Identifying suppressors of the bps-1 phenotype through a mutagenesis screen in Arabidopsis thaliana

Every year, there is about ten billion dollars of crop loss in the US alone. For this reason, gene regulation and a plant's ability to prosper under environmental pressures is extremely important to our economy. In Arabidopsis thaliana, the loss of one gene in particular results in extreme phenotypes.

Identifying Suppressors of the bps-1 Phenotype Through a Mutagenesis Screen in Arabidopsis thaliana Avery Dain Leslie Sieburth, PhD Alex Cummins, PhD Sieburth Lab University of Utah I. Background Every year, there is about ten billion dollars of crop loss in the US alone. For this reason, gene regulation and a plant’s ability to prosper under environmental pressures is extremely important to our economy. In Arabidopsis thaliana, the loss of one gene in particular results in extreme phenotypes. BYPASS1 is a gene in Arabidopsis that regulates synthesis of a signaling molecule called dalekin. Dalekin is a novel, root-toshoot signaling molecule that increases drought stress resistance in Arabidopsis. Bps1-2 mutants produce non-physiological levels of dalekin resulting in seedling growth arrest. We are interested in understanding the entire signaling pathway as well as how BPS1 regulates dalekin. To make sense of this complex pathway, we have used EMS mutagenesis to generate mutations throughout the genome in hopes of hitting a gene that is involved in the biosynthesis, perception, or response of dalekin. Altering any of these will likely suppress the phenotype of bps1-2. Through this suppressor screen, we have identified several suppressors. Bps1-2 bps2-2 BPS2A DOC 1050 II. Methods To identify suppressors, we grew M2 seedlings in a sensitized growth condition, GM+12.5mM CaCl2 . This low amount of calcium causes extremely low root growth and small cotyledon. Suppressors of this phenotype will have very long roots and large cotyledons. They will also have an elongation zone – an area on the root tip lacking root hairs. The M2 were transplanted onto soil and placed in a growth room. To test whether the mutation was dominant or recessive, we grew the seedlings for until flowering and then backcrossed them with the parental genotype. Crossing entailed emasculating the M2 flower and taking the stamen from the parental genotype for pollination. As a control, we emasculated another flower on the same line without fertilizing it to ensure our crosses were done properly. If we find a dominant mutant, the heterozygotes should show the exaggerated calcium response. Conversely, F1s with a recessive mutation would present the regular parental phenotype. from a back cross but not the control, we collected it to analyze its phenotype. This analysis will help us with molecular characterization. Figure 1: Diagram showing method timeline. Created with BioRender.com. DOC 1050 III. Results In our screens, we found several suppressors varying in degree and much more frequently than expected. We expected to find one suppressor in every one hundred lines. Instead, we found 21 suppressors in the 180 lines we screened. Meaning we are finding suppressors in 11.67% of lines screened. We have also began backcrossing the M2 suppressor lines. Although some crosses have been unsuccessful due to improper pollination, we have had at least one successful cross in each line. Strong Suppressor DOC 1527 DOC 1527 Moderate Suppressor DOC 514 DOC 514 IV. Conclusions and Next Steps Weak Suppressor Figure 3: Pictures showing a strong suppressor (DOC 1050) with a control from same line, a moderate suppressor (DOC 1527) with a control from same line, and a weak suppressor (DOC 514) with a control from same line. Dalekin Dalekin Dalekin Dalekin The EMS mutagenesis produced suppressor containing lines. This tells us that these genes function in the dalekin signaling pathway. Making sense of these genes will allow us to create new agricultural methods to develop stress resistant plants. Since we have had successful backcrosses, our next steps will be to analyze and backcross the F1 generation, screen the F2 generation, and sequence their gene pools. This will surely bring us a great deal closer to understanding dalekin and the complex pathway it takes it takes to travel from root to shoot in Arabidopsis thaliana. V. Acknowledgements Response 1 Dalekin No dalekin Figure 2: Target areas for EMS mutations including biosynthesis, perception, and response. Created with BioRender.com. Response 3 Response 2 Response 1 Response 3 Response 2 Thank you to Leslie Sieburth and Alex Cummins for their guidance and mentorship, to Alexa Little and Shelbi Laub for their friendship and suppressor photos, and to Alex Shelton for sharing the horcrux. Avery Dain University of Utah School of Biological Sciences [e-mail]