Stem cells have fascinated humans for a long time because of their fundamental importance in building and sustaining many forms of life. Stem cells are able to divide and maintain themselves while producing new cell types. Like animal stem cells, plant stem cells are a fascinating research object. They provide the cells for plant growth and allow many plants to continue growing throughout their lives, thereby producing enormous amounts of plant biomass on this planet. In this study, I specifically focussed on the plant stem cells that produce wood and bast. These stem cells form a cylindrical stem cell niche, the vascular cambium, which is found in the root, the stem and the tissue that connects the root and stem, the hypocotyl. In my analyses, I focused on the hypocotyl of the model plant Arabidopsis thaliana, since wood and bast production is very active there and the tissue patterning resembles the patterning of the vascular cambium of trees.
A vascular cambium stem cell produces both, wood progenitor cells inward and bast progenitor cells outward. Thus, it needs to “choose” between the following cell fates: becoming a wood progenitor cell, becoming a bast progenitor cell, or remaining a stem cell. The basis on which this first cell fate decision is made has not yet been clarified. To better understand this decision-making process, in a first step I mathematically modelled gene regulatory networks in collaboration with the Mironova laboratory. This has shown that a network of just three genes that regulate each other in a specific way is sufficient to bring about the three cell fate possibilities mentioned above. I then hypothesized that this network could consist of two mutually inhibitory auxin and cytokinin signalling pathway components. Furthermore, these components could be additionally regulated by WUSCHEL-RELATED HOMEOBOX4 (WOX4), an important transcription factor for stem cell regulation in the vascular cambium. Integrated into a 1D model, this network generated bidirectional growth. Using in planta analysis, I was able to show that both auxin and cytokinin signalling levels are low specifically in the vascular cambium. To test my hypothesis, I investigated which genes of the auxin and cytokinin pathways are regulated by WOX4, also in comparison to WUSCHEL, a stem cell regulator of the shoot apical meristem. In the course of my investigations, I identified specific type-A ARABIDOPSIS RESPONSE REGULATORs (ARRs), which negatively regulate cytokinin signalling, as putative WOX4 targets. I found that the expression of the type-A ARR genes ARR5, ARR6, ARR7 and ARR15 is reduced in the hypocotyl vascular cambium. Furthermore, I observed that the expression of ARR6 and ARR15 is downregulated by WOX4. WUSCHEL also exerts this type of regulation on type-A ARRs in the shoot apical meristem and therefore this could represent a general concept for the regulation of plant stem cells. In addition, findings in this study propose that WOX4 itself is regulated by cytokinin via the DNA BINDING WITH ONE FINGER 2.1 (DOF2.1) protein. Importantly, when ARR7 and ARR15 are mutated, the cell fate decisions of vascular cambium stem cells are altered and more wood cells are produced than in wild type plants. In conclusion, this study suggests that a mechanism of cell fate decision making in vascular cambium stem cells is based on the regulation of cytokinin signalling by WOX4 and by type-A ARRs.
A plants capacity to continuously generate new organs and tissues is fueled by pluripotent stem cells, embedded in specialized niche tissues, called meristems, which provide strict regulation of stem cell fate to balance the need for cell proliferation and cell differentiation. In the Arabidopsis shoot apical meristem (SAM), maintenance of a stable stem cell population largely depends on the non-cell autonomous activity of the homeodomain transcription factor WUSCHEL (WUS). WUS mRNA is expressed in the organizing center (OC) within the L3 of the shoot meristem. From here, WUS protein moves several cell layers upwards towards the overlying L2 and L1 and into the stem cells in the central zone (CZ). While it has been shown that short-ranged cell-to-cell movement of the WUS protein, via cytoplasmic bridges called plasmodesmata, is essential for WUS function and shoot stem cell maintenance, many aspects of WUS mobility, including its mechanism and regulation, remain unclear or under debate. Here, I chose a quantitative approach to characterize WUS mobility: For this, I have developed a semi-automated analysis pipeline for layer-specific quantification of fluorescence intensity along the apical-basal axis of the SAM, taking into account meristem curvature. I have then used this analysis in combination with high-resolution live-cell imaging on dissected, unfixed shoot apices to systematically characterize the mobility of differently tagged WUS fusion proteins in complementation lines as well as transcriptionally inactivated mutant alleles in wildtype meristems. From these data, I hypothesize that the mechanism for WUS mobility in the SAM involves a component of active transport that is not directional but regulated via protein retention in the L3. Considering previous studies, my data further indicates that specificity of active WUS movement may be mediated by the WUS homeodomain (HD) and suggests the presence of regulatory sequences not only in the unstructured region between WUS HD and WUS box, but also at the N- and C-terminus.
Even though Nα-terminal acetylation (NTA) is conserved throughout the three domains of life, knowledge about its biological function remains limited. In Arabidopsis thaliana, NTA affects up to 80 % of all soluble cytosolic proteins and is catalyzed by a set of Nα-terminal acetyltransferases (NatA-G). AtNatA is the major Nat complex and targets approximately 40 % of the plant proteome. The core AtNatA complex consists of the catalytic subunit AtNAA10 and the ribosome-binding subunit AtNAA15. In humans, this complex associates with the regulatory subunits HsNAA50 and HsHYPK to form the quaternary HsNatA/E complex. Recently, homologs of NAA50 (AT5G11340) and HYPK (AT3G06610) were identified in A. thaliana. While AtHYPK is tethered to the core AtNatA complex and modulates its activity, the interaction of AtNAA50 with AtNatA is still controversially discussed.
This work provides evidence for the association of AtNAA50 with the core AtNatA components and the ribosome. AtNAA50 was found in the proximity of AtNAA10, AtNAA15 and AtHYPK in vivo. Co-immunoprecipitation experiments independently confirmed these findings and suggested novel potential binding partners of AtNAA50.
Unlike AtHYPK, AtNAA50 did not modulate AtNatA activity. This was evidenced by the absence of significant shifts in the NTA yield of known NatA substrates in NAA50-depleted mutants. In line with this finding, the depletion of AtNAA50 did not impact NatA-mediated proteome stability, either. Nevertheless, AtNAA50 seemed to exert a critical NatA-independent biological function since its knockout resulted in severe growth retardation and infertility.
The generation of aminaa50 knockdown mutants revealed that minimal amounts of AtNAA50 were sufficient to ensure proper plant growth. Global transcriptome and proteome analyses of the mutants showed that the depletion of AtNAA50 caused a constitutive upregulation of the plant defense response against pathogens. In line with this finding, aminaa50 plants were resistant to the bacterium Pseudomonas syringae. However, the abundance of phytohormones that usually trigger this resistance was not increased in aminaa50. Apart from its role in vegetative growth and the regulation of plant defense, complementation studies suggested that NAA50 was required for pollen formation.
In summary, this thesis demonstrates that even though AtNAA50 interacted with the core AtNatA complex, it executed functions beyond regulating AtNatA activity.
Plants dynamically adjust their development and growth pattern to maximize their chances to reproduce even under challenging conditions. The cellular basis for this remarkable phenotypic plasticity are stem cell populations that are maintained throughout the whole life of a plant. Intricate sensing and signaling mechanisms are required to instruct stem cell behavior according to current needs. Consequently, light- and nutrient signals are integrated and modulate the core molecular circuits underlying regulation of the shoot apical meristem (SAM). We know that expression of the stem cell master regulator WUSCHEL (WUS) is modulated by photoreceptor mediated light signaling pathways together with sugar and nitrate. Activity of the evolutionary conserved TOR kinase is required to integrate and relay these signals, but we do not understand how TOR activity is conveyed to modulate WUS expression. In this work, I identified CK signaling in an RNAseq approach as the major downstream effector of TOR activity controlling WUS expression and shoot development. I demonstrate that TOR activity stabilizes trans-Zeatin which is known to be one of the major determinants influencing shoot growth and meristem maintenance. Mechanistically, this is achieved by TOR dependent translational repression of CKX catabolic enzymes, allowing plants to swiftly adjust their growth factor milieu in response to dynamic environments. This study is the first example for TOR mediated translational repression of a catabolic enzyme and thus provides a mechanistic framework integrating the contributions of central metabolism and core developmental regulators towards stemness.
Plants have a sessile lifestyle and cannot run away when attacked. They have therefore developed various defense systems to deal with potential predators or bacteria and fungi. But heat, drought or high concentrations of salt in the water also mean stress for the plant. Calcium plays an important role as a signaling molecule in the transmission of the stress stimulus within the plant. Plants of the species Arabidopsis thaliana can be genetically modified so that their calcium concentration is visible under the microscope. These plants were stimulated by my cooperation partners with various biotic and abiotic stress stimuli and recorded with the camera. As a response, the time-lapse recordings show a locally limited increase in the intracellular calcium concentration close to the root tip, which runs in waves through the plant.
The aim of my work was to analyze the wave qualitatively and quantitatively and to investigate the connection between the type of stimulus and the spatio-temporal pattern of the wave, the calcium signature. By means of modeling, I investigated how this wave can propagate at high speed across cell boundaries and how different responses to a calcium signal can be triggered at the protein level. The course of the calcium wave can be displayed as a kymograph, a space-time diagram. Using the kymograph, I managed to quantify the wave and to determine characteristic parameters such as start time, start position, and speed. The image series of the individual experiments show a high variance with respect to intensity and shape of the calcium wave. To the purpose of convenient evaluation, I designed an analysis script that quantifies the calcium wave automatically. Firstly, the root is detected in an image series. Secondly, a kymograph is created from the mean calcium concentration along the root. Finally, the calcium wave is plotted as a thin, sharply outlined line. This so-called crestline plot highlights the characteristic properties of the individual wave and allows an easy approximation of aforementioned wave parameters. The analysis of the experiments revealed that stimulation with salt leads to an immediate, short-term increase in the calcium concentration, while bacteria or fungi trigger a delayed calcium wave that propagates at a speed of a few µm/s.
It is known from the literature that after stimulation of the root with elevated levels of salt, the wave moves through the plant at a high speed of around 400 µm/s. A combined signal transmission of intracellular calcium and extracellular reactive oxygen species (ROS) is suggested as a possible explanation. Together with my cooperation partner, I designed a corresponding mathematical model and adapted it to the different cell sizes in the root tip. We were able to show that wave propagation based only on intracellular calcium is sufficient for the much slower calcium wave after stimulation with bacteria or fungi. However, the calcium wave after stimulation with salt requires additional components. Based on a simulation, I was able to demonstrate that the plasmodesmata, the narrow tubes between adjacent cells, slow down the expansion of the wave considerably and should not be ignored.
The plant can use calcium-dependent protein kinases (CPKs) to decode the calcium signal and translate it into protein phosphorylations as a starting point for further reactions. For example, the closing process of the stomata is based on the calcium-regulated activation of CPKs. Based on experimental data, I developed a CPK protein model for different CPKs. In a computer simulation, I coupled calcium time series from a stimulation experiment of guard cells and epidermal cells to my protein model and examined the activity of the CPK proteins. I was able to show that by varying the calcium signal, different CPK proteins can be addressed and the stress response of the plant can be adapted to the type of stimulation.
Stilbenes are plant secondary metabolites from the polyphenols group found in 72 unrelated plant species. Among those plants is Vitis vinifera, an important crop plant in wine regions all across the world. The natural functions of stilbenes include the reaction to stress factors like UV-radiation and mechanical injury or defense reactions against bacterial, fungal or other pathogens. One of the most problematic pathogens in the wine regions in Germany is Plasmopara viticola, causing downy mildew, which leads to great crop losses. Aside from that, stilbenes have gotten attention in the recent years for their potent health beneficial properties. A multitude of studies, mainly pre-clinical trials, have indicated that stilbenes can delay aging parameters, help in the prevention of diseases including cancer or diabetes and are effective against cardiovascular diseases. While much attention is given to the functions of stilbenes, both in planta and in the medical or nutritional supplement areas, and the structures and occurrences of stilbenes are mostly known, the biosynthetic pathway leading to this wide variety of modified stilbenes remains largely unknown. The precursors of the stilbenes derive from the phenylpropanoid pathway, where the stilbene synthase, as first dedicated enzyme of the pathway produces trans-resveratrol. Apart from that, only one other enzyme, a resveratrol-O-methyl transferase and the two transcription factors (TFs) VvMYB14 and VvMYB15 are known and characterized. Other enzymes, for example for glycosylation, polymerization or other modification reactions remain yet to be found, as do dedicated transport proteins. Different V. vinifera tissues overexpressing VvMYB15, one of the two TFs that were shown to be important regulators of the stilbene biosynthesis pathway, were used to run two microarrays prior to this project. From this database, candidate genes with potentially important predicted functions were identified by their upregulation. The selected genes were then filtered by the means of in silico analysis and promoter induction assays before the correlation of their gene expression with the content of modified stilbenes, expected to be produced by their reaction, was investigated. This was done in V. vinifera berries of different developmental stages as well as leaf discs infected with P. viticola and allowed for the closer connection of several candidate genes with their expected products, while also giving valuable insight into the potential role of the stilbenes, and thus the genes, during development or in a defense against infection. In the last part of this thesis, the most promising candidate genes, three glycosyltransferases and a laccase, underwent closer biochemical characterization by expression in bacteria, yeast or tobacco and subsequent enzymatic assay or direct HPLC or UHPLC/MS analysis. While the full characterization of the candidate genes remains to be completed, promising results for the glycosyltransferases were obtained concerning the metabolization of resveratrol and other substrates and for the involvement of the laccase and viniferin in defense reactions.
Cell polarity is a prerequisite for the formation of distinct cell shapes, which allow different cell types to fulfill their specialized functions. In plants, the family of Rho-Of-Plants (ROPs) controls cellular pathways required for the initiation symmetry breaking including cytoskeleton rearrangements and targeted vesicle transport. Because of their decisive role in these fundamental processes, the spatio-temporal regulation of ROP activation is a delicate task. Efficient ROP activation is mediated by plant-specific ROP guanine nucleotide exchange factors (RopGEFs), which share the central Plant-specific Rop Nucleotide Exchanger (PRONE) domain. While the PRONE domain of RopGEFs is conserved, the flanking N- and C-termini are variable in sequence and do not contain known functional domains. In this PhD thesis, selected RopGEFs were studied for their roles in the establishment of cell polarity during root hair development in the model plant Arabidopsis thaliana. I investigated the functions and regulation of RopGEFs, which were reported to polarize at the Root Hair Initiation Domain (RHID) early (RopGEF3, RopGEF14) or during the onset of polar growth (RopGEF4). In this thesis, I showed that the early polarizing RopGEF3 is crucial for early events including ROP2 recruitment and timing of growth initiation, while the late polarizing RopGEF4 is required for root hair elongation. Furthermore, the unrelated N-termini of early and late polarizing RopGEFs were characterized by exchange of N-termini in RopGEF3. I showed that the N-termini of early polarizing RopGEFs are functionally related in distinction to the N-terminus of RopGEF4. While N-termini of RopGEF3 and RopGEF14 promote RopGEF removal from the RHID, absence of these N-termini or presence of RopGEF4 N-terminus resulted in root hair phenotypes reminiscent of ROP overexpression as well as protein stabilization at the RHID. Cross-species and pairwise RopGEF N-termini sequence alignments combined with in silico prediction for phosphorylation sites in the RopGEF3 N-terminus revealed promising candidate amino acid residues possibly being involved in RopGEF3 regulation at the RHID. Taken together, in this PhD thesis evidence is provided for a putative task-sharing mechanism of early and late polarizing RopGEFs during root hair development as well as for the involvement of RopGEF N-termini in RopGEF protein abundance regulation at the RHID most likely involving regulatory phosphorylations.
Functional specialization of singles cells but also of whole tissues requires the division of labor and leads to the establishment of unequal distribution and polar organization. As a consequence, a diverse spectrum of cells with polar shapes has arisen in all kingdoms of life and conceptual similarities between these cells indicate the presence of a general unifying machinery. Even though we have a good understanding of the process of polar growth itself, we still know very little about the initial steps that lead to cellular asymmetry and polarity establishment. The root hair system of Arabidopsis thaliana is an excellent model to study all aspects of polarity and especially its initiation. One of the first proteins to localize at the root hair initiation domain (RHID) in Arabidopsis is the small GTPase RHO-OF PLANTS 2 (ROP2), which is a central regulator of the root hair growth machinery. ROP2 is a molecular switch that shuffles between an active (GTP-bound) and an inactive (GDP-bound) state. It is obvious that the spatio-temporal control of ROP2 positioning and its activation is of critical importance for proper root hair development, however to date it remains unclear how ROP2 is recruited to the RHID, how ROP2 polarization is timed and how its polar accumulation is maintained. In this thesis I could show that the poly-basic region close to the C-terminus of ROP2 is necessary, but not sufficient for its accumulation at the RHID. I could further demonstrate that polar accumulation of ROP2 depends on its N-terminus, which we have reported to be involved in the interaction with its putative activator and landmark protein GEF3. I found that the ability to shuffle between its active and inactive state is critical for ROP2 polarization and have presented evidence for an activation-dependent immobilization of ROP2 that involves the interaction with GEF3, activation of GDP-ROP2 and differential protein mobility. Using single-molecule localization microscopy I was able to show that ROP2, GEF3 and other proteins of the root hair growth machinery localize into nanoclusters at the PM. To fully characterize the proteome of these nanoclusters, I established biotin-ligase based proximity labelling in Arabidopsis root hairs. This will allow us to elucidate whether the structuring of the PM (at the RHID) into nanoclusters plays a role in the establishment, maintenance and plasticity of cellular polarity.
The Macaronesian laurel forest is characterized by humidity-adapted, evergreen trees with glossy, entire and elongated leaves. Based on fossil data, this vegetation type has been regarded as a relict of Tertiary, European/Mediterranean forests since at least the middle of the 19th century. In contrast to that, more recent studies indicate that the Macaronesian laurel forest species may be much younger than previously thought, with the majority of the analyzed species dating to the Plio-/Pleistocene. Furthermore, they recovered a rather heterogeneous geographical origin, suggesting that the Mediterranean region, other Macaronesian vegetation zones as well as tropical areas have served as source areas for the corresponding species. Although previous analyses included quite characteristic taxa, e.g. all of the Macaronesian Lauraceae, only a small number (around 26%) of laurel forest genera has been studied to this day, most of them are woody.
In this dissertation, the biogeography of six typical and widespread Macaronesian laurel forest genera (Daucus, Geranium, Gesnouinia, Phyllis, Semele and Visnea) is studied, covering different life-forms and ecologies. Conducting molecular phylogenetic and dating analyses as well as ancestral area estimations, a) the timeframes for the colonization of Macaronesia and the laurel forest, b) the geographical origin of the colonizers and c) the timeframes for inter-archipelago and inter-island dispersal were studied. Furthermore, the usefulness of stem ages and crown ages for inferring the colonization times is tested. Additional analyses were conducted for Gesnouinia and Visnea. In Gesnouinia, the wood anatomy was studied as the genus was considered as potentially insular woody in previous studies, which would contradict a relict status. For Visnea, fossils of the extinct V. germanica from the Miocene to Pliocene of Germany and Italy were analyzed regarding their affinity to laurel forest V. mocanera using MicroCT scans.
The results obtained here provide further support for the heterogeneous origin of the Macaronesian laurel forest and indicate that stem ages should be preferred over crown ages for inferring the relict status. A relict origin of Visnea (Oligocene age) and the laurel forest taxa of Geranium (Miocene age) is very likely, whereas the situation is ambiguous in Semele and Daucus. The latter two are of Miocene age, but their phylogenetic position is poorly resolved. Laurel forest Gesnouinia and Phyllis originated within Macaronesia and are clearly no relicts from the Tertiary by their source area. Dispersal from or into the dry infra-Canarian vegetation is indicated for both genera, with the time frames differing. In Phyllis, dispersal falls into the Early Pliocene, whereas in Gesnouinia, an overlap with range-shifts associated with the Pleistocene glaciation cycles is recovered. The non-relictual trait of insular woodiness could not be unambiguously inferred for Gesnouinia. While woodiness in Gesnouinia probably is derived, it may have evolved prior to island colonization. Interarchipelago colonization between Madeira and the Canary Islands is inferred to be young in most taxa, overlapping with Pleistocene sea-level fluctuations and the timeframes recovered for species from other Macaronesian vegetation zones. The same is found for inter-island colonization within the archipelagos.
For the Macaronesian laurel forest as a whole, the newly generated data as well as literature data indicate that there is likely no obvious relationship between time of colonization and life-form or time of colonization and the extant ecological niche occupied within the forest. Instead, data points towards a link between time of colonization and the main source area of the colonizers. In the humid climate of the Late Miocene, habitat conservative dispersal from the Mediterranean/Europe to newly emerged islands and habitat space created by catastrophic events seems to have predominated. In the still humid Early Pliocene, the influx from the Mediterranean/Europe decreased and the majority of colonizers originated within Macaronesia. During the Late Pliocene climatic deterioration (cooler, drier and increasing seasonal), dispersal from the Mediterranean, probably non-habitat conservatively, was prevalent. In the course of the Pleistocene (Early and Middle), climatic changes and range shifts associated with the glaciation cycles possibly promoted the arrival of a large amount of Macaronesian taxa. Pleistocene establishment is also indicated for a number of Mediterranean/European taxa, but restricted to the Early Pleistocene. Colonization events from Asia, the New World and (Eastern) Africa seem to be rare and likely occurred prior to the Pleistocene. They may have been facilitated by the lack of e.g. climatic, tectonic or marine barriers during certain periods of time.
During post-embryonic development, plants rely on the integrity of phloem within their root systems. The phloem is part of the vasculature and transports energy metabolites from leaves into mitotically active regions such as the root apical meristem (RAM). Loss of function of genes regulating phloem development can result in severe changes in root growth and plant body architecture. The redundantly active genes SUPPRESSOR OF MAX2 1-LIKE3 (SMXL3), SMXL4 and SMXL5 are central regulators of early phloem formation. However, molecular mechanisms underlying SMXL3/4/5 gene activities during early phloem development are mostly unknown. The functional relevance of SMXL3/4/5 protein domains including the EAR motif is also unclear. The aim of my dissertation was to characterise the mode of action of SMXL3/4/5 during early events of phloem development in detail using Arabidopsis thaliana roots as model organ to investigate spatiotemporal tissue formation.
First, I investigated at which developmental steps SMXL3/4/5 genes are required to promote phloem development in the RAM, how they interact genetically with positive regulators (OPS, BRX) and how their function is affected by negative regulators (CLE26, CLE45). I found that SMXL4/5 function is required to initiate and promote the activities other of of genes regulating phloem development (OPS, BRX, BAM3, CVP2 and APL), and that SMXL4/5 protein functions are possibly required to attenuate CLE-mediated suppression of phloem differentiation. Furthermore, I examined whether the highly conserved EAR motif of SMXL5 is functionally relevant to promote early phloem development. Here, I tested whether protein accumulation was altered for EAR motif-mutated SMXL5 proteins (SMXL5mEAR) in planta, and if phloem formation could be restored in smxl4;smxl5 double mutants complemented with SMXL5mEAR proteins. My data suggest that SMXL5 protein function is independent from the EAR motif indicating that SMXL5 proteins do not act as canonical EAR repressors in the context of phloem development. Last, I aimed at identifying new genes that are functionally related to SMXL3/4/5 during early phloem development. Therefore, I performed an ethyl methanesulfonate (EMS)-based mutagenesis of smxl4;smxl5 double mutants to screen for genetic suppressors that alleviate the phloem defects characteristic for smxl4;smxl5 mutants. I found that mutagenesis of yet unknown suppressor genes in the smxl4;smxl5 background could indeed restore phloem development. Further analysis including genome mapping is required to identify candidate genes that result in the suppression of the smxl4;smxl5 mutant phenotype.
In conclusion, I postulate that SMXL3, SMXL4 and SMXL5 genes are required to establish the post-embryonic phloem lineage and regulate the phloem-specific developmental program in the RAM. Together, a complex, tightly balanced network of molecular players depending on SMXL3/4/5 activities ensures the formation of phloem within the root system.
The field of plant cell wall signaling is one that has seen tremendous growth in the last few decades. One result of this has been the identification of many uncharacterized proteins putatively involved in the cell wall signaling network. This thesis outlines the initial characterization of three previously unstudied genes of interest in the model organism Arabidopsis thaliana which are loosely tied together by their apparent links, either directly or indirectly, with cell wall signaling. The first gene investigated, named PHYTOSULFOKINE LIKE 1 (PSKL1), is what seems to be a potential preproprotein with possible links to Phytosulfokine (PSK) singling and RECEPTOR-LIKE-PROTEIN 44 (RLP44). PSKL1 was found to contain two of the YIYTQ amino acid motifs found in PSK genes and to affect vascular cell identity. This was shown by an increase in the average number of metaxylem cells in the roots of pskl1 mutant seedlings, a phenotype that was rescued back to wildtype by exogenous PSK treatment. The inverse of this phenotypic trend was observed in a mutant line where both PSK motifs in the gene remained intact, suggesting that PSKL1 might function as more than a possible source of mature PSK’s. The second gene investigated is a member of the F-Box/RNI Like family. This gene was identified in a forward genetic screen looking for suppressors of the RLP44ox phenotype, where it emerged as the most likely candidate for a Brassinosteroid (BR) signaling independent RLP44ox suppressor. Generation of a f-box/rni like line in the RLP44ox background using the CRISPR/Cas9 system recapitulated the suppression phenotype, supporting its role as the causative mutant gene in the original forward genetic screen. Use of a F-Box/RNI Like:RFP line showed F-Box/RNI Like to be localized to the cytosol and a lack of colocalization with RLP44:GFP, suggesting that it doesn’t directly associate with RLP44. The third gene investigated is a receptor like protein named RECEPTOR-LIKE- PROTEIN 46 (RLP46), which has a high degree of genetic conservation throughout the plant kingdom. Available data showed highest RLP46 expression in mature root tissue, and a strong upregulation of expression upon exposure to the elicitor elf18, suggesting RLP46 might have a role in plant innate immune response. The CRISPR/Cas9 system was used to generate a rlp46 mutant line which was tested for biotic and abiotic stress phenotypes. rlp46 at first showed a possible NaCl resistance phenotype, but repetitions of the experiment gave conflicting results. There was however a subtle but consistent phenotype of resistance to elicitor induced growth inhibition in the rlp46 line. Co-immunoprecipitation and Western Blot showed evidence for an association between RLP46 and SUPPRESSOR-OF-BIR 1 (SOBIR1), providing a strong clue for how RLP46 might interact with the plant immune response signaling network. The experiments outlined in this thesis proved successful in characterizing these three proteins and provide a firm foundation for future research.
For implementation of a sustainable bioeconomy, biorefineries will play a crucial role in converting different biomasses into various platform molecules. For biorefineries using lignocellulose biomass, Miscanthus is one of the ideal sources because of high-yield potential, low-input requirements, and high energy outcome ratios. However, with respect to the lignin component, the demand for high-value products from isolated lignin requires lignin feedstocks with unique properties. Therefore, a better understanding of lignification and monolignol biosynthesis is mandatory. For several model plants (Arabidopsis, rice, poplar), the lignin biosynthesis pathway has been elucidated in extensive detail. In particular, the transcriptional regulatory network of lignin biosynthesis as well as of secondary cell wall formation has attracted attention of worldwide research. However, how transcriptional repressors are involved in regulating lignin biosynthesis in Miscanthus has remained largely unknown. In this study, two R2R3-MYB transcription repressors, MsMYB31 and MsMYB42 were identified from Miscanthus sinensis. Sequence and expression analysis revealed their close structural relationship with AtMYB4, ZmMYB31 and ZmMYB42, transcription factors which have been identified as negative regulators of lignin biosynthesis and the phenylpropanoid pathway. Further characterization of both repressors has been performed via subcellular localization and functional analysis, e.g. via dual-luciferase-assays (DLA) and electrophoretic mobility shift assays (EMSA) to confirm their mode of action and specificity of binding to certain cis-elements in target gene promoters, i.e. MsC4H, MsCCR and MsCAD. Inducible expression of MsMYB31 or MsMYB42 in wild type Arabidopsis Col-0 further confirmed repression of phenylpropanoid metabolic pathway by both repressor proteins. Additionally, transforming the Arabidopsis myb4 mutant with MsMYB31 or MsMYB42 under control of the AtMYB4 promoter revealed that both repressors do not complement the function of AtMYB4, indicating similar but different mechanisms of these repressors. Finally, while showing target redundancy, the differential developmental expression patterns of MsMYB31 and MsMYB42 indicate specific regulatory functions during lignification in planta. Possible physiological functions of both repressors are discussed.
Calcium (Ca2+) is an essential second messenger in plant cells linking the perception of stresses at the plasma membrane to the appropriate defense response. The calcium signature theory states that for each perceived stress there is a unique calcium transient that triggers specific downstream responses. It is thought that the signaling specificity is encoded in the spatio-temporal pattern of cytosolic calcium concentration, which is in turn decoded by various intracellular calcium binding proteins. For 25 years now the calcium signature theory has not been conclusively proven, and alternative theories are now appearing. One of the problems remaining is that there is no standard method to quantify these spatio- temporal signals. The aim of this thesis was to develop a standard method to quantify calcium signatures in plants and start constructing a library of calcium signatures in response to different stresses. As a model system I used Arabidopsis thaliana roots expressing the R-GECO calcium sensor. To quantify the spatio-temporal calcium response, the calcium signature was divided into six quantifiable parameters: (a) delay of the first detected calcium signal, (b) location of the first calcium signal, (c) duration of the calcium signal, (d) distance that the calcium wave traveled along the root, (e) velocity with which the calcium wave travels towards the root tip, and (f) velocity with which the calcium wave travels towards the shoot. Principle component analysis (PCA) was used to look for similarities and analyze the data. Responses to eleven elicitors (ATP, chitin, cellobiose, cold, D-serine, elf18, flg22, glutamate, NaCl, nlp20 and PG3) were tested. The results showed that, indeed, each elicitor resulted in a unique composition of the six parameters that together form the calcium signature. Moreover, calcium signatures in response to biotic versus abiotic elicitors formed two distinct groups. While biotic stress caused delayed calcium responses specific to the elongation zone of plant roots, abiotic stresses resulted in immediate and systemic calcium signatures. Further experiments suggested that ROS play a key role in restricting calcium signatures to the elongation zone in response to biotic stress and in propagation of calcium signals through the root in response to abiotic stress, indicating that there is crosstalk between reactive oxygen species (ROS) and calcium signatures to prioritize distinct stresses.
Post-embryonic growth and development tailored to the environmental condition is a distinguishing characteristic of plants imposed by their sessile lifestyle. Lifelong growth from seed to plant death is enabled by pluripotent stem cells encompassed in the meristems, which continuously generate new plant material with high proliferation rates in the periphery, and slowly dividing stem cells in the centre. This gradient of varying proliferation rates is tightly controlled to balance cell proliferation and replenishment with differentiation. A plethora of signalling networks consisting of peptides, phytohormones and transcriptional regulators are crucial to control all these processes in a spatio-temporal manner. Particularly in the shoot apical meristem (SAM), these processes have to be tightly monitored, as cells in the SAM acquire cell identities along their trajectory from the centre of the meristem through the periphery irrespective of clonal lineage. In this study, we attempted to achieve three main aims using genetic characterization, live-cell imaging, and transcriptome profiling. We tried to understand how cell wall properties influence cell identity and differentiation by means of pectin modifications in the shoot apical meristem. We could reveal that imbalancing the pectin modifications in the whole SAM leads to disruption of cell size control, cell shapes, and overall meristem size. Second, we attempted to decipher the role of malectin-like containing RLP4 and its RLP4-like subgroup in cell wall signalling. We show that the evolutionarily conserved RLP4 is specifically located in cell edges. In addition, the extracellular domain of RLP4 associates with th cell wall, suggesting RLP4 could be a novel component of cell wall signalling. Last, we wanted to characterize a newly identified mutant, named cle signalling component1 (csc1) and its function in maintenance of the root and shoot apical meristem. csc1 displayed an enlarged SAM, defects in flower development and elevated xylem cells in the vasculature. We identified, that CSC1 determines meristem size, excerting negative control over both cytokinine response and the expression of the stem cell fate inducing transcription factor WUSCHEL (WUS) in the SAM. Together, our findings expand the wiring networks in maintaining stem cells by one essential player and elucidate the importance of cell wall signalling and cell wall properties in the meristems of Arabidopsis.
Growth and body shape of complex multicellular organisms is largely determined by a functional long-distance transport of energy metabolites that fuels stem cell activity. In vascular plants, sugars are photosynthetically produced in source tissues and delivered via the phloem to sink tissues for allocation into storage organs or to sustain distinct stem cell niches, called meristems. In the root apical meristem (RAM), which drives longitudinal root growth, sugar supply is ensured by a tight interplay between proto- and metaphloem. Formation of proto- and metaphloem starts with a single stem cell whose daughter cells divide and differentiate in a controlled spatio-temporal manner. Protophloem differentiates first within the RAM to enable sugar unloading close to the stem cell niche. Impaired or delayed protophloem formation has detrimental consequences for plant growth and vitality. Understanding the regulatory mechanisms behind (proto-)phloem formation is an important hub to enhance sink strength and thereby crop yield in the near or further future. In this study I report novel key-components in phloem regulation called SUPPRESSOR OF MAX2 1-LIKE3 (SMXL3), SMXL4 and SMXL5. Unlike most SMXL family members, SMXL3/4/5 act independently from strigolactone (SL) or karrikin (KAR) signaling as positive regulators of phloem formation. They are the first described phloem-specific genes that show promoter activity already in provascular tissues of the embryo, the first phloem stem cell in the RAM and along the whole phloem tissue in adult plants. SMXL3/4/5 promote protophloem initiation and differentiation in a dose-dependent manner. Deficiency of all three gene functions results in complete absence of phloem tissue and seedling lethality. In comparison, double mu-tants show reduced phloem-dependent transport and sugar accumulation in leaves. Moreover, SMXL3/5 play an additional and SMXL4-independent role in radial root growth by promoting procambial cell divisions. Interestingly, SMXL5 activity is sufficient to induce secondary phloem formation at the stem base, but acts redundantly with SMXL4 in suppressing radial stem thickening. This functional specialisation suggests that SMXL3/4/5 play distinct roles in molecular networks of phloem and/or (pro-)cambium formation. To integrate SMXL3/4/5 into such molecular networks, I characterized protein-protein interaction partners of SMXL5. The plant homeodomain (PHD)-finger protein OBERON 3 (OBE3) is the first interaction partner that genetically interacts with SMXL3/4/5 in protophloem formation. Previous studies reported that OBEs are important components in meristem maintenance and, potentially, chromatin remodelling. SMXL3/4/5 are nuclear localized, chap-eron-like proteins with conserved AAA ATPase and ETHYLENE-RESPONSE FACTOR Amphiphilic Repression (EAR) domain, which makes them perfect candidates to act in transcriptional regulation of downstream targets. This study and the characterization of SMXL3/4/5 and OBE3 as novel and fundamental phloem regulators enabled a deeper understanding of phloem development and sugar allocation in plants.
Eukaryotic cells can have a plethora of different shapes. To develop these, cells have to grow polarly and therefore concentrate their resources at a defined domain at the plasma membrane. We have a great understanding how such polar growth is achieved and regulated. However, the early processes that initiate the formation of a new polar domain at the plasma membrane, especially the timing of protein recruitment, remain largely elusive. The aim of this thesis was to understand how a polar domain is established and how the machinery for polar growth is assembled. As a model system I used Arabidopsis thaliana root hairs, which grow tube-like protrusions out of epidermal cells and I established a timeline of protein recruitment to the root hair initiation domain (RHID). To enable sensitive measurements of protein localization, I quantified properties of 10 fluorescent proteins in planta. Using the brightest fluorophore, I was able to detect low amounts of protein and quantitatively measure accumulation at the RHID at endogenous levels. The combined analysis of timing and localization of 30 different markers showed that different proteins accumulate specifically at different phases of root hair development and led to the identification of four novel candidates that act during root hair initiation, along with the established RHID markers RhoGTPases OF PLANTS (ROPs). For ROP2 I could show that its activity, as well as the interaction domain and the C-terminus are important for membrane association and recruitment to the RHID, while posttranslational S-acylation had no effect in this process. Two of the candidates found during RHID initiation, the actin-regulating SCAR/WAVE complex and the undescribed POLLEN RECEPTOR-LIKE KINASE 7 (PRK7,) both led to the establishment of independent projects. Additionally, I identified two ROP activating GEFs, GEF3 and GEF14, that precede ROP accumulation at the RHID. I found that GEF3 is necessary for root hair initiation and sufficient to form polar domains at the plasma membrane, which recruit ROP2. After RHID assembly, tip growth is initiated. This process is regulated by an oscillating apical [Ca2+]cyto gradient. In a collaboration we could show that at least three CNGC Ca2+ channels are regulating these Ca2+ oscillations in root hairs.
Glutathione (GSH) has been reported for its crucial roles in maintaining plant growth as well as responding to environmental stresses. The multiple functions of glutathione require a tight control of GSH levels. Glutamylcysteine ligase (GCL) catalyzes the first rate-limiting step of glutathione biosynthesis. However, the mechanism of redox-dependent regulation on GCL is still largely unknown in plants. Previous findings have demonstrated that formation of an intramolecular disulfide bond followed by homodimerization is unique to the redox-mediated activation of plant GCL. To address whether the disulfide bond formation is sufficient for GCL activation or the subsequent homodimerization is a necessary step, we generated recombinant mutated GCLs unable to form dimers. Enzyme activity assays showed that disrupting dimer formation did not prevent redox-activation of GCL. Substrate affinities were similar among recombinant GCL variants. The dissociation constant of GCL was estimated by FPLC analysis to be less than 10-6 M; additionally, the GCL concentration in plastids was estimated to be approximately 5 mM. Therefore, the GCL dimer is likely to occur in vivo. Taken together, this study reveals that GCL activation relies primarily on intramolecular disulfide bridge whereas dimerization has little contribution. Whether dimerization affects other enzyme properties, e.g. GCL stability in vivo, remains to be investigated. Mitogen-activated protein kinase (MAPK) cascades mediate signal transduction of diverse extracellular stimuli including pathogen attack and oxidative stress. Arabidopsis MAPK3 and MAPK6 can be deactivated by MAPK phosphatase2 (MKP2) which may be involved in oxidative stress-related responses. Therefore, the MKP2-inducible transgenic Arabidopsis lines were used to explore the redox dependency of MKP2 regulation. After induction, an attenuated MKP2 accumulation was observed under sustained oxidative stress conditions. MKP2 may act as a potential target for plants to perceive oxidative stress and enhance MAPK signaling. It is conceivable that the post-translational modification of MKP2 exerts such regulation.
Seit der Intensivierung der Glazial-Interglazial-Zyklizität ab dem späten Pliozän reagierte das Klimasystem sehr sensibel auf Veränderungen in der Atmo-, Kryo- und Ozeanosphäre. Diese führten nicht nur zu ausgeprägten Klimaschwankungen über orbitalskalige Zeitintervalle, sondern resultierten auch in massiver kurzfristiger (d.h. jahrhundert- bis jahrtausendskaliger) Klimavariabilität. In Anbetracht des erheblichen Einflusses des Menschen auf das Klimasystem ist ein Verständnis dieser kurzzeitigen Klimavariabilität dringend erforderlich. Zeitlich hochauflösende Analysen des 1,35 Myr umfassenden Klima- und Vegetationsarchivs von Tenaghi Philippon (NE-Griechenland, NE Mittelmeerraum) können einen wichtigen Beitrag zu einem verbesserten Verständnis kurzzeitiger Klimavariabilität leisten. Das Marine Isotopenstadium 19 (MIS 19; 790–761 ka) ist hierbei von besonderem Interesse, da es als bestes orbitales Analogon des heutigen Holozän-Interglazials gilt. Die in der vorliegenden Arbeit durchgeführten Pollen- und Röntgenpulverdiffraktometrieanalysen des MIS-19-Interglazials von Tenaghi Philippon dokumentieren eine massive kurzzeitige Klimavariabilität. Die zeitliche Einordnung der Vegetationsveränderungen wurde durch magnetostratigraphische Methoden ermöglicht. Klimawechsel hin zu einem kalttrockenen Klima während zum Teil vollinterglazialer Bedingungen führten fünf Mal zu einer Kontraktion temperater Wälder und der Ausbreitung von Kältesteppen innerhalb weniger Jahrzehnte bis Jahrhunderte. Die Erholung der Wälder mit der Rückkehr warmgemäßigter Bedingungen erfolgte ähnlich abrupt. Ein Vergleich der Klima- und Vegetationsdynamik von Tenaghi Philippon mit Klimaveränderungen im Nordatlantik zeigt, dass die insolationsgetriebenen Temperatur- und Eisschild-Veränderungen der hohen nördlichen Breiten und des Nordatlantiks sowie Verschiebungen der Innertropischen Konvergenzzone die Klimavariabilität im nordöstlichen Mittelmeerraum steuerten. Die genannten Faktoren beeinflussten über die Klimasysteme des Nordatlantiks und der hohen nördlichen Breiten indirekt die Vegetation in Tenaghi Philippon. Bei einer starken „bipolar seesaw“ im Atlantik gelangten warmfeuchte Luftmassen über die Westwinde in den Mittelmeerraum und führten zu warmgemäßigten Phasen in Tenaghi Philippon. Demgegenüber sind „ice-rafted debris events“ im Nordatlantik synchron mit kalttrockenen Events in Tenaghi Philippon, da südliche Ausbrüche des Sibirienhochs gleichzeitig kalttrockene Luftmassen in die Mittelmeerregion leiteten. Eine Ausnahme repräsentiert das kalttrockene Event CDE-19/1, das als massives intra-interglaziales Event in Tenaghi Philippon während des MIS-19c-Interglazials nur ein schwach ausgeprägtes Analogon im Nordatlantik und im Mittelmeerraum besitzt. Die abnehmende boreale Sommerinsolation führte in diesem Zeitintervall zur Ausdehnung des Meereises und des Polarwirbels der hohen nördlichen Breiten. Während der Nordatlantik und der westliche Mittelmeerraum jedoch nur eine geringfügige Abkühlung verzeichneten, führten intensive Ausbrüche des Sibirienhochs zu kälteren und trockeneren Bedingungen in Tenaghi Philippon. In einer Gegenüberstellung der Pollendaten von MIS 19 und dem Holozän von Tenaghi Philippon wird deutlich, dass sich im derzeitigen Interglazial temperate Wälder deutlich stärker ausdehnten als während MIS 19. Dies spricht für lokal warmgemäßigtere Bedingungen und somit eine stärkere Intensität des Holozäns gegenüber MIS 19. Zudem unterscheidet sich das heutige Interglazial von MIS 19 durch das Fehlen von Analoga zum intra-interglazialen kalttrockenen Events CDE-19/1 während MIS 19c und zum kalttrockenen Event CDE-19/2, wie es das Ende von MIS 19c in Tenaghi Philippon markiert. Hätte das Holozän ein weniger warmgemäßigteres Klima ähnlich dem des MIS-19c-Interglazials charakterisiert, dann wäre bei einer Übereinanderlegung der Präzessions-Kurvenverläufe beider Zeitintervalle ein Äquivalent zum kalttrockenen Event CDE-19/2 vor ca. 1.600 Jahren eingetreten. Eine Ursache für das im Holozän humidere Klima und die deutlich schwächere kurzzeitige Klimavariabilität könnten die im Vergleich zu MIS 19 deutlich kleinerennördlichen Eisschilde des heutigen Interglazials sein, die möglicherweise aufgrund von Veränderungen in der Glazial-Interglazial-Zyklizität im Zuge der Mittelpleistozänen Übergangsphase und dem „Mid-Brunhes Event“ nicht die Größe des älteren Analogons erreichten. Abgesehen von den kleineren Eisschilden verhinderte auch die spätholozän ansteigende atmosphärische CO2-Konzentration das Kippen des Klimasystems und das Eintreten abrupter Klimavariabilität.
Cell wall recalcitrance is a major limitation hindering the exploitation of the enormous potential of lignocellulosic biomass as a renewable resource for energy and bio-based products. In the last decades, C4 grasses from the genus Miscanthus have emerged as a most promising energy crop for the production of lignocellulosic biomass in temperate climates. Secondary cell walls, which represent the largest proportion of lignocellulosic biomass are formed in specialised cells after cessation of growth. In higher plants, the formation of secondary cell walls is tightly regulated, both spatially and temporally, by a complex network of transcription factors. However, in Miscanthus only little is known about molecular players regulating secondary cell wall formation and lignin biosynthesis. In this study, application of the herbicide isoxaben was shown to trigger ectopic lignification in Miscanthus seedling roots. The chemical treatment may be exploited in the future to identify regulators and biosynthetic genes involved in lignification in Miscanthus. In addition, Miscanthus sinensis transcription factors related to SECONDARY WALLASSOCIATED NAC DOMAIN1 (SND1) and SECONDARY CELLWALL MYBs 1-4 (SCM1-4) were identified in the Miscanthus transcriptome, which act as regulators of secondary cell wall formation and lignin biosynthesis. During Miscanthus development, expression of MsSND1 and MsSCMs coincided with the onset of secondary cell wall formation and lignification of vascular tissue and sclerenchyma fibers. MsSND1 and MsVND7 were capable to fully restore growth in a SCW-deficient Arabidopsis thaliana mutant, suggesting they are the Miscanthus orthologues of the well characterised Arabidopsis determinants of SCW formation in fibers and vessels. Ectopic expression of MsSND1 and MsVND7 in tobacco leaves prompted the formation of patterned deposition of lignin, cellulose, and hemicellulose reminiscent of xylem elements. This observation was in sharp contrast to uniform lignification after transient expression of various MsSCMs. Expression of particular Miscanthus TFs led to specific cell wall compositions, providing attractive targets for biomass improvements. Transgenic Arabidopsis lines carrying an inducible system of MsSND1 revealed that MsSND1 regulates directly or indirectly the expression of a broad range of genes involved in secondary cell wall formation.
An important developmental mechanism utilized by metazoans is the establishment of specialized cells, or organizers that act to pattern tissues in a non-cell autonomous manner. In plants, for which multicellularity evolved independently, the mechanisms that organize tissue level patterning are not well understood. In this thesis, I investigate the role of adaxial-abaxial boundaries as putative organizers of leaf development. Firstly, I show that boundaries between adaxial Class III HD-ZIP and abaxial KANADI gene expression in the shoot apical meristem play an instructive role in positioning new leaves and determining their subsequent patterns of morphogenesis. These data, together with previous results, suggest a model in which ad-ab boundaries influence organogenesis due to the presence of a domain in between Class III HD-ZIP and KAN expression domains where neither set of genes is expressed. Using mosaic analysis I test this model by generating ectopic REV / KAN boundaries in which the Class III HD-ZIP and KAN expression domains are directly juxtaposed. My results indicate that the presence of a region in between REV and KAN expression is important for localized growth at the boundary and lamina morphogenesis, supporting the proposed model. However I also find evidence of non- cell autonomous signaling from REV expressing clones that promotes boundary identity regardless of whether a separated boundary region is present or not. Taken together, these findings reveal striking similarities and differences in the development of plants and animals and start to provide an integrative conceptual framework that helps clarify broad aspects of plant architecture.
Background: Glutaredoxins (GRXs) are small proteins which bind glutathione to either reduce disulfide bonds or to coordinate iron sulfur clusters. Whereas these well-established functions are associated with ubiquitously occurring GRXs that encode variants of a CPYC or a CGFS motif in the active center, land plants also possess CCxC/S-type GRXs (named ROXYs) for which the biochemical functions are yet unknown. ROXYs physically and genetically interact with bZIP transcription factors of the TGA family. In Arabidopsis, ectopically expressed ROXY19 (originally named GRX480 or GRXC9) negatively regulates expression of jasmonic acid/ethylene-induced defense genes through an unknown mechanism that requires at least one of the redundant transcription factors TGA2, TGA5 or TGA6. Results: Ectopically expressed ROXY19 interferes with the activation of TGA-dependent detoxification genes. Similar to the tga2 tga5 tga6 mutant, 35S:ROXY19 plants are more susceptible to the harmful chemical TIBA (2,3,5-triiodobenzoic acid). The repressive function of ROXY19 depends on the integrity of the active site, which can be either CCMC or CPYC but not SSMS. Ectopic expression of the related GRX ROXY18/GRXS13 also led to increased susceptibility to TIBA, indicating potential functional redundancy of members of the ROXY gene family. This redundancy might explain why roxy19 knock-out plants did not show a phenotype with respect to the regulation of the TIBA-induced detoxification program. Complementation of the tga2 tga5 tga6 mutant with either TGA5 or TGA5C186S, in which the single potential target-site of ROXY19 had been eliminated, did not reveal any evidence for a critical redox modification that might be important for controlling the detoxification program. Conclusions: ROXY19 and related proteins of the ROXY gene family can function as negative regulators of TGA-dependent promoters controlling detoxification genes.
Background: Positive selection is recognized as the prevalence of nonsynonymous over synonymous substitutions in a gene. Models of the functional evolution of duplicated genes consider neofunctionalization as key to the retention of paralogues. For instance, duplicate transcription factors are specifically retained in plant and animal genomes and both positive selection and transcriptional divergence appear to have played a role in their diversification. However, the relative impact of these two factors has not been systematically evaluated. Class B MADS-box genes, comprising DEF -like and GLO -like genes, encode developmental transcription factors essential for establishment of perianth and male organ identity in the flowers of angiosperms. Here, we contrast the role of positive selection and the known divergence in expression patterns of genes encoding class B-like MADS-box transcription factors from monocots, with emphasis on the family Orchidaceae and the order Poales. Although in the monocots these two groups are highly diverse and have a strongly canalized floral morphology, there is no information on the role of positive selection in the evolution of their distinctive flower morphologies. Published research shows that in Poales, class B-like genes are expressed in stamens and in lodicules, the perianth organs whose identity might also be specified by class B-like genes, like the identity of the inner tepals of their lily-like relatives. In orchids, however, the number and pattern of expression of class B-like genes have greatly diverged. Results: The DEF -like genes from Orchidaceae form four well-supported, ancient clades of orthologues. In contrast, orchid GLO -like genes form a single clade of ancient orthologues and recent paralogues. DEF -like genes from orchid clade 2 (OMADS3 -like genes) are under less stringent purifying selection than the other orchid DEF -like and GLO -like genes. In comparison with orchids, purifying selection was less stringent in DEF -like and GLO -like genes from Poales. Most importantly, positive selection took place before the major organ reduction and losses in the floral axis that eventually yielded the zygomorphic grass floret. Conclusion: In DEF -like genes of Poales, positive selection on the region mediating interactions with other proteins or DNA could have triggered the evolution of the regulatory mechanisms behind the development of grass-specific reproductive structures. Orchidaceae show a different trend, where gene duplication and transcriptional divergence appear to have played a major role in the canalization and modularization of perianth development.
Background: Simple Sequence Repeats (SSRs) are widely used in population genetic studies but their classical development is costly and time-consuming. The ever-increasing available DNA datasets generated by high-throughput techniques offer an inexpensive alternative for SSRs discovery. Expressed Sequence Tags (ESTs) have been widely used as SSR source for plants of economic relevance but their application to non-model species is still modest. Methods: Here, we explored the use of publicly available ESTs (GenBank at the National Center for Biotechnology Information-NCBI) for SSRs development in non-model plants, focusing on genera listed by the International Union for the Conservation of Nature (IUCN). We also search two model genera with fully annotated genomes for EST-SSRs, Arabidopsis and Oryza, and used them as controls for genome distribution analyses. Overall, we downloaded 16 031 555 sequences for 258 plant genera which were mined for SSRsand their primers with the help of QDD1. Genome distribution analyses in Oryza and Arabidopsis were done by blasting the sequences with SSR against the Oryza sativa and Arabidopsis thaliana reference genomes implemented in the Basal Local Alignment Tool (BLAST) of the NCBI website. Finally, we performed an empirical test to determine the performance of our EST-SSRs in a few individuals from four species of two eudicot genera, Trifolium and Centaurea. Results: We explored a total of 14 498 726 EST sequences from the dbEST database (NCBI) in 257 plant genera from the IUCN Red List. We identify a very large number (17 102) of ready-to-test EST-SSRs in most plant genera (193) at no cost. Overall, dinucleotide and trinucleotide repeats were the prevalent types but the abundance of the various types of repeat differed between taxonomic groups. Control genomes revealed that trinucleotide repeats were mostly located in coding regions while dinucleotide repeats were largely associated with untranslated regions. Our results from the empirical test revealed considerable amplification success and transferability between congenerics. Conclusions: The present work represents the first large-scale study developing SSRs by utilizing publicly accessible EST databases in threatened plants. Here we provide a very large number of ready-to-test EST-SSR (17 102) for 193 genera. The cross-species transferability suggests that the number of possible target species would be large. Since trinucleotide repeats are abundant and mainly linked to exons they might be useful in evolutionary and conservation studies. Altogether, our study highly supports the use of EST databases as an extremely affordable and fast alternative for SSR developing in threatened plants.
Pflanzen bilden nahezu alle oberirdischen Strukturen wie Blätter und Blüten post-embryonal. Hierzu besitzen sie eine Stammzellpopulation in einer speziellen Nische am Sprossapex, dem sogenannten apikalen Sprossmeristem (SAM). Durch Teilung der sich selbst erhaltenden Stammzellen entstehen Tochterzellen, die in die Peripherie des SAM verdrängt und dort in Blatt- und Blütenprimordien aufgenommen werden. Die einzelnen Bereiche des SAM werden durch verschiedene molekulare Signale definiert und stehen über Signalpeptide, Hormone und mobile Transkriptionsfaktoren miteinander in Kontakt. Von entscheidender Bedeutung für den Erhalt der Stammzellpopulation ist der Transkriptionsfaktor WUSCHEL (WUS). Er wird in den Zellen direkt unterhalb der Stammzellen exprimiert und definiert so das Organisierende Zentrum (OC). Vom OC gelangt er als Protein durch cytoplasmatische Verbindungen, den Plasmodesmata, in die Stammzellen. Seine Funktion in den Stammzellen ist notwendig, um ihre Identität zu erhalten. Im Rahmen dieser Arbeit wurden die molekularen Grundlagen und das Potential der WUS-abhängigen Genregulation in Arabidopsis thaliana untersucht. Durch die Erzeugung eines umfassenden Datensatzes aus DNA-Bindedaten, Transkriptom- und Epigenomdaten, konnte das globale Genregulationspotential von WUS untersucht werden. Es konnte gezeigt werden, dass WUS mehrere Tausend Gene direkt, durch Sequenz-spezifische Interaktion mit deren Promoter, regulieren kann. Die duale Funktion als Aktivator und Repressor konnte bestätigt werden. Wie seit langem vermutet, konnte hier erstmals global gezeigt werden, dass die Repressor- Funktion über eine Histon-Deacetylase (HDAC) vermittelte Reduktion das Acetylierungszustands der Genloci erfolgt. Zusätzlich konnten Hinweise gefunden werden, die auf eine ebenfalls HDAC-vermittelte Transkriptions-Aktivierung hindeuten. Bei großer Ähnlichkeit in den Bindeprofilen unterscheiden sich die Regulationsprofile von WUS und anderen Transkriptionsfaktoren deutlich. WUS wirkt v.a. auf die Expression von Transkriptionsfaktoren und reguliert biologische Prozesse wie Differenzierung und Signaltransduktion. Die direkte Regulation der verschiedenen Signaltransduktionskomponenten in den Hormon-Signalwegen spricht für eine bedeutende Funktion von WUS als Koordinator des Hormongleichgewichts im SAM. Anhand des Pflanzenhormons Auxin wird ein Modell für die WUS abhängige Musterbildung im SAM entwickelt.
Apomixis refers specifically to asexual reproduction through seed in plants. Like other modes of asexual reproduction it has received much attention from evolutionary biologists and has been subject of many studies throughout the last decades. At the same time, it attracts interest from an economic point of view, as the long-term goal to technologically induce apomixis in major crop plants offers the prospect of a potential agricultural revolution. Hence, interests have been growing in the scientific community in order to elucidate the evolution and underlying molecular genetic mechanisms of apomixis. Here I present a multifaceted approach to the problem by (1) the development of biotechnological tools in order to (2) apply molecular evolutionary methods to narrow down the possible causes and consequences of asexual reproduction in plants. In this work, representatives of two genera (Ranunculus L. and Boechera Á. LÖVE & D. LÖVE) were studied in order to advance current apomixis knowledge from different perspectives. In the framework of a microarray based transcriptomic analysis of ovules extracted from sexual and apomictic Boechera, a list of housekeeing genes (HKGs) was selected based on a stability of expression analysis subsequently conducted in different vegetative and reproductive tissues of apomictic and sexual species. Using a GeNorm algorithm, different combinations of HKGs were identified, including Ribosomal Subunit 18 (BoechRPS18), Elongation Factor Alpha 1 (BoechEfα1), Actine 2 (BoechACT2) and Ubiquitine (BoechUBQ), based on their pairwise stability in ovules, anthers, and vegetative tissue in apomictic and sexual species. These genes, specifically chosen to be reproduction mode- and tissue-specific, have subsequently been used for normalization in the experimental validation of two candidates genes related to apomixis in Boechera Next, molecular evolutionary causes and consequences of apomixis were investigated by analyzing the transcriptomic effects of asexual reproduction and its correlated traits (i.e. hybridization, polyploidy and mutation accumulation). Flower-specific RNA from sexual and apomictic species of the wild apomictic Ranunculus auricomus complex was used for high throughput transcriptome sequencing (RNAseq). The first de novo assembled transcriptome for these species was used as a reference sequence for mining Single Nucleotide Polymorphism (SNP) and insertion-deletion xii (indel) polymorphisms. The data were further used to design and manufacture a custom 3 x 1.4 million spot expression microarray. Comparative SNP analysis between apomictic and sexual individuals (specifically, two apomicts from two populations and three sexuals from three populations) corroborated the hybrid origin of apomictic Ranunculus, as proposed by Paun et al. (2006b), and could furthermore elucidate the Pleistocene origin and subsequent divergence of the apomictic individuals. In addition, sites of divergent selection were detected with the analysis of non-synonymous (dN) to synonymous (dS) substitution rates, strengthening the idea of rapid divergence in the hybrids. Finally, the custom microarray was used for the hybridization of RNA from live-microdissected ovules (four developmental stages) from the three apomictic and four sexual individuals used in the SNP analysis. The comparative stage specific transcriptome analysis was used to detect stage specific differentially expressed genes in ovules, in order to identify signatures of apomixis and to produce a list of potential candidates underlying the reproductive switch. 555 stage specific genes were found to be differentially expressed throughout ovule development, and eight genes showed a significant shift in expression pattern throughout ovule development in apomicts compared to sexuals. A further classification was conducted following the predictions made from Nogler’s extensive work in Ranunculus in which different genetic factors were proposed for the induction and penetrance of apomixis. In that light, differentially expressed homoeologous genes were classified into three categories based on their relative expression in apomicts compared to their phylogenetic sexual parent, with the final aim of classifying the number of genes potentially responsible for apomixis. In doing so, we have provided a solid base for future studies in wild (i.e with little or no genetic information available) Ranunculus species. By developing biotechnical tools for their study, identifying genes potentially involved in the establishment of apomixis, and analyzing their evolutionary history, this work presents an important step towards a more comprehensive understanding of the processes and patterns connected to apomixis in model and non-model plants, and has far-reaching potential for agricultural use.
Der Botanische Garten im Neuenheimer Feld ist weit über Heidelberg hinaus bekannt. Seit 100 Jahren bereichert er den Heidelberger Campus mit seiner biologischen Vielfalt. Er bietet nicht nur optimale Forschungsbedingungen für angehende Mediziner, Biologen und Pharmazeuten, sondern ist auch ein Magnet für eine interessierte Öffentlichkeit, die den Garten sechs Tage die Woche besuchen kann.
The Canary Islands' laurel forest is a montane evergreen forest formation of an outstanding floristic and biogeographic value. It is endemic to several islands of the Macaronesian biogeographic region, confined to the humid areas limited by the influence of the trade wind clouds. In recent times this relict forest vegetation, which once covered large proportions of the Canary Islands, suffered from a massive range reduction and fragmentation due to human exploitation. This thesis evaluates the phylogeography and population structure of the laurel forest on the basis of two characteristic plant taxa representative for the whole vegetation complex: The widespread and dominant canopy-building tree species Laurus novocanariensis and the more constrictive Canary Island endemic Ixanthus viscosus. Analyses exhibit low levels of differentiation on within- as well as between-island level. Especially for Laurus the exchange between populations from different islands, even over longer distances, is obvious. On island level the forest fragmentation has low impact on population divergence up to now. Furthermore, we localised the genetic hotspots of both model species within the laurel forest distribution range on Tenerife. A small-scale grid square sampling strategy combined with approaches from population and landscape genetics enabled the development of detailed maps showing the centres of genetic diversity and thus revealing the connection between forest fragmentation and diversity loss. Additionally, we analysed the ecological preferences of the two species, recognising not abiotic factors, but rather past forest degradation as having the most important impact on the current distribution range of the endemic Ixanthus. Based on these findings we discuss the necessity and opportunities for conservation strategies of the Canary Islands' laurel forest in the future.
The Brassicaceae (Mustards, Cruciferae) are a cosmopolitan family comprising 370 genera and around 3660 species assigned to lately 50 tribes. The tribal system was originally based on solely homoplasious morphological character traits and reaches back to the early 19th century. De Candolle introduced the first tribal classification of the family nearly 200 years ago (1821) containing 21 partly still utilised classifications nowadays. Although labelling seems to be up to date, generic delimitations have been under permanent significant substitution and replacement. The tribes are arranged in three major monophyletic lineages and some additional small groups. The relationships within and between these lineages have not been resolved very clearly yet, as the Brassicaceae are characterised by frequently occurring hybridisation and polyploidisation events. This could be either the result of early and rapid radiation events or perhaps be the product of reticulate evolution, lediang to conflicting gene trees (KOCH & AL-SHEHBAZ 2009). This lack of resolution could in parts be resolved via the application of the nuclear encoded chalcone synthase gene (chs) on 39 of the Cruciferous tribes. Several small-scale tribal-specific duplication events, including age estimations, could be detected giving insight into the evolutionary history of this molecular single- or low-copy gene. Most definitely a tendency towards diploidisation is proven by purifying selection as well as accelerated synonymous substitution rates among this family resulting sooner or later in the reduction of preliminary multiplied chs loci. Supposedly, chs is single-copy in most diploid mustard taxa. The determination of orthologous and paralogous gene copies exposed to be of essential cause as it could be proven that yet functional but fluctuating DNA sequences demonstrate a huge impact on divergence time estimates as well as on any other extrapolation applying nucleotide or amino acid data. However, all crown age estimations calculated with diverse approaches resulted in reasonable output, dating the most recent common ancestor (tmrca) of the family to the Late Miocene or Oligocene. Adjustments of the DNA sequences resulted in a well-resolved thoroughgoing gene tree phylogeny facilitating established taxonomic as well as phylogenetic achievements and do, moreover, hint to further ambiguities which have to be clarified by the commitment of additional marker systems.
Die Weinrebe (Vitis vinifera L. ssp. vinifera) gehört zu den bedeutendsten Kulturpflanzen weltweit. Ihre hohe Anfälligkeit für eine Vielzahl von Pathogenen, insbesondere dem Echten (Erysiphe necator) und Falschen Mehltau (Plasmopara viticola), ist für den Weinbau eine besondere wirtschaftliche Bedrohung und kann zu erheblichen Ernteausfällen führen. Nicht zuletzt darum werden in Europa zwei Drittel der Gesamtmenge an Fungiziden im Weinbau eingesetzt. Um den Fungizideinsatz zu reduzieren, sollen neue resistente Rebsorten (z.B. `Regent´) gezüchtet werden, wobei es sich bei der angewendeten klassischen Kreuzungszüchtung um ein langwieriges Verfahren handelt. Die Mechanismen, welche in den resistenten Rebsorten die gesteigerte Resistenz vermitteln, sind jedoch größtenteils unbekannt. Die Identifikation sowie die funktionelle Charakterisierung von Genen, welche an der Regulation von Resistenzmechanismen beteiligt sind, könnte somit zur Verbesserung und Beschleunigung der Züchtung neuer resistenter Rebsorten beitragen. Mittels Mikroarrays konnte eine Reihe von Genen identifiziert werden, darunter auch Rezeptoren und Transkriptionsfaktoren (TF), welche in resistenten Rebsorten im Vergleich zu anfälligen Rebsorten eine schnellere und stärkere Induktion zeigten, von denen aber bislang nur vereinzelte Gene funktionell charakterisiert werden konnten. Im Rahmen dieser Arbeit wurden daher die Funktion einer Rezeptorfamilie bei der Abwehr von P. viticola nachgewiesen sowie anschließend die Unterschiede der transkriptionellen Regulation der Resistenzantwort durch TFs zwischen einer anfälligen und einer resistenten Rebsorte untersucht. Hierzu wurden im ersten Teil des Projektes die Gene der VRP1 (Vitis Resistance to Plasmopara 1) Rezeptorfamilie charakterisiert. Der in silico Vergleich der drei chimären VRP1 Rezeptoren zwischen der anfälligen Rebsorte `Lemberger´ und der resistenten Rebsorte `Regent´ zeigte keine Sequenzunterschiede. Die Lokalisation der VRP1 Proteine nach Fusion mit dem Grün Fluoreszierenden Protein (GFP) in Protoplasten einer Suspensionskultur (V. vinifera cv. `Chardonnay´) mittels konfokaler Mikroskopie konnte belegen, dass sich alle drei VRP1 Konstrukte im Cytoplasma befanden. Zusätzlich konnte mittels qPCR Analyse nachgewiesen werden, dass die Rezeptoren im resistenten `Regent´ im Vergleich zum anfälligen `Lemberger´, spezifisch durch Infektion mit P. viticola induziert wurden, was auf eine Funktion bei der Abwehr von P. viticola schließen lies. Die transiente Transformation der VRP1 Gene in Weinrebenblätter mit anschließender P. viticola Infektion zeigte, dass die Expression von VRP1-3 eine Steigerung der Resistenz um bis zu 50 % bewirkte. Darüber hinaus wurden die VRP1 Gene stabil in Arabidopsis thaliana transformiert. In Folge der Überexpression von VRP1-3 konnte ebenfalls eine Verbesserung der Resistenz der transgenen Arabidopsis Pflanzen gegen Hyaloperonospora arabidopsidis um bis zu 50 % detektiert werden. Im zweiten Teil der Arbeit wurde die Induzierbarkeit des Resistenzgens VvPR10.1 (Pathogenesis Related 10.1) durch TFs und ihre Auswirkung auf die Resistenz untersucht. Mittels Promotor Induktionsanalyse konnte gezeigt werden, dass die TFs VvWRKY33, VvERF5 und VvCZF1 den Promotor von VvPR10.1 induzieren konnten, was auf eine Rolle in der Abwehr hindeutete. Es konnte zusätzlich in vivo gezeigt werden, dass eine durch P. viticola Infektion hervorgerufene Induktion von VvWRKY33 in Weinrebenblättern von Gewächshausreben wiederum zur Steigerung der Expression von VvPR10.1 führte. Ektopische Expression der TFs in Weinrebenblättern mit anschließender P. viticola Infektion zeigte, dass die Expression von VvWRKY33 und VvERF5 eine Steigerung der Resistenz um 50-70 % bewirkte. Außerdem konnte durch Komplementierung der A. thaliana knock out Mutante wrky33-1 mit VvWRKY33 nachgewiesen werden, dass die Expression im heterologen System Arabidopsis den Phänotyp des Wildtyps, in Bezug auf die Resistenz, wiederherstellen konnte. Darüber hinaus konnte sogar eine signifikante Steigerung der Resistenz gegen H. arabidopsidis und Botrytis cinerea im Vergleich zum Wildtyp Col-0 erreicht werden. Im Rahmen dieser Arbeit konnte somit zum ersten Mal nachgewiesen werden, dass die gezielte Expression des VRP1-3 Rezeptors sowie der TFs VvWRKY33 bzw. VvERF5 in der Weinrebe, eine gesteigerte Resistenz gegen P. viticola induzieren konnten. Dies stellt eine Basis für die Aufklärung der zugrunde liegenden Resistenzmechanismen, und somit für die Entwicklung neuer molekularer Marker zur Verbesserung und Beschleunigung der Züchtung neuer resistenter Rebsorten, dar.
1869 brach auf den Kaffeeplantagen in der britischen Kolonie Ceylon (Sri Lanka) die Pflanzenkrankheit "Kaffeerost", verursacht durch den Rostpilz Hemileia vastatrix, aus. Innerhalb von nur knapp 15 Jahren beendete die Krankheit den kommerziellen Kaffeeanbau auf Ceylon nahezu vollständig. Die Bemühungen der botanischen Gärten in Kew (London) und dem örtlichen botanischen Garten in Peradeniya, den Pflanzern zu helfen, scheiterten. Stattdessen führte die Erfolglosigkeit der Wissenschaftler zu Konflikten zwischen Pflanzern und Wissenschaftlern. Kernproblem war die Frage, ob Wissenschaft mit Blick auf den praktischen Nutzen für die Pflanzer betrieben werden sollte, oder ob die botanischen Gärten ihre eigenen Forschungsinteressen verfolgen sollten.
In sexual plants meiosis gives rise to recombined and reduced gametes. In diplosporous accessions of the North American genus Boechera meiotic processes are circumvented (i.e. apomeiosis) which lead to a supressed recombination and to production of clonal unreduced male and female gametes. Unreduced male gametes in diplosporous Boechera are required to produce balanced endosperm. The objective of this study was to identify and characterize candidate genetic factors for unreduced pollen formation, and to analyze their genus-wide dynamics in order to contrast the hypotheses whether apomeiosis expression was induced through interspecific hybridization or if it could be an older characteristic of the genus (i.e. pre-Pleistocene). Apomictic Boechera demonstrated high variability for reduced and unreduced meiocyte production. Early flower developmental staging and flow-cytometric analyses together led to the selection of a single pollen mother cell stage at the onset of meiosis for microarray-based comparative gene expression analyses between diploid sexual and diploid apomictic genotypes, and led to the identification of a single highly-upregulated factor (BspUPG-2) which is highly conserved among apomictic Boechera but has no homologue in sexual Boechera or in other taxa. BspUPG-2 exhibits four intron-exon structure variants which suggest alternative splicing, and lack of a prominent open reading frame and no overall sequence homology to known genes suggests that BspUPG-2 belongs to the novel class of long regulatory non-coding mRNA-like RNAs. BspUPG-2 has apparently arisen through a three-step process initiated by ancestral gene duplication of the original non-genic BspUPG-1 locus, followed by sequential insertions of segmentally duplicated gene fragments which led to its chimeric structure and neofunctionalization in apomictic Boechera. Its genesis reflects the hybridization history which characterizes the genus Boechera. Computational analysis demonstrated that portions of BspUPG-2 form secondary structures which were classified as potential primary microRNAs (pri-miRNAs) according to their minimal folding free energy index (MFEI≥0.70) and their A+U content (≥56.70%). One such structure is highly similar to the third exon of a GTP-binding elongation factor Tu/1-A family homolog from Arabidopsis (AT4G02930, E-value=7.00E-24) which could be a potential regulatory target. Two other sequence fragments at the 5`-end of BspUPG-2 are also homologous to known protein-coding genes (AT5G19960 and AT1G18260), hence supporting the hypothesized regulatory function of BspUPG-2. The apomixis-specificity of BspUPG-2 enabled a genus-wide analysis which demonstrated its ubiquity in all Boechera lineages, including the ancient AB haplotype and two single individuals each of one closely-related genus. These results attest to BspUPG-2’s hypothesized importance for unreduced pollen formation and hence for balanced endosperm formation, and point to a single origin of this factor which coincides with the root of the genus Boechera dating to the middle of the Pleistocene.
Because of its ability to catalyse oxidation/reduction reactions, iron (Fe) is an essential microelement in living beings. However, high levels of Fe in the cell can lead to detrimental effects, as this metal can also catalyse the production of harmful reactive oxygen species. Therefore, tight regulation of cellular Fe concentration is required. In this thesis, two independent genes with a putative role in Fe homeostasis were characterized in the model organism Arabidopsis thaliana. AtRAI1 is homolog to the yeast Rai1p (Rat1 Interacting protein 1), an activator of 5´-to-3´ exoribonucleases, enzymes that degrade decapped RNAs. In Arabidopsis protoplasts, AtRAI1 co-localized with the decapping enzyme DCP2 (Decapping 2) in processing bodies, which are structures where mRNAs are processed for degradation. DK9, a loss-of function mutant for AtRAI1, had phenotypic traits attributable to impaired Fe homeostasis (chlorosis, reduced size) and to homeostasis of the phytohormone auxin (curly leaves and low fertility). Assessment of the activity of the Fe-dependent anti-oxidant enzymes catalase and SOD (SuperOxide Dismutase), multi-element analysis and determination of transcript levels of genes involved in Fe homeostasis showed that AtRAI1 is an upstream regulator of Fe uptake and transport in A. thaliana. The studied protein probably activates the cytosolic exoribonuclease XRN4, which is a component of the ethylene signalling pathway that is known to control Fe uptake in roots. Since homozygous knockout alleles of the analysed gene were not viable, it is concluded that AtRAI plays an essential role for Fe acquisition and proper growth of A. thaliana. The second gene studied in this thesis is BTS2 (BRUTUS2). It encodes a protein that shares similarity with BTS, an E3 ubiquitin ligase with putative roles in Fe regulation. A loss-of-function allele was more tolerant to excess Zn, a condition that leads to physiological Fe deficiency in A. thaliana. Under this condition, the mutant accumulated less Fe in the root than wild-type plants, indicating that the protein probably induces Fe uptake under Fe deficiency conditions. Furthermore, low Zn concentrations and reduced transcript levels of marker genes for excess Zn in shoots suggest an additional function in Zn translocation to the shoot. Therefore, BTS2 may play a critical role in positively regulating Fe deficiency response in roots and Zn translocation to the shoot by targeting negative regulators for degradation.
Maize is an important cereal crop that provides staple food to many populations. It is a major source of income for the farmers and is grown all over the world. Drought tolerance is the most important trait in maize, since limitation of water supply limits yield at most. The enhanced production of ROS during drought requires an increased GSH production for the efficient detoxification of ROS, thus the regulation of sulfur assimilation during drought is vital due to the dependency of GSH synthesis on the sulfur assimilation pathway. In this study I analysed the impact of drought on the sulfur assimilation pathway in maize. Maize seedlings exposed to drought for 10 and 12 days were severely affected in leaf and root biomass due to a decrease in plant water content and caused elevated levels of H2O2. The drought-induced increase in the ROS formation altered the redox state of GSH pool towards a more oxidized state and indicated oxidative stress in leaves and roots of drought-treated plants compared to control. Moreover, induction of GR transcription in leaves and roots and an increase in GR activity in leaves under drought imply an important role of GR in ROS detoxification and maintaining reduced GSH during drought. The lower steady state level of thiols in leaves is a consequence of decreased rate of GSH biosynthesis during drought. A decrease in the sulfate contents was observed indicating low availability of sulfur in the shoot during drought. Accordingly, the up-regulation in the Sultr1;1 and Sultr4;1 that is responsible for sulfate efflux from the vacuole and a decrease in the steady state levels of sulfate most likely indicate sulfur-starved situation in leaves during drought. Moreover, the transcriptional up-regulation of more than two-fold in ATPS isoforms and an increase in steady state level of APS reflects that ATPS is also rate limiting and regulated by sulfur status during drought. A reduction in the incorporation of 35S into cysteine and GSH suggests that drought limits the availability of sulfate to shoot, thus causing lower flux through the sulfur assimilation pathway into GSH. On the other hand in roots, thiols, sulfate, APS and sulfide were increased relative to the control. A reduction in the incorporation of 35S into cysteine and GSH during drought and the down-regulation of Sultr4;1 indicate the storage of sulfate in the vacuole that might contribute to reduced flux into cysteine and GSH. A strong reduction was observed in the transport of labeled sulfate in the stem of drought stressed plants. This clearly indicates that drought limits the availability of sulfate to shoot, thereby causing the down regulation of sulfur assimilation pathway and ultimately elevated levels of H2O2 in leaves.
The aim of this thesis was to functionally characterize the Cysteine Three Histidine 2 (CTH2)gene in the model plant Arabidopsis thaliana, which is a candidate for a role in plant iron homeostasis. AtCTH2 belongs to the family of tandem zinc-finger proteins (TZF) which are known to bind and initiate the degradation of mRNAs in other organisms. The closest homologue of AtCTH2 in yeast is negatively regulating the stability of a group of specific transcripts under iron-deficient conditions. Heterologous expression of an Arabidopsis CTH2 cDNA can complement a yeast cth1Δcth2Δ mutant. Here it was shown that AtCTH2 partly co-localizes with a marker of plant stress-granules in Arabidopsis protoplasts. Localization to these sites of transcript degradation is an indication that CTH2 plays a role in post-transcriptional regulation of gene expression by influencing transcript stability. Two mutants carrying T-DNA insertions in CTH2 were identified and characterized. The two mutants showed different phenotypes, which was attributed to different partial CTH2 transcripts originating at the CTH2 locus. The results suggested that the partial transcript found in cth2-1 caused a dominant hypersensitivity to iron deficiency, and possibly represents a gain-of-function allele. Most affected were the youngest leaves, which showed a drastic reduction of chlorophyll concentrations and reduced growth, when compared to wild-type plants grown under the same conditions. The total content of iron in the youngest leaves was not affected by the cth2-1 mutation, which showed that long-distance iron reallocation under Fe-deficient conditions is not disturbed. The cth2-2 allele caused sporophytic male sterility, and is recessive. In homozygous cth2-2 plants tetrads of microspores are found, but instead of separating into individual microspores, the cells enlarged, showed granular structures, and eventually degenerated. In accordance with this, the activity of the CTH2 promoter was localized to the connective tissue during the release of microspores from tetrads. The developmental defect might be caused by a disturbed iron homeostasis, since iron content was lower in cth2-2 anthers then in wild-type anthers. A microarray analysis identified several candidate pathways and categories of genes, in which transcript levels are over-proportionately misregulated in cth2-2 anthers compared to wildtype anthers. In summary, it was shown that CTH2 has a role in Arabidopsis thaliana iron homeostasis and is critical for anther development. In both roles CTH2 is the first described RNA-binding protein in a plant to act in these roles.
Das Wort Bionik ist in aller Munde. Egal ob bei von Haihaut inspirierten Schwimmanzügen, die den Wasserwiderstand reduzieren oder dem Lotus-Effekt, der dafür sorgt, dass kein Schmutz an modernen Fassaden haftet. Das Kunstwort kombiniert die Begriffe Technik und Biologie. Dabei will die Bionik die belebte Natur entschlüsseln und die Erkenntnisse anschließend in technische Lösungen umsetzten. Welche Rolle botanische Gärten dabei spielen, erläutert Professor Marcus Koch von der Universität anhand einer aktuellen Ausstellung in Heidelberg.
"Campus-Report" heißt die Radiosendung der Universitäten Heidelberg, Mannheim, Karlsruhe und Freiburg. Die Reportagen über aktuelle Themen aus Forschung und Wissenschaft werden montags bis freitags jeweils um ca. 19.10h im Programm von Radio Regenbogen gesendet. (Empfang in Nordbaden: UKW 102,8. In Mittelbaden: 100,4 und in Südbaden: 101,1) Uni-Radio Baden: ein gemeinsames Projekt der Universitäten Freiburg, Heidelberg, Karlsruhe und Mannheim in Zusammenarbeit mit Radio Regenbogen – unterstützt von der Landesanstalt für Kommunikation. Sendung vom 14. September 2011
"Campus-Report" heißt die Radiosendung der Universitäten Heidelberg, Mannheim, Karlsruhe und Freiburg. Die Reportagen über aktuelle Themen aus Forschung und Wissenschaft werden montags bis freitags jeweils um ca. 19.10h im Programm von Radio Regenbogen gesendet. (Empfang in Nordbaden: UKW 102,8. In Mittelbaden: 100,4 und in Südbaden: 101,1) Uni-Radio Baden: ein gemeinsames Projekt der Universitäten Freiburg, Heidelberg, Karlsruhe und Mannheim in Zusammenarbeit mit Radio Regenbogen – unterstützt von der Landesanstalt für Kommunikation. Sendung vom 14. Juli 2010
"Campus-Report" heißt die Radiosendung der Universitäten Heidelberg, Mannheim, Karlsruhe und Freiburg. Die Reportagen über aktuelle Themen aus Forschung und Wissenschaft werden montags bis freitags jeweils um ca. 19.10h im Programm von Radio Regenbogen gesendet. (Empfang in Nordbaden: UKW 102,8. In Mittelbaden: 100,4 und in Südbaden: 101,1) Uni-Radio Baden: ein gemeinsames Projekt der Universitäten Freiburg, Heidelberg, Karlsruhe und Mannheim in Zusammenarbeit mit Radio Regenbogen – unterstützt von der Landesanstalt für Kommunikation. Sendung vom 19. August 2009
Diese Arbeit fasst meine Arbeit auf dem Phylogeographie und Phylogenie der größte Gattung in der Brassicaceae, Draba. Es gibt über 370 Draba Arten, weltweit verteilt sind (mit Ausnahme von Australien, alle Tiefland südlichen Asien, Afrika und alle abzüglich der Atlas-Gebirge) in Ökosystemen, die extremen Höhen und Breiten sind. Draba ist ein hervorragendes Modell für die Erforschung der globalen Migration von Arten im alpinen/sauren Lebensräume, die derzeit oder in der Vergangenheit relativ stetigen Pfaden der großen Bergregionen und Polarregionen auftreten. Artenreichtum Daten erhoben und Arten Verteilungsmuster verglichen wurden. Hier präsentieren Ich die erste geograsche Verteilung Zusammenstellung der gesamten Gattung auf globaler Ebene mit einer Anwesenheit/Abwesenheit Matrix und Karten mit ArcView 9 generiert. Chloroplast und nukleare Sequenzen wurden für 580 Proben aus Blattmaterial von Herbaria gesammelt werden. Molekulare evolutionären Methoden zur Ableitung Stammbäume und Gen-Stammbäume verwendet wurden. Diese Arbeit liefert die erste erfolgreiche Gattung - große Phylogenie der Draba, und konnte drei wichtige Kerne Draba, die geographischen Regionen entsprechen, zu identizieren. Ich habe auch festgestellt Arten, die historisch gesehen haben Draba, sind nicht Teil der Kern und vorschlagen, aber sie umbenannt werden. Ich habe unter welchen Arten von Umständen eine Art entwickelt, erforscht, mit der Diskussion der Auswirkungen von Umweltfaktoren auf die Migrationspolitik der Linien Muster im Laufe der Zeit. Ich untersucht, warum die Gattung Draba zu sein scheint mehr artenreicher als andere, und festgestellt, dass seine bevorzugte Lebensraum der Höhenlage und Breite ihrer Fähigkeit zur Anpassung beeinusst haben, und die betroffenen Speziation und Polyploidisierung bewerten. Die Speziation Preise berechnet wurden, und die genetische Mutation Preise wurden korreliert mit der Zeit, in der ich in der Lage, eine Schätzung des Alters der Kern Draba Arten machen zu 2,3 Millionen Jahre alt sein und zuletzt wichen von ihren engsten Vorfahren zwischen 11 und 18 mya. Die Wanderrouten sind zahlreiche globale Muster gefolgt, aber speziell kann mit Eiszeit-Zyklen korreliert während des Pleistozän. Draba angepasst hat, um am Rande dieser Ökosysteme durch die Entwicklung solcher reproduktiven Verhalten als asexuell Saatgut Entwicklung (Apomixis), Polyploidisierung, Selbstbefruchtung zu überleben, und Hybridisierung. Darüber hinaus ist eine langjährige Hypothese über die Entstehung einer Gattung, die die Position der höchsten Vielfalt, sowohl bei der Zahl der Arten in einem Gebiet und in den entsprechenden genetischen Vielfalt, auch ist die gleiche wie ihre Herkunft. Im Falle von Draba, habe ich festgestellt, dass der Bereich der höchsten genetischen Vielfalt mit den Herkunftsregionen, die hier festgestellt hat, zwischen dem Kaukasus und in Zentralasien Berge werden korreliert ist, aber nicht den Bereich der höchsten Arten (alpha) Vielfalt oder den Reichtum, die als den zentralen Rocky Mountains identiziert wurde. Dies steht im Widerspruch zu den anerkannten Theorie, dass die Identizierung der Vielfalt 'hot-spots' in die Bemühungen um die Erhaltung anzu greifen, ist durch die Artenzahlen, wenn hier zeigen wir die genetische Vielfalt ist auch entscheidend für die Erhaltung von Arten.
Hybridisierung und Polyploidisierung tragen wesentlich zur Artbildung im Pflanzenreich bei. Innerhalb der Gattung Arabidopsis ist Hybridisierung nur von Arabidopsis suecica aus Fennoskandinavien und Arabidopsis kamchatica aus Japan bekannt. Diese Studie befasst sich mit den Artkomplexen von Arabidopsis lyrata und Arabidopsis arenosa. Unser Ziel war es, herauszufinden, ob und in welchem Ausmaß Hybridisierung an der Artbildung beteiligt war, und ob Polyploidisierung durch Selbstverdopplung des Genoms stattfand. Zudem waren wir an der evolutionären Historie von Di- und Tetraploiden der beiden Artkomplexe interessiert. Wir näherten uns der Lösung dieser Fragestellungen sowohl auf weltweiter Ebene der Gesamtverbreitungsareale beider Artkomplexe als auch auf regionaler Ebene einer mitteleuropäischen Kontaktzone. Im ersten Kapitel „Amphi-beringische, allopolyploide Arabidopsis und die evolutionäre Historie des Arabidopsis lyrata Komplexes“ charakterisierten wir drei genetische Hauptlinien, eine eurasiatische, nordamerikanische und amphi-pazifische, mit den molekularen Markern ntDNA ITS, ntDNA PgiC und cpDNA trnL/F. Allopolyploidisierung zwischen eurasiatischer Arabidopsis lyrata ssp. petraea und ostasiatischer Arabidopsis halleri ssp. gemmifera in der amphi-pazifischen Linie ereignete sich dreimal unabhängig voneinander in Japan, China und Kamtschatka. Wir identifizierten die unvergletscherten Bereiche der ostösterreichischen Alpen und das arktische Eurasien einschließlich Beringias als eiszeitliche Hauptrefugialgebiete der eurasiatischen Linie. Die nordamerikanische Linie überdauerte die Vereisungen im Südosten Nordamerikas. Genfluss zwischen der eurasiatischen und nordamerikanischen Linie fand wahrscheinlich sowohl zwischen den Perioden der Vergletscherung als auch nach der letzten Vereisung statt. Im zweiten Kapitel „Autopolyploidisierung in Arabidopsis und die evolutionäre Historie des Arabidopsis arenosa Komplexes“ fanden wir das Zentrum der Artbildung innerhalb des Arabidopsis arenosa Komplexes auf der Balkanhalbinsel und in den Karpaten mit vorwiegend diploiden neben vereinzelten tetraploiden Populationen. Polyploidisierung erfolgte durch Selbstverdopplung des Genoms. Sowohl die unvergletscherten ostösterreichischen Alpen als auch die Westkarpaten dienten als pleistozäne Refugialgebiete, was anhand des molekularen Markers cpDNA trnL/F ermittelt wurde. Diese beiden Gebirgsstöcke wurden von Arabidopsis arenosa einst unabhängig voneinander von der Balkanhalbinsel aus kolonisiert. Im dritten Kapitel „Retikulate Evolution in eiszeitlichen Refugialgebieten – die Gattung Arabidopsis im ostösterreichischen Donautal (Wachau)“ fanden wir rezente Introgression von Arabidopsis arenosa in Arabidopsis lyrata ssp. petraea in zwei Hybridzonen, eine in der nördlichen Wachau, die andere am Fuße der Ostalpen. In diesen beiden Gebieten liegen die Populationen beider Arten nahe beieinander, was zur Annahme von aktuellem Genfluss zwischen ihnen führte. Die Hybridzone in der nördlichen Wachau wurde sowohl mit molekularen Markern (cpDNA trnL/F Sequenzdaten und sieben Mikrosatelliten) als auch morphologischen Daten charakterisiert. Die Hybridzone am Fuße der Ostalpen wurde mit Hilfe von Mikrosatelliten entdeckt. Tetraploide, besonders von Arabidopsis lyrata ssp. petraea, zeigten stark erhöhte geno- und phänotypische Plastizität im Gegensatz zu Diploiden. Polyploidisierung innerhalb von Arabidopsis arenosa und Arabidopsis lyrata ssp. petraea fand vermutlich durch Selbstverdopplung des Genoms statt.
Das pflanzliche Retromer besteht aus einem großen Subkomplex aus den vakuolären Proteinsortierungsproteinen VPS26, VPS29 und VPS35 und einem kleinen Subkomplex, der sich vermutlich aus den Sorting Nexinen (SNX) SNX1, SNX2a und SNX2b zusammensetzt. Rezeptor-vermittelte Sortierungsprozesse im sekretorischen Transportweg eukaryontischer Zellen beruhen auf dem Mechanismus des Rezeptor-Rezyklierens, nachdem der Transport beendet wurde. In Pflanzen ist der vakuoläre Sortierungsrezeptor (VSR) BP80 an dem Transport von Molekülen zur lytischen Vakuole beteiligt. BP80 bindet vermutlich seine Liganden in dem Donor-Kompartiment trans-Golgi Netzwerk. Dieser Rezeptor-Liganden-Komplex wird danach mit Hilfe von Clathrin-Vesikeln zum prävakuolären Kompartiment (PVC) transportiert. Während die Liganden freigesetzt und zur lytischen Vakuole transportiert werden, wird der Rezeptor Retromer-vermittelt zurück zum TGN transportiert, um erneut Liganden transportieren zu können. Zur Analyse des Retromer-vermittelten Rezyklierens der VSRs wurden die Retromer-Komponenten SNX1, SNX2a, VPS29 und VPS35 zunächst lokalisiert. Diese Retromer-Komponenten befanden sich ausschließlich am trans-Golgi Netzwerk (TGN). Die Inhibierung der Retromer-Funktion durch die transiente Expression von SNX1- oder SNX2a-Mutanten führte zu einer Akkumulation des VSR BP80 im TGN. Quantitative Proteintransportuntersuchungen sowie konfokal-mikroskopische Analysen mit fluoreszierenden, vakuolären Markerproteinen zeigten, dass die Liganden unter diesen Bedingungen weiterhin die Vakuole erreichen konnten. Anhand dieser Ergebnisse erscheint das TGN als der Ort des Retromer-vermittelten Rezyklierens des VSRs. Außerdem ist der Transport zur lytischen Vakuole ab dem TGN Rezeptor-unabhängig und geschieht möglicherweise durch Reifung. Die komplette Hemmung der Retromer-Funktion entweder durch RNAi-„knock-down“ der SNXs oder durch Koexpression der SNX1- und SNX2a-Mutanten inhibierte spezifisch den ER-Export der VSRs und löslicher, vakuolärer Frachtmoleküle. Der COPII-vermittelte Transportweg wurde dabei nicht beeinflusst. Durch die Expression ER-verankerter BP80-Konstrukte konnte untersucht werden, ob die VSRs dazu in der Lage sind, ihre Liganden bereits im Lumen des Endoplasmatischen Retikulums (ER) zu binden. Diese Experimente führten zu einer Akkumulierung löslicher, vakuolärer Frachtmoleküle im ER. Dadurch konnte gezeigt werden, dass die Rezeptor-Liganden-Interaktion bereits im ER stattfindet und nicht erst im TGN. Darüber hinaus rezykliert Retromer die VSRs vermutlich vom TGN zurück zum ER.
Glutamate cysteine ligase (GCL) is catalyzing the rate-limiting step in glutathione (GSH) synthesis. A complex regulation of this enzyme is required to integrate various signals as GSH is fulfilling a plethora of functions in housekeeping metabolism, stress defence, and in the regulation of development. In this thesis the post-translational redox regulation of plant GCL and closely related proteobacterial enzymes was studied. The crystal structure of Brassica juncea GCL (BjGCL) revealed the presence of two intramolecular disulfide bridges. Biochemical analyses of the wild-type enzyme and of mutants lacking cysteines required for the formation of either disulfide bridge showed that both bridges are involved in the in vitro redox regulation of BjGCL. One disulfide bridge (CC1) is apparently controlling access to the active site and knock-out results in a slower overall catalysis rate without changes in Km-values. The second disulfide bridge (CC2) controls the formation of a GCL homo-dimer and reduction of this disulfide bridge leads to monomerization and almost complete deactivation of the enzyme. Sequence analysis showed that only CC2 is conserved in all higher plants while the occurrence of CC1 is restricted to the Rosids clade. Characterization of the redox regulation of GCL from the (non-Rosid) Nicotiana tabacum confirmed the presence of only the dimerization-dependent mechanism of redox regulation. Furthermore, it could be shown that feedback-inhibition of plant GCL by GSH is mechanistically independent from redox regulation. A model is presented on how these different mechanisms interact to control GSH synthesis in vivo. Comparative sequence analysis of plant GCL and with related enzymes from proteobacteria revealed that the amino acid residues forming the dimer interface in BjGCL are conserved in higher plants only, while the catalytic residues are highly conserved among all sequences. The characterization of recombinantly produced GCL from Agrobacterium tumefaciens and Xanthomonas campestris confirmed that these enzymes show kinetics and susceptibility to inhibitors similar to the plant enzyme but completely lack redox regulation and are active as monomers. In a second project, the influence of soluble thiols on the GSH metabolism of different types of cultured plant cells was studied, revealing a specific induction of GCL expression by cysteine. This observation may hint at a role of GSH synthesis in the control of the cellular concentrations of this amino acid, preventing an accumulation which might lead to oxidative stress.
Pektin Methylesterasen (PMEs) und Pektin Methylesterase Inhibitor verwandte Proteine (PMEI-RPs) im Maispollen: Genexpression, subzelluläre Lokalisation und funktionelle Charakterisierung Während der Pollenentwicklung und dem Pollenschlauchwachstum spielen dynamische Veränderungen der Pektinzusammensetzung in der Zellwand eine entscheidende Rolle. Besonders die durch Pektin Methylesterasen vermittelte Demethylierung der Pektinmatrix hat Auswirkungen auf die physikalischen Eigenschaften der Zellwand. Durch MALDI- bzw. ESI-TOF Untersuchungen von Maispollenproben und Datenbankrecherchen wurden unterschiedliche Pektin Methylesterasen identifiziert. Nach Erhalt der vollständigen cDNSs wurden diese in bakterielle und eukaryotische Expressionsvektoren kloniert. Von den vier identifizierten PMEs besitzen zwei Isoformen eine für Typ I charakteristische Prodomäne. Die anderen zwei Isoformen besitzen keine Prodomäne und gehören zu den Typ II PMEs. Mit Hilfe der rekombinanten Proteine sollen, mittels in vitro Untersuchungen, die biologische(n) Funktion(en) aufgeklärt werden. Mittels semiquantitativer RT-PCR wurden die Expressionsstärken und die Pflanzenorgane, in denen die entsprechenden Gene transkribiert werden, ermittelt. PMEI-RPs konnten nur in Antheren und sich entwickelnden Pollen nachgewiesen werden. Die PMEs konnten sowohl in Maisblättern, Pollen, als auch in weiteren Pflanzenorganen nachgewiesen werden. Die subzelluläre Lokalisation der PMEI-RPs wurde zusätzlich durch Fraktionierungen, durch immunocytochemischen Untersuchungen und durch transiente Expression von PMEI-RP::Reportergen-Chimären untersucht. Erstaunlicherweise führten die rekombinanten und nativen PMEI-RPs weder zu einer Inhibition der PME-Aktivität noch zu einer Inhibition mit Invertasen (Zellwand und Vakuolen-Isoformen). Zusätzlich wurden ß-1,3-Glucanasen, Polygalacturonasen und Inulasen durch in vitro Tests als mögliche Zielproteine ausgeschlossen. In Größenausschluss-Chromatographien unterschiedlicher Maispollen-Proben und rekombinanter PMEI-RPs konnten nur monomere PMEI-RPs bei ihrem erwarteten Mr ohne Hinweis auf einen Bindepartner nachgewiesen werden. Versuche mit Chemikalien, die Proteinkomplexe quervernetzen, führten ebenfalls zu keinen nachweisbaren PMEI-RP Zielproteinkomplexen. Es liegt nahe, dass einige Isoformen der kürzlich identifizierten komplexen PMEI-RP Familien entweder andere Zielproteine als Pektin Methylesterasen und Invertasen haben, oder alternativ, weitere noch unbekannte Funktionen aufweisen. Die Expression der PMEI-RP2 Isoform führte zu einer Reduktion des Pollenschlauchwachstums, was auf eine Interaktion von PMEI-RPs mit PMEs deuten könnte.