Reactive oxygen species signalling in plant stress responses.

Reactive oxygen species (ROS) are key signalling molecules that enable cells to rapidly respond to different stimuli. In plants, ROS play a crucial role in abiotic and biotic stress sensing, integration of different environmental signals and activation of stress-response networks, thus contributing to the establishment of defence mechanisms and plant resilience. Recent advances in the study of ROS signalling in plants include the identification of ROS receptors and key regulatory hubs that connect ROS signalling with other important stress-response signal transduction pathways and hormones, as well as new roles for ROS in organelle-to-organelle and cell-to-cell signalling. Our understanding of how ROS are regulated in cells by balancing production, scavenging and transport has also increased. In this Review, we discuss these promising developments and how they might be used to increase plant resilience to environmental stress.

H2O2-dependent oxidation of the transcription factor GmNTL1 promotes salt tolerance in soybean.

Reactive oxygen species (ROS) play an essential role in plant growth and responses to environmental stresses. Plant cells sense and transduce ROS signaling directly via hydrogen peroxide (H2O2)-mediated posttranslational modifications (PTMs) on protein cysteine residues. Here, we show that the H2O2-mediated cysteine oxidation of NAC WITH TRANS-MEMBRANE MOTIF1-LIKE 1 (GmNTL1) in soybean (Glycine max) during salt stress promotes its release from the endoplasmic reticulum (ER) membrane and translocation to the nucleus. We further show that an oxidative posttranslational modification on GmNTL1 residue Cys-247 steers downstream amplification of ROS production by binding to and activating the promoters of RESPIRATORY BURST OXIDASE HOMOLOG B (GmRbohB) genes, thereby creating a feed-forward loop to fine-tune GmNTL1 activity. In addition, oxidation of GmNTL1 Cys-247 directly promotes the expression of CATION H+ EXCHANGER 1 (GmCHX1)/SALT TOLERANCE-ASSOCIATED GENE ON CHROMOSOME 3 (GmSALT3) and Na+/H+ Antiporter 1 (GmNHX1). Accordingly, transgenic overexpression of GmNTL1 in soybean increases the H2O2 levels and K+/Na+ ratio in the cell, promotes salt tolerance, and increases yield under salt stress, while an RNA interference-mediated knockdown of GmNTL1 elicits the opposite effects. Our results reveal that the salt-induced oxidation of GmNTL1 promotes its relocation and transcriptional activity through an H2O2-mediated posttranslational modification on cysteine that improves resilience of soybean against salt stress.

Structure-function study of a Ca2+-independent metacaspase involved in lateral root emergence.

Metacaspases are part of an evolutionarily broad family of multifunctional cysteine proteases, involved in disease and normal development. As the structure-function relationship of metacaspases remains poorly understood, we solved the X-ray crystal structure of an Arabidopsis thaliana type II metacaspase (AtMCA-IIf) belonging to a particular subgroup not requiring calcium ions for activation. To study metacaspase activity in plants, we developed an in vitro chemical screen to identify small molecule metacaspase inhibitors and found several hits with a minimal thioxodihydropyrimidine-dione structure, of which some are specific AtMCA-IIf inhibitors. We provide mechanistic insight into the basis of inhibition by the TDP-containing compounds through molecular docking onto the AtMCA-IIf crystal structure. Finally, a TDP-containing compound (TDP6) effectively hampered lateral root emergence in vivo, probably through inhibition of metacaspases specifically expressed in the endodermal cells overlying developing lateral root primordia. In the future, the small compound inhibitors and crystal structure of AtMCA-IIf can be used to study metacaspases in other species, such as important human pathogens, including those causing neglected diseases.

ERFVII-controlled hypoxia responses are in part facilitated by MEDIATOR SUBUNIT 25 in Arabidopsis thaliana.

Flooding impairs plant growth through oxygen deprivation, which activates plant survival and acclimation responses. Transcriptional responses to low oxygen are generally associated with the activation of group VII ETHYLENE-RESPONSE FACTOR (ERFVII) transcription factors. However, the exact mechanisms and molecular components by which ERFVII factors initiate gene expression are not fully elucidated. Here, we show that the ERFVII factors RELATED TO APETALA 2.2 (RAP2.2) and RAP2.12 cooperate with the Mediator complex subunit AtMED25 to coordinate gene expression under hypoxia in Arabidopsis thaliana. Respective med25 knock-out mutants display reduced low-oxygen stress tolerance. AtMED25 physically associates with a distinct set of hypoxia core genes and its loss partially impairs transcription under hypoxia due to decreased RNA polymerase II recruitment. Association of AtMED25 with target genes requires the presence of ERFVII transcription factors. Next to ERFVII protein stabilisation, also the composition of the Mediator complex including AtMED25 is potentially affected by hypoxia stress as shown by protein-complex pulldown assays. The dynamic response of the Mediator complex to hypoxia is furthermore supported by the fact that two subunits, AtMED8 and AtMED16, are not involved in the establishment of hypoxia tolerance, whilst both act in coordination with AtMED25 under other environmental conditions. We furthermore show that AtMED25 function under hypoxia is independent of ethylene signalling. Finally, functional conservation at the molecular level was found for the MED25-ERFVII module between A. thaliana and the monocot species Oryza sativa, pointing to a potentially universal role of MED25 in coordinating ERFVII-dependent transcript responses to hypoxia in plants.

Scywalker: scalable end-to-end data analysis workflow for long-read single-cell transcriptome sequencing.

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.

Adenosine monophosphate deaminase modulates BIN2 activity through hydrogen peroxide-induced oligomerization.

The Arabidopsis thaliana GSK3-like kinase, BRASSINOSTEROID-INSENSITIVE2 (BIN2) is a key negative regulator of brassinosteroid (BR) signaling and a hub for crosstalk with other signaling pathways. However, the mechanisms controlling BIN2 activity are not well understood. Here we performed a forward genetic screen for resistance to the plant-specific GSK3 inhibitor bikinin and discovered that a mutation in the ADENOSINE MONOPHOSPHATE DEAMINASE (AMPD)/EMBRYONIC FACTOR1 (FAC1) gene reduces the sensitivity of Arabidopsis seedlings to both bikinin and BRs. Further analyses revealed that AMPD modulates BIN2 activity by regulating its oligomerization in a hydrogen peroxide (H2O2)-dependent manner. Exogenous H2O2 induced the formation of BIN2 oligomers with a decreased kinase activity and an increased sensitivity to bikinin. By contrast, AMPD activity inhibition reduced the cytosolic reactive oxygen species (ROS) levels and the amount of BIN2 oligomers, correlating with the decreased sensitivity of Arabidopsis plants to bikinin and BRs. Furthermore, we showed that BIN2 phosphorylates AMPD to possibly alter its function. Our results uncover the existence of an H2O2 homeostasis-mediated regulation loop between AMPD and BIN2 that fine-tunes the BIN2 kinase activity to control plant growth and development.

Roadmap for the next decade of plant programmed cell death research.

Programmed cell death (PCD) is fundamentally important for plant development, abiotic stress responses and immunity, but our understanding of its regulation remains fragmented. Building a stronger research community is required to accelerate progress in this area through knowledge exchange and constructive debate. In this Viewpoint, we aim to initiate a collective effort to integrate data across a diverse set of experimental models to facilitate characterisation of the fundamental mechanisms underlying plant PCD and ultimately aid the development of a new plant cell death classification system in the future. We also put forward our vision for the next decade of plant PCD research stemming from discussions held during the 31st New Phytologist workshop, 'The Life and Death Decisions of Plant Cells' that took place at University College Dublin in Ireland (14-15 June 2023). We convey the key areas of significant progress and possible future research directions identified, including resolving the spatiotemporal control of cell death, isolation of its molecular and genetic regulators, and harnessing technical advances for studying PCD events in plants. Further, we review the breadth of potential impacts of plant PCD research and highlight the promising new applications of findings from this dynamically evolving field.

Redox regulation of chromatin remodelling in plants.

Changes in the cellular redox balance that occur during plant responses to unfavourable environmental conditions significantly affect a myriad of redox-sensitive processes, including those that impact on the epigenetic state of the chromatin. Various epigenetic factors, like histone modifying enzymes, chromatin remodelers, and DNA methyltransferases can be targeted by oxidative posttranslational modifications. As their combined action affects the epigenetic regulation of gene expression, they form an integral part of plant responses to (a)biotic stress. Epigenetic changes triggered by unfavourable environmental conditions are intrinsically linked with primary metabolism that supplies intermediates and donors, such acetyl-CoA and S-adenosyl-methionine, that are critical for the epigenetic decoration of histones and DNA. Here, we review the recent advances in our understanding of redox regulation of chromatin remodelling, dynamics of epigenetic marks, and the interplay between epigenetic control of gene expression, redox signalling and primary metabolism within an (a)biotic stress context.

Cysteine thiol sulfinic acid in plant stress signaling.

Cysteine thiols are susceptible to various oxidative posttranslational modifications (PTMs) due to their high chemical reactivity. Thiol-based PTMs play a crucial role in regulating protein functions and are key contributors to cellular redox signaling. Although reversible thiol-based PTMs, such as disulfide bond formation, S-nitrosylation, and S-glutathionylation, have been extensively studied for their roles in redox regulation, thiol sulfinic acid (-SO2H) modification is often perceived as irreversible and of marginal significance in redox signaling. Here, we revisit this narrow perspective and shed light on the redox regulatory roles of -SO2H in plant stress signaling. We provide an overview of protein sulfinylation in plants, delving into the roles of hydrogen peroxide-mediated and plant cysteine oxidase-catalyzed formation of -SO2H, highlighting the involvement of -SO2H in specific regulatory signaling pathways. Additionally, we compile the existing knowledge of the -SO2H reducing enzyme, sulfiredoxin, offering insights into its molecular mechanisms and biological relevance. We further summarize current proteomic techniques for detecting -SO2H and furnish a list of experimentally validated cysteine -SO2H sites across various species, discussing their functional consequences. This review aims to spark new insights and discussions that lead to further investigations into the functional significance of protein -SO2H-based redox signaling in plants.

Redox regulation of gene expression: proteomics reveals multiple previously undescribed redox-sensitive cysteines in transcription complexes and chromatin modifiers.

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.

Glutathione: a key modulator of plant defence and metabolism through multiple mechanisms.

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.

The Plant PTM Viewer 2.0: in-depth exploration of plant protein modification landscapes.

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.

Arabidopsis transcription factor ANAC102 predominantly expresses a nuclear protein and acts as a negative regulator of methyl viologen-induced oxidative stress responses.

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 ß-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.

The Arabidopsis mediator complex subunit 8 regulates oxidative stress responses.

Signaling events triggered by hydrogen peroxide (H2O2) regulate plant growth and defense by orchestrating a genome-wide transcriptional reprogramming. However, the specific mechanisms that govern H2O2-dependent gene expression are still poorly understood. Here, we identify the Arabidopsis Mediator complex subunit MED8 as a regulator of H2O2 responses. The introduction of the med8 mutation in a constitutive oxidative stress genetic background (catalase-deficient, cat2) was associated with enhanced activation of the salicylic acid pathway and accelerated cell death. Interestingly, med8 seedlings were more tolerant to oxidative stress generated by the herbicide methyl viologen (MV) and exhibited transcriptional hyperactivation of defense signaling, in particular salicylic acid- and jasmonic acid-related pathways. The med8-triggered tolerance to MV was manipulated by the introduction of secondary mutations in salicylic acid and jasmonic acid pathways. In addition, analysis of the Mediator interactome revealed interactions with components involved in mRNA processing and microRNA biogenesis, hence expanding the role of Mediator beyond transcription. Notably, MED8 interacted with the transcriptional regulator NEGATIVE ON TATA-LESS, NOT2, to control the expression of H2O2-inducible genes and stress responses. Our work establishes MED8 as a component regulating oxidative stress responses and demonstrates that it acts as a negative regulator of H2O2-driven activation of defense gene expression.

Improving oxidative stress resilience in plants.

Originally conceived as harmful metabolic byproducts, reactive oxygen species (ROS) are now recognized as an integral part of numerous cellular programs. Thanks to their diverse physicochemical properties, compartmentalized production, and tight control exerted by the antioxidant machinery they activate signaling pathways that govern plant growth, development, and defense. Excessive ROS levels are often driven by adverse changes in environmental conditions, ultimately causing oxidative stress. The associated negative impact on cellular constituents have been a major focus of decade-long research efforts to improve the oxidative stress resilience by boosting the antioxidant machinery in model and crop species. We highlight the role of enzymatic and non-enzymatic antioxidants as integral factors of multiple signaling cascades beyond their mere function to prevent oxidative damage under adverse abiotic stress conditions.

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 signaling remain unclear. The role of the vasculature, in particular the phloem, in facilitating inter-organ communication is critical and poorly understood. Combining genetic, proteomic and physiological approaches, we investigated the role of AtMC3, a phloem-specific member of the metacaspase family, in osmotic stress responses in Arabidopsis thaliana. Analyses of the proteome in plants with altered AtMC3 levels revealed differential abundance of proteins related to osmotic stress pointing into a role of the protein in water-stress-related responses. Overexpression of AtMC3 conferred drought tolerance by enhancing the differentiation of specific vascular tissues and maintaining higher levels of vascular-mediated transportation, while plants lacking the protein showed an impaired response to drought and inability to respond effectively to the hormone abscisic acid. 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.

The heat is on: a simple method to increase genome editing efficiency in plants.

BACKGROUND: Precision genome mutagenesis using CRISPR/Cas has become the standard method to generate mutant plant lines. Several improvements have been made to increase mutagenesis efficiency, either through vector optimisation or the application of heat stress. RESULTS: Here, we present a simplified heat stress assay that can be completed in six days using commonly-available laboratory equipment. We show that three heat shocks (3xHS) efficiently increases indel efficiency of LbCas12a and Cas9, irrespective of the target sequence or the promoter used to express the nuclease. The generated indels are primarily somatic, but for three out of five targets we demonstrate that up to 25% more biallelic mutations are transmitted to the progeny when heat is applied compared to non-heat controls. We also applied our heat treatment to lines containing CRISPR base editors and observed a 22-27% increase in the percentage of C-to-T base editing. Furthermore, we test the effect of 3xHS on generating large deletions and a homologous recombination reporter. Interestingly, we observed no positive effect of 3xHS treatment on either approach using our conditions. CONCLUSIONS: Together, our experiments show that heat treatment is consistently effective at increasing the number of somatic mutations using many CRISPR approaches in plants and in some cases can increase the recovery of mutant progeny.

Field and saccharification performances of poplars severely downregulated in CAD1.

Lignin is one of the main factors causing lignocellulosic biomass recalcitrance to enzymatic hydrolysis. Glasshouse-grown poplars severely downregulated for CINNAMYL ALCOHOL DEHYDROGENASE 1 (CAD1), the enzyme catalysing the last step in the monolignol-specific branch of lignin biosynthesis, have increased saccharification yields and normal growth. Here, we assess the performance of these hpCAD poplars in the field under short rotation coppice culture for two consecutive rotations of 1 yr and 3 yr. While 1-yr-old hpCAD wood had 10% less lignin, 3-yr-old hpCAD wood had wild-type lignin levels. Because of their altered cell wall composition, including elevated levels of cinnamaldehydes, both 1-yr-old and 3-yr-old hpCAD wood showed enhanced saccharification yields upon harsh alkaline pretreatments (up to +85% and +77%, respectively). In contrast with previous field trials with poplars less severely downregulated for CINNAMYL ALCOHOL DEHYDROGENASE (CAD), the hpCAD poplars displayed leaning phenotypes, early bud set, early flowering and yield penalties. Moreover, hpCAD wood had enlarged vessels, decreased wood density and reduced relative and free water contents. Our data show that the phenotypes of CAD-deficient poplars are strongly dependent on the environment and underpin the importance of field trials in translating basic research towards applications.

Contemporary proteomic strategies for cysteine redoxome profiling.

Protein cysteine residues are susceptible to oxidative modifications that can affect protein functions. Proteomic techniques that comprehensively profile the cysteine redoxome, the repertoire of oxidized cysteine residues, are pivotal towards a better understanding of the protein redox signaling. Recent technical advances in chemical tools and redox proteomic strategies have greatly improved selectivity, in vivo applicability, and quantification of the cysteine redoxome. Despite this substantial progress, still many challenges remain. Here, we provide an update on the recent advances in proteomic strategies for cysteine redoxome profiling, compare the advantages and disadvantages of current methods and discuss the outstanding challenges and future perspectives for plant redoxome research.