, and then cooled
rapidly to room temperature and absorbance was recorded at 560 nm. Cysteine content
was calculated from the standard curve prepared using known concentration of cysteine
(L-cysteine hydrochloride, sigma) and is expressed as nmol g-1 fw.
3.16 ANATOMICAL STUDIES:
Plant material was fixed in FAA [formaldehyde + acetic acid + ethyl alcohol 50%
(in ratios 5:5:90)] for suitable time. It was then dehydrated using graded series of alcohol,
xylene and lastly in paraffin wax blocks having plant tissue were cut using rotatary
microtome. Thickness of sections was kept at 50µM. Permanent slides were prepared for
studying zinc and iron accumulation. For Zn, dithiozone staining alcohol and for Fe
perl’s Prussian blue was used. Section was analyzed using image analyzer (Nikon
Eliijse).
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Mechanism Of Oxidative Stress In Plants And Its Regulation
3.17 STATISTICAL ANALYSIS:
The significant difference between the tested and the control sample was analyzed by
taking least significant difference (LSD). * Value significant at P<0.05 and ** value
significant at P<0.01 levels. The significant values of all the parameter were mean of 5
measurements each. The following statistical formulas were used for the analysis of the
data obtained in present studies.
(I)
(II)
(III)
(IV)
Where:
X=∑X/n
SE=SD/√n
SD=(X1-X)/n
X1=individual values
X=arithmetic mean of individual
values
n= number of observations
Where:
LSD= tn-2 √2*MSwithin/n = tn-2*q’
MS = Mean square
q = Studentized range
n = treatment
Mean or X
Standard Error (SE)
Standard Deviation (SD)
LSD value
49