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The Arp2/3 complex is an actin nucleator shown to be required throughout plant morphogenesis, contributing to processes such as cell expansion, tissue differentiation or cell wall assembly. A recent publication demonstrated that plants lacking functional Arp2/3 complex also present defects in auxin distribution and transport. This work shows that Arp2/3 complex subunits are predominantly expressed in the provasculature, although other plant tissues also show promoter activity (e.g., cotyledons, apical meristems, or root tip). Moreover, auxin can trigger subunit expression, indicating a role of this phytohormone in mediating the complex activity. Further investigation of the functional interaction between Arp2/3 complex and auxin signaling also reveals their cooperation in determining pavement cell shape, presumably through the role of Arp2/3 complex in the correct auxin carrier trafficking. Young seedlings of arpc5 mutants show increased auxin-triggered proteasomal degradation of DII-VENUS and altered PIN3 distribution, with higher levels of the protein in the vacuole. Closer observation of vacuolar morphology revealed the presence of a more fragmented vacuolar compartment when Arp2/3 function is abolished, hinting a generalized role of Arp2/3 complex in endomembrane function and protein trafficking.

The ARP2/3 complex and formins are the only known plant actin nucleators. Besides their actin-related functions, both systems also modulate microtubule organization and dynamics. Loss of the main housekeeping Arabidopsis thaliana Class I membrane-targeted formin FH1 (At3g25500) is known to increase cotyledon pavement cell lobing, while mutations affecting ARP2/3 subunits exhibit an opposite effect. Here we examine the role of FH1 and the ARP2/3 complex subunit ARPC5 (At4g01710) in epidermal cell morphogenesis with focus on pavement cells and trichomes using a model system of single fh1 and arpc5, as well as double fh1 arpc5 mutants. While cotyledon pavement cell shape in double mutants mostly resembled single arpc5 mutants, analysis of true leaf epidermal morphology, as well as actin and microtubule organization and dynamics, revealed a more complex relationship between the two systems and similar, rather than antagonistic, effects on some parameters. Both fh1 and arpc5 mutations increased actin network density and increased cell shape complexity in pavement cells and trichomes of first true leaves, in contrast to cotyledons. Thus, while the two actin nucleation systems have complementary roles in some aspects of cell morphogenesis in cotyledon pavement cells, they may act in parallel in other cell types and developmental stages.

Microtubules (MTs) are involved in key processes in plant cells, including cell division, growth and development. MT-interacting proteins modulate MT dynamics and organization, mediating functional and structural interaction of MTs with other cell structures. In addition to conventional microtubule-associated proteins (MAPs) in plants, there are many other MT-binding proteins whose primary function is not related to the regulation of MTs. This review focuses on enzymes, chaperones, or proteins primarily involved in other processes that also bind to MTs. The MT-binding activity of these multifunctional MAPs is often performed only under specific environmental or physiological conditions, or they bind to MTs only as components of a larger MT-binding protein complex. The involvement of multifunctional MAPs in these interactions may underlie physiological and morphogenetic events, e.g., under specific environmental or developmental conditions. Uncovering MT-binding activity of these proteins, although challenging, may contribute to understanding of the novel functions of the MT cytoskeleton in plant biological processes.

Forest subshrubs Pyroloideae are important models to study mixotrophy - a strategy where both carbon from photosynthesis and fungal one is used. Due to their initial mycoheterotrophy it was impossible to germinate these plants outside natural habitats neither for study of their physiology, medicinal purposes nor for their conservation. Thanks to our novel cultivation method which yields high germination percentages, it was possible to study germination and post-germination ontogenesis of Pyroloideae. It is necessary to break strong dormancy of seeds by bleach and acid and to sow seeds on complex media amended by soluble carbohydrates. Cold stratification turned out to be unnecessary while cultivation in dark was crucial. Many convergencies with orchids were observed, mainly that dust seeds with tiny embryo arrested at globular stage germinate into undifferentiated structures functionally similar to orchid protocorms, from which only one meristem emerges. On the contrary to orchids, the first meristem formed is the root meristem and much later, adventitious shoots appeared. Inability of young plants to grow in the absence of soluble carbohydrates and elevated amount of 13 C stable isotope in tissues of adult plants growing in natural habitats compared to surrounding autotrophic controls suggests,...

Nucleation of microtubules co-determines organization of this cytoskeleton component in cells and makes a significant contribution to shaping its dynamics. In plant cells, micro- tubules are mainly nucleated on preexisting ones and nucleation takes place in the cortex and also within the mitotic spindle and the fragmoplast. Recruiting the γTuRC, a pre- served universal nucleator, to the wall of microtubules is provided by augmin in cooperation with NEDD1/GCP-WD. The function of the γTuRC is at least in the case of nucleation in the spindle, but apparently in other situations as well further enhanced by XMAP215/MOR1, which raises efficiency of the γTuRC through its own polymerization activity, and TPX2, or its homologs, which for one thing, directly activates the complex and for another, locally increases concentration of tubulins by forming condensate with them, which also augments the probability of success of nucleation. Not much is known about regulatory pathways controlling this process, with the exception of the TTP complex, which is functional in the cortex. Overall, knowledge covering nucleation in plants is rather meager and information concerning the molecular mechanisms of functioning of mentioned factors comes mainly from research in animals. Keywords augmin, cytoskeleton, γTuRC,...

ARP2/3 is an evolutionarily conserved heteroheptameric protein complex. Its main activity lies in the nucleation of dendritic actin filaments that are involved in membrane remodeling. ARP2/3 takes part in plasma membrane remodeling and the formation of cytoplasmic protrusions that serve in the amoeboid motion of mammalian cells and some protists and plays role in exocytosis and endocytosis of animal and yeast cells. The main objective of this work was to find a connection between the ARP2/3 complex and the regulation of the plant endomembrane system. Using TIRF microscopy we visualized the localization of the ARP2/3 complex in the cortical layer of plant cells and compared it to the localization of several endomembrane markers from the Rab family and an exocytotic marker Exo84b. In the vicinity of the plasma membrane, the ARP2/3 complex subunits localized to dynamic dots very similar to the localization of Exo84b protein. Colocalization analysis showed that a small portion of Exo84b marker and ARP2/3 complex signals colocalize and this result was seconded by the biochemical approach of coimmunoprecipitation. Key words: ARP2/3, endomembrane system, cortical layer, RabA1g, RabC1, RaD2a, Exo84b

The cuticle is a lipidic structure covering plant aerial organs, providing mechanical rigidity and acting as a protective barrier. It contains the cutin polyester and waxes, which are derived from very-long-chain fatty acids. These compounds are synthesised in two separate pathways. The cuticular biosynthetic machinery is incredibly complex and employs a multitude of enzymes, some of which are functionally redundant, are present in different tissues or catalyse reactions with substrates of various chain lengths. The mechanisms of how these compounds are transported and how the cuticle is assembled rely on ABC transporters, LTP lipid carrier proteins, cutin synthases, and cutinsomes. Knowledge of these highly dynamic processes is very fragmented and the integrated model of cutin synthesis is yet to be elucidated. A tight connection between the cuticle and the cell wall, conventionally seen as two separate entities, has also been implied. The complexity of these mechanisms is also reflected in their transcriptional and post-transcriptional regulation. While SHINE and MIXTA-like transcriptional factors and the WW-domain protein CFL1 regulate the cuticle's synthesis throughout a plant's development, ABA-dependent MYB transcriptional factors are important during abiotic stress. Recent research also...

Plant cell is a complex and complicated environment where countless biochemical processes and metabolic pathways take place independently of each other. Some of them could pose a threat to a plant cell or be a cause of organism lethality altogether, mainly because of their antagonistic character or by their toxicity. To eliminate this danger and ensure the most effective course of these reactions, a separation into properly separated and safe compartments is being applied. This phenomenon is called cellular compartmentalization and these sections are called organelles. Peroxisomes are one of them. They are highly dynamic and metabolically diverse vesicles of spherical nature, formed by a single-layered membrane. They contain crucial enzymes that operate in processes such as reactive oxygen species (ROS) metabolism, photorespiration, storage oils breakdown followed by β-oxidation of fatty acids, glyoxylate cycle, phytohormones biosynthesis, and more and more of other metabolic pathways. By that, they stand as an essential imaginary crossroad connecting these critical processes in plant development and physiological responses to abiotic and biotic stresses. This work is focused on reviewing these peroxisomal functions in plant cells, as well as the peroxisomal structure, its proteome and biogenesis....

Microtubules, which are made of polymers of α- and β-tubulin, are an integral part of the cytoskeleton. Both types of tubulin share a considerable sequential homology across eukaryotic organisms. Tubulins are encoded by relatively large gene families. The expression of these genes produces different tubulin isotypes, some of which may exhibit different properties. Tubulin isotypes can be further posttranslationally modified. The best known posttranslational modifications of tubulin include acetylation, phosphorylation, tyrosination, polyglutamylation and polyglycylation. The tubulin code arises from the combination of expression of different tubulin isotypes and their posttranslational modifications. As a consequence, microtubules in cells can be composed of a "mixture" of different tubulins with distinct functions and properties. Even though the existence of the tubulin code has been proven in every model organism, plants included, the precise understanding of the meaning of microtubules being composed of different tubulins is still subjected to research. Much of the research on the tubulin code has been carried out on animal models. In contrast, relatively little is known about the existence of the tubulin code in plants. This theses summarizes current knowledge on the localization and regulation...

Lignin is an essential compound of all vascular plants. Recent studies have also shown its presence in non-vascular plants. Lignin is important for plant growth and development, provides a mechanical support to plant tissues and is also a major player in the response of plants to various stresses. Lignin is an aromatic heteropolymer and comprises classical lignin units - guaiacyl (G), syringyl (S) and p-hydroxyfenyl (H). The amount and ratio of these lignin units varies among plant species, organs and cell types. Nontraditional monomer units that can be deposited in lignin also increase the variety of lignin. Lignification, i.e. the formation and deposition of lignin, is a complex and precisely controlled process involving the synthesis of monolignols in the cytoplasm, their transport into the cell wall and subsequent polymerization. A number of enzymes are involved in the lignification process, and recent studies are gradually revealing the mechanisms of lignification regulation in various cells of plant body. This bachelor thesis summarizes the knowledge of the importance, synthesis and deposition of lignin, and also compares the mechanisms regulating lignification in different types of plant cells - tracheary elements, sclerenchyma and endodermal cells. An interesting phenomenon of lignification...

The paper deals with the possibilities of detection fungal disease resistance genes in the original European cereals, such as wheat, barley, rye and oats. It provides an overview of the most important resistance genes for breeding. The significance of the resistance genes is evaluated on the basis of the harmfulness of the individual cereal fungal diseases, as well as the importance of the individual cereal species. It describes the possibilities of detecting resistance genes using molecular markers and compares various types of molecular markers. It seeks answers to the question of where the breeding on resistance and therefore the use of molecular markers brings the greatest benefit.

Evolutionary-developmental study of membrane proteins Mgr. Stanislav Vosolsobě Abstract Using a plethora of experimental approaches for phylogenetical and functional study on several membrane signalling proteins, I brought new evidences supporting a hypothesis that the molecular evolution of protein families is a highly dynamic, not conservative, process. In DREPP family of calcium-binding peripherally-associated plasma-membrane proteins I found a broad flexibility in protein-membrane binding manners coupled with a many independent duplication of this Euphyllophyta-clade specific plant gene. In three families of auxin transporting proteins, PIN-FORMED, LAX and PILS, I showed that emergences of these proteins are uncorrelated and placed on different levels of the plant kingdom phylogenetic tree. However these proteins ensure very fundamental plant morphogenetic processes, like cell differentiation, organ formation or tropisms, with strong effects of their deleterious mutations, I found many gene radiations and losses on a all taxonomic levels in these families, evidencing that key and shared physiological processes may be realised by genes touched by a recently undergoing evolution. Evolutionary-developmental synthesis of a functional and phylogenetic data must be done with caution due to high risk of...

Adhesion between the plasma membrane and the cell wall and the existence of the continuum of these two compartments is needed for signal transmission, e.g. under pathogen attack, during cell expansion and cell wall growth, or in response to environmental conditions. This adhesion is, in addition to the turgor, provided by physical connection of both compartments. One of the best-known examples of physical connection is found in the root system, in the Caspary strip region, where it is required to maintain apoplastic barriers of the root system, even under adverse conditions and consequent plasmolysis. There is little information about the physical interconnection and the participating macromolecules but there are candidates, which could participate in this interaction. The diploma thesis deals with arabinogalactan (AGP) proteins with fasciclin-like domain (namely FLA9 and FLA2). These proteins may play a role in the adhesion of plasma membrane and cell wall and may be involved in the growth regulation and development of the root system. Both genes are are relatively strongly expressed in the roots, especially in the elongation zone and in the cortex, including the endodermis (according to chip data). Also, to a smaller extent, the thesis deals with integrin-like At14A protein. The most interesting...

Cytoskeletal proteins form very important structures in eukaryotic cells. It was demonstrated in last years that cytoskeletal proteins are also an important part of prokaryotic cells. One of the cytoskeletal families is the tubulin family. The tubulins are known to form dynamic microtubules which are important for cell division and other processes in eukaryotic cells. Proteins homologous to tubulin were discovered in Prokaryotes, based on their similarity to the tubulin GTP-binding region. This thesis describes the structure and function of known tubulin homologs from bacteria and archaea and compares them to their eukaryotic counterplayers.

The basis of this study are mutant plants with ARP2/3 complex lacking in one of its subunits (arpc5 and arp2). These plants also express CSC subunit CESA6 of primary cell wall tagged by YFP. Thanks to modern imaging technologies, it is possible to observe the movement of tagged cellulose synthase complexes in vivo at plasmatic membrane. Kymograph analyses was used to measure the velocity of CESA complexes. In addition to observing CESA complexes directly on the plasma membrane, experiments were made to regenerate cell walls of protoplasts of Arabidopsis thaliana plants arpc5 and WT. It was found, that observed mutants arpc5 and arp2 have reduced velocity of CESA complexes in comparison to WT and arpc5 protoplasts regenerate cellulose mesh of cell wall slower. Keywords: Cellulose synthesis, ARP2/3 complex, CESA, CSC velocity, arpc5, arp2, Arabidopsis thaliana.

Aluminium toxicity is the main factor limiting plant growth on acid soils. Aluminium inhibits root growth within few minutes after aluminium treatment. The mechanism and primary target of his action is still unknown. In this diploma thesis the effect of aluminium toxicity on dynamics of cortical microtubules WT and pldα1 plants was studied using the EB1a-GFP marker. Polymerization rate in both the transition and the elongation zone increased immediately after the aplication of aluminium. Nevertheless, microtubules in the transition zone are much more sensitive to aluminium, because the aluminium-induced increase in the polymerization rate was higher than in the elongation zone. Plants lacking PLDα1 showed higher dynamics on plus ends of cortical microtubules compared to WT during aluminium stress, which enabled them to react faster to stress stimuli. Mutants showed lower sensitivity to aluminium and 100 μM concentration of aluminium ions has beneficial effect on root growth in pldα1. These results suggest that PLDα1 influences microtubule dynamics. Microtubules in pldα1 plants were more dynamic and they polymerized faster in the response to aluminium, which was accompanied by decreased sensitivity to aluminium stress compared to WT. Changes in microtubule dynamics may play a role in aluminium...

This thesis focuses on the study of TTL protein family in the model species Arabidopsis thaliana. It sums up available published data on this so far poorly studied genes and presents newly gained experimental data. Main emphasis is given to TTLs cellular localization in possible relationship to their physiological function. Presented thesis comprises of in silico analysis of TTL proteins, but also the creation and microscopy analysis of TTL-mRuby2 and TTL-GFP fusion proteins in transient and permanent transformants. Powered by TCPDF (www.tcpdf.org)

In plant cells, actin filaments are nucleated in two different ways: The growth of single filaments or their bundles is enabled by various types of formins, whereas branched meshworks emerge due to Arp2/3 complex activity. Mutations in genes of these nucleators lead to various phenotypic traits. This thesis deals in the first place with influence of Arp2/3 complex subunits' dysfunction on intracellular motility (cytoplasmic streaming, stop-and-go movements of Golgi apparatus cisternae), since it had not been extensively studied before, and also attempts to quantify the already known impacts of mutations in genes for ARP2 and ARPC5 subunits on the vacuolar morphogenesis. For comparison, a few experiments with plants which carried a mutation in gene for FH1 formin were also realised when measuring the cytoplasmic streaming. The experiments were conducted with a model plant Arabidopsis thaliana. The methods particularly included transformation with fluorescent markers by Agrobacterium tumefaciens (or usage of a fluorescent dye), microscopy (both standard and confocal) and subsequent evaluation of the acquired data using a computer. During the cytoplasmic streaming research, effects of cytoskeletal drug latrunculin B were studied, too. The outputs did not prove that the Arp2/3 complex defects would manifest...