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A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2015. / External pollen grain morphology has been widely used in the taxonomy and systematics of flowering plants. The eurypalynous family Acanthaceae is a notable example of a group where these pollen diversities have proved useful in determining relationships between taxa. However, internal pollen wall features have received far less attention due to the difficulty of examining the underlying exine from which the external sculpturing is derived. Consequently, internal wall features have thus far not been used in formulating existing classifications. A new technique involving precise cross sectioning or slicing of pollen grains at a selected position, using a focused ion beam-scanning electron microscope (FIB-SEM), was used on 39 species of Acanthaceae to examine the internal pollen wall structure and identify features of potential systematic relevance. Five basic internal wall structures were described in this study. The study also showed that similar external pollen wall features may have distinctly different underlying structures.

Glutamine (Gln), a major nitrogen source in plants, is considered a central intermediate that coordinates carbon-nitrogen assembly for plant growth and development. To maintain a sufficient Gln supply, plant cells employ glutamine synthetases (GS), including cytosolic GS1 and plastidic GS2 for Gln production. Previous work has shown that the <i>GS1</i> is responsive to various environmental stresses. This study demonstrated the involvement of <i>GS1</i>s in Gln homeostasis and the role of GS1 in abiotic stress tolerance in <i>Arabidopsis</i>. The <i>GS1</i> family is comprised of five isoforms in <i>Arabidopsis thaliana</i>. Gene expression profiling showed that <i>GLN1;1, GLN1;3</i> and <i>GLN1;4</i> had similar expression patterns and were upregulated by abiotic (salinity and cold) stresses, whereas <i>GLN1;2</i> exhibited constitutive expression and no <i>GLN1;5</i> transcript was detected under any of the conditions tested. Null T-DNA insertion mutants for the five <i>GS1</i> genes were obtained. Only the <i>gln1;1</i> mutant displayed enhanced sensitivity to a GS inhibitor, phosphinothricin, and to cold and salinity treatments, suggesting a nonredundant role for GLN1;1. Increased stress sensitivity in <i>gln1;1</i> was associated with accelerated accumulation of reactive oxygen species (ROS), particularly in chloroplasts. To better understand the role of cytosolic GS isoforms, we generated two different triple mutant combinations. Triple mutant <i>gln1;1/gln1;2/gln1;3</i> showed reduced growth at an early stage. The <i>gln1;1/gln1;3/gln1;4</i> mutant is pollen lethal, indicating an essential role of Gln in plant gametophyte development. Collectively, our results establish a link between cytosolic Gln production, ROS accumulation, plant stress tolerance and development.

Glutamine (Gln), a major nitrogen source in plants, is considered a central intermediate that coordinates carbon-nitrogen assembly for plant growth and development. To maintain a sufficient Gln supply, plant cells employ glutamine synthetases (GS), including cytosolic GS1 and plastidic GS2 for Gln production. Previous work has shown that the <i>GS1</i> is responsive to various environmental stresses. This study demonstrated the involvement of <i>GS1</i>s in Gln homeostasis and the role of GS1 in abiotic stress tolerance in <i>Arabidopsis</i>. The <i>GS1</i> family is comprised of five isoforms in <i>Arabidopsis thaliana</i>. Gene expression profiling showed that <i>GLN1;1, GLN1;3</i> and <i>GLN1;4</i> had similar expression patterns and were upregulated by abiotic (salinity and cold) stresses, whereas <i>GLN1;2</i> exhibited constitutive expression and no <i>GLN1;5</i> transcript was detected under any of the conditions tested. Null T-DNA insertion mutants for the five <i>GS1</i> genes were obtained. Only the <i>gln1;1</i> mutant displayed enhanced sensitivity to a GS inhibitor, phosphinothricin, and to cold and salinity treatments, suggesting a nonredundant role for GLN1;1. Increased stress sensitivity in <i>gln1;1</i> was associated with accelerated accumulation of reactive oxygen species (ROS), particularly in chloroplasts. To better understand the role of cytosolic GS isoforms, we generated two different triple mutant combinations. Triple mutant <i>gln1;1/gln1;2/gln1;3</i> showed reduced growth at an early stage. The <i>gln1;1/gln1;3/gln1;4</i> mutant is pollen lethal, indicating an essential role of Gln in plant gametophyte development. Collectively, our results establish a link between cytosolic Gln production, ROS accumulation, plant stress tolerance and development.

Orientador: Simone de Pádua Teixeira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T13:42:31Z (GMT). No. of bitstreams: 1 Capucho_LianaCarneiro_D.pdf: 7485279 bytes, checksum: b54a3fba348638fec00612447ae9cdb5 (MD5) Previous issue date: 2014 / Resumo: Dentre os diversos tipos de agrupamento polínico, encontrados em 42 famílias de angiospermas, as políades são de interesse especial, pois são registradas para apenas quatro destas famílias e sua ocorrência pode ser associada a uma redução no número de grãos de pólen por antera em uma espécie. Em Leguminosae, a maior em número de espécies e a mais amplamente distribuída dentre as quatro famílias com políades, essas estruturas ocorrem na subfamília Mimosoideae. Este trabalho apresenta dados sobre a origem, o desenvolvimento e a diversidade morfológica das políades, em nível estrutural e ultraestrutural (Capítulo 1); sobre a origem do adesivo polínico em Calliandra brevipes, substância encontrada tipicamente em políades de espécies do gênero; além de dados sobre a origem e desenvolvimento da políade nesta espécie (Capítulo 2, já publicado); a morfologia e fertilidade polínica em espécies poliembriônicas de Inga (Capítulo 3); e um estudo aprofundado da morfologia incomum das políades em Parkia, em nível estrutural e ultraestrutural (Capítulo 4). As políades são estruturas peculiares e ainda muito pouco estudadas, e o presente trabalho vem prover dados essenciais para a compreensão da origem e morfologia destas estruturas, e de sua funcionalidade na reprodução de espécies da subfamília Mimosoideae em Leguminosae. Para um entendimento mais completo acerca da função, valor adaptativo e seleção dessas estruturas, com ocorrência tão restrita a determinados grupos de plantas, estudos acerca da fisiologia do pólen, interação pólen-pistilo e de viabilidade de embriões formados após a fertilização dos óvulos, são requeridos / Abstract: Among all different types of pollen aggregation, reported for 42 angiosperm families, polyads are of great interest, because they are reported for only four of these families and it is associated to a reduction on number of pollen grains per anther in a species. Among those four families, Leguminosae stands out because it is the most species-rich family and widely spread. In Leguminosae, polyads often occur in the subfamily Mimosoideae. This study highlighted new information on the origin, development and morphological diversity of the polyads, employing anatomic and ultrastructural analyses (Chapter 1); origin of pollen adhesive in Calliandra brevipes, sticky substance tipically found in Calliandra polyads, in addition to data on polyad origin and development (Chapter 2, already published); polyad morphology and fertility in polyembrionic species of Inga (Chapter 3); and a meticulous analysis of the peculiar morphology of Parkia polyads (Chapter 4). Polyads are peculiar and still not well-known structures, and this study aims to contribute with essential data for its origin and morphology understanding, and its functionality in the reproduction of species comprised by subfamily Mimosoideae, in Leguminosae. For a more complete understanding on the function, adaptive value and selection of these structures, that are restricted to certain groups of plants, studies are required on the physiology of pollen, pollen-pistil interaction and viability of embryos formed after fertilization of the ovules / Doutorado / Biologia Vegetal / Doutora em Biologia Vegetal

Cornichon (CNI) proteins are a conserved family of proteins among eukaryotes, from Erv14 in the yeast Saccharomyces cerevisiae to CNI homologs (CNIHs) in mammals and plants. Erv14 functions as a cargo receptor of coat protein complex II (COPII) for protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus, en route to their final destinations. By interacting with specific cargo proteins, CNI proteins regulate key steps of embryo polarity in Drosophila, budding in yeast, and synaptic transmission in the mammalian brain. However, we have very limited understanding of plant CNIHs. Positive-strand RNA viruses assemble their viral replication complexes (VRCs) at specific host organelle membranes. With a better understanding of host factors involved in targeting viral replication proteins to the preferred organelles, we expect to block trafficking of viral replication proteins and thus, viral infection, by manipulating the required host proteins. Brome mosaic virus (BMV) is a model of positive-strand RNA viruses and its replication can be recapitulated in yeast. Importantly, BMV replication protein 1a is the only required viral protein to form VRCs at the perinuclear ER membrane in yeast. I demonstrate that Erv14 and COPII coat proteins are required for targeting BMV 1a to the perinuclear ER in yeast, suggesting a novel function of COPII vesicles in protein trafficking to the perinuclear ER membrane and in the BMV VRC formation. As for cellular functions, I show that plant CNIHs complement the defective distribution of BMV 1a in yeast mutant lacking Erv14. Taking advantage of Arabidopsis thaliana knockout mutants and knockdown of gene expression in Nicotiana benthamina, I also discover that CNIHs unexpectedly play crucial roles in pollen development, infection of a bacterial pathogen, and maintenance of ER tubules. I further confirm that CNI proteins are also required for maintaining ER tubules in yeast, suggesting a novel and conserved role in shaping ER morphology. Therefore, these findings indicate the functional diversity and redundancy of CNI proteins in key cellular processes and suggest a novel strategy to control plant pathogenic viruses and bacteria by manipulating plant CNIHs. / Ph. D. / Many cellular proteins play important roles in plant development but unfortunately are hijacked by plant viral, bacterial, and/or fungal pathogens for their infections. Cornichon (CNI) proteins are a conserved family of proteins and a great example that is involved in both plant development and plant pathogen infection. CNI protein was first described in a <i>Drosophila</i> mutant. Only 3% of mutant cells survived, but showed abnormal phenotype in abdominal segmentation with a similar shape of “pickle” during embryo development. Later on, this family of proteins was well studied in yeast and mammals but rarely studied in plants. Erv14, one of CNI proteins in yeast, is a cargo receptor of coat protein complex II (COPII) vesicles that participate in cellular early secretory pathway. COPII vesicles serve as cellular carriers to recruit cargo proteins from the endoplasmic reticulum (ER) membrane and depart for the Golgi apparatus, en route to their final destinations for proper cellular processes. In this dissertation, I have discovered that Erv14 and COPII components are unexpectedly involved in targeting a replication protein of a plant RNA virus to the perinuclear ER membrane, instead of the Golgi apparatus, suggesting a novel function of COPII in targeting proteins to the perinuclear ER. Erv14 has never been shown as involved in viral infection and thus, my work has identified a new host protein required for viral infection. I have further explored the cellular functions of CNI proteins in plants, and found that plant CNI proteins play significant roles in maintaining cellular ER network, supporting normal pollen development, and bacterial pathogen infection. Therefore, plant CNI proteins function similarly as Erv14 to recruit various cargo proteins into COPII vesicles en route to their final destinations for proper cellular processes. These cellullar processes may include, but are not limited to: ER morphology maintenance, pollen development, and plant immune response to pathogen infection. Furthermore, it is possible to develop a novel strategy to make plants resistant to plant viruses and/or bacteria by manipulating plant CNIHs.