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Date of Award

2-2010

Document Type

Campus Access

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Plant Biology

First Advisor

Tobias I. Baskin

Second Advisor

Patricia Wadsworth

Third Advisor

Magdalena Bezanilla

Subject Categories

Plant Biology

Abstract

To understand the molecular mechanisms that regulate expansion in plants, I analyzed a mutant, rsw4 , previously isolated on the basis of temperature-dependent root swelling. Map based cloning had identifiedRSW4 as At4g22970, which contains a domain homologous to separase, an enzyme demonstrated to be required for sister chromatid disjunction during mitosis in animals and fungi.

I confirmed a role for separase in segregation of sister chromatids in plants. However, with defects in chromosome disjunction, I detected no DNA damage. In addition, the non-disjoined chromosomes did not trigger detectable cell cycle arrest, based on the expression of cell cycle marker genes examined by real time qRT-PCR. Interestingly, the degradation of cyclin B1;1 was prevented in rsw4 , based on the extensive accumulation of a reporter construct based on GUS.

The transverse alignment of cortical microtubules, which are widely believed to define the directionality of expansion, were disrupted in rsw4 . The disorganization of cortical microtubules was concomitant with root swelling. In addition, polarity of cortex cells appeared to be affected in rsw4 . The polar targeting of the auxin efflux protein, PIN2, reversed polarity in cortex cells of rsw4 .

One micro-array run showed a variety of genes with changed expression level in rsw4 . By real rime PCR, I confirmed some genes to have altered expression, including enzymes involved in cell wall metabolism, proteins involved in ethylene signaling, and proteins related to calcium signaling. The finding of changes in the expression of these genes provides potential connections between defective mitosis and aberrant expansion.

This study not only confirmed the conserved roles of plant separase in chromosome disjunction, but also advanced our understanding of regulation of plant cell cycle and expansion. My results and following research should provide further insight into the relationship between chromosome disjunction, cell cycle regulation, and expansion.

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