Calcium’s Effect on Arabidopsis thaliana RNA Silencing and Growth Patterns

1. Calcium’s Effect on Arabidopsis thaliana RNA Silencing and Growth Patterns Elizabeth Krey, Elizabeth Lyons, Alana White Observations Feb 25, 2009 - Seeds Planted March 2, 2009 - starting to show growth but no differences between wild type, DCL4, and RDR6 March 16, 2009 - Added treatment (2 calcium tablets) adding fertilizer to control and treatment. March 19, 2009 - two days with the calcium, treated seem to have bigger leaves then the control March 24, 2009 - no visible change between the control and treatment - treatment leaves are bigger then control March 30, 2009 - starting to develop bolts - wild type leaves are round shape - DCL4 and RDR6 mutant leaves are growing at a 45 degree angle April 1, 2009 - wild type treatment 3 bolts, control 1 bolt - RDR6 treatment 2 bolts, control 3 bolts - DCL4 treatment 3, control 3 bolts April 3, 2009 - one big stem is growing taller (wild type treated) - two stems are poking out of RDR6 control April 7, 2009 - RDR6 treatment 7, control 5 - DCL4 treatment 4, control 4 - Wild type treatment 6, control 2 - treatment looks bigger and greener then control - treatment leaves are curling on themselves April 9, 2009 - DCL4 showing more than one bolt per rosette April 13, 2009 - Wild type treatment bolt long 34.3 cm - Wild type control blot 25 cm - RDR6 and DCL4 have 4 bolts in one rosette - treatment plants look darker green than control April 17, 2009 - wild type treatment bolt 35.7 - last day going on spring break April 28, 2009 - treated seem to die faster then the control after a week of no water AbstractArabidopsis thaliana is a weed by standard definitions, growing geographically in temperate climates, and having a relatively short life span of about six weeks. Despite being only a “weed,” it is used as the model organism for plant genetics. With a fully mapped genome, we can not only practice Forward Genetics, but Reverse Genetics as well. Reverse Genetics is used by scientists to “knockout” a gene or mutate it intentionally, then using the newly created mutation they grow a crop which will be observed for any phenotypic differences. Using mutated genes RDR 6 (RNA dependent RNA polymerase) and DCL 4 (Dicer Like 4), we grew 48 plants, 16 plants of each: Wild Type, RDR 6 and DCL 4. Half of thesewere subjected to the treatment of an upped amount of calcium in the water that already had fertilizer present. We choose calcium as our stress because the known benefits of calcium for humans and other animals are huge; we were curious to see how it would affect Arabidopsis and if it would show any noticeable phenotypic changes. We measured the size of the leaves, the height of each bolt, and the number of flowers on each plant. Background Arabidopsis thaliana is used in the lab mainly because of its use of RNAi (RNA Interference), which is the production of non-functional RNA or, the destruction of RNA. This mechanism is useful for: gene expression regulation, it helps resist retro-viruses, it is also a very effective stress response.Andrew Fire and Craig Mello successfully indentified the structure of mRNA, dsRNA, and how RNAi alters the production of amino acids by breaking down the dsRNA (double stranded). Scientists have determined two of the many families of genes, RDR and DCL; RDR, or RNA Dependent RNA polymerase, copies the RNA into more RNA. The DCL (dicer) then “reads” the RNA, and cuts it into small pieces that are still double stranded. The RNA induced silencing complex (Risc) then polymerizes the small interfering RNA (siRNA) and breaks them down into even smaller pieces of mRNA that is nonfunctional by the cell. RDR and DCL are both genes that have been linked to a rapid stress response, when mutated. This response usually involves a shorter time period for a plant to transfer from its juvenile stage into its adult stage. They also make the plants have longer leaves, and even purple leaves. Mutations on these genes create a stronger pigment and more mature plants. Faster. Above: Wild Type, DCL and RDR plants that have had calcium treatment Below: RDR treatment with multiple bolts, 4 bolts were observed. Results This experiment was testing the affects of calcium on Arabidopsis. It was found that both of the RDR and DCL plants, the control and treated plants, had leaves that curled inward on themselves. The wild type control plants grew the slowest, while the treated wild type grew faster. All of the treatment plants grew faster and greener than the control. Average Leaf Width Conclusion In conclusion, the calcium treated plants grew better than the plants just grown in fertilizer. This proves our hypothesis, which was that the plants with calcium would grow better – stronger, taller, greener, and with more flowers. The DCL mutants were struggling to keep up with the rest of the plants, even with the calcium. Future If calcium was added to general fertilizer, it would improve the life of plants, and perhaps even humans. Calcium made the plants grow taller, faster, and greener, which are all favorable traits when harvesting food. With a faster growth rate, more people can acquire more food faster. Taller and greener plants may produce more O2, and help absorb the excess CO2 in the air, and even if the impact is minimal, every little bit counts. Future experiments could change the general fertilizer, used by many farmers. These changes could include removing a current substance, or even just adding calcium to see if corn, wheat, and other essential foods grow better when exposed to more calcium. Another experiment could be conducted to discover if a little more calcium in lakes and oceans affect any of the wild life in a negative way – that definitely is not the goal. Number of Bolts Acknowledgements Special thanks to Dr. Johanson for all of her ideas, and to Mrs. Gay for her continual support! www.retrovirology.com/