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The iron-containing porphyrin heme is of high interest for the food industry for the production of artificial meat as well as for medical applications, e.g. for anemia treatment. Recently, the biotechnological platform strain Corynebacterium glutamicum has emerged as a promising host for animal-free heme production. Beyond engineering of complex heme biosynthetic pathways, improving heme export offers significant yet untapped potential for enhancing production strains. In this study, a growth-coupled biosensor was designed to impose a selection pressure on the increased expression of the hrtBA operon encoding an ABC-type heme exporter in C. glutamicum. For this purpose, the promoter region PhrtB was replaced with that of the growth-regulating genes pfkA (phosphofructokinase) and aceE (pyruvate dehydrogenase), creating biosensor strains with a selection pressure for hrtBA activation. Resulting sensor strains were used for plate-based selections and for a repetitive batch f(luorescent)ALE using a robotics platform. Genome sequencing of isolated clones featuring increased hrtBA expression revealed three distinct mutational hotspots: (i) chrS, (ii) chrA, and (iii) cydD. Mutations in the genes of the ChrSA two-component system, which regulates hrtBA in response to heme levels, were identified as a promising target to enhance export activity. Furthermore, causal mutations within cydD, encoding an ABC-transporter essential for cytochrome bd oxidase assembly, were confirmed by the construction of a deletion mutant, which showed strongly increased hrtBA expression as well as increased cellular heme levels. These results further support the proposed role of CydDC as a heme transporter. Mutations identified in this study therefore underline the potential of biosensor-based growth coupling and provide promising engineering targets to improve microbial heme production.

Fungal pathogens depend on sophisticated gene expression programs for successful infection. A crucial component is RNA regulation mediated by RNA-binding proteins (RBPs). However, little is known about the spatiotemporal RNA control mechanisms during fungal pathogenicity. Here, we discover that the RBP Khd4 defines a distinct mRNA regulon to orchestrate membrane trafficking during pathogenic development of Ustilago maydis. By establishing hyperTRIBE for fungal RBPs, we generated a comprehensive transcriptome-wide map of Khd4 interactions in vivo. We identify a defined set of target mRNAs enriched for regulatory proteins involved, e.g., in GTPase signaling. Khd4 controls the stability of target mRNAs via its cognate regulatory element AUACCC present in their 3′ untranslated regions. Studying individual examples reveals a unique link between Khd4 and vacuole maturation. Thus, we uncover a distinct role for an RNA stability factor defining a specific mRNA regulon for membrane trafficking during pathogenicity.

MAdLand Project - Schippers Lab

Evolutionary conserved and divergent responses to copper zinc superoxide dismutase inhibition in plants

After initial evolution in a reducing environment, life got successively challenged by reactive oxygen species (ROS), especially during the great oxidation event (GOE) that followed the development of photosynthesis. Therefore, ROS are deeply intertwined into the physiological, morphological and transcriptional responses of most present-day organisms. Copper-zinc superoxide dismutase (CuZnSOD) evolved during the GOE and are present in charophytes and extant land plants, but nearly absent from chlorophytes. The chemical inhibitor of CuZnSOD, lung cancer screen 1 (LCS-1), could greatly facilitate the study of SODs in diverse plants. Here, we determined the impact of chemical inhibition of plant CuZnSOD activity, on plant growth, transcription and metabolism. We followed a comparative approach by using different plant species, including Marchantia polymorpha and Physcomitrium patens, representing bryophytes, the sister lineage to vascular plants, and Arabidopsis thaliana. We show that LCS-1 causes oxidative stress in plants and that the inhibition of CuZnSODs provoked a similar core response that mainly impacted glutathione homeostasis in all plant species analyzed. That said, Physcomitrium and Arabidopsis, which contain multiple CuZnSOD isoforms showed a more complex and exacerbated response. In addition, an untargeted metabolomics approach revealed a specific metabolic signature for each plant species. Our comparative analysis exposes a conserved core response at the physiological and transcriptional level towards LCS-1, while the metabolic response largely varies. These differences correlate with the number and localization of the CuZnSOD isoforms present in each species.

Plant, Cell & Environment (in submission)

Microtubule-dependent endosomal transport is crucial for polar growth, ensuring the precise distribution of cellular cargos such as proteins and mRNAs. However, the molecular mechanism linking mRNAs to the endosomal surface remains poorly understood. Here, we present a structural analysis of the key RNA-binding protein Rrm4 from Ustilago maydis. Our findings reveal a different type of MademoiseLLE domain (MLLE) featuring a seven-helical bundle that provides a distinct binding interface. A comparative analysis with the canonical MademoiseLLE domain of the poly(A)-binding protein Pab1 disclosed unique characteristics of both domains. Deciphering the MLLE binding code enabled prediction and verification of previously unknown Rrm4 interactors containing short linear motifs. Importantly, we demonstrated that the human MLLE domains, such as those of PABPC1 and UBR5, employed a similar principle to distinguish among interaction partners. Thus, our study provides detailed mechanistic insights into how structural variations in the widely distributed MLLE domain facilitate mRNA attachment during endosomal transport.

Quantification of cell growth is central to any study of photoautotrophic microorganisms. However, cellular self-shading and limited CO2 control in conventional photobioreactors lead to heterogeneous conditions that obscure distinct correlations between the environment and cellular physiology. Here we present a microfluidic cultivation platform that enables precise analysis of cyanobacterial growth with spatio-temporal resolution. Since cyanobacteria are cultivated in monolayers, cellular self-shading does not occur, allowing homogeneous illumination and precise knowledge of the photon-flux density at single-cell resolution. A single chip contains multiple channels, each connected to several hundred growth chambers. In combination with an externally applied light gradient, this setup enables high-throughput multi-parameter analysis in short time. In addition, the multilayered microfluidic design allows continuous perfusion of defined gas mixtures. Transversal CO2 diffusion across the intermediate polydimethylsiloxane membrane results in homogeneous CO2 supply, with a unique exchange-surface to cultivation-volume ratio. Three cyanobacterial model strains were examined under various, static and dynamic environmental conditions. Phase-contrast and chlorophyll fluorescence images were recorded by automated time-lapse microscopy. Deep-learning trained cell segmentation was used to efficiently analyse large image stacks, thereby generating statistically reliable data. Cell division was highly synchronized, and growth was robust under continuous illumination but stopped rapidly upon initiating dark phases. CO2-Limitation, often a limiting factor in photobioreactors, was only observed when the device was operated under reduced CO2 between 50 and 0 ppm. Here we provide comprehensive and precise data on cyanobacterial growth at single-cell resolution, accessible for further growth studies and modeling.

In the present study we aimed at the highest possible resolution of this network by combining analysis of transcriptome, proteome and subcellularly resolved metabolome of plants that were exposed to rising carbon dioxide concentrations over a time course of one week.

Host preference and invasiveness of commensal bacteria in the Lotus and Arabidopsis root microbiota

Traits linked to natural variation of sulfur content in Arabidopsis thaliana

Physiology, biochemistry and anatomy of young fully developed leaves from Brassicaceae species with C3 and C3-C4 (C2) photosynthesis

This ARC contains CPlantBox simulations

MAdLand Project - Höcker Lab

Co-action of COP1, SPA and cryptochrome in light signal transduction and photomorphogenesis of the moss Physcomitrium patens

The Arabidopsis COP1/SPA ubiquitin ligase suppresses photomorphogenesis in darkness. In the light, photoreceptors inactivate COP1/SPA to allow a light response. While SPA genes are specific to the green lineage, COP1 also exists in humans. This raises the question of when in evolution plant COP1 acquired the need for SPA accessory proteins. We addressed this question by generating Physcomitrium Ppcop1 mutants and comparing their visible and molecular phenotypes with those of Physcomitrium Ppspa mutants. The phenotype of Ppcop1 nonuple mutants resembles that of Ppspa mutants. Most importantly, both mutants produce green chloroplasts in complete darkness. They also exhibit dwarfed gametophores, disturbed branching of protonemata and absent gravitropism. RNA-sequencing analysis indicates that both mutants undergo weak constitutive light signaling in darkness. PpCOP1 and PpSPA proteins form a complex and they interact via their WD repeat domains with the VP motif of the cryptochrome CCE domain in a blue light-dependent manner. This resembles the interaction of Arabidopsis SPA proteins with Arabidopsis CRY1, and is different from that with Arabidopsis CRY2. Taken together, the data indicate that PpCOP1 and PpSPA act together to regulate growth and development of Physcomitrium. However, in contrast to their Arabidopsis orthologs, PpCOP1 and PpSPA proteins execute only partial suppression of light signaling in darkness. Hence, additional repressors may exist that contribute to the repression of a light response in dark-exposed Physcomitrium.

The Plant Journal 114: 159–175; https://doi.org/10.1111/tpj.16128

MAdLand Project - Erika Csicsely, Oguz Top, Wolfgang Frank et al.

This ARC accompanies the publication in Plant Journal: https://doi.org/10.1111/tpj.17236

DICER-LIKE (DCL) proteins play a central role in plant small RNA (sRNA) biogenesis. The genome of the early land plant Marchantia polymorpha encodes four DCL proteins: MpDCL1a, MpDCL1b, MpDCL3, and MpDCL4. While MpDCL1a, MpDCL3 and MpDCL4 show high similarities to their orthologs in Physcomitrium patens and Arabidopsis thaliana, MpDCL1b shares only a limited homology with PpDCL1b, but it is very similar, in terms of functional domains, to orthologs in other moss and fern species. We generated Mpdclge mutant lines for all MpDCL genes with the CRISPR/Cas9 system and conducted phenotypic analyses under control, salt stress, and phytohormone treatments to uncover specific MpDCL functions. The mutants displayed severe developmental aberrations, altered responses to salt and phytohormones, and disturbed sexual organ development. By combining mRNA and sRNA analyses, we demonstrate that MpDCLs and their associated sRNAs play pivotal roles in regulating development, abiotic stress tolerance and phytohormone response in M. polymorpha. We identified MpDCL1a in microRNA biogenesis, MpDCL4 in trans-acting small interfering RNA generation, and MpDCL3 in the regulation of pathogen-related genes. Notably, salt sensitivity in M. polymorpha is dependent on MpDCL1b and Mpdcl1bge mutants display enhanced tolerance and reduced miRNA expression in response to salt stress. We propose that M. polymorpha employs specific mechanisms for regulating MpDCL1b associated miRNAs under high salinity conditions, potentially shared with other species harboring MpDCL1b homologs.

MAdLand Project - Wolfgang Hess Lab

Charophyceae are the most complex streptophyte algae, possessing tissue-like structures, rhizoids and a cellulose-pectin-based cell wall akin to embryophytes. Together with the Zygnematophyceae and the Coleochaetophycae, the Charophyceae form a grade in which the Zygnematophyceae share a last common ancestor with land plants. The availability of genomic data, its short life cycle, and the ease of non-sterile cultivation in the laboratory have made the species Chara braunii an emerging model system for streptophyte terrestrialization and early land plant evolution. In this study, tissue containing nodal cells was prepared under the stereomicroscope, and an RNA-seq dataset was generated and compared to transcriptome data from whole plantlets. In both samples, transcript coverage was high for genes encoding ribosomal proteins and a homolog of the putative PAX3- and PAX7-binding protein 1. Gene ontology was used to classify the putative functions of the differently expressed genes. In the nodal cell sample, main upregulated molecular functions were related to protein, nucleic acid, ATP- and DNA binding. Looking at specific genes, several signaling-related genes and genes encoding sugar-metabolizing enzymes were found to be expressed at a higher level in the nodal cell sample, while photosynthesis-and chloroplast-related genes were expressed at a comparatively lower level. We detected the transcription of 21 different genes encoding DUF4360-containing cysteine-rich proteins. The data contribute to the growing understanding of Charophyceae developmental biology by providing a first insight into the transcriptome composition of Chara nodal cells.

Physiologia Plantarum / Volume 175, Issue 5 / e14025, https://doi.org/10.1111/ppl.14025

Pangenome of Camellia sinensis

Algae cultures were grown mixotrophically (TAP). After 24h of 35°C/40°C the cells were shifted back to room temperature for 48h. 'omics samples were taken.

ARC repo for MARS testing

The trace element iron is essential for life, but elevated levels can rapidly cause cellular damage through oxidative stress. Bacteria, like Corynebacterium glutamicum, tightly regulate iron and heme homeostasis via the global regulators DtxR and HrrA. This study provides the first analysis of the genome-wide binding patterns of these two regulators demonstrating significant differences in binding dependent on the tested iron regimes. Overall, we identified 25 new DtxR targets and 210 previously unknown HrrA targets, including genes with crucial roles in central metabolism and DNA repair. Notably, DtxR was shown to link iron metabolism to methionine synthesis, which might be important to protect the cell from oxidative stress. Our findings highlight the interconnected nature of DtxR and HrrA networks and underscore the value of condition-specific analysis to deepen the understanding of how bacteria adapt to environmental changes.