Biology subjects

Schwenkert, S.

Publications and source records attributed to Schwenkert, S..

14 recordsLinked to original sources

bioRxiv2026

Multi-omics dissection of transcriptional and post-transcriptional responses in cyanobacterial high-light adaptation

Understanding how photosynthetic organisms acclimate to excess light is essential for the development of robust chassis for synthetic biology approaches aimed at expanding the photosynthetically active absorption spectrum. High-light (HL) tolerant strains were previously generated by laboratory evolution from a laboratory type (LT) strain of Synechocystis sp. PCC 6803, with tolerance attributed to a small number of specific point mutations. Key mutations affected the NDH-1L complex F1-subunit (NdhF1F124L) and translation elongation factor G2 (EF-G2R461C). Reintroduction of these mutations into the LT background was sufficient to confer HL tolerance. However, the mechanisms by which a limited set of point mutations mediates HL tolerance have remained unclear. Here, integrated transcriptomic and proteomic analyses of HL-tolerant strains reveal a coordinated network of responses underlying HL tolerance. The NdhF1F124L mutation increased the accumulation of NDH-1 complex subunits, likely accounting for the previously observed enhancement of cyclic electron flow. In contrast, EF-G2R461C increased the abundance of multiple functional classes of proteins associated with HL tolerance, while post-transcriptionally reducing the level of the phycobilisome linker protein CpcC2, resulting in a decreased antenna size. Integrated analyses further demonstrated that HL tolerance involves transcriptional regulation of protein abundance, including the maintenance of phosphate metabolism. Consistently, overexpression of two genes from the Pho regulon increased HL tolerance. Overall, this study demonstrates how a small number of point mutations in genes with central cellular functions can reprogram the cyanobacterial cell to achieve enhanced tolerance to HL.

evolutionary biology↗

bioRxiv2026

RNA-dependent association of the pyruvate dehydrogenase complex with mtDNA-containing assemblies supports mitochondrial translation

Mitochondrial DNA (mtDNA) encodes core subunits of the oxidative phosphorylation machinery, and its expression is spatially organized, with the genome packaged into nucleoids around which transcription and mitoribosome assembly are concentrated. How this architecture is built, and how it is linked to the metabolic state of the organelle, remains poorly understood. Here we show that the pyruvate dehydrogenase complex (PDHc), which supplies acetyl-CoA to the tricarboxylic acid cycle, is a component of this machinery in Saccharomyces cerevisiae. PDHc co-purifies with mtDNA and resides in high-molecular-weight assemblies whose integrity requires RNA rather than DNA, and proximity labeling places it selectively adjacent to the mitoribosome. The E1 subunit Pda1 concentrates into discrete foci that depend on mtDNA and disperse reversibly upon inhibition of mitochondrial translation. Loss of Pda1, Pdb1 or Lat1, but not of the E3-binding protein Pdx1, compromises the maintenance of mtDNA; deletion of PDA1, PDB1 or LAT1 also reduces output from a mitochondrially encoded reporter, and PDHc-deficient cells are hypersensitive to translational inhibition. Catalytically inactive Pda1 and Lat1 variants rescue both defects as effectively as the wild-type proteins, demonstrating a function genetically separable from acetyl-CoA synthesis. PDHc therefore acts as a non-catalytic component of the RNA-dependent architecture that supports mitochondrial gene expression.

cell biology↗

bioRxiv2025

Role of Chloroplast Lipid-Remodelling Protein 23 During Cold Acclimation in Arabidopsis thaliana

Cold acclimation is a crucial physiological process that enables plants to adapt to low temperatures. A key aspect of this adaptation is lipid remodeling, which preserves membrane fluidity and integrity under cold stress. Proteins of the chloroplast envelope membranes are increasingly recognized for their role in acclimation to changing environmental conditions. While lipid synthesis occurs at the inner envelope membrane, little is known about specific proteins involved in lipid remodeling during cold acclimation. In this study, we investigate the role of Chloroplast Lipid Remodeling Protein 23 (CLRP23) as a component of the inner chloroplast envelope membrane. Subcellular fractionation combined with protease protection assays provided evidence for its orientation toward the intermembrane space. To explore its function, we analyzed the physiological performance and lipid composition in CLRP23-deficient mutant plants. Under cold stress, we observed significant impairments in photosynthesis and exaggerations in galactolipid response, suggesting CLRP23 is involved in lipid remodeling. Lipid overlay assays, supported by in silico docking analyses, demonstrate that CLRP23 can directly interact with chloroplast lipids, including galactolipids. Complementary transcriptomic and proteomic analyses reveal broader effects on cold-responsive pathways, supporting the view that CLRP23 contributes to the integration of membrane and metabolic responses during acclimation. These findings expand our understanding of protein-mediated processes during cold acclimation.

plant biology↗

bioRxiv2025

High-light adaptation in Synechocystis by accumulating NDH proteins and depleting specific phycobilisome linker proteins

Photosynthetic organisms have evolved mechanisms to manage excess light, crucial for maximizing photosynthetic efficiency. High-light (HL) tolerant Synechocystis sp. PCC6803 strains were developed through laboratory evolution, with tolerance attributed to specific point mutations. Key mutations affected the NDH-1L complex F1-subunit (NdhF1F124L) and translation elongation factor G2 (EF-G2R461C). Reintroducing these mutations into laboratory strains conferred HL tolerance. Comparisons with knockout and overexpressor lines showed NdhF1F124L and EF-G2R461C result in gain of function. Transcriptomic and proteomic analysis unveiled a network of responses contributing to HL tolerance, including maintenance of phosphate metabolism and decreased antenna size by depleting a specific linker protein in EF-G2R461C cells. Consequently, overexpression of Pho regulon genes increased HL tolerance. NdhF1F124L enhances cyclic electron flow (CEF) by increasing NDH-1 complex subunit accumulation. Other HL-adapted strains demonstrated that increased CEF and decreased antenna size are recurring outcomes, achievable through various mutations. This study demonstrates how limited mutations can reconfigure cells for enhanced HL tolerance, offering insights for improving photosynthetic efficiency.

physiology↗

bioRxiv2025

The evolutionary history of plastid outer envelope proteins - a structure-sequence comparison

Plant metabolism heavily relies on chloroplasts, derived from once free-living organisms. However, how two distinct organisms merged into one remains currently only partially understood. Protein-mediated metabolite exchange across the double-membrane chloroplast envelope is essential for plant cell function. Here, we investigate the evolutionary origins of outer envelope proteins (OEPs) involved in these transport processes. The mosaic nature of the nuclear genome and the deep evolutionary distance since plastid acquisition are major challenges. To address them, we combine sequence-based analyses with emerging structure-based tools, which together enable more sensitive evolutionary comparisons than traditional methods alone. To uncover distinct evolutionary trajectories, we focused on five OEP families: four {beta}-barrel proteins involved in metabolite transport and JASSY, the first published OPDA transporter. We found that the {beta}-barrel proteins were recruited in a stepwise manner and structural homologs of some OEPs point to early recruitment via endosymbiotic gene transfer (EGT). Notably, OEP40 shows a recent structural rearrangement, lacking clear structural homologs in plants other than Arabidopsis, yet retaining sequence conservation across all major land-plant lineages. The JASSY-like family is found across plastid-bearing species, while true JASSY orthologs emerged in embryophytes likely via a stable recruitment of the characteristic lipid-binding domain. Overall, our findings highlight the dynamic nature of the chloroplast outer envelope and show how new functions evolved through structural reshaping and novel domain recruitment. Structure-based approaches thus powerfully complement sequence data, offering more in-depth insight into the evolution of plastid transport systems.

evolutionary biology↗

bioRxiv2025

Expression levels of the Band-7 protein FLOTILLIN modulate salt tolerance, growth and development in the moss Physcomitrium patens

The Band-7 proteins, known as FLOTILLINs (FLOT), are present at the plasma membranes of most land plants. They function in clathrin-independent endocytosis and contribute to nodule formation following symbiotic infections. This study reveals that the single FLOT variant in Physcomitrium patens is located at the thylakoid membranes in chloroplasts, serving an unanticipated function. Phenotypic analysis of knockout and overexpression lines demonstrates that PpFLOT overexpression significantly impairs the high salinity tolerance of P. patens. Additionally, liquid protonema cultures of PpFLOT-OEX lines exhibited a distinct color change due to necrotic events and developed brachycyte-like cells. These changes correlate with the strength of PpFLOT expression and do not occur when these lines are cultivated on solid medium. Our study found that PpFLOT-OEX lines display increased chlorophyll and H2O2 production. We also discovered that PpFLOT is regulated by ABA and light, and its high expression can potentially affect retrograde signaling. Metabolomics and proteomics analyses revealed changes in the pigment and lipid composition as well as differentially accumulated proteins in PpFLOT mutant lines. We also observed changes in the expression of ion-transport related genes, accumulation of lipids crucial during pathogen defense, and differentially accumulated proteins taking part in multiple metabolomic pathways. Consequently, our study suggests a novel role for chloroplastic PpFLOT in plant terrestrialization, as it is putatively involved in Ca2+ and reactive oxygen species (ROS) signaling in response to abiotic and biotic stress, along with the light-dependent regulation of chlorophyll biosynthesis.

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↗

bioRxiv2025

Mrx6 binds the Lon protease Pim1 N-terminal domain to confer selective substrate specificity and regulate mtDNA copy number

Mitochondrial DNA (mtDNA) copy number regulation remains incompletely understood, despite its importance in cellular function. In Saccharomyces cerevisiae, Mrx6 belongs to the Pet20-domain-containing protein family, consisting of Mrx6, Pet20, and Sue1. Notably, absence of Mrx6 leads to increased mtDNA copy number. Here, we identify the C-terminus of Mrx6 as essential for its stability and interaction with the mitochondrial matrix protein Mam33. Deletion of Mam33 mimics the effect of Mrx6 loss, resulting in elevated mtDNA copy number. Bioinformatics, mutational analyses, and immunoprecipitation studies reveal that a subcomplex of Mam33 and Mrx6 trimers interacts with the substrate recognition domain of the conserved mitochondrial Lon protease Pim1 through a bipartite motif in the Pet20 domain of Mrx6. Loss of Mrx6, its paralog Pet20, Mam33, or mutations disrupting the interaction between Mrx6 and Pim1 stabilize key proteins required for mtDNA maintenance, the RNA polymerase Rpo41 and the HMG-box-containing protein Cim1. We propose that Mrx6, alongside Pet20 and Mam33, regulates mtDNA copy number by modulating substrate degradation through Pim1. Additionally, Mrx6 loss alters Cim1s function, preventing the detrimental effect on mtDNA maintenance observed upon Cim1 overexpression. The presence of three Pet20-domain proteins in yeast implies broader roles of Lon protease substrate recognition beyond mtDNA regulation.

cell biology↗

bioRxiv2024

Delayed protein translocation protects mitochondria against toxic CAT-tailed proteins

Ribosome-associated quality control (RQC) protects cells against toxic effects of faulty polypeptides produced by stalled ribosomes. However, mitochondria are vulnerable to C-terminal alanyl-and-threonyl (CAT)-tailed proteins that are generated in this process and faulty nuclear-encoded mitochondrial proteins are handled by the recently discovered mitoRQC. Here, we performed a genome-wide screen in yeast to identify additional proteins involved in mitoRQC. We found that Pth2, a peptidyl-tRNA hydrolase in the mitochondrial outer membrane, influences aggregation of CAT-tailed proteins without majorly affecting the CAT-tailing process itself. Peptidyl-tRNA hydrolase activity is essential during this process, yet the activity of Pth2 can be substituted by another peptidyl-tRNA hydrolase, upon proper localization. Our data suggest that Pth2 acts through modulating protein translocation and that mitochondrial proteostasis network is relieved through increased access of CAT-tailed proteins to cytosolic chaperones. Other hits obtained in the screen show that, in general, delayed protein translocation protects mitochondria against toxic CAT-tailed proteins.

cell biology↗

bioRxiv2024

Regulation of plant metabolism under elevated CO2

Plant responses to changing environments afford complex regulation at transcriptome and proteome level to maintain metabolic homeostasis. Homeostasis itself constitutes a complex and dynamic equilibrium of metabolic reactions and transport processes among cellular compartments. In the present study, we aimed at the highest possible resolution of this network by combining analysis of transcriptome, proteome and subcellular resolved metabolome of plants exposed to rising carbon dioxide concentrations over a time course of one week. To prove suitability of our approach, we included mutants affected in photorespiratory metabolism and, thus, should deviate from the wildtype in their response to elevated CO2. Our multi-omics analysis revealed that the hpr1-1 mutant, defective in peroxisomal hydroxypyruvate reduction, is also affected in cytosolic pyruvate metabolism, reaching out to cysteine synthesis, while the hexokinase mutant hxk1 displays a disturbed redox balance upon changing CO2 levels. For the third mutant, defective in the mitochondrial protein BOU, we found compelling evidence that the function of this transporter is related to lipoic acid metabolism, thus challenging current interpretations. This demonstrates that the combined omics approach introduced here opens new insights into complex metabolic interaction of pathways shared among different cellular compartments.

plant biology↗

bioRxiv2024

Flavonoid metabolism and its different physiological roles in plant cold acclimation

Flavonoids represent a diverse group of plant secondary metabolites which are also discussed in context of dietary health and inflammatory response. Numerous studies have revealed that flavonoids play a central role in plant acclimation to abiotic factors like low temperature or high light, but their structural and functional diversity frequently prevents a detailed mechanistic understanding. Further complexity in analysing flavonoid metabolism arises from the different subcellular compartments which are involved in biosynthesis and storage. In the present study, non-aqueous fractionation of Arabidopsis leaf tissue was combined with metabolomics and proteomics analysis to reveal subcellular regulation during cold acclimation in two flavonoid-deficient mutants. During the first three days of a two-week cold acclimation period, flavonoid deficiency was observed to affect pyruvate, citrate and glutamate metabolism which indicated a role in stabilising C/N metabolism and photosynthesis. Also, tetrahydrofolate metabolism was found to be affected which had significant effects on regulation of the proteome of the photorespiratory pathway. In the late stage of cold acclimation, flavonoid deficiency was found to affect protein stability, folding and proteasomal degradation which resulted in a significant decrease of total protein amounts in both mutants. In summary, these findings suggest that flavonoid metabolism plays different roles in early and late stages of plant cold acclimation and significantly contributes to establishing a new protein homeostasis in a changing environment.

plant biology↗

bioRxiv2023

UPP affects chloroplast development by interfering with chloroplast proteostasis

Arabidopsis uracil phosphoribosyltransferase (UPP) is an essential enzyme which appears to have a previously unknown, moonlighting activity. Our analysis of UPP amiRNA mutants has confirmed that this vital function is crucial for chloroplast development and growth. Interestingly, this function appears to be unrelated to nucleotide homeostasis since nucleotide levels were not altered in the studied mutants. Transcriptomics and proteomic analysis suggest that UPP plays a role in chloroplast proteostasis, especially under high light (HL). Immunoblots of mature plants and a de-etiolation experiment with young seedlings revealed PetC, the iron-sulfur protein of the cytochrome b6f complex, as a putative UPP target. In addition, UPP and PetC were identified in a high molecular weight complex. Consistently, we show that PetC is massively reduced in UPP knock-down plants. The block observed in photosynthetic electron transport, as evidenced by reduced high light-induced non-photochemical quenching (NPQ) but increased unregulated energy dissipation (NO), might be therefore a consequence of reduced PetC. After HL treatment, UPP amiRNA mutants showed impaired photosynthesis and reduced carbohydrate contents, resulting in an inability to induce flavonoid biosynthesis. In addition, the levels of the osmoprotectants raffinose, proline and fumarate were found to be reduced. Proteases, including thylakoid filamentation temperature-sensitive 1, 5 (FtsH), caseinolytic protease proteolytic subunit 1 (ClpP1), and processing peptidases, as well as components of the chloroplast protein import machinery, were up-regulated. In sum, our work suggests that UPP assists in stabilizing and protection of PetC during the assembly of the Fe-S cluster and targeting to the thylakoid.

molecular 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

The Arabidopsis AAC Proteins CIL and CIA2 Are Sub-functionalized Paralogs involved in Chloroplast Development

The Arabidopsis gene Chloroplast Import Apparatus 2 (CIA2) encodes a transcription factor that positively affects the activity of nuclear genes for chloroplast ribosomal proteins and chloroplast protein import machineries. CIA2-like (CIL) is the paralogous gene of CIA2. We generated a cil mutant by site-directed mutagenesis and compared it with cia2 and cia2cil double mutant. Phenotype of the cil mutant did not differ from the wild type under our growth conditions, except faster growth and earlier time to flowering. Compared to cia2, the cia2cil mutant showed more impaired chloroplast functions and reduced amounts of plastid ribosomal RNAs. In silico analyses predict for CIA2 and CIL a C-terminal CCT domain and an N-terminal chloroplast transit peptide (cTP). Chloroplast (and potentially nuclear) localization was previously shown for HvCMF3 and HvCMF7, the homologs of CIA2 and CIL in barley. We observed nuclear localization of CIL after transient expression in Arabidopsis protoplasts. Surprisingly, transformation of cia2 with HvCMF3, HvCMF7 or with a truncated CIA2 lacking the predicted cTP could partially rescue the pale-green phenotype of cia2. These data are discussed with respect to potentially overlapping functions between CIA2, CIL and their barley homologs and to the function of the putative cTPs of CIA2 and CIL. HIGHLIGHTThe nucleus-localized CCT domain proteins CIA2 and CIL in Arabidopsis and the homologous chloroplast-localized HvCMF3 and HvCMF7 in barley retained partially overlapping functions in chloroplast development.

plant biology↗