Biology subjects

Engstler, C.

Publications and source records attributed to Engstler, C..

3 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

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↗