The Effects of Green Tea on the Stress of Radish Plants PDF

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The Effects of Green Tea on the Stress of Radish Plants...

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Effects of Green Tea on the levels of Peroxidase Concentration in Radish Plants Purdue University - Biology 232 November 16, 2015

Abstract or summary Peroxidases are enzymes that are universal to plants. Within plants, peroxidase has a multilevel function in aiding in the synthesis of the plant cell wall by strengthening the cell wall components. This process mimics other processes of healing the cell that along with peroxidases ability to destroy toxins illustrates how peroxidase may play a vital role in the defense mechanism of plants. This paper explores research that tests and analyzes levels of stress through concentrations of peroxidase within radish plants, or Raphanus sativus when solely exposed to green tea, a substance that is considered healthy for humans. This test is important to understanding peroxidase, because not much further is known about the full functions of peroxidase. This test is also important because it gives insight into how a substances that is beneficial for humans, may affects the plant cells’ stress and thus peroxidase levels. Within the experiment the degree of stress the radish plant was under was judged based on the amount of peroxidase concentration present in the radish root in comparison to a control radish plant, which is solely given water. The hypothesis stated that due to the sole exposure to the green tea, the experimental plant would be stressed and thus has a higher level of plant peroxidase in comparison to the control plant. To analyze both of the radish plants, samples from the plans’ roots underwent tissue printing and DOT blot assays were performed to compare which plant contained higher concentration levels of peroxidase. Gel electrophoresis and spectroscopy were also performed to collect data on the relation between the concentration levels of the control and experimental plant. However, disproving the hypothesis, the results garnered from these tests performed illustrated minimal to no difference in the Peroxidase concentration between the control plant and the experimental plant exposed to green tea. Ultimately it was concluded that the isolated exposure to the green tea did not negatively affect the experimental radish plant significantly enough to stress the plant. Introduction Peroxidases are enzymes within plants known for biosynthesizing lignin and are also involved in the physiological defense responses to biotic and abiotic stresses within plants. The lignin the Peroxidase produces allows for the plant to become stronger and more rigid through aid in production of hydrophobic cell walls (Vicuna). They are heme-containing protein that utilizes hydrogen peroxide to oxidize substances into water

(Prasad). They catalyze the oxidation of the phenolic compounds through hydrogen peroxide. Peroxidase comes from a large family of isoenzymes, amounting to more than one hundred isoenzymes (Anderson). Despite their innate necessity and vastness in number, peroxidase’s exact function still maintains a level of uncertainty even in this day and age. Thus it is pertinent to better understand peroxidase that researchers continue to further explore and design experiments based around peroxidase In this experiment the control plant was only exposed to water in a controlled environment by a window. The experimental plant, which was only exposed to the green tea instead of the water, was also exposed to the same control environment that the control plant was exposed too. The purpose of the experiment was to analyze the degree to which the plant was stressed when only hydrated with green tea as opposed to water. To quantify the degree to which the plant was stressed, the concentration of the peroxidase within the root of both the control and the experimental were compared and analyzed. The peroxidase was used as a judgment because the higher the stress the plant is under due to exposed to the green tea, the higher the peroxidase concentration should be in comparison the control plant which would have minimal stress. The tissue blotting method and DOT blot assay were used to observe at the localized level the degree to concentration of peroxidase has increased. The gel electrophoresis was used to compare the amounts of peroxidase directly between the two plants. Also spectroscopy and was used to see the absorbance levels, which are indicative to the concentration of the peroxidase within the plants. The hypothesis stated that due to the sole exposure to the green tea, which would damage the plant cells and introduce toxins, the experimental plant would be stressed and thus has a higher level of plant peroxidase in comparison to the control plant. Materials and Methods The experiment spanned over two weeks. To prepare the radish plant samples, one control and one experimental radish plant were obtained, two total radish plants. The control environment remained constant for both plants. The control environment was a windowsill with fresh air, and access to sunlight in a temperature controlled room. The control plant received ¼ cup of room temperature water everyday in the morning. The experiment on the other hand, was hydrated with ¼ cup of green tea solution every morning. To

make the green tea solution, place 1 bag of green tea into 2 cups of boiling water. Let the water cool to room temperature before watering the plant with it. For Materials and Methods to analyze the vegetable samples’ concentration of peroxidase through tissue prints, DOT blot assays, electrophoresis, and spectrometry, please refer to pages 90-109 in the Laboratory in Cell Biology: Laboratory Manual (Anderson). Results- Figures and Tables Tissue Print/Dot Blot

Spectrophotometric Determination Table 1. Spectrophotometric Results- displays the collected data on the absorbance values standards and extracts from control and green tea plant at the spectroscopy machine set to 575 nm Tube Number Additions Absorbance at 575 1 No addition 0 40 ul Peroxidase Standard (0.08 ug/ml) 2 .023 40 ul Peroxidase Standard (0.4 ug/ml) 3 .054 40 ul Peroxidase Standard (2.0 ug/ml) 4 .202 40 ul Peroxidase Standard (10.0 ug/ml) 5 .598 6 5 ul extract 1 .061 7 40 ul extract 1 .378 8 5 ul extract 2 .054 9 40 ul extract 2 .364

Graph 1- Absorbance values of the peroxidase concentration in the Standards

Standard- Absorbance VS Peroxidase Concentration 0.7

Absorbance at 575 nm

0.6

f(x) = 0.06 x + 0.03 R² = 0.98

0.5 0.4 0.3 0.2 0.1 0 0

2

4

6

8

10

12

Peroxidase Concentration (ug/ml)

Graph 2- the Absorbance in comparison to peroxidase concentration for experimental based on Standards’ equation from Graph 1

Extract- Absorbance VS Peroxidase Concentration 0.4

Absorbance at 575 nm

0.35 0.3

f(x) = 17.3 x − 0.54 R² = 1

0.25 0.2 0.15 0.1 0.05 0 0.03

0.04

0.04

0.05

Peroxidase Concentration (ug/ml)

0.05

0.06

Electrophoresis Table 2. Electrophoresis (Refer to Figure 2) Substance Distance migrated (mm) Hemoglobin (lane 3) 10 Albumin (lane 3) 22 Cytochrome C (lane 4) 21

Direction Migrated +

Table 3. Electrophoresis to detect Peroxidase Isoenzymes (Refer to Figure 3) Source of Peroxidase Distance from Sample Well Direction Migrated (mm) HRP- Basic (lane 7) 10 14 + HRP-Mixture (lane 8) 13 13 + Vegetable Extract 1 (lanes 1 & 5) 1) 9, 9 -,+ 5) 10, 10 -,+ Vegetable Extract 2 (lanes 2 & 6) 2) 14, 10 -,+ 6) 14, 10 -,+

Discussion and Conclusion Through the results collected from the experiment run, the original hypothesis, or expectation, was to observe an increase in peroxidase concentration in the experimental radish plant hydrated only with green tea. However, the data collected illustrated that there was no difference in the levels of peroxidase between the control and the experimental radish plants. This signifies that ultimately, green tea has no significant affects on increasing the stress levels of radish plants, which would be indicated through high concentrations of peroxidases. Within the spectroscopy we did, extract 1 and 2 at 5ul had only a .007 difference in the absorbance. Similarly, at 40 ul, extract 1 and 2 only had a difference of .012 (Table 1). The differences were so minimal that there cannot be a significant change observed. Within the absorbance, it is important to remember that the darker the sample is, the more peroxidase is present, and the higher the absorbance value is. The standards were then used to create an equation (had a high R2 of 0.98) to understand the relationship between absorbance and peroxidase concentration, with the equation from Graph 1, the data was analyzed for Graph 2. Even within the graph that showed the different absorbance’s of the extracts (Graph 2), the two points for extract one were relatively located in the same place on the graph and the two point for extract 2 were also located relatively in the same location. Because of this the experimental and the control also had comparatively the exact same equation with a R2 of 1, meaning perfect match. Thus the spectroscopy simply illustrated that the substitution of green tea instead of water did not affect the stress levels of the radish plant. Furthermore, within electrophoresis we did, the two lanes containing the vegetable extracts, were ultimately the same, with bands at 13mm and 9mm (Table 2 & 3). The purple bands of the extracts travelled

the same distance regardless of whether they were from the sample with green tea or water (Figure 1 & 2). Because they had close to no change between the two samples, it is clear that both the experimental and the control had relatively the same levels of peroxidase concentration. Finally in the Tissue Print/ Dot Blot assay experiment, the results showed the same visual colorations of purple between the green tea and water (control) extracts (Figure 3). Therefore, there was similar concentration of the peroxidase in both the experimental and control tissue samples. The coloration was also pretty evenly spread throughout the tissue print of each sample too, thus there were no locations within the tissue of an individual plant that had higher concentrations of peroxidase (Figure 3). According to previous experiments other researchers have conducted, green tea contains fluoride, a toxic substance to certain monocot plants due to accumulation within their xylem (Hershey, 2000). However, because radishes are dichotic plants, fluoride would have minimal affect, thus not stressing the radish plant. These results coincide with results from this current experiment in Bio 232; which, reports no change stress through no increase in the experimental plant’s peroxidase concentration. These results can be further explained through the understanding of ultimately what tea is; it is simply water and supplemental extracts from another plants leafs. If anything, the tea contains mineral, organic acids, and microbial byproducts, which are very beneficial to plant growth (Mahaffee). Because of this, it is highly possible that the green tea did not harm the experimental plant in any way, and thus did not increase the levels of peroxidase. The peroxidase would have only increased in concentration due toxins or damage within the plant. While our data is justifiable, there could have been a possible error due to the concentration of green tea. If we further this experiment, I believe we would need to increase the green tea concentration, simply because the concentration in this test may have been too low to actually impact the plant significantly enough to affect its composition. We could also continue this experiment over a longer span of time instead of the two weeks to gain a better understanding of the long term effects the green tea would have on the stress levels of the radish plants.

Bibliography Anderson, John. Laboratory in Cell Biology: Biology 232, Laboratory manual: Fall 2015. “Lab 8/9 Characterization of Peroxidase in Plants.” Pages 90-109 Print. 10 Nov. 2015 Hershey, D. (2000). What effects does tea have on plants? In MadSci Network: Botany. Web. 12 Nov. 2015 Mahaffee, W., & Scheuerell, S. (n.d.). Compost teas: Alternative approaches to the biological control of plant diseases. Microbial Ecology of Aerial Plant Surfaces, 165-179. Prasad, Tottempudi K., Marc D. Anderson, and Cecil R. Stewart. “Localization and Characterization of Peroxidases in the Mitochondria of chilling-Acclimated Maize Seeding.” Plant Physiology 108 (1995):1597-1605. Web. 12 Nov. 2015 Vicuna, Deborah. "The role of peroxidases in the development of plants and their responses to abiotic stresses." (2005). Doctoral. Paper 15. Web. 12 Nov. 2015...