Chemical Stress On The Tonoplast Of Beta

Vulgaris Taproot Cells. Essay, Research Paper

Abstract

This is an experiment on how chemical stress effects cell membranes, specifically the tonoplast of Beta vulgaris taproot cells. This experiment involved the treatment of the aforesaid cells with different concentrations of an acetone solution, and testing the resulting solution for absorbance of light by betacyanine, using a spectrophotometer. It had been hypothesized that the higher the concentration of acetone in the solution, the higher the concentration of betacyanine in the resulting solution. For the most part the hypothesis was correct, however there was an error involving the blanking of the samples in the spectrophotometer.

Introduction

The experiment, betacyanine leakage due to chemical stresses on taproot cells of Beta vulgaris, was a direct follow-up of the previous experiment, betacyanine leakage due to temperature stresses on Beta vulgaris taproot cells. In the temperature experiment it was discovered that extremes in temperature caused betacyanine leakage, these extremes were anywhere below the freezing point of water, and above approximately 40?C. One speculation as to the cause of the breakage of the tonoplast in the temperature experiment was the expansion of water (when the temperature was below freezing) and by the denaturing of proteins in the membrane, thus weakening the membranes structure and causing it to break (when the temperature was above 40?C).

Based upon the previous results and speculation it was decided to test for the responses by the membrane to different chemicals. The chemical chosen was acetone. It was chosen for its harshness, acetone is used as the primary ingredient in nail polish and paint removers. It was thought that a clear and concise picture of how chemical stress effects cell membranes would be achieved by using different amounts of acetone in an acetone-water solution. As beginning scientists a strong chemical would yield sharp results, if a weaker chemical were to be used results may have been present but harder to detect, thus they may have been misinterpreted or missed completely. Using a strong chemical allows that minor errors will not completely skew our results.

Allowing acetone to react with the taproot of Beta vulgaris begins the experiment. The acetone is in different concentrations in water ranging from 0% (control) to 100%, the solutions reacted with the sample of Beta vulgaris for a period of 30 minutes. The solution was decanted, leaving the Beta vulgaris sample behind. The decanted solution was allowed to rest for five minutes; this was to allow the betacyanine a chance to diffuse throughout the entire solution. The solution would simply be measured for absorbance of light using the spectrophotometer.

It was hypothesized that the solutions with a higher concentration of acetone would yield a higher absorbance of light. The basis of the hypothesis is that the acetone would react with the tonoplast and cause it to breakdown. This breakdown would cause a leak of betacyanine, which would absorb more light.

Materials and Methods

This experiment uses easily attainable materials; the only device of any special note is the spectrophotometer. The spectrophotometer is a device that measures the amount of light allowed to pass through a sample (transparency) in a test tube. The spectrophotometer used for this experiment automatically converts the transparency of the sample to its absorbance. Other materials needed are 14 10-ml test tubes, a cork borer, one Beta vulgaris taproot (garden beet), a pipette, 14 stoppers for test tubes, and 40 ml of acetone.

Using the cork borer obtain seven cylinders from the Beta vulgaris taproot and cut them into 1.5 cm lengths. Separate the test tubes into two sets and label each set one through seven. After obtaining the cylinders of Beta vulgaris taproot, rinse them in a beaker of water three times, and them blot them dry. Place one cylinder in each test tube of the first set. In this order add water to each test tube, 1 add 6 ml of water, 2 add 5 ml of water, 3 add 4 ml of water, 4 add 3 ml of water, 5 add 2 ml of water, 6 add 1 ml of water, and 7 add no water. In this order add acetone to the test tubes, 1 add no acetone, 2 add 1 ml of acetone, 3 add 2 ml of acetone, 4 add 3 ml of acetone, 5 add 4 ml of acetone, 6 add 5 ml of acetone, and 7 add 6 ml of acetone. Place the stoppers upon the test tubes. Allow the acetone to act upon the Beta vulgaris taproot for a half of an hour, allow the reaction at room temperature.

After a half of an hour, decant the solution into the second set of test tubes, be sure to decant into the test tube labeled the same. Allow the solution to sit for five minutes. Finally place each sample in the spectrophotometer and find the absorbance of the solution.

Results

The control showed a very small absorbance of light, and progressively as the concentration of acetone increased so did the absorbance of the solution, see figure 1. In the higher concentrations of acetone erosion of the sample cylinder was clearly viable. The decanted solution of the 100% acetone solution was so dense that when held up to a light it was translucent, but in no way transparent. The values of the data can be found in figure 2.

Discussion

The results support our hypothesis as predicted the acetone broke the tonoplast and caused betacyanine to leak from the Beta vulgaris cells. The acetone reacted so strongly with the cells that in the higher concentrations it could be speculated that the acetone was dissolving the cells themselves. This may explain why the solution in the 100% acetone tube was so absorbent; perhaps other parts of the cell had dissolved making the solution cloudy. Acetone is miscible with water, alcohol, and most oils, which would lead to the conclusion that the acetone did in fact dissolve the membrane of the cell (ref 1).

An error was made in this experiment, when the spectrophotometer was set to 100% transparency. The spectrophotometer was set with a blank containing water; however, the blank should have contained water only when measuring the control sample. Then as each sample was changed so should have the blank. So when the solution tested was 16.67% acetone, the spectrophotometer should have been blanked with a 16.67% solution. If this experiment is repeated, special note should be taken to this error. Luckily this error may not have made the data erroneous, due to the fact that acetone is colorless, like water, thus the betacyanine should have been the only particles measured in the spectrophotometer.

Based upon the results of the previous experiment, these results were not shocking. As predicted in the hypothesis as the percent of acetone increased in the solution, so did the betacyanine. This would suggest that when chemical stress is applied to the cell membranes, in sufficient amounts, the membranes would break down. This is exactly what was observed in the temperature experiment. This is why we observe in mammals, the body regulates temperature and pH levels, and in plants the individual species tend to grow in specific climates, and at specific times of year. In addition plants can only survive if the conditions of the air, soil, and water have appropriate levels of pH and nutrients (ref 2).

The graph in figure 1 clearly demonstrates that when sufficient levels of acetone were present, the membranes broke down. Surprisingly the amount of acetone required to breakdown the membranes was quite high, almost 50%, to see a notable in crease in betacyanine leakage. This was rather remarkable, that meant that a Beta vulgaris taproot could be exposed to as much as 15% acetone in its water supply, and have only mild effects on the organism. I would attribute this hardiness of the organism to the error in the blanking of the spectrophotometer. Nevertheless the graph of the chemical stress on the Beta vulgaris taproot cell (figure 1) was much more exponential than that of the temperature stress (figure 3).

Literature Cited

Anonymous 1996. Acetone. Http://www.apnet.com/inscight/03231999/acetone1.html. Academic press. (Ref 1).

Solomon, Berg, and Martin 1999. Biology fifth edition. Orlando: Saunders College Publishing and Harcourt Brace College Publishers. (Ref 2).