The role of actin isoforms in somatic embryogenesis in Norway spruce

<p>Abstract</p> <p>Background</p> <p>Somatic embryogenesis in spruce is a process of high importance for biotechnology, yet it comprises of orchestrated series of events whose cellular and molecular details are not well understood. In this study, we examined the role of actin cytoskeleton during somatic embryogenesis in Norway spruce line AFO 541 by means of anti-actin drugs.</p> <p>Results</p> <p>Application of low doses (50-100 nM) of latrunculin B (Lat B) during the maturation of somatic embryos predominantly killed suspensor cells while leaving the cells in meristematic centres alive, indicating differential sensitivity of actin in the two cell types. The treatment resulted in faster development of more advanced embryos into mature somatic embryos and elimination of insufficiently developed ones. In searching for the cause of the differential actin sensitivity of the two cell types, we analysed the composition of actin isoforms in the culture and isolated four spruce actin genes. Analysis of their expression during embryo maturation revealed that one actin isoform was expressed constitutively in both cell types, whereas three actin isoforms were expressed predominantly in suspensor cells and their expression declined during the maturation. The expression decline was greatly enhanced by Lat B treatment. Sequence analysis revealed amino-acid substitutions in the Lat B-binding site in one of the suspensor-specific actin isoforms, which may result in a different binding affinity for Lat B.</p> <p>Conclusions</p> <p>We show that manipulating actin in specific cell types in somatic embryos using Lat B treatment accelerated and even synchronized the development of somatic embryos and may be of practical use in biotechnology.</p

The humidity level matters during the desiccation of Norway spruce somatic embryos

In Norway spruce, as in many other conifers, the germination capacity of somatic embryos is strongly influenced by the desiccation phase inserted after maturation. The intensity of drying during desiccation eminently affected the formation of emblings (i.e., seedlings developed from somatic embryos). Compared to non-desiccated embryos, the germination capacity of embryos desiccated at 100% relative humidity was about three times higher, but the reduction of relative humidity to 95 and 90% had a negative effect on the subsequent embryo development. The water loss observed in these embryos did not lead to an increase in lipid peroxidation, as shown by malondialdehyde levels. Another metabolic pathway in plants that mediates a response to abiotic stresses is directed toward the biosynthesis of polyamines (PAs). The activities of PA biosynthetic enzymes increased steadily in embryos during desiccation at 100% relative humidity, whereas they decreased at lower humidity. The total content of free PAs in the embryos gradually decreased throughout desiccation. The increase in free putrescine (Put) and perchloric acid-insoluble Put conjugates was observed in embryos desiccated at lower humidity. These changes were accompanied to some extent by the transcription of the genes for the PA biosynthesis enzymes. Desiccation at 100% relative humidity increased the activity of the cell wall-modifying enzymes β-1,3-glucanases and chitinases; the activities of these enzymes were also significantly suppressed at reduced humidity. The same pattern was observed in the transcription of some β-1,3-glucanase and chitinase genes. Desiccation treatments triggered metabolic processes that responded to water availability, suggesting an active response of the embryo to the reduction in humidity. A positive effect was demonstrated only for desiccation at high relative humidity. Some of the physiological characteristics described can be used as markers of inappropriate relative humidity during somatic embryo desiccation

Mutually opposing activity of PIN7 splicing isoforms is required for auxin-mediated tropic responses in Arabidopsis thaliana

Summary Advanced transcriptome sequencing has uncovered that the majority of eukaryotic genes undergo alternative splicing (AS). Nonetheless, little effort has been dedicated to investigating the functional relevance of particular splicing events, even those in the key developmental and hormonal regulators. Combining approaches of genetics, biochemistry and advanced confocal microscopy, we describe the impact of alternative splicing on the PIN7 gene in the plant model Arabidopsis thaliana. PIN7 encodes a polarly localized transporter for the phytohormone auxin and produces two evolutionary-conserved transcripts PIN7a and PIN7b. PIN7a and PIN7b, differing in a 4-amino acid motif, exhibit almost identical expression pattern and subcellular localization. We reveal that they closely associate and mutually influence their mobility within the plasma membrane. Phenotypic complementation tests indicate that the functional contribution of PIN7b per se is minor, but it markedly reduces the prominent PIN7a activity, which is required for correct seedling apical hook formation and auxin-mediated tropic responses. Our results establish alternative splicing of the PIN family as a conserved, functionally relevant mechanism, unveiling an additional regulatory level of auxin-mediated plant development.Advanced transcriptome sequencing has revealed that the majority of eukaryotic genes undergo alternative splicing (AS). Nonetheless, little effort has been dedicated to investigating the functional relevance of particular splicing events, even those in the key developmental and hormonal regulators. Combining approaches of genetics, biochemistry and advanced confocal microscopy, we describe the impact of alternative splicing on the PIN7 gene in the model plant Arabidopsis thaliana. PIN7 encodes a polarly localized transporter for the phytohormone auxin and produces two evolutionarily conserved transcripts, PIN7a and PIN7b. PIN7a and PIN7b, differing in a four amino acid stretch, exhibit almost identical expression patterns and subcellular localization. We reveal that they are closely associated and mutually influence each other's mobility within the plasma membrane. Phenotypic complementation tests indicate that the functional contribution of PIN7b per se is minor, but it markedly reduces the prominent PIN7a activity, which is required for correct seedling apical hook formation and auxin-mediated tropic responses. Our results establish alternative splicing of the PIN family as a conserved, functionally relevant mechanism, revealing an additional regulatory level of auxin-mediated plant development.Peer reviewe

The Response of Picea abies Somatic Embryos to UV-B Radiation Depends on the Phase of Maturation

Ultraviolet-B (UV-B) radiation is a key environmental signal which initiates diverse responses that affect the metabolism, development, and viability of plants. In keeping with our previous studies, we concentrated primarily on how UV-B radiation affects Norway spruce [Picea abies (L.) Karst.] somatic embryo maturation and how phenolics and polyamines (PAs) are linked to the defense response invoked by UV-B irradiation. We treated clusters of Norway spruce embryogenic culture (EC) with UV-B during the five stages of embryo maturation (early, cylindrical, precotyledonary, cotyledonary, and mature embryos). For the first time, we take an advantage of the unique environmental scanning electron microscope AQUASEM II to characterize somatic embryos in their native state. The severity of the irradiation effect on embryonal cell viability was shown to be dependent on the intensity of radiation as well as the stage of embryo development, and might be related to the formation of protoderm. The response of early embryos was characterized by an increase in malondialdehyde (MDA), a marked decrease in PA contents and a decline in phenolics. The reduced ability to activate the defense system seems to be responsible not only for the severe cell damage and decrease in viability but also for the inhibition of embryo development. The significant reduction in spermidine (Spd), which has been reported to be crucial for the somatic embryo development of several coniferous species, may be causally linked to the limited development of embryos. The pronounced decrease in cell wall-bound ferulic acid might correspond to failure of somatic embryos to reach more advanced stages of development. Embryos at later stages of development showed stress defense responses that were more efficient against UV-B exposure

The importance of time, space and genotype for the intensity of plant-soil feedback

Plant-soil feedback (= zpětná vazba mezi rostlinou a půdou) je důležitý mechanismus fungující v rostlinném společenstvu, který ovlivňuje jeho druhové složení. Plant-soil feedback je studován pro získání informace o vzájemném působení rostlin ve společenstvu skrz změnu půdních charakteristik. Vliv plant-soil feedback může být jak pozitivní, negativní, tak neutrální a díky těmto změnám dochází ke kompetici či vzájemné koexistenci rostlinných druhů. Plant-soil feedback je často studován v rámci sukcese rostlinných druhů, u invazních rostlin nebo u dominantních druhů ve společenstvu, kde se zjišťuje, jak silně se vzájemně druhy ovlivňují. Navzdory velkému množství předchozích studií na toto téma víme velmi málo o změně síly plant-soil feedback v čase u jednoho druhu, tedy jak se mění síla ovlivnění půdy a tedy následná biomasa druhu rostoucího v této půdě v závislosti na různé délce působení rostlinného druhu. Podobně málo informací máme o vlivu vzdálenosti od rostliny na intenzitu plant-soil feedback a možné vnitrodruhové variabilitě v těchto interakcích. Cílem mé práce tedy bylo sledovat změnu síly plant-soil feedback v čase u druhu rostoucího v půdě ovlivněné stejným či jiným rostlinným druhem a identifikovat, které abiotické faktory mohou být zodpovědné za změnu rostlinné biomasy. Dále jsem...Plant-soil feedback is an important mechanism in plant communities affecting their species composition. Understanding plant-soil feedback is crucial for describing the interactions between plant communities and their soil communities. The effects of plant-soil feedback can be positive, negative or neutral and because of these changes plants are either coexisting or competing. Plant-soil feedback is often studied within plant succession, plant invasion and plant dominance in a plant community. In these cases, the nature and strength of influence of each species is studied. Despite the large number of previous studies on plant-soil feedback, very little is known about the temporal and spatial changes of the intensity of plant-soil feedback and the intraspecific variability in plant-soil feedback. The aim of my work was to observe the temporal changes in the plant-soil feedback of a species growing in soil which was conditioned by the same or other plant species. Another goal was to explore spatial changes in plant-soil feedback. The last objective was to test differences in the intensity of plant-soil feedback among different genotypes of one species. The results of my work demonstrated that the intensity of intraspecific plant-soil feedback of Rorippa austriaca is negative and increases with...Department of BotanyKatedra botanikyPřírodovědecká fakultaFaculty of Scienc

Anaemia disease models

Hematopoiesis is a process by which blood cells are generated. All vertebrates have two phases of hematopoiesis - primitive and definitive. The main purpose of primitive hematopoiesis is the production of red blood cells, which provide oxygenation to the developing embryo. Other blood cell lineages are established by definitive hematopoiesis. The main function of erythrocytes is oxygen transport to all tissues. When erythrocyte production is decreased or they are damaged due to the membrane, enzyme or hemoglobin impairment, the condition called anemia arises. Sickle cell disease and β-thalassemia are called hemoglobinopathies as they are caused by the damaged hemoglobin. Fanconi anemia is caused by mutations in one of 21 genes of Fanconi anemia pathway, which plays an essential role in DNA repair. Diamond Blackfan anemia is caused by mutations gene for ribosomal proteins. Human cells, Mus musculus, Gallus gallus, Xenopus laevis and Danio rerio seem to be good models for study of this diseases and they are also useful for achieving therapeutical goals

The importance of time, space and genotype for the intensity of plant-soil feedback

Plant-soil feedback is an important mechanism in plant communities affecting their species composition. Understanding plant-soil feedback is crucial for describing the interactions between plant communities and their soil communities. The effects of plant-soil feedback can be positive, negative or neutral and because of these changes plants are either coexisting or competing. Plant-soil feedback is often studied within plant succession, plant invasion and plant dominance in a plant community. In these cases, the nature and strength of influence of each species is studied. Despite the large number of previous studies on plant-soil feedback, very little is known about the temporal and spatial changes of the intensity of plant-soil feedback and the intraspecific variability in plant-soil feedback. The aim of my work was to observe the temporal changes in the plant-soil feedback of a species growing in soil which was conditioned by the same or other plant species. Another goal was to explore spatial changes in plant-soil feedback. The last objective was to test differences in the intensity of plant-soil feedback among different genotypes of one species. The results of my work demonstrated that the intensity of intraspecific plant-soil feedback of Rorippa austriaca is negative and increases with..

The nursing process for children with ear disease

Ear diseases in childhood are relatively frequent. Of these, the most affecting and threatening the children in infant, toddler and preschool age is an infection of the middle ear. Despite the increasing quality of healthcare and development of vaccines that contribute to a reduction of the number of children diagnosed with acute otitis media, occurrences of these inflammations are still frequent. Inflammation of the ear is accompanied by a number of unpleasant symptoms and as well as other diseases it requires specific treatment and care. Going through an inflammation of the middle ear is for the child an unpleasant and often very painful experience. This disease also brings its own risks in terms of complications. Although its course is mostly without complications and treatment is directed by a pediatrician, we are dealing with cases where it is necessary to hospitalize a child patient. This work in its theoretical part refers to the types of otitis media, prevention, risk factors and complications. It describes the diagnosis, treatment and care for a child with otitis media. It also deals with the awareness of parents, points to a nursing problems associated with inflammation of the middle ear and the specifics of child care. In the empirical part of this thesis, two objectives were set. The first objective was to map the state of awareness of parents about the risk factors and preventive measures to prevent otitis media. The second objective was to determine the incidence of the most common problems in nursing care for children with otitis media. Four research questions were set for these objectives and it was: How are parents being educated regarding risk factors that can lead to ear disease of children? How are parents being informed about preventive measures to prevent ear disease in children? How parents perceive education on inflammatory diseases of ears? What are the most common problems in nursing care for a child with inflammatory diseases of the ears? For the processing of the empirical part of the research it was chosen quantitative research. The survey was carried out using the methods of data collection through semi-structured interviews. It was designed for two nurses working in GP for children and adolescents and four nurses working in the pediatric department. Another research group consisted of parents of children. Half of them had experience with caring for their own child who has undergone at least one ear infection. Respondents were before the start of the interview informed about the reason and purpose of the research. Interviews were held up with the express consent of each respondent addressed. Interviews were carried out on a pre-arranged location in the period March-May 2014. They were recorded using a voice recording and a written record. After that, the interviews were transcribed and processed into categories and subcategories. This gave rise to four categories: Awareness of Parents, Education of Parents, Knowledge of Parents and Inflammation of the Middle Ear

Role of Rnf207b in zebrafish hematopoiesis

Hematopoiesis is the process of proliferation, differentiation and self-renewal of hematopoietic stem cells. Regulation of hematopoiesis is a complex process, which takes place on many different levels and is directed by many signals. RNF207 is one of the perspective genes chosen based on a screen in chicken model, where obtained data show its role in hematopoiesis. The aim of this work was to confirm the role of rnf207b as a new regulator of hematopoiesis in Danio rerio and to find out on which level of hematopoiesis is active. Danio rerio is an excellent model to study the function of genes in vivo, thanks to the easy manipulation of genetic expression and wide range of phenotypes during the development. To study the effect of rnf207b in hematopoiesis of Danio rerio we performed the knock-down of this gene by microinjection of morpholino oligonucleotides into one cell stage embryos. In these injected fish, we saw the effect in both thrombocyte and erythroid lineage, suggesting that rnf207b could be a regulator at the hierarchical level of progenitors or even more upstream. The results of developmental and tissue specific expression analysis then show that expression of rnf207b begins as early as 18 hpf, at the time of primitive hematopoiesis. Although rnf207b is expressed in the kidney (an...Hematopoéza (krvetvorba) je proces proliferace, diferenciace a sebeobnovy hematopoetických kmenových buněk. Regulace hematopoézy je komplexní děj, který probíhá na mnoha úrovních a je řízen mnoha signály. RNF207 je jedním z perspektivních genů vybraných na základě experimentu v kuřecím modelu, ve kterém získaná data ukazují na jeho roli v krvetvorbě. Cílem této práce je určit úlohu rnf207b jako nového regulátoru krvetvorby Dánia pruhovaného (Danio rerio) a případně tedy zjistit, na jaké úrovni hematopoézy funguje. Dánio pruhované je výborným modelem pro studium funkce genů in vivo, a to díky snadné manipulaci genové exprese a širokým možnostem analýzy fenotypů během vývoje. Pro studium vlivu rnf207b na hematopoézu Dánia pruhovaného jsme snížili expresi tohoto genu mikroinjikací morfolino oligonukleotidů do jednobuněčných embryí. U takto injikovaných embryí jsme viděli efekt jak v trombocytární, tak v erytroidní linii, z čehož vyplývá, že by rnf207b mohl být regulátor na hierarchické úrovni progenitorů, případně by mohl být v hierarchii ještě výše. Výsledky analýzy vývojově a tkáňově specifické exprese pak ukazují, že exprese rnf207b začíná již v 18 hpf, tedy ještě v době primitivní hematopoézy. Přestože je rnf207b v dospělosti exprimován v ledvinách (anatomické místo definitivní hematopoézy ryb),...Katedra buněčné biologieDepartment of Cell BiologyPřírodovědecká fakultaFaculty of Scienc

Plant-soil feedback as a mechanism affect species diverzity of communities

Plant-soil feedback is known as the interaction between plant and soil. There is the feedback, when plant affect soil during their roots and on the other side soil affect growth and development through the change of their biotic and abiotic conditions. The plant-soil feedback is important mechanism of affecting plants or their competitive plant species through the soil. In this thesis I would like to summarize the functioning of the plant-soil feedback and factors which influence the feedback, the effect of feedback on the species diversity of plant communities, role in secondary succession, invasion and during coexistence of different species. I would like to point out, in which cases this feedback was confirmed experimentally and which experimental methods are used in the study of plant-soil feedback