Biology subjects

Eirich, J.

Publications and source records attributed to Eirich, J..

14 recordsLinked to original sources

bioRxiv2025

HDA19-mediated deacetylation of histone H3.3 lysine 27 and 36 regulates plant sensitivity to salt stress

Plants survive extreme environments through rapid chromatin reprogramming, yet the epigenetic marks that confer stress resilience remain poorly understood. Histone deacetylase HDA19 is a key epigenetic regulator in Arabidopsis, and hda19-deficient mutants display tolerance to multiple abiotic stresses, including drought, heat, and salinity. Using lysine acetylome profiling, we identified a non-canonical K27/K36 di-acetylation mark on histone H3.3, among nine H3 variants, as a specific substrate of HDA19. Under salinity stress, this mark decreased in wild-type plants but increased in hda19 mutants, while other known H3 modifications were similarly affected in both genotypes. Mimicking constitutive di-acetylation of H3.3 K27/K36 through lysine-to-glutamine substitutions promoted accumulation of stress-responsive late embryogenesis abundant (LEA) proteins and conferred salinity tolerance in seedlings, phenocopying hda19 mutants. Furthermore, generating the lea7-1/lea29-1/rab18-1 triple mutant abolished hda19-dependent salinity tolerance, confirming the LEA proteins role downstream of HDA19. Our findings demonstrate that H3.3 K27/K36 di-acetylation, modulated by HDA19, drives LEA protein accumulation and enables plants to withstand environmental stress, revealing a previously unknown core mechanism of plant stress resilience. Significance statementHistone acetylation is crucial for regulating chromatin states and transcription, yet the specific histone codes controlled by histone deacetylases and acetyltransferases during plant stress acclimation remain unclear. Arabidopsis mutants lacking histone deacetylase 19 show enhanced tolerance to drought, heat, and salinity. Using acetylome profiling and genetic analyses, we identify a previously unknown di-acetylation of histone H3.3 at lysines 27 and 36 as a key epigenetic switch activating late embryogenesis abundant proteins essential for salt tolerance in seedlings. This discovery reveals a novel mechanism linking histone H3.3 K27/K36 di-acetylation to stress-resilient protein accumulation, offering new insights into epigenetic control of plant stress responses and informing strategies to improve crop resilience under climate change.

plant biology↗

bioRxiv2025

Arabidopsis root lipid droplets are hubs for membrane homeostasis under heat stress, and triterpenoid synthesis and storage.

O_LIPlant lipid droplets (LDs) and their associated proteins have numerous subcellular and physiological functions. While considerable progress has been made for LDs in many tissues, the function and composition of LDs in roots remains largely unexplored. C_LIO_LIWe investigated the changes of the number of LDs and of the lipidome in heat-stressed Arabidopsis thaliana roots. Furthermore, we isolated root LDs from the Arabidopsis mutant trigalactosyldiacylglycerol 1-1 sugar dependent 1-4 and investigated their proteome and lipidome. C_LIO_LIHeat stress lead to a degradation of membrane lipids and an increase in TAGs and LDs. while, fatty acid SEs decreased, probably acting as precursors for acylated sterol glycosides. A variety of proteins were enriched in root LDs, which are thus far not described as LD proteins. Transient expression of these proteins in many cases confirmed their LD localization, for example of the triterpene biosynthetic enzymes thalianol synthase and marneral synthase. We could furthermore show that the educts and products of these enzymes are enriched in root LDs, too. C_LIO_LIWe conclude that root LDs simultaneously act as a sink and source during heat stress-induced membrane remodeling. Furthermore, root LDs play a pivotal role in triperpene synthesis and storage, thereby highlighting LDs as hubs in specialized metabolism. C_LI

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

Immune priming in the insect gut: a dynamic response revealed by ultrastructural and transcriptomic changes

Research on forms of memory in innate immune systems has recently gained momentum with the study of trained immunity in vertebrates and immune priming in invertebrates. Immune priming provides protection against previously encountered pathogens. However, causes and mechanisms of immune priming are still not well understood in most organisms. In this work, we combine RNA sequencing with transmission electron microscopy to investigate the dynamic processes during priming in the gut of a well-established model for oral immune priming, consisting of the host Tribolium castaneum and its entomopathogen Bacillus thuringiensis tenebrionis (Btt). We show that priming with specific, pathogen-derived virulence-relevant factors causes damage in the gut of T. castaneum larvae, which leads to an early physiological stress response as well as the upregulation of a specific set of immune genes. This response diminishes over time yet enables the gut to upregulate genes known to interfere with Btt virulence when T. castaneum larvae are later exposed to infectious Btt spores. The identification of these processes increases our understanding of immune priming as a dynamic process where cellular responses in concert with specific gene regulation prepare the gut tissue and thereby enable more efficient protection against infection. Such work can further help us understand the origin and mechanism of innate immune memory. Author summaryInvertebrate animals do not possess a classical adaptive immune system. And yet, many of these animals show forms of immune memory, collectively called immune priming. In this work, we investigate the causes of oral immune priming in the red flour beetle Tribolium castaneum in response to its bacterial entomopathogen Bacillus thuringiensis tenebrionis. Primary exposure to Btt spent culture media supernatants enables the larvae of T. castaneum to better withstand a subsequent infection with infectious Btt spores. We show that exposing T. castaneum larvae to those supernatants leads to damage in the gut resulting in a strong stress and immune response early after priming and a targeted up-regulation of beneficial genes upon the secondary re-encounter with Btt. These results suggest fundamental stress responses can be involved in innate immune memory phenomena, which has implications in translational research across species.

evolutionary biology↗

bioRxiv2024

Regulation of Crassulacean Acid Metabolism at the protein level in the CAM plant Kalanchoë laxiflora

Crassulacean acid metabolism (CAM) is an adaptation to environments where water availability is seasonal or extremely low. It serves to ensure survival and/ or maintain productivity in these adverse environments. CAM has repeatedly evolved although it requires a large and complex set of enzymes and transporters and regulatory processes to control metabolite flux and pools. To test potential regulatory levels, we analyze the CAM plant Kalanchoe laxiflora embedded in the context of available CAM genome and transcriptome sequences. We show that CAM associated transcripts and proteins do not show a binary on/off pattern between day and night in K. laxiflora. Instead, we observe that many CAM plants display shared amino acid changes compared to C3 plants especially in starch metabolism. Phosphoproteomics identifies phosphoproteome changes in K. laxiflora between day and night. Taken together, the analyses demonstrate the CAM photosynthesis is regulated at the levels of transcripts and proteins. One sentence summaryRegulation of CAM cannot be explained by transcript and protein abundance alone but is also dependent on adaptive changes in proteins and posttranslational modifications.

plant biology↗

bioRxiv2024

DOG1 prevents AFPs activation by AHG1 to control dormancy separately from ABA core signaling

Seed dormancy determines germination timing and thereby critically influences seed plant adaptation and overall fitness. DELAY OF GERMINATION1 (DOG1) is a conserved central regulator of dormancy acting in concert with the phytohormone abscisic acid (ABA) through negative regulation of ABA HYPERSENSITIVE GERMINATION (AHG) 1 and AHG3 phosphatases. The current molecular mechanism of DOG1 signaling proposes that it regulates the activation state of central ABA-related SnRK2 kinases. Here, we unveil DOG1s functional autonomy from the regulation of ABA core signaling components and unravel its pivotal control over the activation of ABSCISIC ACID INSENSITIVE FIVE BINDING PROTEINs (AFPs). Our data revealed a DOG1-AHG1-AFP relay in which AFPs genuine activation by AHG1 is contained by DOG1 to prevent the breakdown of maturation-imposed ABA responses independently of ABA-related kinase activation status. This work offers a molecular understanding of how plants fine-tune germination timing, while preserving seed responsiveness to adverse environmental cues, and thus represents a milestone in the realm of conservation and breeding programs. One-Sentence SummaryAutonomous control of maturation-imposed ABA responses by DOG1 enables seeds to regulate dormancy and stress-reactivity traits independently.

plant biology↗

bioRxiv2024

The major nucleoid-associated protein WHIRLY1 promotes chloroplast development in barley

WHIRLY1 is a DNA-binding protein of high abundance in chloroplast nucleoids, which have a complex proteome consisting of proteins involved in gene expression and unexpected proteins indicating links to energy production and biosynthetic activities of chloroplasts. In addition, WHIRLY1 has a second localization in the nucleus making it an excellent candidate for chloroplast-to-nucleus communication. To unravel the role of WHIRLY1 for structure and protein composition of nucleoids and its potential involvement in retrograde signaling during chloroplast development, knockout mutants of HvWHIRLY1 were prepared by site-directed mutagenesis using Cas9 endonuclease. In contrast to mutants of rice and maize, which die after the seedling stage, the barley why1 mutants survive and produce grains. Leaves of the mutants are initially pale and get green with time (xantha-to-green phenotype). However, the chlorophyll content of primary leaves stayed distinctly lower than that of the wild-type leaves, coinciding with a rather heterogeneous plastid population, whereby only 50% developed a rather normal thylakoid membrane system. For comparison, mature foliage leaves had almost normal levels of chlorophyll but a severely reduced photosynthetic capacity. A proteome analysis of chloroplasts isolated from mature foliage leaves revealed that in the absence of WHIRLY1, the abundances of a considerable fraction of proteins were downregulated. The fraction included multiple nucleoid-associated proteins including components of the transcriptional apparatus. Furthermore, ribosomal proteins, subunits of pyruvate dehydrogenase, CLP protease, ATP synthase, Rubisco and chaperons/chaperonins were found to be downregulated. In conclusion, the characterization of the barley why1 mutant plants revealed that WHIRLY1 is not essential for chloroplast development. Rather, it ensures a fast and failure-free progression of chloroplast development by remodeling nucleoids, which serve as assembly platforms for a concerted workflow of the numerous processes required for chloroplast development. Gene expression analyses revealed that the disturbance of chloroplast development is signaled to the nucleus, indicating that WHIRLY1 is not part of the biogenic retrograde signaling of plastids.

plant biology↗

bioRxiv2023

Proteoform-level deconvolution reveals a broader spectrum of ibrutinib off-targets

Over the last decade, proteome-wide mapping of drug interactions has revealed that most targeted drugs bind to not only their intended targets, but additional proteins as well. However, the majority of these studies have focused on analyzing proteins as encoded by their genes, thus neglecting the fact that most proteins exist as dynamic populations of multiple proteoforms. Here, we addressed this problem by combining the use of thermal proteome profiling (TPP), a powerful method for proteome analysis, with proteoform detection to refine the target landscape of an approved drug, ibrutinib. We revealed that, in addition to known targets, ibrutinib exhibits an intricate network of interactions involving multiple different proteoforms. Notably, we discovered affinity for specific proteoforms that link ibrutinib to mechanisms in immunomodulation and cellular processes like Golgi trafficking, endosomal trafficking, and glycosylation. These insights provide a framework for interpreting clinically observed off-target and adverse events. More generally, our findings highlight the importance of proteoform-level deconvolution in understanding drug interactions and their functional impacts, and offer a critical perspective for drug mechanism studies and potential applications in precision medicine.

molecular biology↗

bioRxiv2023

A large-scale sORF screen identifies putative microproteins and provides insights into their interaction partners, localisation and function

The human genome contains thousands of potentially coding short open reading frames (sORFs). A growing set of microproteins translated from these sORFs are known to have important cellular functions. However, the majority remains uncharacterised. Thus, larger screens to find functional microproteins have become more vital. Here, we performed a high-throughput CRISPR/Cas9 knock-out screen with a customised library of 11,776 sORFs, curated from literature and databases to identify microproteins essential for cancer cell line growth. 16/17 tested candidates displayed a reproducible knockout phenotype. We selected our top six hits, consisting of 11 to 63 amino acids. Various of these candidates localised to distinct subcellular compartments and the majority showed specific interaction partners. Endogenous tagging demonstrated translation of an sORF in the CENPBD2P pseudogene that bears no resemblance to the CENPBD2P name-giving CENPB DNA binding domains. For two candidates, uORFs in the DSE and NUTF2 genes, the microprotein supplied in trans ameliorated the growth defect of the respective knock-out. RNA-seq analysis revealed however that gene expression changes in the knock-out could only partially be rescued. Overall, we identified various putative microproteins and a microprotein-producing pseudogene that might be involved in cancer cell growth, but also illustrate the limitations and caveats of sORF functional screening and characterisation.

molecular biology↗

bioRxiv2023

A eukaryote-specific factor mediates an early step in the assembly of plant photosystem II

ABSTRACTThe initial step of oxygenic photosynthesis is the thermodynamically challenging extraction of electrons from water and the release of molecular oxygen. This light-driven process, which is the basis of life on Earth, is catalyzed by the photosystem II (PSII) within the thylakoid membrane of photosynthetic organisms. The biogenesis of PSII requires a controlled step-wise assembly process of which the early steps are considered to be highly conserved between plants and their cyanobacterial progenitors. This assembly process involves auxiliary proteins, which are likewise conserved. In the present work, we show that in plants, the early assembly step, in which the PSII reaction center (RC) is associated with the intrinsic antenna protein CP47 to form the RC47 intermediate, is facilitated by a novel eukaryote-exclusive assembly factor. This factor, we named DEAP2 for DECREASED ELECTRON TRANSPORT AT PSII, works in concert with the conserved PAM68 assembly factor. The deap2 and pam68 mutants showed similar defects in PSII accumulation and assembly of the RC47 intermediate. The combined lack of both proteins results in a loss of functional PSII and the inability of plants to grow photoautotrophically on soil. While overexpression of DEAP2 partially rescued the pam68 PSII accumulation phenotype, this effect was not reciprocal. DEAP2 accumulates at 20-fold higher levels than PAM68, together suggesting that both proteins have distinct functions. In summary, our results uncover eukaryotic adjustments to the PSII assembly process, which involve the addition of DEAP2 for the rapid progression from RC to RC47.

plant biology↗

bioRxiv2023

A bi-kinase module sensitizes and potentiates plant immune signaling

Systemic signaling is an essential hallmark of multicellular life. Pathogen encounter occurs locally but triggers organ-scale and organismic immune responses. In plants, elicitor perception provokes systemically expanding Ca2+ and H2O2 signals conferring immunity. Here, we identify a Ca2+ sensing bi-kinase module as becoming super-activated through mutual trans-phosphorylation and imposing synergistically enhanced NADPH oxidase activation. A combined two-layer bi-kinase/substrate phospho-code allows for sensitized signaling initiation already by near-resting elevations of Ca2+ concentration at the infection site. Subsequently, it facilitates further signal wave proliferation with minimal amplitude requirement, triggering protective defense responses throughout the plant. Our study reveals how plants build and perpetuate trans-cellular immune signal proliferation while avoiding disturbance of ongoing cellular signaling along the path of response dissemination. One sentence summaryMutual trans-activation of a Ca2+ sensing bi-kinase module potentiates NADPH oxidase activation to facilitate systemic immunity.

plant biology↗

bioRxiv2021

A versatile mitochondria isolation- and analysis-pipeline generates 3D nano-topographies and mechano-physical surface maps of single organelles

Living eukaryotic cells typically contain large quantities of highly dynamic mitochondria, which sustain the cells energy and redox homeostasis. Growing evidence suggests that mitochondria can functionally differ among but also within cells. The extent and biological significance of mitochondrial diversity is still largely unexplored, due to technical limitations that hamper profiling of individual organelles. Previous measurements of the cells interior have shown that membrane-bound compartments respond to metabolic manipulation by changes in their surface stiffness, suggesting that mechano-physical properties are a valuable readout of mitochondrial function. We here present the establishment of a robust multi-step analysis pipeline that allows one to profile mechano-physical properties of single mitochondria at the nanoscale using Atomic Force Microscopy (AFM). Firstly, we developed a rapid cell-type specific isolation protocol (mRACE), which selectively functionalizes mitochondria with biotin, facilitating isolation by streptavidin decorated microbeads. We established the technique for human and rat cell cultures, the invertebrate Caenorhabditis elegans, and the model plant Arabidopsis thaliana. Based on this versatile tool, we detected diversity of mitochondrially associated proteins among different tissues, reflecting the trophic condition of the source material. Secondly, a rapid filtration-based mitochondria isolation protocol was established, which was combined with mRACE. Lastly, we established an AFM analysis platform, which generates 3D maps of the nano-topography and mechano-physical properties of individual mitochondria. The comparison of mitochondria with each other revealed an unprecedented diversity in their mechano-physical properties and suggests that shape is not the sole determining parameter for outer membrane stiffness. We expect our results to not only introduce a new dimension for basic mitochondrial research, but in addition to open the door for the exploitation of individual mitochondria for diagnostic characterization.

plant biology↗

bioRxiv2021

Inhibition of the ubiquitin-proteasome system by a bioactivatable compound

Malignant cells display an increased sensitivity towards drugs that reduce the function of the ubiquitin-proteasome system (UPS), which is the primary proteolytic system for destruction of aberrant proteins. Here, we report on the discovery of the bioactivatable compound CBK77, which causes an irreversible collapse of the UPS, accompanied by a general accumulation of ubiquitylated proteins and caspase-dependent cell death. CBK77 caused accumulation of ubiquitin-dependent, but not ubiquitin-independent, reporter substrates of the UPS, suggesting a selective effect on ubiquitin-dependent proteolysis. In a genome-wide CRISPR interference screen, we identified the redox enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a critical mediator of CBK77 activity, and further demonstrated its role as the compound bioactivator. Through affinity-based proteomics, we found that CBK77 covalently interacts with ubiquitin. In vitro experiments showed that CBK77-treated ubiquitin conjugates were less susceptible to disassembly by deubiquitylating enzymes. In vivo efficacy of CBK77 was validated by reduced growth of NQO1-proficient human adenocarcinoma cells in nude mice treated with CBK77. This first-in-class NQO1-activatable UPS inhibitor suggests that it may be possible to exploit the intracellular environment in malignant cells for leveraging the impact of compounds that impair the UPS.

cell biology↗

bioRxiv2018

Metal-independent ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens

Ribonucleotide reductase (RNR) catalyzes the only known de-novo pathway for production of all four deoxyribonucleotides required for DNA synthesis. In aerobic RNRs, a di-nuclear metal site is viewed as an absolute requirement for generating and stabilizing an essential catalytic radical. Here we describe a new group of RNRs found in Mollicutes, including Mycoplasma pathogens, that possesses a metal-independent stable radical residing on a modified tyrosyl residue. Structural, biochemical and spectroscopic characterization reveal a stable DOPA radical species that directly supports ribonucleotide reduction in vitro and in vivo.\n\nThe cofactor synthesis and radical generation processes are fundamentally different from established RNRs and require the flavoprotein NrdI. Several of the pathogens encoding this RNR variant are involved in diseases of the urinary tract and genitalia. Conceivably, this remarkable RNR variant provides an advantage under metal starvation induced by the immune system. We propose that the new RNR subclass is denoted class Ie.

biochemistry↗