L'auxine régule la croissance et le développement des plantes grâce aux facteurs de transcription de la famille des "AUXIN RESPONSE FACTOR" (ARF). Chez Arabidopsis thaliana en particulier, ARF5, 6, 7, 8 et 19 activent l'expression de gènes cibles en réponse à l'auxine. Ces cinq ARF activateurs contrôlent de façon plus ou moins redondante des processus divers au cours du développement de la plante, notamment la régulation des croissances au niveau des méristèmes racinaires et aériens ainsi que la formation des racines latérales ou des méristèmes axillaires.Chacun de ces cinq ARF activateurs présente des patrons d'expression uniques dans chacun des tissus racinaires et aériens, en association avec leurs fonctions particulières. Il est probable que cette expression tissu-spécifique trouve son origine dans un contrôle différencié de leur transcription. Dans cette étude, des régulateurs amonts de la transcription de ARF5, 6, 7, 8 et 19 ont été identifiés par une méthode haut-débit de crible simple hybride en levure (Y1H). Une procédure d'expression transitoire en protoplastes a permis de confirmer que l'expression de chaque ARF activateur est contrôlée par des régulateurs spécifiques, dont la majorité se comportent comme des répresseurs de la transcription des ARF in planta. Parmi les régulateurs identifiés, les facteurs de transcription ont été étudiés grâce à des mutants pour préciser les interactions in planta. Ces mutants montrent des phénotypes développementaux typiques de perturbations de l'auxine dans les racines et les tiges : altérations des cinétiques de croissance, de l'émergence des organes latéraux ou de réponses à l'auxine et modification de l'expression des ARF activateurs.Par ailleurs, ce travail aborde également les dialogues entre les voies de signalisation de l'auxine et des cytokinines, et en particulier le rôle de ces interactions dans le développement des racines et des tiges. Une des interactions identifiées dans le crible Y1H est la répression de ARF7 par CRF10, un gène membre de la famille des "Cytokinin Response Factors". Nous avons mis en évidence l'importance de cette interaction pour le maintien de l'architecture du méristème apical racinaire, pour la sénescence des feuille et pour la réponse phototropique à la lumière bleue dans les hypocotyles. / Auxin regulates plant growth and development through the transcription factors of the AUXIN RESPONSE FACTOR (ARF) gene family. Most notably in Arabidopsis thaliana ARF5, 6, 7, 8 and 19 activate expression of target genes in response to auxin. These five ARF activators control both variable and overlapping processes during plant development including regulation of growth at the root and the shoot apical meristems, lateral root and axillary shoot formation. Each of the five ARF activators shows unique tissue-specific expression patterns in the root and the shoot associated with their distinct functions. This tissue-specific expression is likely derived from the differences in the control of ARF activator transcription. In this study the upstream regulators of ARF5, 6, 7, 8 and 19 transcription were identified. This was achieved by utilizing a high-throughput yeast one-hybrid (Y1H) method. The transient protoplast assay revealed that each ARF activator is controlled by specific transcriptional regulators and that the majority of these regulators are repressors of ARF transcription in planta. Mutants of the regulatory transcription factors were utilized to additionally investigate the interactions in planta. These mutants display auxin-related developmental phenotypes in the root and the shoot including alternations in growth kinetics, emergence of lateral organs, responses to auxin and altered expression of ARF activators. Furthermore, this study additionally focuses on cross-talk between the auxin and cytokinin signaling pathways and its role in root and shoot development. One of the interactions identified in the Y1H screen is a repression of ARF7 by CRF10, a member of the Cytokinin Response Factors gene family. The importance of this interaction in maintaining architecture of the root apical meristem, in leaf senescence and in the phototropic response to blue light in hypocotyls is studied.

Made available in DSpace on 2015-03-26T13:39:57Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1667701 bytes, checksum: 773fc3ac273d03aa28a80662f100d0a8 (MD5) Previous issue date: 2013-03-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The present research is about flowering control of chrysanthemum (Dendranthema grandiflora), variety "Eliot" in the greenhouse, by th technique called "night break" with artificial lighting using light bulb incandescent 100 Watt, LED 3 and 1 Watt power with different lighting cycles in pot chrysanthemum. The experimental design was randomized blocks with the following treatments: incandescent light cycles in 10 minutes of light for every 20 minutes of dark, light for 20 minutes every 10 minutes of dark and continuous illumination in the period from 22 to 02 hours. 3 Watt LED light cycles in 10 minutes of light to dark every 20 minutes, 20 minutes of light for every 10 minutes of dark and continuous illumination in the period from 22 to 02 hours. 1 Watt LED light cycles in 10 minutes of light to dark every 20 minutes, 20 minutes of light for every 10 minutes of dark and continuous illumination in the period from 22 to 02 hours. Finally no artificial lighting, totaling 10 treatments, each plot consisting of 3 pots. At the end of treatment the shoot apical meristem were collected and analyzed by anatomical longitudinal cuts measuring the follow features: first and second transversal length, meristem height, width and number of cells across the pith meristem and the number of cells with dense cytoplasm in the meristematic apex. At the peak of bloom the pots were analyzed and measured, plant height, average diameter of the plant in the pot, number of stems per pot, number of nodes per stem, length of the 3rd internode, number of inflorescences, dry mass of stem, leaf, flower and matter air. The results showed that the LED lighting keeps the characteristics of incandescent lighting getting quality plants and within the standards of marketing, but with reduced power consumption and improved durability reducing production costs. / A presente pesquisa trata sobre o controle do florescimento de crisântemo (Dendranthema grandiflora), variedade ―Eliot‖ em casa de vegetação, pela técnica chamada ―noite interrompida‖ com iluminação artificial utilizando lâmpada incandescente de 100 Watts, lâmpada LED de 3 Watts e 1 Watt de potência com diferentes ciclos de iluminação em crisântemo de vaso. O delineamento experimental utilizado foi em blocos casualizados com os seguintes tratamentos: lâmpada incandescente nos ciclos de iluminação 10 minutos de luz para cada 20 minutos de escuro, 20 minutos de luz para cada 10 minutos de escuro e iluminação contínua no período das 22 às 02 horas. Lâmpada LED 3 Watts nos ciclos de iluminação 10 minutos de luz para cada 20 minutos de escuro, 20 minutos de luz para cada 10 minutos de escuro e iluminação contínua no período das 22 às 02 horas. Lâmpada LED 1 Watt nos ciclos de iluminação 10 minutos de luz para cada 20 minutos de escuro, 20 minutos de luz para cada 10 minutos de escuro e iluminação contínua no período das 22 às 02 horas. E por fim sem iluminação artificial, totalizando 10 tratamentos, sendo cada parcela constituída por 3 vasos. Ao final do experimento do controle do florescimento foram coletados os meristemas apicais das hastes e analisadas através de cortes anatômicos longitudinais as características, comprimento transversal 1 e 2, altura do meristema, largura e número de células transversais do meristema medular e número de células com citoplasma denso do ápice meristemático. No auge do florescimento analisou-se altura de planta, diâmetro médio da planta no vaso, número de hastes por vaso, número de nós por haste, comprimento do 3º entrenó, número de inflorescências, massas seca de caule, folha, inflorescência e massa seca da parte aérea. Os resultados obtidos mostraram que a iluminação LED se equipara as características da iluminação incandescente obtendo plantas de qualidade e dentro dos padrões de comercialização, mas com redução no consumo de energia elétrica e maior durabilidade reduzindo assim os custos de produção.

La phyllotaxie, arrangement d’éléments botaniques autour de l’axe primaire de la plante, suit unpatron spatio-temporel robuste. Elle est établie au niveau du Méristème Apical Caulinaire (MAC),qui est la niche de cellules souches post-embryonnaires des parties aériennes de la plante.L’accumulation locale de la phytohormone auxine déclenche la formation des organes latéraux auniveau du MAC. En même temps, la déplétion d’auxine dans les cellules environnantes crée unchamp inhibiteur, où toute nouvelle organogenèse est impossible. La croissance permet auxanciens organes de constamment s’éloigner du MAC, ce qui libère l’espace nécessaire auxnouvelles organogenèses. C’est un exemple frappant de processus biologique auto-organisé etitératif. Des mécanismes moléculaires et génétiques régulant la phyllotaxie ont été identifiés,majoritairement dans le contexte d’une phyllotaxie commune, qu’est la spirale de Fibonacci. Iln’est pas actuellement démontré que ces mécanismes expliquent aussi d’autres types dephyllotaxies. Nous avons identifié DRB27, un mutant d’Arabidopsis thaliana présentant une fortetendance à générer des pseudo-verticilles. Cela rappelle la phyllotaxie observée chez presquetoutes les fleurs et quelques tiges d’espèces non parentes. La quantification de la phyllotaxie deDRB27 ainsi que des expériences d’imagerie in vivo, ont révélé que ces groupes d’organes ne sontpas des verticilles, mais correspondent à des éruptions d’organogenèses se produisant en croissantà la périphérie du MAC quand, à l’inverse, de larges domaines restent inactifs. Ces observationsvont à l’encontre des règles classiques de positionnement d’organes lors de la mise en place de laphyllotaxie. De façon surprenante, nous avons identifié deux mutations candidates affectant lesrégulateurs abaxiaux FILAMENTOUS FLOWER et MIR166A, potentiellement toutes deuximpliquées dans le phénotype phyllotactique de DRB27. Alors que ces gènes sont exprimés dansles organes en développement, cela suggère des retours d’information sur la phyllotaxie, de façonnon autonome. Nous avons identifié des séries d’anomalies dans le MAC de DRB27, incluant despatrons de signalisation d’auxine anormaux, la perturbation des frontières entre les organes et leméristème, la modification des domaines WUS-CLV3 et de la géométrie du MAC, ainsi qu’unemodification de la dureté de la surface du méristème. Toutes ces données suggèrent que l’identitéet le développement des organes latéraux agissent sur l’homéostasie du MAC et l’établissement dupatron phyllotactique. / Phyllotaxis, the arrangement of botanical elements around plant axis,conforms to a robust spacial-temporal pattern. It is primarily established atthe shoot apical meristem (SAM), the post-embryonic aerial stem-cellniche. Local accumulation of the phytohormone auxin locally triggersorgan formation at the SAM, while depletion of auxin in the surroundingcells creates an inhibitory field, where no new organ can be initiated.Growth constantly moves older organs away from the SAM, clearingspace for new organogenesis. This is a striking example of an iterative and self-organized process driven by inhibitory fields. Molecular and geneticmechanisms regulating phyllotaxis are now being identified, but mostly inthe context of the most common Fibonacci spiral. Whether or not thesame mechanisms explain other types of phyllotaxis remains to beexplored. We identified DRB27, an Arabidopsis thaliana mutant with astrong tendency to generate clusters of organs. This is reminiscent of thewhorled phyllotaxis, observed in almost all angiosperm flowers and insome shoots of unrelated species. Quantification of DRB27 phyllotaxisand live imaging revealed that clusters are not whorls but correspond toburst of organs initiating in crescent domains at the periphery of the SAM.Conversely, large crescent domains remain devoid of organ initiation.Organogenesis in these clusters violates classical rules of organ spacing inphyllotactic systems. Surprisingly, we identified two candidate mutationsaffecting the two abaxial regulators FILAMENTOUS FLOWER andMIR166A, which likely combines to produce DRB27 peculiar phyllotaxis.Since these genes are expressed in developing organs, it suggests non-cellautonomous feedbacks on phyllotaxis. We identify a series of anomaliesin DRB27 SAM, including abnormal patterns of auxin signalling,perturbation of organ boundary formation, modification of CLV3/WUSdomains and SAM geometry and increase in cell wall stiffness. Takentogether our data questions how lateral organ identity and developmentfeedbacks on SAM homeostasis and phyllotaxis patterning.

Dans l’optique d’une seconde Révolution Verte, visant, à la différence de la première, à accroître les rendements des cultures dans un contexte de faible fertilité, les stratégies mises en place par les plantes pour une assimilation optimale des nutriments du sol se trouvent au cœur du problème. Afin de le résoudre et d’identifier les variétés idéales parmi la diversité génétique des plantes cultivées, les systèmes racinaires, leur développement et leur architecture, sont appelés à jouer le premier rôle. La variabilité au sein des racines latérales semble s’avérer une caractéristique cruciale pour l’optimisation de l’exploration du sol et de l’acquisition de ses ressources mobiles et immobiles, mais ce phénomène est encore mal appréhendé.Le travail présenté dans cette thèse se concentre sur les racines latérales du maïs (Zea mays L.) dans un effort pour révéler les processus à l’origine des variations intrinsèques dans le développement racinaire. Il s’appuie en particulier sur le phénotypage des racines latérales à une échelle sans précédent, suivant la croissance journalière de milliers d’entre elles à haute résolution spatiale, pour caractériser précisément les variations spatio-temporelles entre et au sein des individus racinaires. Les profils individuels de vitesse de croissance ont été analysés à l’aide d’un modèle statistique qui a identifié trois principales tendances temporelles dans les vitesses de croissance menant à la définition de trois classes de racines latérales avec une vitesse et durée de croissance distinctes. Des différences de diamètre à l’émergence de ces racines (dont l’origine remonte au stade du primordium) conditionnent probablement la tendance ultérieur de croissance mais ne suffisent pas à déterminer le destin de la racine. Finalement, ces classes racinaires sont distribuées aléatoirement le long de la racine primaire, ce qui suggère qu’aucune stimulation ou inhibition locale n’existe entre racines voisines.Pour expliquer l’origine des variations observées dans la croissance, ce travail a été complété par une caractérisation multi-échelle de groupes de racines latérales présentant une croissance distincte, à un niveau cellulaire, anatomique et moléculaire. Un effort particulier a été dirigé à l’analyse des profils de longueur de cellules dans des apex racinaires pour lequel nous avons introduit un modèle de segmentation pour identifier des zones développementales. Grâce à cette méthode, une forte modulation dans la longueur des zones de division et d’élongation a été mise en évidence, en lien avec les variations de la croissance des racines latérales. Le rôle régulateur de l’auxine sur l'équilibre entre les processus de prolifération et d’élongation cellulaire a été montré avec l’utilisation de lignées mutantes. En fin de compte, les variations de la croissance entre racines latérales sont remontées jusqu’à l’allocation d’assimilats carbonés et la capacité de transport de la racine, ce qui suggère l’existence d’un mécanisme de rétroaction qui pourrait jouer un rôle déterminant dans la mise en place de tendances contrastées dans la croissance des racines latérales. / In the perspective of a second Green Revolution, aiming, unlike the first one, to enhance yields of crops in a low fertility context, the strategies used by plants for an optimal uptake of soil nutrients are at the core of the problem. To solve it and identify ideal breeds among the genetic diversity of crops, plant root systems, their development and their architecture, are called upon to play the leading role. The variability among secondary roots appears as a crucial feature for the optimality of soil exploration and acquisition of mobile and immobile resources, but this phenomenon remains poorly understood. The work presented in this thesis focuses on the lateral roots of maize (Zea mays L.) and attempts to unravel the processes at the origin of intrinsic variations in lateral root development. It relies notably on the phenotyping of individual lateral roots at an unprecedented scale, tracking the daily growth of thousands of them at a high spatial resolution, in order to characterize precisely the spatio-temporal variations existing both between and within root individuals. Individual growth rate profiles were analyzed with a statistical model that identified three main temporal trends in growth rates leading to the definition of three lateral root classes with contrasted growth rates and growth duration. Differences in lateral root diameter at root emergence (originating at the primordium stage) were likely to condition the followed growth trend but did not seem enough to entirely determine lateral root fate. Lastly, these lateral root classes were randomly distributed along the primary root, suggesting that there is no local inhibition or stimulation between neighbouring lateral roots. In order to explain the origin of the observed differences in growth behaviour, we complemented our study with a multi-scale characterization of groups of lateral roots with contrasted growth at a cellular, anatomical and molecular level. A particular focus is set on the analysis of cell length profiles in lateral root apices for which we introduced a segmentation model to identify developmental zones. Using this method, we evidenced strong modulations in the length of the division and elongation zones that could be closely related to variations in lateral root growth. The regulatory role of auxin on the balance between cellular proliferation and elongation processes is demonstrated through the analysis of mutant lines. Ultimately, variations in lateral root growth are traced back to the allocation of carbon assimilates and the transport capacity of the root, suggesting that a feedback control loop mechanism could play a determinant role in the setting out of contrasted lateral root growth trends.

Proteínas que se ligam a RNAs geralmente regulam estabilidade, localização e tradução de mensageiros por interação com seqüências específicas na região 3 não traduzida. A proteína PUMILIO foi descrita pela primeira vez em Drosophila, apresentando a função de controlar a tradução de mensageiros alvo durante o desenvolvimento. Homólogos podem ser encontrados em outras espécies filogeneticamente distantes com funções similares. O seqüenciamento do genoma de Arabidopsis thaliana indicou a presença de quatro genes que codificam homólogos ao PUMILIO de Drosophila. Três deles estão no cromossomo II (APUM-1, 2 e 3), com alto grau de identidade e situados muito próximos uns dos outros. A terceira cópia está no cromossomo IV (APUM-4) e sua seqüência tem menor similaridade em relação aos três anteriores. Neste trabalho, foi clonado e caracterizado APUM-2. Ensaios de northern blot e RT-PCR indicaram que o mensageiro de Apum-2 pode ser encontrado em amostras de raiz, caule, folha, flores e frutos. Entretanto, sistemas repórteres, APUM-2::GUS e APUM-2::GFP, foram introduzidos no vegetal e a expressão foi observada nos ápices do caule e raízes, especificamente nas regiões meristemáticas. Também foram feitas construção para ensaios de genética reversa e as plantas contendo construções constitutivas (35S::APUM-2) se desenvolveram sem dominância apical e com grande quantidade de ramos, folhas e raízes secundárias, mostrando perturbações nos meristemas. Os resultados obtidos sugerem que APUM-2 possui papel relevante durante o desenvolvimento meristemático, possivelmente através de interações com mRNAs. / RNA-binding proteins often regulate the stability, localization, and translation of mRNAs by interaction with specific motifs in the 3-UTR. The PUMILIO protein from Drosophila was shown to control translation of specific mRNAs during development. PUMILIO homologs were found in several species and constitute the PUF family. The Arabidopsis thaliana sequencing project revealed four genes homolougs to PUMILIO. Three are situated in chromosome II (APUM-1, 2, and 3) that are nearly identical among themselves, and that contain a region that is 52% homologous to the PUMILIO. RNA-binding domain. The fourth is in chromosome IV (APUM-4), and shows low similarity to the other three. In this work, we characterized APUM-2 in further detail. Northern blots indicated that Apum-2 is expressed in shoot and root apices. Reporter transgenic plants were made that contained either APUM-2::GUS or Apum2::GFP constructs. Reporter gene expression confirmed that APUM-2 is active in shoot and root apices, specifically in the meristematic regions. Finally transgenic plants containing the 35S::APUM-2 construct were created. Constitutive APUM-2 expression resulted in plants with no apical dominance and a high number of leaves, stems and lateral roots. These results suggest that APUM-2 plays an important role during meristem development, possibly through a mRNA interaction.

La protéine kinase TOR (Target Of Rapamycin) a été identifiée chez la levure et les mammifères comme participant à deux complexes protéiques qui servent de carrefour entre la perception des facteurs endogènes et exogènes et la stimulation de la croissance cellulaire. Depuis la découverte de la kinase AtTOR chez Arabidopsis thaliana, des études ont été menées afin de mieux caractériser son rôle chez les plantes et l’influence de son niveau d’expression sur la régulation du métabolisme et du développement.Au cours de ce travail, j’ai contribué à l’étude de cette kinase en étudiant l’influence de l’inactivation de TOR sur la composition du ribosome au niveau protéique et sur le niveau de phosphorylation de ces protéines, ainsi que sur l’organisation du méristème au niveau moléculaire et cytologique Au cours de cette étude, j’ai montré que certaines protéines constitutives du ribosome pourraient être des cibles de l’activité TOR au niveau de leur abondance et/ou de leur état de phosphorylation. Ainsi, l’inactivation de TOR entraine une diminution du niveau de phosphorylation des protéines RPS6 et pourrait influencer l’abondance des protéines acides constitutives du stalk ribosomal, une structure importante dans la régulation de la traduction. Les résultats obtenus suggèrent également que l’activité TOR est nécessaire au maintien du méristème à l’état fonctionnel en régulant les voies importantes contrôlant la division et la différentiation au sein de cette structure. / The TOR (Target Of Rapamycin) kinase has first been identified in yeast and mammals as being part of two different protein complexes that are implicated in the stimulation of cell growth in response to endogenous and exogenous stimuli. Since the discovery of this kinase in Arabidopsis, some studies have been led to characterize its role in plants and the influence of its expression level on the metabolism and development regulation.In this study, I worked on the influence of the TOR inactivation on the composition of the ribosome on its protein composition and on the phosphorylation status of these proteins and also on the organisation of the meristem at a molecular and cellular level.Regarding to the results I have obtained, I showed that TOR may regulate the abundance and/or the phosphorylation status of some proteins involved in the ribosome composition. Hence, TOR inactivation leads to a decrease of the phosphorylation level of RPS6 proteins and could regulate the abundance of acid proteins constitutive of the ribosomal stalk, a structure important for the translation regulation. The results obtained also suggest that TOR activity may be necessary to keep the meristem functional by the regulation of the main important pathways controlling division and differentiation in that structure.

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.

Muitas plantas apresentam crescimento indeterminado e são capazes de produzir novos órgãos e tecidos ao longo de todo seu ciclo de vida. Essa capacidade é devida parcialmente à expressão altamente regulada de genes específicos, tais como os genes SQUAMOSA Promoter-Binding Protein-Like (SPLs). SPLs codificam fatores de transcrição específicos de plantas que desempenham papéis importantes em diferentes aspectos do desenvolvimento, tais como mudança de fase juvenil-adulto, definição da arquitetura da planta e amadurecimento do fruto. A maioria dos genes SPLs são pós-transcricionalmente regulados pelo microRNA (miRNA) miR156. Apesar de alguns aspectos regulados pelas SPLs serem bem estudados, suas funções moleculares durante o desenvolvimento do fruto são pouco compreendidas. Neste trabalho, nós geramos 22 eventos transgênicos de Solanum lycopersicum cv. Micro-Tom (MT) superexpressando o precursor AtMIR156b. Plantas transgênicas exibiram morfologia foliar e floral anormal além de alteração na arquitetura vegetal. Interessantemente, a maioria dos eventos apresentou frutos de crescimento indeterminado, os quais não apresentam sementes sendo caracterizados pelo crescimento de frutos secundários além da presença de estruturas vegetativas e meristemas florais ectópicos. Fazendo uso da técnica de RT-qPCR, nós encontramos uma robusta correlação entre expressão do miR156 e o fenótipo dos frutos, sugerindo que esta rota regula o desenvolvimento e determinação do fruto de tomateiro. O mutante de tomateiro Mouse ears (Me) apresenta um fraco nível de indeterminação no fruto, sendo que os níveis do transcrito miR156 maduro são maiores no fruto mutante quando comparado aos controles MT, mas significativamente menores que nos frutos transgênicos. Oito SPLs de tomateiro foram silenciadas em diferentes níveis em frutos de diferentes eventos transgênicos e no mutante Me. O fator de transcrição do tipo MADS-box MACROCALYX (MC), o qual é ortólogo ao gene APETALA1 (AP1) de arabidopsis (alvo direto in vivo da proteína SPL3), afeta a determinação da inflorescência e o desenvolvimento das sépalas. A expressão de MC foi severamente reduzida nos frutos dos eventos transgênicos, mas não no mutante Me. Este dado sugere que a desregulação da expressão de MC deve ser responsável pelo forte fenótipo de indeterminação do fruto observados nos eventos transgênicos. Tomados em conjunto, nossos dados sugerem uma nova função para a via genética mir156/SPL na regulação do desenvolvimento e determinação de frutos carnosos, provavelmente através da regulação da expressão de MC. / Many plants have indeterminate growth and are capable of producing new organs and tissues throughout their life. This capability is partially due to the highly regulated expression of specific genes such as SQUAMOSA Promoter-Binding Protein-Like (SPL) genes. SPLs encode plant-specific transcription factors that play important roles in development, such as phase transition, plant architecture, and fruit ripening. Most SPL genes are post-transcriptionally regulated by the microRNA (miRNA) miR156. Although some developmental aspects regulated by SPLs have been well studied, their molecular roles during fruit development are poorly understood. In this work, we generated 22 transgenic events of Solanum lycopersicum cv. Micro-Tom (MT) overexpressing AtMIR156b precursor. Transgenic plants exhibit abnormal leaf and flower morphology and altered vegetative architecture. Interestingly, most events display navel-like fruits that are seedless and characterized by the growth of secondary fruits and present leaf-like structures as well as ectopic flowering meristems. By using RT-qPCR, we found a robust correlation between miR156/SPL expression and the fruit phenotype, suggesting that this pathway regulates tomato fruit development and determinacy. The tomato mutant Mouse ears (Me) displays a weak level of fruit indeterminacy. Levels of mature miR156 transcripts are higher in fruits from the mutant as comparing to MT fruits, but significantly lower than in transgenic fruits. Eight tomato SPLs were downregulated at variable levels in fruits from distinct transgenic events and Me plants. The MADS-type of transcription factor Macrocalyx (MC) gene is an orthologue of Arabidopsis AP1 (a direct in vivo target of SPL3) and affects tomato inflorescence determinacy and sepal development. MC was severely downregulated in fruits from transgenics but not in fruits from Me mutant. This data suggests that the MC misregulation may lead to the strong fruit indeterminacy phenotype observed in the transgenic events. Taken together, our data suggest a new function for miR156/SPL pathway in regulating fruit development and determinacy likely through the regulation of MC expression.

Muitas plantas apresentam crescimento indeterminado e são capazes de produzir novos órgãos e tecidos ao longo de todo seu ciclo de vida. Essa capacidade é devida parcialmente à expressão altamente regulada de genes específicos, tais como os genes SQUAMOSA Promoter-Binding Protein-Like (SPLs). SPLs codificam fatores de transcrição específicos de plantas que desempenham papéis importantes em diferentes aspectos do desenvolvimento, tais como mudança de fase juvenil-adulto, definição da arquitetura da planta e amadurecimento do fruto. A maioria dos genes SPLs são pós-transcricionalmente regulados pelo microRNA (miRNA) miR156. Apesar de alguns aspectos regulados pelas SPLs serem bem estudados, suas funções moleculares durante o desenvolvimento do fruto são pouco compreendidas. Neste trabalho, nós geramos 22 eventos transgênicos de Solanum lycopersicum cv. Micro-Tom (MT) superexpressando o precursor AtMIR156b. Plantas transgênicas exibiram morfologia foliar e floral anormal além de alteração na arquitetura vegetal. Interessantemente, a maioria dos eventos apresentou frutos de crescimento indeterminado, os quais não apresentam sementes sendo caracterizados pelo crescimento de frutos secundários além da presença de estruturas vegetativas e meristemas florais ectópicos. Fazendo uso da técnica de RT-qPCR, nós encontramos uma robusta correlação entre expressão do miR156 e o fenótipo dos frutos, sugerindo que esta rota regula o desenvolvimento e determinação do fruto de tomateiro. O mutante de tomateiro Mouse ears (Me) apresenta um fraco nível de indeterminação no fruto, sendo que os níveis do transcrito miR156 maduro são maiores no fruto mutante quando comparado aos controles MT, mas significativamente menores que nos frutos transgênicos. Oito SPLs de tomateiro foram silenciadas em diferentes níveis em frutos de diferentes eventos transgênicos e no mutante Me. O fator de transcrição do tipo MADS-box MACROCALYX (MC), o qual é ortólogo ao gene APETALA1 (AP1) de arabidopsis (alvo direto in vivo da proteína SPL3), afeta a determinação da inflorescência e o desenvolvimento das sépalas. A expressão de MC foi severamente reduzida nos frutos dos eventos transgênicos, mas não no mutante Me. Este dado sugere que a desregulação da expressão de MC deve ser responsável pelo forte fenótipo de indeterminação do fruto observados nos eventos transgênicos. Tomados em conjunto, nossos dados sugerem uma nova função para a via genética mir156/SPL na regulação do desenvolvimento e determinação de frutos carnosos, provavelmente através da regulação da expressão de MC. / Many plants have indeterminate growth and are capable of producing new organs and tissues throughout their life. This capability is partially due to the highly regulated expression of specific genes such as SQUAMOSA Promoter-Binding Protein-Like (SPL) genes. SPLs encode plant-specific transcription factors that play important roles in development, such as phase transition, plant architecture, and fruit ripening. Most SPL genes are post-transcriptionally regulated by the microRNA (miRNA) miR156. Although some developmental aspects regulated by SPLs have been well studied, their molecular roles during fruit development are poorly understood. In this work, we generated 22 transgenic events of Solanum lycopersicum cv. Micro-Tom (MT) overexpressing AtMIR156b precursor. Transgenic plants exhibit abnormal leaf and flower morphology and altered vegetative architecture. Interestingly, most events display navel-like fruits that are seedless and characterized by the growth of secondary fruits and present leaf-like structures as well as ectopic flowering meristems. By using RT-qPCR, we found a robust correlation between miR156/SPL expression and the fruit phenotype, suggesting that this pathway regulates tomato fruit development and determinacy. The tomato mutant Mouse ears (Me) displays a weak level of fruit indeterminacy. Levels of mature miR156 transcripts are higher in fruits from the mutant as comparing to MT fruits, but significantly lower than in transgenic fruits. Eight tomato SPLs were downregulated at variable levels in fruits from distinct transgenic events and Me plants. The MADS-type of transcription factor Macrocalyx (MC) gene is an orthologue of Arabidopsis AP1 (a direct in vivo target of SPL3) and affects tomato inflorescence determinacy and sepal development. MC was severely downregulated in fruits from transgenics but not in fruits from Me mutant. This data suggests that the MC misregulation may lead to the strong fruit indeterminacy phenotype observed in the transgenic events. Taken together, our data suggest a new function for miR156/SPL pathway in regulating fruit development and determinacy likely through the regulation of MC expression.

Plant reproductive development begins when vegetative shoot apical meristems change their fate to inflorescence meristems which develop floral meristems on the flanks. This process of meristem fate change and organ development involves regulated activation and/or repression of many cell fate determining factors that execute down-stream gene expression cascades. Flowers are formed when floral organs are specified on the floral meristem in four concentric whorls. In the model dicot plant Arabidopsis, the identity and pattern of floral organs is determined by combined actions of MADS-domain containing transcription factors of the classes A, B, C, D and E. Rice florets are produced on a compact higher order branch of the inflorescence and have morphologically distinct non-reproductive organs that are positioned peripheral to the male and female reproductive organs. These unique outer organs are the lemma and palea that create a closed floret internal to which are a pair of lodicules that are asymmetrically positioned fleshy and reduced petal-like organs. The unique morphology of these rice floret organs pose intriguing questions on how evolutionary conserved floral meristem specifying and organ fate determining factors bring about their distinct developmental functions in rice. We have studied the functions for two rice MADS-box proteins, OsMADS2 and OsMADS1, to understand their role as master regulators of gene expression during rice floret meristem specification and organ development. OsMADS2; a transcriptional regulator of genes expression required for lodicule development Arabidopsis B-function genes AP3 and PI are stably expressed in the whorl 2 and 3 organ primordia and they together with other MADS-factors (Class A+E or C+E) regulate the differentiation of petals and stamens (Jack et al, 1992; Goto and Meyerowitz, 1994). Rice has a single AP3 ortholog, SPW1 (OsMADS16) but has duplicated PI-like genes, OsMADS2 and OsMADS4. Prior studies in our lab on one of these rice PI-like genes OsMADS2 showed that it is needed for lodicule development but is dispensable for stamen specification (Kang et al., 1998; Prasad and Vijayraghavan, 2003). Functional divergence between OsMADS2 and OsMADS4 may arise from protein divergence or from differences in their expression patterns within lodicule and stamen whorls. In this study, we have examined the dynamic expression pattern of both rice PI-like genes and have examined the likelihood of their functional redundancy for lodicule development. We show OsMADS2 transcripts occur at high levels in developing lodicules and transcripts are at reduced levels in stamens. In fully differentiated lodicules, OsMADS2 transcripts are more abundant in the distal and peripheral regions of lodicules, which are the tissues that are severely affected in OsMADS2 knock-down florets (Prasad and Vijayraghavan, 2003). The onset of OsMADS4 expression is in very young floret meristems before organ primordia emergence and this is expressed before OsMADS2. In florets undergoing organogenesis, high level OsMADS4 expression occurs in stamens and carpels and transcripts are at low level in lodicules (Yadav, Prasad and Vijayraghvan, 2007). Thus, we show that these paralogous genes differ in the onset of their activation and their stable transcript distribution within lodicules and stamens that are the conserved expression domains for PI-like genes. Since the expression of OsMADS4 in OsMADS2 knock-down florets is normal, our results show OsMADS2 has unique functions in lodicule development. Thus our data show subfunctionalization of these paralogous rice PI-like genes. To identify target genes regulated by OsMADS2 that could contribute to lodicule differentiation, we have adopted whole genome transcript analysis of wild-type and dsRNAiOsMADS2 panicles with developing florets. This analysis has identified potential down-stream targets of OsMADS2 many of which encode transcription factors, components of cell division cycle and signalling factors whose activities likely control lodicule differentiation. The expression levels of few candidate targets of OsMADS2 were examined in various floret organs. Further, the spatial expression pattern for four of these down-stream targets of OsMADS2 was analysed and we find overlap with OsMADS2 expression domains (Yadav, Prasad and Vijayraghvan, 2007). The predicted functions of these OsMADS2 target genes can explain the regulation of growth and unique vascular differentiation of this short fleshy modified petal analog. OsMADS1, a rice E-class gene, is a master regulator of other transcription factors and auxin and cytokinin signalling pathways In Arabidopsis four redundant SEPALLATA factors (E-class) are co-activators of other floral organ fate determining MADS-domain factors (classes ABCD) and thus contribute to floral meristem and floral organ development (Krizek and Fletcher, 2005). Among the grass-specific sub-clade of SEP-like genes, rice OsMADS1 is the best characterized. Prior studies in our lab showed that OsMADS1 is expressed early throughout the floret meristem before organ primordia emergence and later is restricted to the developing lemma and palea primordia with weak expression in carpel (Prasad et al, 2001). Stable expression continues in these floret organs. OsMADS1 plays critical non-redundant functions to specify a determinate floret meristem and also regulates floret organ identities (Jeon et al., 2000; Prasad et al, 2001; 2005; Agarwal et al., 2005; Chen et al., 2006). In the present study, we have adopted two different functional genomic approaches to identify genes down-stream of OsMADS1 in order to understand its mechanism of action during floret development. We have studied global transcript profiles in WT and dsRNAiOsMADS1 panicles and find OsMADS1 is a master regulator of a significant fraction of the genome’s transcription factors and also a number of genes involved in hormone-dependent cell signalling. We have validated few representative genes for transcription factors as targets regulated by OsMADS1. In a complementary approach, we have determined the consequences of induced-ectopic over-expression of a OsMADS1:ΔGR fusion protein in shoot apical meristems of transgenic plants. Transcript levels for candidate target genes were assessed in induced tissues and compared to mock-treated meristems and also with meristems induced for OsMADS1:ΔGR but blocked for new protein synthesis. These analyses show that OsMADS55 expression is directly regulated by OsMADS1. Importantly, OsMADS55 is related to SVP that plays an important role in floral transition and floral meristem identity in Arabidopsis. OsHB3 and OsHB4, homeodomain transcription factors, with a probable role in meristem function, are also directly regulated by OsMADS1. The regulation of such genes by OsMADS1 can explain its role in floret meristem specification. In addition to regulating other transcription factors, OsMADS1 knock-down affects expression of genes encoding proteins in various steps of auxin and cytokinin signalling pathways. Our differential expression profiling showed OsMADS1 positively regulates the auxin signalling pathway and negatively regulates cytokinin mediated signalling events. Through our induced ectopic expression studies of OsMADS1:ΔGR, we show OsMADS1 directly regulates the expression of OsETTIN2, an auxin response transcription factor, during floret development. Overall, we demonstrate that OsMADS1 modulates hormonal pathways to execute its functions during floret development on the spikelet meristems. Functional studies of OsMGH3; an auxin-responsive indirect target of OsMADS1 To better understand the contribution of auxin signalling during floret development, we have functionally characterized OsMGH3, a down-stream indirect target of OsMADS1, which is a member of the auxin-responsive GH3 family. The members of this family are direct targets of auxin response factors (ARF) class of transcription factors. GH3-proteins inactivate cellular auxin by conjugating them with amino acids and thus regulate auxin homeostasis in Arabidopsis (Staswick et al., 2005). OsMGH3 expression in rice florets overlaps with that of OsMADS1 (Prasad et al, 2005). In this study, we have demonstrated the consequences of OsMGH3 over-expression and knock-down. The over-expression of OsMGH3 during vegetative development causes auxin-deficient phenotypes such as dwarfism and loss of apical dominance. Its over-expression in developing panicles that was obtained by driving its expression from tissue-specific promoters created short panicles with reduced branching. The latter is a phenotype similar to that observed upon over-expression of OsMADS1. In contrast, the down-regulation of endogenous OsMGH3 through RNA-interference produced auxin over-production phenotypes such as ectopic rooting from aerial nodes. Knock-down of OsMGH3 expression in florets affected carpel development and pollen viability both of which affect floret fertility. Taken together, this study provides evidence for the importance of auxin homeostasis and its transcriptional regulation during rice panicle branching and floret organ development. Our analysis of various conserved transcription factors during rice floret development suggest that factors like OsMADS2, OsMADS4 and OsMADS1 are master regulators of gene expression during floret meristem specification and organ development. The target genes regulated by these factors contribute to development of morphologically distinct rice florets.