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In eukaryotic organisms, polyubiquitination is the modification of a protein with polymerized ubiquitin (Ub) chain. This process is well known for its function in targeting proteins for degradation by the 26S proteasome. However, a polyUb chain assembled through the lysine 63 residue of the Ub moiety (Lys63-linked polyubiquitination) has been shown to play a signaling role rather than targeting proteins for degradation. In plants, the functions of Lys63-linked polyubiquitination are currently not well understood. Ub-protein ligase (E3) catalyzes the last step in the ubiquitination reactions, and to a large extent it also determines the substrate specificity of protein ubiquitination. In order to study the roles of Lys63-linked polyubiquitination in plants, two E3s of Arabidopsis thaliana, proteins encoded by AtCHIP and At1g74370 (tentatively named E3-A1), were chosen for functional studies, since they interacted with AtUbc13A protein. Sequence analysis showed that AtCHIP is the only member in the family. A T-DNA insertion mutant line (Atchip-1) was obtained to study the AtCHIP gene knock-out effect. The mutant line was grown in normal conditions and further tested in a variety of conditions: hormonal treatments, osmotic stress, seed deterioration, high temperature stress, high-intensity light stress, oxidative stress and DNA damaging stress. However, no clear difference was observed between the mutant and wild type plants based on the several parameters measured. Sequence analysis of E3-A1 indicated two closely related proteins, tentatively named E3-A2 and E3-A3. As E3-A1 and E3-A2 appeared to share more sequence similarity, RNA interference (RNAi) transformants, with the level of transcripts for either of the two E3-A genes reduced by over 90% were generated. Selected RNAi mutant lines for E3-A1 and E3-A2 were crossed with each other, and double RNAi mutants were obtained. However, no distinct phenotype was detected under normal, high-sucrose or hormonal conditions for either single or double RNAi lines. Although various assays did not reveal a significant phenotype in the mutants in this study, the materials generated and the assays used will benefit a wider range of phenotypic survey in the future.

Glutamine (Gln), a major nitrogen source in plants, is considered a central intermediate that coordinates carbon-nitrogen assembly for plant growth and development. To maintain a sufficient Gln supply, plant cells employ glutamine synthetases (GS), including cytosolic GS1 and plastidic GS2 for Gln production. Previous work has shown that the <i>GS1</i> is responsive to various environmental stresses. This study demonstrated the involvement of <i>GS1</i>s in Gln homeostasis and the role of GS1 in abiotic stress tolerance in <i>Arabidopsis</i>. The <i>GS1</i> family is comprised of five isoforms in <i>Arabidopsis thaliana</i>. Gene expression profiling showed that <i>GLN1;1, GLN1;3</i> and <i>GLN1;4</i> had similar expression patterns and were upregulated by abiotic (salinity and cold) stresses, whereas <i>GLN1;2</i> exhibited constitutive expression and no <i>GLN1;5</i> transcript was detected under any of the conditions tested. Null T-DNA insertion mutants for the five <i>GS1</i> genes were obtained. Only the <i>gln1;1</i> mutant displayed enhanced sensitivity to a GS inhibitor, phosphinothricin, and to cold and salinity treatments, suggesting a nonredundant role for GLN1;1. Increased stress sensitivity in <i>gln1;1</i> was associated with accelerated accumulation of reactive oxygen species (ROS), particularly in chloroplasts. To better understand the role of cytosolic GS isoforms, we generated two different triple mutant combinations. Triple mutant <i>gln1;1/gln1;2/gln1;3</i> showed reduced growth at an early stage. The <i>gln1;1/gln1;3/gln1;4</i> mutant is pollen lethal, indicating an essential role of Gln in plant gametophyte development. Collectively, our results establish a link between cytosolic Gln production, ROS accumulation, plant stress tolerance and development.

<p>RPL23a is one of the ~80 ribosomal proteins (r-proteins) of the cytoplasmic ribosome in the model plant <i>Arabidopsis thaliana</i>. The objectives of this research were to establish Arabidopsis RPL23a as a functional r-protein, characterize expression patterns for the two genes (RPL23aA and B) encoding RPL23a using reverse transcription PCR (RT-PCR), and identify regulatory elements controlling the expression of RPL23aA and B. Complementation of a yeast l25 mutant with AtRPL23aA demonstrated that AtRPL23aA can fulfill all the essential functions of L25 in vivo. A survey of various Arabidopsis tissue types showed that, while RPL23aA and B expression patterns both showed increased transcript abundance in mitotically active tissues, RPL23aB transcript levels were generally lower than those of RPL23aA and responded differently to abiotic stresses. In order to determine cis regulatory elements controlling RPL23aA and B expression, the 5 regulatory region (RR) of each gene was characterized via plants carrying a series of 5 RR deletion fragments upstream of a reporter. Transcript start sites and 5 untranslated regions (UTRs) for both RPL23aA and B were also characterized using primer extension, and transcripts from 5 deletion transgenics were amplified using RT-PCR. No correlation was observed between putative cis-acting elements and the expression patterns from the RPL23aA and B deletion transgenics, although a 102 bp sequence in the RPL23aB 5 RR was found to contain pollen-specific elements. 5 leader introns were found in each RPL23a gene, and amplification of transgene transcripts from deletion series plants indicated the importance of post-transcriptional and translational regulation in RPL23aA and B expression. This thesis work is the first demonstration of a plant RPL23a protein as a functional member of the L23/L25 (L23p) conserved r-protein family, and is one of the few in-depth studies of the regulation of r-protein genes in plants. While the majority of previous research on plant r-protein gene expression has focused solely on transcript levels, I show herein that post-transcriptional mechanisms have a critical role in regulating these genes, and thus plant r-protein genes more strongly resemble their mammalian counterparts than those of yeast in terms of structure and regulation.

Eukaryotic cytosolic ribosomes are composed of two distinct subunits consisting of four individual ribosomal RNAs and, in Arabidopsis thaliana, 81 ribosomal proteins. Functional subunit assembly is dependent on the production of each ribosomal component. Arabidopsis thaliana r-protein genes exist in multi-gene families ranging in size from two to seven transcriptionally active members. The cytosolic RPS15a gene family consists of four members (RPS15aA, -C, -D and -F) that, at the amino acid level, share 87-100% identity. Using semi-quantitative RT-PCR I have shown that RPS15aC is not expressed and that transcript abundance differs both spatially and temporally among the remaining RPS15a genes in non-treated Arabidopsis tissues and in seedlings following a variety of abiotic stresses. A comprehensive analysis of the RPS15a 5' regulatory regions (RRs) using a series of deletion constructs was used to determine the minimal region required for gene expression and identify putative cis-regulatory elements. Transcription start site mapping using 5' RACE indicated multiple sites of initiation for RPS15aA and -F and only a single site for RPS15aD while all three genes contain a leader intron upstream of the start codon. Analysis of reporter gene activity in transgenic Arabidopsis containing a series of 5' RR deletion::GUS fusions showed that, similar to previous RT-PCR results, there was a trend for mitotically active tissues to stain for GUS activity. Putative cis-elements including the TELO box, PCNA Site II motif and pollen specific elements were identified. However, there was not always a clear correlation between the presence of a putative element and RPS15a transcript abundance or GUS activity. Although variation in transcriptional activity of each RPS15a gene has been observed, subcellular localization of both RPS15aA and -D in the nucleolus has been confirmed in planta by confocal microscopy. The results of this thesis research suggest while all three active RPS15a genes are transcriptionally regulated, additional post-transcriptional and/or translational regulation may be responsible for final RPS15a levels while differential isoform incorporation into ribosomal subunits may be the final point of r-protein regulation.

Ribosomal proteins (RPs), synthesized in the cytoplasm, need to be transported from the cytoplasm to the nucleolus (a nuclear compartment), where a single molecule of each RP assembles with rRNAs to form the large and small ribosomal subunits. The objectives of this research were to identify nuclear/nucleolar localization signals (NLSs/NoLSs; generally basic motifs) that mediate the transport of Arabidopsis RPL23aA, RPL15A and RPS8A into the nucleus and nucleolus, and to study transcriptional regulation and subcellular localization of RPs. While all previous research has shown that nucleolar localization of proteins is mediated by specific basic motifs, in this study, I showed that a specific number of basic motifs mediated nucleolar localization of RPL23aA, rather than any specific motifs. In this protein, single mutations of any of its eight putative NLSs (pNLSs) had no effect on nucleolar localization, however, the simultaneous mutation of all eight completely disrupted nucleolar localization, but had no effect on nuclear localization. Furthermore, mutation of any four of these pNLSs had no effect on localization, while mutation of more than four increasingly disrupted nucleolar localization, suggesting that any combination of four of the eight pNLSs is able to mediate nucleolar localization. These results support a charge-based system for the nucleolar localization of RPL23aA. While none of the eight pNLSs of RPL23aA were required for nuclear localization, in RPS8A and RPL15A, of the 10 pNLSs in each, the N-terminal two and three NLSs, respectively, were absolutely required for nuclear/nucleolar localization. Considering the presence of only a single molecule of each RP in any given ribosome, which obligates the presence of each RP in the nucleolus in equal quantities, I studied transcriptional regulation of Arabidopsis RP genes and the subcellular localization of five RP families to determine the extent of coordinated regulation of these processes. Variation of up to 300-fold was observed in the expression levels of RP genes. However, this variation was drastically reduced when the expression level was considered at the RP gene family level, indicating that coordinate regulation of expression of RP genes, coding for individual RP isoforms, is more stringent at the family level. Subcellular localization also showed differential targeting of RPs to the cytoplasm, nucleus and nucleolus, together with a significant difference in the nucleolar import rates of RPS8A and RPL15A. Although one could expect coordinated regulation of the processes preceding ribosomal subunit assembly in the nucleolus, my results suggest differential regulation of these processes.

The branching or tillering of crops is an important agronomic trait with a major impact on yield. Maintaining an appropriate number of branches allows the plant to use limited light resources and to produce biomass or yield more effectively. The branching process includes the initiation of the axillary meristem leading to bud formation and the further outgrowth of the axillary buds. Phytohormones, including cytokinins and auxin, are known to play major roles in regulating axillary bud outgrowth. Light signals, including light quantity and light quality, are among the most important factors regulating plant growth and are perceived by the action of specialized photoreceptors, including phytochromes. Phytochromes sense red (R) and far-red (FR) light and allow some plants to perceive and respond to competing neighbors by evoking the shade avoidance syndrome (SAS). One component of the SAS is inhibition of branching. Phytochrome B (phyB) is especially important in sensing shade signals and loss of phyB function results in a constitutive shade avoidance phenotype, including reduced branching. While it has been anecdotally reported that phyB-deficient Arabidopsis branches less than wild type, a detailed study of the defects in the process is lacking. In this research, the interactions between light signals, phytochromes and phytohormones in the regulation of branching were assessed using an integrated physiological, molecular and genetic approach.

Thesis (M.S.)--Auburn University, 2008. / Abstract. Includes bibliographical references (p. 39-42).

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 204-224). Also available in print.