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Molecular and cellular analysis of lipid kinases involved in signalling and membrane trafficking in pollen tubes
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In flowering plants fertilization occurs when a pollen tube delivers the sperm cells to the female gametes within floral tissues. Therefore understanding of pollen tube development is fundamental for comprehension of plant reproduction. Pollen tube elongation involves many interactions, including cell-cell recognition and intracellular and intercellular signalling. These responses involve the activation of complex signalling networks and rapid synthesis and release of specific molecules. Phosphoinositides are emerging as novel second messengers in plant cells. In the classical pathway phosphatidylinositol monophosphate kinases (PIP kinases) transform phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], which has been implicated in vesicle trafficking and cytoskeletal rearrangements, crucial events in pollen tip growth. A reverse genetics approach was used to investigate the function of the Arabidopsis thaliana pollen-expressed gene encoding phosphatidylinositol-4- monophosphate 5-kinase 4 (PIP5K4). Pollen germination, tube growth, and polarity were significantly impaired in homozygous mutant plants lacking PIP5K4 transcript. In vitro, supplementation with PtdIns(4,5)P2 rescued these phenotypes. In vivo, pip5k4 null mutant allele was transmitted through the pollen at a reduced frequency and exhibited a competitive disadvantage when compared to wild-type pollen. Nevertheless pollen from pip5k4 mutant plants fertilized ovules leading to normal seed set and silique length. Analysis of endocytic events using FM1-43 (or FM4-64) suggested a reduction in endoexocytosis and membrane recycling in pip5k4 null mutant pollen tubes. Imaging of elongating tobacco (Nicotiana tabacum) pollen tubes transiently transformed with a PIP5K4-green fluorescent protein fusion construct revealed that the protein localized to the plasma membrane, particularly in the sub-apical region. Over-expression of PIP5K4-GFP delocalized the protein to the apical region of the plasma membrane, perturbed pollen tube growth, and caused apical cell wall thickening. Consequently PIP5K4 seams to play an important role in regulating the polarity of pollen tubes supporting a model for membrane secretion and recycling where the apical and sub-apical regions appear to contain the components required to promote and maintain growth.
Resumo alargado em português disponível no documento
Resumo alargado em português disponível no documento
Descrição
Tese de doutoramento, Biologia (Biologia Molecular), 2009, Universidade de Lisboa, Faculdade de Ciências
Palavras-chave
Endocitose Exocitose Mutante de T-DNA Biologia molecular Teses de doutoramento