Browsing by Subject "Arabidopsis"

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External nutrition stimuli induced proteome and phosphoproteome responses of maize root hairs and arabidopsis root microsomal fraction
(2021) Li, Zhi; Schulze, Waltraud
This work studied how the proteome from young maize root hair cells responds to different nutrition deprivation, and gives perspectives to the possible involvement of NRT1.1 and NRT2.1 in regulating root membrane phosphoproteome responses. This work also proposes a phospho-switch model that may explain how the NRT2.1 activity was regulated.
Prediction of protein-protein complexes by combining size exclusion chromatography and mass spectrometric analysis
(2021) Gilbert, Max; Schulze, Waltraud
Two major objectives were pursued and met in this study. First, the goal was to add to the scientific toolbox a diligent method for uncovering PPi dynamics on a proteomic scale, with a focus on plant membranes. There are large-scale or high-throughput approaches, but they rely on genetically modified proteins or heterologous expression systems to describe PPi outside of their natural context. Similarly, those methods are incapable of describing the dynamics of protein interactions. In course of this study, a co-elution based approach was combined with modern mass spectrometric label free quantification in order to investigate PPi and interaction dynamics on a proteomic scale. A rigorous data processing pipeline was developed to not only address known fallacies of using co-elution based methods (such as for example random co elution), but also to access and utilize meta-information in form of protein abundance and protein network connectivity to draw conclusions not only on proteomic scale, but also for individual proteins. In total, 6.928 individual proteins extracted from Arabidopsis thaliana root membranes were detected under different nutritional conditions (full nutrition, nitrogen starvation and nitrogen resupply). The data processing pipeline described in this study was used to predict and discover connectivity information for at least 2.058 of these proteins. Each step in data processing was validated by comparison to database confirmed interactions to improve filtering criteria. Protein abundance was evaluated through a unique ranking system, allowing a seamless integration as network attributes for each condition. From the suggested interaction data, an interactome network of the various nutritional conditions was reconstructed. Using different network parameters from graph theory, protein significance and dynamic conditional changes were described. Second, this study applied the aforementioned approach to identify relevant proteins involved in nitrogen signaling in Arabidopsis thaliana root membranes. Through correlation analysis and network reconstruction, receptor kinase AT5G49770 was identified as a component of the nitrogen signaling network that collaborates with co-receptor QSK1, BAK1, the nitrogen transporter NRT2.1 and proton pump AHA2. In response to nitrogen deficiency, the network parameters of AT5G49770 reacted strongly and its involvement was demonstrated by a phenotypic similarity to knock-out lines of NRT2.1, NRT1.1 and AHA2 during a root growth assay of Arabidopsis seedlings. The interaction between QSK1 and BAK1 was further confirmed using FRET/FLIM microscopy and pulldown assays. These findings show that combining a co-elution based approach with a rigorous data processing pipeline and network analysis is suitable to study the protein interaction environment and signal response dynamics in plant root membranes. The modular experimental design allows for a simple adaptation to study different stimuli and the unbiased proteomic approach yields results for proteins regardless of the individual scientific focus. Meta-information such as protein abundance and network connectivity parameters can be used to prospect and identify important proteins involved in stress response dynamics. The author of this study is confident that the proteomic data produced can be utilized in further research and contributes to the understanding of nitrogen signaling in plant root membranes. Through integration of the data processing pipeline and adaptation to different scientific scenarios, valuable information beyond protein interaction is gained. Thus, this work makes an important contribution to the advancement of proteomic analysis and data interpretation methodology.
The AtIREGs - characterization of a new family of metal transporters in Arabidopsis thaliana
(2009) Kirchner, Silvia; von Wirén, Nicolaus
Essential transition metals are required in all plant cells for the activities of numerous metal-dependent enzymes and proteins, but can become toxic when present in excess. For the detoxification of heavy metals and to adjust to changes in micronutrient concentrations in the environment, plants possess a tightly controlled metal homeostasis network. In this regard, transition metal transporters are of central importance. Many metal transporters have already been identified, but a large number of candidates for heavy metal transport proteins still have to be analyzed at the biochemical level and within the plant metal homeostasis network. Based on the description of the animal IREG1 metal transporter as an iron exporter in vertebrates, a phylogenetic analysis of eukaryote and prokaryote sequences with similarity to IREG1 showed three homologous genes in Arabidopsis, which were named AtIREG1, AtIREG2 and AtIREG3. As these AtIREG family members were candidates for yet uncharacterized metal transporters, the main objective of this thesis was to investigate the physiological function of this newly identified transporter family in plants.
Transcriptional and proteomic responses towards early nitrogen depletion in Arabidopsis thaliana
(2016) Menz, Jochen; Ludewig, Uwe
Plant roots acquire nitrogen predominantly as ammonium and nitrate, which besides serving as nutrients, also have signaling roles. Re-addition of nitrate to starved plants rapidly and di-rectly transcriptionally re-programs the metabolism and induces root architectural changes, but the earliest responses to nitrogen deprivation are unknown. In this thesis, the early transcriptional response of developed roots to nitrate or ammonium deprivation were analyzed in two Arabidopsis ecotypes contrasting in their nitrogen use efficiency: the inefficient genotype Col-0 and the efficient Tsu-0. The rapid transcriptional repression of known nitrate-induced genes proceeded the tissue NO3- concentration drop, with the transcription factor genes LBD37/38 and HRS1/HHO1 among those with earliest significant change. Some transcripts were stabilized by nitrate, but similar rapid transcriptional repression occurred in loss-of-function mutants of the nitrate response factor NLP7. In contrast, an early transcriptional response to ammonium deprivation was almost completely absent. In Col-0, the analysis was extended with the proteome and phospho-proteome resulting in a rapid and transient perturbation of the proteome induced by ammonium deprivation and a differential phosphorylation pattern in proteins involved in adjusting the pH and cation homeostasis, plasma membrane H+, NH4+, K+ and water fluxes. Fewer differential phosphorylation patterns in transporters, kinases and other proteins occurred with nitrate deprivation. The deprivation responses are not just opposite to the resupply responses, identify NO3--deprivation induced mRNA decay and signaling candidates potentially reporting the external nitrate status to the cell. Transcrip-tome comparison revealed only few N-nutrition related genes between both ecotypes contributing the increased NUE of Tsu-0, which probably relies on higher biomass accumulation. Besides, Tsu-0 confirmed the transcriptional depletion response of Col-0.