The N-degron pathways of ubiquitin mediated proteolysis target proteins for degradation dependent on the amino terminal residue, often produced after endopeptidase 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), a master 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. ### Competing Interest Statement The authors have declared no competing interest. BBSRC Ghent University, https://ror.org/00cv9y106, Research Project (01J00819) “DESTINY

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 (H 2 O 2 ) 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.

Abstract Deubiquitylases are key proteolytic regulators of ubiquitin-dependent cellular processes, catalyzing the removal or remodelling of ubiquitin modifications on substrate proteins, including those targeted for proteasomal degradation. UBIQUITIN PROTEASE (UBP)6 is a deubiquitylase that promotes the abundance of NONEXPRESSOR OF PATHOGENESIS RELATED GENES (NPR)1, a conserved master regulator of plant immunity. Here, we show that the Arabidopsis thaliana protease METACASPASE (MC)9 site-specifically processes UBP6, generating the E157-UBP6 proteoform, whose stability is controlled by the Arginyl-transferase (ATE) N-degron pathway. We observed that pathogen recognition both triggers UBP6 cleavage and leads to conditional stabilisation of E157 UBP6, which is enhanced as the defence response intensifies. Our data suggest that E157 UBP6, which lacks deubiquitylating activity, may induce inhibition of the proteasome, elevating NPR1 levels and enhancing salicylic acid (SA) induced gene activation, all of which collectively contribute to restricting pathogen growth. Thus, UBP6 cleavage and N-degron pathway regulation provide distinct proteoforms of UBP6 with specific effects on the immune processes.

Proteolysis is a universal process, as proteases play a pivotal role in modulating numerous signaling pathways. Proteases control the fate and function of their target proteins by hydrolyzing peptide bonds within these proteins. Understanding the temporal and spatial dynamics of proteolytic events, including the proteases that execute them, is crucial for elucidating their particular roles across diverse biological processes. In this study, we developed and characterized a set of genetically encoded Förster resonance energy transfer (FRET)-based reporters for the detection of various proteolytic activities in plants. Our sensors reliably reported the activity of specific proteases, exhibiting a performance comparable to previously established detection systems. In addition, we engineered variants capable of detecting the spatial dynamics of metacaspase-triggered proteolysis after wounding and during programmed cell death in roots. We demonstrated the feasibility of these FRET-based sensors for detecting various activities in vivo with high spatiotemporal resolution. The implementation of these tools in plant research opens opportunities to explore proteolytic mechanisms with enhanced precision. Overall, these biosensors constitute a versatile toolbox for probing protease function within its native cellular context, paving the way for deeper insights into plant biology and signaling.

Abstract 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 H 2 O 2 -triggered salicylic acid (SA) signaling we identified the cytosolic G6PDH isoform G6PD5 as a key determinant of redox homeostasis and SA-dependent defense activation (Trémulot 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.

Hydrogen sulfide (H2S) functions as a gaseous signaling molecule in plant stress responses through the persulfidation of protein cysteine (Cys) residues. A comprehensive, Cys site-specific map of the plant persulfidome has been lacking, despite its importance for achieving a systems-level understanding of the biological roles of Cys persulfidation. Using a state-of-the-art N-ethylmaleimide-biotin-based proteomics strategy, we generate a dynamic map of 1,691 persulfidated Cys sites in the rice (Oryza sativa) leaf proteome. Our results reveal a global dynamic changes in protein persulfidation during prolonged salt stress, with notable impacts on proteins involved in metal-dependent catalysis, redox metabolism, and the pentose phosphate pathway (PPP). Based on these patterns, we investigated the functional relevance of persulfidation within the nonoxidative PPP. H2S-mediated persulfidation decreased the activity of the representative nonoxidative PPP enzyme ribose-5-phosphate isomerase, leading to increased NADPH production and subsequent activation of NADPH-dependent redox enzymes, including monodehydroascorbate reductase (MDHAR) isoforms of the ascorbate-glutathione (AsA-GSH) cycle. Persulfidation protected MDHAR3/5 from oxidative inhibition and degradation, thereby sustaining AsA-GSH cycle capacity and supporting reactive oxygen species scavenging. This site-specific persulfidome provides a valuable resource for exploring plant redox regulation, and our functional analyses offer mechanistic insight into how H2S-dependent protein persulfidation modulates redox metabolic fluxes to bolster NADPH availability and maintain redox homeostasis during salt-stress adaptation.

Grass pea (Lathyrus sativus L.) is a grain legume of increasing importance in the Mediterranean region due to its outstanding tolerance to abiotic stresses such as salinity, heat, drought, and flooding, outperforming many other legume species. Despite established natural phenotypic variation in response to water-related stresses, the genetic basis of this resilience remains poorly understood, hindering precision breeding for single and combined stress tolerance. A genome-wide association study was conducted here to investigate the genetic architecture of water stress responses in grass pea. Previously, phenotypic data, including gas exchange, chlorophyll a fluorescence, photosynthetic pigments, leaf water status, and biomass partitioning traits, were assessed under well-watered, mild drought, and partial submergence conditions across 194 representative grass pea accessions worldwide. The data were associated with 5,651 single nucleotide polymorphisms (SNPs) using linear mixed models under a restricted maximum likelihood framework, incorporating population structure and the newly assembled L0007 genome. A total of 130 unique SNPs associated with at least one trait-treatment combination or with trait variation between stress and control conditions, providing a valuable resource for precision breeding of multi-stress tolerance in grass pea. The loci associated with drought and waterlogging were largely non-overlapping, suggesting distinct genetic bases for the two stress tolerances. However, some common mechanisms, such as redox regulation and carbohydrate metabolism, emerged among the identified candidate genes, highlighting some interconnectedness of biological pathways involved in grass pea responses to water stress.

Abstract 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 H 2 O 2 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.

The antioxidative enzyme monodehydroascorbate reductase (MDHAR) is represented by five genes in Arabidopsis, including four that encode cytosolic and peroxisomal proteins. The in planta importance of these specific isoforms during oxidative stress remain to be characterised. T-DNA mutants for MDAR genes encoding cytosolic and peroxisomal isoforms were studied. To examine their roles in conditions of intracellular oxidative stress, mutants were crossed with a cat2 line lacking the major leaf catalase. Enzyme assays in mdar mutants and of recombinant MDHARs suggest that peroxisomal MDHAR1 and cytosolic MDHAR2 are major players in leaf NADH- and NADPH-dependent activities, respectively. All mutants showed a wild-type phenotype when grown in standard conditions. In the cat2 background, loss of peroxisomal MDHAR functions decreased growth whereas loss of the cytosolic MDHAR2 function had no effect on growth but annulled a large part of transcriptomic and phenotypic responses to oxidative stress. The effects of the mdar2 mutation included decreased salicylic acid accumulation and enhanced glutathione oxidation, and were reverted by complementation with the MDAR2 sequence. Together, the data show that the cytosolic MDHAR2 is dispensable in optimal conditions but essential to promote biotic defence responses triggered by oxidative stress.

Hydrogen sulfide (H2S) is increasingly recognized as a crucial signaling molecule in plants that plays key roles in regulating physiological processes and enhancing stress tolerance. This review provides an updated summary of H2S signaling in plant stress responses, and discusses its uptake from external environmental sources, its endogenous biosynthesis, and its broader functions in stress adaptation. We summarize the impact of H2S on plants under various stress conditions and review the mechanisms through which it mediates signaling functions, with a particular focus on H2S-mediated protein persulfidation. In addition, we provide an overview of the current understanding of protein persulfidation in regulating physiological processes and stress responses in plants, offering both a general discussion of its effects under different stress conditions and specific examples to highlight its significance. Finally, we review recent proteomic studies on protein persulfidation in plants, comparing the identified persulfidated proteins across studies and highlighting shared biological processes and pathways. This review aims to consolidate our current understanding of H2S signaling and its roles as mediated by protein persulfidation in plants, while also offering insights to inspire future research in this rapidly evolving field.

In aerobic life forms, reactive oxygen species (ROS) are produced by the partial reduction of oxygen during energy-generating metabolic processes. In plants, ROS production increases during periods of both abiotic and biotic stress, severely overloading the antioxidant systems. Hydrogen peroxide (H2O2) plays a central role in cellular redox homeostasis and signalling by oxidizing crucial cysteines to sulfenic acid, which is considered a biologically relevant post-translational modification (PTM). Until now, the impact of the nucleus on cellular redox homeostasis has been relatively unexplored. The regulation of histone-modifying enzymes by oxidative PTMs at redox-sensitive cysteine or tyrosine residues is particularly intriguing because 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 (YAP1) probe and identified 225 potential redox-active proteins undergoing S-sulfenylation. Mass spectrometry analysis further confirmed the S-sulfenylation of GCN5 at Cys293, Cys368, and Cys400, and their functional significance and impact on the GCN5 protein-protein interaction network were assessed using cysteine-to-serine mutagenesis.

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. Considering also 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.

Redox signalling is crucial for regulating plant development and adaptation to environmental changes. Proteins with redox-sensitive cysteines can sense oxidative stress and modulate their functions. Recent proteomics efforts have comprehensively mapped the proteins targeted by oxidative modifications. The nucleus, the epicentre of transcriptional reprogramming, contains a large number of proteins that control gene expression. Specific redox-sensitive transcription factors have long been recognized as key players in decoding redox signals in the nucleus and thus in regulating transcriptional responses. Consequently, the redox regulation of the nuclear transcription machinery and its cofactors has received less attention. In this review, we screened proteomic datasets for redox-sensitive cysteines on proteins of the core transcription complexes and chromatin modifiers in Arabidopsis thaliana. Our analysis indicates that redox regulation affects every step of gene transcription, from initiation to elongation and termination. We report previously undescribed redox-sensitive subunits in transcription complexes and discuss the emerging challenges in unravelling the landscape of redox-regulated processes involved in nuclear gene transcription. This review highlights essential transcription machinery complexes and chromatin modifiers targeted by redox regulation, filling knowledge gaps in the intricate field of gene transcription.

Post-translational modifications (PTMs) greatly increase protein diversity and functionality. To help the plant research community interpret the ever-increasing number of reported PTMs, the Plant PTM Viewer (https://www.psb.ugent.be/PlantPTMViewer) provides an intuitive overview of plant protein PTMs and the tools to assess it. This update includes 62 novel PTM profiling studies, adding a total of 112 000 modified peptides reporting plant PTMs, including 14 additional PTM types and three species (moss, tomato, and soybean). Furthermore, an open modification re-analysis of a large-scale Arabidopsis thaliana mass spectrometry tissue atlas identified previously uncharted landscapes of lysine acylations predominant in seed and flower tissues and 3-phosphoglycerylation on glycolytic enzymes in plants. An extra 'Protein list analysis' tool was developed for retrieval and assessing the enrichment of PTMs in a protein list of interest. We conducted a protein list analysis on nuclear proteins, revealing a substantial number of redox modifications in the nucleus, confirming previous assumptions regarding the redox regulation of transcription. We encourage the plant research community to use PTM Viewer 2.0 for hypothesis testing and new target discovery, and also to submit new data to expand the coverage of conditions, plant species, and PTM types, thereby enriching our understanding of plant biology.

MOTIVATION:Existing nanopore single-cell data analysis tools showed severe limitations in handling current data sizes. RESULTS:We introduce scywalker, an innovative and scalable package developed to comprehensively analyze long-read sequencing data of full-length single-cell or single-nuclei cDNA. 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. AVAILABILITY AND IMPLEMENTATION:Scywalker is available on github.com/derijkp/scywalker under the GNU General Public License (GPL) and at https://zenodo.org/records/13359438/files/scywalker-0.108.0-Linux-x86_64.tar.gz.

Plants, being sessile organisms, constantly need to respond to environmental stresses, often leading to the accumulation of reactive oxygen species (ROS). While ROS can be harmful, they also act as second messengers guiding plant growth and stress responses. Because chloroplasts are sensitive to environmental changes and are both a source and a target of ROS during stress conditions, they are important in conveying environmental changes to the nucleus, where acclimation responses are coordinated to maintain organellar and overall cellular homeostasis. ANAC102 has previously been established as a regulator of β-cyclocitral-mediated chloroplast-to-nucleus signaling, protecting plants against photooxidative stress. However, debates persist about where ANAC102 is located—in chloroplasts or in the nucleus. Our study, utilizing the genomic ANAC102 sequence driven by its native promoter, establishes ANAC102 primarily as a nuclear protein, lacking a complete N-terminal chloroplast-targeting peptide. Moreover, our research reveals the sensitivity of plants overexpressing ANAC102 to severe superoxide-induced chloroplast oxidative stress. Transcriptome analysis unraveled a dual role of ANAC102 in negatively and positively regulating genome-wide transcriptional responses to chloroplast oxidative stress. Through the integration of published data and our own study, we constructed a comprehensive transcriptional network, which suggests that ANAC102 exerts direct and indirect control over transcriptional responses through downstream transcription factor networks, providing deeper insights into the ANAC102-mediated regulatory landscape during oxidative stress.

Redox reactions are fundamental to energy conversion in living cells, and also determine and tune responses to the environment. Within this context, the tripeptide glutathione plays numerous roles. As an important antioxidant, glutathione confers redox stability on the cell and also acts as an interface between signalling pathways and metabolic reactions that fuel growth and development. It also contributes to the assembly of cell components, biosynthesis of sulfur-containing metabolites, inactivation of potentially deleterious compounds, and control of hormonal signalling intensity. The multiplicity of these roles probably explains why glutathione status has been implicated in influencing plant responses to many different conditions. In particular, there is now a considerable body of evidence showing that glutathione is a crucial player in governing the outcome of biotic stresses. This review provides an overview of glutathione synthesis, transport, degradation, and redox turnover in plants. It examines the expression of genes associated with these processes during pathogen challenge and related conditions, and considers the diversity of mechanisms by which glutathione can influence protein function and gene expression.