1. Emi Birch, Lilly Hoff, Julia Kuczkowski, Lucas Stover Arabidopsis thaliana – the Effects of pH on RDR6 and DCL4 Mutants Steamboat Springs High School Throughout the process of Central Dogma, DNA becomes a protein. DNA within an organism is transcribed from DNA to RNA within the nuclear membrane. The RNA is then transferred outside the membrane, messenger RNA (mRNA), and experiences translation into a protein. However, RNA interference (RNAi) disrupts this process, making the RNA non-functional, which eliminates translation. RNAi silences gene expression by the presence of a double-stranded RNA homologous to the selection of a gene being expressed. There are two types of gene families that act on the double-stranded RNA. The first is called RNA Dependent RNA Polymerase or RDR; there are seven genes of RDR. RDR copes RNA to more RNA creating the dsRNA. The second then acts on the dsRNA to dice it into smaller sections. DCL is the dicer, it is broken up into four genes. The small section of dsRNA called siRNA binds to a protein; this complex is considered to be a RNS-induced silencing complex. The complex then binds to the homologous mRNA strand and demolishes the section of mRNA. In the case that RNAi occurs in the Arabidopsis plant, phenotypic ally, the leaves will be elongated and may become purple- colored. The Arabidopsis Thaliana will be used in the experiment because its’ development is very rapid allowing for the experimentation process to be reduced. Because the Arabidopsis’ genome is only 25,500 genes long, scientists have been able to sequence the entire genome. These plants have similar stress responses to that of many crop plants. By experimenting on a plant similar to those of popular crop plants, information can be discovered that can aid to the survival and production of these plants Abstract The purpose of this experiment is to discover what kind of stress will make the RDR6 and DCL4 genes react. Specifically, it will be studied whether a change in the pH of the water will act as an environmental stressor to induce the reaction of RDR6 and DCL4. This method of forward genetics on the Arabidopsis plant will phenotypic ally show whether RDR6 and DCL4 reacted to the stressor. Based on the phenotype that is produced through the changed level of pH, it will be apparent whether these two genes reacted, and thus determine if the purpose of this lab was achieved. Specific Aims Acknowledgements This work was supported by Dr. Lisa K. Johansen at the University of Colorado at Denver http://nobelprize.org/nobel_prizes/medicine/laureates/2006/illpres/imag es/3.gif Results As was expected, the baking soda in the water triggered a stress response. After only one month, the bolts in all of the experimental plants were almost twice as tall as the control group, and the DCL4 leaves were visibly smaller than the other leaves. Because the rosettes began to form so quickly and, in some cases, reached a height of over 30 centimeters, they clearly tried to reproduce faster. This is also shown by the lack of leaves in the experimental containers. After about six weeks, the DCL4 plants began to turn a slight purple color. By the end of the experiment, part of each of the experimental plants had a small amount of purple tint while the control remained green. Although each of the experimental plants showed signs of stress response, the DCL4 showed the most prominent signs. The experimental plants produced a greater amount of pods than the control in a quicker amount of time. Conclusion After careful analysis of this Arabidopsis experiment, it can be said that in the end, the DCL4 had the most significant stress response to treatment. The rosettes and bolts located on the DCL4 turned purple which is a picture perfect definition of a stress response. The RDR6 and wild type in the experimental group showed signs of stress as well: reproducing quickly and eventually turning a slight purple color. All in all, the plants did not respond well to an increase in the pH level of the water. The next phase of this experiment would be to use a substance with an acidic pH to determine whether or not a stress response was present.