In Arabidopsis thaliana (Arabidopsis), by screening for the inability to germinate on low concentrations of exogenous abscisic acid (ABA), loss-of-function mutations in the β-subunit of a protein farnesyltransferase (FTase) were identified (Cutler et al., 1996). Designated era1-2, these mutants are pleiotropic and show a hypersensitive ABA response at the level of germination and stomatal closure, thereby conferring drought resistance, besides having particular developmental phenotypes (Pei et al., 1998; Bonetta et al., 2000). Although a number of proteins have been shown to be farnesylated in plants, which has provided some insight into how farnesylation regulates various processes, there is still no clear understanding of how loss of farnesylation can confer ABA hypersensitivity, for example. The simplest interpretation is that farnesylation acts as a negative regulator of ABA signal transduction. The primary goal of this thesis is to carry out several reverse genetic screens using a Arabidopsis homozygous T-DNA knockout collection to discover potential targets of farnesylation as well as to determine the overall function of these farnesylated targets in plant growth and development. This included screening for morphological changes related to era1-2, altered responses to ABA at the level of germination, and altered drought responses. In total, 15 unique mutants were identified from the aforementioned reverse genetic screens. A knockout in the gene At3g30180 became particularly interesting for further study since it exhibited several phenotypes that resemble era1-2, including ABA hypersensitivity in germination, drought resistance, protruding carpels, reduced fertility, and round and broadened leaves. At3g30180, otherwise known as CYP85A2, is a cytochrome P450 that mediates the final step in the biosynthesis of brassinolide (BL), a brassinosteroid (Kim et al., 2005). At3g30180 was also identified through a bioinformatic screen (Brady and Provart, 2009; Usadel et al., 2009). Overall, ERA1 positively regulates CYP85A2 function through farnesylation, and therefore BL production, which negatively regulates ABA signaling.

The enzyme 3-deoxy-D-arabino-heptulusonate 7-phosphate synthase (DHS) catalyzes the first step of the shikimate pathway. In bacteria, the regulation of the pathway is mediated by allosteric inhibition of DHS by the aromatic amino acids tyrosine, phenylalanine and tryptophan. The regulation of the pathway in plants remains elusive but the aromatic amino acids are involved as suggested by the hypersensitivity of dhs1 knockout mutant to tyrosine. In this study the effects of the dhs1 mutation on endogenous levels of aromatic amino acids and of downstream metabolites are explored. HPLC analysis is used to measure levels of tyrosine and phenylalanine and 5-methyltryptophan sensitivity is used to probe levels of tryptophan. Additionally, the auxin content of whole seedlings was quantified by LC/MS and its local levels at the root apex are visualized with the DR5::GUS reporter system.

The enzyme 3-deoxy-D-arabino-heptulusonate 7-phosphate synthase (DHS) catalyzes the first step of the shikimate pathway. In bacteria, the regulation of the pathway is mediated by allosteric inhibition of DHS by the aromatic amino acids tyrosine, phenylalanine and tryptophan. The regulation of the pathway in plants remains elusive but the aromatic amino acids are involved as suggested by the hypersensitivity of dhs1 knockout mutant to tyrosine. In this study the effects of the dhs1 mutation on endogenous levels of aromatic amino acids and of downstream metabolites are explored. HPLC analysis is used to measure levels of tyrosine and phenylalanine and 5-methyltryptophan sensitivity is used to probe levels of tryptophan. Additionally, the auxin content of whole seedlings was quantified by LC/MS and its local levels at the root apex are visualized with the DR5::GUS reporter system.

In Arabidopsis thaliana (Arabidopsis), by screening for the inability to germinate on low concentrations of exogenous abscisic acid (ABA), loss-of-function mutations in the β-subunit of a protein farnesyltransferase (FTase) were identified (Cutler et al., 1996). Designated era1-2, these mutants are pleiotropic and show a hypersensitive ABA response at the level of germination and stomatal closure, thereby conferring drought resistance, besides having particular developmental phenotypes (Pei et al., 1998; Bonetta et al., 2000). Although a number of proteins have been shown to be farnesylated in plants, which has provided some insight into how farnesylation regulates various processes, there is still no clear understanding of how loss of farnesylation can confer ABA hypersensitivity, for example. The simplest interpretation is that farnesylation acts as a negative regulator of ABA signal transduction. The primary goal of this thesis is to carry out several reverse genetic screens using a Arabidopsis homozygous T-DNA knockout collection to discover potential targets of farnesylation as well as to determine the overall function of these farnesylated targets in plant growth and development. This included screening for morphological changes related to era1-2, altered responses to ABA at the level of germination, and altered drought responses. In total, 15 unique mutants were identified from the aforementioned reverse genetic screens. A knockout in the gene At3g30180 became particularly interesting for further study since it exhibited several phenotypes that resemble era1-2, including ABA hypersensitivity in germination, drought resistance, protruding carpels, reduced fertility, and round and broadened leaves. At3g30180, otherwise known as CYP85A2, is a cytochrome P450 that mediates the final step in the biosynthesis of brassinolide (BL), a brassinosteroid (Kim et al., 2005). At3g30180 was also identified through a bioinformatic screen (Brady and Provart, 2009; Usadel et al., 2009). Overall, ERA1 positively regulates CYP85A2 function through farnesylation, and therefore BL production, which negatively regulates ABA signaling.

Human enteric pathogen Salmonella contaminates raw produce and triggers significant economic loss and illness. Under a natural environment, Salmonella resides in soil and enters the interior of plants without causing disease or eliciting symbiotic growth. Upon being consumed by humans, complex virulence mechanisms are elicited by the specific intestine conditions, such as high temperature and humidity and lead to profound infection. The lack of effective prevention and drug treatment are largely attributed to the unclear mechanistic understanding on Salmonella association with environmental media, and in vivo host and pathogen factors required for persistent infection. We have explored the potential of deploying the model plant organism Arabidopsis thaliana to tackle this fundamental yet clinically challenging question, as Arabidopsis possesses many advantages as a model system, including enriched genomic resources, powerful genetic tools, low maintenance cost and a large collection of individual gene deletion mutants. Our preliminary data demonstrated Arabidopsis seedlings under liquid culture conditions mimicking the intestine environment were infected and killed by salmonella within 2 days upon inoculation. The Arabidopsis system possesses well-developed genetic information and the resources to study host factors required for infection on very short time scales, thus complementing traditional animal genetic studies. We aim to define the pathogen factors required for this infection. By merging the fields of extremely powerful Arabidopsis genetics and bacterial genetics/genomics, we hope to provide insight into possible new paradigms for addressing salmonella-mediated food born infection.

Allopolyploids are formed by the combination of evolutionarily-diverged genomes, the union of which leads to dynamic changes in gene expression and genome organization. Expression patterns of orthologous genes are rapidly and stochastically established in newly created allotetraploids, where gene silencing is maintained by microRNAs, DNA methylation, and other chromatin modifications. Among them, DNA methylation has been known as an important mechanism of epigenetic regulation of gene expression and chromatin structure. However, it is unclear how DNA methylation affects genome-wide expression of homoeologous genes in the natural polyploid Arabidopsis suecica that contains genome of both A. thaliana and A. arenosa. To understand the role that DNA methylation plays in the polyploidization process, a comparative analysis was performed comparing up- or down-regulated genes in met1-RNAi A. suecica lines with the non-additively expressed genes in the synthetic allotetraploids, i.e., different from the mid-parent value. The previous studies indicated that decreased DNA methylation in A. suecica induces A. arenosa-specific demethylation in centromere regions and differentially alters expression of >200 genes encoding many transposons, unknown proteins and some other functional proteins that are located along chromosomes, whereas >1,300 non-additively expressed genes in the synthetic allotetraploids are distributed randomly along the chromosomes and encode various proteins in metabolism, energy, cellular biogenesis, cell defense and aging, and hormonal regulation. The origins of the progenitors of the genes whose expressions are altered in both met1-RNAi A. suecica and resynthesized allotetraploid were analyzed with single strand conformation polymorphism (SSCP) analysis. Reactivated genes in met1-RNAi A. suecica lines were predominately derived from the A. thaliana genome in euchromatic regions, whereas the suppressed genes were mainly derived from the A. Arenosa genome, indicating that changes in DNA methylation are genome-sensitive. The data suggest that allotetraploids incidentally display chromosome-specific changes and genomedependent regulation of homoeologous genes in response to DNA methylation perturbations.

Phosphate is an essential but limited macronutrient for all plants. In response to limited levels of phosphate, plants have developed highly specialized developmental, biochemical, and molecular responses. To further expand our knowledge of the phosphate starvation induced signal transduction pathway in plants, the expression of the phosphate starvation inducible Purple Acid Phosphatase 1 (PAP1) gene was studied in transgenic Arabidopsis. While few components have been identified regulating gene expression under phosphate starvation conditions in plants, one cis regulatory element recognized by the MYB transcriptions factor Phosphate Starvation Response 1 (PHR1) has been identified in many phosphate starvation induced (PSI) genes. PAP1 and many other genes examined during the course of the mutant characterization contain this cis element. Using the GUS reporter gene under control of the PAP1 promoter, a mutant screen was devised for plants showing abnormal PAP1 response to phosphate nutrition. Three mutant lines were identified and subsequently characterized for the phosphate starvation-induced signal-transduction pathway in Arabidopsis. Two mutants, BT1 and BT2, both with dominant mutations, showed increased GUS staining. The mutations in BT1 and BT2 are tightly linked to the transgene and to each other, but complementation analysis suggested that they are in different genes. Characterization of these mutants indicated that the PSI genes PAP1 and At4 (in BT1 roots), and RNS1 (in BT2 leaves) have alternative or additional methods of regulation other than PHR, even though these genes all contain PHR1 binding sites. A third mutant, BT3, had a phenotype similar to the PAP1 null-mutant and did not show PAP1 phosphatase activity under normal soil-grown conditions. Characterization of BT3 indicates that PAP1, RNS1, and AtIPS1 are not exclusively regulated by PHR1. In an attempt to map the BT3 mutant in a Columbia background by crossing with Landsberg erecta (Ler), it was discovered that the Ler ecotype does not show PAP1 phosphatase activity under normal soil-grown conditions. The PAP1 phosphatase regulatory trait, named BT5, was mapped to a 15,562 bp-region area containing only two genes between the GPA1 and ER markers on Chromosome 2.

Telomeres are critical for the integrity of eukaryotic genomes. They function to protect chromosome ends from DNA damage surveillance and inappropriate repair. Telomeres are maintained by the specialized ribonucleoprotein complex telomerase. Without telomerase, telomere shortening would ultimately lead to compromised genome stability and cellular senescence. Therefore, telomerase function is necessary for extension of the proliferative capacity of the cell. In this dissertation, we describe the characterization of core components of telomerase ribonucleoprotein complex in the flowering plant, Arabidopsis thaliana. We find that dyskerin, one of the core telomerase components in humans is also conserved in Arabidopsis telomerase. Arabidopsis dyskerin associates with the telomerase RNP in an RNA-dependent manner and is required for telomere length maintenance in this organism. We also describe the characterization of another core telomerase component, the telomerase RNA subunits (TERs). Unexpectedly, we uncovered two distinct TER subunits that share a region of high identity. The two TERs named TER1G7 and TER5G2, based on their chromosomal positions, display differences in their expression levels and their association with telomere-related proteins. Both TERs can serve as templates for telomerase in vitro. Through genetic analyses, we show a templating function for TER1G7 in vivo and a novel role for TER5G2 as a negative regulator of telomerase. Finally, the presence of TER genes in other plant species was investigated and evidence for duplication of TER genes in plants closely related to Arabidopsis was obtained. We also show evidence for a template mutation in Asparagus TER that could lead to variant repeats in this organism. In summary, the studies presented in this dissertation reveal that Arabidopsis telomerase shares both similarities and differences with other telomerase RNPs, making it an exciting model system for study of telomere biology.

Thesis (Ph. D.)--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, June 2001. / Includes bibliographical references. Also available on the Internet.