Browsing by Subject "Proteome"

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Adaptations of maize to low phosphate availability : establishing regulatory networks from large-scale quantitative proteomic profiling
(2022) He, Mingjie; Schulze, Waltraud
Maize (Zea mays) is an important crop in global for human food, animal feed and industrial usage. Suboptimal phosphorus (P) availability is one of the primary constraints for maize growth and productivity (Jianbo Shen et al., 2011; L.pez-Arredondo et al., 2014). Over 70% arable land suffers from P-deficiency, and plants can take up small amounts of P from the soil due to P-fixation. However, over-application of P fertilizer has frequently happened in last decades and resulted in environmental pollution (L.pez- Arredondo et al., 2014). Modern agriculture calls for maintaining productivity while reducing synthetic-P fertilizer inputs and losses, thus, requiring breeding of novel cultivars to increase phosphate use efficiency (PUE) (Balemi and Negisho, 2012; X., Li, Mang, et al., 2021; Mardamootoo et al., 2021). Understanding the regulation of maize to low phosphate(LP)-availability at the molecular level will offer unlimited potential for the development of selection markers and engineering targets in breeding programs. Nowadays, “OMIC” approaches and computational science are developing rapidly. They are advanced tools for investigation of molecular adaptations on a large-scale and in a systemic view. Thereby, the major research task within this thesis is to reveal P-deficiency induced responsive components and regulations at protein level based on proteomic profiles, aiming to provide promising candidate genes/proteins for research on the molecular mechanisms of adaptation to LP-stress, and potentially to provide promising candidate gene/proteins for development of selection markers and engineering targets to obtain desired traits, in the long term goal of improving PUE in novel cultivars. In Chapter 1, we focused on six genotypes (EP1, F2, F142, F160, SF1, SM1) with close genetic background but several contrasting traits to LP-stress, such as PUE (X., Li, Mang, et al., 2021). They were cultured in pot with either sufficient or inefficient P-fertilizer in a climate chamber for one month. The young seedlings were sampled by root and shoot for analysis of multiple traits, transcriptome and proteome. Firstly, we constructed the co-expression network of proteins and transcripts separately using WGCNA method (Langfelder and Horvath, 2008), which predicted potential protein-protein interactions or their co-regulations. Secondly, we categorized proteins/transcripts to modules according to their different coexpression patterns, thus, identified potential determining relationships of modules-traits. Thirdly, we compared the responses between transcripts and proteins, presenting their responses being concordant or dis-concordant. Fourthly, we identified common and genotype-specific P-starvation response modules and biological processes. Finally, we focused on protein kinases, which play roles as regulators, to demonstrated protein kinases-centered network and validated protein interactions between mitogenactivated protein kinase-kinase 1 (MEK1, Zm00001d043609) either with sucrose synthase1 (SH1,Zm00001d045042) or translation elongation factor 1-gamma 3 (eEF1B-γ, Zm00001d046352). MEK1 is a potential genotype-specific regulator via sucrose metabolism and translation elongation process. In Chapter 2, we aimed to adapted an experimental workflow for phosphoproteome analysis in maize, addressing the interference to phosphoproteome quantification by fibers, secondary metabolites and low abundant of phosphorylated proteins. In this manuscript, we described a rapid and universal protocol for both proteome and phosphoproteome analysis that is suitable for cereal crops. The results of phosphoproteome in maize root testing samples showed that proteins within kinase-centered network in Chapter 1 can be largely quantified based on this workflow. It provides a possible way to analyze phosphorylation dynamics to P-starvation responses, it allows further investigation for kinase-centered 1 network in Chapter 1 to identify phosphorylation pairs of “protein kinase – protein substrate”, which will largely expand a view on P-starvation regulations through posttranslational modifications.
Einfluss verschiedener Getreidearten und Herstellungsverfahren auf den Gehalt immunogener Substanzen in Brot sowie in vivo auf die Verträglichkeit an der Maus und im Menschen
(2022) Zimmermann, Julia; Bischoff, Stephan C.
There are three medical conditions that are triggered by consumption of cereals. Celiac disease, wheat allergy and non-celiac wheat sensitivity (NCWS). While the underlying triggers and mechanisms of the first two entities have been extensively studied, there is still uncertainty in this regard for NCWS. Symptoms are nonspecific and diagnostic markers are lacking. Besides bacterial fermentable carbohydrates (FODMAPs), selected cereal proteins such as gluten or α-amylase trypsin inhibitors (ATIs) are in the focus of research as triggers. The aim of the present work was firstly to investigate the influence of the choice of cereal (common wheat, spelt, rye) and the production of bread (degree of milling and choice between yeast and sourdough) on the presence of potentially immunogenic proteins based on proteomic analysis. In a second step, the tolerability of selected breads should be investigated in a transgenic mouse model with intestinal inflammation and in a human study in patients with NCWS and subjective spelt tolerance. This was to narrow down possible triggers of NCWS and to investigate underlying mechanisms. Within the project, protein composition of bread and flour samples was analyzed based on a quantitative proteomics method (nano-UHPLC-ESI-MS based). In addition, a list of known and potentially immunogenic cereal proteins was generated based on Pfam annotation, which was used for the analysis of allergens in flour and bread. This showed that neither the absolute number nor the abundance of these allergenic proteins were dependent on the degree of milling of the flour or the fermentation process of the dough, which means that they are not selectively degraded during bread production. However, such proteins were identified in higher numbers and higher relative amounts in spelt and wheat samples compared to rye samples. Furthermore, different bread types from the proteome analysis were investigated in a mouse model with intestinal inflammation. This did not demonstrate better tolerability of rye bread compared to spelt and wheat bread. Instead, there was a trend for sourdough bread to have less negative effects on intestinal inflammation compared to yeast dough bread. It also turned out that inflammation was increased independently of gluten. No differences were found between wheat and spelt in either the proteomic analysis or the animal studies in this project. However, in a subgroup of NCWS patients, spelt bread is subjectively better tolerated than wheat bread, which could be due to both genetics and the different production of wheat and spelt bread. In order to verify the phenomenon and identify underlying mechanisms, if any, a clinical study was conducted in patients of this subgroup. The aim of the blinded study was to investigate whether spelt bread is actually better tolerated than wheat bread and whether the production process (16h dough or 1h dough + baking agent) has an influence. After each bread (4 days each + 3 days washout), gastrointestinal symptoms were assessed using the Irritable Bowel Syndrome Severity Scoring System (IBS-SSS) questionnaire. Extraintestinal symptoms and various blood and stool parameters were also analyzed. It was found that spelt bread was not better tolerated compared to wheat bread after blinded consumption and that FODMAP-rich bread was not worse tolerated compared to FODMAP-poor bread.
Escherichia coli O157:H7 und seine Adaption an verschiedene Wachstumsbedingungen in vitro : Untersuchung des intrazellulären Proteoms und Kohlenhydratmetabolismus
(2014) Polzin, Sabrina; Schmidt, Herbert
The aim of this study was to investigate the linkage of metabolism and pathogenicity of EHEC bacteria under certain environmental conditions in vitro. During the investigations the influence of environmental conditions could be shown on the protein as well as on the gene expression level. In experiments using the culturel media SIEM and SCEM compared to TSB as a reference, differential expression of proteins and genes were shown. For example, decreasing motility and the formation of biofilm-like substances were observed. Furthermore, differences in metabolism and expression of chaperons and stress response systems were detected. After culturing EHEC bacteria in experimental media, increased glucose and pyruvate metabolism, increased amino acid biosynthesis, as well as increased chaperon levels were found, in contrast to decreased expression levels of stress associated proteins. The experimental procedure also affected the presence of pathogenicity factors, like EspP and EspB. Notably, the environmental conditions induced the glutamic acid resistance, providing the EHEC bacteria an unhindered passage through the human gastrointestinal tract, especially the acidic environments like the stomach. Additionally, the sialic acid esterase Z1466 along with shiga toxin, was strongly expressed. This could provide an advantage over the intestinal micro flora, in respect of growth. Effects onto glucose metabolism and amino acid biosynthesis were investigated by isotopoloque profiling experiments, prepared thankworthy by Dr. rer. nat. Wolfgang Eisenreich (Munich). To conclude, stress response and expression of pathogenicity factors could be induced in vitro in the cultural media elected, and without the need of a human host. The experiment also advices the complex regulation of different processes in bacterial cells as response to external factors.
Microbiological and proteome analysis to gain insights into the pathogenesis of the highly adapted not-cultivated hemotrophic Mycoplasma suis
(2016) Dietz, Stefanie; Hölzle, Ludwig
The aims of this work were to establish a chronic pig infection model and to clarify unrecognized transmission pathways of M. suis. In addition, proteomics-based investigations of M. suis should be performed to improve the knowledge on the host pathogen interactions and host adaptation in IAP. Based on the succeeded and frequently applied splenectomized M. suis pig model (acute infection model) it was possible to establish a novel non-splenectomized M. suis pig model. This infection model enables the experimental investigation of the chronic M. suis infection. To this end, the piglets were infected intramuscularly with a highly virulent M. suis strain. Infected animals exhibited clinical signs (e.g. Morbus maculosus) including the typical cyclic course of chronic IAP. In the next step, potential transmission pathways of M. suis were analyzed during acute and chronic M. suis infection based on these two pig infection models. Feces, urine, air and dust as well as nasal, vaginal and saliva excretions were collected during the course of infection and examined for M. suis DNA by Rt-PCR. For the first time it was possible to detect M. suis in urine with and without erythrocytes as well as in nasal, vaginal and saliva excretions during acute and chronic infections. These results indicate blood-independent M. suis transmission via vaginal discharge, nasal excretions, saliva, and urine. The non-culturability limited the improvement of proteomic-based investigations of M. suis-related host-pathogen interactions. Therefore, we used modern and sophisticated proteome analysis to solve this problem. Blood samples from experimentally infected pigs at different time points of infection were investigated. For this, novel enrichment methods for M. suis proteins (especially membrane proteins) were established. These methods enabled an improved resolution of the protein expression profile of M. suis and thereby deeper insights into the pathogenesis of this microorganism. Despite of the missing cultivation system it was possible to identify more than 50% of the predicted M. suis proteins during acute infection. This identification ratio is similar to the one found in cultivable bacteria. Furthermore, the results of the proteome analysis indicate that nutrients such as glucose, hexose-6-phosphate, spermidine, putrescine, phosphate, amino acids, magnesium, potassium, sodium and iron are taken up by M. suis from the host leading to the high degree of host adaptation. Therefore, gained information on expressed M. suis proteins involved in transport are helpful in the establishment of an in vitro cultivation system in future. Particularly the supplementation of individual nutrients can play key functions in the media to support growth. Besides the M. suis proteome the acquired dataset firstly enables also the quantitative identification of Sus scrofa proteins differentially expressed during M. suis infection. This information can be used to unravel infection-relevant processes in the host in further studies.
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.