The morphogenetic changes involved with the differentiation of a unicellular fertilized egg to an organised multicellular embryo pass through a cycle of events. Manifestation of differentiation in structural diversity starts from the physiological differences In the tissue. These differences can be demonstrated through biochemical variations. These ideas are examined In the gametophyte-embryo complex of Ginkgo biloba and Pinus resinose. It is shown that the embryo experiences different physiological conditions during the various phases of development. In addition, the potentialities of embryonic cells to grow and differentiate vary during these phases. Alternative developmental sequences can occur if the nutritional environment is altered. Radiosensitivity of the embryo as measured by abortion and abnormal differentiation is shown to be dependent on the stage of embryonic development. / Thesis / Doctor of Philosophy (PhD)

Gametophytic selection was examined as a breeding tool in developing Phalaenopsis hybrids that are more cool or warm temperature tolerant. Two hybrid Phalaenopsis, P. (Taisoco Windian à Sogo Sogo Yukidian) by P. hybrid unknown, were reciprocally cross-pollinated and exposed to 14°C/9°C for 7 days as a cold pollination treatment. Plants were pollinated again and exposed to 30°C/25°C for 3 days for the warm pollination treatment. Each cultivar was placed in either of two growth chambers during the pollination treatments and exposed to the selected temperatures, an 11-h photoperiod with an irradiance of 180 Mmol·m-2·s-1 and a relative humidity of 70%. The plants were returned to the greenhouse after pollination and the green capsules were collected after 150 days. Seeds obtained from these treatments were surface-sterilized and equal volumes were placed on Phytamax® medium. Evaluation of protocorm development was done after 73 days on a thermogradient table ranging from 10 to 30ºC. For the first family for which reciprocal crosses were available, the number of protocorms per plate ranged from 0 in the coldest treatments to 290 at 28°C. For cold pollinated seeds, protocorm development was optimum at 22 and 28°C (means of 290 and 250 protocorms per plate, respectively) whereas the greatest protocorm development for warm pollinated seeds occurred at 20°C (103 protocorms per plate). Of the 1471 total protocorms obtained 1095 were from cold pollinations, whereas 376 were from the warm pollinations. Protocorms were evaluated for leaf and root formation 125 days after initial plating. Transfer to warm or cold incubators occurred as protocorms developed leaves and roots. Seedlings were finally transferred to dried sphagnum and placed in growth chambers set to original pollination temperatures. One year after initial plating seedlings were evaluated on the following criteria: wet weight, number of leaves, leaf area, number of roots, and root length. The pollination treatment significantly affected the number of roots per seedling whereas germination temperature during germination significantly affected the weight (g). Weight of the seedlings, number of roots and the average root length were significantly affected by the interaction between pollination treatment and germination temperature. The weight, number of leaves, and average root length were significantly affected by the interaction between pollination treatment and incubator/growth chamber. These differences indicated that seedlings derived from warm pollination were more vigorous under warm growing conditions and those derived from cold pollination were more vigorous under cold growing conditions. The significance of the interaction between pollination treatment and incubator/growth chamber indicates that gametophytic selection for thermotolerance in Phalaenopsis can be successfully used as a plant breeding tool. Additional replication is required to confirm the greater germinability of seed derived from pollination occurring over a greater range of temperatures. / Master of Science

The placenta of the anthocerote, Dendroceros tubercularis Hatt., consists of long and branched haustorial cells, that arise from the foot and gametophyte transfer cells. Both cell types contain electron‐dense vacuolar deposits that were digested by pronase and therefore are assumed to be protein. These deposits were negative to the PATAg test for carbohydrates. Protein bodies were also found in the parenchyma cells of the foot and younger meristematic cells at the base of the capsule. Vacuolar deposits of osmiophilic material in the gametophyte cells external to the placenta were stained non‐specifically with PATAg method and were not affected by pronase. The haustorial cells have pleomorphic plastids lacking starch and a thylakoid system, whereas the transfer cells have well developed chloroplasts. No pronase‐sensitive material was detected in the apo plastic space separating gametophyte and sporophyte cells. These results suggest that protein is synthesized in the haustorial cells, perhaps from precursors provided by transfer cells, and is then transferred, via plasmodesmata, to the parenchyma cells of the foot and eventually to the cells of the growing capsule.

The placenta of two Sphagnum species was examined by electron microscopy. In contrast to all mosses so far investigated, neither sporophyte nor gametophyte placental cells of Sphagnum develop wall ingrowths. The sporophyte cells are highly vacuolate and the gametophyte cells close to them degenerate to produce a system of spaces filled with mucilage. Whether this type of placenta represents a primitive or derived condition in mosses is discussed.

Plants have developed intricate mechanisms to control growth in response to a variety of environmental cues, to compensate its immobility and to survive in both normal and adverse conditions. The TCP proteins are a family of plant-specific, basic helix-loop-helix (bHLH) transcription factors that involve in different aspects in plant growth and developmental control. The Arabidopsis TCP20 has been shown to involve in coordinating cell growth and proliferation, and in growth arrest in response to DNA double-stranded breaks (DSB). In this thesis, the main interest is to examine the function of Arabidopsis TCP6, which shares the highest homology with TCP20, and like TCP20, contains a putative ATM phosphorylation motif that suggests potential involvement in the ATM/ATR-mediated DSB responses. Expressional analysis including transcript measurement and reporter gene tagging demonstrated that TCP6 is expressed in flowers, in particular in the first mitotic event of pollen and ovule/embryo sac development, indicating that TCP6 potentially involves in regulating the mitotic cell cycle during gametophyte development. Yet no gametophytic or fertility-affecting mutant phenotype was observed in the tcp6 single and tcp6/tcp20 double mutants, which may be due to high functional redundancy. The tcp6/tcp20 double mutant seedlings exhibited significantly higher growth performances in true leaf growth compared to wild type when treated with gamma radiation, implying that both functional TCP6 and TCP20 are involved in response to gamma radiation-generated DSBs. The work of this thesis provides the first expressional and functional characterizations of TCP6, with the results suggesting that TCP6 and other class I TCPs play a role in regulating growth under both normal and stress conditions.

Bromeliaceae owns a huge diversity of species in the Neotropical region, comprising about 3140 species, in which 551 consist of the genus Tillandsia. The article aims the descritption, of the anther development and ovule as the esporogenesis and gametogenesis characterization as well, in order to make easier both the taxonomy and philogeny of the family, which is still in process of change. Tillandsia aeranthos (Lois.) L. B. Sm. contains six stamens flowers; superior, tricarpellate and trilocular ovary. The pattern of development of the anther wall is characterized as mixed-type. Androsporangium is formed by epidermis, middle layers, endothecium and tapetum. The tapetum is the secretor-type. Meiosis is the successive-type with cleavage of the centrifugal type. The tetrads formed are decussate or isobilateral. The first division of the pollen is preceded by vacuolation, it is assymmetric and produce both generative and vegetative cells. The two cells formed are separated by a callose wall. The mature grain pollen is bicelullar. The ovule of Tillandsia aeranthos is anatropus, bitegmic and crassinucellate, with axial placentation. The ovule originates in the subdermal layer (zone II) of the placentae. The integument nucellar epidermis is originated by divisions in the dermal layers. One to three ginospore mother celss, which are originated by divisions in archesporial cell, undergo meiotic divisions development a linear tetrad, with presence callosic wall. Only the chalazal ginospore becomes functional. The functional ginospore differs in that a gametophyte uninucleate after mitosis and yields a two-nucleate and four-nucleate gametophyte. The female gametophyte has a monosporic origin and a Polygonum-type development. The female gametophyte consists of two synergids, an egg cell, three antipodes and two polar nuclei. Polar nuclei fuse prior to fertilization. / Bromeliaceae possui grande diversidade de espécies na região neotropical, compreendendo cerca de 3140 espécies, sendo 551 do gênero Tillandsia. O presente trabalho teve como objetivo descrever o desenvolvimento da antera e do rudimento seminal, além da caracterização da espogênese e gametogênese, com a finalidade de auxiliar na taxonomia e filogenia da família, ainda em fase de mudança. Tillandsia aeranthos, apresenta flores com estames em número de seis e ovário súpero, tricarpelar. Na antera, o desenvolvimento dos estratos parietais é do tipo misto , formado por epiderme, endotécio, camada média e tapete. A epiderme é papilada e o tapete é do tipo secretor. A microsporogênese é sucessiva com clivagem do tipo centrífuga, onde são formadas tétrades isobilaterais e decussadas. Após a liberação do andróspro da tétrade, ocorre a primeira divisão do grão de pólen e é precedida por vacuolação. Após mitose, é formada a célula generativa e a célula vegetativa, separadas por uma parede de calose. O andrófito é liberado bicelular. O rudimento seminal de Tillandsia aeranthos é anátropo, bitegumentado e crassinucelado, com placentação axial. O rudimento seminal tem origem na camada subdérmica da placenta. Os tegumentos são de origem dérmica. A inicial arquesporial dá origem á célula mãe de ginósporo (CMG). A CMG sofre o primeiro ciclo meiótico que origina uma díade de ginósporo com presença de calose ao redor. O segundo ciclo meiótico dá origem a tétrade linear de ginósporo, tornando-se o ginósporo calazal funcional. O ginósporo funcional diferencia-se em um gametófito uninucleado que após mitoses origina um gametófito bi e tetranucleado. Esse tipo de desenvolvimento do ginófito é do tipo monospórico e Polygonum, onde o ginófito apresenta sete células e oito núcleos, com a presença de duas sinérgides, uma oosfera, formando o aparelho oosférico e três antípodas. Os núcleos polares de fusionam antes da fecundação.

In plants, translation regulation plays an important role during progamic phase, fertilization and seed development. The process of translation is mostly regulated in its initiation phase, where Eukaryotic translation initiation factor 3 (eIF3) is the largest and most complex initiation factor, consisting of 12 different subunits. In plants, single eIF3 subunit mutants caused various growth and development defects, depending on the particular subunit that was mutated. However, not all the plant eIF3 subunits were characterized to this date. The objective of this work was to functionally characterize the eIF3 subunit A using Arabidopsis thaliana as the main model plant. We described in this work that plant eIF3A proteins share high levels of homology and domain organization with eIF3A subunits from non-plant eukaryotic species but contain regions specific only to plants. Next we described that Arabidopsis thaliana AteIF3A gene is transcribed in highly proliferating tissues, its protein product localizes to cytoplasm and around pollen vegetative cell nucleus and observed an increased frequency of defective pollen grains and defects in seed formation in plants with T-DNA insertion localized to the AteIF3A gene. We also produced stable transgenic Nicotiana tabacum lines expressing heterologous AteIF3A...

Les cascades de Mitogen-Activated Protein Kinases (MAPKs) sont présentes chez tous les eucaryotes et permettent la transduction des signaux de l’extérieur vers l’intérieur de la cellule. Chez les végétaux, elles sont très abondantes et actives dans une multitude de processus, autant lors de la réponse aux stress que lors du développement. Elles fonctionnent comme un système de phosphorelais, se transférant un groupement phosphate d’une protéine à l’autre, de la MAPKKK à la MAPKK (MKK), puis de la MKK à la MAPK (MPK) et finalement, de la MPK vers des facteurs de transcription ou toute autre protéine qui permettra un changement au niveau de la réponse cellulaire. Depuis quelques années, plusieurs membres de la grande famille des MAPKs ont été étudiés pour leur rôle dans la reproduction sexuée des végétaux. Des mutants ont été caractérisés, mais jusqu’à maintenant, peu de voies complètes ont été décelées. Des précédents travaux dans le laboratoire ont démontré que deux MAPKKKs, de la sous-famille des MEKKs, ScFRK1 et ScFRK2, sont importantes pour le développement normal de l’ovule et du pollen chez Solanum chacoense, une espèce de pomme de terre sauvage diploïde. Sachant que les mutants des gènes les plus orthologues chez Arabidopsis thaliana ne possèdent pas les mêmes phénotypes, nous avons émis l’hypothèse que les Solanacées, du moins S. chacoense, possèdent une famille de MAPKKKs différente, qui n’est pas présente chez A. thaliana. Nous avons donc analysé les génomes/transcriptomes/protéomes de 15 espèces issues de différents clades du règne végétal afin d’étudier les relations phylogénétiques à l’intérieur de la sous-famille des MEKKs. Cela nous a permis d’observer que ScFRK1 et ScFRK2 ne sont pas seuls, mais sont inclus dans un groupe monophylétique que nous avons nommé la classe des FRKs (FRK pour Fertilization-Related Kinase). De plus, nous avons observé une expansion considérable de cette classe chez les Solanacées, comparativement à d’autres dicotylédones comme le peuplier, la vigne ou le coton. La classe des FRKs est absente chez les monocotylédones étudiées (riz et maïs) et ne possède qu’un seul membre (une FRK primitive) chez l’angiosperme basal Amborella trichopoda. Cette analyse phylogénétique des MEKKs nous a poussés à nous poser des questions sur l’origine de la classe des FRKs ainsi que sur son rôle au sein des Solanacées. Dans un deuxième temps, nous avons fait la caractérisation fonctionnelle de ScFRK3, un troisième membre de la classe des FRKs chez S. chacoense, aussi impliqué dans le développement des gamétophytes mâle et femelle. Du patron d’expression jusqu’à l’établissement d’une voie de signalisation potentielle, en passant par la caractérisation phénotypique des mutants, plusieurs expériences ont été réalisées dans le but de comprendre le rôle de ScFRK3 au niveau de la reproduction chez S. chacoense. Dans un contexte plus global, il est important de se questionner sur les rôles semblables, mais forcément différents, des trois membres de la famille FRKs qui ont été caractérisés jusqu’à présent. / Mitogen-Activated Protein Kinases (MAPKs) signaling cascades are found in all Eucaryotes and allow signal transduction from the outside of the cell to the inside. In plants, they are particularly numerous and play roles in several signaling processes, including stress responses and response to developmental cues. Their system involves a phosphorelay: they interact with each other to transfer a phosphate group. It starts with an activated MAPKKK, which transfers the phosphate group to a MAPKK (MKK), then this MKK transfers the signal to a MAPK (MPK), which ends this relay by phosphorylating transcription factors or any other proteins that will, in a way or an other, change the cell response according to the signal. During the last few years, many MAPKs members have been studied for their role in plants sexual reproduction. Some mutants were characterized, but until now, our knowledge of complete signaling cascades is very limited. Previous studies in our lab have shown that two MAPKKKs from the MEKK subfamily, ScFRK1 and ScFRK2, are important for male and female gametophytes development in Solanum chacoense, a wild diploid potato species. Genes that are the most orthologous to ScFRK1 and ScFRK2 in Arabidopsis thaliana, AtMAPKKK19, 20 and 21, do not seem to play the same roles in reproduction, which led us to make the hypothesis that in solanaceous species, at least in S. chacoense, there is one MAPKKK family that is different and not present in A. thaliana. At first, we did analyze the genomes/transcriptomes/proteomes of 15 species from different clads of the plant kingdom to find all the members of the MEKK subfamily of MAPKKKs in order to study their phylogenetic relationship. We then observed that ScFRK1 and ScFRK2 are included in a large monophyletic group which was called the FRK class (Fertilization Related Kinase). Moreover, we also observed that this class has considerably expanded within the solanaceous species, compared to other species like A. thaliana, poplar, cotton or grape vine. The FRK class is totally absent in the monocot species studied (rice and maize) and only one member is found in the basal angiosperm Amborella trichopoda. This phylogenetic analysis led us to ask questions about the origins of the FRK class and its role inside the Solanaceae family. Secondly, we characterized ScFRK3, a third member of the FRK class in S. chacoense, which is also involved, as its two FRK sisters, in male and female gametophytes development. From its expression pattern to the establishment of a potential signaling cascade, analysis and phenotyping of ScFRK3 mutant lines, many experiments were realized in order to understand the role of ScFRK3 in S. chacoense sexual reproduction. Overall, the appearance of this new and expanded class of MEKKs questions its specific role in comparison to other species that have much lesser members, mainly when compared to the model plant A. thaliana, which harbor only a fifth of the FRKs found in solanaceous species.

N-acylethanolamines (NAEs) with C12-C18 acyl chain are ubiquitous in seed plants and play a role in mediating abscisic acid (ABA)-dependent or -independent responses to stress. In moss Physcomitrella patens, using selective lipidomics approach, we recently identified the occurrence of anandamide or N-arachidonylethanolamide (NAE 20:4) and its precursors that were previously not reported in plants. Occurrence of anandamide in moss provides us with a unique opportunity to address if early land plants retained NAE-mediated signaling mechanism that is akin to animals but not to vascular plants. It is hypothesized that a distinctive NAE profile and metabolic pathway occurs in P. patens. To this extent, putative genes that might be responsible for anandamide metabolic pathway were identified and their expression levels were determined for three developmental stages of moss. The NAE metabolite levels and transcript levels for putative genes were higher in protonema stage and anandamide showed higher growth inhibitory effects, chlorophyll reduction, and putative gene induction than NAE 12:0, compared to ABA, when applied exogenously.

myo-Inositol-1,2,3,4,5,6-hexakisphosphate (InsP6), also called phytic acid, is ubiquitous in eukaryotic cells and the most abundant inositol phosphate derivative. Loblolly pine (LP, Pinus taeda) constitutes the primary commercial species in the southern forest of U.S. Somatic embryogenesis (SE) is an effective technique to maintain the desirable genetic composition of the progeny and to accomplish the efficiency of propagation. SE can also serve as a tool for study of plant development. Unlike angiosperm embryos with attached cotyledons as seed storage organs, the diploid conifer embryo is surrounded by the unattached haploid female gametophyte (FG). In LP SE, FG tissue is absent in the embryogenic tissue culture. We found that extracts from early-stage FG stimulate growth and multiplication of early-stage somatic embryos, whereas FG water extracts from late stage contain substance(s) inhibitory to early-stage somatic embryo growth (DeSilva et al., 2007). We now present the isolation and identification of the inhibitory substance as InsP6 by means of water extraction, two gel filtrations and two ion exchange FPLC chromatographies. The results represent the first complete structural characterization of InsP6 from a natural product using LC/MS, LC/MS/MS, exact MS, 1D- and 2D-NMR analyses. We also report that there is a good correlation between the amount of InsP6 purified from FG tissue (1.3 nmoles per full-term FG) and the amount of InsP6 which inhibits somatic embryo growth. This novel approach of isolating and characterizing InsP6 from plant tissue, and investigating its role on SE can allow us to improve SE technology by circumventing current bottleneck, to elucidate enigmatic functions of InsP6 in plants, and most importantly, to utilize this molecule properly.