The Stress Effects on RNAi Expression when Stressing Wild-type and Mutant Strains of Arabidopsis With Calcium Nitrate By: Brennan Ramos, Matthew Newell Alex Jackson Monarch High School

Question What are the effects on RNAi expression when stressing wild type and mutant strains (RDR126, DCL234) of Arabidopsis with calcium nitrate?

Why use Arabidopsis? Short life cycle 6-8 weeks Small convenient to grow plant Easily matched environmental conditions Small genome Has been completely genetically mapped Closely resembles genes in crop plants

RDR6 and DCL4 Mutants Arabidopsis contains the genes for RDR 1-6, and DCL genes 1-4. RDR - RNA dependant polymerase. DCL - Dicer Like

RNAi is the immune response of the plant. The RISC complex cleaves viral looking double stranded RNA within the cytoplasm of the cell so that the enzyme/protein can never be produced.

Hypothesis If there is a relationship between RNAi expression and environmental conditions, and we stress wild type and two other mutant Arabidopsis strains with calcium nitrate, we should see a difference in phenotypic expression. We predict that under the same stress conditions we will see differences in the development of the wild type and mutant strains. These differences may be in the form of changes in length of developmental stages, number of leaves, size and shape of leaves, and color of leaves.

Methods Used a magenta box system for wild type and two triple knockout mutant plants to stress both using calcium nitrate. Our controls will be all three types of Arabidopsis plants grown in agar, and our test groups will be all three types of plants stressed with calcium nitrate. All plants will receive 16 hours of light and 8 hours of dark. We will stress with 1 and.25 molar calcium nitrate. We will follow the MSDS Safety Guidelines for calcium nitrate. All plants will be autoclaved and disposed of through the University of Colorado Denver.

Data Plant TypeTallest Height (mm)# of LeavesColorBolting? DCL-23412GreenNo 1RDR-12622GreenNo 1/28/2010Wild Type00NoneNo DCL-23452GreenNo 2RDR-12662GreenNo 2/1/2010Wild Type12NoneNo DCL-23462GreenNo 3RDR-12682GreenNo 2/6/2010Wild TypeMold DCL-23482PurpleNo 4RDR GreenNo 2/15/2010Wild TypeMold DCL PurpleNo 5RDR PurpleNo 2/25/2010Wild TypeMold

Data DCL PurpleYes 6RDR PurpleNo 3/1/2010Wild TypeMold DCL PurpleYes 7RDR PurpleYes 3/5/2010Wild TypeMold DCL PurpleYes 8RDR PurpleYes 3/8/2010Wild TypeMold DCL PurpleYes 9RDR PurpleYes 3/11/2010Wild TypeMold DCL PurpleYes 10RDR PurpleYes 3/15/2010Wild TypeMold

Data DCL PurpleYes 11RDR PurpleYes 3/19/2010Wild TypeMold DCL PurpleYes 12RDR PurpleYes 4/1/2010Wild TypeMold

Height of Plants

Pictures

Conclusion It seems that there was a lot of mold growth in only the Wild Type plants which leads us to believe that mutations in RDR and DCL give those plants more resistance against mold.. Our RDR and DCL Arabidopsis plants grew straight through mold at times which leads us to believe that the genes that were missing in these plants may have played a role in mold resistance. A few of the RDR and DCL plants exhibited leaf color changes from green to purple as well. This leads us to believe that the triple knockout mutant exhibits stress in plain agar. Those genes that were knocked out play a role in leaf color and development.

Conclusion cont… We found that a calcium nitrate concentration.25 molar and higher would completely suppress the plant’s development. If we had been given more time we would have kept using lower concentrations of the Calcium Nitrate in order to see its effects on Arabidopsis.

We’d like to thank… Dr. Lisa Johansen for helping us by supplying us with seeds and soil, and for checking in with us to see how our plants are growing. Mrs. Kristin Donley for helping us with our ideas for how to grow our plants and for her overall guidance.

Bibliography http//: hydroponics-gardening.com; September 2008 Andrew Fire and Craig Mellow, (2006) the Nobel prize in physiology/medicine (http//:nobelprize.org) Elliot Meyerowitz, Prehistory and History of Arabidopsis Research, Plant Physiology, January 2001, vol. 125, pp Sabina Leonelli, Arabidopsis, the Botanical Drosophilia: from Mouse Cress to Model Organism; March 2007, Endeavor, vol. 31 No. 1, pp.34-38

Bibliography Franck Vazquez, Arabidopsis endogenous small RNAs: Highways and Byways, Trends in Plant Science Vol. 11 No. 9 pp Elliot M. Meyerowitz, Prehistory and History of Arabidopsis Research, Plant Physiology, January 2001, Vol. 125 pp Thomas Mitchell-Olds, Arabidopsis thaliana and its wild relatives: a model system for ecology and evolution, Trends in Ecology and Evolution Vol. 16 No. 12 December Sabina Leonelli, Arabidopsis, the botanical Drosophilia: from mouse cress to model organism, Endeavor Vol 31 no.1, pp Maria J. Clauss and Marcus A. Koch, poorly known relatives of Arabidopsis thaliana, trends in Plant Science, vol. 11 No.9, pp Douglas C. Boyes, et al., Growth Stage-Based phenotypic analysis of Arabidopsis: A Model for High Throughput Functional Genomics in Plants, The Plant Cell, Vol. 13, , July Michael Wasenegger and Gabi Krczal, Nomenclature and functions of RNA-directed RNA polymerases, Trends in Plant Science, Vol. 11 No. 3 pp , March Fralin Biotechnology Center. “PREP Experiment Guide”. Virginia Polytechnic Institute and State University. Dec. 1,

Bibliography Andrew Fire and Craig Mello. “The Nobel Prize In Physiology or Medicine for 2006.” NobelPrize.org. November 20, Fralin Biotechnology Center. “PREP Experiment Guide”. Virginia Polytechnic Institute and State University. Dec. 1,