Thesis (B.S.) Summa Cum Laude--Butler University, 2009. / Includes bibliographical references (leaves 30-31).

Thesis (Ph. D.)--University of Oregon, 1999. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 87-98). Also available for download via the World Wide Web; free to University of Oregon users. Address: http://wwwlib.umi.com/cr/uoregon/fullcit?p9947979.

The shoot apical meristem (SAM) is the structure that shapes the aerial architecture of the plant, by producing lateral organs throughout development. In the model plant Arabidopsis thaliana, the SAM is always identifiable as a characteristic dome, whether it is found in the centre of a rosette of leaves or at the tip of an inflorescence. When senescence occurs and organogenesis ceases, the now inactive SAM still retains its characteristic appearance and it is never consumed into a terminal structure, such as a flower. Mutant plants that undergo termination represent a valuable tool to understand how the SAM structure and function are maintained during plant life. The aim of this work was to investigate the dynamics of meristem development through morphological and genetic studies of three Arabidopsis mutants that exhibit distinct modes of SAM termination: distorted architecture 1 (dar1), adenosine kinase 1 (adk1) and terminal flower 2 (tfl2). The dar1 mutation is characterised by a severely distorted cellular architecture within the SAM. We propose that dar1 affects the pattern of cell differentiation and/or cell proliferation within the SAM apical dome, resulting in termination by meristem consumption. Instead, the adk1 mutation affects the organogenic potential of the SAM, without altering its structure. The adk1 mutant has increased levels of cytokinins and, as a consequence of this, cell division is enhanced and cell differentiation is prevented in the apex, causing termination by meristem arrest. Finally, tfl2 is mutated in the conserved chromatin remodelling factor HP1, a transcriptional repressor with multiple roles during plant development. The tfl2 SAM terminates by conversion into a floral structure, due to de-repression of floral identity genes. Interestingly, tfl2 mutants also show an altered response to light, an indication that TFL2 might act as a repressor also in the context of light signalling.

Although cottonseed is an important source of oil and fiber, the development of cotton embryos has not been investigated as well as development of cotton fiber. The development of cotton embryos in late heart-stage and early cotyledonary stage is less well investigated than the first 10-14 days after anthesis, or the late stages of embryo development during seed-fill and desiccation. This analysis focused on cotton embryos in the late heart-stage and early cotyledonary stage of development (1.5-4.0 mm or about 13-18 DPA). In vitro analyses are important tools for studying embryos in isolation from the endosperm and fiber and when it is necessary to monitor the developing embryo continuously. The original goal of this work was to develop an in vitro culture method that would support continued development of excised zygotic embryos from the early cotyledonary stage into complete plants with true shoots, i.e. true leaves or visible buds and then to use this method to study aspects of developmental regulation during cotyledonary stage and the transition to later stages. Not all embryos were competent to develop true shoots (an apical bud or a leaf plus a bud) in culture. A number of cultural variables were tested and eliminated. Embryo maturity at the time embryos were excised and the presence or absence of light during the first 14 days of culture affected the competence of immature embryos to developed true shoots. The effect of light was verified in several large replicated experiments. Morphological changes occurring during in vivo development were examined microscopically. The transition from heart-stage to early cotyledonary stage and the development of the first leaf from initials to a large structure were identified. Embryonic shoot apices continued to grow in cultured 1-3 mm embryos. The size and shape of light-treated and dark-treated embryonic apices was compared. A germination test of mature seeds identified seedlings with a similar phenotype occurring at similar rates in seedlings and light-cultured embryos and possible causes were discussed.

Genomic instability underlies diseases of unregulated cell growth that result in cancers and developmental abnormalities in humans. Similar genome destabilizing mechanisms are used to create genetic variety in crops for use in breeding and trait development. Errors that occur during DNA replication may cause mutations if they are not corrected before further cell divisions. DNA mismatch repair (MMR) corrects misinsertions and insertion/deletion DNA loop-outs that arise during DNA replication in plants, animals, prokaryotes, and some archaea, all of which incur mutations at rates 100 to 1,000-fold greater when subjected to inherited or somatic-mismatch repair deficiencies. An understanding of the effects of mismatch repair on somatic and germ-line cells in Arabidopsis thaliana is critical to the development of this plant as a model system for the study of genomic instability. Insertions and deletions of multiples of two base pairs in dinucleotide repeat sequences (microsatellites) occur more frequently in the absence of mismatch repair, and the mismatch-repair status of an individual, tissue, or cell may be inferred on the basis of microsatellite mutation frequency. Single-template PCR analysis measured microsatellite mutation frequencies in leaves and shoot-apical-meristem stem cells, and allowed me to address for the first time an important question: Do plants relax mismatch repair in vegetative tissues relative to meristematic germ-line and floral tissue? Analyses of four microsatellite loci in mismatch repair-deficient and wild type plants surprisingly suggest that there is little difference in mismatch repair activity between leaves and seeds. Mismatch-repair-deficient leaves displayed only two-fold higher microsatellite mutation frequency compared to wild type, and wild-type leaves also displayed a two-fold higher microsatellite mutation frequency compared to shoot-apical- meristems. The high frequency of microsatellite mutation in these wildtype tissues is unexpected, and it suggests that plants relax mismatch repair in differentiated tissues while maintaining genetic fidelity in a small set of stem cells in the shoot apical meristem (SAM). Genome sequencing of msh2⁻/⁻ mutation accumulation A. thaliana lines provides an estimated germ-line mutation rate of 3.9 × 10⁻⁷ in the absence of mismatch repair. Comparison of the rates of base substitution mutation per chromosome in mismatch repair-deficient plants with rates reported for wild-type plants suggests mismatch repair is more efficient on chromosome 5 than on chromosomes 1-4. Bias towards G:C → A:T mutations among transitions is maintained but increased nearly 100-fold in the absence of mismatch repair. / Graduation date: 2012

Die vorliegende Arbeit liefert neue Kenntnisse über das Konkurrenzverhalten der Sprossscheitelschichten bei der Blattmesophyllbildung und trägt dadurch zum Verständnis der Entwicklungsgeschichte höherer Pflanzen bei. Weißbunte Pflanzen von Peperomia serpens SW. LOUD, Sedum rubrotinctum R. T. CLAUSEN, Pedilanthus tithymaloides (L.) POIT. und Plectranthus coleoides BENTH wurden verwendet, um den Bau des Sprossscheitels und die chimärische Natur des Laubblattes zu analysieren. Durch die Untersuchungen zum Bau des Sprossscheitels und zur Blattanatomie wurden die Anzahl initialer Sprossscheitelschichten und die periklinalchimärische Natur der untersuchten Pflanzen bestätigt. Mit Hilfe von Mittelwertvergleichen der Mächtigkeit L2- und L3-bürtiger Mesophyllgewebe wurde die Beteiligung genetisch verschiedener internaler Sprossscheitelschichten an der Bildung des Blattmesophylls bei Sedum rubrotinctum, Pedilanthus tithymaloides und Peperomia serpens erfasst. Die Existenz histogenetisch grüner L2- oder L3-bürtiger Gewebe verursacht eine Zunahme der Blattquerschnittfläche (Sedum rubrotinctum) und eine Vergrößerung der Blattmesophyllhöhe (Peperomia serpens und Pedilanthus tithymaloides). Es wurden Regenerationsversuche an Blattstecklingen der Periklinalchimäre von Peperomia serpens und Sedum rubrotinctum durchgeführt. Durch In-vivo-Provozierung von Adventivsprossen an Blattstücken und achselknospenfreien Sprossen gelang es, die zwei untersuchten heterohistischen Musterpflanzen von Peperomia serpens ('GGW' und 'GWG') in grüne und weiße Nachkommen zu zerlegen. An Blattstecklingen bildeten sich in der Mehrzahl L3-bürtige Regenerate (ca. 75 %). Eine Beteiligung der L2-bürtigen Gewebe bei der Regeneration war an den Blattrandexplantaten zu beobachten. Das L1-bürtige Hypoderm konnte nur in der In-vitro-Blattregeneration deutlich seine Fähigkeit zur Adventivsprossbildung zeigen. Die Blattregenerationsergebnisse bei Peperomia serpens demonstrieren deutlich, dass sich alle drei Sprossscheitelschichten (L1, L2, L3) an der Blattmesophyllbildung beteiligen können. An Blattstecklingen von Periklinalchimären bei Sedum rubrotinctum bildeten sich grüne, weiße und neue chimärische Adventivsprosse. Aus den Regenerationsergebnissen lässt sich die entscheidende Rolle der L2-bürtigen Gewebe bei der Adventivsprossbildung ablesen. Die Regenerationsergebnisse sprechen dafür, dass die Bildung der Adventivsprosse durch die Beteiligung der L2- und L3-bürtigen Gewebe hervorgerufen wurde und die L1-bürtigen Gewebe an der Adventivsprossbildung nicht beteiligt sein konnten. Demzufolge sind tiefer liegende Gewebe (L2- und L3-bürtige) des Laubblattes beider Arten bei der Bildung der Adventivsprosse entscheidender als die L1-bürtige Epidermis. Das Ausmaß der Beteiligung an der Adventivsprossbildung bei Peperomia serpens und Sedum rubrotinctum wird nicht von der genetischen Herkunft (weiß oder grün) des L2- bzw. L3-bürtigen Gewebes gesteuert, sondern durch die Lage und damit durch die Abstammung der Gewebe aus der entsprechenden Sprossscheitelschicht bestimmt. Die abschließenden Untersuchungen an Plectranthus coleoides, dessen Chlorophyll- und Ploidiechimären quantitativ analysiert wurden, verdeutlichen die Erkenntnisse über die Beteiligung der Sprossscheitelschichten an der Bildung des Blattmesophylls. Es wurde deutlich, dass die Gewebekonkurrenz im Beisein einer doppelten Markierung nicht lagebedingt sein kann, sondern aufgrund verschiedener Ploidiestufen stattfindet. / The studies presented in this thesis provide new insights into the competitive reaction of the shoot apical layers during the foliar mesophyll formation and thus contribute to understanding of plant development. The variegated plants of Peperomia serpens SW. LOUD, Sedum rubrotinctum R. T. CLAUSEN, Pedilanthus tithymaloides (L.) POIT. and Plectranhus coleoides BENTH were used to analyse the cellular organisation of shoot apex and the histogenetic constitution of the leaf. Shoot apex and leaves structural analyses confirm the number of initial shoot apical layers and the periclinal chimeric nature of investigated plants. Quantitative analysis of foliar mesophyll of Sedum rubrotinctum, Peperomia serpens and Pedilanthus tithymaloides have been used to deduce patterns of meristem layers intercellular interaction during mesophyll formation. The expression of the histogenetic green meristem layer (L2 or L3) causes a increase of "mesophyll area" (Sedum rubrotinctum) and a enlargement of "mesophyll height" (Peperomia serpens and Pedilanthus tithymaloides) in leaves. Four periclinal chimeric forms of Peperomia serpens ('GGW' and 'GWG') and of Sedum rubrotinctum ('GGW' and 'GWG'), each of which possesses normal green cell layers but a genetically different chlorophyll-deficient cell layer, were utilized to study the effect of genotype on the ability of the cell layers of in vivo and in vitro leaf cutting to regenerate adventitious shoots and to analyse the competition between apical layers and their derivatives in the plant ontogeny. Among the in vivo adventitious shoots of the leaf cuttings and leaf of Peperomia serpens, shoots were green, white and variegated. The L3-derived cell layer is alone responsible for the formation of ca. 75 % of adventitious shoots. The relative significant contribution of L2-derived cell layers to mesophyll formation increases in margin of leaf. The L1-derived hypoderm in foliar mesophyll of Peperomia serpens were apparently incapable of shoot regeneration of in vivo leaf cutting, yet in both periclinal forms clearly produced green shoots in vitro. Results demonstrate that all initial apical meristem layers in Peperomia serpens can contribute with different ability to foliar mesophyll formation. Adventitious shoots were in vivo induced on leaf of periclinal chimeric plants of Sedum rubrotinctum. Plants derived from leaf culture were three types: green, white and variegated. Among the adventitious shoots of green- and white-margined leaf of Sedum rubrotinctum, most adventitious shoots (ca. 90 %) were L2-derived, a few were L3-derived. Results demonstrate that the L1 derivatives can not contribute to foliar mesophyll formation. According to these results the internal tissues (L2- and L3-derived cell layers) of leaf are more competitive than the epidermis. The lineage of adventitious shoot is not controlled by the genetic origin of L2- and L3-derived tissues, but by the position of these derived tissues according to the shoot apical meristem layer. The last experiments on Plectranthus coleoides which have combined quantitative analysis of variegated- leaf chimeras with quantitative analysis of cytochimeras have begun to shed more light on the contribution of apical meristem layers to foliar mesophyll formation. It has revealed how the ploidy degree of apical layers derivatives in a cytochimera control leaf cell fate more than their position in the meristem.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Eugenia involucrata DC. and Handroanthus chrysotrichus (Mart. ex DC) Mattos are native species with economic, ecological and silvicultural. They have problems with their spread via seeds, because they are recalcitrant and lose viability within a few weeks after collection. In addition, there is little information about the propagation of these species. Therefore, the general objective of this study was to evaluate methodologies which may contribute to micropropagation of E. involucrata and H. chrysotrichus. For multiplication of E. involucrata were tested different sources (BAP, KIN, 2iP and TDZ) and concentrations (0, 16 and 32 μM) of cytokinins added to the nutrient medium MS/2. Also, we tested the effect of TDZ (16 or 32 μM) combined with GA3 (0; 10; 20 and 40 μM) added in medium MS/2, on in vitro multiplication of nodal segments. Even for E. involucrata, we examined the effect of NAA (0; 0,5 and 1 μM) combined with TDZ (0; 16 and 32 μM), added to the medium MS/2. Finally, for E. involucrata, we tested the effect of different concentrations of TDZ (0; 16 and 32 μM) added to the nutrient medium MS/2, on the multiplication of shoot apices of seedlings germinated in vitro. For H. chrysotrichus were tested different types of explants (shoot apical segments and epicotyl) and nutrient media (WPM, MS, WPM/2 and MS/2) in the presence and absence of activated charcoal as well as the effect of BAP (0; 2; 4; 8 and 16 μM) on in vitro multiplication of this species. For in vitro multiplication of nodal segments of E. involucrata the cytokinins tested are dispensable; cytokinins favor the growth of bacterial contamination, high levels of contamination reduce in vitro survival and establishment of nodal segments; the in vitro establishment for this species is impaired by the occurrence of leaf chlorosis and swelling of the explants. As for H. chrysotrichus, the shoot apical segments show the highest development in the in vitro survival and establishment than of epicotyl ; there is a high in vitro swelling of the explants in the absence of growth regulators; in the in vitro establishment in medium WPM/2 in the presence of 0,05 μM shoot apical segments are more efficient under low concentrations of BAP; there isgreater calli formation in epicotyl with increase the concentration of BAP, the opposite was true for the shoot apical segment. / Eugenia involucrata DC. e Handroanthus chrysotrichus (Mart. ex DC) Mattos são espécies florestais nativas com importância econômica, silvicultural e ecológica. Possuem problemas quanto a sua propagação via sementes, pois estas são recalcitrantes, perdendo a viabilidade em poucas semanas após a coleta. Além disso, existem poucas informações sobre a propagação vegetativa dessas espécies. Considerado o exposto, o objetivo geral do presente estudo foi avaliar metodologias que possam contribuir para a micropropagação in vitro de E. involucrata e de H. chrysotrichus. Para a multiplicação de E. involucrata foram testadas diferentes fontes (BAP, CIN, TDZ e 2iP) e concentrações (0; 16 e 32 μM) de citocininas adicionadas ao meio nutritivo MS/2. Também foi testado o efeito de TDZ (16 e 32 μM) combinado a GA3 (0; 10; 20 e 40 μM), acrescentados em meio MS/2, na multiplicação in vitro de segmentos nodais. Ainda para E. involucrata, foi avaliado o efeito de ANA (0; 0,5 e 1 μM) combinado a TDZ (0; 16 e 32 μM), acrescidos ao meio MS/2. Por fim, para esta espécie, testou-se o efeito de diferentes concentrações de TDZ (0; 16 e 32 μM) adicionadas ao meio MS/2, na multiplicação de segmentos apicais caulinares obtidos de plântulas germinadas in vitro. Para H. chrysotrichus, foram testados diferentes tipos de explantes (segmento apical caulinar e epicótilo) e meios nutritivos (WPM, MS, WPM/2 e MS/2), na presença e ausência de carvão ativado, bem como o efeito de BAP (0; 2; 4; 8 e 16 μM) na multiplicação in vitro dessa espécie. Para multiplicação in vitro de segmentos nodais de E. involucrata, as citocininas testadas são dispensáveis, uma vez que favorecem o crescimento de contaminação bacteriana. Elevados índices de contaminação acarretam reduzida sobrevivência e estabelecimento in vitro de segmentos nodais; o estabelecimento in vitro para essa espécie é comprometido pela ocorrência de clorose foliar e intumescimento dos explantes. Já para H. chrysotrichus, o segmento apical caulinar apresenta desenvolvimento superior ao do epicótilo na sobrevivência e no estabelecimento in vitro; há elevado intumescimento in vitro dos explantes na ausência de fitorreguladores. No estabelecimento in vitro em meio WPM/2 na presença de 0,05 μM de ANA, os segmentos apicais caulinares são mais eficientes sob concentrações reduzidas de BAP; há maior calogênese em epicótilos à medida que aumenta a concentração de BAP, ocorrendo o contrário para o segmento apical caulinar.

Le contrôle moléculaire du patterning au cours des processus développementaux est aujourd’hui bien décrit chez les organismes multicellulaires. A l’inverse, la contribution de la croissance dans l’émergence des patterns reste peu explorée, et est souvent réduite à un rôle passif. Au cours de cette thèse, j’ai étudié cette question en utilisant le méristème apical caulinaire (MAC) d’Arabidopsis comme modèle. Le méristème est un groupe de cellules en divisions situé à l’extrémité de toutes les tiges et les branches et qui génère tous les organes aériens de la plante selon un patron stéréotypé, aussi appelé phyllotaxie. Dans une première partie, j’ai étudié comment la croissance de la tige pouvait influencer le patron phyllotactique. Plus précisément, en découplant dépôt de la cellulose dans la paroi et l’orientation des microtubules, j’ai montré que le patron de phyllotaxie devenait bimodal en raison de l’induction d’une torsion lors de la croissance de la tige. Dans une seconde partie, j’ai analysé le lien entre forme du MAC et expression génétique. En particulier, j’ai pu corréler l’expression d’un gène maître : SHOOTMERISTEM LESS (STM) au degré de courbure dans le MAC. De plus, en utilisant des approches de micromécaniques, j’ai aussi pu montrer que l’expression de STM pouvait être induite par le patron de contraintes localement généré par la courbure. Pour finir, j’ai aussi étudié comment la taille du méristème influence la robustesse du pattern de phyllotaxie sur la tige en modulant la fréquence d’initiation des organes. L’ensemble de ce travail met ainsi en avant le rôle de la croissance dans le patterning, notamment via des mécanismes de rétrocontrôles géométriques et mécaniques. / The molecular mechanisms behind the emergence of patterns during developmental processes have been well described in multicellular organisms. However, the contribution of growth in patterning is still poorly understood; growth is often seen as a passive output of the activity of the patterning signals. In this PhD, I have studied the relation between growth and patterning using the shoot apical meristem of Arabidopsis as a model system. The meristem is a group of dividing cells located at the tip of every stems and branches that generates all the aerial organs of the plant following a typical spatio-temporal pattern also called phyllotaxis. In a first part, the influence of post-meristematic growth on phyllotaxis was assessed. More precisely, by uncoupling cellulose deposition from the orientation of the microtubule array, I showed that the resulting stem torsion induces the emergence of a new and robust bimodal phyllotactic pattern. In a second part, the relation between meristem shape and gene expression was analyzed. More precisely, I correlated the expression of a master regulatory gene: SHOOT MERISTEMLESS (STM) to tissue curvature in the boundary domain that separates the emerging organ from the meristem. Furthermore, I showed that STM expression can be induced by micromechanical perturbations thus suggesting that shape-derived mechanical stresses in the meristem boundary contribute to STM expression. Finally, I also studied how meristem size can influence the robustness of the pattern of phyllotaxis along the stem through a modulation of the frequency of organ initiation. Altogether, this work highlights the important contribution of growth in patterning, notably thanks to the existence of geometrical and mechanical feedbacks.

No description available.

Among the major phytohormones, the cytokinin exhibits unique features for its ability to positively affect the developmental status of plastids. Even early on in its research, cytokinins were known to promote plastid differentiation and to reduce the loss of chlorophyll in detached leaves. Since the discovery of the components of cytokinin perception and primary signaling, the genes involved in photosynthesis and plastid differentiation have been identified as those directly targeted by type-B response regulators. Furthermore, cytokinins are known to modulate versatile cellular processes such as promoting the division and differentiation of cells and, in concert with auxin, initiating the de novo formation of shoot apical meristem (SAM) in tissue cultures. Yet how cytokinins precisely participate in such diverse cellular phenomena, and how the associated cellular processes are coordinated as a whole, remains unclear. A plausible presumption that would account for the coordinated gene expression is the tight and reciprocal communication between the nucleus and plastid. The fact that cytokinins affect plastid developmental status via gene expression in both the nucleus and plastid is interpreted here to suggest that cytokinin functions as an initiator of anterograde (nucleus-to-plastid) signaling. Based on this viewpoint, we first summarize the physiological relevance of cytokinins to the coordination of plastid differentiation with de novo shoot organogenesis in tissue culture systems. Next, the role of endogenous cytokinins in influencing plastid differentiation within the SAM of intact plants is discussed. Finally, a presumed plastid-derived signal in response to cytokinins for coupled nuclear gene expression is proposed.