DEGRADING STRUCTURAL EFFECTS SEEN IN GATORADE® TREATED BRASSICA RAPA, ANALYZED BY SUGAR, PROTEIN AND ACTION TESTS
By:
Ansari, Lubna
Argenti, Candace
Crumrin, Trasi
Hurst, Rebecca
Figure 8. Height of Brassica rapa vs. Time. The height of the plants increased with time. The plants watered with the Gatorade ® solutions grew slower than the plants watered with DI water.
Abstract
In this experiment, we wanted to compare the chemical structures/components of Brassica rapa , fast plants, when treated with different concentrations of Gatorade® to see if the Gatorade® would have a positive effect on plants like it does on humans (Campbell, 1999). Our control group was watered with de-ionized water (DI water). We had two different treatments of diluted solutions of Gatorade® in DI water; the dilutions were 3% and 30%. The plants were divided up to run three replications of each test at a 10% extract concentration for all three treatments. For all of the carbohydrate tests and the protein test, we predicted the plants watered with the Gatorade® solutions would have the same chemical components but more of them, because of the addition of several nutrients, such as sucrose and potassium (Schwartzkopf 1972). We predicted the plants watered with Gatorade® would grow taller accordingly (Lebouf, J., et al, 2003) and that plants grown with added potassium from Gatorade ® would have higher protein concentrations (Croley, C., 1962). We used Benedict's, Barfoed's, Selivanoff's, Bial's, an Iodine test, the Bradford Assay, Action spectrum, and average height to analyze the Brassica rapa . From the carbohydrate tests, it was found that both the control and 3% treatments contained reducing sugars, disaccharide ketoses, furanose rings, and starch. Bradford's showed the 3% produced more protein than the control treatment. Measurements show the control treatment's average growth was 0.496cm/day, the 3% averaged 0.304cm/day and the 30% averaged 0.187cm/day. We also found that Gatorade® alters leaf color.
Discussion
In our experiment, we tested the affect of the addition of 3% and 30% Gatorade® solutions on the sugars present and growth of Brassica rapa . Our hypothesis for the carbohydrate and protein tests were both treatments watered with Gatorade® would have the same chemical components as the control treatment, except the treatments watered with Gatorade® would contain more chemical components due to the added nutrients. For the Bradford test we predicted the Gatorade® treated plants would produce more protein than the control treatments. For our plant growth test, we predicted the treatments watered with Gatorade® would grow taller and be greener than the control treatment. Our hypotheses were based on information stating the ingredients of Gatorade® are similar to what is found naturally occurring in plants (Schwartzkopf, C. 1972). A basis for these predictions was another experiment done by LBS 145 students who added caffeine to their fast plants. These students found the addition of caffeine had significant degenerative effects on the plants (Lebeouf, J., et al., 2003). We predict the Gatorade® will have the opposite effect of the caffeine; the Gatorade® will enhance the growth of the plants instead of having a degenerative effect. In each of the tests done, positive and negative controls were used as comparisons.
Our hypothesis for Benedict's test, a reducing sugar test, was free aldehydes and ketones would be present in all three treatments because of the sugar already present in the plant and the extra added sugar from the Gatorade®. Our prediction was there would be a red precipitate formed when the two experimental treatments were tested because of glucose, fructose, and sucrose being saccharides and listed as ingredients in Gatorade®. We predicted the control treatments, the plants watered with DI water, would likely yield precipitates because of the presence of glucose and fructose (Campbell 1999). After performing one replication with the 10% extract, we found the control and 3% treatment had a turquoise liquid with red precipitate, and the 30% treatment had a dark green liquid with red precipitate. This indicated there were free aldehydes or ketones present. Since the 30% treatment was dark green, the color indicated only a slight positive reaction had occurred. We performed another replication of the test, which yielded the same results.
The second test we ran for reducing sugars was Barfoed's test; our hypothesis was we would find different sugars in the experimental treatments, than in the control treatment. Our prediction was a red/orange precipitate would form for all three treatments because glucose and fructose are monosaccharides. The additional sugar in Gatorade®, sucrose, is a disaccharide, therefore this test won't indicate its presence. After one replication, the control treatment produced a turquoise solution with a red precipitate indicating there were monosaccharides present. Our 3% treatment, after one replication, resulted in a blue solution with a small red precipitate. Therefore the 3% treatment had trace amounts of monosaccharides. We performed another replication of the test, which yielded the same results.
The third test, Selivanoff's, differentiates between ketoses and aldoses. Our hypothesis was the three treatments would have the same structural groups and react in approximately one minute. We made the prediction that both ketoses and aldoses would be present in all three treatments. This positive reaction would produce a red solution for all treatments. The control treatment turned olive-green in about one minute, while the 3% treatment turned an orange color in approximately one minute; both indicated the presence of disaccharide ketoses. We performed another replication of the test, which yielded the same results.
Bial's test, the fourth test conducted, indicated if furanoses were present and if they were pentoses or hexoses. The hypothesis was we would find different structural compounds in the Gatorade® treated plants compared to the control treatment. Our prediction was we would get an olive-green/brown solution because of the fructose, glucose and sucrose present in all three treatments. The control treatment produced an olive-green solution indicating the presence of a hexose-furanose. The 3% treatment turned olive-green also representing a hexose-furanose. We performed another replication of the test, which yielded the same results.
Our fifth test, the Iodine test for Coiled Polysaccharides, distinguishes a polysaccharide from a mono/di – saccharide. Our hypothesis was the experiment would yield the same results for the Gatorade® treatments and the control treatment. Our prediction for this test was a bluish-black liquid would form for all three treatments. All plants have a basic concentration of starch, and we didn't introduce any more into the soil by adding the Gatorade®. The same results were obtained for all three treatments, indicating Gatorade® had no effect on the starch of the plants (adding it or removing it). The control treatment resulted in a dark orange solution, indicating there was a small positive reaction; this verified that starch was present in the treatment. The 3% treatment turned olive green and the 30% treatment turned reddish brown. This, also, confirmed the presence of starch. We performed another replication of the test, which yielded the same results.
The test done to determine protein concentration was the Bradford Assay. Our hypothesis for this test was the Gatorade® treated plants would have a different protein concentration than the control treatment. After running this test, we found the 3% treatment produced more protein than the control treatment. This was due to the added potassium from the Gatorade® (Schwartzkopf, 1972).
The second set of labs involved photosynthesis tests. Chromatography pigmentation was one of these. Our hypothesis was there would be some change between the experimental treatments and control treatment. Our prediction was all pigments would be present for both the 3% Gatorade® treatment and the control treatment. The pigments we expected were: xanthophyll, chlorophyll b, chlorophyll a, and carotene (Khra, et al., 2005). The second part of our hypothesis was the bands of the Gatorade® treated plants would be lighter in comparison to the control treatment. This prediction was based on research that indicated increasing sucrose decreased chlorophyll concentrations (Cha-um S., et al. 2004). After conducting the chromatography test, we found that our results confirmed our hypothesis.
The second test, the Hill Reaction, tested for the amount of white light absorbed in the treatments. Our hypothesis was the control treatment would absorb more white light than the 3% treatment. After observing the growth of the plants, we made an educated guess that the plants being watered with DI water (control treatment) were performing photosynthesis better than the plants being watered with the 3% Gatorade®. After completing the test, it showed that the control treatment absorbed more white light than the 3% treatment. This indicated the control treatment was able to perform photosynthesis more efficiently in white light.
Our hypothesis for the third, independent lab was the plants treated with Gatorade® would grow taller than the plants watered with DI water. The prediction was the plants watered with a Gatorade® concentration would utilize the added nutrients and be able to do more photosynthesis than the plants watered with just DI water. Results refuting our prediction were found in an on-line journal, Scientia Horticulturae . These results were, the net photosynthetic rate and chlorophyll concentration of the plantlets on day 90 increased with decreasing sucrose concentration in the medium (Cha-um S., et al. 2004). Overall, the plants watered with DI water appeared healthier looking to the eye and were on average taller than the plants watered with the Gatorade® concentrations. An online source discussed the relative elemental growth rate; this tells us the rate of expansion of any particular point on the root and where the most rapidly expanding regions are located (Erickson and Sax, 1956). It, also, discussed the limited factor of growth, which in our case would have been the Gatorade® solution. This is also why the 3% treatments were healthier and taller than the plants watered with the 30% concentration (which seem to be purple, indicating photosynthesis was not using red and blue light, but reflecting those wavelengths). These observations refuted our hypothesis; hence, our results were the exact opposite of what we predicted.
An interesting observation made during our third lab was that the leaves of each of the treatments were different colors; the control treatment had healthy, green leaves, the 3% treatment had flimsy, brown leaves, and the 30% treatment had purple leaves. In order to take this further and find out why this was happening, we researched material regarding the cause of this occurrence.
We know that during the autumn months, leaves change colors. Research done on this color change showed that the foliar starch, glucose and fructose concentrations are positively correlated with a red leaf color change; the concentrations of glucose, fructose, sucrose and stachyose were all positively correlated with the red color expressed as a percent of total leaf area (Donnelly, J. R. et al., 2003). Considering that our Gatorade® treated plants had added carbohydrates, such as starch, sucrose, and glucose, this could be why their color change was more drastic than the control treatment. Another study we found discussed the change in leaf color and how it is related to the change of chlorophyll contents. The change in color was due to degeneration in the chloroplast structure; this was caused by the many structural alterations found that in the green zone of the green-yellow banded leaf. Two changes were the decrease in the number of granum thylakoids and the disordered grana arrangement in the chloroplast; they found that the structural alterations were reversible in the recovery of the yellow part of the leaves to green, which would then allow the chloroplast structure to renew itself (Shao, J. R. et al., 1999). Consequently, another reason the leaves of each treatment were different colors could have dealt with each plant having a unique chloroplast structure due to the diverse structural alterations. The last theory as to why the leaves were different colors could have been because of the divergent concentrations of Chlorophyll A and Chlorophyll B located in each treatment. An experiment that was done previously found that nuclear mutations in peas led to a much greater decrease in Chlorophyll A than Chlorophyll B; the high Chlorophyll A/ Chlorophyll B ratio and reduction of membrane stacking indicated a decrease in the content of the light-harvesting Chlorophyll A/ Chlorophyll B-protein complex in chloroplasts of the mutant lines (Gostimskii, S. A., et al., 1991). This could, also, explain why the plants watered with the 3% Gatorade® treatments had lighter chromatography strips as well as less Chlorophyll A and B.
In order to test why our plants were not growing properly, we decided to test the pH of the Gatorade® solution. The pH was 3; this meant the Gatorade® solution was highly acidic. A method of furthering our research would have been to test what is actually in the Gatorade®, and if what is in it is different from what it listed in the ingredients.
We encountered some errors throughout all of our experiments. Our first error that could have affected our test results was we didn't have enough plant in order to use a 10% and 20% plant extract solution for our tests. Therefore we only used a 10% extract solution and it may not have been enough to react properly in the different tests. Also, the replications for each test were limited due to the amount of leaves produced by each treatment. For all of the carbohydrate and protein tests an error we could have encountered was the extraction of our 10% solution. The solution might not have been centrifuged enough, or plant stems might have been mixed into the blending process by accident, instead of just leaves. Another error could have occurred during any of the pipette measuring. Errors that could have occurred throughout the whole experiment were measuring the heights, because different people were measuring the plants each time; the other error was, the Gatorade® was coating the leaves when the plants were getting watered which could have hindered photosynthesis the absorption of light, air, or CO2. Another error could have been the plants were not receiving equal amounts of liquid when they were watered, because more than one plant occupied each quadrant of the planters. Inconsistencies in the plant growth could be skewed due to plants dying, and new plants sprouting throughout the growing process. The positive control for Barfoed's test turned dark blue instead of red as it should have. This let us to believe the Barfoed solution was contaminated. However, other trials were run after with a different batch of the solution which gave us better results.
In order to make our experiment work more efficiently, we could have planted more seeds. Then, we would have been able to make more of the 10% extract solution as well as the 20% extract solution; and therefore, it would have allowed us to conduct more replications of all the tests. This would have given us more substantial evidence; hence, leading to better results overall.