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Androgenesis
Kočová, Helena ; Honys, David (advisor) ; Kocábek, Tomáš (referee)
(anglicky) Androgenesis in the plant kingdom is an interesting phenomenon, in which a new individual is regenerated from male gametophyte. Having gametophytic, i.e. haploid number of chromosomes, these plants are potentially useful in research as well as for the generation of new genotypes. Duplication of their genetic information then results in fully homozygous plants, that can be used for breeding. At the same time, microspores represent a unique system for studying totipotency, cell proliferation, differentiation and embryogenesis. However, in many important crops as well as in some model species, such technology has not yet been efficiently managed. The aim of this thesis is to summarize the knowledge about androgenesis, from the historical context to the latest discoveries, including methods, development, complications and at the end also the possible use of obtained doubled haploid plants. Keywords: androgenesis, male gametophyte, microspore embryogenesis, pollen, totipotency, cell differentiation, stress, organogenesis, haploid
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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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The role of protein phosphorylation during progamic phase of tobacco male gametophyte development
Fíla, Jan
v angličtině (English abstract) Tobacco male gametophyte has a strongly dehydrated cytoplasm and represents a metabolically inactive stage. Upon cytoplasm rehydration, pollen grain becomes metabolically active and after the activation is finished, the pollen tube growth through a selected pollen aperture starts. The rehydration together with metabolic activation are accompanied by the regulation of translation and post-translational modifications (mainly phosphorylation) of the existing proteins. In this Ph.D. thesis, there were identified phosphopeptides from tobacco (Nicotiana tabacum) mature pollen, pollen activated in vitro 5 min and pollen activated in vitro 30 min. The total proteins from the above male gametophyte stages were extracted. The protein extract was trypsinized and the acquired peptide mixture was enriched by MOAC (metal oxide/hydroxide affinity chromatography) with titanium dioxide matrix. The enriched fraction was subjected to liquid chromatography coupled with tandem mass spectrometry (LC- MS/MS). Totally, there were identified 471 phosphopeptides, carrying 432 exactly localized phosphorylation sites. The acquired peptide identifications were mapped to 301 phosphoproteins that were placed into 13 functional categories, dominant of which were transcription, protein synthesis,...
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The role of protein phosphorylation during progamic phase of tobacco male gametophyte development
Fíla, Jan ; Honys, David (advisor) ; Paleček, Jan (referee) ; Smýkal, Petr (referee)
v angličtině (English abstract) Tobacco male gametophyte has a strongly dehydrated cytoplasm and represents a metabolically inactive stage. Upon cytoplasm rehydration, pollen grain becomes metabolically active and after the activation is finished, the pollen tube growth through a selected pollen aperture starts. The rehydration together with metabolic activation are accompanied by the regulation of translation and post-translational modifications (mainly phosphorylation) of the existing proteins. In this Ph.D. thesis, there were identified phosphopeptides from tobacco (Nicotiana tabacum) mature pollen, pollen activated in vitro 5 min and pollen activated in vitro 30 min. The total proteins from the above male gametophyte stages were extracted. The protein extract was trypsinized and the acquired peptide mixture was enriched by MOAC (metal oxide/hydroxide affinity chromatography) with titanium dioxide matrix. The enriched fraction was subjected to liquid chromatography coupled with tandem mass spectrometry (LC- MS/MS). Totally, there were identified 471 phosphopeptides, carrying 432 exactly localized phosphorylation sites. The acquired peptide identifications were mapped to 301 phosphoproteins that were placed into 13 functional categories, dominant of which were transcription, protein synthesis,...
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Revealing phosphoproteins playing role in tobacco pollen activated in vitro
Fíla, Jan ; Honys, David (advisor) ; Fischer, Lukáš (referee)
5 Abstract Tobacco mature pollen rehydrates in vivo on a stigma tissue, and develops into the rapidly-growing pollen tube. This rehydration process is accompanied by the de-repression of stored mRNA transcripts, resulting in the synthesis of novel proteins. Furthermore, such metabolic switch is also likely to be regulated on the level of post-translational modifications of the already-present proteins, namely via phosphorylation, since it was shown to play a significant regulatory role in numerous cellular processes. Since only a minor part of proteins is phosphorylated in a cell at a time, the employment of various enrichment techniques is usually of key importance. In this diploma project, metal oxide/hydroxide affinity chromatography (MOAC) with aluminium hydroxide matrix was applied in order to enrich phosphoproteins from the mature pollen and the 30-minute in vitro activated pollen crude protein extracts. The enriched fraction was separated by both 2D-GE and gel-free liquid chromatography (LC) approaches with subsequent mass spectrometric analyses. Collectively, 139 phosphoprotein candidates were identified. Additionally, to broaden the number of phosphorylation sites identified, titanium dioxide phosphopeptide enrichment of trypsin-digested mature pollen crude extract was performed. Thanks to the...
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