Date of Graduation

2009

Document Type

Dissertation/Thesis

Abstract

The ubiquitin/26S proteasome system (UPS) is conserved in all eukaryotic species. The 26S proteasome serves as the protease in this pathway and is responsible for the degradation of abnormal and short-lived proteins after their modification with a polyubiquitin tag. It consists of one or two ATP-dependent, 19S regulatory particles and an ATP-independent 20S core protease. PAG1 serves as one of at least 14 different subunits of the 20S core protease. In this study, the role of the 20S core protease in general and PAG1, the α7 subunit of the 26S proteasome, in particular during Arabidopsis growth and development was investigated using a reverse genetic approach. Homozygous pag1-1 mutant individuals were not identified because the pag1-1 allele could not be transmitted through pollen. Transmission was not restored by reducing competition by wild-type pollen grains suggesting that pag1-1 mutant pollen is incapable of successful pollination. Pollen function was restored, however, by the introduction of a wild-type PAG1 transgene. The particular cause for the defect in pollen transmission was not readily apparent because different mutant pollen grains displayed different phenotypic characteristics. Some pag1-1 mutant pollen was inviable, some failed to undergo pollen mitosis II, and some failed to germinate and grow pollen tubes. However, more than 50% of the pollen grains produced by PAG1/pag1-1 plants appeared normal according to viability, nuclear DNA staining and in vitro pollen germination assays. Nevertheless, no evidence for successful pollination by a mutant pollen grain lacking a wild-type PAG1 allele was ever obtained, indicating that PAG1 is essential for pollen function. The ubiquitin-specific proteases (UBPs) are essential for proper UPS function. They are responsible for processing the newly-translated polyubiquitin and ubiquitin extension proteins, and for dismantling polyubiquitin chains to help maintain a supply of ubiquitin monomers for use as protein tags. Some UBPs are responsible for the deubiquitination of specific target proteins including histones and ubiquitin protein ligases. Whereas the general functions of the UBP enzyme family are known, the particular functions performed by specific UBPs remain, for the most part, unknown. This study investigates the roles of the UBP12/13 and UBP3/4 subfamilies in Arabidopsis thaliana . Homozygous ubp12, ubp13, ubp3, and ubp4 single gene mutants all appear wild-type in phenotype. However, ubp12; ubp13 double homozygous mutant individuals display several aberrant phenotypes, including delayed seed germination, and an extreme reduction in plant size and fertility. These characteristics are shared by various Arabidopsis mutants defective in gibberellic acid biosynthesis/signal transduction suggesting that this UBP subfamily may play a role in responses to this phytohormone. ubp3; ubp4 double homozygous mutant individuals have not been identified. One contributing factor is that the germination of double mutant pollen is significantly reduced compared to that of wild-type or single mutant pollen. Thus, there is a functional redundancy for UBP12 and UBP13 as well as UBP3 and UBP4. Further, investigation revealed that only a single wild-type UBP12 or UBP13 allele is required for a plant to display the wild-type phenotype. However, genotyping of their progeny revealed that a lower fraction than expected were of the same genotype as their parent; this was due to a lower than expected contribution of double mutant pollen. Overall, our results indicate that the UBP12/13 and UBP3/4 subfamilies perform important functions during plant growth and development.

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