Biology subjects

Hoehenwarter, W.

Publications and source records attributed to Hoehenwarter, W..

11 recordsLinked to original sources

bioRxiv2026

Dynamic Patterns of Nuclear Transcription Factor Abundance in Plant Basal Immunity Revealed by Spatial Proteomics of Arabidopsis Nuclei

O_LIThe control of amount of nuclear proteins is fundamental in regulating plant gene expression, but the mechanisms of quantitative dynamics of the nuclear proteome are largely unstudied during adaptive responses to pathogens. C_LIO_LIHighly specific labeling, enrichment and measurement of the nuclear proteome was performed using TurboID LC-MS of Arabidopsis thaliana leaves treated with the pathogen-associated molecular pattern (PAMP), flg22, and/or cycloheximide. The chosen experimental approach allowed discrimination of the effects of translation, nuclear protein import, trafficking of preexisting proteins, derepression, and nuclear protein turn-over upon elicitation of basal immunity. C_LIO_LIThe highly specific, deep coverage of proteins in the nucleus makes this study a resource for anyone interested in plant nuclear proteome dynamics and defense. C_LIO_LIAround 2,000 nuclear proteins were repeatedly quantified, including more than 300 transcription factors or other proteins related to transcription. Several proteins with documented activity in endosomes were newly synthesized and imported into nuclei upon PAMP challenge, suggesting alternative nuclear functions in PAMP-triggered immunity (PTI). Circadian clock components, including the transcription factor, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)-HIKING EXPEDITION (CHE), were depleted upon PAMP challenge, suggesting a safeguard against untimely induction of systemic acquired resistance (SAR). C_LIO_LIBased on proteomic patterns, proteins moonlighting in the nucleus as well as trafficking and turn-over regulation of the proteome are common elements during plant immunity. C_LI

plant biology↗

bioRxiv2025

CENTRA: Knowledge-Based Gene Contexuality Graphs Reveal Functional Master Regulators by Centrality and Fractality

Deciphering gene function via context-aware approaches is limited by various means. Especially static gene sets used in enrichment analyses and the lack of single-gene resolution in such analyses restrains the flexible association of genes with specific context. Here, we introduce CENTRA (Centrality-based Exploration of Network Topologies from Regulatory Assemblies), a framework that models gene contextuality through topic-specific gene co-occurrence networks derived from curated gene sets and associated literature. Using Latent Dirichlet Allocation on 12,045 abstracts linked to MSigDB C2 gene sets, we uncovered 27 biological topics and constructed corresponding topic-specific networks that reflect distinct biological states, perturbation conditions, and disease-related regulatory programs. Graph-topological metrics, including centrality, local fractality, and perturbation sensitivity, were computed for each gene to capture structural relevance within these topic-specific contexts. We demonstrate that topological profiles distinguish well-characterized regulators, identify emerging functional candidates, and reveal context-specific roles. Thereby, our framework enables the prioritization of understudied genes by assessing the robustness of their topological signatures across topic-specific networks. To support exploration of these results, we developed a publicly accessible interactive browser application, CENTRA, which enables dynamic navigation of networks and their functional annotations. CENTRA provides an interpretable, scalable framework for investigating context-dependent gene function and hypothesis generation, offering a novel entry point beyond traditional enrichment approaches. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/662180v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@10e4eeorg.highwire.dtl.DTLVardef@125d5b4org.highwire.dtl.DTLVardef@f137aeorg.highwire.dtl.DTLVardef@7ea4e5_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2023

Changes in Protein Turn-over Rates Regulate Tryptophan and Glucosinolate Biosynthesis, IAA Transport and Photosynthesis in Arabidopsis Growth and Defense Transitions

An organism continuously experiences shifts in biological states necessitating extensive rearrangement of physiology and molecular order of the cell. Here we model transitions between optimal growth conditions (homeostasis), fully induced pattern triggered immunity (PTI) and back in Arabidopsis thaliana, chronologically measuring changes in protein synthesis and degradation rates, transcript, protein and phytohormone abundance of 99 targets using qPCR and LC-MS parallel reaction monitoring (PRM). Temporally changing synthesis and degradation rates were primary determinants of abundance, next to changes in mRNA levels, of tryptophan, glucosinolate (GS) biosynthesis and photosynthesis associated (PAP) proteins particularly in the earlier establisher phases but also in fully induced PTI. While transcripts returned to growth levels 3 to 16 hours post elicitation, protein levels remained at fully induced PTI levels up to 16 hours into the transitory phase back to optimal growth. A notable exception were polar auxin transporters PIN3 and PIN7 levels which decreased in PTI but quickly returned to initial homeostasis levels after transition, although global auxin levels only decreased by 20%. Tryptophan, GS and JA biosynthesis proteins all increased in abundance in the wild type and the myc234 mutant background linking induction of the tryptophan and GS biosynthesis pathways to flg22 treatment and PTI independent of MYC2 and homologs. PAPs abundance was depleted in fully induced PTI however not in the myc234 mutant linking this active immune response to these bHLH transcription factors. FERREDOXIN-NADP(+)-OXIDOREDUCTASE (FNR1) synthesis rates decreased while its degradation rate increased in the earlier PTI establisher phase. FNR1 is the penultimate protein in the photosynthetic electron transfer chain and imparts electrons onto NADP+ however in its absence electrons are used for oxygen photoreduction and H2O2 production, an active defense compound. Thus FNR1 may be a molecular switch that switches photosystem activity between growth and defense under post-transcriptional control. The myc234 mutation generally led to delayed changes in transcript and protein abundance and also abolished IAA depletion. Protein turn-over rates of a set of PAPs were affected in the mutant suggesting a possible positive role of the transcription factors in controlling post-transcriptional regulatory processes in PTI induction.

plant biology↗

bioRxiv2021

Bacterial-type plant ferroxidases tune local phosphate sensing in root development

Fluctuating bioavailability of inorganic phosphate (Pi), often caused by complex Pi-metal interactions, guide root tip growth and root system architecture for maximizing the foraged soil volume. Two interacting genes in Arabidopsis thaliana, PDR2 (P5-type ATPase) and LPR1 (multicopper oxidase), are central to external Pi monitoring by root tips, which is modified by iron (Fe) co-occurrence. Upon Pi deficiency, the PDR2-LPR1 module facilitates cell type-specific Fe accumulation and cell wall modifications in root meristems, inhibiting intercellular communication and thus root growth. LPR1 executes local Pi sensing, whereas PDR2 restricts LPR1 function. We show that native LPR1 displays specific ferroxidase activity and requires a conserved acidic triad motif for high-affinity Fe2+ binding and root growth inhibition under limiting Pi. Our data indicate that substrate availability tunes LPR1 function and implicate PDR2 in maintaining Fe homeostasis. LPR1 represents the prototype of an ancient ferroxidase family, which evolved very early upon bacterial colonization of land. During plant terrestrialization, horizontal gene transfer transmitted LPR1-type ferroxidase from soil bacteria to the common ancestor of Zygnematophyceae algae and embryophytes, a hypothesis supported by homology modeling, phylogenomics, and activity assays of bacterial LPR1-type multicopper oxidases.

plant biology↗

bioRxiv2021

LC-MS Based Draft Map of the Arabidopsis thaliana Nuclear Proteome and Protein Import in Pattern Triggered Immunity

Despite its central role as the ark of genetic information and gene expression the plant nucleus is surprisingly understudied. We isolated nuclei from the Arabidopsis thaliana dark grown cell culture left untreated and treated with flg22 and nlp20, two elicitors of pattern triggered immunity (PTI) in plants, respectively. An LC-MS based discovery proteomics approach was used to measure the nuclear proteome fractions. An enrichment score based on the relative abundance of cytoplasmic, mitochondrial and golgi markers in the nuclear protein fraction allowed us to curate the nuclear proteome producing high quality catalogs of around 3,000 nuclear proteins under untreated and both PTI conditions. The measurements also covered low abundant proteins including more than 100 transcription factors and transcriptional co-activators. Protein import into the nucleus in plant immunity is known. Here we sought to gain a broader impression of this phenomenon employing our proteomics data and found 157 and 73 proteins to be putatively imported into the nucleus upon stimulus with flg22 and nlp20, respectively. Furthermore, the abundance of 93 proteins changed significantly in the nucleus following elicitation of immunity. These results suggest promiscuous ribosome assembly and retrograde signaling from the mitochondrion to the nucleus including Prohibitins and Cytochrome C, in the two forms of PTI.

plant biology↗

bioRxiv2020

A modular two yeast species secretion system for the production and preparative application of fungal peroxygenases

Fungal unspecific peroxygenases (UPOs) are biocatalysts of outstanding interest. Providing access to novel UPOs using a modular secretion system was the central goal of this work. UPOs represent an enzyme class, catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and solely rely on hydrogen peroxide as the oxygen source. Fungal peroxygenases are widespread throughout the fungal kingdom and hence a huge variety of UPO gene sequences is available. However, the heterologous production of UPOs in a fast-growing organism suitable for high throughput screening has only succeeded once--enabled by an intensive directed evolution campaign. Here, we developed and applied a modular Golden Gate-based secretion system, allowing the first yeast production of four active UPOs, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be broadly applicable and consists of the three module types: i) a signal peptide panel guiding secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. We show that optimal signal peptides could be selected for successful UPO secretion by combinatorial testing of 17 signal peptides for each UPO gene. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid workflow from putative UPO gene to preparative scale enantioselective biotransformations.

synthetic biology↗

bioRxiv2020

Reshaping of the Arabidopsis thaliana proteome landscape and co-regulation of proteins in development and immunity

Proteome remodeling is a fundamental adaptive response and proteins in complex and functionally related proteins are often co-expressed. Using a deep sampling strategy we define Arabidopsis thaliana tissue core proteomes at around 10,000 proteins per tissue and absolutely quantify (copy numbers per cell) nearly 16,000 proteins throughout the plant lifecycle. A proteome wide survey of global post translational modification revealed amino acid exchanges pointing to potential conservation of translational infidelity in eukaryotes. Correlation analysis of protein abundance uncovered potentially new tissue and age specific roles of entire signaling modules regulating transcription in photosynthesis, seed development and senescence and abscission. Among others, the data suggest a potential function of RD26 and other NAC transcription factors in seed development related to desiccation tolerance as well as a possible function of Cysteine-rich Receptor-like Kinases (CRKs) as ROS sensors in senescence. All of the components of ribosome biogenesis factor (RBF) complexes were co-expressed tissue and age specifically indicating functional promiscuity in the assembly of these little described protein complexes in Arabidopsis. Treatment of seedlings with flg22 for 16 hours allowed us to characterize proteome architecture in basal immunity in detail. The results were complemented with parallel reaction monitoring (PRM) targeted proteomics, phytohormone, amino acid and transcript measurements. We obtained strong evidence of suppression of jasmonate (JA) and JA-Ile levels by deconjugation and hydroxylation via IAA-ALA RESISTANT3 (IAR3) and JASMONATE-INDUCED OXYGENASE 2 (JOX2) under the control of JASMONATE INSENSITIVE 1 (MYC2). This previously unknown regulatory switch is another part of the puzzle of the as yet understudied role of JA in pattern triggered immunity. The extensive coverage of the Arabidopsis proteome in various biological scenarios presents a rich resource to plant biologists that we make available to the community.

plant biology↗

bioRxiv2019

Prioritization of abiotic and biotic stress responses by direct linkage of ABI1 phosphatase and CPK5 calcium-dependent protein kinase

In nature plants are constantly challenged by simultaneous abiotic and biotic stresses, and under conflicting stress scenarios prioritization of stress responses is required for plant survival. Calcium-dependent protein kinase CPK5 is a central hub in local and distal immune signaling, required for hormone salicylic acid (SA)-dependent immunity and pathogen resistance. Here we show that CPK5-dependent immune responses and pathogen resistance are inhibited upon abscisic acid (ABA) treatment or in genetic mutant backgrounds lacking PP2C phosphatase activities including abi1-2, whereas immune responses are enhanced by co-expression of active ABI1 phosphatase variants. Biochemical studies and mass spectrometry-based phospho-site analysis reveal a direct ABI1 phosphatase-catalyzed de-phosphorylation of CPK5 auto-phosphorylation site T98. Mimicking continuous de-phosphorylation in CPK5T98A leads to enhanced ROS production and more resistant plants, mimicking the auto-phosphorylated status in CPK5T98D, reduces CPK5-mediated immune responses. Mechanistic insight identifies differential phosphorylation at T98 in the N-terminal domain of CPK5 to control the level of interaction between the kinase and its substrate protein rather than CPK5 catalytic activity. Thus, CPK5-catalyzed immune signaling may become discontinued even at an elevated cytoplasmic calcium concentration. Our work reveals an elegant mechanism for stress response prioritization in plants: The ABA-dependent phosphatase ABI1, negative regulator of abiotic responses, functions as positive regulator of biotic stress responses, stabilizing CPK5-dependent immune signaling in the absence of ABA. Continuous pathogen survey activates plant immunity in environmentally friendly conditions, whereas under severe abiotic stress the phosphatase/kinase pair prohibits immune signaling through a direct biochemical switch involving two key regulatory enzymes of these antagonistic pathways. Significance StatementPlants challenged by simultaneous abiotic and biotic stresses must prioritize in conflicting scenarios to guarantee survival. Pathogen resistance and immune memory depends on the phytohormone salicylic acid (SA). Adaptation to abiotic stress signaling involves the phytohormone abscisic acid (ABA). We identify a direct biochemical switch by which ABA-mediated abiotic signaling prioritizes over SA-dependent immune responses via reversible phosphorylation at a single protein mark involving two key regulatory enzymes of these antagonistic pathways. Phosphatase ABI1 de-phosphorylates calcium-dependent protein kinase CPK5 at an auto-phosphorylation site T98, which effects the interaction efficiency between the kinase and its substrate. Under abiotic stress ABA mediates phosphatase inhibition, which facilitates prolonged auto-phosphorylation of CPK5, preventing CPK5 substrate interaction and ultimately stop CPK5-mediated immune signaling.

plant biology↗

bioRxiv2019

Phosphorylation of the CAMTA3 transcription factor triggers its destabilization and nuclear export.

The calmodulin-binding transcription activator 3 (CAMTA3) is a repressor of immunity-related genes but an activator of cold-induced genes in plants. Post-transcriptional or -translational mechanisms have been proposed to control CAMTA3s role in the crosstalk between immune and chilling responses. Here, we show that treatment with the bacterial flg22 elicitor, but not cold stress, induces a phospho-mobility shift of CAMTA3 proteins. Correspondingly, CAMTA3 is directly phosphorylated by two flg22-responsive mitogen-activated protein kinases (MAPKs), MPK3 and MPK6, which triggers CAMTA3 nuclear export and destabilization. SR1IP1, a substrate E3 ubiquitin ligase adaptor required for pathogen-induced CAMTA3 degradation, is shown here to be likely plasma-membrane-localized and therefore cannot physically interact with the nuclear CAMTA3. Despite the flg22-inducible re-localization of CAMTA3 to the cytoplasm, we failed to detect CAMTA3-SR1IP1 complexes. Hence, the role of SR1IP1 for CAMTA3 degradation needs to be re-evaluated. Surprisingly, flg22 elicitation can still induce nuclear export and phospho-mobility shift of a phospho-null CAMTA3 that cannot be phosphorylated by MAPKs, suggesting the participation of additional flg22-responsive kinase(s). A constitutively-active calcium-dependent protein kinase, CPK5, can stimulate a phospho-mobility shift in CAMTA3 similar to that induced by flg22. Although CPK5 can interact with CAMTA3, it did not directly phosphorylate CAMTA3, suggesting the requirement of a still unidentified downstream kinase or additional components. Overall, at least two flg22-responsive kinase pathways target CAMTA3 to induce degradation that presumably serves to remove CAMTA3 from target promoters and de-repress expression of defence genes.\n\nOne sentence summaryTreatment with flg22 activates two independent kinase pathways that effect CAMTA3 phosphorylation and degradation.

plant biology↗

bioRxiv2019

Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin receptor assemblies

Cullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5 and their ubiquitylation targets, AUX/IAAs, sense auxin concentrations in the nucleus. TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, we resolved TIR1{middle dot}auxin{middle dot}IAA7 and TIR1{middle dot}auxin{middle dot}IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degrons vicinity, cooperatively position AUX/IAAs on TIR1. The AUX/IAA PB1 interaction domain also assists in non-native contacts, affecting AUX/IAA dynamic interaction states. Our results establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation, and might provide conformational flexibility for adopting a multiplicity of functional states. We postulate IDRs in distinct members of the AUX/IAA family to be an adaptive signature for protein interaction and initiation region for proteasome recruitment.

plant biology↗

bioRxiv2018

Phosphorylation of the exocyst subunit Exo70B2 contributes to the regulation of its function

The exocyst is a conserved hetero-octameric complex that mediates early tethering of post-Golgi vesicles during exocytosis. Its Exo70 subunit functions as a spatiotemporal regulator by mediating numerous interactions with proteins and lipids. However, a molecular understanding of the exocyst functions remains challenging. Exo70B2 localized to dynamic foci at the plasma membrane and transited through Brefeldin A (BFA)-sensitive compartments, indicating that it participates in conventional secretion. Conversely, treatment with the immunogenic peptide flg22 or the salicylic acid (SA) defence hormone analogue Benzothiadiazole (BTH), induced Exo70B2 transport into the vacuole where it colocalized with autophagic markers AUTOPHAGY-RELATED PROTEIN 8 (ATG8) and NEIGHBOR OF BRCA1 GENE 1 (NBR1). According with its role in immunity, we discovered that Exo70B2 interacts with and is phosphorylated by the MITOGEN-ACTIVATED PROTEIN KINASE 3 (MPK3). Mimicking phosphorylation inhibited Exo70B2 localization at sites of active secretion. By contrast, lines expressing phosphonull variants displayed higher Effector-Triggered Immunity and were hypersensitive to BTH, conditions known to induce the secretory pathway. Our results suggest a molecular mechanism by which phosphorylation of Exo70B2 regulates interaction with the plasma membrane, and couples the secretory pathway with cellular signalling.

cell biology↗