This item is under embargo and not available online per the author's request. For access information, please visit http://libanswers.wustl.edu/faq/5640.

Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Plant and Microbial Biosciences

Language

English (en)

Date of Award

Summer 9-1-2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Ram Dixit

Abstract

The cell wall is essential for plant growth and development. Cell wall deposition depends on the secretory system and the cortical microtubule cytoskeleton. However, the molecular mechanisms that link these two systems are unknown. I hypothesized that the Arabidopsis FRA1 kinesin acts as such a molecular linker by transporting vesicles containing cell wall components along cortical microtubules to facilitate their secretion.

I first studied the motility properties of FRA1 in vitro. Using bulk biochemical assays and single molecule studies, I showed that that FRA1 moves long distances as a dimer towards the plus-ends of microtubules. These findings indicated that FRA1 has the potential to drive long-distance transport of cargo along cortical microtubules.

To understand the function of FRA1 in vivo, I characterized a knockout mutant of FRA1, designated as fra1-5. I found that this mutant has pleiotropic phenotypes including shorter, wider and fragile inflorescence stems and seed pods. Correlating with these phonotypes, the fra1-5 mutant is thinner in both primary and secondary cell wall but has essentially unchanged wall composition. These results suggested that FRA1 is essential for the secretion of all types of wall components. Consistent with this interpretation, the delivery of cellulose synthase complexes to the plasma membrane and secretion of pectin is decreased in fra1-5 mutant. In addition, transmission electron microscopy showed that there are more and bigger vesicles accumulated in the cell cortex of fra1-5 mutant. Using live-cell imaging of a functional FRA1-3GFP marker, I demonstrated that the FRA1 kinesin moves processively along cortical microtubules. Together, these results indicated that FRA1 contributes to secretion of cell wall components in a cortical microtubule-dependent manner.

To identify potential molecular mechanisms that might recruit FRA1 to cargo and regulate FRA1 motility, I have characterized two Arabidopsis kinesin light chain related proteins called KLCR1 and KLCR2, which were identified as putative FRA1-interacting proteins in a yeast 2-hybrid screen. In mammals, kinesin light chains directly bind to kinesin-1 and regulate motor activity and cargo binding. However, the function of light chain related proteins in plants remains unknown. I showed that FRA1 and KLCR1/2 genetically interact and both KLCR1 and KLCR2 are localized on cortical microtubules. Based on these findings, I hypothesize that cortical microtubule-bound KLCR proteins recruit FRA1 to cargo and activate their movement along cortical microtubules.

In conclusion, my work showed that FRA1 kinesin is essential for cell wall formation by moving along cortical microtubules and likely transporting cell-wall-related cargo for their exocytosis. In addition, identification of KCLR1 and KLCR2 as FRA1 interacting proteins has shed light on potential mechanisms that regulate FRA1 activity and cargo recruitment.

DOI

https://doi.org/10.7936/K7PN93N5

Comments

This work is not available online per the author’s request. For access information, please contact digital@wumail.wustl.edu or visit http://digital.wustl.edu/publish/etd-search.html.

Permanent URL: http://dx.doi.org/10.7936/K7PN93N5

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