Discipline: Biological Sciences
Subcategory: Plant Research
Zoe Yeoh - Gettysburg College
Co-Author(s): Elizabeth Luscher and Patricia Springer, University of California-Riverside, Riverside, CA
Transcription factors (TFs) help ensure proper gene expression in developing tissues, and thus play a role in plant development and plant architecture. LATERAL ORGAN FUSION1, or LOF1, is a TF expressed in the organ boundaries of Arabidopsis thaliana. Mutants of LOF1 have fused axillary branches and cauline leaves, which indicates importance in boundary development. Because transcription factors are known to act in complexes, we wanted to examine what other proteins interact with LOF1. We conducted a yeast-2-hybrid (Y2H) screen that identified several TFs as potential interactors: WHIRLY 3 (WHY3), MYB DOMAIN PROTEIN32 (MYB32), HOMEOBOX-LEUCINE ZIPPER PROTEIN4 (HB4), and LIGHT RESPONSE BTB2 (LRB2). As WHIRLY1 (WHY1) and HOMEOBOX ARABIDOPSIS THALIANA3 (HAT3) are thought to be redundant with WHY3 and HB4, respectively, they are also included in our study. To gain evidence that the interactions between the potential protein interactors and LOF1 are biologically relevant in planta, we characterized Salk T-DNA insertion lines in which the genes that encode these interactors are disrupted. Our goal was three-fold: genotype the T-DNA lines to identify homozygous mutants using PCR; pinpoint the insertion sites using sequencing data; and identify null alleles using RT-PCR. We successfully identified several different Salk lines that were homozygous mutant for our genes of interest by using two sets of primers. Gene-specific primers amplified a wild-type product if a wild-type allele is present, while a T-DNA-specific primer and one gene-specific primer amplified a T-DNA product if insertion allele is present. By comparing amplified PCR products from both reactions, we deduced the genotype of the plant. We further confirmed T-DNA insertion sites in each of our genes of interest through sequencing of our T-DNA product. Lastly, we characterized one of our homozygous mutant lines, lrb2-2, through RT-PCR as a null allele, allowing us to better understand its function in our mutant. Using the findings from this project, our ultimate goal is to create double- and triple-mutants between lof1 and the other TFs, and to characterize their phenotypes in comparison to lof1 mutants. Because the boundary region is involved in determining leaf angle and leaf angle affects planting density, changes in leaf angle have the potential to impact crop yield. In the future, we may be able to apply the knowledge we obtain in the model plant Arabidopsis thaliana to crop species in order to improve crop yield.
Funder Acknowledgement(s): This work was supported by the National Science Foundation REU grant 1461297 to the UC-Riverside Center for Plant Cell Biology.
Faculty Advisor: Patricia Springer, email@example.com
Role: Genotype the T-DNA lines to identify homozygous mutants using PCR; pinpoint the insertion sites using sequencing data; and identify null alleles using RT-PCR.