The Use of Terbacil as a Photosynthetic Inhibitor in Lemna minor

By:
The Lac Operons
Oliver Abela
Brent Atkinson
Leah Jenkins
Robin Shrestha

LBS 145 Cell and Molecular Biology
Tuesday 7:00-10:00pm
Dr. Douglas Luckie
October 15th, 2002
http://www.msu.edu/~shresth8/lacoperons.html

Abstract: Brent Atkinson (Revised by Oliver Abela) (Second Revision by Leah Jenkins)

The purpose in this study was to develop an understanding of the effects that impurities in water sources have on the life processes of aquatic plants. Lemna minor (duckweed) was placed under three tests to determine the effects of constant lighting and foreign chemicals. Four samples of Lemna minor were observed. One containing only the original pond water, one with cupric sulfate added to the pond water, one with terbacil added to the pond water and one which was kept under a light for a period of 24 hours.

The sample in which the Lemna minor was kept in the original pond water was used as a control for all of the experiments. We found both the duckweed treated with cupric sulfate and control contained no detectable levels of polyphenoloxidase (PPO), but the duckweed grown in the CuSO4 was not able to break down starch into monosaccharides, while the control duckweed was. It was also found that adding terbacil to the medium did not affect the plant's ability to carry on photosynthesis. We also concluded that the plant which was kept under light for a period of 24 consecutive hours had more detectable levels of monosaccharides.
The effect of foreign chemicals was also tested using Nymphaea spp. (lily pads), one that was found upstream from an industrial park and another that was found downstream of that same park. We found that the lily pad found upstream had significantly more photosynthetic pigments and both samples contained no detectable levels of PPO.

Figure 16: Enzyme Test with starch after Benedict's Test after 1 Hour.

This is the after picture of our enzyme test with starch solution after Benedict's test was run. The starch solution was mixed for 1 hour before we ran this test. The first three test tubes (left to right) contain the control in the starch solution and the next three contain CuSO4 in the starch solution. All test tubes received a slight precipitate.

Discussion: Leah Jenkins (Revised by Robin Shrestha) Second Revision by Brent Atkinson

Our overall goal of this experiment was to determine if growing plants in pollutants would change the types of sugars, photosynthetic pigments, and/or enzymes normally present. Our results did not support our hypothesis which led us to believe that there might have been some sort of error with our plants and/or the tests we performed on them.

In this experiment, we varied the conditions in which we grew four duckweed samples. We grew one sample naturally, one exposed to light for a period of twenty four consecutive hours, one with terbacil added to the water, and one with cupric sulfate added to the water. We hypothesized that, exposing one sample to twenty-four hours of pure light would increase the amount of monosaccharides present in the duckweed, the addition of terbacil would inhibit the photosynthetic pigments present, and the addition of copper would increase the amount of active PPO. A test was also run to see whether the cupric sulfate would effect duckweed's ability to break down starch into glucose. We hypothesized that the duckweed grown in cupric sulfate would be able to break down starch faster than naturally grown duckweed. We also performed tests on two of the same types of plant, lily pads. One of the lily pads was grown in the run off area of a waste water treatment center in Walled Lake, Michigan, and one was grown in a natural wetland area located on Park Lake road in East Lansing, Michigan. We wanted to see if the different growth environments would affect the types of sugars, photosynthetic pigments and activities, and/or enzymes present. We hypothesized that there would be monosaccharides present in the industrial plant and none present in the natural plant, the photosynthetic pigments and activities would be less prominent in the industrial plant than the natural one, and the amount of active PPO would be greater in the plant grown in the industrial run off than the one grown naturally.

Our results did not support our original hypothesis that the excess amount of chemicals in the environment of the plant grown by the waste water treatment plant caused free aldehyde and ketone groups to be present in the leaves, while there were no free aldehyde and ketone groups found in the plant grown in the natural wetlands. We received a negative test for both the plants grown in the natural wetlands and the plants grown by the wastewater treatment plant for Benedict's test. This tells us that both plants under their respective conditions both had undetectable levels of free ketones and aldehydes. These plants have apparently adapted to their environment and are capable of living under these conditions. Our hypothesis also did not hold true for Barfoed's test. We hypothesized that the industrial plant would test positive for monosaccharides and the natural plant would not. Although, we did get a slightly positive test for the industrial plant, the natural plant tested highly positive for monosaccharides. We suspected that the added chemicals in the water would have an added effect on breaking down the sugar, but this was not the case, contradicting our hypothesis. For the iodine test, there was no starch present in the naturally or industrial grown lily pads. This test might have been done incorrectly by not having enough starch substance to give detectable positive test for the iodine test.

Our original hypothesis was, again, not supported by our results from the sugar test done between the natural duckweed and the duckweed exposed to twenty-four hours of light. We expected the duckweed exposed to twenty-four hours of light to test positive for free aldehydes or ketones, while the natural duckweed would not. Both the duckweed samples tested negative for Benedict's test, as with the lily pads. Due to all of the test runs with the lily pads and the duckweed testing negative for a free aldehyde or ketone group, we believe that our plants do not require reducing sugars to function. When testing for monosaccharides, we expected no precipitate for the duckweed grown naturally and precipitate for the duckweed grown under the light. This was not case either. When we ran the tests both the control duckweed and the duckweed exposed to twenty-four hours of light they both tested positive for Barfoed's test, the duckweed having the least amount of precipitate when compared to the duckweed grown in light for twenty-four hours. This supports our hypothesis that the duckweed exposed to the light would have a larger amount of monosaccharides present because of its increased time to use light for photosynthesis. Finally, for the Iodine test we expected a positive test with the duckweed grown naturally and a negative test with the duckweed grown under the light for 24 hours. Instead we had a negative test for the duckweed grown naturally and a slightly positive test with the duckweed grown under the light for 24 hours. We figured that starch was not detected because we did not have a high enough starch concentration in our test. The specimens should have both had starch, since plants store starch as food.

Additionally, the presence of terbacil in the other duckweed plant did not seem to hinder the photosynthetic process, which went against our predictions. The chromatography strips for natural duckweed and terbacil treated duckweed were almost identical, and all pigments were detectable. The absorbance spectrum graphs had almost identical curvature, showing that both samples of duckweed were absorbing the same wavelengths of light. We felt that these were not accurate results, and we contributed this to the amount of terbacil we added to the water. We felt that the concentration wasn't high enough to have a great enough effect on the photosynthetic activity over the week in which the plant was treated. We feel this way because terbacil is a known photosynthetic inhibitor, and at the right concentration, it should have had a greater effect on the duckweed.

The absorbance spectrum for natural and industrial lily pads were not identical in curvature, showing that both plants were not absorbing the same wavelengths of light. The industrial lily pad had an absorbance spectrum curve that was just a slope from high absorbance at low wavelength to low absorbance at high wavelengths. There seemed to be no increase in absorbance between wavelengths of 550 and 650, like in the naturally grown lily pad's absorbance spectrum. This supports our hypothesis that industrial grown lily pads would show less photosynthetic activity. The industrial lily pad is not absorbing as much light as the naturally grown lily pad, therefore less photosynthesis can be done. We also found that the lily pad grown near the wastewater treatment plant had pigments that were not detectable using paper chromatography, while the lily pad that was grown in a natural environment had very prominent pigments. We think this is due to the various chemicals that the industrial lily pad had been subject to. We feel that these chemicals were inhibiting the plants photosynthetic pigments, therefore affecting photosynthesis. To make these results more concrete, we should have run the paper chromatography more than once, to ensure that the lack of pigments was not due to error on our part, but truly due to the environment in which the two samples were grown.

Our results did not support our original prediction that the amount of active PPO would be higher in the duckweed grown with copper ions than the duckweed grown naturally. We were unable to detect any levels of PPO in any of our samples. We came to the conclusion that neither duckweed nor lily pads need PPO to function.
Our results also did not support our hypothesis in our test of duckweed's ability to break down starch into glucose. We found that the duckweed that was grown naturally was able to break enough starch down to glucose within one hour so the glucose was detectable in Benedict's test. The cupric sulfate duckweed was unable to break enough starch into glucose for detection by benedict's test in two hours. This means that the enzymes which break apart starch in the naturally grown duckweed were able to work at least twice as fast as the enzymes in the duckweed grown in cupric sulfate.

Overall, we have concluded that the addition of pollutants to growing plants does change the sugars, photosynthetic pigments, and enzymes that are on average present in a plant growing in normal conditions. The sugars are clearly broken down from polysaccharides to monosaccharides in the plants exposed to the pollutants. This disables their ability to store food in the form of starch and may be hurtful over time. The photosynthetic pigments present are also hindered in the plants grown with the pollutants. This is hurtful to the plants because it inhibits their ability to perform photosynthesis quickly and efficiently. Finally, cupric sulfate was found to hinder the plant's ability to break down starch, which would allow the plant to store more starch in vacuoles for later use, possibly enabling the plant to live longer without having to make sugar. Therefore, we have concluded that polluted environments are only helpful in hindering certain enzymes, such as the enzymes which break apart starch in duckweed, that can be harmful to the plant if they are not controlled, but overall, chemicals are hurtful to a plant's development.

If we were to do this lab over again, our group would have made sure to have enough duckweed and lily pads for the duration of the laboratory. Although we used duckweed and lily pads from constant locations, we had to harvest more of each plant before our enzyme lab. This could have created a problem, because the other tests were run on duckweed and lily pads that were harvested in August, at the peak of their growing season, while the samples for use in the enzyme lab were harvested in mid October, when the plants were slowing down their respective reactions due to cold nights. We also would have done more extensive research on the effects of terbacil and cupric sulfate on aquatic plants, so we knew how long the plants had to be treated with each chemical for the best results.