<p>In this thesis, we have firstly cloned and expressed the domains that harbours the putative catalytic GC domain in these receptor molecules and demonstrate that these molecules can convert GTP to cGMP in vitro. Secondly, we show that exogenous application of both Phytosulfokine and Brassinosteroid increase changes of intracellular cGMP levels in Arabidopsis mesophyll protoplast demonstrating that these molecules have GC activity in vivo and therefore provide a link as second messenger between the hormones and down-stream responses. In order to elucidate a relationship between the kinase and GC domains of the PSK receptor, we have used the AtPSKR1 receptor as a model and show that it has Serine/Threonine kinase activity using the Ser/Thr peptide 1 as a substrate. In addition, we show that the receptor`s ability to phosphorylate a substrate is affected by the product (cGMP) of its co-domain (GC) and that the receptor autophosphorylates on serine residues and this step was also observed to be affected by cGMP. When Arabidopsis plants are treated with a cell permeable analogue of cGMP, we note that this can affect changes in the phosphoproteome in Arabidopsis and conclude therefore that the cGMP plays a role in kinase-dependent downstream signalling. The obtained results suggest that the receptor molecules investigated here belong to a novel class of GCs that contains both a cytosolic kinase and GC domains, and thus have a domain organisation that is not dissimilar to that of atrial natriuretic peptide receptors NPR1 and NPR2. The findings also strongly suggest that cGMP has a role as a second messenger in both Brassinosteroid and Phytosulfokine signalling. We speculate that other proteins with similar domain organisations may also have dual catalytic activities and that a significant number of GCs, both in plants and animals, remain to be discovered and characterised.</p>

Abscisic acid (ABA) is a key plant hormone regulating agronomically important processes including seed maturation and dormancy, stomatal opening and closure, along with the transcriptional and physiological response of plants to abiotic and biotic stresses. The current study sought to functionally characterize members of an ABA-responsive gene family encoding GRAM (Glucosyltransferases, Rab-like GTPase activators and Myotubularins) domain proteins in Arabidopsis thaliana. Utilizing reverse genetics loss- and gain-of-function lines associated with GEM-RELATED 5 (GER5) were obtained, which displayed several defects in reproductive development. Gene expression profiling, RNA in situ hybridization and immunohistochemical techniques were utilized to evaluate GER5 and two closely related GRAM genes, GEM-RELATED 1 (GER1) and GLABRA2 EXPRESSION MODULATOR (GEM) in reproductive structures. Microarray profiling of seeds from ger5-2 mutants and wild-type plants revealed transcriptional changes in carbohydrate metabolism, hormone signaling and catabolic processes accompanied seed development defects of ger5-2 mutants. Seed germination assays further revealed ger5-2 mutants exhibited reduced sensitivity to ABA. In assessing GER5, GER1 and GEM as putative ABA-response genes, a second study evaluated the expression of GRAM, AuTophaGy-related (ATG), and ABA-response genes in source and sink organs exposed to abiotic stress or within mutant backgrounds deficient in sugar signaling. Monodansylcadaverine (MDC) staining was also utilized to localize autophagosomes or autophagic bodies within vegetative or reproductive organs during plant development, or in response to carbon starvation or abiotic stress. In a third study transcriptional differences in ABA metabolism, transport and homeostasis were examined within reproductive organs (cauline leaves, inflorescence meristem, developing siliques) exposed to cold and heat stress. This study revealed reproductive organs are characterized by unique patterns of ABA metabolism which differ from tissues typically associated with classical ABA responses. Together, these studies indicate GER5, an uncharacterized ABA-responsive GRAM domain gene, plays a novel role in the reproductive development of plants and that ABA metabolism and signaling are uniquely regulated in reproductive organs.

The cytoskeleton is a remarkable system of filaments that helps in the organisation and functioning of living cells. In plant cells, this cytoskeleton comprises actin microfilaments and microtubules that polymerise from actin and tubulin respectively. While these proteins are highly conserved in eukaryotes, the plant cytoskeleton performs many plant-specific functions. The organisation and functions of the cytoskeleton are determined by a plethora of accessory proteins (actin-binding proteins, microtubule-associated proteins) that link the cytoskeletal filaments to other cell components and to each other. While there is extensive data for the subcellular localisation of actin-binding proteins with actin microfilaments in animal cells, surprisingly few experiments of this type have previously been tried in plants, and the subcellular localisation of most plant actin-binding proteins remains unknown. Such information is important in assessing functions of these proteins to give a better understanding of the actin cytoskeleton. In this study, an attempt was made to visualise the association of actin microfilaments and actin-binding proteins. A range of antibodies raised against various plant and animal actin-binding proteins were screened in two model systems for plant cytoskeleton research, the root of Arabidopsis thaliana and in whole cells of the tobacco BY2 liquid cell culture. Further, because previous data in the localisation of the actin-binding protein tropomyosin have suggested that the localisation of this actin-biding protein with the finer cortical actin microfilaments in Arabidopsis roots might not be discerned due to high cytoplasmic background, immunolabelling experiments were also conducted on plasma membrane ghosts generated from tobacco BY2 from which any non-specific cytoplasmic labelling could be washed away. There experiments gave some preliminary suggestions for the association of the actin-binding proteins to the actin cytoskeleton in plant cells. The most intriguing observations were obtained with antibodies against the β-subunit of capping protein which colocalised with larger microfilament bundles in tobacco BY2 cells. No colocalisation was observed on membrane ghosts on which these bundles are not well retained. However, the previous experiments in which there were suggestions of tropomyosin-related proteins associated with fine cortical microfilaments in Arabidopsis could not be replicated. As no cytoskeletal localisation was observed in either Arabidopsis or tobacco with antibodies raised against known actin-bundling proteins from Arabidopsis such as villin and fimbrin, it is speculated that the labelling protocols, currently optimised for visualising the actin cytoskeleton, might not to be modified to allow visualisation of actin-binding proteins in plant cells.

All nascent polypeptides synthesized in bacteria, mitochondria, or chloroplastsstart with a N-formylmethionine. Peptide deformylase (PDF) is a mononuclear metal ionprotein that is responsible for removing the N-formyl group of nascent proteins found inbacteria and chloroplasts in order for them to become mature proteins. It is possible, asseen from the literature with actinonin, to chelate the enzyme's metal ion and inhibit thefunction of protein production essentially resulting in death of the bacteria, or plant. Thisstudy examines the active site of Arabidopsis thaliana (At) types of PDF (AtDEF1 andAtDEF2, respectively) as well as bacterial DEF2 using sequence alignments andcomputational modeling. This work also investigates the biological efficacy of designingand synthesizing inhibitors that mimic actinonin or the D1 substrate that will halt, orseverely retard, the activity of the PDF enzyme in vitro and in vivo. Through thisresearch, we were able to determine specific residues that were conserved amongst theplant DEF2 sequences that were present less than 20% of the time in plant DEF1 andbacteria DEF2. This data allowed us to hypothesize plant DEF2's substrate specificity aswell as a possible design that is selective towards plants and not bacteria. Also, based onpreliminary results, the novel thiol-actinonin chimera that was synthesized showedinhibition activity of AtDEF2 during in vitro enzyme assays.

A fundamental tenet of classical Mendelian genetics is that allelic information is stably transmitted from parent to progeny. Work in our laboratory has revealed a novel exception to this law where Arabidopsis thaliana plants homozygous for the recessive organ-fusion mutation hothead (hth) gave rise to phenotypically and genotypically wild-type (HTH) progeny at high frequencies. We have coined the term restoration to describe this phenomenon, since the reverted HTH allele was not detectable in the parental genome but was present in a recent ancestor (the grandparent). Recent work in our laboratory has demonstrated that 45-94 bp insertions and deletions (indels) can also restore, irrespective of their genomic location. The work described in this thesis expands our understanding of restoration by characterizing previously identified non-parental loci at the molecular level, and monitoring the inheritance of native and transgenic alleles in hth mutant and wild-type genetic backgrounds. Two ??? eight hundred bp genomic intervals containing non-parental loci were cloned and sequenced. This revealed that the tracts of sequence which had been reinstated were identical in phase and sequence composition to the corresponding grandparental sequences. Furthermore, molecular markers flanking non-parental loci were profiled across 80-90 kb chromosomal regions. In all cases, the flanking markers reverted concurrently, suggesting that restoration can affect comparatively large genomic regions. However, it is not clear if flanking markers revert as a result of multiple independent events or, alternatively, are the result of one continuous restoration event. A number of individuals studied in this thesis are genetic mosaics, wherein the restoration events are localized within a single individual. Genetic mosaicism cannot be attributed to pollen contamination, and provides the strongest evidence to-date that restoration is a genuine and novel biological phenomenon. The inheritance of a transgenic allele and two native alleles was monitored in pedigrees compromising a number of distinct ancestries in hth-4, hth-8, and wild-type genetic backgrounds. Although none of the F3 progeny exhibited atypical segregation of the investigated alleles, molecular screening may have revealed localized (mosaic) restoration of the transgenic marker. However, these results remain inconclusive based on results obtained in a negative control experiment. Several significant conclusions can be derived from the work described in this thesis: (1) Restoration is a highly specific template-directed process. The template is likely of ancestral origin, although the nature of the template and the precise mechanism of restoration remain unclear. (2) Restoration frequently gives rise to individuals that are genetic mosaics, a finding that cannot be attributed to outcrossing. (3) Restored sequences are more readily identified by molecular genotyping than phenotypic screening. Possible mechanisms and recommended future studies are discussed.

How patterns arise from an apparently uniform group of cells is one of the classical problems in developmental biology. The mechanism is complicated by the fact that patterning occurs on a growing medium. Therefore, changes in an organism’s size and shape affect the patterning processes. In turn, patterning itself may affect growth. This interaction between growth and patterning leads to the generation of complex shapes and structures from simpler ones. Studying such interactions requires the possibility to monitor both processes in vivo. To this end, we developed a new technique to monitor and quantify vein patterning in a growing leaf over time using the leaves of Arabidopsis thaliana as a model system. We used a transgenic line with fluorescent markers associated with the venation. Individual leaves are followed in many samples in vivo through time-lapse imaging. Custom-made software allowed us to extract the leaf surface and vein pattern from images of each leaf at each time point. Then average spatial maps from multiple samples that were generated revealed spatio-temporal gradients. Our quantitative description of wild type vein patterns during leaf development revealed that there is no constant size at which a part of tissue enclosed by vasculature will become irrigated by a new vein. Instead, it seemed that vein formation depends on the growth rate of the tissue. This is the first time that vein patterning in growing leaves was quantified. The techniques developed will later be used to explore the interaction between growth and patterning through a variety of approaches, including mutant analysis, pharmacological treatments and variation of environmental conditions.

Many environmental stresses result in increased generation of active oxygen species (AOS) in plant cells, leading to the induction of protective mechanisms. In this study, signalling components linking AOS perception to downstream responses were examined, with particular emphasis on H₂O₂ signalling. All AOS investigated had an early [Ca²⁺]_cyt peak in common, but differed in other aspects of their Ca²⁺ signatures, indicating that the plant is able to discriminate between different types of AOS. An early event in AOS signal transduction may involve changes in the cellular redox balance as reduction of glutathione levels prior to stress application increased the height of the first [Ca²⁺]_cyt peak. Inhibiting or enhancing the height of the H₂O₂-triggered Ca²⁺ signature lead to inhibition or enhancement of GST1 and APX1 induction, respectively, demonstrating that the Ca²⁺ signature is required for induction of genes encoding antioxidant enzymes. OX1, encoding a putative ser/thr kinase, was shown to be involved in signal transduction in response to H₂O₂-generating stresses. Transcript levels of OX1 were increased upon treatment with H₂O₂ and a range of abiotic and biotic stresses as well as ABA, all of which have been shown to result in H₂O₂ accumulation. Inhibition of stress-induced [Ca²⁺]_cyt elevations inhibited OX1 induction, placing the OX1 kinase downstream of Ca²⁺ in the signalling chain. OX1 is required for full activation of AtMPKS and AtMPK6 in response to ozone fumigation, indicating that OX1 functions upstream of these MAP kinases. An ox1 null-mutant displayed enhanced susceptibility to infection with a virulent Peronospora parasitica isolate as well as reduced induction of several defence genes. In addition, the ox1 mutant exhibited shorter root hairs and an early flowering phenotype. AOS treatment induced several genes encoding AtERF transcription factors, but did not have an effect on other members of this family. Induction occurred in an ethylene-independent but Ca²⁺-dependent manner.

The sequencing of eukaryotic model organisms has provided us an unprecedented opportunity for a genome-wide characterization of Transposable Elements (TEs) and the study of TE-host relationships. By developing methodologies on database mining, we explored the existence of MUtator (Mu)-Like Elements (MULEs) in Arabidopsis thaliana and Oryza sativa. Mu elements were first discovered in Zea mays; so far, a dozen of the elements have been identified in the genome. We identified a total of 1392 MULEs from the sequenced Arabidopsis genome. They represent one of the most abundant, diversified, yet still mobile DNA transposon families in eukaryotes. The Arabidopsis MULEs are composed of not only the elements showing the typical Mu-family-specific terminal structure (that is the long Terminal Inverted Repeat, TIR), but also a novel type of non-TIR MULEs. Some of this latter type of elements was found to be active both transcriptionally and transpositionally. To understand host-mediated genome-wide regulation(s) on the MULE system in Arabidopsis, we characterized 235 MULE mobility-specific genes (or mudrA-like genes) by mapping them on the sequenced Arabidopsis chromosomes and performing a genome-wide expression assay utilizing Arabidopsis METHYLTRANSFERSE1 (MET1) mutant (met2) plants, we showed that MET1-mediated global CpG methylation can only repress a portion of the gene family; its efficiency depends largely on the gene locations within the context of Arabidopsis chromatin remodeling: stronger in heterochromatic regions but weaker in euchromatic ones. This finding suggests that the Arabidopsis heterochromatic regions are not just a graveyard for the accumulation of defective elements; rather, they may have been playing an important role on the repression of TE activity via, at least in part, exerting MET1-mediated silencing effect. Our expression analysis also suggested that a TIR structure is not necessarily required for the MET1-mediated si

The brevipedicellus (bp) mutant, caused by a loss-of-function mutation in the KNAT1 homeobox gene, is known to affect the stem morphogenesis. BP and ERECTA (ER) genes are required to promote internode and pedicel development and delineate nodal boundaries to maintain the radial symmetry of stems and pedicels. My research aims to identify genes acting on the BP pathway by utilizing a forward genetics approach. The suppressor4 mutant, identified from the bp er mutant screen, exhibits moderate length and perpendicularly-oriented pedicels with partially formed distal pedicel bulges, absent in the bp mutant. The kinky mutant, identified from the bp mutant screen, develops severe bends at the floral nodes and enhanced achlorophyllous stripes. These results suggest the SUPPRESSOR4 gene contribution in inhibiting the development of distal pedicel bulge and influencing both pedicel angle and length whereas the KINKY gene might act with BP in regulating proper inflorescence development.

Auxins are plant hormones involved in virtually all aspects of plant life. Despite long-term commercial and horticultural use of the auxin Indole-3-Butyric Acid (IBA), a full recognition of its natural occurrence in plants was made only recently. I have used multiple approaches to dissect the role of IBA in Arabidopsis thaliana. This thesis includes the first characterization of a mutant with an altered response to IBA that retains wild-type sensitivity to Indole-3-Acetic Acid (IAA), the most studied endogenous auxin. This mutant, named resistant to IBA ( rib1), has modified root architecture and gravitropism and is resistant to auxin transport inhibitors. As these phenotypes are reminiscent of those of characterized auxin transport mutants, movement of IAA and IBA was studied in wild-type and mutant plants. IBA is transported in seedlings in three distinct flows, like IAA, and this transport is saturable, indicating it is carrier mediated. However, unlike IAA, IBA is not polarly transported in inflorescence axes, and IBA transport is not sensitive to IAA transport inhibitors. These results suggest IAA and IBA transport could be mediated or regulated by different mechanisms. In rib1 seedlings, all flows of IBA transport are modified, while IAA transport levels are unchanged. Modifications in IBA transport match phenotypic differences between rib1 and wild-type, and analyses of the physiological effects of IBA also suggest IBA has a role in defining wild-type seedling morphology in Arabidopsis. Though IAA transport levels are not changed in rib1, one flow of IAA transport is rendered insensitive to IAA transport inhibitors, perhaps revealing cross-talk between IAA and IBA transport regulation. Additionally, double mutant analyses reveal that IAA transport and response mutants can suppress some phenotypes of rib1, and some mutant combinations produce novel phenotypes, further suggesting cross-talk between IBA and IAA transport and response p