Presenting my research work with molecular as well as biochemical perspectives.

1. 06/08/17 1
Alteration in photosynthetic pigments,
antioxidant enzymes and protein profile
in imparting lead stress tolerance to
two varieties of Triticum aestivum L.
G. B. PANT ENGINEERING COLLEGE, PAURI ,
UTTARAKHAND
PRESENTED BYPRESENTED BY
DIVYA SRIVASTAVADIVYA SRIVASTAVA
M.TECH BIOTECHNOLOGYM.TECH BIOTECHNOLOGY
IV SEMIV SEM
Under the Supervision ofUnder the Supervision of
DR. MAMTA BAUNTHIYALDR. MAMTA BAUNTHIYAL
Associate ProfessorAssociate Professor
Head Of DepartmentHead Of Department
BiotechnologyBiotechnology
G. B. Pant Engineering College, Pauri.G. B. Pant Engineering College, Pauri.
06/08/17 1

2. 06/08/17 2
Environmental damage to plants
◇ Biotic stress
◇ Abiotic stress
Environment Stress On Plant
❤❤
ABIOTIC STRESSES
Environmental, non-
biological
• Temperature (high /
low)
• Water (high / low)
• Salt
• Radiation
• Chemical
BIOTIC STRESSES
Caused by living
organisms
• Fungi
• Bacteria
• Insects
• Herbivores
• Other
plants/competition

3. 06/08/17 3
Stress avoidance
In the whole growth process does not meet with the face of
adversity
Stress tolerance
Plant has a capacity of environmental stress defense, and a
variety of physiological processes remain normal
❤❤

4. 06/08/17 4
The most widespread visual evidence of heavy metal toxicity
is a reduction in plant growth (Sharma and Dubey, 2007)
including leaf chlorosis, necrosis, a decrease in the rate of seed
germination, often correlated with progressing senescence
processes or with plant death (Dalcarso et al., 2010; Carrier et
al., 2003).

5. 06/08/17 5
In my work I have selected two varieties of wheat : I randomly
selected two normal varieties to find out tolerant variety.
 PBW-373: PBW 373 is a wheat variety suitable for late sown
and irrigated conditions. It gives an average yield of 41-45
qtls/ha. The variety normally takes early (126-134 days) to
mature. On maturity the plants of the variety attains a height of 80-
90 cms. The variety is very good for chapati making, biscuit
making. Its protein contents is 12-13 percent.
 PBW-343: PBW 343 is a wheat variety suitable for timely sown
and irrigated conditions. It gives an average yield of 46-50
qtls/ha. The variety normally takes early (126-134 days) to
mature. On maturity the plants of the variety attains a height of 80-
90 cms. Although care should be taken to save it from the attack
of loose smut. The variety grows well in high fertility condition. Its
protein contents is 11-12 percent.

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 To study the accumulation of Pb in two varieties of T. aestivum
under various concentrations of Pb in soilrite medium.
 To study the effect of various concentrations of Pb on physiological
parameters viz. seed germination percentage, root length, shoot
length, growth rate and tolerance index of two varieties of T.
aestivum.
 To study the effect of various concentrations of Pb on different
biochemical parameters viz. chlorophyll, carbohydrates, proline and
protein concentrations.
 To study the effect of various concentrations of Pb on different
antioxidant enzyme activities viz. catalase, peroxidase, glutathione
peroxidase and superoxide dismutase.
 To study the effect of various concentrations of Pb on protein
profile.

7. Lead accumulation in two varieties of Triticum aestivum
i. Lead Accumulation in Plants
ii.Bioaccumulation of lead
Determination of physiological parameters
i. Quantitative Determination of Germination Percentage, Root
Length and Shoot Length
 Germination percentage
 Root length
 Shoot Length
ii. Growth Measurement
 Growth Ratio Percentage
 Pb tolerance index (TI)
Determination of Biochemical parameter06/08/17 7

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The plant and soilrite samples were sent to ICRISAT- Hyderabad
for accumulation studies.
Accumulation of lead in samples = lead concentration (µg/g) in
samples x their biomass.

9. 06/08/17 9
Lead accumulation was directly proportional to the increasing
concentration of lead treatment. It was increased by 63% in PBW
373 whereas 30 % lead accumulation was observed in PBW 343
as compared to control. That means both wheat varieties
accumulated lead but the capacity of lead accumulation was
different in both varieties. PBW 373 showed 50 fold more
accumulation of lead as compared to PBW 343. According to
this result PBW 373 is a good lead accumulator.

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The BF of Triticum aestivum was calculated with the following
formula as per Zhao et al. (2003):
BF= [Pb concentration in plant]/ [Pb concentration in soilrite culture]

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Bioaccumulation factor was increased in both varieties, the BF
of PBW 373 was increased by 60% and 65% increase in BF of
PBW 343 was seen as compared to control of both varieties
(Wierzbicka et al., 2007).

12. 06/08/17 12
1.1Germination percentage
Germination percentage = Total number of seeds germinated/total
number of seeds sown x100

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It is clear that as compared to control (C), the germination of
seeds in both varieties decreased. 57% decrease in seed
germination of PBW 343 variety and 50 % decrease in seed
germination of PBW 373 variety was observed. PBW 373
showed a good accumulation capacity and its
bioaccumulation factor was less than other variety PBW
343. The decrease in seed germination was less in PBW 373
as compared to PBW 343. According to all results presented
till this section PBW 373 variety showed tolerance as it
accumulated more lead still showed less decrease in the seed
germination as compared to PBW 343.

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Root length (cm) = measured at the time when the plants of
two wheat varieties grown under four lead treatments were
harvested.

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The Pb concentration in the growing medium decreased the
root length of both T. aestivum varieties significantly. Root
length was reduced due to inhibition of cell division in
meristematic zone of root. Pb is a powerful inhibitor of root
growth and accumulates largely in the roots (Nakano et. al.
1981). With respect to control 75% decrease in root length of
PBW 373 was seen whereas in PBW 343 it was 52% at the
highest concentration of lead acetate (8 g/kg). The decrease
in root length was more in PBW 373 as it accumulated lead
50 times more than PBW 343, so the roots are highly
affected in this variety as compared to other one.

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1.3. Shoot Length
Shoot length (cm) = measured at the time when the plants of
two wheat varieties grown under four lead treatments were
harvested.

17. 06/08/17 17
Pb has inhibitory effect on morphological parameters of T.
aestivum varieties in present study. Shoots were negatively
affected by increasing concentration of lead, same results were
seen in earlier study by Akinci et al., 2010 for tomato crops.
70% decrease in roots length of PBW 373 was seen whereas
80% decrease was observed in PBW 343. This decrease was
due to reduction of meristematic cells in the shoot region by
the accumulation of Pb. These findings are similar to another
study in which there was also reduction in shoot length of
wheat due to Pb contaminated soil (Egley et. al., 1983).

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The growth ratio of plant was calculated with the following formula:
GR= [Plant biomass with Pb]/ [Plant biomass without Pb] x 100

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The growth ratio was decreased with the increasing lead
concentration. 68% decrease and 55% decrease were observed
in PBW 373 and PBW 343 respectively. The decrease in the
growth ratio of PBW 373 was 20 fold to PBW 343 at the
highest concentration of lead. This drastic decrease in PBW 373
is related to the lead accumulated by this variety, as it
accumulated high amount of lead so its growth ratio decreased
more than PBW 343 at the highest concentration of lead as
compared to control of both varieties.

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The tolerance index of plant is calculated with the following formula:
TI= [Root length with Pb]/ [Root length without Pb]

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The tolerance index was decreased with the increasing lead
concentration. 76% decrease and 53% decrease were seen in
PBW 373 and PBW 343 respectively.

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III. DETERMINATION OF BIOCHEMICAL PARAMETERS
1. Effect of Lead on Chlorophyll
2. Effect of Lead on Carbohydrates
3. Effect of Lead on Proline
4. Effect of Lead on Protein Content
5. Effect of Lead on Antioxidant Enzyme Activity
6. Protein profile

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Chlorophyll was estimated using the protocol of Holden (1960).
•Each of the plant samples were weighed 0.5 g and homogenized
separately in a mortar in the presence of excess of 80% acetone
until all the color was released from the tissue.
•CaCO3 was added to prevent pheophytin formation.
• Centrifuged at 5000 rpm for 10 minutes at room temperature.
•The clear supernatant was collected and then made up to a known
volume (10 ml).
•The test tubes were wrapped with black paper to protect
chlorophyll degradation.
•The spectronic colorimeter (Bausch and Lomb) was adjusted at
wavelength of 663 nm for chlorophyll ‘a’ and 645 nm for
chlorophyll ‘b’ set at 100% transmittance using 80% acetone as
blank before taking the readings of the samples respectively.
Chlorophyll Test

24. 06/08/17 24
•The optical density was measured and the chlorophyll
contents in the original extract was estimated using the
formula:
Total chlorophyll (mg/L) = 20.20A645 + 08.02 A663
Chlorophyll ‘a’ (mg/L) = 12.70A663 – 02.69 A645
Chlorophyll ‘b’ (mg/L) = 22.90A645 – 04.68 A663
•These can be converted to chlorophyll content in mg/g
dry weight as follows:
Total Chlorophyll = a + b
Chlorophyll Test

25. 06/08/17 25

26. 06/08/17 26
•Pb addition reduced the photosynthetic pigments (chlorophyll a
and chlorophyll b) significantly. Chlorophyll contents were
reduced with increasing concentration of Pb because Pb
prevents the incorporation of Fe (iron) in phytoporphyrin ring of
chlorophyll molecule and this leads to reduction in chlorophyll
contents (Foyer C.H. et. al. 1994).
•Pb is known to inhibit chlorophyll synthesis either due to
impaired uptake of Mg and Fe by plants (Malanga G. et. al.
1995) or because of increased chlorophyllase activity.
•Heavy metal stress such as caused by Pb reduced
photosynthetic pigments by either reducing their synthesis or
enhancing the process of biodegradation.
•57% decrease in chlorophyll was seen in PBW 373 and 58%
decrease in chlorophyll was seen in PBW 343.

27. 06/08/17 27
CARBOHYDRATES
•100 mg of leaf sample was taken into a boiling tube and then
hydrolyzed by keeping in a boiling water bath for 3 hours with 5ml
of 2.5N HCl. It was then cooled to room temperature. It was
neutralized with solid Na2
CO3.
•The volume was made up to 100ml and then it was centrifuged.
Supernatant was collected and 0.5 ml and 1 ml of aliquots were
taken for analysis.
•Carbohydrates are first hydrolysed into simple sugars using dilute
hydrochloric acid. In hot acidic medium glucose is dehydrated to
hydroxymethyl furfural. This compound forms with anthrone a green
coloured product with an absorption maximum at 630 nm.

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Amount of carbohydrate present in 100mg of the sample
= mg of glucose X 100 Volume of test sample

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In the present investigation, total carbohydrate of both
varieties of wheat plant was negatively affected by the
increasing concentration of lead in soilrite medium. The
negative effect of lead on carbon metabolism is a result of
their possible interaction with the reactive centre of
ribulose bisphosphate carboxylase. In the present study
53% decrease in carbohydrates was seen in PBW 373
where as 65% decrease in carbohydrates was seen in
PBW 343.

30. 06/08/17 30
1 gm of leaf samples were extracted in 5 ml of 3 % salfosalicyclic
acid at 950
C for 15 minutes. After filtration, 2 ml of supernatant was
transferred to a new tube containing 2 ml of acetic acid and 2 ml of
acidified ninhydrin reagent.
After 30 minutes of incubation at 950
C, samples were kept at room
temperature for 30 minutes and 5 ml of toluene was added to the
tube with shaking at 150 rpm to endure red product. The absorbance
of toluene layer was determined at 532 nm.
Determination of proline content

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Proline concentration increased in leaves of both
varieties of wheat exposed to increasing lead
concentrations. In wheat plant, proline concentration
peak was significantly increased at 8 g/kg lead acetate
in both varieties PBW 373 and PBW 343 by 81.35%
and 93% respectively as compared to control. Both
varieties showed significant increase but PBW 343
was increased 10 folds of PBW 373. Proline increases
as the result of lead toxicity to plants, present work
shows that PBW 343 got more toxicity as compared to
PBW 373.

33. 06/08/17 33
Determination of protein
The method developed by Lowry et al., (1951) and is sensitive
enough to give a moderately constant value.
1 gm of sample was taken and macerated in pestle mortar in 5 ml of
phosphate buffer. The homogenate was centrifuged at 8000 rpm for
20 minutes. The supernatant was collected and extraction was
repeated 4-5 times. The supernatants were combined and the
volume was made to 50 ml with phosphate buffer. 1 ml of the above
was taken and to it 1 ml of 20% TCA was added. It was then kept
for half an hour and centrifuged at 8000 rpm for 20 minutes. The
pellet was washed with acetone twice and it was again centrifuged.
Supernatant was then discarded.

34. 06/08/17 34

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The protein concentration was decreased with the increasing
concentration of lead.81.4% reduction was observed in PBW373
at the highest concentration of lead but 84% reduction was
observed in PBW373 at the highest concentration of lead as
compared to control. The decrease in protein concentration is
observed because lead damages the genetic material which causes
disturbance in central dogma so proteins get damaged due to
higher concentration of lead.

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Catalase activity was assayed following the method of Luck
(1974).
PRINCIPLE : The UV absorption of hydrogen peroxide can be
measured at 240nm, whose absorbance decreases when
degraded by the enzyme catalase. From the decrease in
absorbance, the enzyme activity can be calculated.
REAGENTS
1. Phosphate buffer : 0.067 M (pH 7.0)
2. Hydrogen peroxide (2mM) in phosphate buffer

37. 06/08/17 37
 PREPARATION OF ENZYME EXTRACT
A 20% homogenate of the different parts of B. monnieri was
prepared in phosphate buffer. The homogenate was centrifuged and
the supernatant was used for the enzyme assay.
 ASSAY
H2O2-phosphate buffer (3.0ml) was taken in an experimental
cuvette, followed by the rapid addition of 40µl of enzyme extract
and mixed thoroughly. The time required for a decrease in
absorbance by 0.05 units was recorded at 240nm in a
spectrophotometer (Genesys 10-S, USA). The enzyme solution
containing H2O2-free phosphate buffer served as control.
RH2 + H2O2 R + 2H2O

38. 06/08/17 38

39. 06/08/17 39
Lead action determined either CAT decline (Dey et al.,
2007) or moderate increases of this enzyme. Here 79.33%
decrease in CAT activity of PBW 373 and 66% decrease in
PBW 343 was observed as compared to controls of both
varieties.

40. 06/08/17 40
Peroxidase catalyses the dehydrogenation of a large number of
organic compounds such as phenols, aromatic amines,
hydroquinones etc. Peroxidase occurs in animals, higher plants
and other organisms. Guaiacol is used as substrate for the
assay of peroxidase.
AH2 + H2O2 A +2H2O

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The resulting oxidized (dehydrogenated) guaiacol is probably
more than one compound and depends on the reaction
conditions. The rate of formation of guaiacol dehydrogenation
product is a measure of the POD activity and can be assayed
spectrophotometrically at 436nm.
Peroxidase catalyses the dehydrogenation of a large number of
organic compounds such as phenols, aromatic amines,
hydroquinones etc. Peroxidase occurs in animals, higher plants
and other organisms. Guaiacol is used as substrate for the
assay of peroxidase.

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Enzyme units/liter= activity 3.18 x 0.1 x 1000/6.39 x 1 x Δt x 0.1=
500/Δt

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In the present study, POD was increased in both varieties PBW
373 and PBW 343 by 84.5 % and 83.13% respectively at the
highest concentration of lead as compared to control.

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Glutathione peroxidase
1 gm of sample was taken and homogenized in 5 fold volume
of phosphate buffer (pH 9.5). It was then centrifuged at 7000
rpm for 15 minutes. The supernatant was collected. This
process was repeated once or twice by adding phosphate buffer
in the residue.
The test tubes were labeled. 1.44 ml of phosphate buffer, 0.1
ml of EDTA, 0.1 ml of NADPH and 0.01 H2
O2
were taken in
all test tubes and mixed well. Then 0.1ml of sample was added
in all test tubes except ‘blank’. The volume was made up to 2
ml and took the reading at 340 nm.
Specific activity=A340 x Volume of reaction mixture/6.22 X 106
x Volume of sample

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46. 06/08/17 46
Enzyme activity decreases in both varieties PBW 373 and PBW
343 by 69.23% and 64.4% respectively at the highest
concentration of lead as compared to control. The decrease in
glutathione peroxidase of both varieties did not show
significant difference as compared to each other but it was
observed to be decreased as compared to control in both
varieties.

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SOD enzyme activity
1 gm of leaves was taken and was homogenized in 5 fold
volume of phosphate buffer (pH 9.5). It was then
centrifuged at 7000 rpm for 15 minutes. The supernatant
was collected. This process was repeated once or twice by
adding phosphate buffer in the residue. All of the test tubes
were labeled properly. 1.5ml of phosphate buffer, 0.1ml of
NBT, 0.1 ml of Na2CO3, 0.2 ml of methionine, 0.1 ml of
EDTA and 0.1ml of sample were added in all tubes except
blank. After that 0.7 ml of distilled water was added in all
test tubes. 0.1ml of riboflavin was added in the dark. 60
minutes incubation of the prepared samples was followed
by absorbance at 560 nm and readings were taken.
Specific activity=A560 x volume of reaction mixture/0.025 x
volume of sample x protein content

48. 06/08/17 48
2O2
¯
+ 2H+
H2
O2
+ O2

49. 06/08/17 49
70% and 21% increase in SOD enzyme activity was observed in
PBW 373 and PBW 343 respectively. According to the present
result PBW 373 showed an drastic increase in its SOD activity
which provides first line of defense to this plant. PBW 373
accumulated high amount of lead and SOD increased in same
variety, which means it can tolerate lead at high concentration.

50. 06/08/17 50
Determination of Protein Profile of Both
Varieties
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) is the molecular technique used for the characterization of
macromolecules.
Lane 1
Lane 2
Lane 3
Lane 4
Lane 5
Lane 6
Lane 7
Lane 8
SAMPLES
Molecular marker
PBW 373 C
PBW 373 T3
PBW 373 T1
PBW 343 T2
PBW 343 C
PBW 343 T1
PBW 373 T2
PBW 343 T3

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There are different band intensities which were measured by
GelQuant.net software. At 245 kDa no band was seen and at
190 kDa molecular weight all eight samples showed band
intensity in which L4 which was PBW 343 treated with 4g/kg
lead acetate showed the highest intensity, this band intensity
decreased with increasing concentration of lead acetate and
decreased to 97% in PBW 343 treated with 8 g/kg lead acetate.
The intensity of 135 kDa protein was more in control of PBW
373 as compared to other and it was seen to be decreased with
the increasing concentration of lead acetate treatment and 95%
decreased in PBW 373 treated with 8g/kg lead acetate. The
intensity of band of 17kDa was the highest for PBW 373

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kDa M L1 L2 L3 L4 L5 L6 L7 L8
245 0.09181 - - - - - - - -
190 0.09405 0.04117 0.01152 0.04309 0.46098 0.26425 0.14275 0.19992 0.0128
135 0.06245 0.06201 0.00146 0.00528 - 0.31198 - - 0.03869
100 0.00245 - - - - 0.01269 - - -
76 0.11216 0.03487 0.03419 0.0130 - 0.14947 0.19636 0.01186 -
63 - - - - - - - - -
48 0.0545 - 0.03753 0.16505 0.57034 0.17877 - 0.21837 0.32816
35 0.12493 - 0.15079 0.18079 0.1721 0.23895 0.37207 0.10264 0.26744
25 0.13266 0.00065 - - - - 0.10126 0.18652 0.16993
20 0.11002 0.14189 0.19496 0.07648 0.0815 0.06167 0.14955 0.09001 -
17 0.12691 0.21529 0.3265 0.17145 0.14295 - - 0.03715 0.06913

54. 06/08/17 54
Conclusion
•At the highest lead concentration (8g/kg) lead accumulation was
increased by 63% in PBW 373 whereas 30 % PBW 343 as
compared to control, bioaccumulation factor was 7% more in
PBW 373 than PBW 343.
•A drastic change was observed in chlorophyll of both varieties,
which was highly reduced in PBW 373 by 76%, this decrease
was 30 folds of chlorophyll of PBW 343 whereas 53% decrease
in carbohydrates was seen in PBW 373 where as 65% in PBW
343.
• Proline concentration peak was significantly increased in PBW
343 12 fold of PBW 373 at 8 g/kg lead acetate.
•There was no significant difference between both varieties in
respect to their protein content but lead showed toxic effects on
protein of both varieties.

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•Catalase activity was drastically decreased in PBW 373 by
79.33% and 66% PBW 343.
•A significant difference of 70% was observed in both
varieties, PBW 373 showed more decrease in SOD enzyme
activity.
•Protein profiling showed variation in intensity of all bands
of both varieties.
•Overall these results showed that both varieties are lead
tolerant but PBW 373 has more Pb tolerance capacity but
PBW 373 accumulates lead in high amount so should not
be used at land which are highly lead contaminated.

56. 06/08/17 56
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Kopittke PM, Asher CJ, Kopittke RA, Menzies NW (2008)
Prediction of Pb speciation in concentrated and dilute nutrient
solutions. Environ Pollut 153(3):548–554
Wierzbicka MH et. al., 2007. Comparison of the toxicity and
distribution of cadmium and lead in plant cells. Protoplasma
231, 99–111
Srivastava D, Singh A, Baunthiyal M. Lead Toxicity and
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