This dissertation describes the cloning and characterization of the TOUSLEDlike kinases genes of maize (ZmTLKs). The TOUSLED-like kinases (TLKs) are a conserved family of nuclear Ser/Thr kinases in higher eukaryotes. The maize genome has three TOUSLED-like kinase genes (ZmTLK1, ZmTLK2, and ZmTLK3). Based upon sequence similarity, the ZmTLKs are divided into two classes, the ZmTLK1 and the ZmTLK2/3 class. The origins of these genes can be inferred from their map positions and relationships with TLKs in other Zea species. The ZmTLK1 and ZmTLK2 genes occupy syntenous positions on chromosome arms 1L and 5S in the maize genome. There are two equivalent classes of TLK genes in other Zea species, altogether indicating that the two ZmTLK classes are orthologous genes from the precursor species of maize, an ancient allotetraploid. Gene expression studies of ZmTLKs show that there is a higher level of expression in tissues undergoing DNA synthesis. This is consistent with studies of TLKs in animal systems that show involvement in chromatin assembly/remodeling activities during DNA replication and repair, as well as in transcription. The highest level of gene expression for the ZmTLK2/3 class was observed during development of the endosperm, in a period of massive nuclear endoreduplication. ZmTLK1 is not upregulated in endoreduplicating endosperm, suggesting functional divergence between the two classes of ZmTLK genes. The function of the ZmTLKs was examined by testing whether maize TLK genes could complement the tousled mutant of Arabidopsis. In Arabidopsis thaliana, recessive mutations in the single copy TOUSLED (TSL) gene cause moderate vegetative and severe floral defects, suggesting that TLKs may play a role in gene expression modulation through chromatin remodeling. The ZmTLK proteins are 84% identical to TSL in the catalytic region and 45 - 49% at the N-terminal regulatory domain. However, structural features of the N-terminal region domains of the ZmTLKs are similar to that of TSL. Arabidopsis tsl-1 mutant plants were transformed with ZmTLK2, under the control of the CaMV 35S promoter. These plants showed wild-type Arabidopsis phenotype, indicating that in spite of their sequence differences, ZmTLK2 and TSL interact with the same substrates and regulatory partners and are functionally equivalent. / Graduation date: 2005

Graduation date: 2001

Aluminum (Al)stress is a major limitation to crop productivity on acidic soils. To help understand the cellular mechanisms underlying the toxicity and resistance of plants to Al, this thesis involved a large-scale, transcriptomic and proteomic analysis of roots of Arabidopsis thaliana and reports on comparative analysis of transcriptome and proteome of Al stress responses. Using a microarray representing ~93% of the predicted genes in Arabidopsis, a relatively small proportion (3%) of transcripts were detected as Al- responsive. More changes in the transcriptome were detected after long-term (48 h; 1,114 genes), than short-term (6 h; 401 genes) with relatively little overlap of transcripts detected for each time point. These results suggest that Al toxicity is progressive over time and poses some unique challenges to plants. Further, using two dimensional differential in gel electrophoresis (DiGE), 12 (6 h) and 17 (48 h) proteins were found differentially abundant after Al exposure. Most of the identified proteins were involved in primary metabolism and oxidative stress. Cytosolic-malate dehydrogenase (cyt-MDH) was one of the novel Al-responsive protein identified in this study. Transcript abundance of cyt-MDH correlated well with protein abundance, suggesting that cyt-MDH is regulated in part at transcriptional level. Furthermore, homozygous mdh-1 and mdh-2 mutants were more resistant to Al as compared to WT suggesting that regulation of cyt-MDH could play a role in Al resistance. In general, comparative analysis of proteomics data and transcriptomics data showed a poor correlation for both 6 h (r2 = 0.155) and 48 h (r2 = 0.083). The potential role of five class III peroxidases (PER2, PER27, PER34, PER42, PER69) in resistance of roots to Al was explored using quantitative reverse transcriptase PCR and a reverse genetics approach. A diverse range of patterns of transcript abundance was detected using QRT-PCR in response to Al. Furthermore, per2, per21, and per69 mutants showed greater increases in root lengths as compared to WT after Al stress suggesting that regulation of PER might play a role in Al resistance. These results contribute to the identification of candidate genes for the generation of Al-resistant transgenic plants. / Plant Biology

Abstract In silico analysis showed that Arabidopsis thaliana gene AT5G09530 encodes a uniquely repetitive, proline-enriched protein that is conserved across species, and is likely secreted to the cell wall. Based on its most common amino acid repeat motif, I named the gene PELPK1 and its putative paralog PELPK2 (AT5G09520). Reporter (GUS) expression showed that the PELPK1 upstream genomic region is sufficient for expression in the aleurone layer during seed germination, and is induced throughout the plant by biotic factors (especially Pseudomonas syringae infection), defense chemicals (MeJa, salicylic acid), and mechanical wounding, consistent with the presence of conserved regulatory elements. Sub-cellular localization of a translational fusion of PELPK1 with GFP showed that the protein was secreted into seed-coat aleurone cells and to the cell walls of other tissues. Based on these results, it was concluded that the PELPK1 is a cell wall-associated protein and is most actively transcribed during radicle penetration of the seed coat and during pathogen and wounding responses. A proteomic survey of aleurone proteins failed to identify PELPK1, although several proteins not previously associated with this tissue were identified. Mutational analysis demonstrated that RNAi silencing significantly down-regulated the transcript abundance of PELPK1. Phenotypic analysis showed that RNAi plants exhibited significantly slower germination and root growth when the medium was supplemented with sucrose (100mM). Conversely, constitutive overexpression (OX) of PELPK1 enhanced seed germination and root elongation as compared to wild-type (WT). Analysis of soil-grown plants showed slower emergence and slower vegetative growth for RNAi lines, while OX plants exhibited faster emergence and enhanced vegetative growth and flowering as compared to WT. However, PELPK1 RNAi and OX lines did not differ from WT in response to treatment with pathogens. These results show that the abundance of PELPK1 is positively correlated with plant growth rate under some conditions. PELPK1 may influence growth through CW modification or other independent pathways. / Plant Biology

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, August 1999. / Includes bibliographical references. Also available on the Internet.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 153-163).

In transgenic research the precise control of transgene expression is crucial in order to obtain transformed organisms with expected desirable traits. A broad range of transgenic plants use the constitutive cauliflower mosaic virus (CaMV) 35S promoter to drive expression of selectable marker genes. Due to its strong enhancer function, this promoter can disturb the specificity of nearby eukaryotic promoters. When inserted immediately downstream of the 35S promoter in transformation vectors, special DNA sequences called insulators can prevent the influence of the CaMV35S promoter/enhancer on adjacent tissue-specific promoters for the transgene. Insulators occur naturally in organisms such as yeasts and animals but few insulators have been found in plants. Therefore, the goal of this study is to identify DNA sequences with insulator activity in Arabidopsis thaliana. A random oligonucleotide library was designed as an initial step to obtain potential insulators capable of blocking enhancer-promoter interactions in transgenic plants. Fragments from this library with insulator activity were identified and re-cloned into pB31, in order to confirm their activity. To date, one insulator sequence (CLO I-3) has been identified as likely possessing enhancer-blocking activity. Also, two other oligonucleotide sequences (CLO II-10 and CLO III-78) may possess insulator activity but more sampling is needed to confirm their activity. Further studies are needed to validate the function of plant insulator(s) and characterize their associated proteins.

Plant form and structure is remarkably diverse encompassing a myriad of shapes and sizes, and is a result of coordinated instances of cell growth and division. This varied form and structure represents adaptive strategies enabling plants to exploit and endure their environment maximizing fitness. Common to both cell growth and division processes, is the requirement of growth material and effectors via the secretory system. The secretory system is a dynamic pathway of organelles connected via vesicular traffic, responsible for protein modification and delivery. Challenges in studying secretion are related to its' dynamic flow between organelles, and the prevalence of redundancy and lethality, which cause difficulties in interpretation and genetic modulation. To further our understanding of the secretory process, we employed a chemical genetic approach to identify small molecule probes of secretion by first screening for inhibitors of cell expansion, followed by a microscopy based screen of GFP marker lines for perturbagens of subcellular structures. Small molecules offer the advantages of temporal and spatial application, have been shown to overcome redundancy and can be varied in concentration to control the severity of effects. We identified twenty-five small molecule probes of secretion and pursued two chemicals with striking properties, polarazine and eroonazole. Polarazine was identified as a cell division inhibitor, affecting phragmoplast structure and function, while eroonazole remodels the endoplasmic reticulum into small vesicles and is implicated in auxin signaling. This approach shows the utility of small molecule probes in furthering our understanding of secretory processes.

The Arabidopsis TGA family of basic leucine zipper transcription factors regulate the expression of pathogenesis-related genes and are required for resistance to disease. Members of the family possess diverse properties in respect to their ability to transactivate and interact with NPR1, the central regulator of systemic acquired resistance in Arabidopsis. Two TGA factors, TGA1 and TGA2, have 83 % amino acid similarity but possess differing properties. TGA1 does not interact with NPR1 but is able to transactivate, while TGA2 interacts with NPR1 but is unable to transactivate. This study uses these two TGA factors to identify amino acids that are responsible for their function. <p>Four cysteines residues within TGA1 were targeted for study by site-directed mutagenesis and the resulting mutants were tested for interaction with NPR1 in yeast. The construct containing a mutation of cysteine 260 (Cys-260) interacted well with NPR1, while those with mutations at Cys-172 or Cys-266 interacted poorly. The Cys-260 mutant also displayed the greatest decrease in transactivation potential in yeast, while mutation of Cys-172 or Cys-266 resulted in smaller decreases. Mutation of Cys-287 had no effect on NPR1 interaction or transactivation. Combining various point mutations in a single protein did not increase NPR1 interaction or transactivation levels, indicating that Cys-260 is crucial for regulating TGA1 properties. Cysteines possess the unique ability of forming reversible disulfide bonds which have been shown to regulate several mammalian cellular processes. The observation that mutation of a single TGA1 cysteine (Cys-260) greatly alters the proteins properties provides a convincing argument that oxidoreduction of this residue is important for its regulation, possibly through the formation of a disulfide bond with either Cys-172 or Cys-266. <p>To test whether other members of the TGA family could be regulated by oxidoreduction, several TGA2 constructs were created that introduced Cys at positions corresponding to those found in TGA1. When tested in yeast none were able to transactivate but continued to interact with NPR1.