Discipline: Biological Sciences
Subcategory: Plant Research
Elizabeth Gamillo - Alverno College
Co-Author(s): Nicole Gibbs, University of Wisconsin-Madison, WI; Dr. Patrick Masson, Ph.D., University of Wisconsin-Madison, WI
Studying the processes involved in plant development and root growth allow us to develop techniques to produce stress resistant crops. As sessile organisms, plants remain where they have germinated their entire life and use their roots to take up essential nutrients, water and to gather information provided by the environment to guide their growth. One environmental signal that alters plant growth are polyamines. Polyamines, such as cadaverine, alter processes such as signaling, stress response and plant development. Cadaverine modifies root architecture by decreasing primary root growth and promoting lateral root development. To characterize how this process occurs, mutants in cadaverine response were assessed.
The root growth change is seen in A. thaliana mutants, spds1-1 and plc4-1 when grown on agar containing cadaverine. Cadaverine causes a short root phenotype in the mutant spds1-1. SPDS1 is responsible for encoding spermidine synthase in A. thaliana. When plc4-1 mutants are grown on cadaverine media, a longer root phenotype is observed. PLC4 is responsible for coding phospholipid breakdown and stress response. To observe the effect cadaverine has on these genes, a double mutant of spds1-1 and plc4-1 was produced by crossing two plants containing the individual knockout genes resulting in the F1 plant. The F1 plant self-pollinated and F2 seeds were produced. In work presented here, the F2 plant population was grown and root phenotype was noted. The F2 plants were screened using PCR techniques to confirm the identification of a double mutant. First, seedlings were genotyped for PLC4, five of 24 plants showed potential to be a double mutant due to homozygous bands showing up in the T-DNA region for plc4-1. Next, these plants were screened for spds1-1. Homozygous bands in the T-DNA region showed up for 3 plants for spds 1-1. The controls of the spds 1-1 PCR for this screen did not amplify likely because of an issue with primers. However, the short root phenotype of one of the plants suggests this plant could be a double mutant because it shows spds1-1 phenotype, likely suggesting it is homozygous at the spds1-1 locus. Therefore, the double mutant obtained from the F2 population cross of plc4-1xspds1-1 likely has a short root phenotype. Future work includes troubleshooting the spds1-1 wild type PCR by designing alternative primers, and further analyzing the double mutant produced by the F2 population. Ultimately, future work will characterize whether cadaverine may also leave lasting effects on the growth and development of the double mutants.
References:. Jancewicz, A. L., Gibbs, N. M., & Masson, P. H. (2016). Cadaverine’s Functional Role in Plant Development and Environmental Response. Frontiers in Plant Science, 7, 870.
Mai, A., Matsuyama, T., Hanzawa, Y., Akiyama, T., Tamaoki, M., Saji, H., Takahashi, T. (2004). Spermidine Synthase Genes Are Essential for Survival of Arabidopsis. Plant Physiology, 135(3), 1565?1573.Not Submitted
Funder Acknowledgement(s): I would like to thank Dr. Patrick Masson for hosting me in his lab for this 10- week summer research experience, Nicole Gibbs and Dr. Shih-Heng Su for their guidance and support while in the lab, Justin LaManna and Amal El-Sheikh for their help and guidance, Angela Frey for her guidance and support and the Wisconsin Louis Stokes Alliance for Minority Participation for their support. Funding was provided by an NSF grant provided through the NSF REU Biological SIGNALS Summer Research Program (NSF Award 1659159), The University of Wisconsin-Madison CALS HATCH grant, and The Alverno College Center for Academic Excellence.
Faculty Advisor: Dr. Patrick Masson, firstname.lastname@example.org
Role: I screened the F2 population of the A. thaliana double mutants using PCR techniques and then looked at the phenotype of the F2 population to correlate the genotype shown through PCR with the root phenotype observed on the agar plates containing cadaverine.