Small RNAs (sRNAs), circa 21-26nt RNA molecules, are a novel class of regulatory molecules that influence many aspects of plant biology. The first objective of this thesis was to utilize computational approaches both to investigate how microRNAs (miRNAs), a type of sRNA, as a class affect their target transcripts’ accumulation and to identify novel miRNAs in Arabidopsis thaliana. The second objective of this thesis was to examine the regulation of protein coding (PC) cis natural antisense transcripts (cis-NATs), which have the potential to make double stranded RNA. Computational analysis of the expression of miRNA-regulated genes demonstrated that the transcriptomes of the inflorescences of plants defective in miRNA biogenesis were similar to normal leaf tissues and dissimilar to normal pollen and seed. Thus, miRNAs cause the plant transcriptome to shift from a vegetative to reproductive state. Known miRNA targets fail to explain miRNA-defective mutant transcriptome patterns. Novel computational approaches were used to discover five new mature miRNAs. Interestingly, two miRNAs have different functions but are encoded by perfect complements of the same precursor molecule. Genome-wide analysis of cis-NAT abundances revealed that protein coding (PC) cis-NATs tend to be co-expressed, broadly expressed, and highly expressed across diverse abiotic stress conditions. These expression patterns were negatively associated with sRNAs because sRNAs were under-represented within PC cis-NATs compared to PC non-cis-NATs. sRNAs also mapped to cis-NATs and non-cis-NATs at similar frequencies in mutants defective in nat-siRNA biogenesis relative to other genotypes. We suggest a common euchromatin environment and possibly antisense RNA stabilization of mRNA transcripts may contribute to the high level, breadth, and co-expression of cis-NATs. However, cis-NATs are correlated less frequently than expected, and cis-NAT transcript abundances often differ more than expected. In addition, sRNAs matched PC cis-NATs relative to PC non-cis-NATs more frequently in abiotic stress conditions than in control conditions. Thus, although sRNAs do not have a widespread role in regulating cis-NATs, sRNAs may have a focused role in regulating cis-NAT transcript abundances. / PhD thesis / NSERC

Upregulation of intracellular and secreted acid phosphatases (APases) is a universal response of orthophosphate-starved (-Pi) plants. APases hydrolize Pi from a broad spectrum of phosphomonoesters at an acidic pH. Plant APases belong to a relatively large multigene family whose specific functions in Pi metabolism are poorly understood. This study focuses on the identification and characterization of cell wall (CW) localized purple acid APases (PAPs) upregulated by -Pi Arabidopsis thaliana. Three glycosylated PAP isozymes secreted into the CW of -Pi Arabidopsis suspension cells were purified and identified by peptide mass fingerprinting using mass spectrometry (MALDI-TOF MS) and N-terminal microsequencing as AtPAP12 (At2g27190; subunit size 60-kDa), AtPAP25 (At4g36350; subunit size 55-kDa) and AtPAP26 (At5g34850; subunit size 55-kDa). Both AtPAP12 and AtPAP26 were previously shown to be upregulated and secreted by –Pi Arabidopsis to scavenge Pi from extracellular organic-P. However, the novel AtPAP25 has never been suggested to be involved in the plant Pi-starvation response. Biochemical characterization of AtPAP25 revealed a monomeric 55 kDa protein. Similar to other PAPs it was purple-in-solution and insensitive to tartrate. Glycoprofiling via LC MS/MS revealed highly complex NXS/T glycosylation motifs at Asn172, Asn367 and Asn424. I hypothesize that these motifs play a role in AtPAP25 targeting and function. Kinetic characterization revealed a broad pH optimum centered at 5.6 and inhibition of activity by several common APase inhibitors. AtPAP25 exhibited broad substrate selectivity, low Vmax, and a Km (phosphoenolpyruvate) value of 0.52 mM. Immunoblot and semi-quantitative RT-PCR transcript analysis indicated that AtPAP25 is exclusively synthesized under –Pi conditions. Deduced amino acid sequences were compared using multiple sequence alignment and phylogenetic analysis. Growth of atpap25 T-DNA insertion mutant knockout seedlings was completely arrested when transferred to a soluble Pi deficient organic-P containing soil mix, pointing to a potential regulatory function of AtPAP25 during nutritional Pi stress. Overall, this research is helping to shed light on the functional importance of specific PAP isozymes in facilitating plant acclimation to nutritional Pi deficiency. This is important because there is an urgent need to engineer Pi-efficient transgenic crops to minimize the huge input of expensive, non-renewable, and polluting Pi fertilizers in agriculture. / Thesis (Master, Biology) -- Queen's University, 2012-09-10 08:28:21.631

Despite the ubiquity of transposons in eukaryotic genomes, their evolutionary role remains controversial. The discovery of several domesticated genes has suggested that transposons can gain host functions, and thus contribute to the evolution of their host. Here, I present the results of a genome-wide screen for transposon-derived host genes, which was based on the idea that, once domesticated, the open reading frame of such elements would be maintained, while terminal structures necessary for transposition would be lost. Eight-hundred-and-sixty-three such transposon-dissociated elements were mined from the genome of Arabidopsis thaliana var. Columbia-0, of which less than 10% are associated with expression data. Phylogenetic analysis of Mutator superfamily genes in the genomes of Oryza sativa ssp. japonica (cv Nipponbare) and Arabidopsis, including 121 Mutator-derived transposon-dissociated elements, found that only two gene families are taxonomically widespread. MUSTANG1, a member of one of these families, appears to be under purifying selection. Thus, despite the dearth of taxonomically widespread and/or expressed transposon-dissociated elements, MUSTANG1, as well as three transposon-dissociated elements that may be associated with mutant phenotypes, might be newly discovered transposon-derived host genes.

The response of Arabidopsis thaliana (Columbia) to infection with turnip mosaic virus (TuMV) was characterized at the level of: disease symptom expression, cell content and protein composition. Visual symptoms observed were chlorotic and mottled leaf colouring, severely stunted growth, distortion of leaf blades and delayed bolting. All plants died before seed cases dehisced. Electron microscopy revealed three types of cylindrical inclusion bodies: pinwheels, scrolls and laminated aggregates, in the cytoplasm of infected plants similar to those observed in other plants infected with TuMV. Inoculation of Arabidopsis with TuMV resulted in quantitative changes in several proteins in both soluble and membrane proteins, as revealed by electrophoresis on 12% polyacrylamide gels. Antibodies were made to both infected membrane and soluble proteins. Western blots of infected and uninfected, soluble and membrane proteins probed with antibodies revealed quantitative changes in the same proteins identified by polyacrylamide gels. A CNBr 4B activated sepharose column was used to make infection-specific antibodies to infected soluble proteins. No infection-specific host proteins were detected in Arabidopsis infected with TuMV.

<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.