Sugar, Photosynthesis, and pH Tests Reveal Inhibitory Effect of Acid Rain on Hedera helix L.

Discussion

Though acid rain was found to have no effect on the concentration of carbohydrates in Scots pine needles (Shumejko et al., 1996), our results might suggest that there are no starches in the plants that were watered with the 3.0 pH rain. We believe this would be due to the denaturing of enzymes, facilitated by the increased pH, that perform condensation reactions to form glucose molecules in photosynthesis. This would be consistent with the findings of Velikova et al. (1999), who showed that photosynthetic rate decreased in bean plants treated with stimulated acid rain. This most likely will not occur in the plants that were watered with 4.0 pH rain and 5.6 pH rain because the buffers in the plants would be able to maintain the internal pH of the plant (Soares et al., 1995).

The Iodine test results indicate that no starch is present in any of the samples. We this is a somewhat surprising result. Some starch should have been detected in at least the tap water treatment, since the cellulose of the cell wall is not likely to have been degraded in this sample. The implication of this result is that our sampling method was unable to liberate enough starches to be detectable in solution. Therefore we find the results of this test to be inconclusive.
Similarly, Benedict’s test for the presence of reducing sugars revealed the presence of sugars for all samples. Since the test is qualitative in nature, we can draw no conclusions about the relative amounts of sugar present, only that all samples did show evidence of sugar. We believe this would be due to the buffers’ ability to maintain the internal pH of the plant. In addition, physical changes to the plant may alter the ability of the plant to take in CO2 for photosynthesis, which would inhibit production of glucose (Velikova et al., 1999).

The Biuret test was inconclusive. The presence of pigments in the sample solutions overwhelmed the color of the Biuret reagent, making it impossible to discern any color change in the sample treatments. Future experiments should take this into account, and would benefit from extracting the pigments before performing the Biuret analysis. With a clearer solution, it would be possible to detect the color change results of this test. Since the Biuret would detect free peptides in addition to complex proteins, one could view any apparent discrepancy between it and the Bradford assay as evidence that the altered pH inside the plant cell was sufficient to break the larger proteins apart.

The Bradford assay shows no significant trend, though all samples do show the presence of protein. We can guess, based on these results, that the change in pH has no significant effect on the amount of proteins present. This supports our original prediction, that protein synthesis would be largely unaffected.
Examining the pH of each treatment by grinding leaves into solution showed a very narrow range of variation among the samples. This suggests that the plants were all capable of maintaining internal homeostasis. However, it is worth noting that the tap water treatment had the lowest cellular pH, at 5.93, while the other treatments all hovered between 6.2 and 6.3. It is possible that the non-neutral treatments caused the plant to overcompensate for the presence of acid, thus slightly raising the internal pH.

The photosynthetic action test showed the expected results. The tap water treatment showed the most evidence of photosynthesis, with the treatment at pH 5.6 showed slightly less, but similar. The treatments at pH 4.0 and 3.0 also had very similar results, but both had significantly higher absorbencies than the other treatments. Since this test specifically measures the production of NADPH by substituting indophenol for NADP, we can only suggest that acid rain interferes with photosystem I. The higher acidity treatments may degrade the proteins in the electron transport chain, or they may degrade NADP+ reductase. Alternatively, the increased acidity may simply raise the activation energy of the NADPH synthesis reaction, rendering NADP+ reductase unable to catalyze the reaction.
Several limitations to this investigation should be acknowledged. First, there may only be a marginal amount of carbohydrates present in the ivy leaves and analysis by means of the Benedict’s and iodine tests may not have been be sensitive enough to reveal all of the carbohydrates. What is more, if there’s only a trivial amount of carbohydrates to begin with, then any changes due to the increased acidity will not be salient. Second, there are many factors that contribute to the overall pH of the ivy leaves and, thus, no claims can specifically be made about the plant’s ability to maintain homeostasis by just measuring the pH of a leaf-water mixture. Third, the pH of simulated acid rain that might negatively affect the ivy plants in this study, 3.0, was significantly lower than the average pH of rainfall in Michigan, 4.6 to 4.8. Thus, the direct correlation between these results and the effects of acid rain in the environment are not as clear.

These data suggest that simulated acid rain interferes with the ivy plant’s normal biological processes. The increased plant acidity may lead to the denaturing of proteins, structural and enzymatic, which could lead to the break down of many biologically important macromolecules. Though the Bradford assay demonstrated that the protein concentration was largely unaffected by the acid rain treatment, more effective use of the Biurette assay could determine whether acid rain leads to the denaturing of proteins into peptides, most likely by altering the acidity of the cell, which would lead to the break down of proteins. Finally, acid rain seems to attenuate the photosynthetic activity of ivy plants, as was revealed by the photosynthesis action test, and this could be due to the altering of enzymatic proteins involved in photosynthesis or by altering the reaction conditions beyond the tolerance of those proteins. [top]

References

Anonymous. 2003. 2003 Isopleth Maps. http://nadp.sws.uiuc.edu/isopleths/maps2003/
Accessed 1/23/05.

Driscoll, C.T., Driscoll, K.M., Mitchell, M.J., Raynal, D.J. (2003). Effect of Acidic
Deposition on Forest and Aquatic Ecosystems in New York State. Environmental
Pollution, 123, 327-336.

Driscoll, C.T., Lawrence, G.B., Bulger, A.J., Butler, T.J., Cronan, C.S., Eager C.,
Lambert, K.F., Likens, G.E., Stoddard, J.L., Weathers, K.C. (2001). Acidic Deposition in Northeastern United States: Sources and Inputs, Ecosystem Effects,
and Management Strategies. BioScience, 51, 180-198.

Khra, M., Maleszewski, J., Wilterding, J., Sayed, M., Luckie, D. (2005). LBS-145: Cell and Molecular Biology Lecture/Lab Spring 2005 Course Pack. East Lansing:
MSU Printing Services.

Koricheva J., Sashwati R., Vranjic J., Haukioja E., Hughes P. Hanninen O. (1997)
Antioxidant Responses to Stimulated Acid Rain and Heavy Metal Deposition in
Birch Seedlings. Environmental Pollution, 95, 249-258

Mader, S. (2002). Laboratory Manual: Human Biology Seventh Edition. New York:
McGraw-Hill.

Neufeld H.S., Jernstedt J.A., Haines B.L. (1985). Direct Foliar Effects of Simulated
Acid Rain: I. Damage, Growth, and Gas Exchange. New Phytol, 99,389-405.

Schumejko P., Ossipov V., Neuvonen S. (1996). The Effect of Simulated Acid Rain on the Biochemical Composition of Scots Pine (Pinus sylvestris L.) Needles.
Environmental Pollution, 92, 315-321

Soares, A., Ming, J., Pearson, J. (1995). Physiological Indicators and Susceptibility of Plants to Acidifying Atmospheric Pollution: A Multivariate Approach.
Environmental Pollution, 87, 159-166.

Velikova, V., Yordanov, I., Kurteva, M., Tsonev, T. (1997). Effects of simulated acid
rain on the photosynthetic characteristics of Phaseolus vulgaris L.
Photosynthetica, 34, 523-535.

Velikova, V., Tsonev, T., Yordanov, I. (1999). Light and CO2 responses of
photosynthesis and chlorophyll fluorescence characteristics in bean plants after
simulated acid rain. Physiologia Plantarum, 107, 77.

[top]

Abstract Discussion References	Sugar, Photosynthesis, and pH Tests Reveal Inhibitory Effect of Acid Rain on Hedera helix L. by: The Flying Hellfish Dan Shapiro, Giuseppe Valentini, Patrick McKeown, and Kevin Ogden Visible foliar damage occurs to ivy plants treated with simulated acid rain at pH 4.0 and 3.0. The most visible damge occured in pH 4.0. Tap water and simulated acid rain at pH 5.6 did not visibly damage the plants. Abstract Acid rain is known to have a detrimental effect on plant life We were interested in finding out exactly what the detrimental effects are in ivy plants (Hedera helix L.). We hypothesized that the pH change, which is greatly attributed to the presence of sulfuric acid, would impede a plant’s ability to synthesize vital structures and substances (Neufaeld et al, 1985). Individual ivy plants were exposed to simulated rain at pHs of 3.0, 4.0, and 5.6, along with a control plant exposed to only tap water. Leaves were collected and used to make solutions that could be tested to ascertain the effects of the treatments. An iodine test showed that little or no starch was present in all treatments. Benedict’s tests revealed that the amount of reducing sugars present increased as the pH level treatments declined. A Biuret test yielded a negative result in testing for the presence of both proteins and pepsins in the plants. The Bradford assay showed no significant trend among the treatments, though all treatments had protein present. A photosynthetic action spectrum test revealed a relative trend that as the pH of the water treatment increased the photosynthetic activity of the specimen increased. Using a pH meter to measure the pH of each treated plant, we showed that the pH of each plant differed slightly, but all remained relatively similar, despite the range in the pH of their treatment water. The similarity in the plants’ pHs suggests that the pH buffers in the plants were able to maintain relatively normal internal pH levels. [top] Discussion Though acid rain was found to have no effect on the concentration of carbohydrates in Scots pine needles (Shumejko et al., 1996), our results might suggest that there are no starches in the plants that were watered with the 3.0 pH rain. We believe this would be due to the denaturing of enzymes, facilitated by the increased pH, that perform condensation reactions to form glucose molecules in photosynthesis. This would be consistent with the findings of Velikova et al. (1999), who showed that photosynthetic rate decreased in bean plants treated with stimulated acid rain. This most likely will not occur in the plants that were watered with 4.0 pH rain and 5.6 pH rain because the buffers in the plants would be able to maintain the internal pH of the plant (Soares et al., 1995). The Iodine test results indicate that no starch is present in any of the samples. We this is a somewhat surprising result. Some starch should have been detected in at least the tap water treatment, since the cellulose of the cell wall is not likely to have been degraded in this sample. The implication of this result is that our sampling method was unable to liberate enough starches to be detectable in solution. Therefore we find the results of this test to be inconclusive. Similarly, Benedict’s test for the presence of reducing sugars revealed the presence of sugars for all samples. Since the test is qualitative in nature, we can draw no conclusions about the relative amounts of sugar present, only that all samples did show evidence of sugar. We believe this would be due to the buffers’ ability to maintain the internal pH of the plant. In addition, physical changes to the plant may alter the ability of the plant to take in CO2 for photosynthesis, which would inhibit production of glucose (Velikova et al., 1999). The Biuret test was inconclusive. The presence of pigments in the sample solutions overwhelmed the color of the Biuret reagent, making it impossible to discern any color change in the sample treatments. Future experiments should take this into account, and would benefit from extracting the pigments before performing the Biuret analysis. With a clearer solution, it would be possible to detect the color change results of this test. Since the Biuret would detect free peptides in addition to complex proteins, one could view any apparent discrepancy between it and the Bradford assay as evidence that the altered pH inside the plant cell was sufficient to break the larger proteins apart. The Bradford assay shows no significant trend, though all samples do show the presence of protein. We can guess, based on these results, that the change in pH has no significant effect on the amount of proteins present. This supports our original prediction, that protein synthesis would be largely unaffected. Examining the pH of each treatment by grinding leaves into solution showed a very narrow range of variation among the samples. This suggests that the plants were all capable of maintaining internal homeostasis. However, it is worth noting that the tap water treatment had the lowest cellular pH, at 5.93, while the other treatments all hovered between 6.2 and 6.3. It is possible that the non-neutral treatments caused the plant to overcompensate for the presence of acid, thus slightly raising the internal pH. The photosynthetic action test showed the expected results. The tap water treatment showed the most evidence of photosynthesis, with the treatment at pH 5.6 showed slightly less, but similar. The treatments at pH 4.0 and 3.0 also had very similar results, but both had significantly higher absorbencies than the other treatments. Since this test specifically measures the production of NADPH by substituting indophenol for NADP, we can only suggest that acid rain interferes with photosystem I. The higher acidity treatments may degrade the proteins in the electron transport chain, or they may degrade NADP+ reductase. Alternatively, the increased acidity may simply raise the activation energy of the NADPH synthesis reaction, rendering NADP+ reductase unable to catalyze the reaction. Several limitations to this investigation should be acknowledged. First, there may only be a marginal amount of carbohydrates present in the ivy leaves and analysis by means of the Benedict’s and iodine tests may not have been be sensitive enough to reveal all of the carbohydrates. What is more, if there’s only a trivial amount of carbohydrates to begin with, then any changes due to the increased acidity will not be salient. Second, there are many factors that contribute to the overall pH of the ivy leaves and, thus, no claims can specifically be made about the plant’s ability to maintain homeostasis by just measuring the pH of a leaf-water mixture. Third, the pH of simulated acid rain that might negatively affect the ivy plants in this study, 3.0, was significantly lower than the average pH of rainfall in Michigan, 4.6 to 4.8. Thus, the direct correlation between these results and the effects of acid rain in the environment are not as clear. These data suggest that simulated acid rain interferes with the ivy plant’s normal biological processes. The increased plant acidity may lead to the denaturing of proteins, structural and enzymatic, which could lead to the break down of many biologically important macromolecules. Though the Bradford assay demonstrated that the protein concentration was largely unaffected by the acid rain treatment, more effective use of the Biurette assay could determine whether acid rain leads to the denaturing of proteins into peptides, most likely by altering the acidity of the cell, which would lead to the break down of proteins. Finally, acid rain seems to attenuate the photosynthetic activity of ivy plants, as was revealed by the photosynthesis action test, and this could be due to the altering of enzymatic proteins involved in photosynthesis or by altering the reaction conditions beyond the tolerance of those proteins. [top] References Anonymous. 2003. 2003 Isopleth Maps. http://nadp.sws.uiuc.edu/isopleths/maps2003/ Accessed 1/23/05. Driscoll, C.T., Driscoll, K.M., Mitchell, M.J., Raynal, D.J. (2003). Effect of Acidic Deposition on Forest and Aquatic Ecosystems in New York State. Environmental Pollution, 123, 327-336. Driscoll, C.T., Lawrence, G.B., Bulger, A.J., Butler, T.J., Cronan, C.S., Eager C., Lambert, K.F., Likens, G.E., Stoddard, J.L., Weathers, K.C. (2001). Acidic Deposition in Northeastern United States: Sources and Inputs, Ecosystem Effects, and Management Strategies. BioScience, 51, 180-198. Khra, M., Maleszewski, J., Wilterding, J., Sayed, M., Luckie, D. (2005). LBS-145: Cell and Molecular Biology Lecture/Lab Spring 2005 Course Pack. East Lansing: MSU Printing Services. Koricheva J., Sashwati R., Vranjic J., Haukioja E., Hughes P. Hanninen O. (1997) Antioxidant Responses to Stimulated Acid Rain and Heavy Metal Deposition in Birch Seedlings. Environmental Pollution, 95, 249-258 Mader, S. (2002). Laboratory Manual: Human Biology Seventh Edition. New York: McGraw-Hill. Neufeld H.S., Jernstedt J.A., Haines B.L. (1985). Direct Foliar Effects of Simulated Acid Rain: I. Damage, Growth, and Gas Exchange. New Phytol, 99,389-405. Schumejko P., Ossipov V., Neuvonen S. (1996). The Effect of Simulated Acid Rain on the Biochemical Composition of Scots Pine (Pinus sylvestris L.) Needles. Environmental Pollution, 92, 315-321 Soares, A., Ming, J., Pearson, J. (1995). Physiological Indicators and Susceptibility of Plants to Acidifying Atmospheric Pollution: A Multivariate Approach. Environmental Pollution, 87, 159-166. Velikova, V., Yordanov, I., Kurteva, M., Tsonev, T. (1997). Effects of simulated acid rain on the photosynthetic characteristics of Phaseolus vulgaris L. Photosynthetica, 34, 523-535. Velikova, V., Tsonev, T., Yordanov, I. (1999). Light and CO2 responses of photosynthesis and chlorophyll fluorescence characteristics in bean plants after simulated acid rain. Physiologia Plantarum, 107, 77. [top]
Abstract Discussion References