Flowering plants or angiosperms have developed a fertilization mechanism that involves a female egg and central cell, as well as two male sperm cells. A male gametophyte carries the two non-mobile sperm cells, as they need to be delivered to the female gametophyte, the embryo sac. This transport is initiated by a pollen grain that is transmitted onto the stigma of the angiosperm flower. Here it hydrates, germinates, and forms a pollen tube, which navigates through the female plant tissue towards the ovary. The pollen tube grows into an ovule through the funiculus and into one of the two synergid cells. There, growth arrests and the pollen tube bursts, releasing the two sperm cells. One of the sperm cells fuses with the egg cell, giving rise to the embryo, the other one fuses with the central cell, developing into the endosperm, which nourishes the embryo during its development. After a successful fertilization, each ovule develops into a seed and a fruit is formed. This usually consists of several fertilized ovules. The directional growth of the pollen tube through the maternal tissues towards the ovule, as well as sperm cell release, requires a complex communication between the male and the female gametophyte to achieve reproductive success. Over the last years many studies have been performed, contributing to the understanding of cell-cell communication events between the two gametophytes, nevertheless still many aspects remain to be elucidated. This work focused on two topics: i.) Analysis of biological processes affected by pollination and fertilization in the Nicotiana tabacum flower and identification of cysteine rich proteins (CRPs) expressed via isolating and sequencing RNA from the tissue and analyzing the resulting data. ii.) Identification of the defensin-like protein (DEFL) responsible for pollen tube attraction towards the ovule in tobacco. First, tissue samples of pollen tubes and mature ovules were taken at different stages of the fertilization process (unpollinated ovules, after pollination, and after fertilization of the flower). RNA was then isolated and a transcriptome was created. The resulting reads were assembled and transcriptome data analysis was performed. Results showed that pollen tubes and mature ovules differ severely from each other, only sharing about 23 % of the transcripts, indicating that different biological processes are dominant in the two gametophytes. A MapMan analysis revealed that in the pollen tube the most relevant biological processes are related to the cell wall, signaling, and transport, which supports the fact that the pollen tube grows fast to reach the ovule. On the other hand, in the ovule the values of highest significance were obtained for processes related to protein synthesis and regulation. Upon comparing the transcripts in the ovule before and after pollination, as well as after fertilization, it showed that pollination of the flower causes a bigger alteration in the ovule on the transcriptomic level compared to the step from pollination to fertilization. A total of 953 CRPs were identified in Nicotiana tabacum, including 116 DEFLs. Among those, the peptide responsible for pollen tube attraction towards the ovule should be found. Based on in-silico analysis four candidate peptides were chosen for further analysis, two of which had increased expression levels upon pollination and fertilization and the other two displayed an opposite expression. Quantitative real time PCR experiments were performed for the candidates, confirming the in-silico data in vivo. The candidate transcripts were then expressed in a cell free system and applied to pollen tubes in order to test their effect on the growing cells. Positive controls were used, where pollen tubes grew towards freshly dissected ovules. The four candidates did not provoke a pollen tube attraction towards the peptide, leaving open the chance to work on the 112 remaining DEFLs in the future.

The Venus flytrap, \textit{Dionaea muscipula}, with its carnivorous life-style and its highly specialized snap-traps has fascinated biologist since the days of Charles Darwin. The goal of the \textit{D. muscipula} genome project is to gain comprehensive insights into the genomic landscape of this remarkable plant. The genome of the diploid Venus flytrap with an estimated size between 2.6 Gbp to 3.0 Gbp is comparatively large and comprises more than 70 % of repetitive regions. Sequencing and assembly of genomes of this scale are even with state-of-the-art technology and software challenging. Initial sequencing and assembly of the genome was performed by the BGI (Beijing Genomics Institute) in 2011 resulting in a 3.7 Gbp draft assembly. I started my work with thorough assessment of the delivered assembly and data. My analysis showed that the BGI assembly is highly fragmented and at the same time artificially inflated due to overassembly of repetitive sequences. Furthermore, it only comprises about on third of the expected genes in full-length, rendering it inadequate for downstream analysis. In the following I sought to optimize the sequencing and assembly strategy to obtain an assembly of higher completeness and contiguity by improving data quality and assembly procedure and by developing tailored bioinformatics tools. Issues with technical biases and high levels of heterogeneity in the original data set were solved by sequencing additional short read libraries from high quality non-polymorphic DNA samples. To address contiguity and heterozygosity I examined numerous alternative assembly software packages and strategies and eventually identified ALLPATHS-LG as the most suited program for assembling the data at hand. Moreover, by utilizing digital normalization to reduce repetitive reads, I was able to substantially reduce computational demands while at the same time significantly increasing contiguity of the assembly. To improve repeat resolution and scaffolding, I started to explore the novel PacBio long read sequencing technology. Raw PacBio reads exhibit high error rates of 15 % impeding their use for assembly. To overcome this issue, I developed the PacBio hybrid correction pipeline proovread (Hackl et al., 2014). proovread uses high coverage Illumina read data in an iterative mapping-based consensus procedure to identify and remove errors present in raw PacBio reads. In terms of sensitivity and accuracy, proovread outperforms existing software. In contrast to other correction programs, which are incapable of handling data sets of the size of D. muscipula project, proovread’s flexible design allows for the efficient distribution of work load on high-performance computing clusters, thus enabling the correction of the Venus flytrap PacBio data set. Next to the assembly process itself, also the assessment of the large de novo draft assemblies, particularly with respect to coverage by available sequencing data, is difficult. While typical evaluation procedures rely on computationally extensive mapping approaches, I developed and implemented a set of tools that utilize k-mer coverage and derived values to efficiently compute coverage landscapes of large-scale assemblies and in addition allow for automated visualization of the of the obtained information in comprehensive plots. Using the developed tools to analyze preliminary assemblies and by combining my findings regarding optimizations of the assembly process, I was ultimately able to generate a high quality draft assembly for D. muscipula. I further refined the assembly by removal of redundant contigs resulting from separate assembly of heterozygous regions and additional scaffolding and gapclosing using corrected PacBio data. The final draft assembly comprises 86 × 10 3 scaffolds and has a total size of 1.45 Gbp. The difference to the estimated genomes size is well explained by collapsed repeats. At the same time, the assembly exhibits high fractions full-length gene models, corroborating the interpretation that the obtained draft assembly provides a complete and comprehensive reference for further exploration of the fascinating biology of the Venus flytrap.

The internal transcribed spacer 2 (ITS2) of the ribosomal gene repeat is an increasingly important phylogenetic marker whose RNA secondary structure is widely conserved across eukaryotic organisms. The ITS2 database aims to be a comprehensive resource on ITS2 sequence and secondary structure, based on direct thermodynamic as well as homology modelled RNA folds. Results: (a) A rebuild of the original ITS2 database generation scripts applied to a current NCBI dataset reveal more than 60,000 ITS2 structures. This more than doubles the contents of the original database and triples it when including partial structures. (b) The end-user interface was rewritten, extended and now features user-defined homology modelling. (c) Other possible RNA structure discovery methods (namely suboptimal and shape folding) prove helpful but are not able to replace homology modelling. (d) A use case of the ITS2 database in conjunction with other tools developed at the department gave insight into molecular phylogenetic analysis with ITS2.

Insect microbiota plays an essential role on the hosts’ health and fitness, regulating their development, nutrition and immunity. The natural microbiota of bees, in particular, has been given much attention, largely because of the globally reported bee population declines. However, although the worker honey bee has been associated with distinctive and specialized microbiota, the microbiota of solitary bees has not been examined in detail, despite their enormous ecological importance. The main objectives of the present thesis were a) the bacterial community description for various solitary bee species, b) the association of the solitary bee microbiota with ecological factors such as landscape type, c) the relation of the bee foraging preferences with their nest bacterial microbiota, d) the examination of the nest building material contribution to the nest microbiota, e) the isolation of bacterial strains with beneficial or harmful properties for the solitary bee larvae and f) the pathological investigation of bacteria found in deceased solitary bee larvae. The findings of the present study revealed a high bacterial biodiversity in the solitary bee nests. At the same time, the bacterial communities were different for each bee host species. Furthermore, it was shown that the pollen bacterial communities underwent compositional shifts reflecting a reduction in floral bacteria with progressing larval development, while a clear landscape effect was absent. The examination of the nest pollen provisions showed different foraging preferences for each included bee species. Both the pollen composition and the host species identity had a strong effect on the pollen bacteria, indicating that the pollen bacterial communities are the result of a combinatory process. The introduced environmental material also contributed to the nest natural microbiome. However, although the larval microbiota was significantly influenced by the pollen microbiota, it was not much associated with that of the nest material. Two Paenibacillus strains isolated from O. bicornis nests showed strong antifungal activities, while several isolated strains were able to metabolize various oligosaccharides which are common in pollen and nectar. Screening for potential pathogenic bacteria in the nests of O. bicornis unveiled bacterial taxa, which dominated the bacterial community in deceased larvae, while at the same time they were undetectable in the healthy individuals. vi Finally, larvae which were raised in vitro developed distinct bacterial microbiomes according to their diet, while their life span was affected. The present thesis described aspects of the microbiota dynamics in the nests of seven megachilid solitary bee nests, by suggesting which transmission pathways shape the established bacterial communities and how these are altered with larval development. Furthermore, specific bacterial taxa were associated with possible services they might provide to the larvae, while others were related with possible harmful effects. Future studies should integrate microbiota examination of different bee generations and parallel investigation of the microbiota of the nests and their surrounding environment (plant community, soil) to elucidate the bacterial transmission paths which establish the nest microbiota of solitary bees. Functional assays will also allow future studies to characterize specific nest bacteria as beneficial or harmful and describe how they assist the development of healthy bees and the fitness of bee populations.

The greatest problems faced during the 21st century is climate change which is a big threat to food security due to increasing number of people. The increase in extreme weather events, such as drought and heat, makes it difficult to cultivate conventional crops that are not stress tolerant. As a result, increasing irrigation of arable land leads to additional salinization of soils with plant-toxic sodium and chloride ions. Knowledge about the adaptation strategies of salt-tolerant plants to salt stress as well as detailed knowledge about the control of transpiration water loss of these plants are therefore important to guarantee productive agriculture in the future. In the present study, I have characterized salt sensitive and salt tolerant plant species at physiological, phenotypic and transcriptomic level under short (1x salt) and long-time (3x) saline growth conditions. Two approaches used for long-time saline growth conditions (i.e increasing saline conditions (3x salt) and constant high saline conditions (3x 200 mM salt) were successfully developed in the natural plant growth medium i.e soil. Salt sensitive plants, A. thaliana, were able to survive and successfully set seeds at the toxic concentrations on the increasing saline growth mediums, with minor changes in the phenotype. However, under constant high saline conditions they could not survive. This was due to keeping low potassium, and high salt ions (sodium and chloride) in the photosynthetic tissue i.e leaf. Similarly, high potassium and low salt ions in salt tolerant T. salsuginea on both saline environments were the key for survival of this plant species. Being salt tolerant, T. salsuginea always kept high potassium levels and low sodium (during 1x) and chloride levels (during both 1x and 3x) in the leaf tissue. A strict control over transpirational water loss via stomata (formed by pair of guard cells) is important to maintain plant water balance. Aperture size of the stomata is regulated by the turgidity of the guard cells. More turgid the guard cells, bigger the apertures are and hence more transpiration. Under osmotic stress, the water loss is reduced which was evident in the salt sensitive A. thaliana plants under both short and long-time saline growth conditions. As the osmotic stress was only increased during long time saline growth conditions in T. salsuginea therefore, water loss was also decreased only under these saline conditions. Environmental CO2 assimilation also takes place via stomata in plants which then is used for photosynthesis. Stomatal apertures also influence CO2 assimilation. As the light absorbing photosynthetic pigments were more affected in A. thaliana, therefore photosynthetic activity of the whole plant was also reduced. Similarly, both short and long-time saline growth conditions also reduced the effective quantum yield of A. thaliana guard cells. Growth of the plant is dependent on energy which comes from photosynthesis. Reduced environmental CO2 assimilation would affect photosynthesis and hence growth, which was clearly observed in A. thaliana guard cells under long-time saline growth conditions. Major differences in both guard cells types were observed in their chloride and potassium levels. Energy Dispersive X-Ray Analysis (EDXA) suggested strict control of chloride accumulation in T. salsuginea guard cells as the levels remain unchanged under all conditions. Similarly, use of sodium in place of potassium for osmotic adjustments seems to be dependent on Na+/K+ rations in both guard cell types. Increased salt ions and reduced potassium levels in A. thaliana guard cells posed negative effect on photochemistry which in turn increased ROS metabolism and reduced energy related pathways at transcriptomic level in this plant species. Moreover, photosynthesis was strongly affected in A. thaliana guard cells both at transcriptomic and physiological levels. Similarly, global phytohormones induced changes were more evident in A. thaliana guard cells especially on 3x salt medium. Among all phytohormones, genes under the control of auxin were more differentially expressed in A. thaliana guard cells which suggests wide changes in growth and development in this plant species under salinity. Phytohormone, ABA is vital for closing the stomata under abiotic stress conditions. Increased levels of ABA during saline conditions led to efflux of potassium and counter anions (chloride, malate, nitrate) from the guard cells which caused the outward flow of water and hence reduction in turgor pressure. Reduced turgor pressure led to reduced water loss and CO2 assimilation especially in A. thaliana. Guard cells of both plant species synthesized ABA during saline conditions which was reflected from transcriptomic data and ABA quantification in the guard cells. ABA induced signaling in both plant species varied at the ABA receptor (PYL/PYR) levels where totally contrasting responses were observed. PYL2, PYL8 and PYL9 were specific to A. thaliana, furthermore, PYL2 was found to be differentially expressed only under 3x salt growth conditions thus suggesting its role during long term salt stress in this plant species. Protein phosphatases, which negatively regulate ABA signaling on one hand and act as ABA sensor on the other hand were found to be more differentially expressed in A. thaliana than T. salsuginea guard cells, which suggests their diverse role in both plant species under saline conditions. Differential expression of more ABA signaling players in long time saline conditions was prominent which could be because of darkness, as it is well known that rapid closure of stomata under dark conditions require ABA signaling. Moreover, representation of these components in dark also suggests that plants become more sensitive to dark under saline conditions which is also evident from the transpiration rates. Altogether, increased salt ions in A. thaliana guard cells and leaves led to pigment degradation and ABA induced reduction in transpiration which in turn influenced its growth. In contrast, T. salsuginea is the salt excluder and therefore keeps low levels of salt ions especially the chloride both in leaves and guard cells which mildly affects its growth. Guard cells of A. thaliana encounter severe energy problems at physiological and transcriptomic level. Main differences in the ABA signalling between both plant species were observed at the ABA receptor level.