TWO DIMENSIONAL DIFFERENCE IN GEL ELECTROPHORESIS (DIGE) PROTEOMIC ANALYSIS OF ARABIDOPSIS LEAF HETEROTRIMERIC G-PROTEIN MUTANTS IN RESPONSE TO ABSCISIC ACID TREATMENT

Open Access
Author:
Auker, Erik Christopher
Area of Honors:
Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Sarah Mary Assmann, Thesis Supervisor
  • Bernhard Luscher, Honors Advisor
Keywords:
  • DIGE
  • G-Proteins
  • proteomics
  • ABA
  • arabidopsis
Abstract:
Heterotrimeric G proteins have been studied extensively because of their ability to generate cellular responses due to signal transduction cascades initiated by the binding of ligands to G protein coupled receptors (GPCRs). Mutaions of one or more ¦Á, ¦Â, and ¦Ã subunits of this heterotrimeric protein have been shown to display a plethora of phenotypic changes in a plant¡¯s development and response to environmental stimuli. Most studies to date have focused on the model organism Arabidopsis thaliana because of its fully sequenced genome and ease of growth. Comparing various knockout mutants to the wild type Columbia continues to be an active area of research. However, little work has been done to compare such plants on the proteomic level. In this study, the G¦Â mutant agb1-2, G¦Á mutant gpa1-4, double mutant agb1-2 /gpa1-4, and Columbia wild type were treated with abscisic acid (ABA) and analyzed using two dimensional Difference in Gel Electrophoresis (DIGE). This technique allows for the separation of two samples in one gel based on their proteins' various isoelectric charges and molecular weights of proteins. Analytical software was used to compute relative abundances of proteins within the gels. The significance of these relative abundance values was determine by 2-way ANOVA (p-value ¡Ü 0.01) comparing the genotypes, treatments, and the interactions between the two. 186 spots were targeted and picked from the gels and the confidence intervals for accurate protein identification by Mass Spectrometry were recorded. Proteins with C.I values above 80% were described. By observing the relative abundance of identified proteins, it was possible to draw conclusions as to which subunits were regulating the levels of specific proteins. Many of the identified proteins were found to perform actions involving response to ABA, drought, or heat while others had more general functions.