Biology subjects

Brandt, B.

Publications and source records attributed to Brandt, B..

12 recordsLinked to original sources

bioRxiv2026

pamiR: INVESTIGATING PLANT CELLS ONE ORGANELLE AT A TIME

Functional genetic redundancy (FGR) within gene families limits the discovery of gene function in plants because single-gene perturbations often fail to produce informative phenotypes. Artificial microRNAs (amiRNAs) provide a strategy to silence multiple related genes simultaneously. However, the existing amiRNA-based libraries used for genetic gene function discovery in plants do not account for the subcellular localization of gene products, which can lead to pleiotropic or difficult-to-interpret phenotypes. Plastids are essential plant cell organelles that integrate central metabolic and signaling processes, including photosynthesis, hormone biosynthesis, and environmental responses. Here we introduce pamiR, a plastid-targeted amiRNA library designed to enable organelle-specific gene function discovery in Arabidopsis thaliana. Using plastid proteomic datasets, we identified high-confidence plastid-localized proteins and designed amiRNAs to target their gene(s) (families) minimizing FGR. This amiRNA library was introduced in a vector with fluorescence-accumulating seed technology enabling rapid, herbicide-free selection and screening in the first generation. Validation by next-generation sequencing, confirmed high representation and uniform distribution of amiRNAs within pamiR. Proof-of-concept screens recovered mutants affecting known and additional candidate genes involved in photosynthesis and abscisic acid biosynthesis. Therefore, the pamiR library provides a fast platform for plastid-focused genetic screens that is compatible with existing mutant collections. One-sentence summaryThe plastid amiRNA (pamiR) library enables organelle-specific forward genetics without functional genetic redundancy.

plant biology↗

bioRxiv2025

Conserved and Lineage-Specific Roles of KEA-Mediated Ion Homeostasis in Chlamydomonas

In Arabidopsis thaliana, seamless plastid gene expression and development depend on finely balanced ion homeostasis across the inner envelope (IE) membrane, maintained by the K/H antiporters AtKEA1/2. To assess whether these functions are retained across mono- and polyplastidic representatives of the green lineage, we studied CrKEA1, the sole IE KEA homolog in the unicellular alga Chlamydomonas reinhardtii. Using CRISPR/Cas9, we generated a Cr-kea1 knockout mutant that exhibits impaired photoautotrophic growth, chloroplast deformation, and photoinhibition. Transcriptomics revealed strong induction of ribosome biogenesis genes and reduced abundance of transcripts associated with cell and plastid division. Further RNA analyses confirmed defects in stromal rRNA maturation of Cr-kea1, paralleling observations from Arabidopsis kea1kea2 mutants. Expression of CrKEA1 in Arabidopsis rescued growth and rRNA maturation in At-kea1kea2, demonstrating functional continuity after the ancient divergence between the two lineages. Cross-species transcriptomic comparisons further revealed that IE KEA loss elicits both shared and species-specific transcriptional responses: PhANG repression was conserved between algae and plants, whereas activation of the chloroplast unfolded protein response (cpUPR) and reduced expression of genes tied to cell-cycle and plastid fission occurred only in Chlamydomonas. Single-cell time-lapse imaging confirmed that Cr-kea1 exhibits an increased frequency of aberrant cytokinesis, unequal division, and division failure. Our findings demonstrate that while IE KEA transporters fulfill conserved roles in maintaining the conditions for plastid gene expression, their integration into broader cellular networks has diverged between unicellular chlorophytes and embryophytes (land plants). This underscores a lineage-dependent tuning of plastid-nucleus communication shaped by organismal complexity and plastid number. One-sentence summaryDisruption of KEA-mediated chloroplast ion homeostasis in Chlamydomonas reinhardtii reveals conserved and lineage-specific control of plastid rRNA processing and cell cycle progression.

plant biology↗

bioRxiv2025

Catch & Release - rapid cost-effective protein purification from plants using a DIY GFP-Trap-protease approach

The purification of proteins is the foundation to study their structure, function, biochemical properties, and interaction partners. In plant research, unique challenges arise from the complexity of plant tissues, interference of secondary metabolites, and sometimes the low abundance of target proteins. Many conventional plant protein purification methods rely on expensive reagents, multi-step procedures, and labor-intensive workflows, limiting their feasibility for many applications. Here, we present the "Catch & Release" system, a cost-effective, fast and reliable one-step purification workflow for the isolation of soluble and membrane-bound proteins from plant tissues. The Catch & Release toolbox includes a vector set, a homemade GFP-trap and homemade proteases. Catch & Release vectors streamline cloning and transgenic plant selection through the Fluorescence-Accumulating Seed Technology (FAST), which marks positive transformants with a strongly fluorescing seed coat. Each plasmid consists of four, easy to exchange, modules: a plant promoter, a cloning dropout marker, protease cleavage sites, and seven different epitope tags, including an innovative dual-fluorescent tag, providing flexibility for diverse experimental needs. The in vivo functionality of all modules has been confirmed. Besides enabling standard molecular biological experimentation, our vector set in combination with homemade GFP-trap and proteases enables efficient and rapid isolation of soluble and high molecular weight membrane proteins directly from plants. By following our detailed reagent preparation instructions, purification costs can be decreased hundred-fold compared to the commercially available options.

plant biology↗

bioRxiv2025

Plant cells tolerate high rates of organellar mistranslation

Bacteria can trigger protein mistranslation to survive stress conditions 1. Mitochondria and plastids evolved from bacteria and therefore also use prokaryotic-type expression machineries to synthesize proteins. However, fungi and animal mitochondria are highly sensitive to mistranslation, which for instance manifests in lethal mitochondrial cardiomyopathy disorder 2. The response in plant cells is unknown. Glutaminyl-transferRNAs (Gln-tRNAGln) of bacteria, mitochondria, and plastids are synthesized indirectly 3,4. Initially, tRNAGln gets charged with glutamate. Subsequently, Gln is produced through trans-amidation by the aminoacyl-tRNA amido-transferase complex GatCAB. Consequentially, affected GatCAB activity results in pools of misloaded Glu-tRNAGln. Here we show that Arabidopsis mutants with decreased GatCAB level provide global insights into organellar mistranslation in plants. Proteomics revealed Gln-to-Glu misincorporation in plastid- and mitochondrially-expressed protein complexes with only modest abundance changes in mutant plants. Plastids appear more lenient to mistranslation as they exhibit much higher Gln-to-Glu misincorporation. Through efficient compensatory mechanisms, mutant plants display surprisingly subtle phenotypes. However, their acclimation to temperature stress differs. Interestingly, wild-type plants under similar stress also have altered Gln-to- Glu misincorporation. Our study shows that the response towards organellar mistranslation varies among eukaryotes. In plants, this knowledge can be used to improve stress tolerance.

plant biology↗

bioRxiv2024

Multi-way modelling of oral microbial dynamics and host-microbiome interactions during induced gingivitis

Gingivitis - the inflammation of the gums - is a reversible stage of periodontal disease. It is caused by dental plaque formation due to poor oral hygiene. However, gingivitis susceptibility involves a complex set of interactions between the oral microbiome, oral metabolome and the host. In this study, we investigated the dynamics of the oral microbiome and its interactions with the salivary metabolome during experimental gingivitis in a cohort of 41 systemically healthy participants. We use Parallel Factor Analysis (PARAFAC), which is a multi-way generalization of Principal Component Analysis (PCA) that can model the variability in the response due to subjects, variables and time. Using the modelled responses, we identified microbial subcommunities with similar dynamics that connect to the magnitude of the gingivitis response. By performing high level integration of the predicted metabolic functions of the microbiome and salivary metabolome, we identified pathways of interest that describe the changing proportions of Gram-positive and Gram-negative microbiota, variation in anaerobic bacteria, biofilm formation and virulence.

systems biology↗

bioRxiv2023

Probing the physiological role of the plastid outer-envelope membrane using the oemiR plasmid collection

Plastids are the site of complex biochemical pathways, most prominently photosynthesis. The organelle evolved through endosymbiosis with a cyanobacterium, which is exemplified by the outer envelope (OE) membrane that harbors more than 40 proteins in Arabidopsis. Their evolutionary conservation indicates high significance for plant cell function. While a few proteins are well-studied as part of the protein translocon complex the majority of OE protein (OEP) functions is unclear. Gaining a deeper functional understanding has been complicated by the lack of observable loss-of-function mutant phenotypes, which is often rooted in functional genetic redundancy. Therefore, we designed OE-specific artificial micro RNAs (oemiRs) capable of downregulating transcripts from several loci simultaneously. We successfully tested oemiR function by performing a proof-of-concept screen for pale and cold-sensitive mutants. An in-depth analysis of pale mutant alleles deficient in the translocon component TOC75 using proteomics provided new insights into putative compensatory import pathways. The cold stress screen not only recapitulated three previously known phenotypes of cold-sensitive mutants, but also identified four mutants of additional oemiR OE loci. Altogether our study revealed a role of the OE to tolerate cold conditions and showcasts the power of the oemiR collection to research the significance of OEPs.

plant biology↗

bioRxiv2021

Perception of a conserved family of plant signalling peptides by the receptor kinase HSL3

Plant genomes encode hundreds of secreted peptides; however, relatively few have been characterised. We report here an uncharacterised, stress-induced family of plant signalling peptides, which we call CTNIPs. Based on the role of the common co-receptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) in CTNIP-induced responses, we identified the orphan receptor kinase HAESA-LIKE 3 (HSL3) as the CTNIP receptor via a proteomics approach. CTNIP binding, ligand-triggered complex formation with BAK1, and induced downstream responses all involve HSL3. Notably, the HSL3-CTNIP signalling module is evolutionarily ancient, predating the divergence of extant angiosperms. The identification of this signalling module will help establish its physiological role and provides a resource to understand further receptor-ligand co-evolution.

plant biology↗

bioRxiv2020

Single-nucleus transcriptomics reveals functional compartmentalization in syncytial skeletal muscle cells

Syncytial skeletal muscle cells contain hundreds of nuclei in a shared cytoplasm. We investigated nuclear heterogeneity and transcriptional dynamics in the uninjured and regenerating muscle using single-nucleus RNA-sequencing (snRNAseq) of isolated nuclei from muscle fibers. This revealed distinct nuclear subtypes unrelated to fiber type diversity, completely novel subtypes as well as the expected ones at the neuromuscular and myotendinous junctions. In fibers of the Mdx dystrophy mouse model, new subtypes emerged, among them nuclei expressing a repair signature that were also abundant in the muscle of dystrophy patients, and a nuclear population associated with necrotic fibers. Finally, modifications of our approach revealed the compartmentalization in the rare and specialized muscle spindle. Our data identifies new nuclear compartments of the myofiber and defines a molecular roadmap for their functional analyses; the data can be freely explored on the MyoExplorer server (https://shiny.mdc-berlin.net/MyoExplorer/).

developmental biology↗

bioRxiv2019

Molecular mechanism for the recognition of sequence-divergent CIF peptides by the plant receptor kinases GSO1/SGN3 and GSO2.

Plants use leucine-rich repeat receptor kinases (LRR-RKs) to sense sequence diverse peptide hormones at the cell surface. A 3.0 [A] crystal structure of the LRR-RK GSO1/SGN3 regulating Casparian strip formation in the endodermis reveals a large spiral-shaped ectodomain. The domain provides a binding platform for 21 amino-acid CIF peptide ligands, which are tyrosine sulfated by the tyrosylprotein sulfotransferase TPST/SGN2. GSO1/SGN3 harbors a binding pocket for sulfotyrosine and makes extended backbone interactions with CIF2. Quantitative biochemical comparisons reveal that GSO1/SGN3 - CIF2 represents one of the strongest receptor-ligand pairs known in plants. Multiple missense mutations are required to block CIF2 binding in vitro, and GSO1/SGN3 function in vivo. Using structure-guided sequence analysis we uncover novel CIF peptides conserved among higher plants. Quantitative binding assays with known and novel CIFs suggest that the homologous LRR-RKs GSO1/SGN3 and GSO2 have evolved unique peptide binding properties to control different developmental processes. A quantitative biochemical interaction screen, a CIF peptide antagonist and genetic analyses together implicate SERK LRR-RKs as essential co-receptor kinases required for GSO1/SGN3 and GSO2 receptor activation. 0ur work provides a mechanistic framework for the recognition of sequence-divergent peptide hormones in plants.\n\nSignificance StatementTwo sequence-related plant membrane receptor kinases and their shape-complementary co-receptors are shown to selectively sense members of a small family of secreted peptide hormones to control formation of an important diffusion barrier in the plant root.

plant biology↗

bioRxiv2018

Mechanistic insights into the evolution of DUF26-containing proteins in land plants

Large protein families are a prominent feature of plant genomes and their size variation is a key element for adaptation in plants. Here we infer the evolutionary history of a representative protein family, the DOMAIN OF UNKNOWN FUNCTION (DUF) 26-containing proteins. The DUF26 first appeared in secreted proteins. Domain duplications and rearrangements led to the emergence of CYSTEINE-RICH RECEPTOR-LIKE PROTEIN KINASES (CRKs) and PLASMODESMATA-LOCALIZED PROTEINS (PDLPs). While the DUF26 itself is specific to land plants, structural analyses of Arabidopsis PDLP5 and PDLP8 ectodomains revealed strong similarity to fungal lectins. Therefore, we propose that DUF26-containing proteins constitute a novel group of plant carbohydrate-binding proteins. Following their appearance, CRKs expanded both through tandem duplications and preferential retention of duplicates in whole genome duplication events, whereas PDLPs evolved according to the dosage balance hypothesis. Based on our findings, we suggest that the main mechanism of expansion in new gene families is small-scale duplication, whereas genome fractionation and genetic drift after whole genome multiplications drive families towards dosage balance.

plant biology↗

bioRxiv2018

ALDH1-positive intratumoral stromal cells indicate epithelial differentiation and good prognosis in prostate cancer

Aldehyde dehydrogenase 1 (ALDH1) characterizes tumor-initiating cells in solid tumors, however little is known about its expression in intratumoral stromal cells. Herein, we aimed to dissect its potential dual relevance in prostate cancer (PCa).\n\nALDH1 expression was evaluated immunohistochemically in tumor and stromal cells in primary PCa and metastasis. It was correlated with clinico-pathological parameters, outcome of patients, and selected protein expression (CK5/6, CK14, CK8/18, CK19, EpCAM, Ki-67, E-cadherin, N-cadherin, and vimentin).\n\nALDH1 protein was detected in tumor and stromal cells in 16% and 67% of 348 primary PCa, respectively. Tumor cell ALDH1 expression was associated with advanced tumor (T) status (p=0.009), higher Gleason score (p=0.016), shorter time to biochemical recurrence (BR) (p=0.010) and CK14 expression (p=0.023). Stromal cell ALDH1 expression correlated with lower T status (p=0.008), N0 status (p=0.017), lower Gleason score (p=0.016) and longer time to BR (p=0.017). In the subgroup of dAmico high-risk patients it occurred even to be an independent predictor of good prognosis (multivariate analysis, p=0.050). ALDH1 was found in stroma of tumors characterized by CK8/18 (p=0.033) or EpCAM expression (p<0.001) and rarely by epithelial-mesenchymal transition defined as CK8/18(-)vimentin(+) phenotype (p=0.003). ALDH1 was detected in tumor cells and stroma of 33% and 41% of hormone naive lymph node metastases (n=63), 52% and 24% of castration resistant bone metastases, as well as 89% and 28% of castration resistant visceral metastases (n=21), respectively.\n\nWe have determined that contrary to tumor cell ALDH1, the presence of stromal ALDH1 is associated with a more differentiated tumor epithelial phenotype in primary PCa, improved clinical outcome, and is less frequent in PCa metastases.\n\nAbbreviations

cancer biology↗

bioRxiv2018

Somatic aberrations of BRCA1 gene are associated with progressive and stem cell-like phenotype of prostate cancer

BackgroundBRCA1 is a pivotal tumor suppressor. Its dysfunction is known to play a role in different tumor entities. Among others, BRCA1 germline mutations account for higher risk and more aggressive course of prostate cancer (PCa). In addition, somatic BRCA1 gene loss was demonstrated to be a signature of PCa dissemination to regional lymph nodes and peripheral blood, and indicate worse clinical outcome. In order to substantiate the data for BRCA1 gene loss in PCa and to reveal its phenotypical background, BRCA1 gene status was assessed in a large cohort of PCa patients and compared to different molecular factors.\n\nMethodsBRCA1 gene dosage was assessed in 2398 tumor samples from 1199 PCa patients using fluorescent in situ hybridization. It was compared to clinic-pathological parameters, patients outcome as well as selected proteins (Ki-67, apoptosis marker, cytokeratins, vimentin, E- and N-cadherin, ALDH1 and EGFR) examined by immunohistcohemistry.\n\nResultsBRCA1 losses were found in 10%, whereas gains appeared in 7% of 603 informative PCa patients. BRCA1 losses correlated to higher T status (p=0.027), Gleason score (p=0.039), shorter time to biochemical recurrence in patients with Gleason score >7 independently of other factors (multivariate analysis, p=0.005) as well as expression of proteins regulating stemness and epithelial-mesenchymal transition i.e. ALDH1 (p=0.021) and EGFR (p=0.011), respectively. BRCA1 gains correlated to shorter time to metastasis (p=0.012) and expression of ALDH1 (p=0.014).\n\nConclusionsThe presented results support the assumption that BRCA1 gene losses contribute to a progressive and stem cell-like phenotype of PCa. Furthermore, they reveal that also BRCA1 gain might mark more invasive tumors.

cancer biology↗