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Biologické účinky a metabolismus cytokininů typu cis-zeatinu v rostlinách
Gajdošová, Silvia ; Motyka, Václav (advisor) ; Brzobohatý, Břetislav (referee) ; Doležal, Karel (referee)
Cytokinins (CKs) are plant hormones that affect a wide range of developmental processes. The most important group of isoprenoid CKs represent zeatin and its derivatives occurring in two, cis and trans, positional isomers. Whereas trans-zeatin (transZ) was found to be a highly bioactive substance, cis-zeatin (cisZ) has been viewed for years as inactive or weakly active adjunct to its corresponding trans counterpart playing only an insignificant physiological role in plants. The occurrence of cisZ-type CKs was found in a great number of plant species with especially high levels identified in species of family Poaceae. All tested derivatives of cisZ, surprisingly including also zeatin-N9-glucoside, delayed dark-induced chlorophyll degradation in oat and wheat leaf segments. Additionally, cisZs effectively induced cell division in CK-dependent tobacco callus. The most pronounced activity was exhibited by cisZ riboside (cisZR) in the two types of CK bioassays. Metabolism of both zeatin isomers differed in short-term as well as in long-term experiments, which was supported also by various affinity of CK degrading enzyme, CK oxidase/dehydrogenase (CKX), to individual cis and trans isomers. Primary root enlargement of Arabidopsis seedlings was inhibited by cisZR in the same or similar way as by transZR,...
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The thermostability of photosystem II photochemistry is related to maintenance of thylakoid membranes organization
Karlický, Václav ; Kurasová, I. ; Špunda, Vladimír
For higher plant photosynthetic reactions, responses to the temperature changes are important, particularly if we consider global warming and the increasing frequency of extreme temperature fluctuations. High temperature stress decreases photosynthetic assimilation through the inactivation of photosystem II (PSII), the most heat-sensitive component of the oxygen-evolving complex. We have recently found higher thermostability of spruce PSII photochemistry compared to such control plants as Arabidopsis species and barley. In this work, we have therefore attempted to describe the causes of this effect on the level of the organization of pigment–protein complexes (PPCs) in spruce thylakoid membranes using circular dichroism (CD) spectroscopy. We have confirmed higher maximum efficiency of PSII photochemistry (FV/FM) for spruce needles in comparison to barley leaves. Temperature-dependent CD spectra have also demonstrated higher (by about 6°C) PSII thermostability of chiral macro-organization of PPCs in spruce thylakoid membranes compared to those in barley. However, thermal disruption of PPCs did not reveal significant differences. Our results demonstrate that the stability of PSII macro-organization in different plant species correlates with the thermostability of PSII photochemistry in intact needles/leaves and so effective PSII photochemistry is related to the maintenance of PSII macro-organization under high temperature stress.
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Isoflavonsynthasa: přítomnost a aktivita v bobovitých a nebobovitých rostlinách
Pičmanová, Martina ; Honys, David (advisor) ; Vaňková, Radomíra (referee)
Isoflavone synthase (IFS; CYP93C) plays a key role in the biosynthesis of the plant secondary metabolites, isoflavonoids. These phenolic compounds, which are well-known for their multiple biological effects, are produced mostly in leguminous plants (family Fabaceae). However, at least 225 of them have also been described in 59 other families, without any knowledge of orthologues to hitherto known IFS genes from legumes (with the single exception of sugar beet - Beta vulgaris, from the family Chenopodiaceae). In view of these facts, this masters thesis has focused on two main objectives: (1) to identify isoflavone synthase genes in selected leguminous and non-leguminous plants exploiting the PCR strategy with degenerate and non-degenerate primers, and (2) to find a system for the verification of the correct function of these genes. Our methodology for the identification of IFS orthologues was successfully demonstrated in the case of two examined legumes - Phaseolus vulgaris L. and Pachyrhizus tuberosus (Lam.) Spreng, in the genomic DNA of which the complete IFS sequences have been newly identified. To design a procedure for ascertaining the correct function of these genes and others once they have been completely described, a pilot study with IFS from Pisum sativum L. (CYP93C18; GenBank number...
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Characterization of the PTEN domain of selected Arabidopsis class II formins
Přerostová, Sylva ; Cvrčková, Fatima (advisor) ; Havelková, Lenka (referee)
Formins are proteins facilitating formation of actin filaments. They affect structure of cytoskeleton and participate in cytokinesis and tip growth. There are 2 classes of formins in Arabidopsis thaliana, which include FH1 and FH2 (Formin Homology 1 and 2) domain. Formins of the class I have usually a transmembrane domain on N-terminus. Due to this fact they can interact with membranes. Some formins from the class II include PTEN domain (Phosphatase and Tensin Homolog) derived from sequences of PTEN proteins which has lost the function of phosphatase. It is assumed this domain can bind on a membrane via the phosphatase section or C2 domain. This thesis was focused on the formin AtFH13 from the class II in Arabidopsis thaliana and on its PTEN domain. There were analyzed differences between mutants and wild-types in length of roots in seedlings and in size of seeds and seed coats, and observed the effect of dexamethasone on the length of roots on AtFH13. PTEN domain of the formin was isolated from cDNA, cloned to a vector and fused with YFP. The tagged protein was visualized by the method of transient expression in epidermal cells in the leaves of Nicotiana benthamiana. No big differences were observed between plants mutant in the gene AtFH13 and wild-type in choice parameters. Dexamethasone did't influence...
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Biological effects and metabolism of cis-zeatin-type cytokinins in plants
Gajdošová, Silvia
9 Summary Cytokinins (CKs) are plant hormones that affect a wide range of developmental processes. The most important group of isoprenoid CKs represent zeatin and its derivatives occurring in two, cis and trans, positional isomers. Whereas trans-zeatin (transZ) was found to be a highly bioactive substance, cis-zeatin (cisZ) has been viewed for years as inactive or weakly active adjunct to its corresponding trans counterpart playing only an insignificant physiological role in plants. The occurrence of cisZ-type CKs was found in a great number of plant species with especially high levels identified in species of family Poaceae. All tested derivatives of cisZ, surprisingly including also zeatin-N9-glucoside, delayed dark-induced chlorophyll degradation in oat and wheat leaf segments. Additionally, cisZs effectively induced cell division in CK-dependent tobacco callus. The most pronounced activity was exhibited by cisZ riboside (cisZR) in the two types of CK bioassays. Metabolism of both zeatin isomers differed in short-term as well as in long-term experiments, which was supported also by various affinity of CK degrading enzyme, CK oxidase/dehydrogenase (CKX), to individual cis and trans isomers. Primary root enlargement of Arabidopsis seedlings was inhibited by cisZR in the same or similar way as by transZR,...
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The role of evolutionarily conserved proteins BIR-1/Survivin and SKP-1 in the regulation of gene expression
Kostrouch, David ; Kostrouch, Zdeněk (advisor) ; Dráber, Pavel (referee) ; Pacák, Karel (referee)
SKIP and BIR/Survivin are evolutionarily conserved proteins. SKIP is a known transcription and splicing cofactor while BIR-1/Survivin regulates cell division, gene expression and development. Loss of function of C. elegans SKIP (SKP-1) and BIR-1 induces overlapping developmental phenotypes. In order to uncover the possible interactions of SKP-1 and BIR-1 on the protein level, we screened the complete C. elegans mRNA library using the yeast two-hybrid system. These experiments identified partially overlapping categories of proteins as SKP-1 and BIR-1 interactors. The interacting proteins included ribosomal proteins, transcription factors, translation factors and cytoskeletal and motor proteins suggesting involvement of the two studied proteins in multiple protein complexes. To visualize the effect of BIR-1 on the proteome of C. elegans we induced a short time pulse BIR-1 overexpression in synchronized L1 larvae. This led to a dramatic alteration of the whole proteome pattern indicating that BIR-1 alone has the capacity to alter the chromatographic profile of many target proteins including proteins found to be interactors in yeast two hybrid screens. The results were validated for ribosomal proteins RPS-3, RPL-5, non-muscle myosin and TAC-1, a transcription cofactor and a centrosome associated...
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Proteomika CDK inhibice v nádorových buňkách
Kovářová, Hana ; Skalníková, Helena ; Halada, Petr ; Strnad, M. ; Hajdúch, M.
In order to improve our understanding of the biochemical basis of the anti-cancer activity of olomoucine-derived synthetic cyclin-dependent kinase inhibitors (CDKIs) and to search for novel proteins associated with these biological effects we applied complex proteomic approaches. To analyse cellular responses to the CDKI we used two cancer models: the CEM T-lymphoblastic leukemia cell line representing hematological malignancy, and the A549 lung adenocarcinoma cell line as a solid tumor model. Cancer cells of these lines were cultured in both the presence and absence (controls) of the CDKI, bohemine (BOH). Cellular proteins of both of these lines were then extracted and fractionated using conventional two-dimensional gel electrophoresis (2-DE), and for the CEM T-lymphoblastic leukemia cell line we also used a 2-D liquid phase fractionation system ProteomeLab PF 2D ( Beckman Coulter). Computer-assisted data analysis of the resulting 2-D protein expression maps was applied to determine the similarity/dissimilarity of the maps and to select characteristic protein spots or bands based on the quantitative differences between BOH-treated and control cells. Many of these differentially expressed proteins were identified by mass spectrometry, since they represent candidate biomarkers of cancer cell responses to CDK inhibition and cellular pathways that are relevant to the anti-cancer activity of the CDKIs. Subsequently, we focused directly on these proteins in confirmatory studies using various techniques (including quantitative immunoblotting, immunocytochemistry and functional activity analyses) to demonstrate the validity of the proteomic results and extend our knowledge of the CDKI effects.
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