Date of Award

Spring 2017

Project Type

Dissertation

Program or Major

Genetics

Degree Name

Doctor of Philosophy

First Advisor

Estelle M Hrabal

Second Advisor

Richard H. Cote

Third Advisor

Kevin Culligan

Abstract

Protein phosphatase 2A (PP2A) is a key regulator of many biological pathways. The PP2A heterotrimer is composed of a scaffolding/regulatory A subunit, regulatory B subunit, and catalytic C subunit. In Arabidopsis thaliana, the A, B and C subunits are encoded by 3, 17, and 5 genes, respectively. Despite the ubiquitous expression and high sequence conservation of the A and C subunits, plants with mutations in specific subunits display phenotypes. A subset of the A and C subunit mutants exhibited root skewing or curling phenotypes when grown on vertically-oriented plates supplemented NaCl. I characterized the root response of a1rcn1 and c4 mutants and found that the root phenotype was sodium specific and influenced by NaCl concentration, photoperiod, and temperature. In response to NaCl, roots of PP2A mutants showed an obvious skewing response within 24 hours, likely due to the right-handed rotation of root cell files. A physical interaction was detected between the A1RCN1 and C4 subunits in root tips by co-immunoprecipitation. Taken together, these results indicate that PP2A is involved in the response of Arabidopsis seedlings to sodium stress.

Underlying mechanisms involved in the root response to sodium stress was examined using a multistep mutant screen. A mutant collection was screened representing 21,520 T-DNA insertions. Out of the 138,000 seedlings visually examined for root skewing, fifteen putative mutants were classified as sodium-induced root skewers. In addition, eight mutants were described with other aberrant root phenotypes. Thermal Asymmetric Interlaced PCR was used to determine the insertion site of the T‑DNA mutagen and the mode of inheritance was determined.

I used a genetic approach to further analyze the functional interaction of PP2A subunits. Single and double mutants were created and confirmed by PCR genotyping. To improve PCR genotyping efficiency and our ability to store DNA long-term, I optimized a method to capture DNA on a cellulose matrix as a substitute for commercially available FTA® Cards and FTA® Purification Reagent. Single and double mutants were assayed to quantitate three phenotypes (root angle, root length, and hypocotyl length) and different growth conditions. The quantitative data from wildtype, single mutants, and corresponding combinatorial mutants were used to describe 109 genetic interaction inequalities and modes. Each phenotype and growth condition genetic interaction network was unique and both redundant and distinct functions for the A and C subunits were revealed. For the root angle phenotype, the a1rcn1 and c4 subunits functionally interact in the same network to regulate the response of seedling roots to NaCl stress. Overall, these results describe novel root and shoot functions for the A and C subunits of PP2A.

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