Date of Award

Winter 2017

Project Type

Thesis

Program or Major

Genetics

Degree Name

Master of Science

First Advisor

Estelle M Hrabak

Second Advisor

Kevin M Culligan

Third Advisor

David H Townson

Abstract

As sessile organisms, plants must use molecular mechanisms to cope with stressors, such as NaCl stress. Protein Phosphatase 2A (PP2A) is a highly-conserved enzyme that regulates many processes including auxin transport, gravitropic responses, ABA responses, ionic stress responses, and microtubule organization. PP2A is a heterotrimer comprised of one scaffolding A subunit, one regulatory B subunit, and one catalytic C subunit. Roots of both the A subunit mutant a1rcn1 and the C subunit mutant c4 skewed when grown on vertically-oriented agar plates containing NaCl. Quantitation of cell file orientation in roots indicated that the root skewing phenotype was due to cell file rotation. Changes in cell shape are usually regulated by the cytoskeleton so microfilaments and microtubules were studied in depth. In wildtype seedlings, treatment with an actin polymerization inhibitor during NaCl stress weakly phenocopied the PP2A mutant phenotype with roots that skewed to the left and exhibited right-handed cell file rotation. In the PP2A mutants, microfilament orientation, bundling, and organization were not correlated with the NaCl-induced root skewing phenotype. In contrast, in a1rcn1 and c4 seedlings experiencing NaCl stress, microtubules were remodeled into left-handed oblique arrays that are correlated with right-handed cell file rotation rather than the predominantly transverse arrays and linear cell files in wildtype root cells. Drugs that either stabilize or destabilize microtubule arrays were used to investigate the relationship between PP2A, NaCl, and microtubule remodeling. In general, microtubule stabilization caused by taxol decreased the impact of NaCl stress on PP2A mutants, leading to less cell file rotation and root skewing. Conversely, propyzamide destabilization of microtubules increased remodeling in a manner similar to NaCl stress. In fact, propyzamide treatment in concert with NaCl resulted in root angle, cell file, and microtubule phenotypes that generally recapitulated the effect of salt stress alone. A few inconsistencies in the microtubule inhibitor experiments suggested that further experiments are warranted. The results indicate that the function of PP2A may be to stabilize microtubules under NaCl stress conditions since PP2A deficiency can be overcome using taxol. Future studies should investigate both cytoskeletal proteins as potential PP2A substrates and microtubule dynamics to gain a greater understanding of how PP2A influences cortical microtubule orientation under NaCl stress.

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