Cycloheximide and its effect on Impatiens Pollen Elongation in Relation to the Central Dogma of Biology

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1 Cycloheximide and its effect on Impatiens Pollen Elongation in Relation to the Central Dogma of Biology Rixin Wang 2005 University of Toronto ABSTRACT Impatiens pollen germination and pollen tube elongation are driven by the central-dogma of biology (M. Olaveson, 2005). Cycloheximide is a protein synthesis inhibitor that inhibits the production of certain proteins required for such process, thus the presence of such chemical will likely result in fewer pollen germinations and shorter pollen tubes. The purpose of this laboratory was to test this hypothesis using statistical methods involving t-tests. The data was obtained by comparing Impatiens pollen growth in two cultures, one contained the chemical cycloheximide and the other acting as control with no chemicals. The observation from did agree with the hypothesis and prediction that the presence of cycloheximide will decrease the production of proteins needed for pollen growth thus result in fewer germination, this is further supported by the t-test which succeeded in rejecting the Null hypothesis which stated that cycloheximide has no effects on pollen germination and growth. INTRODUCTION The purpose of this lab was to investigate biological inhibitors effects on pollen tube elongation in Impatiens, which are popular ornamental greenhouse crops due to their wide range of bright colours and large-flowered cultivars (L. Stephens, 1998). Pollens are small grains produced by meiosis in male plants and perform the duty of delivering male nuclei to female ovary by elongation/tip growth after landing on the stigma (H. Hao et al., 2004; N. Campbell and J.B. Reese, 2005). In flowering plants, it seems that the synthesis of new proteins is required for the elongation of pollen tubes after germination shown by studies done by Mascarehas and Capkova from the 1980s which concluded that pollen grains are similar in protein content compare to pollen tubes and only differ in concentration (D. Fernando et al., 2001). Protein synthesis is part of the central dogma of biology that involves the translation of mrna into proteins using enzymes; cycloheximide is an enzyme inhibitor used in this lab to demonstrate the relationship between protein synthesis and pollen tube elongation. In addition, research suggests that the introduction of cycloheximide resulted in pollen tubes of shorter lengths (O.D. Que and X.H. Tang, 1988; D. Fernando et al., 2001). The goal was to test the hypothesis that the inhibition of protein synthesis caused by cycloheximide stops the production of certain vital proteins required for the elongation of Impatiens pollen tubes, and the prediction that the treatment with cycloheximide will result in pollen tubes of shorter lengths comparing to those under normal conditions. Using statistical method, two cultures of Impatiens pollens are studied. With one of the cultures acting as control with out any biological inhibitors; the other was treated 1

2 with the addition of cycloheximide. Each culture of pollens was allowed to grow for a certain period then counted and measured. For the purpose of performing the statistical test, it is hypothesized here that the two cultures shouldn t show any significant differences in length if cycloheximide does not affect production of the proteins needed for pollen tube elongation. However, base on the researches previously done, it is expected to observe some difference between the two cultures (Tang, 1988; Fernando, 2001). MATERIALS & METHODS Cycloheximide (at a physiologically meaningful concentration) was prepared in a test tube; another test tube was prepared with distilled water to ensure that no unknown chemicals are involved in this experiment. Pollens grains from Impatiens, a red-pink flowering plant, are collected by lightly brushing the tips of the pistils and distributed to each test tube resulting in two different pollen germination media. After stirring the two test tube in order to ensuring that the pollen grains are evenly distributed and then one slide was prepared for each medium. (after five minutes since the media was prepared) Using the Leica DME compound microscope and field of view vs. magnification technique, it was possible to estimate the size of the pollens (use this as the standard) while observing and recording the number of pollens germinated and those did not. After thirty minutes, prepare another set of slides from the test tubes and observe the number of pollens germinated. The same process is repeated again after sixty minutes (see figure 1). At the end of sixty minutes, prepare another set of slides in order to test for the elongation of pollen tubes by counting the number of pollens germinated and estimate its length by comparing them to the standard and the values were recorded for further investigation (see figure 2). From the obtained data, several statistical tests were performed in order to test the Null hypothesis that the presence of cycloheximide is of no effect to the elongation of Impatiens pollens (Olaveson, 2005). 2

3 Time (min) Germination with control Treatment Group % Germination 10.13% 30.11% 57.14% Germination with Cycloheximide Treatment Time (min) Group % Germination 1.27% 13.13% 28.57% Figure 1. Germination of Impatiens from the two treatments, the percentage germination was calculated by dividing the of pollens germinated by the total of pollens observed. Length (micrometer Pollen Tube Growth Pollen Tube Cycloheximide Control Figure 2. Thirty pollens of each media were observed and their lengths were estimated 3

4 RESULTS Two cultures of impatiens pollens were studied in this experiment. The media containing no chemical treatment showed a significant increase in the percentage pollen germination, as shown from figure 1; whereas the percentage growth in germination with was significant lower in comparison with the cycloheximide treatment. This difference was expected since cycloheximide acts as an protein synthesis inhibitor that block the germination of pollen grains by stopping the production of required proteins. Graph I best represent this relation: 60% Percentage Germination vs. Time Treat w /h cycloheximide Control 50% Germination (%) 40% 30% 20% 10% 0% Time (minutes) Graph I. Germination vs. Time As shown, at every time interval, the percentage of pollen germination in cycloheximide treatment ranges from 10~20% lower that of control treatment. This is due to that fact that the pollens from control treatment were able to synthesis proteins required for germination/elongation at a normal pace, therefore they have the building blocks for elongation to occur properly. A series of statistical test performed based on figure 2 resulted in the following chart: 4

5 STATISTIC CONROL Figure 3. Data Analysis TREATMENT CYCLOHEXIMIDE Sample Size Mean Standard Deviation Variance SEM t - Statistic Degrees of Freedom 58 t Statistic for two-tail test Since p < 0.025, therefore Null Rejected The t-test further disproves the validity of the Null hypothesis which states that the presence of cycloheximide has no effect on the germination/elongation of Impatiens pollens. Again, the relation between two media is most visible when construction a graph that compares the average length of germinated pollens from the two cultures: Pollen Tube Growth 1200 Average Length (micrometers) Treat w /h cycloheximide Control 0 Samples Graph II. Average pollen tube growth of two cultures From the above statistical tests and the graph showing significant difference in pollen tube lengths, it is concluded that the Null hypothesis do not stand and 5

6 cycloheximide does have an effect on Impatiens pollen germination. In the case of this laboratory, the presence of cycloheximide in the culture resulted in fewer germinated pollens and significantly shorter pollen tube lengths. DISCUSSION Among the two cultures of Impatiens pollen grains studied, the treatment of cycloheximide resulted in the decrease on percentage germination and tube length. This was as expected base on the knowledge that cycloheximide is a protein synthesis inhibitor that disrupts the translation process from mrna (M. Olaveson, 2005). Also, the differences between the two cultures of pollens were quite significant, that the data obtained from the control treatment greatly surpasses those obtained from the cycloheximide treatment (almost double in values for both the percentage of germination and the average pollen tube lengths). After performing the t-test base on data presented in Figure 2, it is apparent that the Null hypothesis should be rejected since the critical t Statistic from t Test Table is considerable less than the value calculated from obtained data. This was further proved by the researches done by Fernando (2001) and Hao (2004), both concluded in their research that the presence of cycloheximide affected the germination of Impatiens pollens due to the very fact that it inhibits the production of several proteins that are required in the process. This makes sense since a functional body would likely require the functioning of the central-dogma of biology; in every biological process, there are certain proteins (either act as enzymes or for other purposes) required in order to proceed to the next step. In the case of pollen germination, the elongation of pollen tubes require a constant supply of required proteins, but with the presence of a protein synthesis inhibitor, the central-dogma can not proceed to completion, therefore the building of pollen tubes can not continue; and in this case cycloheximide was the inhibitor. However, it was unfortunate that although many scientists have stated that cycloheximide inhibited the production of some proteins needed for germination (H. Hao, et al., 2004). Yet none of the articles specified exactly which proteins are involved in the germination process and the actual mechanism of which cycloheximide inhibits the production Overall, this experiment was done quite well with most results came out as expected. The only flaw lies within the sample size, since it is relatively small and can not accurately represent the whole population of Impatiens pollens. It would be interesting to see what a larger sample size would show us. Also, the time intervals during which the pollens are measured were not long enough and do little justice in representing what may actually be happening in a natural environment where complete germination may take days to occur (D. Fernando et al., 2001). So an alternative design to this lab is to increase the sample size and time intervals, thus increasing the similarity of data comparing to the natural world. 6

7 REFERENCES Olaveson, M. University of Toronto Stephens, Loren C Pollen fertility among BC2 offspring of Impatiens interspecific hybrids of New Guinea and Indonesian ancestry. Euphytica 103: Hao, H., Li, Y., Hu, Y., Lin, J Inhibition of RNA and protein synthesis in pollen tube development of Pinus Bungeana by actinomycin D and cycloheximide. Fernando, Danilo D., Owens, John N., Yu, X., Ekramoddoullah. Abul K.M RNA and protein synthesis during vitro pollen germination and tube elongation in Pinus monticola and other conifers. Sex Plant Reprod 13: