Arabidopsis and Gene Silencing; Effects of Acid Rain

1. Arabidopsis and Gene Silencing; Effects of Acid Rain By: Becky Rupnow, Jennifer Kosmicki, Margot Binetti, and Annie Kuczkowski Abstract: Arabidopsis thaliana is an ideal plant organism to be used for studying and experimenting because it behaves extremely similar to many crop plants found throughout the United States and Canada. It is also useful to study because it grows fast and matures quickly. In our experiment, we are testing the gene silencing that occurs in the Arabidopsis plant. When the plant is subjected to stress, the genes that will produce a response are silenced when the mRNA is chopped up by a dicer protein. In gene silencing, DCL acts as a dicer, where it finds a target mRNA and cuts it so that transcription cannot happen. Therefore, the specific gene cannot be expressed. In the wild type of Arabidopsis, these genes that are silenced are stress response genes. When the plant is subjected to a small amount of stress, it wants to avoid going into stress response by silencing these genes. But in the mutant plants, the RDR gene and the DCL dicers are not present, which means that the gene silencing will not occur. Therefore, when subjected to stress, the mutant plants should show more of a stress response than the wild type. This could come through in many different ways; earlier reproductive stalk, smaller leaves, less leaves, or a smaller plant. Our specific stress experiment is the effect of acidity in water on the plants. Acid rain is a significant problem in many places. To test this, we are watering the plants with 4.8 pH water, which is more acidic than natural rainwater. Under this stress, the mutant plants should exhibit a phenotypic stress response that we can measure. We will be measuring phenotypic effects such as stalk height, number of leaves, and overall height of the plant. By separating the control group, getting normal water, and the experiment group, getting acidic water, we should be able to see a difference after a couple of weeks, and may be able to get an idea about what roles the RDR and DCL carry in the plants. Results After two days of cold germination only seventy-one percent of the seeds planted sprouted. Only fifty-six of the RDR6 seeds planted sprouted. The Wild Type leaf growth was stunted by pH levels. Leaf growth was stunted in DCL4 plants treated with 4.8 pH. Leaf growth in RDR6 was not stunted by pH levels. The test Wild Type showed no signs of forming a stalk. pH levels prevented DCL4 from forming a stalk at the average height that the control DCL4 formed a stalk at. RDR6 did not show signs of forming a stalk in either the test plants or the control. Fungus gnats killed the plants before any more conclusive data could be taken. Conclusion Duration of germination period affects the plant’s ability to take root. RDR6 silenced seeds are more fragile in early stages of growth. RDR6 is not affected by pH level pH levels stunt DCL4 and Wild Type plants Formation of a stalk is stunted by increased water acidity. Fungus gnats killed the plants when they began to feed on them. What’s Next? After conducting this experiment, we found many variables that had an effect on our data. For one, the germinating seeds endured two days of germination, which effected the growth. Because of that, only about 70% of our plants came up. Also, the gnats came and destroyed our plants before we could finish the experiment. This may have prevented some data from being expressed, and therefore our experiment was not complete. If this experiment were to be conducted again, we believe that the variables should stay the same. The pH level of 4.8 was enough to send the plants into shock, but was not enough to kill the plants. However, we would need to make sure that the seeds only had one day of cold germination, and we would also need to be careful to administer the acidic water at the right time, when the plants have gotten somewhat strong but can still show an effect. Also, variables such as gnats and other environmental factors need to be kept out of the experiment. In the future, we need to make sure that we keep the plants in an enclosed space where only the variable being tested is subjected to the plants. By doing this experiment in more exact detail, we believe that it would be possible to find a specific thing that the RDR and DCL proteins do. Because of our experimental error, we had an idea of the difference between the test and control groups, but the data was blurred because of the extra variables. By doing the experiment correctly, the experimenters could discover the detailed purposes of the RDR and DCL. • Sources • http://www-plb.ucdavis.edu/labs/chan/images/arabidopsis.jpg • http://www.nature.com/nrg/journal/v4/n1/images/nrg982-f1.jpg Gene silencing involving DCL and RISC Arabidopsis thaliana plant with reproductive stalk