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.

No description available.

During Arabidopsis embryogenesis, cell-cell signaling plays an essential role in establishing an organized body plan centered around two major axes of development: apical-basal and radial. Two topics of great interest are how the layered structure is initiated and maintained during and after embryogenesis and how communication between layers is achieved to allow for coordinated development. Recent research involving Receptor-Like Kinases (RLKs) in plants suggests that their roles in integrating various signals are important in many aspects of development, including embryonic and post-embryonic patterning. The research presented here describes the roles of two pairs of RLKs with independent roles in two different signaling environments. The first RLK pair, GSO1 and GSO2, function in root development at the transition to photoautotrophic nutrition to integrate sugar signals and regulate root growth. GSO1 and GSO2 regulate root epidermal cell identity by controlling the pattern of cell division of stem cells. The second pair of RLKs, RPK1 and TOAD2, function to control root development by regulation of meristem proliferation and a coordinated response to signaling molecules of the CLE family. The response of wild-type roots to treatment with CLE peptides (A-type) is meristem growth arrest, resulting in short roots. toad2 mutants are insensitive to the effect of CLE peptides in reducing meristem size and TOAD2 also regulates RPK1 upon CLE stimulation. Although responding to different signals, the two pairs of RLK share a common output of regulating cell proliferation in and around the root meristem, especially in the epidermis of the root.

No description available.

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.

Transposons, also known as transposable elements (TEs), are genetic elements capable of changing their location in the genome and amplifying in number. Because of their ability to cause mutations in the host genome, often with detrimental consequences to the host, yet avoid being eliminated by natural selection, transposons have been labeled selfish elements or genomic parasites. However, the advent of genomics has allowed the identification of numerous instances where transposons have played a crucial role in host genome evolution. In this thesis, I evaluate the extent to which transposons have influenced the genomes of their hosts, with an emphasis on plant genomes. I review the present knowledge of different mechanisms by which this is achieved and provide examples to illustrate them. Next, I tackle the problem of annotating transposons in the completed genomic sequence of domestic rice by comparing RepeatMasker, the standard approach used in transposon annotation, with an alternative approach employing hidden Markov models. In addition, I perform a genome-wide analysis of gene fragment capture by rice Mutator-like transposons. I conclude that, while this is a widespread phenomenon in rice, it is unlikely to represent a major force in generating novel protein-coding genes. Nevertheless, the duplicated gene fragments that are transcribed may playa role in the regulation of host genes they arose from via an RNAi-like mechanism. Finally, I conduct an in silico analysis of a gene family derived from a domesticated Mutator-like transposase, called MUSTANG (MUG), in conjunction with an experimental characterization of the MUG family in Arabidopsis. The results of the study indicate that the MUG family arose in a common ancestor of flowering plants and that the Arabidopsis genes AtMUG1 and/or AtMUG2 may act as global regulators of mitochondrial function. I conclude that our appreciation of the role of transposons in host function and evolution will undoubtedly continue to grow as our understanding of these processes deepens.

Lignin is a major chemical component of plants and the second most abundant natural polymer after cellulose. The concerns and interests of agriculture and industry have stimulated the study of genes governing lignin content in plants in an effort to adapt plants to human purposes. Arabidopsis thaliana provides a convenient model for the study of the genes governing lignin content because of its short growth cycle, small plant size, and small completely sequenced genome. In order to identify the genes controlling lignin content in Arabidopsis accessions using Quantitative Trait Locus (QTL) analysis, a rapid micro-scale method of lignin determination is required. The acetyl bromide method has been modified to enable the rapid micro-scale determination of lignin content in Arabidopsis. Modifications included the use of a micro-ball mill, adoption of a modified rapid method of extraction, use of an ice-bath to stabilize solutions and reduction in solution volumes. The modified method was shown to be accurate and precise with values in agreement with those determined by the conventional method. The extinction coefficient for Arabidopsis lignin, dissolved using acetyl bromide, was determined to be 23.35 g-iLcm-1. This value is independent of the Arabidopsis accession, environmental growth conditions and is insensitive to syringyl/guaiacyl ratio. The modified acetyl bromide method was shown to be well correlated with the 72% sulfuric acid method once the latter had been corrected for protein contamination and acid-soluble lignin content (R² = 0.988, P < 0.0001). As determined by the newly developed acetyl bromide method and confirmed by the sulfuric acid method, lignin content in Arabidopsis was found to be a divergent property. Lignin content in Arabidopsis was found to be weekly correlated with growth rate among Arabidopsis accessions (R² = 0.48, P = 0.011). Lignin content was also found to be correlated with plant height among Arabidopsis accessions (R² = 0.491, P < 0.0001).

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