O processo de fertilização está intimamente relacionado ao íon cálcio. Há relatos que bloqueadores de canais de íons cálcio voltagem-dependentes, utilizados na terapia anti-hipertensiva, podem levar à infertilidade masculina, mas poucos relatos são os de seus efeitos sobre o oócito. O objetivo deste experimento foi verificar a influência dos bloqueadores de canais de cálcio voltagem-dependentes do tipo-L, verapamil, nifedipina e diltiazem, sobre os gametas masculino e feminino. Machos e fêmeas hamsters foram utilizados em testes in vivo e in vitro. Foram realizados tratamentos in vitro no Experimento I: espermatozóides foram capacitados em meios contendo três concentrações de bloqueadores de canais de cálcio e avaliados quanto à capacitação espermática, hiperatividade e fertilidade in vitro. Oócitos foram tratados previamente por 30 minutos com quatro concentrações de antagonistas de canais de cálcio e submetidos a teste de fertilização in vitro e à avaliação do comportamento dos grânulos corticais. No Experimento II, os bloqueadores de canais de cálcio foram administrados, duas vezes ao dia, por 60 dias nos machos e por 30 dias nas fêmeas, que foram analisados pela fertilidade in vivo, capacitação e hiperatividade espermáticas, fecundação in vitro e exocitose dos grânulos corticais, e microscopia eletrônica de transmissão nos oócitos tratados in vivo. No Experimento III, a concentração intracelular de cálcio foi mensurada em espermatozóides e oócitos na presença dos bloqueadores de canais de cálcio. Como resultados, no Experimento I, os fármacos atuaram de forma dose-dependente, os espermatozóides tratados apresentaram menor taxa de capacitação espermática, de hiperatividade e de fecundação in vitro. Os oócitos tratados mostraram menor taxa de fertilização in vitro em relação ao controle e, nos grupos verapamil e diltiazem, exocitose parcial dos grânulos corticais e ativação não completa dos oócitos. O Experimento II demonstrou que os machos dos grupos tratados tiveram menor taxa de capacitação espermática, de hiperatividade e de fertilidade in vitro em relação ao grupo controle, e nos grupos verapamil e nifedipina houve uma redução da fertilidade refletida no número de fêmeas paridas em relação ao grupo controle e diltiazem. A fecundação in vivo das fêmeas tratadas não variou entre os grupos; porém, a taxa de fertilização in vitro dos oócitos de fêmeas tratadas in vivo com os antagonistas foi menor em relação ao controle; tanto a exocitose dos grânulos corticais quanto a microscopia eletrônica não foram detectadas diferenças entre os grupos. As ninhadas de fêmeas que receberam a terapia no início da gestação apresentaram menor peso ao nascimento e maior taxa de mortalidade. No Experimento III, notou-se que os fármacos causaram um bloqueio do influxo de cálcio extracelular tanto em espermatozóides como em oócitos. Concluiu-se que, tanto espermatozóides como oócitos, possuem em sua membrana plasmática canais de íons cálcio sensíveis aos bloqueadores de canais de cálcio voltagem-dependentes do tipo-L. Nos tratamentos in vitro, estes fármacos atuaram negativamente na capacidade de fecundação dos espermatozóides e dos oócitos. A terapia com os bloqueadores de canais de cálcio reduziu a fertilidade nos machos, mas não nas fêmeas. A administração dos bloqueadores no início da gestação causou efeitos teratogênicos / Fertilization process is closely related with calcium ion. There are reports about voltage-dependent calcium ion channel blockers of anti-hypertension therapeutic use causing male infertility, but there are few reports about such effects on the oocyte. The main goal of this experiment was to check the influence of verapamil, nifedipine and diltiazem (L-type voltage-dependent calcium ion channel blockers) on spermatozoa and oocytes. Male and female hamsters were used for in vivo and in vitro experiment. In vitro treatments were preformed in the Experiment I, in which: spermatozoa were capacitated in three concentrations of the three calcium ion channel blockers and they were evaluated for sperm capacitation, hyperactivity and in vitro fertility. Oocytes were incubated for 30 minutes in four concentrations of the same calcium channel antagonists and submitted to in vitro fertilization and evaluation of cortical granules exocytosis. In the Experiment II, calcium channel blockers were supplied twice per day to males during 60 days and to females during 30 days. After this period, drug effects on the animals were evaluated through in vivo fertility, sperm capacitation and hyperactivity, in vitro fertilization, cortical granules exocytosis and transmission electronic microscopy of in vivo treated oocytes. In the Experiment III, the intracellular calcium concentrations were measured in spermatozoa and oocytes in the presence of calcium ion channel blockers. As results, in the Experiment I, the antagonists presented dose-dependent effects, affecting sperm capacitation rate, hyperactivity and in vitro fertilization rate on treated spermatozoa. Treated oocytes presented lower in vitro fertilization rate when compared to the control group and, in the verapamil and diltiazem groups, cortical granules exocytosis was partial and oocyte activation was not concluded. Experiment II showed that male hamsters of treatment groups presented lower sperm capacitation rate, hyperactivity and in vitro fertilization rate and there were fewer pregnant females, demonstrating a reducing male fertility in verapamil and nifedipine groups when compared to the control and diltiazem groups. In vivo fertility of treated females did not change among groups; however, in vitro fertilization rates in oocytes of calcium antagonists in vivo treated females were lower. Nor cortical granules exocytosis neither electronic microscopy detected differences among groups. Progeny of females that received the therapy in the beginning of pregnancy presented lower birth weight and higher mortality rate. In the Experiment III, the antagonists blocked extracellular calcium influx in spermatozoa and in oocytes. It is concluded that, there were calcium ion channels with sensibility to L-type voltage-dependent calcium ion channel blockers in the plasmatic membrane of spermatozoa and oocytes. In in vitro treatments, these antagonists influenced negatively spermatozoa and oocytes capacity of fertilization. Calcium channel blocker therapy reduced the fertility in males, but not in females. Calcium channel blockers supplied in the beginning pregnancy caused teratogenic effects

Douglas-fir develops a megagametophyte regardless of the pollination state of the ovule, whereas many other conifers develop a megagametophye in response to polli- nation. Megagametophytes in unfertilized ovules degrade two weeks following fertil- ization of the surrounding population. This is mediated by programmed cell death (PCD). Pollinated and unpollinated megagametophytes were dissected from Douglas- fir cones and extracted for RNA, which was then used as input for sequencing. A transcriptome was assembled from this data and expression levels were calculated. The data were fitted to quadratic regressions to produce coexpression groups. There is no clear upregulation of PCD effectors in the unpollinated megagametophyte. Po- tential regulators of megagametophyte fate are present in the data. Some are as- sociated with ABA signalling and proanthocyanadin biosynthesis while others share similarity to known regulators of PCD. Seed development processes are represented in the expression data, which support current knowledge of conifer seed development and provide targets for research. / Graduate / 0369 / 0309 / 0817 / igboyes@gmail.com

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Asteraceae has attracted many embryologists because of its high variability of the embryological features. Nevertheless, nothing is known about the embryology of the species basal subfamily, Barnadesioideae. Thus, this paper aims to describe the processes of ginosporogenesis and ginogametogenesis in two species of Barnadesioideae, Sclechtendalia luzulaefolia, considered basal and sister to the other species that belong to family and Dasyphyllum brasiliense whose genus was also sometimes considered basal. Both species are native to Rio Grande do Sul. S. luzulaefolia was collected in São Pedro do Sul, RS, Brazil and D. brasiliense in Santa Maria, RS, Brazil and in São Pedro do Sul, RS, Brazil. The corolla is the first floral whorl to arise in both species, followed by the androecium, gynoecium and calyx in S. luzulaefolia; calyx, androecium and gynoecium in D. brasiliense. Tenuinucellate incomplete ovule, asymmetric integument and dual origin endothelium (funicule and integument) are new results for Asteraceae. The archesporium is usually unicellular, but ovules with two archesporial cells were observed. In S. luzulaefolia two types of tetrad are present, the linear tetrad being more commom than the tetrad with paired ginospores. The ginophyte development pattern in D. brasiliense can be an indication of its derived position in Barnadesioideae, since it has bisporic ginophyte of the Allium type and in S. luzulaefolia where ginophyte is monosporic of the Polygonum type, integrating these features simultaneity of mitotic cycles and cellularization of ginófito. The mature ginophyte is 4-cell, in both species, because of the degeneration of the antipodes, which can configure a plesiomorphic character for the family. The synergids extend into the micropile canal and they have fibrillar apparatus of typical morphology for Asteraceae. Labyrinthine walls of the middle cell and flange type wall thickening are present only in S. luzulaefolia in cells of the endothelium and micropilar canal. Therefore, common characteristics were identified for S. luzulaefolia and D. brasiliense which may be considered basal for Asteraceae and diagnostic for Barnadesioideae, as well as embryological characters that corroborate the position derived from D. brasiliense in this subfamily. / Asteraceae despertou interesse de muitos embriologistas devido a grande variabilidade das características embriológicas. Apesar disso, nada é sabido sobre a embriologia nas espécies de Barnadesioideae, sua subfamília basal. Sendo assim este trabalho objetiva descrever os processos de ginosporogênese e ginogametogênese em duas espécies de Barnadesioideae, Sclechtendalia luzulaefolia, considerada basal e irmã das demais pertencentes a família, e Dasyphyllum brasiliense cujo o gênero por vezes também foi considerado basal. Ambas as espécies são nativas do Rio Grande do Sul. S. luzulaefolia foi coletada em São Pedro do Sul, RS, Brasil e D. brasiliense em Santa Maria, RS, Brasil e em São Pedro do Sul, RS, Brasil. A corola é o primeiro verticilo floral a surgir em ambas as espécies, seguida pelo androceu, cálice e gineceu em S. luzulaefolia e pelo cálice, androceu e gineceu em D. brasiliense. O rudimento seminal tenuinucelado incompleto, a presença de tegumento assimétrico e de endotélio de origem dupla (funicular e tegumentar) são resultados inéditos para Asteraceae. O arquespório é usualmente unicelular, mas rudimentos seminais com duas células arquesporiais foram observados. Em Sclechtendalia luzulaefolia dois tipos de tétrade estão presentes, sendo a tétrade linear mais comum do que a tétrade com ginósporos pareados. O tipo de desenvolvimento do ginófito em D. brasiliense pode ser um indício de sua posição derivada para Barnadesioideae, já que possui ginófito bispórico do tipo Allium e em S. luzulaefolia o ginófito é monospórico do tipo Polygonum, integrando-se a estas características a simultaneidade dos ciclos mitóticos e da celularização do ginófito. O ginófito maduro é 4-celular, em ambas as espécies, devido degeneração das antípodas, o que pode configurar um caráter plesiomórfico para a família. As sinérgides estendem-se para o interior do canal micropilar e possuem um aparelho fibrilar de morfologia típica para Asteraceae. Somente em S. luzulaefolia foi observada a presença de parede labiríntica da célula média e espessamento de parede tipo flange em células do endotélio e canal micropilar. Portanto, neste trabalho foi possível identificar características comuns à S. luzulaefolia e D. brasiliense que podem ser considerados basais para Asteraceae e diagnósticas para Barnadesioideae, bem como caracteres embriológicos que corroboram a posição derivada de D. brasiliense nesta subfamília.

Astragalus ampullarioides and A. holmgreniorum are two rare endemics of southwestern Utah. Over two consecutive field seasons (2009-2010) we examined pre-emergent reproductive success, based on F/F and S/O ratios, from populations of both Astragalus ampullarioides and A. holmgreniorum, estimated the density of the soil seed bank of A. holmgreniorum as a measure of potential post-emergent reproductive success, and estimated seed persistence within the soil seed bank. Fruit/flower (F/F) ratios and seed/ovule (S/O) ratios varied significantly between populations and among years in both species, and showed low reproductive output in both taxa. In Astragalus ampullarioides F/F and S/O were 0.06±0.01 and 0.16±0.02, respectively (2009), and 0.14±0.01 and 0.41±0.02, respectively (2010). For Astragalus holmgreniorum F/F and S/O ratios were 0.11±0.01 and 0.38±0.02, respectively (2009), and 0.23±0.01 and 0.66±0.02, respectively (2010). Although Astragalus holmgreniorum exhibited a low soil seed bank density (4.3 seeds m-2), seed persistence data showed low a low percentage of seeds germinated during the first year in the soil seed bank. Seeds remaining in the seed bank maintained high percent viability. Soil seed persistence of Astragalus ampullarioides differed from A. holmgreniorum in that a high percentage of seeds germinated during the first year in the soil seed bank. A high percentage of viability in ungerminated seeds was also maintained in A. ampullarioides. Although these species differ in life histories and dependence on soil seed banks, an understanding of the strategies unique to each species will prove useful in management plans.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Le nombre de siliques par plante et le nombre de graines par silique sont les composantes du rendement du colza d'hiver qui présentent la plus grande variabilité. La production d'une graine résulte de la combinaison de plusieurs processus physiologiques, à savoir la formation des ovules et des grains de pollen, la fécondation des ovules et le développement de jeunes embryons. Un problème survenu à n'importe quelles des étapes peut entraîner l'avortement de graines ou de la silique. Le nombre potentiel d'ovules par silique et le nombre graines arrivant la maturité dépendraient de la position du dans l'architecture de plante et le temps de son apparition, mais le mode complexe de développement de colza rend difficile l'analyse des causes et effets. Dans cette étude, la variabilité des composantes du rendement suivantes est étudiée: (a) nombre d'ovules par silique, (b) nombre de graines par silique, et (c) nombre de siliques par axe en fonction d'une part, l'emplacement de la fleur dans l'inflorescence, et la position de cette dernière sur la tige, et l'autre part, le temps d'apparition de la silique, qui affectent la disponibilité d'assimilats. Basé sur les processus biologiques de la fertilité des fleurs, un modèle probabiliste est développé pour simuler le développement des graines. Le nombre de grains de pollen par fleur peut être déduit par le modèle et ainsi que les facteurs qui influent le rendement. Des expériences de terrain ont été menées en 2008 et 2009. Le nombre et la position des fleurs qui s'épanouissaient dans l'inflorescence ont été enregistrés sur la base des observations tous les deux à trois jours pendant la saison de floraison. Différents états trophiques ont été créés par tailler de la tige principale ou des ramifications à étudier l'effet de l'assimilation de la compétition. Les résultats montrent que la quantité d'assimilâtes disponibles a été le principal déterminant de la production de graines et de siliques. La répartition d'assimilâtes a été sensiblement affectée par l'emplacement de silique au sein d'une inflorescence et la location de l'inflorescence sur la tige colza. En outre, le paramètre de la distribution du nombre de pollen a indiqué que la production de graines pourrait être limitée par la pollinisation. La réduction de la viabilité des ovules pourrait entraîner la diminution du nombre de siliques et le nombre de graines par silique à l'extrémité de l'inflorescence. Le modèle proposé pourrait être un outil pour étudier la stratégie de l'amélioration du rendement des plantes à fleurs.

The goals of this research project were to investigate aspects of pollination biology of two native boreal species: Vaccinium myrtilloides (Canadian blueberry) and Vaccinium vitis-idaea (lingonberry) in central Saskatchewan. Accordingly, surveys of insect taxa visiting the flowers were performed, and determination of the effectiveness of these insect taxa to serve as pollinators was measured through pollen deposition and pollen tube growth in the style. Accompanying fieldwork, and morphological and anatomical studies were done for the two food rewards offered by flowers of both Vaccinium species: pollen released through poricidal anthers and nectar secreted from the nectary atop the inferior ovary. Pollen-ovule (P/O) ratios were determined for the two study species in Saskatchewan (V. myrtilloides, V. vitis-idaea) as well as for five other Vaccinium species from eastern Canada (Nova Scotia – V. angustifolium, V. boreale, V. caespitosum, V. corymbosum, and V. uliginosum). Pollen, released at maturity as tetrads, were converted to total pollen grains per flower to yield P/O ratios ranging from 238 (V. caespitosum) to 2,008 (V. vitis-idaea), but 736 for the latter in Saskatchewan. These P/O ratios are indicative of a breeding system ranging from facultative autogamy to facultative xenogamy. Additionally, the structure of mature stamens and pollen tetrads was studied in V. myrtilloides and V. vitis-idaea. Each anther was functionally bilocular; had a single-cell thickness (i.e., epidermis) with regularly occurring papillae; lacked an endothecium; and possessed two distal, hollow tubules each terminating in a pore. Overall pollen grain viability was 76-97% (V. myrtilloides) and 51-93% (V. vitis-idaea), with about 20% of tetrads having only 1-3 grains viable, and 12% and 27% of tetrads entirely non-viable in V. myrtilloides and V. vitis-idaea, respectively. Pollen tetrads occasionally were connected by a sticky substance resembling pollenkitt, but viscin threads were absent. One instance of precocious (in situ) germination of tetrads was recorded within anthers of V. myrtilloides. The floral nectary was a disk of secretory tissue situated between the stamens and the style. The epidermis possessed solitary stomata that were variable in number, but not different between V. myrtilloides and V. vitis-idaea. The nectary was vascularized by phloem alone; many traces were found for V. myrtilloides throughout the nectary, whereas V. vitis-idaea had few traces at the nectary base, concentrated at the inner side of the disk closest to the style base. Young sclerenchyma cells were found throughout the nectary parenchyma. Nectar production started on the day of anthesis for both species, although many flowers of V. vitis-idaea appeared to have no measureable nectar at that time. V. myrtilloides produced a larger range of nectar solutes per flower (0 - 3684.1 μg), than V. vitis-idaea iii (1.29 to 1147.62 μg) over both years; nectar volumes per flower never exceeded 5 μL. Nectar was measured daily in flowers aged 1 – 4 days in 2010 and 1 – 6 days in 2011, however, over the two years at the same study site there was no clear pattern of secretion and reabsorption throughout flower life for V. myrtilloides, and only a gradual increase for V. vitis-idaea as flowers aged. Insect visitors to flowers surveyed in 2010 included a large proportion of honeybees (Apis mellifera) as visitors to both species, whereas in 2011 there were no honeybees present at the field site. There was a larger proportion of hoverflies (Syrphidae) found on the flowers of V. vitis-idaea than on V. myrtilloides. Other visitors to V. myrtilloides were bees (Bombus, Andrena, Osmia, Colletes) and wasps (Vespidae), whereas flowers of V. vitis-idaea were visited by bees (Bombus, Andrena, Osmia, Lasioglossum, Colletes, Hylaeus), an ant (Formicidae) and a butterfly (Lycaenidae). Bombus spp. were shown to be pollinators of V. myrtilloides. Andrena spp. were probable pollinators, whereas honeybees appeared to be poor pollinators. Bombus spp. seemed probable pollinators of V. vitis-idaea and hoverflies to be barely more than visitors, though small sample sizes did not allow for conclusive evidence. The time that an insect spent on a virgin flower had no relationship to the pollination result. Among individuals of various Bombus spp. that did or did not sonicate flowers of V. myrtilloides, the action of “buzz pollination” was shown to result in an increase in the number of pollen tetrads deposited and in pollen tube growth.

Tesis por compendio / [ES] Como precursores de las semillas, los óvulos representan un órgano fundamental durante el ciclo de vida de las plantas. Debido a su importancia, el desarrollo del óvulo ha sido estudiado durante décadas desde un punto de vista morfológico y molecular, lo que ha permitido dilucidar la compleja e intrincada red de regulación genética que lo rige. En concreto, la iniciación del óvulo está controlada por las hormonas vegetales auxinas, citoquininas y brasinoesteroides (BRs), siendo todas ellas reguladoras positivas del número de óvulos. Recientemente demostramos que las giberelinas (GAs) modulan negativamente el número de óvulos mediante la desestabilización de las proteínas DELLA. Sin embargo, aún debe aclararse cómo encajan las GAs y las proteínas DELLA en el modelo regulador de la iniciación de los óvulos. El trabajo presentado en esta tesis doctoral tiene como objetivo aclarar el mecanismo molecular por el cual las GAs actúan en la iniciación del óvulo. Después de una introducción general, en el Capítulo 1 mostramos que tanto las GAs como los BRs regulan el número de óvulos en Arabidopsis independientemente de los niveles de actividad de la otra hormona, lo que sugiere que las GAs y los BRs actúan de forma independiente para controlar la iniciación del óvulo. En el Capítulo 2 proporcionamos evidencias genéticas y moleculares que apuntan a que las proteínas DELLA participan en la iniciación de los óvulos mediante su interacción con el factor de transcripción CUC2 en las células placentarias. En conjunto, los hallazgos presentados aquí nos han permitido integrar a las GAs y proteínas DELLA en la red genética que guía el inicio de los primordios de óvulos. Una discusión final destaca las preguntas abiertas que aún deben abordarse para comprender completamente el control hormonal de la iniciación de los óvulos en las plantas. / [CAT] Com a precursors de les llavors, els òvuls representen un òrgan fonamental durant el cicle de vida de les plantes. A causa de la seva importància, el desenvolupament de l'òvul ha estat estudiat durant dècades des d'un punt de vista morfològic i molecular, el que ha permès dilucidar la complexa i intricada xarxa de regulació genètica que el regeix. En concret, la iniciació del òvul està controlada per les hormones vegetals auxines, citoquinines i brasinoesteroides (BRs), sent totes elles reguladores positives del nombre d'òvuls. Recentment demostrem que les gibberel·lines (GAs) modulen negativament el nombre d'òvuls mitjançant la desestabilització de les proteïnes DELLA. No obstant, encara s'ha d'aclarir com encaixen les GAs i les proteïnes DELLA al model regulador de la iniciació dels òvuls. El treball presentat en aquesta tesi doctoral té com a objectiu aclarir el mecanisme molecular pel qual les GAs actuen a la iniciació de l'òvul. Després d'una introducció general, al Capítol 1 mostrem que tant les GAs com els BRs regulen el nombre d'òvuls a Arabidopsis independentment dels nivells d'activitat de l'altra hormona, cosa que suggereix que les GAs i els BRs actuen de forma independent per controlar la iniciació de l'òvul. Al Capítol 2 proporcionem evidències genètiques i moleculars que apunten que les proteïnes DELLA participen en la iniciació dels òvuls mitjançant la seva interacció amb el factor de transcripció CUC2 a les cèl·lules placentàries. En conjunt, els descobriments presentats ací ens han permès integrar les GAs i proteïnes DELLA a la xarxa genètica que guia l'inici dels primordis d'òvuls. Una discussió final destaca les preguntes obertes que encara cal abordar per comprendre completament el control hormonal de la iniciació dels òvuls a les plantes. / [EN] As precursors of seeds, ovules represent a fundamental organ during the plant life cycle. Due to their importance, ovule development has been studied for decades from a morphological and molecular point of view, allowing the elucidation of a complex and intricate gene regulatory network governing it. Specifically, ovule initiation is controlled by the plant hormones auxins, cytokinins and brassinosteroids (BRs), all of them being positive regulators of ovule number. Recently, we demonstrated that gibberellins (GAs) negatively module ovule number by the destabilization of DELLA proteins. However, how GAs and DELLA proteins fit in the regulatory model for ovule initiation still needs to be clarified. The work presented in this PhD thesis aims to clarify the molecular mechanism by which GAs act in ovule initiation. After a comprehensive introduction, we show in Chapter 1 that both GAs and BRs regulate ovule number in Arabidopsis regardless of the activity levels of the other hormone, suggesting that GAs and BRs act independently to control ovule initiation. In Chapter 2 we provide genetic and molecular evidence pointing to DELLA proteins participating in ovule initiation by the interaction with the CUC2 transcription factor in placental cells. Collectively, the findings presented here allowed us to integrate GAs and DELLA proteins in the gene regulatory network guiding ovule primordia initiation. A final discussion highlights open questions that still need to be addressed to fully understand the hormonal control of ovule initiation in plants. / La realización de esta Tesis Doctoral ha sido posible gracias a un contrato para la Formación de Personal Investigador de la Universidad Politécnica de Valencia (durante un año y medio) y a un contrato para la Formación de Profesorado Universitario (FPU18/00331) del Ministerio de Universidades (durante dos años y medio). Las estancias breves en Chile y Francia fueron posible gracias a la financiación H2020-MSCA-RISE-2014 y a una ayuda EMBO Short-Term (STF 8961), respectivamente. El trabajo experimental ha sido financiado por los proyectos BIO2017-83138-R y PID2020-113920RB-100 del Ministerio de Ciencia e Innovación y AICO/2020/256 de la Generalitat Valenciana. / Barro Trastoy, D. (2022). Gibberellins and ovule number: a molecular mechanism [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/187756 / TESIS / Compendio

Chez les angiospermes, la reproduction passe par la double fécondation. Le tube pollinique délivre deux cellules spermatiques au sein du gamétophyte femelle. Une cellule féconde la cellule œuf pour produire un zygote; l’autre féconde la cellule centrale pour produire l’endosperme. Pour assurer un succès reproductif, le développement du gamétophyte femelle au sein de l’ovule doit établir un patron cellulaire qui favorise les interactions avec le tube pollinique et les cellules spermatiques. Pour ce faire, un dialogue doit s’établir entre les différentes cellules de l’ovule lors de son développement, de même que lors de la fécondation. D’ailleurs, plusieurs types de communications intercellulaires sont supposées suite à la caractérisation de plusieurs mutants développementaux. De même, ces communications semblent persister au sein du zygote et de l’endosperme pour permettre la formation d’un embryon viable au sein de la graine. Malgré les développements récents qui ont permis de trouver des molécules de signalisation supportant les modèles d’interactions cellulaires avancés par la communauté scientifique, les voies de signalisation sont de loin très incomplètes. Dans le but de caractériser des gènes encodant des protéines de signalisation potentiellement impliqués dans la reproduction chez Solanum chacoense, l’analyse d’expression des gènes de type RALF présents dans une banque d’ESTs (Expressed Sequence Tags) spécifiques à l’ovule après fécondation a été entreprise. RALF, Rapid Alcalinization Factor, est un peptide de 5 kDa qui fait partie de la superfamille des «protéines riches en cystéines (CRPs)», dont les rôles physiologiques au sein de la plante sont multiples. Cette analyse d’expression a conduit à une analyse approfondie de ScRALF3, dont l’expression au sein de la plante se limite essentiellement à l’ovule. L’analyse de plantes transgéniques d’interférence pour le gène ScRALF3 a révélé un rôle particulier lors de la mégagamétogénèse. Les plantes transgéniques présentent des divisions mitotiques anormales qui empêchent le développement complet du sac embryonnaire. Le positionnement des noyaux, de même que la synchronisation des divisions au sein du syncytium, semblent responsables de cette perte de progression lors de la mégagamétogénèse. L’isolement du promoteur de même que l’analyse plus précise d’expression au sein de l’ovule révèle une localisation sporophytique du transcrit. La voie de signalisation de l’auxine régule également la transcription de ScRALF3. De surcroît, ScRALF3 est un peptide empruntant la voie de sécrétion médiée par le réticulum endoplasmique et l’appareil de Golgi. En somme, ScRALF3 est un important facteur facilitant la communication entre le sporophyte et le gamétophyte pour amener à maturité le sac embryonnaire. L’identification d’un orthologue potentiel chez Arabidopsis thaliana a conduit à la caractérisation de AtRALF34. L’absence de phénotype lors du développement du sac embryonnaire suggère, cependant, de la redondance génétique au sein de la grande famille des gènes de type RALF. Néanmoins, les peptides RALFs apparaissent comme d’importants régulateurs lors de la reproduction chez Solanum chacoense et Arabidopsis thaliana. / In angiosperms, reproduction occurs through double fertilization. The pollen tube delivers two sperm cells into the female gametophyte. A first sperm cell fertilizes the egg cell to produce a zygote, while the other fertilizes the central cell to produce the endosperm. To ensure reproductive success, the development of the female gametophyte within the ovule must establish a cellular pattern allowing interaction with the pollen tube and sperm cells. To this end, a dialogue must be established amongst the various cells of the ovule during its development, as well as during fertilization. Several types of communication are suggested by the analysis of developmental mutants. These communications must persist in the zygote and endosperm to allow the formation of a viable embryo within the seed. Recent developments have helped to find signaling molecules that support cell interaction models developed by the scientific community, but the signaling pathways are far from complete. In order to characterise genes encoding signaling proteins which are potentially active during reproduction in Solanum chacoense, I undertook the expression analysis of the RALF-like genes present in a bank of ESTs (Expressed Sequence Tags) specific to the ovule after fertilization. RALF, Rapid Alcalinization Factor, is a 5 kDa peptide that is part of the superfamily of Cysteine Rich Proteins (CRPs), which play a wide variety physiological roles within the plant. This expression analysis led to a detailed analysis of ScRALF3, whose expression in the plant is largely restricted to the ovule. The analysis of ScRALF3 RNAi transgenic plants revealed a function during megagametogenesis. The transgenic plants exhibit abnormal mitotic divisions that prevent the maturity of the embryo sac. The positioning of the nuclei, as well as the timing of divisions in the syncytium, appear to be responsible for the arrest of development during megagametogenesis. Isolation of the promoter as well as more accurate analysis of transcript expression reveals localisation within the ovule sporophytic tissue. The auxin signaling pathway is also involved in the regulation of ScRALF3 expression. ScRALF3 is a secreted peptide passing via the endoplasmic reticulum and the Golgi apparatus. In summary, ScRALF3 may be an important factor facilitating communication between the gametophyte and the sporophyte to allow maturation of the embryo sac. The identification of a potential orthologue in Arabidopsis thaliana led to the characterisation of AtRALF34. The lack of a phenotype during embryo sac development, however, suggests that genetic redundancy within the family of RALF-like genes is very complex. Nevertheless, the RALF peptides appear to be important regulators during reproduction in Solanum chacoense and Arabidopsis thaliana.

Thesis (MScAgric (Horticulture))--Stellenbosch University, 2008. / Sweet cherries (Prunus avium L.) have a high chilling requirement and grow best in areas receiving >1 100 Utah chill units during winter. The main production areas in South Africa, and particularly the eastern Free State, frequently receive insufficient winter chilling and late spring frosts leading to problems of poor budburst, flowering, floral abnormalities and poor fruit set. Research was conducted on the cultivar ‘Bing’ to determine the main factors causing its low fruit set. Various trials were conducted to optimize the timing of rest breaking agents, identify suitable cross pollinizers that flower synchronously with ‘Bing’, and evaluate the influence of temperature and pollen-pistil interactions on fertilization and fruit set. Pollen biology studies using 2- to 3-year-old ‘Bing’ sweet cherry trees were conducted near Clarens, eastern Free State, during the 2005 and 2006 seasons to determine the most suitable cross pollinizer/s for ‘Bing’ and to assess the influence of temperature and pollen-pistil interactions on pollen tube growth and ovule longevity. Significant differences in pollen germination (‘rates’ deleted) occurred between pollinizers, although differences were noted in pollen performance on the stigma and style (in vivo) compared to the artificial media (in vitro), indicating a lack of correlation between in vitro germination and in vivo pollen-pistil interactions. Pollen tube growth, following cross pollination, was influenced by pollinizer genotype, temperature, and the number of pollen grains deposited on the stigma. The highest pollen tube growth rates in ‘Bing’ styles were recorded for the pollinizers ‘Black Tartarian’ (2006), ‘Lapins’ and ‘Rainier’ sweet cherry cultivars at temperatures of approximately 21°C. Temperature had the most significant influence on ovule longevity with the lower orchard temperatures extending ovule viability compared to the higher laboratory temperatures, although pollen tube growth rates were also reduced, thus shortening the effective pollination period. Cross pollination was also shown to extend ovule viability. The results indicate that ‘Black Tartarian’, ‘Lapins’ and ‘Rainier’ were the most suitable pollinizers for ‘Bing’. Hand-pollination with pollen from these donors resulted in a several-fold increase in seed set over naturally-pollinated control flowers. It appears that the principle factors causing poor fruit set in ‘Bing’ sweet cherry are premature abortion of the ovule before fertilization and inadequate transfer of sufficient viable pollen under orchard conditions. Rest breaking trials were conducted on 4-year-old ‘Bing’ sweet cherry trees on ‘Gisela® 5’ rootstock near Clarens (28°28’S; 28°19’E, 1860m) and Reitz (28°0’S; 28°28’E; 1717m) in the eastern Free State, South Africa, during the 2005 and 2006 seasons respectively. In 2005 five treatments were evaluated; viz. 1% and 2% Dormex® (hydrogen cyanamide, HCN); 1% Dormex® + 3% mineral oil; and 3% Lift® (thidiazuron and mineral oil) sprayed at three dates (29 July 2005, 5 August 2005 and 12 August 2005) preceding expected the “green-tip” stage of flower development, plus an unsprayed control. In 2006 four treatments were evaluated; viz. 1% Dormex®; 1% Dormex® + 3% mineral oil; 3% Lift® applied on three dates (26 July 2006, 7 August 2006 and 12 August 2006) and an unsprayed control. No interaction was observed between time of application and type of rest breaking agent (RBA). RBAs were effective at improving budburst and yield during both seasons with the time of application of RBAs having the most significant influence on budburst and production efficiency in ‘Bing’ sweet cherry trees. RBAs were most effective at improving vegetative budburst when applied 9 to 16 days before the (‘actual’ deleted) “green-tip” stage of flower development. Floral budburst and yield were increased by 1% Dormex® + 3% mineral oil and 3% Lift®, but results varied between seasons indicating that time of RBA application should be based on chilling accumulation and bud development stage and not based on calendar date. This current research suggests that ‘Bing’ sweet cherry is poorly suited climatically to the current production areas of the eastern Free State and short-term research needs to identify methods of improving chilling and fruit set by means of evaporative cooling and fruit set-enhancing plant growth regulators. Longer term work requires the identification of new, lower chill cultivars with improved climatic adaptation to South African conditions.