Biology subjects

Van Breusegem, F.

Publications and source records attributed to Van Breusegem, F..

At least 19 recordsLinked to original sources

bioRxiv2026

Cytosolic GLUCOSE-6-PHOSPHATE DEHYDROGENASE 5 is a key player in redox homeostasis during oxidative stress and in oxidative stress-triggered activation of the salicylic acid pathway

Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first step of the oxidative pentose phosphate pathway, generating NADPH to sustain redox metabolism and signaling. However, whether individual G6PDH isoforms directly regulate oxidative stress signaling remains unclear. To determine the contribution of the different Arabidopsis G6PDH isoforms to oxidative stress signaling, we introduced single T-DNA mutants into the catalase-deficient cat2 background, a genetic system in which intracellular H2O2 production activates salicylic acid (SA)-dependent cell death and defense pathways. Interestingly, impairment of cytosolic, but not chloroplastic G6PDH activity suppressed cat2-triggered phenotypes, with loss of G6PD5 function fully abolishing lesion formation. The cat2 g6pd5 double mutant phenocopied the SA biosynthesis-deficient mutant cat2 sid2 and showed reversion of defense responses as well as metabolomic and transcriptomic profiles to the wild-type state. Strikingly, despite the suppression of SA-dependent lesions, loss of G6PD5 activity does not appear to reduce stress intensity. On the contrary, cat2 g6pd5 plants exhibit increased glutathione synthesis and oxidation, elevated expression of oxidative stress marker genes, and enhanced accumulation of reactive nitrogen species relative to cat2. Protein-protein interaction analyses revealed that G6PD5 associates with several redox and defense-related proteins. In particular, we confirmed a physical interaction between G6PD5 and thioredoxin h5, a key component of redox-dependent SA signaling. However, analysis of cat2 trxh5 and cat2 npr1 lines indicated that this interaction alone cannot explain the G6PD5-dependent control of SA responses. Our work reveals that cytosolic G6PD5 integrates redox metabolism with immune signaling to control plant responses to oxidative stress.

plant biology↗

bioRxiv2026

Antagonistic functions of the two oxidative pentose phosphate pathway dehydrogenases in shaping oxidative stress responses

The oxidative pentose phosphate pathway (OPPP) is a source of cellular NADPH, generated through the sequential activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). Using the catalase-deficient cat2 background as a model for H2O2-triggered salicylic acid (SA) signaling we identified the cytosolic G6PDH isoform G6PD5 as a key determinant of redox homeostasis and SA-dependent defense activation (Tremulot et al., companion manuscript). However, the mechanisms underlying this function remain enigmatic. In this work, genetic and transcriptomic analyses show that the role of G6PD5 cannot be explained solely by altered NADPH generation for either NADPH oxidases or the ascorbate-glutathione pathway, suggesting other possible links. To identify such links, a forward genetic screen was employed. We searched for mutations that modulate the suppressed lesion phenotype in cat2 g6pd5 in a photorespiration-dependent manner. This screen identified a mutation in PGD2, encoding the cytosolic 6PGDH. Strikingly, functional analyses of mutants and overexpression lines revealed that PGD2 exerts effects opposite to those of G6PD5 in SA signaling. Our observations uncover an unexpected antagonism between the two cytosolic NADPH-producing steps within the OPPP. Pharmacological analyses support a signaling role for the metabolic intermediate 6-phosphogluconolactone in linking the OPPP to SA signaling. These findings indicate that the OPPP is not solely a source of reducing power during oxidative stress but also acts as a signaling module in which metabolic intermediates contribute to the control of stress-induced immune responses.

plant biology↗

bioRxiv2026

Conditional stability of HY5 through the ATE N-degron pathway regulates environmental responses in Arabidopsis thaliana

The N-degron pathways of ubiquitin mediated proteolysis target proteins for degradation dependent on their amino terminal residue, often produced after protease activity. Very few substrates have been identified in plants even though enzymes of these pathways are highly conserved in eukaryotes. Here we identify ELONGATED HYPOCOTYL5 (HY5), an important transcriptional regulator involved in many aspects of plant development, as a target for the endopeptidase METACASPASE (MC)9, producing the carboxy-terminal protein fragment (proteoform) E59-HY5. E59-HY5 is shown to be a substrate of the arginyl transferase (ATE) N-degron pathway, and influences physiological processes known to be controlled by HY5, including photomorphogenesis and the unfolded protein response. Conditional stability of E59-HY5 was shown to result from environmentally controlled ATE function, which may highlight a general mechanism for N-degron pathway regulation of proteoform and proteome function during growth and development.

plant biology↗

bioRxiv2026

A redox-regulated RCC1-like protein controls catalase activity in Arabidopsis

Reactive oxygen species (ROS) are central regulators of plant growth and stress responses. Cellular ROS levels are tightly controlled by antioxidant systems, including the evolutionarily conserved catalases that detoxify hydrogen peroxide (H2O2) predominantly within peroxisomes. Despite their importance, substantial gaps remain in our understanding of catalase biogenesis, regulation, subcellular targeting, and potential extra-peroxisomal functions. Using affinity purification of the UV-B photoreceptor UVR8 coupled with mass spectrometry, we identified a REGULATOR OF CHROMATIN CONDENSATION 1-like protein in Arabidopsis, which we named CATALASE-INTERACTING RCC1-LIKE 1 (CAIR1). CAIR1 interacts with all three catalase isoforms (CAT1-CAT3) as well as their chaperone NO CATALASE ACTIVITY 1 (NCA1). Loss-of-function cair1 mutants partially phenocopy cat2 and nca1, with reduced catalase activity, enhanced sensitivity to oxidative stress and alkaline growth conditions, and impaired primary root elongation. Mechanistically, cytosolic interaction between CAIR1 and CAT2 enhances total cellular catalase activity by facilitating peroxisomal import and proper subcellular localization of CAT2. In the absence of CAIR1, CAT2 forms aggregates, likely accounting for the observed loss of catalase activity. Notably, CAIR1 undergoes reversible, redox-dependent oligomerization that enhances its interaction with catalases. Mutation of CAIR1 at Cys-356 and Cys-545 compromises this interaction under elevated ROS conditions and fails to rescue the oxidative stress sensitivity of cair1 mutants. Moreover, UV-B exposure suppresses catalase activity by weakening the interaction between CAIR1 and catalases, thus linking environmental light signalling to cellular redox regulation. Together, our findings reveal CAIR1 as a dynamic redox-responsive regulator of catalase activity that maintains cellular redox homeostasis by coordinating catalase localization and function through reversible oligomerization.

plant biology↗

bioRxiv2025

Lack of AtMC1 catalytic activity triggers autoimmunity dependent on NLR stability

Plants utilize cell surface-localized pattern recognition receptors (PRRs) and intracellular nucleotide-binding leucine-rich repeat (NLR) receptors to detect non-self and elicit robust immune responses. Fine-tuning the homeostasis of these receptors is critical to prevent their hyperactivation. Here, we show that Arabidopsis plants lacking metacaspase 1 (AtMC1) display autoimmunity dependent on immune signalling components downstream of NLR and PRR activation. Overexpression of a catalytically inactive AtMC1 in an atmc1 background triggers severe autoimmunity partially dependent on the same immune signalling components. Overexpression of the E3 ligase SNIPER1, a master regulator of NLR homeostasis, fully reverts the AtMC1-dependent autoimmunity phenotype, inferring that a broad defect in NLR turnover may underlie the severe phenotype observed. Catalytically inactive AtMC1 localizes to punctate structures that are degraded through autophagy. Altogether and considering previous evidence on the proteostatic functions of AtMC1, we speculate that Wt AtMC1 may either directly or indirectly control NLR protein levels, thereby preventing autoimmunity.

plant biology↗

bioRxiv2024

Shedding light on plant proteolysis: genetically encoded fluorescent sensors as tools for profiling protease activities

Proteolysis, a ubiquitous process in living organisms, is driven by proteases that regulate numerous signaling pathways through the hydrolysis of peptide bonds in protein substrates. Understanding the temporal and spatial dynamics of proteolysis and the activation of proteases is crucial for elucidating their roles in biological pathways. Here, we introduce a suite of genetically encoded FRET reporters designed to detect various proteolytic activities in plants. These sensors effectively reported in planta the specific activity of both Tobacco Etch Virus protease and caspase-3. Furthermore, we developed sensors for detecting plant metacaspase activity, validated through both in vitro and in planta experiments. These experiments revealed the spatial dynamics of proteolysis triggered by metacaspase activation following wounding and programmed cell death in roots. The implementation of these tools in plant biology research opens new avenues for investigating proteolytic mechanisms, significantly enhancing the potential for in-depth studies. Our work demonstrates the feasibility of using these sensors to detect diverse protease activities in vivo with high spatiotemporal resolution. These plant proteolytic biosensors hence represent a valuable toolbox for understanding protease functions within their natural context, paving the way for future advancements in plant biology research.

plant biology↗

bioRxiv2024

The nuclear sulfenome of Arabidopsis: spotlight on histone acetyltransferase GCN5 regulation through functional thiols

Partial reduction of oxygen during energy generating metabolic processes in aerobic life forms results in the production of reactive oxygen species (ROS). In plants, ROS production is heightened during periods of both abiotic and biotic stress, which imposes a significant overload on the antioxidant systems. Hydrogen peroxide (H2O2) holds a central position in cellular redox homeostasis and signalling, playing an important role by oxidising crucial cysteines to sulfenic acid (-SOH), considered as a biologically relevant post-translational modification (PTM). Until now, the role of the nucleus in the cellular redox homeostasis has been relatively underexplored. The regulation of histone-modifying enzymes by oxidative PTMs on redox-active cysteines or tyrosine residues is particularly intriguing as it allows the integration of redox signalling mechanisms with chromatin control of transcriptional activity. One of the most extensively studied histone acetyltransferases is the conserved GENERAL CONTROL NONDEPRESSIBLE 5 (GCN5) complex. This study investigated the nuclear sulfenome in Arabidopsis thaliana by expressing a nuclear variant of the Yeast Activation Protein-1 (YAP) probe, identifying 225 potential redox-active nuclear proteins subject to sulfenylation. Mass spectrometry analysis further confirmed the sulfenylation of GCN5 at specific cysteine residues, with their functional significance and impact on the protein-protein interaction network assessed through cysteine-to-serine mutagenesis. HighlightProtein cysteine thiols are post-translationally modified under oxidative stress. Through the in vivo capturing of nuclear proteins undergoing sulfenylation in Arabidopsis, we highlight the functionality of particular cysteines in the histone acetyltransferase GCN5.

plant biology↗

bioRxiv2024

Scywalker: scalable end-to-end data analysis workflow for nanopore single-cell transcriptome sequencing

We introduce scywalker, an innovative and scalable package developed to comprehensively analyze long-read nanopore sequencing data of full-length single-cell or single-nuclei cDNA. Existing nanopore single-cell data analysis tools showed severe limitations in handling current data sizes. We developed novel scalable methods for cell barcode demultiplexing and single-cell isoform calling and quantification and incorporated these in an easily deployable package. Scywalker streamlines the entire analysis process, from sequenced fragments in FASTQ format to demultiplexed pseudobulk isoform counts, into a single command suitable for execution on either server or cluster. Scywalker includes data quality control, cell type identification, and an interactive report. Assessment of datasets from the human brain, Arabidopsis leaves, and previously benchmarked data from mixed cell lines, demonstrate excellent correlation with short-read analyses at both the cell-barcoding and gene quantification levels. At the isoform level, we show that scywalker facilitates the direct identification of cell-type-specific expression of novel isoforms.

bioinformatics↗

bioRxiv2023

CysQuant: simultaneous quantification of cysteine oxidation and protein abundance using data dependent or independent acquisition mass spectrometry

Protein cysteinyl thiols are susceptible to reduction-oxidation reactions that can influence protein function. Accurate quantification of cysteine oxidation is therefore crucial for decoding protein redox regulation. Here, we present CysQuant, a novel approach for simultaneous quantification of cysteine oxidation degrees and protein abundancies. CysQuant involves light/heavy iodoacetamide isotopologues for differential labeling of reduced and reversibly oxidized cysteines analyzed by data-dependent acquisition (DDA) or data-independent acquisition mass spectrometry (DIA-MS). Using plexDIA with in silico predicted spectral libraries, we quantified an average of 18% cysteine oxidation in Arabidopsis thaliana by DIA-MS, including a subset of highly oxidized cysteines forming disulfide bridges in AlphaFold2 predicted structures. Applying CysQuant to Arabidopsis seedlings exposed to excessive light, we successfully quantified the well-established increased reduction of Calvin-Benson cycle enzymes and discovered yet uncharacterized redox-sensitive disulfides in chloroplastic enzymes. Overall, CysQuant is a highly versatile tool for assessing the cysteine modification status that can be widely applied across various mass spectrometry platforms and organisms.

molecular biology↗

bioRxiv2023

Seed Longevity is Controlled by Metacaspases

To survive extreme desiccation, seeds enter dormancy that can last millennia. This dormancy involves the accumulation of protective but structurally disordered storage proteins through unknown adjustments of proteolytic surveillance mechanisms. Mutation of all six types II metacaspases (MCAs)-II in the model plant Arabidopsis revealed their essential role in modulating these proteolytic mechanisms. MCA-II mutant seeds fail to properly target at the endoplasmic reticulum (ER) the AAA ATPase Cell Division Cycle 48 (CDC48) to dispose of misfolded proteins. MCA-IIs cleave a CDC48 adaptor, the ubiquitination regulatory X domain-containing (PUX) responsible for localizing CDC48 to the lipid droplets. When cleaved, CDC48-PUX is inactivated and allows a lipid droplet-to-ER shuttling of CDC48, an important step in the regulation of seeds lifespan. In sum, we uncover antagonism between proteolytic pathways bestowing longevity. One-Sentence SummaryMetacaspase proteases confer seed longevity by antagonizing CDC48 activity.

plant biology↗

bioRxiv2023

Structure-function analysis of a calcium-independent metacaspase reveals a novel proteolytic pathway for lateral root emergence

Metacaspases are part of an evolutionarily broad family of multifunctional cysteine proteases, involved in disease and normal development. Despite the extensive study of metacaspases in the two decades since their discovery, the structure-function relationship of metacaspases remains poorly understood. Furthermore, previous studies on their function have been thwarted by the redundancy in gene copy number and potential phenotypic suppression of genetic mutations, especially in plants. Here, we have solved the X-ray crystal structure of an Arabidopsis thaliana type II metacaspase (AtMCA-IIf) that belongs to a particular sub-group that does not require calcium ions for activation. Compared to crystal structures of other metacaspases and caspases, the AtMCA-IIf active site is structurally similar and poses a conundrum for the catalytic mechanism of the cysteine-histidine dyad. To study metacaspase activity in plants, we developed an in vitro chemical screen to identify small molecule metacaspase inhibitors. Several hits with a minimal thioxodihydropyrimidine-dione (TDP) structure were identified, some being specific inhibitors of AtMCA-IIf. We provide a mechanistic basis for inhibition by the TDP-containing compounds through molecular docking onto the AtMCA-IIf crystal structure. Finally, a TDP-containing compound (TDP6) was effective at inhibiting lateral root emergence in vivo, likely through the inhibition of metacaspases that are specifically expressed in the endodermal cells overlaying developing lateral root primordia. In the future, the small compound inhibitors and crystal structure of AtMCA-IIf can be used to study metacaspases in various other species, such as important human pathogens including those causing neglected diseases.

plant biology↗

bioRxiv2022

A phloem-localized Arabidopsis metacaspase (AtMC3) improves drought tolerance

Increasing drought phenomena pose a serious threat to agricultural productivity. Although plants have multiple ways to respond to the complexity of drought stress, the underlying mechanisms of stress sensing and signalling remain unclear. The role of the vasculature, in particular the phloem, in facilitating inter-organ communication is critical. Here, we investigated the role of AtMC3, a phloem-specific member of the metacaspase family, in osmotic stress responses in Arabidopsis thaliana. Overexpression of AtMC3 conferred drought tolerance by enhancing the differentiation of the metaphloem sieve elements and maintaining higher levels of vascular-mediated transportation, whilst plants lacking the protein showed an impaired response to drought and inability to respond effectively to the hormone abscisic acid. Analyses of the proteome in plants with altered AtMC3 levels revealed differential abundance of proteins related to osmotic stress. Overall, our data highlight the importance of AtMC3 and vascular plasticity in fine-tuning early drought responses at the whole plant level without affecting growth or yield.

plant biology↗

bioRxiv2022

Functionally annotating cysteine disulfides and metal binding sites in the plant kingdom using AlphaFold2 predicted structures

Deep learning algorithms such as AlphaFold2 predict three-dimensional protein structure with high confidence. The recent release of more than 200 million structural models provides an unprecedented resource for functional protein annotation. Here, we used AlphaFold2 predicted structures of fifteen plant proteomes to functionally and evolutionary analyze cysteine residues in the plant kingdom. In addition to identification of metal ligands coordinated by cysteine residues, we systematically analyzed cysteine disulfides present in these structural predictions. Our analysis demonstrates most of these predicted disulfides are trustworthy due their high agreement (~96%) with those present in X-ray and NMR protein structures, their characteristic disulfide stereochemistry, the biased subcellular distribution of their proteins and a higher degree of oxidation of their respective cysteines as measured by proteomics. Adopting an evolutionary perspective, zinc binding sites are increasingly present at the expense of iron-sulfur clusters in plants. Interestingly, disulfide formation is increased in secreted proteins of land plants, likely promoting sequence evolution to adapt to changing environments encountered by plants. In summary, Alphafold2 predicted structural models are a rich source of information for studying the role of cysteines residues in proteins of interest and for protein redox biology in general.

systems biology↗

bioRxiv2022

Mutation of Arabidopsis SME1 and Sm core assembly improves oxidative stress resilience

Alternative splicing is a key posttranscriptional gene regulatory process, acting in diverse adaptive and basal plant processes. Splicing of precursor-messenger RNA (pre-mRNA) is catalyzed by a dynamic ribonucleoprotein complex, designated the spliceosome. In a suppressor screen, we identified a nonsense mutation in the Sm protein SME1 to alleviate photorespiratory H2O2-dependent cell death in catalase deficient plants. Similar attenuation of cell death was observed upon chemical inhibition of the spliceosome, suggesting pre-mRNA splicing inhibition to be responsible for the observed cell death alleviation. Furthermore, the sme1-2 mutants showed increased tolerance to the reactive oxygen species inducing herbicide methyl viologen. Both an mRNA-seq and shotgun proteomic analysis in sme1-2 mutants displayed a constitutive molecular stress response, together with extensive alterations in pre-mRNA splicing of transcripts encoding metabolic enzymes and RNA binding proteins, even under unstressed conditions. Using SME1 as a bait to identify protein interactors, we provide experimental evidence for almost 50 homologs of mammalian spliceosome-associated protein to reside in the Arabidopsis thaliana spliceosome complexes and propose roles in pre-mRNA splicing for four uncharacterized plant proteins. Furthermore, like in sme1-2, a mutant in the Sm core assembly protein ICLN resulted in a decreased sensitivity to methyl viologen. Taken together, these data show that both a perturbed Sm core composition and assembly results in the activation of a defense response and enhanced resilience to oxidative stress.

plant biology↗

bioRxiv2020

Unraveling transcription factor functions through integrative inference of transcriptional networks in Arabidopsis thaliana yields novel regulators involved in reactive oxygen species stress signaling

Gene regulation is a dynamic process in which transcription factors (TFs) play an important role to control spatiotemporal gene expression. While gene regulatory networks describe the interactions between TFs and their target genes, our global knowledge about the complexity of TF control for different genes and biological processes is incomplete. To enhance our global understanding of regulatory interactions in Arabidopsis thaliana, different regulatory input networks capturing complementary information about DNA motifs, open chromatin, TF binding and expression-based regulatory interactions, were combined using a supervised learning approach, resulting in an integrated gene regulatory network (iGRN) covering 1,491 TFs and 31,393 target genes (1.7 million interactions). This iGRN outperforms the different input networks to predict known regulatory interactions and has a similar performance to recover functional interactions compared to state-of-the-art experimental methods like yeast one-hybrid and ChIP-seq. The iGRN correctly inferred known functions for 681 TFs and predicted new gene functions for hundreds of unknown TFs. For regulators predicted to be involved in reactive oxygen species stress regulation, we confirmed in total 75% of TFs with a function in ROS and/or physiological stress responses. This includes 13 novel ROS regulators, previously not connected to any ROS or stress function, that were experimentally validated in our ROS-specific phenotypic assays of loss- or gain-of-function lines. In conclusion, the presented iGRN offers a high-quality starting point to enhance our understanding of gene regulation in plants by integrating different experimental data types at the network level.

plant biology↗

bioRxiv2020

Identification of sulfenylated cysteines in Arabidopsis thaliana proteins using a disulfide-linked peptide reporter

In proteins, hydrogen peroxide (H2O2) reacts with redox-sensitive cysteines to form cysteine sulfenic acid, also known as S-sulfenylation. These cysteine oxidation events can steer diverse cellular processes by altering protein interactions, trafficking, conformation, and function. Previously, we had identified S-sulfenylated proteins by using a tagged proteinaceous probe based on the yeast AP-1-like (Yap1) transcription factor that specifically reacts with sulfenic acids and traps them through a mixed disulfide bond. However, the identity of the S-sulfenylated amino acid residues remained enigmatic. Here, we present a technological advancement to identify in situ sulfenylated cysteines directly by means of the transgenic Yap1 probe. In Arabidopsis thaliana cells, after an initial affinity purification and a tryptic digestion, we further enriched the mixed disulfide-linked peptides with an antibody targeting the YAP1C-derived peptide (C598SEIWDR) that entails the redox-active cysteine. Subsequent mass spectrometry analysis with pLink 2 identified 1,745 YAP1C cross-linked peptides, indicating sulfenylated cysteines in over 1,000 proteins. Approximately 55% of these YAP1C-linked cysteines had previously been reported as redox-sensitive cysteines (S-sulfenylation, S-nitrosylation, and reversibly oxidized cysteines). The presented methodology provides a noninvasive approach to identify sulfenylated cysteines in any species that can be genetically modified.

plant biology↗

bioRxiv2019

On the move: Redox-dependent protein relocation

Compartmentation of proteins and processes is a defining feature of eukaryotic cells. The growth and development of organisms is critically dependent on the accurate sorting of proteins within cells. The mechanisms by which cytosol-synthesized proteins are delivered to the membranes and membrane compartments have been extensively characterised. However, the protein complement of any given compartment is not precisely fixed and some proteins can move between compartments in response to metabolic or environmental triggers. The mechanisms and processes that mediate such relocation events are largely uncharacterized. Many proteins can in addition perform multiple functions, catalyzing alternative reactions or performing structural, non-enzymatic functions. These alternative functions can be equally important functions in each cellular compartment. Such proteins are generally not dual targeted proteins in the classic sense of having targeting sequences that direct de novo synthesised proteins to specific cellular locations. Accumulating evidence suggests that redox post-translational modifications (PTMs) can control the compartmentation of many such proteins, including antioxidant and/or redox associated enzymes.

cell biology↗

bioRxiv2018

The Plant PTM Viewer, a central resource exploring plant protein modifications. From site-seeing to protein function.

Posttranslational modifications (PTMs) of proteins are central in any kind of cellular signaling. Modern mass spectrometry technologies enable comprehensive identification and quantification of various PTMs. Given the increased number and types of mapped protein modifications, a database is necessary that simultaneouly integrates and compares site-specific information for different PTMs, especially in plants for which the available PTM data are poorly catalogued. Here, we present the Plant PTM Viewer (http://www.psb.ugent.be/PlantPTMViewer), an integrative PTM resource that comprises approximately 200,000 PTM sites for 17 types of protein modifications in plant proteins from five different species. The Plant PTM Viewer provides the user with a protein sequence overview in which the experimentally evidenced PTMs are highlighted together with functional protein domains or active site residues. The PTM sequence search tool can query PTM combinations in specific protein sequences, whereas the PTM BLAST tool searches for modified protein sequences to detect conserved PTMs in homologous sequences. Taken together, these tools facilitate to assume the role and potential interplay of PTMs in specific proteins or within a broader systems biology context. The Plant PTM Viewer is an open repository that allows submission of mass spectrometry-based PTM data to remain at pace with future PTM plant studies.

systems biology↗