Date of Award

Fall 2008

Project Type

Dissertation

Program or Major

Plant Biology

Degree Name

Doctor of Philosophy

First Advisor

Estelle M Hrabak

Abstract

Reversible protein phosphorylation is a highly regulated process that affects almost all cellular activities. Phosphorylation involves two groups of enzymes: protein kinases and protein phosphatases. Considering the central role of these enzymes in cells, elucidating their function is extremely important. My research focuses on protein phosphatase 2A (PP2A) C subunits. PP2A is one of the most abundant types of serine/threonine phosphatases in all eukaryotic cells. Compared with animals, PP2A function is not well known in plants. Early studies using protein phosphatase inhibitors are not useful to demonstrate the function of individual PP2A subunits. A reverse genetic approach can be helpful to gain insight into the function of PP2A in plants since a resulting phenotype is due to the lack of function of a single protein and provides direct evidence to elucidate function.

In this study, 13 homozygous mutant lines generated by the insertion of foreign DNA (T-DNA) in all five Arabidopsis PP2A C subunit genes were identified by the polymerase chain reaction. The precise location of the insertion was determined from DNA sequencing, expressed sequence tags and cDNA data. The presence of a full-length mRNA was not detected from five of the 13 mutant alleles (cl-1, c3-1, c3-3, c4-1 and c4-2) as determined by the reverse transcriptase-polymerase chain reaction. Wild type and mutant plants were compared under different growth conditions and chemical treatments to characterize the effect of the mutations. There was no obvious difference between mutant and wild type plants except in one case: growth in the presence of NaCl.

At greater than 50 mM supplemental NaCl, roots of c4 mutant seedlings grown on vertically-oriented plates had a strong right-skewing growth pattern (when viewed from top of the plate) while wild type seedlings showed only a slight right skewing. Complementation confirmed that the c4 mutant phenotype was due to loss of PP2A C4 function. The c4 mutant phenotype did not appear when seedlings were grown in the presence of KCl, LiCl or CaCl2. Additional experiments showed that Ca2+ and auxin transport might also be involved in the NaCl-induced skewing phenotype of the PP2A c4 mutants.

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