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This bachelor thesis deals with the current state of Volšatrans transport company fleet, captures and evaluates its overall condition and subsequently recommends a proposal for its replacement. The change is made on the basis of the multi-criteria decision-making method, namely the TOPSIS method, the result of which is the output showing particular proposals for the replacement of the vehicle. This output may be in practice used by Volšatrans as a recommendation for selection of a suitable vehicle variant on the basis of their requirements.

The PsbO protein is necessary for the function of the electron-transport chain of the thylakoid membrane in higher plants. In most of the angiosperms, including Arabidopsis thaliana, this protein has two isoforms termed as PsbO1 and PsbO2. Many authors tried to reveal the fundamental difference between the PsbO1 and PsbO2 with the help of the mutant lines which lack one of the isoforms. The problem is that the mutants in psbO isoforms do not possess the same level of PsbO as WT does. So we made psbo1isoL mutants. These lines contain only one isoform but their level of the whole PsbO is comparable to the level of the whole PsbO of WT. Results from these experiments suggest that if a psbo1isoL plant has the same amount of PsbO as WT does, there is no observable phenotype difference. Thus we were not able to identify, in the usual cultivation conditions, if there are any functional differences between PsbO1 and PsbO2 Following the above mentioned results, we would like to know conditions (if there are any) in which T-DNA insertion mutants psbo2 (respectively psbo2cr which are made with the use of the CRISPR/Cas9), which have only PsbO1 isoform, could be phenotypically distinguished from WT. With the use of usual cultivation conditions, we are unable to tell apart the psbo2 and psbo2cr from WT by the...

In context with ongoing climate change, the average temperature is still rising. This is a problem mainly for agriculture and production of groceries, because plants generally have difficulties when coping with ambient temperature higher than 40 řC. If there would be continuing trend in decrease of global food production, it could cause problems with feeding still growing world population. Organisms in general developed many mechanisms of stress adaptation. One of these evolutionary adaptions to high temperature stress is production of heat shock proteins (HSPs). HSPs are very conserved family of proteins present in both prokaryotes and eukaryotes. This thesis summarizes present knowledge about the HSPs and their function (not only) during the high temperature stress in plants, but some of the information presented in this thesis were obtained also from other organisms. The thesis starts with general introduction to the high temperature stress and how plants can sense the ambient temperature. Next is general mechanism of induction of HSPs and their classification, structure, and mechanism of action. The possibility of future use of HSPs in agriculture and medicine is opened in last chapter.

This bachelor thesis is focused on regulating of the activity of the Rho GTPases. Rho GTPases are important signalling proteins that are involved in regulation of many cellular processes, for example dynamic cytoskeleton changes (especially actin fibres) associated with cell adhesion and migration, vesicle trafficking or cell cycle. The activity of the Rho GTPases is primarily regulated by other proteins affecting the presence GTP/GDP or by posttranslational modifications. This thesis is focused on a narrow section of these modifications, namely phosphorylations. Phosphorylation of the Rho GTPases affects for example their activity, localization, or degradation. Understanding the mechanism of regulation of Rho GTPase activity is important for comprehension of cellular processes. Rho GTPases are involved, for example, in tumour cell invasiveness. By studying Rho GTPases and their regulation, it is possible to better target potential treatment or even prevent the occurrence of the metastases.

Plant autophagy is a crucial evolutionary conserved process for recycling cytoplasmic material under stress conditions or during development. The autophagic pathway is negatively regulated by TOR kinase, a versatile molecule modulating a wide range of cellular processes. In mammals, TOR kinase may be activated by phosphatidic acid, a vital signalling lipid. This thesis aims to prove the possible involvement of phospholipids in plant autophagy. I analysed the rate of primary root inhibition in knock-out mutants coding phospholipases in A. thaliana with induced autophagy, measured activity of lipid metabolising enzymes in wild type and atg10 mutant and observed autophagosome formation in selected mutants. Autophagosomes were labelled by fluorescent protein in vivo and by indirect immunolabelling in fixed samples. Using advanced stereological approach, I optimized a method for obtaining an unbiased estimate of autophagosome number in plant root cells.

Plants can encounter many stressors throughout their lives, including low temperatures. This work characterizes the limits of extreme temperatures that have an effect on plants; cold (temperatures ranging from 0 ř C to 15 ř C) and frost (temperatures below 0 ř C accompanied by ice formation in plant tissues). Consequences of plant damage caused by extreme temperatures are also given, as well as the most important plant resistance and defense mechanisms in response to these stressors, especially at the cellular level. These are temperature responding signalling transduction pathways leading to the activation of stress genes and some phytohormones (abscisic acid, salicylic acid, jasmonates), as well as the accumulation of stress proteins (e.g. anti-freezing proteins, dehydrins). At the same time, some of the impacts of climate change, including global warming, on selected plants species are mentioned. Key words: stress, plants resistance, tolerance, low temperature, cold, frost, damage to plants

Autophagy is a cellular mechanism when cell gets rid of its damaged or unwanted components. These are wrapped into a double membrane and creates so-called autophagosome, which is transported to the vacuole, where its contents are degraded or recycled. Autophagy runs essentially through whole life of the plant, but if it is necessary, for example in response to stressful conditions, it is significantly intensified. It is relatively complexly regulated mechanism, which, especially in plants, still has not been completely described. Autophagy plays an important role under the influence of stress conditions. Especially, the effects of abiotic stress play an important role in plant life. Plants are immobile organisms so they must therefore develop mechanisms to enable them to survive in adverse conditions. In response to different types of stress, we can observe different roles in the involvement of autophagy, whether it is disposal of poorly folded proteins, remobilization of nutrients or antioxidant mechanism. Autophagy is also especially important for the process of stress memory and regulation.

Argonaut proteins are RNA binding proteins with RNase activity. They can bind short non-coding RNAs, which are usually 21-24 nt in length. These small RNAs are formed in plants from double-stranded RNAs and are involved in the regulation of gene expression, in the protection of the plant against viruses, transposons, or other invasive genetic elements, and affect the state of chromatin. Using small RNAs, Argonaut proteins participate in a mechanism called RNA interference, a complex process used to regulate gene expression, at the transcriptional or post-transcriptional level. In the plant species Arabidopsis thaliana, the Argonaut family of proteins has 10 genes, while in other plant species they have their functional homologues (orthologs). Individual proteins differ in their preference for different types of small RNAs depending on their length, the 5 'terminal nucleotide and the way in which the source double-stranded RNA is formed. At the same time, the involvement of individual types of Argonaut in the partial processes of RNA interference and in connection with their cellular localization also differs. This work is focused on the interaction of small RNAs with Argonaut proteins, describes the processes of their functioning and further deals with individual paralogs of Argonaut proteins in...

The eukaryotic actin cytoskeleton is required for numerous cellular processes, including cell shape, development and movement, gene expression and signal transduction, and response to biotic and abiotic stress. Actin constitutes a wide family of proteins that are major components of the cytoskeleton. Actin is one of the most abundant proteins in living organisms. Actin has essential functions both in the cytoplasm and in the nucleus, where it has been linked to key nuclear processes. Recent studies have shown that actin is actively transported from the cytoplasm to the nucleus, where it regulates transcriptional aktivity, regulates RNA polymerases, is involved in chromatin remodeling and repair damaged DNA. The presence of typical actin filaments in the nucleus has not been demonstrated directly.but nuclear actin occurs in many forms such as actin rods, short actin polymers, actin monomers, or actin complexes with profilin or cofilin. Most eukaryotic cells also contain at least eleven actin-related proteins (ARPs). Although many ARPs are cytoskeletal, recent biochemical and genetic work has demonstrated that some ARPs function largely or entirely in the nucleus. Nuclear ARPs are recognized as novel key regulators of genome function, and affect not only the remodeling of chromatin but also the...

Potassium is one of the essential elements necessary for plant growth. It is involved in many plant processes, such as osmoregulation, enzymes activaton, etc. These functions are very often closely related to its transport in the cell and the whole plant. Although potassium is abundant in earth's crust, the mount of plant-available form is often insufficient. Potassium deficiency manifests itself on many levels and also contributes to the reduction of yield and quality of agricultural crops. There are many of potassium-transporting proteins in the plant. One of the important families of potassium transporters is the KT/HAK/KUP family. This family includes, among others, the high-affinity transporter HAK5, which is key for the uptake of potassium from the environment with low-potassium availability. One of the not very characterized transporters from the KT/HAK/KUP family is the KUP5 transporter, which I deal with in my diploma thesis. The aim of this work is to analyze the phenotypic manifestations of kup5 T-DNA insertion mutants and to characterize the functions of the transporter KUP5 in Arabidopsis thaliana plants. I analyzed the growth of kup5 insertion mutants in various environmental conditions and performed plant transformation to determine the localization of the KUP5 transporter in the...

Tomosyn is a protein belonging to the Lgl family and conserved across the animal and plant kingdom. Tomosyn is composed of N-terminal domain containing WD40 motif and Cterminal domain, where the R-SNARE motif is located at the end of the C-terminal domain. This motif is classified as homologous to R-SNARE motif of synaptobrevine, which is a protein located on the surface of the vesicles and participating in the formation of SNARE complex and subsequent fusion of the vesicles with the plasma membrane. Thus the role of this tomosyn is mainly the regulation of exocytosis. Apart from the animal tomosyn, its yeast homolog Sro7/Sro77 is also examined, however during the evolution it has lost its R-SNARE motif and therefore the plant tomosyns remain unexplored. The aim of this diploma thesis was to characterize both of the plant tomosyns: AtTYN1 and AtTYN2 in Arabidopsis thaliana plant. The experimental thesis included the bioinformatic analysis, the DNA construct creation, a search for interactors by yeast two-hybrid system and monitoring the localization using the confocal microscope. The bioinformatic analysis results, including the creation of phylogenetic tree, not only revealed the conservation of tomosyns across the different classes, but also the division of both tomosyns into different clusters. There was...

Plants are daily exposed to stress, biotic or abiotic. Both of these types can be divided into several subgroups. This work is focused on plant cuticle, its formation and function in respect to abiotic stress resistance. The main factors, mentioned in this work, are drought, heat, UV radiation, excessive salinity or ozone exposure. The individual chapters focus on the composition, biosynthesis and changes in the build-up construction caused by certain types of stressors. A significant part is also devoted to cutin and cuticular waxes, which play an important role in plant protection and whose structure is also affected by abiotic stress. Phytohormones, especially abscisis acid, which had its application here, are also not omitted. In the final phase of the thesis, cuticular mutants are mentioned in connection with abiotic stress types illustrating the role and the importance of the cuticle layer. Key words: plants, cuticle, osmotic stress, abscisic acid, cutin, waxes

Stomata are structures located in higher plants' epidermis and they regulate mainly CO2 intake and transpiration. The stomata are important for plants' protection against excessive water loss during osmotic stress. This thesis is a literary research summarizing stomatal movements regulation during osmotic stress. The main part of the thesis is focused on stomatal closure with the help of ABA under water deficiency conditions. There are the ABA synthesis, transport and its impact on stomata described in individual chapters. The ABA early signalling pathway and an ionic channel regulation are described in detail on the basis of the published data. The second part of the thesis is about second messengers produced by ABA and their impact on stomatal movement regulation. The special attention is paid to hydrogen peroxide and nitric oxide and their contribution to stomatal closure. The last part is about passive (hydraulic) stomatal closure. Keywords: plants, stomata, osmotic stress, ABA, stomatal movements

Tropospheric ozone is an important pollutant formed by the photochemical reactions of ultraviolet radiation and nitrogenous. Recently, its concentration in the air is increasing due to the promoting climate change and antropogenic activities, mainly because of combustion of the fossil fuels. The ozone is highly reactive molecule degrading to the reactive oxygen species having negative effects on the leaf physiological functions and structure. Ozone penetrates the leaf through the stomata, thereafter it reacts with the cells and their singular compartments. The products formed due to ozone degradation are able to cause the disintegration of plasma membranes and other oxidative damage, for example accumulation of plastoglobuli inside the chloroplasts, changes in the shape of chloroplasts, mitochondria or peroxisomes. At the macroscopic level the visible lesions, chloroses and necroses are observed. In addition, the faster senescence of leaves is observed under the effect of ozone. The main aim of present thesis is to review knowledge of the ozone-induced changes in the leaf structure beginning on the cell structure level and ending at the whole plant body level and to explain physiological mechanisms leading to these damages. Key words: ozone, oxidative stress, leaf structure, mesophyll, reactive...

The Tobacco mosaic virus (TMV) is a simple and frequently used model virus which has been studied already more over than 130 years. Due to the intensive study of this virus the details of its infectious cycle, genomic information and also the structure of the created viral particle as well as the mechanism of its creation are known today. The process of encapsidation (viral particle formation) is sufficiently described in the in vitro conditions. In the in vitro conditions the origin of assembly (OAS) was also described. The OAS was identified in the coding sequence of the gene for the movement protein (MP). The importance of replication centers (replication factories) has also been supposed. The aim of the diploma thesis was to study the specificity of the interaction of RNA and coat protein in the process of the particle assembly taking place directly inside the plants. The experiments were performed to verify the necessity of presence of OAS sequence in process of initiation of viral encapsidation. The effect of the cell compartmentation on this process has also been studied. Based on several viral systems (the Tobacco mosaic virus, the Potato virus X, the Bean yellow dwarf virus and Cowpea mosaic virus) gene constructs were created. These constructs enables to study this idea at the molecular...

Endocytosis is a process typical for eukaryotic cells. It is a mechanism that ensures invagination of the plasma membrane which leads to the creation of a membrane vesicle with specifically selected cargo. The vesicle is then internalized by the cell. Endocytosis has a crucial role in plant growth and development not only thanks to the reception of nutrients from the extracellular space but also by having a huge impact on the membrane composition which influences cellular signalling. There are few types of endocytosis known in plants. They differ from each other by the molecular machinery which ensures their process. The most studied type in plants is the clathrin-mediated endocytosis. Clathrin-coated vesicle formation proceeds through five stages. However, there are less details known about it in plant cells than in animal cells. In the first part of my bachelor thesis I describe recent knowledge of this topic and the most important participants in this process. I also mention the mechanisms of regulations that plant cells use to coordinate clathrin-mediated endocytosis. In the second part I summarize recently discovered types of endocytosis in plants that are independent from clathrin. These are endocytosis associated with microdomains and fluid- phase endocytosis. The details of these endocytic...

Plants as sessile organisms are strongly affected by abiotic and biotic stress factors. Thus, they have developed an array of morfological, biochemical and physiological adaptations to reduce the negative effects of these factors. The membrane trafficking, among others, plays very important role in adaptation to abiotic stress. In my bachelor thesis I have focused on two important protein families involved in this trafficking, namely on RAB GTPases and SNARE proteins. In the first part, the phenomenon of stress is characterized and the strategies how plants cope with the effect of stressors are described, especially the production of reactive oxygen species (ROS) and autophagy. Following chapter deals with an introduction to the membrane trafficking in plants. In the rest of the thesis, I characterize RAB GTPases and SNARE proteins and provide contemporary insight in the mechanism of their function. The aim of the key parts of these chapters is to summarize current knowledge of RAB GTPases' and SNARE proteins' functions in post-Golgi trafficking pathways during response to abiotic stressors or secondary oxidative stress. Key words: plants, abiotic stress, membrane trafficking, secretion, endocytosis, vacuole, RAB GTPases, SNARE proteins

All orchid species studied so far have been shown to participate in orchideoid mycorrhizal symbiosis. Morover, this symbiosis is absolutely vital component of their life cycle. Exchange of nutrients occurs between symbionts where the fungi provides the orchid with energy and carbon supply at least in its early developmental stages. This study focuses on the possible role of trehalose in this transfer. In vitro experiments have showed in five species from three different subfamilies of Orchidaceae family that they can utilize trehalose comparably with sucrose and glucose. Thus, the ability of trehalose utilization seems to be conserved among orchids. Trehalase enzyme activity was localized histochemically in orchid mycorrhizas. The activity strongly colocalized with colonized tissue supporting a hypothesis that trehalose transfer occurs in this site and is mediated by trehalase. Using bioinformatic methods, trehalase gene duplications were identified in many taxons of Embryophyta including three orchid species. Interestingly, highest number of trehalase gene copies was identified in genome of orchid Dactylorhiza majalis. Trehalose utilization, high trehalase activity in mycorrhizas and trehalase gene duplications in some orchids together indicate that trehalose transfer in orchid myccorhizas...

The formation of reactive oxygen species (ROS) in plants is inevitable part of aerobic cellular metabolism, during which ROS are generated as by-products of basal metabolic pathways. ROS participate in many processes in plants. If the ROS are at normal level, they serve as signal molecules. Under stress, however, the ROS level in plants increases. High ROS accumulation can cause severe oxidative damage of cellular components, but simultaneously it activates other signal pathways controlling defence against oxidative stress. Plants protect themselves against increased level of ROS in different ways. Either they try to prevent increased level of ROS, or they try to detoxify them. Plants utilize for detoxification two efficient antioxidative systems - enzymatic and non-enzymatic ones. The main goal of this work is to put saccharides into context of plant non-enzymatic antioxidative system and summarize saccharides-based ROS scavenging mechanisms, which have been invented so far. Research on mechanisms of ROS scavenging is highly important, because oxidative damage through excessive production of ROS is considered to be significant cause of productivity losses in agricultural crops. These losses are likely to increase as a result of increased stress burden connected with expected climatic changes. Key...

Rab GTPases are small signaling molecules that play an important role in vesicle trafficking in eukaryotic cells. Correct signaling through small GTPases allows orchestration of vesicle transport among cellular organelles and also to the cell wall providing cell wall material for cell growth and elongation. Engagement of Rab GTPases in the regulation of endomembrane trafficking is one of the evolutionary conserved aspects of secretion regulation. The network of Rab GTPases interaction includes also various downstream effectors. One of them is the exocyst complex involved in vesicle docking at the plasma membrane. It is a complex composed of eight different subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84). Exocyst was discovered as Sec4p Rab GTPase effector in yeast and also data from animal models describe the Sec15 exocyst subunit as the Rab-interacting partner, but data from plants are missing. On the other hand, numerous studies identified exocyst role in tip growth of pollen tube and root hairs, seed coat formation, cell plate and cell wall formation, hypocotyl elongation, and importantly also PIN auxin efflux carriers recycling and polar auxin transport. There are two paralogues of SEC15 in the Arabidopsis genome, SEC15a and SEC15b, the previous one already shown to be...

Water is one of the main limiting factors of plant growth. Availability of water on our planet is decreasing, and if we add fast-growing human population, we have a problem growing. Scientists from all around the world, especially those from arid regions, are facing a big problem, which is an attempt to make the most efficient use of water by plants. If this problem should be solved, we need to understand the issue of Water Use Efficiency (WUE). This paper attempts to summarize the current knowledge related to the issue of WUE, mainly focused on anatomical and physiological parameters control of WUE, which is focused primarily on the issue of evapotranspiration. Another part focuses on clarifying the issue of WUE at the gene level and the related biotech opportunities. At the end of the work are discussed possibilities of increasing WUE using irrigation techniques. Key words: water use efficiency, evaporation, transpiration, stoma, stomatal density, abscisic acid, ERECTA

8 ABSTRACT Rab GTPases (Rabs) are the most populous branch of eukaryotic Ras GTPase superfamily. In active GTP-binding conformation, they serve as key instruments in defining transient membrane identity and through various effectors regulate formation, transport, conversion, and fusion of membrane vesicles. This is important for upkeep of compartmentalized structure of eukaryotic cells and for facilitating both endo- and exocytic processes. Rabs are converted into GDP-binding conformation by interactions with Rab GTPase activating proteins (Rab GAPs) that possess ability to significantly speed up weak intrinsic GTP hydrolytic activity of Rabs. Through this process, Rab GAPs can limit scope of the Rabs' activity and lay out spatiotemporal boundaries for varying Rab populations. In this thesis, I tried to characterize a Rab GAP, GAP2, seemingly necessary for standard development of thale cress plants. Besides TBC catalytic domain, GAP2 (product of At2g39280 gene) possesses a C-terminal coiled-coil motif, which was previously found to interact with Rab GTPases. Experiments aiming to complement T-DNA insertion mutant in GAP2, elucidate GAP2 intracellular localization, novel interacting partners, and character of interaction with the Rabs discovered in the pilot study were undertaken. The results suggest that...

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...

Quinoa is traditionally grown crop originating from areas adjacent to Andean Mountains in South America, where it is grown by local native population for over 5000 years. Its extraordinal resistance to abiotic stresses as salinity, drought or frost, along with quite exceptional nutritional qualities of the seed, earned it the designation "supercrop" and the year 2013 was even announced by the FAO organization as the International Year of Quinoa. The main aim of this thesis is to identify the main mechanisms responsible for quinoa resistance to frost, salinity and drought. Marginally, this work also focuses on reaction of quinoa against an attack of most common fungal pathogen and also on different sensitivity of quinoa varieties towards the lenght of photoperiods, which are two important factors in expanding the cultivation of quinoa outside the original area.

In these days, genetic modifications of crops are commonly used in the fight against pests and weeds. More and more often there are also efforts to use genetic modifications to increase tolerance to abiotic stresses that affect worldwide yields much more than biotic ones. Manipulation of trehalose metabolism represents one of the frequently studied options of abiotic stress tolerance enhancement. This work summarizes current knowledge on the protective effects of trehalose in cells and describes signaling functions of trehalose and its metabolic intermediate trehalose-6-phosphate in plants. It also describes current achievements in increasing abiotic stress tolerance through genetic modifications of trehalose metabolism together with accompanying problems. Finally, the potential of this method for future exploitation is discussed.

PsbO (manganese-stabilizing protein) is the largest extrinsic protein of photosystem II, located on the lumen side of photosystem. It is present in all known oxyphototrophic organisms. PsbO facilitates photosynthetic water splitting, which takes place in an oxygen evolving center (Mn4CaO5 cluster) of photosystem II. This work is focused on PsbO of higher plants and its isoforms, particularly their evolution and functions. Bioinformatic analyses revealed that majority of higher plants express exactly two psbO isoforms. A phylogenetic tree of PsbO sequences has an unusual topology. The two paralogous isoforms do not diverge at the base of the phylogenetic tree, as anticipated, but rather at the end of particular branches, at the level of family or lower taxonomic unit. In this work we propose and discuss several hypotheses concerning evolution of PsbO isoforms. The work further includes detailed analysis and identification of protein spots assigned to PsbO on 2D IEF-SDS PAGE gels of potato thylakoid proteins. We identified predominant version of PsbO isoform in most of the spots. We did not succeed to find any posttranslational modification. We optimized a method of psbO expression in E. coli and subsequent purification, which yielded relatively big amount of properly folded recombinant protein. Analysis of...