Degenerative Effects of Caffeine to Brassica Rapa using CHO, Photosynthesis, and Protein Analysis

Abstract

Since caffeine originates from plants, we studied the effect of it on other plants. Groups of 16 Wisconsin Fast Plants (Brassica Rapa) were given different concentrations of caffeinated water in order to study the effects of caffeine. We believe that adding caffeine to plants will show significant degradation. This was tested through differing concentrations of caffeine to show the extent of the degenerative effects.

The plants were watered six times weekly, twice a week with different amounts of .005m caffeine solution. The plants were tested with Benedict’s, Barfoed’s, Selivanoff’s, and Bial’s tests. These test for monosaccharides, ketoses vs. aldoses, reducing sugars, and furanose rings. Paper chromatography was also performed, to test for rates of flow of different carotenoids. These plants’ Absorption Spectrum and Action Spectrums were also tested, and a Bradford Assay was performed to test for protein amounts. The vascular tissue was tested to see any structural differences between the plants’ conductive tissue.

It was found that all four plants contained reducing sugars and monosaccharides, but no furanose rings. It was found that caffeine affects the function of chloroplasts. The plants that received the most caffeine had flattened absorption spectrums when compared to those that did not receive any caffeine. Vascular tissue showed a correlation between caffeine and plant degradation. The plants with caffeine added to them had very low amounts of chlorophyll a and b as compared to the control plants with only water. The Bradford assay showed that plants not given caffeine had higher concentrations of proteins.

Since caffeine originates from plants, we studied the effect of it on other plants. Groups of 16 Wisconsin Fast Plants (Brassica Rapa) were given different concentrations of caffeinated water in order to study the effects of caffeine. We believe that adding caffeine to plants will show significant degradation. This was tested through differing concentrations of caffeine to show the extent of the degenerative effects.

The plants were watered six times weekly, twice a week with different amounts of .005m caffeine solution. The plants were tested with Benedict’s, Barfoed’s, Selivanoff’s, and Bial’s tests. These test for monosaccharides, ketoses vs. aldoses, reducing sugars, and furanose rings. Paper chromatography was also performed, to test for rates of flow of different carotenoids. These plants’ Absorption Spectrum and Action Spectrums were also tested, and a Bradford Assay was performed to test for protein amounts. The vascular tissue was tested to see any structural differences between the plants’ conductive tissue.

It was found that all four plants contained reducing sugars and monosaccharides, but no furanose rings. It was found that caffeine affects the function of chloroplasts. The plants that received the most caffeine had flattened absorption spectrums when compared to those that did not receive any caffeine. Vascular tissue showed a correlation between caffeine and plant degradation. The plants with caffeine added to them had very low amounts of chlorophyll a and b as compared to the control plants with only water. The Bradford assay showed that plants not given caffeine had higher concentrations of proteins.

Discussion

The purpose of our experiments and research was to find out the degenerative effects of caffeine when it is added to plants. We experimented on four different sets of fast plants, each with different amounts of caffeine solution added at regular watering intervals. We tested the carbohydrates in the plants, to find if caffeine hindered any synthesis. We tested for the presence of pigments in each sample and absorption of these plant pigments at certain wavelengths. This was to investigate caffeine’s effect on photosynthesis as a whole. We also tested the quantity of protein present in an allotted mass of each sample, to see if the caffeinated plants contained less than the control plants. All of these tests can be found in the LBS 145 Fall 2003 Lab Book (Krha et al, 2003). We also observed the microscopic physical appearance of the plants, to check for any visible physical degradation in the caffeinated plants. We hypothesized that each sample plant will have the same carbohydrates, the plants with caffeine added will not have the same amount or type of pigments that the control plants will have, and the amount of protein in the control plants will be greater than the amount of protein in the caffeinated samples.

In our first set of tests we ran Benedict’s test, Barfoed’s test, Selivanoff’s test and Bial’s test. Benedict’s test is used to test for potential free sugars in tested solutions. We tested sample our four samples, and three controls: glucose, sucrose, and reagent standard. Benedict’s test resulted in what seemed to be no reactions, but upon closer inspection all four samples had a slightly darker appearance than sucrose. The samples did not, however, show any precipitate. We used sucrose and glucose because we knew sucrose did not react with Benedict’s test and glucose did. The slightly darker solutions could mean that the samples did have reducing sugars, however the sugars were not prevalent enough to show the reactions very well (Figure 2). This somewhat followed our prediction for Benedict’s test, which was that that all samples, those containing caffeine and those not, would contain the reducing sugars (Michalovic, 1995). The reason that the predictions were not fully accurate may be because of the minute amount of plant matter used in making our solutions. In Barfoed’s test, the test showed a brownish red precipitate if there was a presence of monosaccharides. This test also showed that the four samples were slightly darker than the control sucrose, which does not react to the test (Figure 2). This could be because of the low concentration of sugars in the plant matter. It was predicted that all samples would show a presence of polysaccharides (Campbell, 2002), and this was shown to be true. In Selivanoff’s test, all four samples reacted in more than one minute (Figure 3, 4). This means that there was no clear majority presence of aldoses and ketoses. This mimicked our prediction: it was predicted that the test would not be very conclusive, as both ketose groups (fructose, which makes up part of sucrose) and aldose groups (beta glucose) are present in plant tissue (Campbell, 2002). In Bail’s test all samples did not react, which tells us that there is not a presence of furanoses in each sample (Figure 5). This shows that our prediction was not accurate, because we predicted that there would be furanoses in all of the samples. The carbohydrate testing was qualitative, so it just showed the effects of caffeine existed, but not to what extent. Further quanitative research could be done to show the extent of degradation through the addition of caffeine.

We also performed physical analyses of the plants. One analysis was the vascular tissue examination, through microscopy. These examinations showed that the samples that received more caffeine had thinner vascular tissue (Figure 8, 9, 10). This shows that caffeine inhibited growth of the walls of the tissue, by not allowing them to develop to the right thickness. This is because the plants could not utilize all their Calcium, since the caffeine absorbed it. This means that they could not organize membranes, hydrolyze starch, or detoxify oxalic acid as well as the control plants (Boucher et. al., 2001). This represents our predictions as accurate, because, as stated in the Introduction, we predicted that the tissues with the most caffeine would look quite different than the controls.

Another test was a measurement of the height of each plant at four-day intervals. The samples that received the most caffeine were the shortest, and the samples that received no caffeine were the largest. Pictures were taken of these plants (Figure 7). The plants with the most caffeine were a purplish brown color and did not grow leaves, like the control plants did. All of this evidence shows that caffeine is in some way slowing and/or hurting the growth of the plants, which follows our predictions. We had predicted that the plants would be smaller because of the absorption of Calcium by caffeine (Boucher et. al. 2002).

The photosynthetic tests showed us many differences between the four samples. We ran the absorption spectrum test, and paper chromatography test out of the LBS 145 Lab Book’s (Krha et al, 2003). The paper chromatography was inconclusive, because the plant samples were not large enough to drop an adequate amount of drops on the paper. The reason the samples were so small is because the plants that had the most caffeine added to them did not grow very much at all. The Absorption Spectrum shows what wavelengths the pigments in the plant absorb. When samples of the four different plant samples were tested in the spectrometer, the absorbencies of the plants subjected to caffeine deviated from the control plants (Figure 6). This means that the caffeine has changed or added different pigments to the plants cells that absorb different wavelengths of light. This could potentially have adverse effects on photosynthesis because the pigments in plant are not taking in the appropriate energy needed to make sugars. These tests on the plant samples’ photosynthetic properties show that caffeine is changing the way the plants go through photosynthesis.

During the protein analysis parts of out experiment, we chose to use the Bradford Assay. The Bradford Assay tests for total protein content. We found that the plants that have not been exposed to caffeine had more absorbency at longer wavelengths meaning that in the same about of mass, the plants not exposed to caffeine had more physical protein in them. The caffeine is stopping or destroying the protein in the caffeinated plants. The smaller amount of protein in the plants with caffeine may be another reason why the plants look so sickly.

Over the process of growing the plants, we had to change the watering procedure three times. This was because the plants with the caffeine were not growing fast enough, if they grew at all. In our first attempt at watering, we watered the plants every other day with set amounts of caffeine solution and water for each of the four samples. We realized that we may have not been adding enough water to grow, so we kept the previously watered plants and planted two more sets of plants and began to water them every day, but only added the set amounts of caffeine twice a week. The results were slightly different. Most of the plants grew, but very slowly. We asked the TAs of the LBS 144 lab they watered Fast Plants in their lab. They told us that the plants grow best when they are in standing water. So we changed our watering, watered from the bottom, and added the caffeine solution from the top to make sure the plants received caffeine. The growing results were better, however, after the four weeks of watering the set of plants, the third and fourth samples that were planted first had not growth at all. It appeared that the high concentrations of caffeine and lack water completely killed them while the first and second sample, which received the same amount of watering as the third and fourth, but no caffeine, were still growing. This shows that the high concentrations of caffeine actually killed the growth of the third and fourth samples. So caffeine definitely had a detrimental effect in the fast plant growing process.

With these experiments it would appear that the caffeine has physically and chemically changed the fast plants. These effects were detrimental to the growth of the plants and often caused major problems with the experiments we tried to perform. In spite of these problems we found how the Fast Plants treated with caffeine solutions differ from Fast Plants treated with only water.