Philipp Westhoff's research while affiliated with Heinrich-Heine-Universität Düsseldorf and other places

Photorespiration is an essential process of phototropic organisms caused by the limited ability of rubisco to distinguish between CO2 and O2. To understand the metabolic flux through the photorespiratory pathway, we combined a mass spectrometry-based approach with a shift experiment from elevated CO2 (3000 ppm) to ambient CO2 (390 ppm). Here, we describe a protocol for quantifying photorespiratory intermediates, starting from plant cultivation through extraction and evaluation.

Drought stress alters the metabolic activity, physiological processes, and plant growth and such responses might differ with the intensity of stress. We evaluated the genotypic diversity on plant morphology, photosynthetic responses, metabolite shift and their relationship in diverse barley inbreds under dry down (DD) and moderate drought (MD) stress using 23 genetically diverse parental inbreds of genetic mapping population of barley. MD stress caused a strong growth reduction, while DD stress triggered inhibition of photosynthetic health. We observed that the induced changes occurred in a genotype-dependent manner. Compared to control conditions, the metabolism of simple sugars and polyhydroxy acids increased in MD and DD, while the maximum accumulation of amino acid, lipids and phosphates occurred in DD stress. Accumulation of hexose and metabolites with unknown classification was the metabolic signature of drought tolerant inbreds. The inbreds tolerant to MD originated from the temperate regions while those tolerant to both MD and DD came from semi-arid regions. Low stomata density, reduced water loss and retarded growth under drought stress were the key features of inbreds with better survival capacity under severe dehydration. We identified drought tolerant barley inbreds and our study offers resources for future genetic research on various drought tolerance strategies.

Photosynthetic organisms use sunlight as energy source but rely on respiration during the night and in non-photosynthetic tissues. Respiration is also active in photosynthetically active cells, where its role is still unclear due to a lack of viable mutants. Plants lacking cytochrome c oxidase (complex IV) activity are generally lethal but were here isolated exploiting the possibility of generating knockout lines through vegetative propagation in the moss Physcomitrium patens . The mutants showed severely impaired growth, with an altered composition of the respiratory apparatus and increased electron transfer through the alternative oxidase. The light phase of photosynthesis remained largely unaffected while the efficiency of carbon fixation was moderately reduced. Transcriptomic and metabolomic analyses showed that the disruption of the cytochrome pathway had broad consequences for carbon and nitrogen metabolism. A major alteration in nitrogen assimilation was observed with a general reduction in amino acid abundance. A partial rescue of the growth could be obtained by growing the plants with an external supply of amino acids but not with sugars, demonstrating that respiration in plant photosynthetic cells plays an essential role at the interface between carbon and nitrogen metabolism and a key role in providing carbon skeletons for amino acid biosynthesis.

Background Sulfur (S) is a mineral nutrient essential for plant growth and development, which is incorporated into diverse molecules fundamental for primary and secondary metabolism, plant defense, signaling, and maintaining cellular homeostasis. Although, S starvation response is well documented in the dicot model Arabidopsis thaliana, it is not clear if the same transcriptional networks control the response also in the monocots. Results We performed series of physiological, expression, and metabolite analyses in two model monocot species, one representing the C3 plants, Oryza sativa cv. kitaake, and second representing the C4 plants, Setaria viridis. Our comprehensive transcriptomic analysis revealed twice as many differentially expressed genes (DEGs) in S. viridis than in O. sativa under S-deficiency, consistent with a greater loss of sulfur and S-containing metabolites under these conditions. Surprisingly, most of the DEGs and enriched gene ontology terms were species-specific, with an intersect of only 58 common DEGs. The transcriptional networks were different in roots and shoots of both species, in particular no genes were down-regulated by S-deficiency in the roots of both species. Conclusions Our analysis shows that S-deficiency seems to have different physiological consequences in the two monocot species and their nutrient homeostasis might be under distinct control mechanisms.

The microbiome greatly affects health and wellbeing. Evolutionarily, it is doubtful that a host would rely on chance alone to pass on microbial colonization to its offspring. However, the literature currently offers inconclusive evidence regarding two alternative hypotheses: active microbial shaping by host genetic factors or transmission of a microbial maternal legacy. To untangle the influence of host genetics and maternal inheritance, we collected 2-cell stage embryos from two representative wildtypes, C57BL6/J and BALB/c, and transferred a mixture of both genotype embryos into hybrid recipient mice to be inoculated by an identical microbiome at birth. Observing the offspring for six generations unequivocally emphasizes the impact of host genetic factors over maternal legacy in constant environments, akin to murine laboratory experiments. Interestingly, maternal legacy solely controlled the microbiome in the first offspring generation. However, current evidence supporting maternal legacy has not extended beyond this initial generation, resolving the aforementioned debate. Graphical abstract

Photosynthesis plays a vital role in acclimating to and mitigating climate change, providing food and energy security for a population that is constantly growing, and achieving an economy with zero carbon emissions. A thorough comprehension of the dynamics of photosynthesis, including its molecular regulatory network and limitations, is essential for utilizing it as a tool to boost plant growth, enhance crop yields, and support the production of plant biomass for carbon storage. Photorespiration constrains photosynthetic efficiency and contributes significantly to carbon loss. Therefore, modulating or circumventing photorespiration presents opportunities to enhance photosynthetic efficiency. Over the past eight decades substantial progress has been made in elucidating the molecular basis of photosynthesis, photorespiration, and the key regulatory mechanisms involved, beginning with the discovery of the canonical Calvin-Benson-Bassham cycle. Advanced chromatographic and mass spectrometric technologies have allowed a comprehensive analysis of the metabolite patterns associated with photosynthesis, contributing to a deeper understanding of its regulation. In this review, we summarize of the results of metabolomics studies that shed light on the molecular intricacies of photosynthetic metabolism. We also discuss the methodological requirements essential for effective analysis of photosynthetic metabolism, highlighting the value of this technology in supporting strategies aimed at enhancing photosynthesis.

Red blood cells (RBC) are the major carriers of sphingosine-1-phosphate (S1P) in blood. Here we show that variations in RBC S1P content achieved by altering S1P synthesis and transport by genetic and pharmacological means regulate glucose uptake and metabolic flux. This is due to S1P-mediated activation of the catalytic protein phosphatase 2 (PP2A) subunit leading to reduction of cell-surface glucose transporters (GLUTs). The mechanism dynamically responds to metabolic cues from the environment by increasing S1P synthesis, enhancing PP2A activity, reducing GLUT phosphorylation and localization, and diminishing glucose uptake in RBC from diabetic mice and humans. Functionally, it protects RBC against lipid peroxidation in hyperglycemia and diabetes by activating the pentose phosphate pathway. Proof of concept is provided by the resistance of mice lacking the S1P exporter MFSD2B to diabetes-induced HbA1c elevation and thiobarbituric acid reactive substances (TBARS) generation in diabetic RBC. This mechanism responds to pharmacological S1P analogues such as fingolimod and may be functional in other insulin-independent tissues making it a promising therapeutic target.

The application of patient-derived (PD) in vitro tumor models represents the classical strategy for clinical translational oncology research. Using these cellular heterogeneous cultures for the isolation of cancer stem cells (CSCs), suggested to be the main driver for disease malignancy, relies on the use of surrogate biomarkers or is based on CSC-enriching culture conditions. However, the ability of those strategies to exclusively and efficiently enrich for CSC pool has been questioned. Here we present an alternative in vitro CSC model based on the oncogenic transformation of single clone-derived human induced pluripotent stem cells (hiPSC). Hotspot mutations in the DNA encoding for the R132 codon of the enzyme isocitrate dehydrogenase 1 (IDH1) and codon R175 of p53 are commonly occurring molecular features of different tumors and were selected for our transformation strategy. By choosing p53 mutant glial tumors as our model disease, we show that in vitro therapy discovery tests on IDH1-engineered synthetic CSCs (sCSCs) can identify kinases-targeting chemotherapeutics that preferentially target tumor cells expressing corresponding genetic alteration. In contrast, neural stem cells (NSCs) derived from the IDH1R132H overexpressing hiPSCs increase their resistance to the tested interventions indicating glial–to-neural tissue-dependent differences of IDH1R132H. Taken together, we provide proof for the potential of our sCSC technology as a potent addition to biomarker-driven drug development projects or studies on tumor therapy resistance. Moreover, follow-up projects such as comparing in vitro drug sensitivity profiles of hiPSC-derived tissue progenitors of different lineages, might help to understand a variety of tissue-related functions of IDH1 mutations.

Mitochondria play central roles in metabolism and metabolic disorders such as type 2 diabetes. MIC26, a MICOS complex subunit, was linked to diabetes and modulation of lipid metabolism. Yet, the functional role of MIC26 in regulating metabolism under hyperglycemia is not understood. We employed a multi-omics approach combined with functional assays using WT and MIC26 KO cells cultured in normoglycemia or hyperglycemia, mimicking altered nutrient availability. We show that MIC26 has an inhibitory role in glycolysis and cholesterol/lipid metabolism under normoglycemic conditions. Under hyperglycemia, this inhibitory role is reversed demonstrating that MIC26 is critical for metabolic adaptations. This is partially mediated by alterations of mitochondrial metabolite transporters. Furthermore, MIC26 deletion led to a major metabolic rewiring of glutamine utilization as well as oxidative phosphorylation. We propose that MIC26 acts as a metabolic rheostat, that modulates mitochondrial metabolite exchange via regulating mitochondrial cristae, allowing cells to cope with nutrient overload.

Hotspot mutations in the DNA encoding for the R132 codon of the enzyme isocitrate dehydrogenase 1 (IDH1) is a common molecular feature of different tumors. The oncogenic potential of IDH1R132 and its clinical prognostic value however, varies strongly between tumors of different tissues. Technologies to conduct functional investigations of isogentic controlled IDH1R132 in dependency of differentiation status offers a chance to understand underlying mechanisms of this heterogeneity or identify new tissue-dependent features of IDH1 mutation. Here we genetically engineered the first IDH1 MUT model using human induced pluripotent stem cells (hiPSC) for inducible overexpression of IDH1R132H or its wildtype paralog. Confirming the known relevance of IDH1R132H, we identified a transcriptomic switch of hiPSC cells towards pro-angiogenetic program meanwhile suppression of p53 signaling upon oncogene induction. We chose neural differentiation of the cells and drug sensitivity testing to compare the influence of IDH1R132H on functional properties of the cells in tissue-specific context. Our results reveal the augmentation of drug resistance levels to clinical approved kinase inhibitors in induced neural stem cells, which was not observed in the pluripotent counterpart. Applying our technology in follow-up projects, such as comparing isogenic progenitor cells of different differentiation lineages, might help to understand a variety of tissue-related functions of IDH1 mutations. Moreover, given the fact that patient-derived human neuronal in vitro models with constitutive active IDH1R132H are challenging to establish, the presented work supports to overcome this limitation.