Plants are continuously exposed to different abiotic and biotic stresses in their natural environment. Their capacity to survive depends on the capacity to perceive external signal and quality amount a defence response for protection from the stress perceived. The purpose of this project was to study the impact of combined abiotic stress and biotic stress on the outcome of the disease inducing Arabidopsis thaliana – Pseudomonas syringae interaction. This study included a focus on the role of ABA in these interactions and also whether 3´-O-β D- ribofuranosyl adenosine (hereafter it called ‘400’ compound), a novel adenosine derived compound induced during compatible interactions, was involved. The later involved the targetted disruption of a putative 400 biosynthetic pathway involving analysis of knockout mutants of enzymes; APD-ribose diphosphatase NAD binding / hydrolases of the NUDIX class, glucosyl transferases, ribosyltransferases, a ribose-phosphate pyrophosphokinase3 and galactosyltransferases. Unfortunately, none of these targeted interventions modified the host response to Pseudomonas infection, nor altered levels of 400 in challenged leaves. The primary research investigated the interaction between abiotic and biotic stresses in Arabidopsis plants focussing on the modulation of plant defence against multiple, and possibly antagonistic, stress responses and the role plant hormones play in this process. We showed that high light caused enhanced susceptibility to the already virulent Pseudomonas syringae DC3000pvsp61. The pathways contributing to this enhanced susceptibility were largely ABA independent. Subsequent characterization of transgenic lines expressing the soluble Arabidopsis abscisic acid receptors, PYRABACTIN RESISTANCE1-LIKE4-6 provided compelling evidence for a role for these receptors in DC3000 virulence strategies, but they contribute to a lesser extent to the enhanced susceptibility under high light. This was corroborated genetically by using mutants of the immediately downstream targets of PYLs, the type two protein phosphatase, specifically the triple mutant hab1-1/abi2-1/abi1-2. A number of epitope and fluorescent constructs were generated to facilitate future studies of the role of ABA signaling. Targetted profiling suggested that SA dynamics were altered under DC3000 challenged Arabidopsis grown under high light. Furthermore, differential accumulation of flavonoids suggested these may also play a role in attenuating host defences under high light. Finally we provide evidence based on comparative analysis of that the photoreceptors phytochrome double mutant phyA-211/phyB-9 and cry1/cry2 behave antagonistically in Arabidopsis response to DC3000. Overall our studies support the conclusion that plants abiotic stress (HL) response takes precedence over biotic stress (DC3000) responses and that abiotic stress is detrimental to plant immunity. The luciferase transgenic PYL lines showed high level of expression of ClucP::PYL5 plant tissues challenged 2hpi of DC3000 (OD600: 0.15) in comparison with C1lucP::PYL6. This result opposes to what RT-PCR reported; which was that three PYLs genes display similar expression level at 6hpi of hrpA or 18hpi of DC3000. The epitope tags of CaMV::HA transgenic plants showed HA-tagged signal with stunted phenotype in a range of PYL4, 5 and 6 plants but none of the plants displayed any differences in susceptibility to DC3000. Although, RT-PCR assay showed high levels of expression in the three PYLs, 6hpi of hrpA but no signal was detected in B8eGFP::PYL5 transgenic line either followed the DC3000 and hrpA infection or by examined plant seedlings at early stages under confocal microscopy.

Pesticides and their excessive use lead to environmental pollution. Violation of the guidelines for their use disposal of empty containers could lead to contamination of water, soil and poisoning of animals and beneficial insects. Honey bee is useful creature on our planet. Good farming depends entirely on the pollination, but whole vegetal kingdom couldn’t exist it form known and used by mankind. Therefore, rules that protect these useful creatures against inadequate use of pesticides have been developed. This study focuses on the evaluation of the possibility to use bee products as bioindicators of the state of environment. Five active substances which are components of pesticides used in the treatment of agricultural field around Tasovice village were analysed in the pollen and honey. For sample preparation QuEChERS and SPE methods were used, gas chromatography with to mass spectrometric detection was employed as final analytical technique.

This diploma thesis entitled "Attractiveness of Planting Species with a Potential to Extend Bee Forage for Bees" discusses pollen and nectar plants planted in the Brník municipality in the Central Bohemian Region. The thesis consists of two parts. The theoretical part describes honeybees, bee plants and their pollen- and nectar production ability as well as bee forage from early spring until late autumn so as to reflect the needs of pollinators as best as possible. The practical part uses pollen analysis to microscopically analyse the representation of pollen grains of plants in the honey produced by local bee colonies. Pollen analyses of honey from own bee colonies are compared with that from bee colonies belonging to neighbouring beekeepers.

The development of plant flowers represents a complex process controlled by numerous mechanisms. The creation of double homozygous mutant of both β subunits (sometimes also referred to as basic transcription factor 3) of nascent polypeptide associated complex in Arabidopsis thaliana (further referred to as nacβ1 nacβ2) caused quite a strong defective phenotype including abnormal number of flower organs, shorter siliques with a reduced seed set, and inferior pollen germination rate together with a lower ovule targeting efficiency. Previously, NAC complex was described to be formed as a heterodimer composed of an α- and β-subunit, which binds ribosome and acts as a chaperone in Saccharomyces cerevisiae. In plants, NACβ is connected to stress tolerance and to plant development as a transcription regulator. However, little is known of NAC heterodimer function in plants. In this thesis, yeast two hybrid system (Y2H) and bimolecular fluorescence complementation (BiFC) assays were used to verify the NAC heterodimer formation in A. thaliana and to establish any potential interaction preferences between both NACβ paralogues and five NACα paralogues. To deepen the understanding about molecular mechanisms behind the nacβ1 nacβ2 phenotype, flower bud transcriptome of the nacβ1 nacβ2 double homozygous mutants...

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...

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...

[EN] ABSTRACT Abscisic acid (ABA) signaling plays a critical role in regulating root growth and root system architecture. ABA-mediated growth promotion and root tropic response under water stress are key responses for plant survival under limiting water conditions. In this work, we have explored the role of Arabidopsis (Arabidopsis thaliana) PYR/PYL/RCAR receptors (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS) for root ABA signaling. As a result, we discovered that PYL8 plays a nonredundant role for the regulation of root ABA sensitivity. Unexpectedly, given the multigenic nature and partial functional redundancy observed in the PYR/PYL family, the single pyl8 mutant showed reduced sensitivity to ABA-mediated root growth inhibition. This effect was due to the lack of PYL8-mediated inhibition of several clade A phosphatases type 2C (PP2Cs), since PYL8 interacted in vivo with at least five PP2Cs, namely HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 as revealed by tandem affinity purification and mass spectrometry proteomic approaches. Membrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calciumdependent interactions of PYR/PYL/RCAR ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA-independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant-specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL/RCAR receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL/RCAR function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, and CAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL/RCAR-interacting partners that mediate a transient Ca2+-dependent interaction with phospholipid vesicles, which affects PYR/PYL/RCAR subcellular localization and positively regulates ABA signaling. / [ES] RESUMEN La señalización por la hormona vegetal ácido abscísico (ABA) desempeña un papel crítico en la regulación del crecimiento de la raíz y en la arquitectura del sistema radical. La promoción de crecimiento de la raíz en condiciones de estrés hídrico mediada por ABA es clave para la supervivencia de las plantas bajo condiciones limitantes de agua. En este trabajo, hemos explorado el papel de los receptores PYR/PYL/RCAR (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/ REGULATORY COMPONENTS OF ABA RECEPTORS) de Arabidopsis (Arabidopsis thaliana) en la ruta de señalización de ABA en raíz. Así, hemos descubierto que el receptor de ABA PYL8 juega un papel no redundante en la regulación de la percepción de ABA en raíz. Inesperadamente, dada la naturaleza multigénica y la redundancia funcional parcial observada en la familia PYR/PYL/RCAR, el mutante pyl8 fue el único mutante sencillo de pérdida de función de los receptores PYR/PYL/RCAR que mostraba una sensibilidad reducida a la inhibición del crecimiento mediada por ABA en raíz. Este efecto se debe a la falta de inhibición mediada por PYL8 de varias fosfatasas del grupo A tipo 2C (PP2Cs), ya que PYL8 es capaz de interactuar in vivo con al menos cinco PP2Cs, denominadas HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 según lo han revelado la purificación por afinidad en tándem (TAP por sus siglas en inglés) y estudios proteómicos de espectrometría de masas. La transducción de la señal del ABA localizada en la membrana plasmática celular juega un papel crucial en los pasos iniciales de la señalización de la fitohormona, pero los mecanismos moleculares que unen los componentes básicos de la señalización y la membrana plasmática no están claros. Estudiando las interacciones de los receptores del ABA PYR/PYL/RCAR con la membrana plasmática hemos encontrado que éstos pueden interaccionar transitoriamente con ella de forma dependiente de calcio gracias a una familia de proteínas con dominios C2 relacionadas con la ruta de señalización de ABA (denominadas C2-domain ABA-related (CAR) proteins). Específicamente, se encontró que PYL4 interacciona de manera independiente de ABA con CAR1 tanto en la membrana plasmática como en el núcleo de las células vegetales. La proteína CAR1 pertenece a una familia multigénica constituida por 10 miembros en Arabidopsis thaliana, desde CAR1 hasta CAR10, y que solo se encuentra en plantas. Los ensayos de complementación bi-molecular de fluorescencia y de co-immunoprecipitación confirmaron la interacción en células vegetales tanto de PYL4-CAR1 como de otras parejas de PYR/PYL-CAR. La cristalización de la proteína CAR4 reveló que, además de un dominio C2 clásico de unión a lípidos dependiente de calcio, las proteínas de la familia CAR presentan un dominio específico que probablemente es responsable de la interacción con los receptores PYR/PYL/RCAR y de su posterior reclutamiento a las vesículas de fosfolípidos. Esta interacción es relevante para la función de los receptores PYR/PYL/RCAR en la señalización del ABA, ya que diferentes mutantes triples car de pérdida de función, que tienen afectados los genes CAR1, CAR4, CAR5, y CAR9, demostraron una reducción de la sensibilidad al ABA en ensayos de establecimiento de plántula y crecimiento de la raíz. En resumen, hemos identificado nueva familia de proteínas que son capaces mediar las interacciones transitorias dependientes de Ca2+ con vesículas de fosfolípidos, lo que a su vez afecta localización de PYR/PYL/RCAR y regula positivamente la señalización de ABA. / [CA] RESUM La senyalització per l'hormona vegetal àcid abcíssic (ABA) exerceix un paper crític en la regulació del creixement de l'arrel i també en l'arquitectura del sistema radical. La promoció del creixement de l'arrel en condicions d'estrés hídric, regulada per ABA és clau per la supervivència de les plantes sota condicions limitants d'aigua. Amb aquest treball, hem investigat el paper dels receptors PYR/PYL/RCAR (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/ REGULATORY COMPONENTS OF ABA RECEPTORS) d'Arabidopsis (Arabidopsis thaliana) en el camí de senyalització d'ABA en arrel. Així, hem descobert que el receptor d'ABA PYL8 exerceix un paper no redundant en la regulació de la percepció d'ABA en arrel. Inesperadament, donada la naturalesa multigènica i la redundància funcional parcial que s'observa en la família PYR/PYL/RCAR, el mutant pyl8 va ser l'únic mutant senzill de pèrdua de funció dels receptors PYR/PYL/RCAR que mostrava una sensibilitat reduïda a la inhibició del creixement mitjançada per l'ABA en l'arrel. Doncs aquest efecte es deu a la falta d'inhibició regulada per PYL8 de diverses fosfatases del grup A tipus 2C (PP2Cs), ja que PYL8 té la capacitat d'interactuar in vivo almenys amb cinc PP2Cs, anomenades HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABAHYPERSENSITIVE GERMINATION3 segons ho han revelat per una banda la purificació per afinitat en tàndem (TAP són les seues sigles en anglés) i per altra banda, estudis proteòmics d'espectrometria de masses. Pel que fa a la transducció del senyal del l'ABA, la qual es localitza en la membrana plasmàtica cel¿lular, juga un paper molt important en els primers instants de la senyalització de la fitohormona, no obstant això els mecanismes moleculars que uneixen els components bàsics d'aquesta senyalització amb la membrana plasmàtica, no es troben del tot clars. Per tant, s'han estudiat les interaccions que tenen els receptors del ABA PYR/PYL/RCAR amb la membrana plasmàtica, i hem trobat que aquests tenen la capacitat d'interaccionar transitòriament amb la membrana de forma dependent al calci, gràcies a una família de proteïnes amb domini C2, les quals es troben relacionades amb la ruta de senyalització d'ABA(anomenades C2domain ABArelated (CAR) proteins).Específicament, es va trobar que PYL4 interacciona d'una manera independent al ABA amb CAR1, tant en la membrana plasmàtica, com en el nucli de les cèl¿lules vegetals. La proteïna CAR1 pertany a la família multigènica constituïda per 10 components en Arabidopsis thaliana, des de CAR1 fins CAR10, que tan sols es troba en plantes. Els assajos de complementació bimolecular de fluorescència i de co-immunoprecipitació, van confirmar la interacció en cèl¿lules vegetals, tant de PYL4CAR1 com d'altres parelles de PYR/PYL-CAR. La cristal¿lització de la proteïna CAR4 va revelar que, a més d'un domini C2 clàssic de unió a lípids dependent del calci, les proteïnes de la família CAR presenten un domini PYR/PYL/RCAR, i del seu posterior reclutament a les vesícules fosfolipídiques. Doncs, aquesta interacció és rellevant en la funció dels receptors PYR/PYL/RCAR, ja que participa en la senyalització del l'ABA. Aquesta interacció es clau per a la funció dels receptors, ja que diferents mutants triples car de pèrdua de funció, els quals posseïxen afectats els gens CAR1, CAR4, CAR5 i CAR9, van mostrar una reducció de la sensibilitat a l'ABA en assajos d'establiment de plàntula i creixement de l'arrel. En conclusió, hem identificat una nova família de proteïnes amb la capacitat d'organitzar les interaccions transitòries dependents del calci amb vesícules de fosfolípids, fet que al seu torn afecta la localització de PYR/PYL/RCAR i regula positivament la senyalització d'ABA. / Rodríguez Solovey, LN. (2015). IDENTIFICATION OF TARGETS AND AUXILIARY PROTEINS OF PYR/PYL/RCAR ABA RECEPTORS: PROTEIN PHOSPHATASES TYPE 2C (PP2Cs) AND C2-DOMAIN ABA-RELATED PROTEINS (CARs) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58862

Systematic studies of basal nonsense suppression, orthogonality of tRNAPyl variants, and cross recognition between codons and tRNA anticodons are reported. E. coli displays detectable basal amber and opal suppression but shows a negligible ochre suppression. Although detectable, basal amber suppression is fully inhibited when a pyrrolysyl-tRNA synthetase (PylRS)-tRNAPyl_CUA pair is genetically encoded. trnaPyl_CUA is aminoacylated by an E. coli aminoacyl-tRNA synthetase at a low level, however, this misaminoacylation is fully inhibited when both PylRS and its substrate are present. Besides that it is fully orthogonal in E. coli and can be coupled with PylRS to genetically incorporate a NAA at an ochre codon, tRNAPyl_UUA is not able to recognize an UAG codon to induce amber suppression. This observation is in direct conflict with the wobble base pair hypothesis and enables using an evolved M. jannaschii tyrosyl-tRNA synthetase-tRNAPyl_UUA pair and the wild type or evolved PylRS-tRNAPyl_UUA pair to genetically incorporate two different NAAs at amber and ochre codons. tRNAPyl_UCA is charged by E. coli tryptophanyl-tRNA synthetase, thus not orthogonal in E. coli. Mutagenic studies of trnaPyl_UCA led to the discovery of its G73U form which shows a higher orthogonality. Mutating trnaPyl_CUA to trnaPyl_UCCU not only leads to the loss of the relative orthogonality of tRNAPyl in E. coli but also abolishes its aminoacylation by PylRS.