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Comprendre la diversité morphologique des fleurs passe par l'étude de son origine moléculaire et développementale et de ses conséquences fonctionnelles et écologiques. Le périanthe est composé d'organes stériles, sépales et pétales, qui jouent un rôle majeur dans le succès reproducteur des plantes pollinisées par les animaux du fait de leur fonction d'attraction.Cette thèse propose une approche multidisciplinaire visant à comprendre l'origine génétique et moléculaire de la diversité morphologique du périanthe et sa signification évolutive, à l'aide du modèle Nigella damascena L. Cette Renonculacée présente un dimorphisme spontané. La forme probablement ancestrale, trouvée en populations naturelles, a un périanthe bipartite composé de cinq sépales pétaloïdes et huit pétales nectarifères. Dans la forme variante, cultivée à des fins d'horticulture, les pétales sont remplacés par un nombre élevé d'organes allant d'une forme proche des sépales à une forme proche des étamines.La première partie de cette thèse est consacrée à l'étude de l'origine développementale, génétique et moléculaire du dimorphisme, par la caractérisation détaillée de la morphologie florale et de son développement dans les deux morphes dans le cadre d'une approche gène candidat. Par analyse d'expression et validation fonctionnelle, nous avons montré que le gène NdAP3-3 est responsable de l'ensemble des aspects du dimorphisme floral de N. damascena, ce qui suggère que ce gène joue un rôle dans l'identité du pétale mais aussi dans l'architecture du méristème, potentiellement via la régulation du nombre d'organes et de la frontière entre périanthe et étamines.La seconde partie de cette thèse concerne l'impact du dimorphisme floral sur le mode de reproduction des deux morphes et leur maintien potentiel. Nous avons caractérisé les stratégies reproductives et la valeur sélective des deux morphes en conditions naturelles dans des populations expérimentales. Le variant sans pétale est peu visité par les pollinisateurs, et se reproduit majoritairement en autogamie. L'analyse de la vigueur de ses descendants suggère une dépression de consanguinité. Par ailleurs, dans notre matériel, il semble que l'allèle donnant le phénotype sans pétale soit lié à un allèle augmentant la valeur sélective. A la lumière de nos résultats, nous discutons les conditions du maintien de ce polymorphisme. / Understanding flower diversity requires on one hand the study of the molecular and developmental origin of floral architecture, and on the other the study of the functional and ecological consequences of flower morphology. A great deal of that diversity can be found at the perianth level which comprises the sepals and petals, sterile and versatile organs that play a major role in the reproductive success of animal pollinated flowering plants through their attractive characteristics.This thesis is the result of a multidisciplinary effort to understand the genetic and molecular origin as well as the evolutionary significance of perianth diversity, using the Nigella damascena L. as a model. This Ranunculaceae species presents a rare naturally occurring floral dimorphism affecting perianth architecture. The putatively ancestral form found in natural populations has a well differentiated bipartite perianth composed of five petaloid sepals and eight nectariferous petals, while the perianth in the alternative apetalous mutant, cultivated for horticultural purpose, has no petals and but is instead composed of numerous organs showing a continuum of forms from outer sepal-like to inner stamen-like.The first part of this thesis was dedicated to the study of the developmental, genetic and molecular origin of this dimorphism, via a detailed characterization of floral morphology and development in both morphs, which laid a foundation for the interpretation of the results of a candidate gene approach. Using expression analysis and functional validation we showed that NdAP3-3 is fully responsible for the complex N. damascena floral dimorphism, suggesting that it plays a role not only in petal identity but also in meristem patterning, possibly through the regulation of perianth organ number and perianth-stamen boundary.The second half of this thesis focused on the impact of the floral dimorphism on the reproduction mode and evolutionary maintenance of the two morphs. We assessed reproduction strategies and reproductive success in the two morphs by studying a polymorphic experimental population in natural conditions. The absence of petals in the mutant form was associated with a qualitative drop in pollinator visitation which resulted in a shift towards selfing. The study of their progeny suggests that selfing had a negative effect on the descendant’s vigor via inbreeding depression. Additionally, in our material, the allele responsible for the apetalous phenotype seems to be linked to a favorable allele increasing fitness. We discuss the mechanisms of the dimorphism maintenance in light of these results.

<p>The reproductive organs of conifers and angiosperms differ in morphology in several fundamental respects. The conifer Norway spruce <i>(Picea abies)</i> form pollen and seed cones from separate meristems whereas angiosperms bear bipartite flowers with sepals and petals surrounding two inner whorls of stamens and carpels. Despite these differences in morphology this thesis present data to suggest that reproductive development in conifers and angiosperms is regulated by a similar molecular mechanism. This implies an evolutionary conservation of the major mechanism for reproductive development since the origin of seed plants. </p><p>Flower organ identity in angiosperms is determined by regulatory genes belonging to the MADS-box gene family of transcription factors. This thesis presents the cloning and characterisation of four novel MADS-box genes from Norway spruce<i>.</i> Three of these genes <i>DAL11</i>, <i>DAL12</i> and <i>DAL13 </i>are most closely related to angiosperm B function genes <i>i.e.</i> genes required for petal and stamen development. <i>DAL11</i>, <i>12</i> and <i>13</i> all are specifically active in developing pollen cones, with different temporal and spatial expression pattern. Functional analysis in transgenic Arabidopsis and yeast suggest that the reproductive aspect of the B-function is conserved between conifers and angiosperms. The results also suggest that the B-function in conifers is separated into one shoot identity and one organ identity determinant. </p><p>A fourth gene presented;<i> DAL10,</i> is specifically expressed in vegetative parts of pollen- and seed cones. Phylogenetically <i>DAL10</i> is not closely related to any of the known angiosperm clades, but rather forms a separate clade with other gymnosperm genes, suggesting a gymnosperm specific function. We suggest that the <i>DAL10</i> activity reflects a function in the determination of the reproductive shoot.</p>

The reproductive organs of conifers and angiosperms differ in morphology in several fundamental respects. The conifer Norway spruce (Picea abies) form pollen and seed cones from separate meristems whereas angiosperms bear bipartite flowers with sepals and petals surrounding two inner whorls of stamens and carpels. Despite these differences in morphology this thesis present data to suggest that reproductive development in conifers and angiosperms is regulated by a similar molecular mechanism. This implies an evolutionary conservation of the major mechanism for reproductive development since the origin of seed plants. Flower organ identity in angiosperms is determined by regulatory genes belonging to the MADS-box gene family of transcription factors. This thesis presents the cloning and characterisation of four novel MADS-box genes from Norway spruce. Three of these genes DAL11, DAL12 and DAL13 are most closely related to angiosperm B function genes i.e. genes required for petal and stamen development. DAL11, 12 and 13 all are specifically active in developing pollen cones, with different temporal and spatial expression pattern. Functional analysis in transgenic Arabidopsis and yeast suggest that the reproductive aspect of the B-function is conserved between conifers and angiosperms. The results also suggest that the B-function in conifers is separated into one shoot identity and one organ identity determinant. A fourth gene presented; DAL10, is specifically expressed in vegetative parts of pollen- and seed cones. Phylogenetically DAL10 is not closely related to any of the known angiosperm clades, but rather forms a separate clade with other gymnosperm genes, suggesting a gymnosperm specific function. We suggest that the DAL10 activity reflects a function in the determination of the reproductive shoot.

The Euphorbiaceae are notable for floral and inflorescence diversity and evolutionary complexity. Croton, is the second largest genus in the family and exhibits particular diversity in its flowers, especially regarding perianth and number of stamens, besides the inflorescences, which are also very diverse. Considering Croton\'s great variability in the reproductive structures, the aim of this thesis was to study flowers and inflorescences with an evolutionary approach, including morphology, ontogeny, vasculature, auxin regulation and genetic expression. Flowers in several stages of development were analyzed using light microscopy and scanning electron microscopy. Inflorescences were analyzed in stereomicroscope and the traits were plotted on the most recent phylogeny of the genus. The genetic expression was tested using RNAseq. In the first chapter the flowers showed similarity in the initiation of sepals and the presence of filamentous, petaloid structures in Croton lundianus (Didr.) Müll. Arg., interpreted here as staminodes. In Croton sphaerogynus Baill., staminodes were described for the first time. The staminodes reported here could be interpreted as transitional structures that we considered as evolutionary reductions. In the second chapter, the staminate flowers showed polystemonous androecium and the delay in petals\' initiation and the antesepalous nectaries development interfered in the development of the stamens, characterizing obdiplostemony. Vasculature corroborated obdiplostemony and revealed a central stamen in C. fuscescens with carpelar features, interpreted here as a homeosis case. Glandular staminodes were registered and interpreted as a heterotopy case. The obdiplostemony may be related to modulation of the free IAA concentrations during floral developmental steps and Croton flowers can be used as good models for obdiplostemony, homeosis and heterotopy. In the third and fourth chapter we studied Croton inflorescences, which showed 17 patterns with differences on the organization and distribution of pistillate flowers. The inflorescence traits analyzed were very homoplastic, most likely determined by convergent evolution in distantly related lineages distributed in similar habitats. The genetic expression of C. fuscescens was particularly analyzed and the transcriptome showed that the different zones have their development guided through the same transcripts set. Each zone has different expression level and these variations and gradient could be interpreted as the boundary between each inflorescence zone. The floral developmental novelties and evolutionary links identified here raise the importance of future floral studies with the genus, what would bring a better understanding on how the reproductive structures evolved in the history of the group / Euphorbiaceae é uma família que recebe destaque quanto à diversidade de flores e inflorescências, além de sua complexidade evolutiva. Croton L. é o segundo maior gênero da família e exibe particular diversidade floral, em especial quanto a o perianto e número de estames, além das inflorescências, que também se apresentam muito diversas. Considerando a grande variação nas estruturas reprodutivas de Croton, o objetivo desta tese foi estudar as flores e inflorescências com abordagem evolutiva, incluindo morfologia, ontogênese, vascularização, regulação hormonal e expressão gênica. Flores em diversos estágios de desenvolvimento foram analisadas em microscopia der luz e varredura. Inflorescências foram estudadas em estereomicroscópio e os caracteres observados foram analisados nas filogenias mais recentes do grupo. A expressão gênica foi analisada com a técnica RNAseq. No primeiro capítulo as flores apresentaram semelhanças na iniciação das sépalas e presença de filamentos, estruturas petaloides em Croton lundianus (Didr.) Müll. Arg., interpretadas como estaminódios. Em Croton sphaerogynus Baill., estaminódios foram descritos pela primeira vez. Estas estruturas podem ser interpretadas como estruturas de transição evolutiva e reduções florais. No segundo capítulo as flores estaminadas apresentaram androceu polistêmone e o retardo na iniciação das pétalas e o desenvolvimento antessépalo dos nectários foram considerados como fatores chave para o desenvolvimento do androceu como obdiplostêmone. A vascularização corroborou a obdiplostemonia e revelou um estame central com características carpelares em C. fuscescens, interpretado aqui como um caso de homeose. Nectários glandulares foram registrados e interpretados como uma mudança heterotópica. A obdiplostemonia pode estar relacionada com as diferentes concentrações de auxina ao longo das etapas de desenvolvimento e as flores de Croton podem ser consideradas como bons modelos de obdiplostemonia, homeose e heterotopia. No terceiro e quarto capítulo nós investigamos as inflorescências de Croton, que apresentaram 17 padrões com diferenças na organização e distribuição das flores pistiladas especialmente. Os caracteres das inflorescências se mostraram homoplásticos e provavelmente determinados por evolução convergente em linhagens distantes distribuídas em habitats semelhantes. A expressão gênica de C. fuscescens foi particularmente analisada e o transcriptoma demonstrou que o desenvolvimento das diferentes zonas é regulado pelo mesmo conjunto gênico. Cada zona, pistilada ou estaminada, apresenta níveis distintos de expressão diferencial e o gradiente na expressão pode ser o delimitador entre as zonas. Os novos relatos quanto ao desenvolvimento floral em Croton e os links evolutivos identificados nesta tese levanta a importância de estudos para uma melhor compreensão sobre a evolução das estruturas reprodutivas neste grupo tão importante

LEAFY (LFY) est un facteur de transcription central pour le développement des plantes, en particulier pour la floraison chez les angiospermes. LFY est très conservée, même chez les espèces ne portant pas de fleurs. On dispose de nombreuses données génétiques sur LFY et son réseau de régulation chez la plante modèle Arabidopsis thaliana, mais les mécanismes moléculaires impliqués dans son fonctionnement ne sont pas entièrement élucidés. LFY possède deux domaines conservés : un domaine de liaison à l'ADN et un domaine de fonction inconnue en position N-terminal. L'objectif a été de comprendre le rôle du domaine N-terminal et d'étudier l'évolution de la spécificité de liaison à l'ADN de LFY. Nous avons obtenu la structure cristallographique du domaine N-terminal de LFY et découvert qu'il s'agissait d'un domaine SAM (Sterile Alpha Motif) permettant l'oligomérisation de la protéine. Nous avons validé l'importance de cette propriété pour la fonction florale de LFY chez A. thaliana. Nous avons ensuite montré, par des analyses in vitro et in vivo en ChIP-seq que l'oligomérisation influençait la liaison à l'ADN en permettant une liaison coopérative sur plusieurs sites de liaison, en assurant la sélectivité de la protéine vis-à-vis de l'ADN et en permettant l'accès de la protéine à des régions génomiques où la conformation de la chromatine est normalement défavorable à la liaison. Cette étude intégrative a permis de mieux comprendre le fonctionnement de LFY. Des modifications dans les réseaux de régulation de l'expression des gènes sont source de nouveauté et d'évolution. LFY étant très conservée et ne faisant pas partie d'une famille multigénique, nous nous sommes demandé si sa spécificité de liaison à l'ADN avait évoluée. Nous avons montré que LFY était apparue chez les algues multicellulaires et que sa spécificité avait connue au moins deux changements majeurs au cours de l'évolution. Nous avons expliqué ces modifications au niveau moléculaire par des approches de biologie structurale et de biochimie. Nous avons identifié une espèce chez qui LFY a une spécificité relâchée et nous proposons qu'une telle forme ait pu permettre les transitions d'une spécificité à une autre. / LEAFY (LFY) is a key transcription factor for plant development, particularly for flowering in angiosperms. LFY is highly conserved in plants, including non-flowering species. Despite a wealth of genetic data about LFY and its regulatory network in the model plant Arabidopsis thaliana, how the protein works at the molecular level is not fully understood. It has two conserved domains: a DNA binding domain and a N-terminal domain of unknown function. My two main projects were to understand the role of the N-terminal domain and to study LFY DNA binding specificity evolution. We obtained LFY N-terminal domain crystal structure and discovered it was a Sterile-Alpha-Motif (SAM) mediating LFY oligomerisation. We validated the importance of that property for flower development in A. thaliana. Using both in vitro analyses and a ChIP-seq experiment, we pointed out that oligomerisation is required for proper DNA binding. It enables cooperative binding on several LFY binding sites, increases the protein selectivity towards DNA and allows LFY to access genomic regions where the chromatin conformation normally prevents binding. This integrative study provides a better understanding of how LFY works. The rewiring of transcriptional networks provides a rich source of evolutionary novelty. As LFY is highly conserved and single copy in most plant genomes, we asked whether its DNA binding specificity had evolved. We showed that LFY was present since multicellular algae and that it underwent at least two major shifts in DNA-binding specificity during plant evolution. We provided a structural explanation for the two newly identified DNA binding modes and we identified a LFY form with a relaxed specificity that could have served as an intermediate between evolutionary transitions.

Les plantes conservent la capacité à former de nouveaux organes tout au long de leur vie grâce au maintien de structures contenant des cellules souches, les méristèmes. La formation des fleurs, structures reproductives de la plante, est une étape essentielle de son cycle de vie. Afin d’assurer un développement floral complet, un méristème doit être formé de novo au sein du jeune bouton floral. Des données éparses de la littérature indiquent que le facteur de transcription LEAFY, en plus d’être un régulateur clé de l’identité florale, est aussi impliqué dans la mise en place du méristème floral.Dans la première partie de ce travail nous explorons le rôle de LEAFY dans l’initiation du méristème floral. Cette étude est concentrée sur un gène cible de LEAFY, le facteur de transcription REGULATOR OF AXILLARY MERISTEMS1 (RAX1). Nous montrons notamment que la voie régulée par LEAFY/RAX1 agit en parallèle du facteur de transcription REVOLUTA pour permettre la mise en place du méristème floral.Dans la deuxième partie de ce travail nous étudions les propriétés du domaine N-terminal de LEAFY. Ce domaine permet l’oligomérisation de LEAFY ainsi que potentiellement sa liaison aux régions fermées de la chromatine. Nous étudions également de manière plus exploratoire le rôle de ce domaine dans la régulation de l’expression du gène AGAMOUS, un important régulateur du développement floral. / Plants have the capacity to continuously produce organs throughout their life because they maintain stem cells containing structures called meristems. The formation of flowers is an essential step of the plant’s life-cycle. In order to ensure flower development, a new meristem must be formed within the young flower bud. Various data across the literature indicate that the transcription factor LEAFY is involved flower meristem formation in addition to its role as a master regulator of flower identity.In the first part of this work we explore the role of LEAFY in the initiation of flower meristem. This study focuses on a LEAFY target gene, the transcription factor REGULATOR OF AXILLARY MERISTEMS1 (RAX1). We show that the LEAFY/RAX1 pathway acts in parallel of the transcription factor REVOLUTA to allow flower meristem formation.In the second part of this work we study the properties of the N-terminal domain of LEAFY. This domain mediates LEAFY oligomerization and potentially its binding to closed chromatin regions. We also study in a more prospective manner the role of this domain in the transcriptional regulation of AGAMOUS, an important regulator of flower development.

Orientador: Marcelo Carnier Dornelas / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T06:46:46Z (GMT). No. of bitstreams: 1 Aizza_LilianCristinaBaldon_M.pdf: 4635864 bytes, checksum: b27d03cf7e934f1060d7a39ebc654ca3 (MD5) Previous issue date: 2010 / Resumo: Algumas estruturas relacionadas à atração de agentes polinizadores foram incorporadas aos órgãos reprodutivos, resultando em inovações florais que aperfeiçoaram a reprodução durante a evolução das angiospermas. Passiflora é um exemplo de diversidade e complexidade floral. A característica mais marcante do gênero é a presença de uma corona de filamentos entre o perianto e o androginóforo, cuja principal função parece ser a atração de polinizadores. Neste estudo, a ontogenia da corona foi investigada em quatro espécies e um híbrido interespecífico artificial, representando os dois maiores subgêneros: P. edulis var flavicarpa Deg, P. coccinea Aubl., P. 'Lady Margaret' (híbrido P. edulis x P. coccinea) pertencentes ao subgênero Passiflora, P. tulae Urban e P. suberosa L. do subgênero Decaloba. A descrição morfo-anatômica comparativa do desenvolvimento da corona foi obtida com a utilização de microscopia de luz e eletrônica de varredura. Fragmentos correspondentes a homólogos dos genes MADS-box foram clonados de P. edulis e usados em análises filogenéticas. Estes fragmentos compartilharam similaridade com as sequências dos fatores de transcrição MADS-box em Arabidopsis: APETALA1, PISTILLATA e AGAMOUS. Estas sequências foram nomeados PeAPETALA1, PePISTILLATA e PeAGAMOUS, respectivamente. Os padrões de expressão destes genes foram investigados em diferentes tecidos vegetais por RT-PCR e em botões florais em diferentes estágios de desenvolvimento por hibridização in situ. / Abstract: Some structures related to the attraction of pollinators were incorporated into the reproductive organs, resulting in floral innovations that have improved plant reproduction during the evolution of angiosperms. Passiflora is an example of floral diversity and complexity. The most striking feature of the genus is the presence of corona filaments between the perianth and androgynophore, whose main function seems to be the attraction of pollinators. In this study, the ontogeny of the corona was investigated in four Passiflora species representing the two major subgenera. Belonging to the subgenus Passiflora are P. edulis var flavicarpa, P. coccinea Aubl. and the artificial interspecific hybrid P. 'Lady Margaret' (P. edulis x P. coccinea). P. tulae Urban and P. suberosa L. belong to subgenus Decaloba. The comparative morpho-anatomical description of the corona development was obtained with the use of light microscopy and scanning electron microscopy. Fragments corresponding to homologs of the MADS-box genes were cloned from P. edulis and used in filogenetic analyzes. These fragments showed similarity with the sequences of the Arabidopsis MADS-box transcription factors: APETALA1, PISTILLATA and AGAMOUS. Thus, these Passiflora sequences were named PeAPETALA1, PePISTILLATA and PeAGAMOUS, respectively. The expression patterns of these genes were investigated in different plant tissues by RT-PCR and in flower buds with different stages of corona development by in situ hybridization. / Mestrado / Mestre em Biologia Vegetal

Dans le domaine des plantes, la formation de la fleur a été un pas crucial dans la capacité des végétaux à coloniser une grande diversité de niches écologiques sur notre planète. Les deux espèces Petunia x hybrida et Arabidopsis thaliana représentent deux groupes majeurs des plantes à fleur. Nous avons montré que les gènes à homéodomaine d’une famille appelée WOX (Wuschel homeobOX) sont fortement impliqués dans le développement des organes dotés de polarité (dont les feuilles et des organes de la fleur : sépales, pétales, carpelles). Un double mutant (maw mawb), chez le Pétunia, développe des pétales en forme de filament, avec disparition du tube floral. De plus, nous avons découvert que ces mêmes gènes interagissent au niveau génétique avec d’autres gènes (appelés gènes à boite MADS) dans la formation des ovules, structures à partir desquelles les graines se forment. Nous avons aussi montré que des gènes de la même famille sont impliqués dans la formation d’autres structures chez le Pétunia : les trichomes ou poils aériens de surface. Ces derniers sont impliqués dans plusieurs taches, qui vont de la protection contre les pathogènes à celles contre les stress abiotiques. Grâce à des études de génétique fonctionnelle nous avons pu montrer un recrutement différentiel des gènes WOX ici étudiés, dépendant de l’organe et de l’espèce. Ces travaux de thèse montrent l’importance de cette famille génique pour les études d’evo-devo (Biologie Evolutionniste du Développement). Finalement, une analyse de RNA-Seq (séquençage du transcriptome), dévoile les réseaux génétiques contrôlés par ces gènes WOX. / In the Kingdom of Plants, the emergence of flowers was a crucial step in their ability to colonize a large variety of ecological niches on our planet. The two species Petunia x hybrida and Arabidopsis thaliana represent two major groups of flowering plants. In this work, we have shown that HOMEOBOX genes from the WOX family (Wuschel homeoboxes) are heavily involved in polar organ development (such as leaves and sepals, petals, and carpels at the flower level). The maw mawb double mutant in Petunia displays string-like petals, with consequent disappearance of the floral tube. Moreover, we found that these two genes genetically interact with genes from a different family (the MADS family) in ovule identity (ovules are the structures from which seeds develop). We have also shown that other genes from the WOX family are involved in development of a different kind of structures in Petunia: the trichomes. Trichomes are involved in different tasks, protecting the plant from pathogens or abiotic stress. Thanks to functional genetics studies, we have shown functional genetic recruitment of these WOX genes among different plant organs and among different species. This PhD thesis provides evidence for the importance of the WOX family in Evo-Devo studies. Eventually, we unravelled genetic networks controlled by MAW and MAWB trough RNA-Seq analysis.