2. 2
Abstract:
Peroxidase is a common enzyme found in many plants. It is believed that it plays a
role in cell wall formation and defense. Previous research has shown that
peroxidase levels increase when a plant is put under stress and that many fertilizers
today cause plants to over grown and break their stems. That is the basis of this
experiment. If the radish plant is watered with an alcohol mixture instead of water,
then a higher level of peroxidase will be observed in the plant. After performing
multiple tests, the results showed that by exposing a plant to alcohol, higher levels
of peroxidase will be observed.
Introduction:
Peroxidase is an enzyme that can be found in most plants. Much is known about
them but their exact function is yet to be revealed. Scientists know that they catalyze
the reduction of hydrogen peroxide and have many different forms called
isoenzymes. They play a role in cell wall formation by linking glycoproteins and cell
wall polysaccharides, kill microorganisms, and destroy chemicals like alcohol, H2O2 ,
and phenol. High levels of peroxidase have been observed when a plant is stressed
with high salt concentrations, microorganisms, and physical wounds. All of these
observed functions have lead scientists to believe that peroxidase is a response to
stress and plays a role in defense and wound healing. (J. Anderson)
With such a high demand for crops today, many farmers use fertilizers to help their
plants grow bigger and faster (Oder). This is essential for a higher yield per acre but
a problem has been generated due to this. The plants are growing so fast that they
become too large and heavy to hold themselves up. (Marshall)
In this experiment, the plant was exposed to alcohol and the effect on peroxidase
production was observed. Knowing that peroxidase production increases with
stress and also is involved with cell wall formation, the peroxidase levels were
observed in a plant exposed to alcohol and a plant exposed to water. If the radish
plant is watered with an alcohol mixture instead of water, then a higher level of
peroxidase will be observed in the plant.
Materials and Methods:
The experiment was carried out with 2 radish plants. For 2 weeks, both plants were
given 1 cup of water every three days but the experimental plant was given a
mixture that had ¾ cup of 70% isopropyl alcohol mixed with ¼ cup of water. They
both were exposed to sunlight and kept at the same temperature. After 2 weeks of
exposure to the experimental conditions, peroxidase levels were then determined in
each plant using procedures described on pages 90-108 in the BIO 232 Lab Manual
by John Anderson. No changes were made to this procedure.
3. 3
Results:
The following images, tables and graphs show the results of our experiments.
-Dot Blot and Tissue Print (figure 1)
5 4 3 2 1
Tables of amount of Peroxidase in samples (0= none, +++ = very high concentration)
Peroxidase Standard Concentrations Relative Blue Intensity
#1 (0.01ug/mL) 0
5. 5
-Gel Electrophoresis (figure 3) – run at 90mV
Data from gel electrophoresis
Source of Peroxidase Distance from sample well
(mm)
Direction migrated
Vegetable Extract (Lanes 1
and 5)
3 To Negative
8 To Negative
7 To Positive
Vegetable Extract 2 (Lanes
2 and 6)
3 To Negative
8 To Negative
7 To Positive
Hemoglobin (Lane 3) 4 To Positive
Albumin (Lane 3) 13 To Positive
Cytochrome C (Lane 4) 11 To Negative
Horse Radish Peroxidase
Basic (Lane 7)
1 To negative
Blur To Positive
8.
7.
6.
5.
4.
3.
2.
1.
Lanes
- electrode + electrode
6. 6
Horse Radish Peroxidase
Mixture (Lane 8)
No Results No Results
Tube Number Addition Absorbance at 575 nm
1 No Addition 0
2 40 uL Peroxidase
Standard (0.08 ug/mL)
0.01
3 40 uL Peroxidase
Standard (0.4 ug/mL)
0.02
4 40 uL Peroxidase
Standard (2 ug/mL)
0.2
5 40 uL Peroxidase
Standard (10 ug/mL)
0.6
6 5 uL Extract 1 0.07
7 40 uL Extract 1 0.6
8 5 uL Extract 2 0.14
9 40 uL Extract 2 1.2
-Spectrophotometric Determination (figure 4)
y = 0.0594x + 0.0177
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 5 10 15 20 25
Absorbanceat575nm
[Peroxidase] ug/mL
Graph of Absorbance and
[Peroxidase]
Series1
Linear (Series1)
7. 7
Data for above graph
Tube Absorbance Concentration
1 0 0
2 0.01 0.08
3 0.02 0.4
4 0.2 2
5 0.6 10
6 0.07 0.88
7 0.6 9.80
8 0.14 2.06
9 1.2 19.9
On the top of the nitrocellulose paper (figure 1), the peroxidase standards
that go 10, 1, 0.1, 0.01 ug/mL from left to right. The more intense the color, the more
peroxidase present so in the first row, a gradient from left to right of high to low
peroxidase concentrations was observed. The second row is the 100% extracts from
our experimental plant on the left and then the 100% extract from our control plant.
The control plant was dark than the experimental plant, showing color intensity
similar to the 10 ug/mL control. The experimental plant showed similar color
intensity but it is not as well distributed. The line below is much more faint. The
experimental plant blot is a little darker, similar to the 0.1 ug/mL control. The
control plant at 10% concentration showed a very faint blot. In the tissue prints, the
one on the top was a section of our control plant, showing characteristics of a dicot.
The tissue print below that was from the experimental plant, showing
characteristics of a dicot as well. The print to the right showed signs of a dicot and it
was from parsnip, a known dicot. The bottom most print was from ginger, a know
monocot.
This nitrocellulose sheet (figure 2) is the same as the one above just with a
different stain and therefore all the locations are the same as above. This stain
showed protein concentration. The areas of red show where protein is present and
the darker the color, the higher the concentration. The stain shows the protein
concentrations and follows the same trend as the stain above.
This is a gel from the experiment (figure 3). The lane numbers go from 1 on
the bottom up to 8 on the top. In lane one, three bands were observed from
vegetable extract 1, 2 going to the negative electrode and one going to the positive.
In lane two, similar bands were observed as lane two but this lane contained
vegetable extract 2. In lane 3, 2 bands (a dark orange on and then a blue one that
traveled further) from the Hemoglobin-Albumin sample traveled to the positive
electrode. Lane 3 showed 2 bands (a blue one that barely traveled and an orange
one that traveled further) that traveled to the negative electrode. Lane 5 was the
same as lane one and lane 6 was the same as lane 2. Lane 7 had basic horse radish
peroxidase and show a heavy band that barely traveled to the negative electrode
and a lighter gradient to the positive electrode. It also had a light band that traveled
far down to the positive electrode.
8. 8
Figure 4 is a graph of absorbance vs. peroxidase concentration. The main line
stems from the standards that were given and then the over values were calculated
from obtained absorbance. It is clear that as absorbance goes up, so does the
peroxidase concentration.
Discussion:
The results from this experiment were somewhat inconclusive. The data from the
protein stain and the peroxidase stain (figures 1 and 2) did not show conclusive
data on an increase in peroxidase concentration. This is likely due to the fact that
these 2 experiments are not very precise. The best results came from the absorption
spectrum (seen in figure 4), which showed that if the radish plant is watered with an
alcohol mixture instead of water, then a higher level of peroxidase will be observed
in the plant. The peroxidase blots of the standard concentrations show a good
gradual increase as expected (Figure 1 and 2). The protein stain showed the location
of higher protein concentrations, which helped show the xylem and phloem in the
roots. This helped distinguish which plants were monocots and dicots. Going off of
the data from the absorbance spectrum (Figure 4), adding alcohol to fertilizers may
help strengthen their stems, thus preventing them from breaking from the weight of
themselves. Further experimentation should focus on whether or not the plant stem
has been strengthened, other ways to increase peroxidase concentration to figure
out peroxidase’s function. Another experiment performed by Filippo Passardi in
2004 showed similar results as this experiment. His results showed that when
exposed to alcohol, the plants became much stronger. They also observed a stunned
growth of the plant that was not observed in this experiment, but that may be due to
the short period of this experiment.
Citations:
Anderson, John. BIO 232 Laboratory Manual. West Lafayette: Purdue Print and
Digital Services, 2014. 90-108. Print.
Marshall, Sam. "NC Cooperative Extension." Brunswick County Center News. N.p., n.d.
Web. 18 Nov. 2014.
Oder, Tom. "Alcohol Keeps Amaryllis, Paperwhites from Flopping over." MNN. N.p.,
n.d. Web. 18 Nov. 2014.
