By: Kristin Watkins, Lindsay Baum, and Kylie Dickson The stress effects of caffeine and ethanol on Arabidopsis wild type and mutant strands By: Kristin Watkins, Lindsay Baum, and Kylie Dickson Monarch High School

Research Question What are the stress effects of caffeine and ethanol on Arabidopsis wild type and mutant strands (RDR6 and DCL4)?

Hypothesis We believe that the strain type of Arabidopsis will be affected adversely by stresses such as alcohol and caffeine. If a wild type, DCL4, or RDR6 Arabidopsis plant is exposed to caffeine, then its growth will be stunted; its stem will be shorter and leaves less abundant and full. If a wild type, DCL4, or RDR6 Arabidopsis plant is exposed to ethanol, then the ethanol will dry out the plants and affect the life cycle.

Background Arabidopsis strands Dicer-like ribonucleases fourth gene silenced (DCL4) RNA-dependent RNA polymerases six gene silenced (RDR6) Wild type

Background How RNAi works RNA interference is a regulatory process where small non-coding RNA molecules inhibit gene expression thus silencing the gene. RNAi stops gene expression at the stage of translation or by stopping the transcription of specific genes. This is because siRNA have matching nucleotide sequences to the target RNA stand. SiRNA then “cleave” the messenger RNA, breaking it down so the protein is untranslatable.

Background Role of RNAi Role in development, virus resistance and stress response May be used to silence parts of cell to help identify areas necessary for a particular gene expression and response Promising tool in biotechnology, medicine, and could help lead to curing diseases by silencing the protein responsible

Background How this relates to our project Caffeine Arabidopsis expresses multiple dicers that react different when the plant is exposed to certain stresses. These reactions can be seen using RNAi silencing.   We are testing the mutant types in order to determine if RNA interference and the silencing of certain genes in mutant plants has an effect on whether the caffeine and ethanol will affect the growth and phenotypes of the plant. Caffeine Central nervous system and metabolic stimulant In humans caffeine is absorbed by the stomach and small intestine within 45 minutes Calcium release inducer Adding caffeine to cells causes internal calcium to be released and this affects the permeability and organization of the membranes

Background Alcohol Central nervous system depressant Death is possible to humans when blood alcohol level reaches .4% Can resist the growth of roots in plants when concentrations are above 50 mM and 100 mM  How this relates to our project Arabidopsis wild type and mutant strands (DCL4 and RDR6) have some of the same genes that we do as humans We wanted to see how alcohol and caffeine affects the gene expression and the DNA expression in the plants. translate this into the possible effects on the genes in humans

Methods Control plants: One tray each of Wild Type, DCL4, and RDR6 Each plant is exposed to light 8 hours a day, with 16 hours a day of dark exposure. Each plant is watered 2- 3 times a week with specially formulated fertilizer water. If time allows, we will collect the seeds from the first generation of plants and research and observe the plants they grow.

Methods Experimental Plants: Once again, each plant is exposed to light 8 hours a day, with 16 hours a day of dark exposure. Each plant is watered 2- 3 times a week with specially formulated fertilizer water. If time allows, we will collect the seeds from the first generation of plants and research and observe the plants they grow. Caffeine: We will water the plants with their normal water as well as the stress of caffeine. The plants will be exposed to .00125 Molar, .0025 Molar, and .005 Molar solution of caffeine. Ethanol: We will water the plants with their normal water as well as the stress of ethanol. The strength of the ethanol solutions we expose to the plants will be 1%, 0.5%, and 0.25%.

RDR6 Control Group Preliminary Data: Number of Leaves

DCL4 Control Group Preliminary Data: Number of Leaves

Ethanol

Ethanol

Number of Leaves on PLants exposed to ethanol Wild Type with exposure to ethanol .005M .0025M .00125M 4 12 10 20 16 9 Average 6.125 7.75 Median Standard Deviation 4.35685 5.897942 RDR6 with exposure to ethanol 13 3 15 11 3.875 10.75 12.5 0.353553391 4.440077 4.399675 T-test 0.350616663 0.21221 0.269682

DCL4 with exposure to ethanol .005M .0025M 0.00125M 4 2 9 12 11 7 14 15 Average 3.75 4.75 10.5 Median Standard Deviation 0.707106781 2.187628 T-test 0.350616663 0.443001 0.303085

Observations Ethanol All Plants .005 morality solution killed all the plants very moldy and algae is growing Plants generally have less leaves and are not as green or prickly as the control plants Tends to amplify the characteristics of the control plants Wild Type Very round leaves Uneven edges and died right away in .005 morality solution RDR6 Lighter green Sprouted straight up Leaves curl under and are narrow DCL4 Darker green .00125 turning purple and growing alright Curled leaves

Observations Caffeine Wild Type Not very different Smaller Leave ends are burning and drying out RDR6 Look similar to a control DCL4 Long stems and leaves Yellowing and burnt ends DCL4 Smallest Most dried out Small and skinny leaves

Conclusions Caffeine and Ethanol both have a effect on the plants, however caffeine had a bigger effect on the phenotypes of the plant. Caffeine seems to have stunted the growth in the diameter of the plants, especially with the DCL4 plants. The effects of the caffeine and ethanol could be due to the epigenome (comprehensive biological unit that controls gene function and expression, comprised of DNA and the histone proteins) and its response to environmental factors such as the ethanol and caffeine The stunted growth could be due to methylated or turned off genes. The environment changes the proteins which thus turns them off. The bolting that we experienced on one plant is an abnormality and temperature could be a reason as to why all our other plants did not bolt.

Conclusions A change in the environment could affect the knocked out gene in the mutant strands, which is a possible explanation for the differences in mutants verses wild type plants. It could be due to mutations in dicer and risc complex in the RNAi process that then affects protein expression and thus phenotypes. DCL4 was hurt the most by caffeine, then wild type, and the RDR6 RDR6 could have been the least effected because of the disappearance of tags due to the knock out gene Wild type was most effected by ethanol, then DCL4, and lastly RDR6 again 1% ethanol and caffeine was toxic to all plant groups .00125 DCL4 could tolerate and this may all be due to epigenomic differences

Thanks Thank you to Mrs. Donley and Dr. Johansen for helping us with our research. Their help and support has allowed our project to be successful.

Bibliography PREP Experiment Guide, Virginia Polytechnic institute & State University, Fralin Biotechnology Center, http://www.prep.biotech.vt.edu/ PREP Classroom Guide, Frequently Asked Questions, Fralin Biotechnology Center, Virginia Polytechnic Institute & State University, http://www.prep.biotech.vt.edu/ Role of Arabidopsis ARGONAUTE4 in RNA-Directed DNA Methylation Triggered by Inverted Repeats .  Current Biology , Volume 14 , Issue 13 , Pages 1214 - 1220 D . Zilberman , X . Cao , L . Johansen , Z . Xie , J . Carrington , S . Jacobsen Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, et al. (2004) Genetic and Functional Diversification of Small RNA Pathways in Plants. PLoS Biol 2(5): e104 doi:10.1371/journal.pbio.0020104 Chan SW, Zilberman D, Xie Z, Johansen LK, Carrington JC, Jacobsen SE. (2004) RNA silencing genes control de novo DNA methylation. Science 303, 1336.