The cyclic nucleotide monophosphates (CNs), cAMP and cGMP, are second messengers that participate in the regulation of development, metabolism and adaptive responses. In plants, CNs are associated with the control of pathogen responses, pollen tube orientation, abiotic stress response, membrane transport regulation, stomatal movement and light perception. In this study, we hypothesize that cAMP and cGMP promote changes in the transcription level of genes related to photosynthesis, high light and membrane transport in Arabidopsis thaliana leaves and, that these changes at the molecular level can have functional biological consequences. For this reason we tested if CNs modulate the photosynthetic rate, responses to high light and root ion transport. Real time quantitative PCR was used to assess transcription levels of selected genes and infrared gas analyzers coupled to fluorescence sensors were used to measure the photosynthetic parameters. We present evidence that both cAMP and cGMP modulate foliar mRNA levels early after stimulation. The two CNs trigger different responses indicating that the signals have specificity. A comparison of proteomic and transcriptional changes suggest that both transcriptional and post-transcriptional mechanisms are modulated by CNs. cGMP up-regulates the mRNA levels of components of the photosynthesis and carbon metabolism. However, neither cAMP nor cGMP trigger differences in the rate of carbon assimilation, maximum efficiency of the photosystem II (PSII), or PSII operating efficiency. It was also demonstrated that CN regulate the expression of its own targets, the cyclic nucleotide gated channels - CNGC. Further studies are needed to identify the components of the signaling transduction pathway that mediate cellular changes and their respective regulatory and/or signaling roles.

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Transposons are a diverse collection of mobile genetic elements and are important components of nearly every genome. Because of their mobile and repetitive nature, transposons can have considerable effects on host gene expression, genome organization and evolution. The recent availability of genomic sequence information has expedited the discovery and study of transposons, as exemplified in this thesis by the complete genome analysis of the model plant system Arabidopsis thaliana. Data mining in Arabidopsis has revealed a rich diversity of transposons, of which Basho and Terminal-repeat Retrotransposons In Miniature (TRIM) elements were previously unknown types. The identification of Related to Empty Sites (RESites) provide evidence for past transposition events. Examples of elements contributing to coding regions, acquiring cellular sequences, along with in-depth analysis of the insertions, their target sites and their distribution illustrate the impact of transposons on gene and genome structures. Computer-based searches of genomic sequences has also improved our understanding of previously identified transposon families, such as the origin, classification and mobilization of Tourist elements. In addition, information on transposons gathered from in silico analysis of genomic sequences has served to design in vivo experiments. In a whole genome strategy, Transposon Display was used to investigate transposition and regulation of mobility of Tourist-like elements in A. thaliana and in the nematode Caenorhabditis elegans.

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.

An alkaline extract of the brown seaweed, Ascophyllum nodosum (ANE) increases plant growth and imparts resistance against biotic stresses. However, little is known of the effects of ANE on insects. Myzus persicae, green peach aphid (GPA), and Arabidopsis model were used to determine whether application of ANE confers protection from GPA infestation. GPA colonization increased in ANE treated plants, associated with improved biomass. However, ANE treated plants exhibited less cell death and also showed a greater ability to recover from GPA injury. Lower expression of SAG13, SAG21 and CHL1 and a higher expression of ARR5 was observed in ANE treated plants. Taken together, gene expression along with lower cell death suggests ANE may delay senescence in Arabidopsis. Delayed senescence in Arabidopsis following ANE treatment may be a result of increased cytokinin activity. Increased GPA numbers could be, at least in part, due to delayed senescence in Arabidopsis following ANE treatment.

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.