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Role of TCTP1 in plant reproduction
Pitoňak, Oliver ; Hafidh, Said (advisor) ; Lafon Placette, Clément (referee)
TCTP is a conserved eukaryotic protein involved in regulation of multiple cellular processes, such as translation, cell cycle and cell death. Apart from its intracellular functions, TCTP is secreted and participates in human immune response. Dimerization via a terminal cysteine residue is critical for its extracellular function. Multiple sequence alignment of eukaryotic TCTPs revealed that terminal cysteine residue is conserved not only in animals but also in green plant lineage. In contrast to animal model organisms, knowledge about TCTP in plants is limited. Arabidopsis thaliana genome harbors two TCTP paralogs, TCTP1 and TCTP2. TCTP1 expression is highly upregulated in pollen. Previously published studies pointed to its role in embryo development, pollen tube growth and targeting. Using mutant characterization and analysis of subcellular localization, the role of TCTP1 in plant reproduction was investigated in this thesis. Recombinant Arabidopsis thaliana TCTP1 protein was produced along with its potential interacting partners BRL2 and TTL3 in heterologous Escherichia coli system. tctp-1 T- DNA line characterization pointed to TCTP1 role in pollen tube growth. It was expressed in mature pollen and pollen tube under native promoter and localized to the cytoplasm. Recombinant AtTCTP1 formed dimers...
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Characterization of eukaryotic translation initiation factor 3 subunits (eIF3) in A. thaliana male gametophyte
Linhart, Filip ; Hafidh, Said (advisor) ; Retzer, Katarzyna (referee)
From RNA-to-protein, translation initiation and protein synthesis is mediated by trans-acting factors that recognize mRNA features common to almost all eukaryotes. Eukaryotic translation initiation factor 3 complex (eIF3) is a highly conserved protein complex that recognizes 5'-CAP elements of the mRNA to initiate translation. eIF3 consists of nine subunits, three of them having two isoforms: eIF3A, eIF2B1, eIF3B2, eIF3C1, eIF3C2, eIF3D, eIF3E, eIF3F, eIF3G1, eIF3G2, eIF3H and eIF3K. This work deals with functional characterization, expression and subcellular localization of eIF3B1, eIF3B2 and eIF3E in Arabidopsis thaliana male gametophyte and interaction of eIF3E with the Constitutive photomorphogenesis 9 (COP9) complex as a regulatory complex of eIF3E post-translational control. Here we show that depletion of eif3b1 or eif3b2 is not gametophytic lethal and that the two protein might function redundantly, whereas, knockout of eIF3E causes male gametophyte lethality. Interestingly, eif3b1 show post-fertilization defects during embryogenesis, suggesting that its redundancy with eIF3B2 is restricted to the gametophyte. Gene expression studies revealed high expression of eIF3 subunits in actively dividing zones of leaf primordia, root meristem and root elongation zones as well as in the vegetative...
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Characterization of the exocyst complex SEC15 subunit in A. thaliana
Aldorfová, Klára ; Hála, Michal (advisor) ; Hafidh, Said (referee)
The final step of secretion termed exocytosis is mediated by the exocyst complex. The exocyst is an evolutionary conserved protein complex that tethers secretory vesicle to the target membrane and consists of eight subunits: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo84, and Exo70. Sec15 exocyst subunit was previously shown to connect the rest of the exocyst complex with a secretory vesicle in yeast, mammals and fruit fly via interaction with Rab GTPase and GEF of Rab GTPase. Here, I show that plant SEC15B potentially functions in evolutionary conserved manner. First, two mutant lines of Arabidopsis thaliana sec15b mutant were tested in characteristics typical for other exocyst mutants. Although some characteristics reach certain level of plasticity, both sec15b-1 and sec15b-2 show similar tendencies, which are mostly consistent with defects with other mutants in exocyst subunits. sec15b-1 has been determined as a stronger allele that is defective in formation of seed coat, elongation of etiolated hypocotyl, growth of stem and primary root, establishment of axillary branches and lateral roots, diameter of rosette and, unexpectedly, growth of pollen tubes. Phenotype of sec15b-1 was rescued by insertion of SEC15B gene under SEC15B promotor. Second, complementation test showed that SEC15B and SEC15A are...
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