Stomata act as small valves that help in the regulation of gas exchange in plants. These valves arise from epidermal stem cells that asymmetrically divide to become meristemoids, which are precursor cells that eventually give rise to guard cells. Our lab used microarray-based gene expression profiling to identify genes that might play a role in maintaining or controlling meristemoid behavior. One gene, At5g60880, encodes a small protein that plays a role in stomata patterning. The sequence of this protein provides little information about its putative function. In order to gain insight into the function of this protein, we obtained mutant plants (SALK_86936) and characterized their phenotype. These plants exhibited abnormal asymmetric divisions resulting in clustered stomata. Since nothing is known about this protein, we determined the At5g60880 gene expression pattern by making transgenic plants carrying a fusion between the native At5g60880 promoter and green fluorescent protein (GFP). Confocal laser scanning microscopy was used to visualize the expression pattern of GFP in developing leaves. At5g60880 appears to be expressed in most epidermal cells. To better understand the possible activity of this protein, we also created transgenic plants used to deduce the protein subcellular localization. These plants constitutively express a translational fusion between the At5g60880 protein and GFP driven by the CaMV 35S promoter. Preliminary results reveal cortical and cytoplasmic localization of this fusion protein. Understanding the function of this novel protein will ultimately contribute to our knowledge of stomata patterning mechanisms, and more generally stern cell differentiation in plants.

Thesis (Ph. D.)--Miami University, Dept. of Chemistry and Biochemistry, 2008. / Title from second page of PDF document. Includes bibliographical references.

The Arabidopsis arenosa species complex is closely related to the model species Arabidopsis thaliana. Distribution range of the diploid lineage covers most parts of Central and South-eastern Europe with a remarkable diversity of ecological niches from sea level to alpine peaks. There is still little known about genetic relationships among the diploid populations of A. arenosa. The evolutionary history of the diploid cytotype across its entire range was explored by using 14 nuclear microsatellite loci. Five lineages occupying biogeographically distinct regions were identified in Arabidopsis arenosa complex. These lineages were labelled as Western Carpathians, Eastern Carpathians, Pannonian Lowland, Dinaric Alps and Baltic Shore (according to geographical regions where these lineages occur). Powered by TCPDF (www.tcpdf.org)

Environmental stress conditions can drastically affect plant growth and productivity. In contrast to soil moisture or salinity that can gradually change over a period of days or weeks, changes in light intensity or temperature can occur very rapidly, sometimes over the course of minutes or seconds. So, in our study we have taken an metabolomics approach to identify the rapid response of plants to light stress. In the first part we have focused on the ultrafast (0-90 sec) metabolic response of local tissues to light stress and in the second part we analyzed the metabolic response associated with rapid systemic signaling (0-12 min). Analysis of the rapid response of Arabidopsis to light stress has revealed 111 metabolites that significantly alter in their level during the first 90 sec of light stress exposure. We further show that the levels of free and total glutathione accumulate rapidly during light stress in Arabidopsis and that the accumulation of total glutathione during light stress is dependent on an increase in nitric oxide (NO) levels. We further suggest that the increase in precursors for glutathione biosynthesis could be linked to alterations in photorespiration, and that phosphoenolpyruvate could represent a major energy and carbon source for rapid metabolic responses. Taken together, our analysis could be used as an initial road map for the identification of different pathways that could be used to augment the rapid response of plants to abiotic stress. In addition, it highlights the important role of glutathione in initial stage of light stress response. Light-induced rapid systemic signaling and systemic acquired acclimation (SAA) are thought to play an important role in the response of plants to different abiotic stresses. Although molecular and metabolic responses to light stress have been extensively studied in local leaves, and to a lesser degree in systemic leaves, very little is known about the metabolic responses that occur in the different tissues that connect the local to the systemic leaves. These could be important in defining the specificity of the systemic response as well as in supporting the propagation of different systemic signals, such as the reactive oxygen species (ROS) wave. Here we report that local application of light stress to one rosette leaf resulted in a metabolic response that encompassed local, systemic and transport tissues (tissues that connect the local and systemic tissues), demonstrating a high degree of physical and metabolic continuity between different tissues throughout the plant. We further show that the response of many of the systemically altered metabolites could be associated with the function of the ROS wave, and that the level of eight different metabolites is altered in a similar way in all tissues tested (local, systemic, and transport tissues). These compounds could define a core metabolic signature for light stress that propagates from the local to the systemic leaves. Taken together, our findings suggest that metabolic changes occurring in cells that connect the local and systemic tissues could play an important role in mediating rapid systemic signaling and systemic acquired acclimation to light stress.

Le système de correction des mésappariements (MMR) des eucaryotes est responsable de la correction des erreurs de réplication de l'ADN et intervient dans la recombinaison méiotique. AtPMS1, un gène du MMR chez Arabidopsis thaliana, est impliqué dans la correction des mésappariements, mais son rôle méiotique n’a pas encore été déterminé. Nous avons caractérisé deux lignées mutantes pour le gène AtPMS1. Une lignée présente une morphologie florale aberrante qui est probablement indépendante de l’inactivation d’AtPMS1 mais serait plutôt attribuable à un phénomène de cosuppression. La seconde lignée présente plusieurs phénotypes vraisemblablement causés par l'inactivation d’AtPMS1 : une baisse de fécondité de 65 à 80 %, une ségrégation biaisée de l'allèle mutant et des anomalies cytogénétiques chez 16 % des microsporocytes (fragmentation et ségrégation anormale des chromosomes). Le biais de ségrégation n’a encore jamais été observé chez les autres eucaryotes et semble particulier aux homologues de MutL chez A. thaliana ou chez les plantes. / The eukaryotic DNA mismatch repair system (MMR) is responsible for the repair of replication errors, and plays a role in genetic recombination. AtPMS1, a MMR gene from Arabidopsis thaliana, has already been shown to play a role in DNA repair but its involvement in meiosis has yet to be examined. We characterised two lines of insertional mutants at AtPMS1. A first mutant line has abnormal flowers, which is probably a phenotype unrelated to the loss of AtPMS1 activity but rather caused by cosuppression. The second mutant line shows many phenotypes, likely linked to AtPMS1 inactivation: a 65 to 80% reduction in seed production, an abnormal segregation of the mutant allele and cytogenetic abnormalities in 16% of the microsporocytes (chromosome fragmentation and missegregation). The observed bias in segregation as not been observed in other eukaryotes, and seems to be a particularity of MutL homologues in Arabidopsis or plants as a whole.

Forward genetic screen was performed in Arabidopsis thaliana to isolate novel genes involved in leaf development. The tarani (tni) mutant was selected for further study based on its unique cup-shaped lamina with +ve Gaussian curvature. We show that the larger size of tni leaves is due to rapid growth rate due to excess and prolonged cell division. We monitored the front of the receding cell division zone as a function of time and showed that the shape of the front is more concave compared to wild type, leading to positive curvature. Application of gibberellic acids (GA) synthesis inhibitor rescued the positive curvature of tni suggesting a role for GA in maintaining leaf flatness. Overexpression of cell cycle inhibitor KRP2 also flattened the leaf, confirming a role of cell division. The floral organs and seed are also larger in the tni mutant. Besides growth, tni trichomes are hyper-branched which usually happens when there is more endoreduplication. We found that the nuclei of tni trichomes are larger than wild type nuclei, suggesting increased DNA content. Genetic interaction studies showed that TNI works independent of other trichome branching genes such as with TRYPTICHON and FURCA1. Map-based cloning showed that tni is positioned on left arm of the 3rd chromosome. Using molecular markers, we narrowed down to interval to a 65 kb region, which codes for 19 genes. Sequencing several of them revealed a G→A transition at the 3rd intron - 4th exon junction of At3g20630 gene. RT-PCR analysis showed the presence of an additional full-length transcript with extra un-spliced 3rd intron. Overexpression of this un-spliced variant in wild type plants produced phenotypes like hyperbranched trichomes and cup-shaped leaves; plus additional phenotypes like organ fusion and organ polarity defects. Complementation and allelic tests confirmed that TNI codes for AtUBP14, an ubiquitin protease. The tni plants have longer stem and roots which grow at faster rate compared to wild type. Confocal microscopic analysis of mature embryos showed that both shoot (SAM) and root apical meristems (RAM) of tni plants are larger in size. In RAM, the numbers of quiescent center (QC) cells and stem cells have increased in tni plants. The tni inflorescence and flowers are bigger than wild type in size. Also the degree of axillary shoots has increased in the tni plants. Overexpression of the splice variant of TNI produced undifferentiated callus-like structures in the shoot apex and in hypocotyl. All these phenotypes show that TNI is involved in meristem proliferation. The tni siliques produced many un-fertilized ovules and shrunken and malformed seeds suggesting gametic and/or embryo lethality. We observed that tni embryos were mis-patterned at various stages of development. Following the cell division pattern shows that cells arising from the ‘basal cell’ of the embryo take apical cell fate in tni embryos. The topmost cell of the suspensor, which is also the precursor cell of RAM, is not specified as hypophysial cell in several tni embryos. In the forward genetic screen, we isolated another mutant called tooth (tth), which has deeper serrations at the leaf margin and narrower leaves compared to wild type. It has been mapped to the longer arm of the 2nd chromosome. Genetic interaction studies show that tth is not allelic to other serration mutants such as serrate and mir164a.

[Truncated abstract] Mitochondria play an essential role in diverse metabolic pathways in plants. Their primary roles are the oxidation of organic acids via the tricarboxylic acid cycle and the synthesis of ATP coupled to the transfer of electrons from reduced NAD+ to oxygen via the electron transport chain. Plant mitochondria also contain nonphosphorylating bypasses of the respiratory chain, catalysed by the alternative oxidase (AOX), type II NAD(P)H dehydrogenases (NDHs) and uncoupling proteins (UCPs). Each of these components bypasses energy conservation by either circumventing the formation or utilization of the electrochemical proton gradient, and each is encoded by a small gene family in Arabidopsis. It is proposed that the alterative pathways are likely to be involved in balancing cellular redox and energy status and in minimizing the production of ROS generated by over-reduction of basal respiratory chain components. Furthermore the alternative respiratory pathways are thought to play a role in plant responses to stress. In this study a transcriptomic approach was taken to investigate the role of the alternative respiratory pathways in Arabidopsis, with a focus on elucidating the role and regulation of the AOX gene family. Analysis of the expression of the five AOX genes in Arabidopsis over development and in a range of tissues revealed a unique spatiotemporal expression pattern for each gene. Expression profiling using quantitative RT-PCR, MPSS and microarrays detected an abundance of the AOX1a transcript throughout the plant and over development. The expression patterns of other AOX genes provide insight into their putative roles, AOX1b was expressed predominantly in the flower, AOX1d was particularly abundant in senescing leaves and AOX2 expression was only observed in the seed.

An understanding of the potential roles as lipid mediators of a family of bioactive metabolites called N-acylethanolamines (NAEs) depends on their accurate identification and quantification. The levels of 18C unsaturated NAEs (e.g. NAE18:2, NAE 18:3, etc.) in wild-type seeds (about 2000 ng/g fw) generally decreased by about 80% during germination and post-germinative growth. In addition, results suggest NAE-degradative fatty acid amide hydrolase (FAAH) expression does not play a major role in normal NAE metabolism as previously thought. Seedlings germinated and grown in the presence of abscisic acid (ABA), an endogenous plant hormone, exhibited growth arrest and secondary dormancy, similar to the treatment of seedlings with exogenous N­lauroylethanolamine (NAE12:0). ABA-mediated growth arrest was associated with higher levels of unsaturated NAEs. Overall, these results are consistent with the concept that NAE metabolism is activated during seed germination and suggest that the reduction in unsaturated NAE levels is under strict temporal control and may be a requirement for normal seed germination and post-germinative growth.

Les travaux effectués dans le cadre de cette thèse ont permis de documenter un nouveau système modèle par la caractérisation et l'utilisation à.''Arabidopsis lyrata, une espèce apparentée à la plante modèle Arabidopsis thaliana. L'intérêt de ce nouveau système modèle tient au fait qu'il pourra contribuer à la compréhension de l'évolution des génomes et de la spéciation chez les plantes. Dans cette étude, la stratégie d'un croisement pseudotestcross réciproque {A. lyrata ssp. lyrata x A. lyrata ssp. petraea) a été employée afin de construire une carte génétique pour chacune des sous-espèces. Les marqueurs AFLP se sont avérés efficaces pour révéler la couverture des cartes tfA. lyrata mais ont surtout permis d'augmenter la densité des marqueurs sur les groupes de liaisons. Ensuite, nous avons développé un système alternatif pour l'étude de la polyploïdie chez Arabidopsis. Un processus en deux étapes comprenant un croisement A. thaliana x A. lyrata ssp. petraea suivi d'un événement spontané de doublement chromosomique a permis d'obtenir des allopolyploïdes. Plusieurs phénomènes observés chez ces allopolyploïdes suggèrent la présence d'un choc génomique. Des changements génétiques et épigénétiques ont été décrits avec ce système modèle. A. lyrata possède un ensemble de caractéristiques intéressantes pour devenir un nouvel outil de travail en génétique moléculaire des plantes. / The work carried out within this thesis resulted in the description of a novel model system by characterizing and using Arabidopsis lyrata, a close relative species of the model plant Arabidopsis thaliana. This new model system could contribute to a better understanding of genome evolution and speciation in plants. In this study, a 2-way pseudo-testcross strategy {A. lyrata subsp. lyrata x A. lyrata subsp. petraea) was used to construct a genetic map for each subspecies. The AFLP markers proved to be effective to reveal the coverage of A. lyrata maps, but mostly increased marker density on the linkage groups. Then, we developed an alternative system for the study of polyploidy in Arabidopsis. A two-step process involving the cross A. thaliana x A. lyrata subsp. petraea followed by a spontaneous chromosomal doubling generated allopolyploids. Several phenomena observed with these allopolyploids suggest the presence of genomic shock. Genetic and epigenetic changes were described with this model system. A. lyrata has several interesting characteristics to become a new working tool in plant molecular genetics.

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