Reactive oxygen species signalling in plant stress responses

1 This is a preprint of an article published in Nature Reviews Molecular Cell Biology. The final authenticated version is available online at: https://doi.org/10.1038/s41580-022-00499-2 Reactive oxygen species (ROS) signalling in plant stress responses Ron Mittler1,2,*, Sara I Zandalinas1,#, Yosef Fichman1,# and Frank Van Breusegem3,4 1 Division of Plant Sciences and Interdisciplinary Plant Group, College of Agriculture, Food and Natural Resources, Christopher S. Bond Life Sciences Center, University of Missouri, 1201 Rollins St, Columbia, MO, 65201, USA. 2 Department of Surgery, University of Missouri School of Medicine, Christopher S. Bond Life Sciences Center, University of Missouri, 1201 Rollins St, Columbia, MO 65201 3 Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium 4 Center for Plant Systems Biology, VIB, 9052 Gent, Belgium * e-mail: [email protected] [corresponding author] # Contributed equally Abstract ׀ 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 defense 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. 2 Introduction Pathogens, insects and different abiotic stresses such as flooding, prolonged droughts and heat waves, result in heavy losses to agricultural production and threaten global food security1,2. The alarming increase in the frequency and intensity of these stresses, an outcome of global warming and climate change3,4 highlights the importance of understanding the mechanisms that increase plant resilience against such stresses. Reactive oxygen species (ROS) play key roles in stress sensing, the integration of different stress-response signalling networks and the activation of plant defense mechanisms and acclimatization. Dissecting and understanding how ROS orchestrate plant responses to stress will allow us to improve plant tolerance to stress and increase our ability to mitigate crop damage when exposed to harsh environmental conditions4. The term ROS describes a group of molecules derived from molecular oxygen (O2). Whereas O2 is generally nonreactive towards most cellular components, ROS can cause the oxidation of lipids, proteins, RNA, DNA and many small molecules in cells. The high reactivity of ROS towards these cellular components is due to their altered chemistry, compared to O2, that allows them to donate an electron or transfer an excited energy state to an acceptor molecule5. The major forms of ROS in cells, which vary greatly in their properties and chemical reactivity, include hydrogen peroxide (H2O2), superoxide (O2.-), singlet oxygen (1O2), hydroxyl radical (HO.) and various forms of organic and inorganic peroxides (FIG 1a; Supplementary Table 1)5–9. As ROS are highly reactive, and independently produced in all or most cell compartments, their levels are kept under control to prevent unintended cellular oxidation. This is achieved by balancing of ROS production, scavenging and transport, which together keeps ROS at low concentrations, as well as controls ROS signalling reactions and their outcomes (FIG 1b). Several hundred genes encode for the different proteins and enzymes that regulate ROS metabolism and signalling in plants (Supplementary Table 2)6–8. ROS are produced ’passively’, by house-keeping enzymes or as byproducts of metabolic pathways (for example, photosynthesis and respiration), or ‘actively’, by dedicated oxidases that generate ROS for the purpose of signalling — for example, RESPIRATORY BURST OXIDASE HOMOLOGs (RBOHs), which are the functional equivalents of mammalian NADPH OXIDASEs (NOXs)5–9. At the same time, ROS are scavenged by an array of enzymatic and non-enzymatic antioxidants also found in most or all cell compartments (Supplementary Table 2; Supplementary Box 1)59. In addition, ROS can be transported between different compartments (for example, by AQUAPORINs; AQPs)10, or to other cells and tissues, for the purpose of signalling, removal, or accumulation. Thus, ROS can function where they are produced, or at a distance. ROS accumulation in cells during stress affects the redox state of many different proteins, including enzymes, receptors, and small molecules, activating, modifying, or integrating multiple stress-response 3 signal transduction pathways (FIG 1b). These alter gene expression and enhance the resilience of plants to stress11–21. Recent advancements in our understanding of these important processes include the identification of specific ROS sensors and regulatory hubs that connect ROS signalling with other stressresponse signal transduction pathways and hormones, the use of artificial intelligence-driven tools to dissect the different regulatory networks triggered by ROS sensing, and the identification of new roles for ROS in organelle-to-organelle and cell-to-cell stress signalling. In this Review, we first describe our current understanding of the mechanisms that control ROS production, scavenging, sensing and transport in plants. We then discuss how plants integrate ROS signalling with different hormone, retrograde, calcium, phosphorylation, and other stress-response signal transduction mechanisms to regulate gene expression and induce stress resilience. We focus mainly on H2O2, as it has a prominent role in the regulation of biological activity in cells. ROS production and scavenging during stress Cellular homeostasis is characterized by a baseline level of ROS that depends on the plant developmental stage, circadian clock, environmental and physiological conditions, and interactions with its microbiome. Different biotic and abiotic stresses can disrupt this homeostasis, uncouple metabolic pathways and lead to the accumulation of ROS in different cell compartments. For example, during excess light stress, when the flux of photons overcomes the plant energy needs to fix CO2, O2.- and 1O2 are primarily produced in the chloroplasts by photosystems I and II, respectively, and if photorespiration is activated (e.g., in C3 plants), H2O2 will also be produced in peroxisomes22–24. The production of ROS could be further elevated during drought stress when CO2 availability is limited due to the closure of stomata, and the excess energy absorbed by the photosynthetic apparatus cannot be channeled into CO2 fixation25–27. During heat stress when membrane complexes involved in different electron transfer chains are disrupted, O2.- and H2O2 are produced in mitochondria and chloroplasts, and increased levels of ROS accumulate in the cytosol and nucleus (Supplementary Box 1)28,29. A different pattern of ROS accumulation appears during responses to pathogens. O2.- and H2O2 are primarily produced in the apoplast due to the activation of specific oxidases such as RBOHs (Supplementary Box 1), as well as in chloroplasts as a consequence of the disruption and imbalance of metabolic pathways30–34. In contrast, virus infection was recently shown to cause the suppression of peroxisomal ROS production due to interactions of viral proteins with glycolate oxidase35. 4 Recent advancements in the use of genetically encoded ROS sensors and dyes revealed that during different stresses different types of ROS accumulate in different compartments of the cell25,28,29,36–45. Therefore, different patterns or signatures of ROS accumulation in cells are induced in a stress-specific manner (FIG 2). Moreover, recent studies have revealed that ROS can be transported in or out of different compartments and/or trigger different retrograde and anterograde signalling pathways between different cell compartments and the nucleus10,34,39,46–49. The different ROS and other signals produced in the different cell compartments in response to different stimuli could trigger stress-specific signal transduction pathways that activate stress-specific acclimatization and defense mechanism (FIG 2). The findings that different stresses result in the formation of different ROS signatures can serve as a working platform for future studies on how specificity in plant responses to stress is achieved. When studying ROS signalling in plant cells it is also important to consider that aerobic life evolved in the presence of ROS (Supplementary Box 2)50,51, suggesting that most cells are able to prevent ROS toxicity, and that ROS are primarily used for stress-sensing and signalling purposes6. To understand how the transient or continuous accumulation of ROS in different compartments during stress triggers defense responses, it is first important to understand how ROS are sensed in cells. ROS perception and redox regulation Unlike most ‘classical’ signal transduction molecules such as hormones or peptides that have a defined set of receptors, changes in ROS levels in cells can alter the structure and function of multiple proteins and therefore impact on many different signal transduction pathways. This ‘multiple-pathway’ signalling property of ROS is primarily mediated through oxidative post-translational modifications (oxi-PTMs)52–56 and allow ROS to be broad and dynamic regulators of multiple responses to stress (FIG 3). Oxi-PTMs of different proteins during stress. Thiols in Cysteine (Cys) and Methionine (Met) residues of many proteins are susceptible to oxidation as they are intrinsically nucleophilic. However, their protein microenvironment, such as the presence of positively charged residues or hydrogen bonds, influences their reactivity57. The first ROS-induced oxidation intermediate of the Cys thiol is sulfenic acid (-SOH) which is highly reactive and reversible (FIG 3a). Sulfenic acid can be further oxidized to sulfinic (-SO2H) and sulfonic (SO3H) acids, both of which are considered to be mostly irreversible modifications triggering protein degradation (-SO2H can in some cases be reversed through the action of sulfiredoxin)58–60. Most common in the context of ROS signalling events are the reactions of sulfenic acids with proximal 5 proteinaceous thiols that are either interor intra-molecular (i.e., mixed disulfides), or with small molecules such as glutathione (GSH; i.e., S-glutathionylation)61,62 (FIG 3a). In addition to ROS, other reactive electrophilic species can modify Cys thiols. For example, nitric oxide (NO) can trigger the formation S-nitrosothiols (–SNO), whereas hydrogen sulfide (H2S) can react with – SOH to form persulfides (–SSH). Methionine residues of proteins can also undergo oxidation to form Met sulfoxides (MetO) that can be reduced back to Met by Met-sulfoxide reductases63. If they are not reduced back, Met-sulfoxide can be further converted into Met sulfone (MetO2). Recent studies have shown that the majority of the oxi-PTMs described above cause protein conformation changes (for example in kinases, phosphatases and transcription factors). ROS can thus induce changes in the properties of these proteins, including their activity, specificity and localization, which can activate or suppress stress-response signal transduction processes. Reversibility in ROS-induced oxi-PTMs as a key feature of ROS signalling. The ability to revert an oxiPTM in a regulated manner adds plasticity to ROS signalling during stress, especially when it comes to integrating different stress or developmental signals, and/or recovering from stress. Glutathionylation events are typically reversed back to the original thiol by GLUTAREDOXINs (GRXs), whereas proteindisulfides are mostly reduced back by THIOREDOXINs (TRXs) (FIG 3a)64–67. TRXs contain at least one conserved redox-active dithiol and form a mixed disulfide bond with their target proteins, regulating their structure and function, whereas GRXs function as oxidoreductases that regulate the redox state of thiol groups or exchange a glutathionylated moiety with a protein. These reactions can be highly selective, adding an extra level of complexity to redox signalling during stress. Depending on the original context of the oxiPTM, reversing it can reactivate or suppress protein function, which can activate, suppress or alter stressresponse pathways. A unique role for THIOL PEROXIDASEs (TPXs) in ROS signalling. Thiol-based peroxidases, such as GLUTATHIONE PEROXIDASEs (GPXs) and PEROXIREDOXINs (PrxRs) can reduce H2O2, peroxynitrites and different organic peroxides68,69. In addition to this peroxiredoxin activity, they can act as redox sensors transducing the H2O2 signal to different regulatory or enzymatic targets (FIG 3a). The high affinity of GPXs for H2O2, combined with their relatively low peroxidase activity, make some GPXs ideal candidates for these signalling functions. It was found, for example, that in yeast GPX3 conveys an H2O2 signal to the transcription factor YAP1 to regulate a multitude of H2O2 transcriptional responses70. In plants, a dual role of scavenging and signalling was proposed for AtGPXL3, as loss-of-function gpxl3 mutants displayed higher sensitivity to H2O2 treatments and, in vitro, AtGPXL3 suppressed the activity of the 2Ctype Ser/Thr protein phosphatase 2A (FIG 3b)71. 6 GSH and the ascorbate-glutathione (ASC-GSH) cycle. The Foyer-Asada-Halliwell pathway (also known as the ASC-GSH cycle)5,8,72 is an NADPH-driven H2O2-scavenging pathway found in many plant subcellular compartments (Supplementary Box 1). Although an integral part of the ASC-GSH cycle, GSH is also used by other pathways; for example, GSH is oxidized by PrxR and/or GPX. Although the ASCGSH cycle was originally considered to be a potent first line of defense against excessive H2O2 accumulation, changes in the oxidation state of the GSH pool (that is, changes in the GSH/GSSG ratio), caused by the function of the ASC-GSH cycle, also act as a sensing mechanism for altered ROS levels and redox perturbations during stress72–76. ROS-induced changes in the GSH/GSSG ratio can induce oxi-PTMs of Cys residues of receptors, signal transducers, RBOHs, transcription factors and other proteins, potentially through S-glutathionylation (FIG 3a). In addition to directly oxidizing Cys residues, H2O2 can therefore impact the GSH/GSSG ratio in cells through the ASC-GSH cycle, PrxRs and GPXs, indirectly regulating GSH-driven oxi-PTMs. Recent examples of ROS-induced oxi-PTMs involved in stress signalling in plants. ROSand redoxdriven oxi-PTMs regulate many metabolic reactions in plant cells (e.g., the Calvin–Benson cycle), as well as the activity of different kinases, phosphatases, transcription factors and chromatin/RNA processing regulators, ion channels, and receptors during stress (FIG 3b-f). Some of the most prominent examples include inhibition of protein phosphatases such as protein TYR PHOSPHATASE (PTP), CLASS 2 PROTEIN PHOSPHATASE (PP2A or PP2C; FIG 3b), the catabolic phosphatase SAL1, and the phosphatase STARCH-EXCESS 4 (SEX4)56,77–79, involved in hormone, metabolic and retrograde signalling. In contrast, ROS-induced oxi-PTMs activate MITOGENACTIVATED PROTEIN KINASE (MAPK) cascades such as the MEKK1-MM1/2-MPK4/6 cascade, and Ser/Thr kinases, required for the full activation of MPK3 and MPK680–82, that play key roles in the induction of pathogen and stress responses. Additional examples for important oxi-PTM targets during stress include transcriptional regulators such as NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1; FIG 3c), HEAT SHOCK TRANSCRIPTION FACTORs (HSFs), C-REPEAT BINDING FACTORs (CBFs), ANAC089, MYB30, and RADICAL-INDUCED CELL DEATH 1 (RCD1)46,83–88, involved in pathogen, heat, cold and retrograde signaling, respectively. Although histones are not typically subjected to oxi-PTMs, chromatin and histone modifiers such as the methyltransferase PROTEIN ARGININE METHYLTRANSFERASE 5 (PRMT5), as well as the DICER proteins DCL3 and DCL4, and RNASE THREE LIKE 1 (RTL1; FIG 3d) are; linking ROS to gene regulation89. In addition, ion channels such as the STELAR K+ OUTWARD RECTIFIER (SKOR) efflux channel, involved in drought and nutrient stress responses, were shown to undergo oxi-PTM90. 7 ROS were also shown to induce the oxidation of BRASSINAZOLE-RESISTANT 1 (BZR1), which functions as a master regulator of brassinosteroid signalling in plants, causing it to bind DNA and alter stress responses (FIG 3e)91. Lastly, receptors such as the leucine-rich-repeat receptor kinase HYDROGENPEROXIDE-INDUCED CALCIUM INCREASES 1 (HPCA1; FIG 3f), were recently shown to undergo oxi-PTMs at their extracellular domains leading to autophosphorylation and subsequent activation of plasma membrane (PM)-localized Ca2+ channels92, that trigger stomatal closure in response to stress. HPCA1 was also identified as CANNOT RESPOND TO DMBQ 1 (CARD1) involved in the signalling response of plants to quinones93, required for the interaction of parasitic plants with their hosts. A recent study has identified QUIESCIN SULFHYDRYL OXIDASE 1 (QSOX1) as a redox sensor that inactivates S-NITROSOGLUTATHIONE REDUCTASE (GSNOR), which leads to increased levels of Snitrosoglutathione (GSNO), S-nitrosylation, and inactivation of RBOHs94. QSOX1 could therefore function as part of a negative feedback loop that decreases ROS production upon ROS accumulation in cells. A recent Cryo-EM analysis of the plant GLUTAMATE RECEPTOR LIKE (GLR) channel GLR3.4, that plays a key role in Ca2+ signalling, revealed that GSH regulates GLR3.4 channel activity by binding to Cys 205 in the amino-terminal domain of each subunit of the protein tetramer95. The redox level of the cell, reflected in the levels of free GSH could therefore impact Ca2+ signalling. The potential of ROS to induce oxi-PTMs of so many different components of numerous signal transduction pathways, as well as different ion channels and other metabolic enzymes, highlights the important part that ROS play in stress sensing and signalling in plants. To understand and potentially modulate these roles, it is important to know how ROS levels are regulated across the different plant subcellular compartments, as discussed below. ROS signalling pathways in plants In the complex subcellular environment of plant cells, the sensing of ROS and activation of different signal transduction pathways can occur at different compartments (FIG 2). In general, ROS signalling can be divided into extrinsic (apoplast and cell wall), intrinsic (cytosol and nucleus) and organellar (chloroplast, mitochondria, peroxisomes and other compartments; FIG 4a). Recent studies revealed that these different routes can interact or remain separate during stress. Extrinsic ROS signalling. The apoplast and cell wall contain multiple enzymes that scavenge or actively produce ROS, as well as several non-enzymatic antioxidants (Supplementary Table 2). RBOHs, AQPs, and cell wall-bound PEROXIDASEs (PRXs) have the greatest role in ROS signalling at the apoplast (FIG 4a). 8 RBOHs are highly regulated transmembrane proteins that use cytosolic NADPH to generate O2.- in the apoplast (converted to H2O2 spontaneously or by SUPEROXIDE DISMUTASEs; SODs)6–8. They are thought to reside at the PM in nano domains together with several ancillary proteins involved in their regulation96–98. ROS production by RBOHs can be regulated by the binding of Ca2+ to EF-hand domains in their cytosolic N-terminal region, phosphorylation/dephosphorylation of their cytosolic Nor C-terminals, binding of phosphatidic acid, and/or binding of RHO OF PLANTS (ROP) small GTP-binding proteins. Recent studies have shown that RBOHs are also regulated by ubiquitination, persulfidation, nitrosylation, glutathionylation, and/or endocytosis99–113. RBOHs have been called ‘the engines of ROS signalling’ and are turned ‘on’ or ‘off’ in response to many different stresses, and/or other stimuli, driving the formation of ROS signatures at the apoplast (FIG 4a)30,114–116. Cell-wall-bound PRXs can also produce or scavenge ROS under different conditions and have been shown to regulate apoplastic ROS levels in response to different stimuli31,117. Moreover, other oxidases localized to the apoplast produce ROS (Supplementary Table 2)118. ROS that accumulate in the apoplast can directly, or indirectly (potentially through redox-transducing proteins), react with different receptors (e.g., HPCA1), oxidize different antioxidants, and/or regulate Ca2+ and/or K+ channels (FIG 4a). However, to directly regulate intracellular pathways, ROS produced at the apoplast must enter cells via AQPs. AQPs are water channels that facilitate the transport of H2O210,119,120. The opening and closing of AQPs is regulated by phosphorylation, acetylation and/or guanidinylation, linking different signalling processes with ROS transport120–125. ROS and/or entire complexes of RBOHs can also enter cells via endocytosis and impact cytosolic ROS levels126. As ROS production via RBOHs and ROS transport via AQPs are regulated processes, ROS levels in the apoplast and cytosol, and their signalling functions, can be actively controlled in response to different stresses. Moreover, because apoplastic ROS production and entry into the cytosol are regulated through PTMs of RBOHs and AQPs at their cytosolic side, and ROS accumulation at the apoplast can trigger cytosolic phosphorylation reactions via receptors and alter Ca2+ fluxes through plasma membrane channels, the apoplastcytosol interface is emerging as a major hub for many ROS-associated signal transduction processes during stress (FIG 4a). Intrinsic ROS signalling. The cytosol contains many ROS scavenging mechanisms, as well as a few ROS producing enzymes (Supplementary Table 2). These are thought to regulate ROS signals generated in the cytosol as well as ROS signals transported from the apoplast or the different organelles to the nucleus, via the cytosol (FIG 4a)127,128. In addition, the cytosol contains many different signalling hubs, such as MAPK cascades, CALCIUM-DEPENDENT PROTEIN KINASEs (CDPKs or CPKs), CALCINEURIN B-LIKE (CBL)-interacting protein kinases (CIPKs), ROP/RAC small GTPases, different phosphatases (PP2As, PP2Cs, PTPs)129 and different redox sensing networks (e.g., PrxRs, GRXs, TRXs) that integrate different ROS signals with other signalling molecules, such as Ca2+ and different hormones (FIG 4a). 9 As AQPs found at the PM and/or organelle membranes facilitate the transport of H2O2 in both directions, cytosolic H2O2 levels can impact H2O2 levels in other compartments and vice versa. In addition, retrograde and anterograde signals between organelles and the nucleus are relayed via the cytosol46,47,49,56. Indeed, manipulating the ability of the cytosol to scavenge ROS can change signalling in response to stress and alter acclimatization and/or defense responses, supporting a key role for the cytosol in regulating ROS signalling127,128,130,131. Furthermore, ROS gradients can form within cells, suggesting that cytosolic ROS scavenging mechanisms attenuate ROS signals132. Thus, the cytosol plays an important role in decoding and integrating different ROS signatures generated in different cell compartments, transferring the information stored in these signatures to the nucleus. Moreover, the ROSand redox-dependent activation of many transcriptional regulators that control plant stress responses, such as NPR1, HSFA, and ANACs, occurs in the cytosol before these proteins enter the nucleus46,133–136. Compared to the cytosol, regulation of ROS and redox levels in the nucleus are poorly understood. The plant nucleus contains several ROS and redox regulating proteins, such as GRXs, TRXs, PrxRs and GPXs, as well as GSH (Supplementary Table 2)137,138. These can regulate oxi-PTMs of different transcription factors, as well as attenuate ROS signals in the nucleus139–141. The findings that many redox-responsive transcriptional regulators are activated in the cytosol before entering the nucleus suggests that ROS levels in the nucleus are maintained under control to prevent extreme fluctuations which could cause DNA damage and mutations. One of the most important questions related to intrinsic ROS signalling is how can different ROS signals, generated in the different subcellular compartments during different stresses, reach the nucleus through the cytosol without losing their specificity?8,9 A possibility that has been put forward in recent studies34,39,143–149, is the inclusion of a separate ROS signalling network, that of organelles. Organelle ROS signalling network. The different plant cell organelles contain multiple ROS scavenging and producing mechanisms that regulate ROS signalling within each organelle as well as participate in organelleto-organelle and organelle-tonucleus communication (Supplementary Table 2)6–8,72,142. The levels of ROS in each compartment are determined by an interplay between three different processes: organelle-autonomous regulation, nucleus-controlled retrograde/anterograde regulation, and direct export/import (FIG 4b). Recent studies have shown that some ROS signals between organelles or from organelles to the nucleus do not cross the cytosol or cross the cytosol only over very short distances34,39. At least three different mechanisms are thought to play a role in this process: physical proximity between organelles (resulting in shorter distances and gradients), physical connections between different organelles and the nucleus, enabled by long tube-like extensions (e.g., stromules, peroxules and matrixules), and organelle-to-organelle protein complexes that form membrane contact sites and may contain aquaporins (FIG 4b)39. Examples to these mechanisms include stress-response ROS signalling mediated by sub- 16 transport’ that becomes activated across cells and along tissues (FIGs 1, 7). This distinction is important because unlike many other signalling molecules in plants, ROS are likely to be scavenged during transport over long distances. However, an auto-propagating state of ‘ROS production, scavenging and transport’ can maintain a certain steady-state ROS level or signature at almost any cellular location along its path. Moreover, it was recently reported that two ROS waves originating from different tissues of the same plant can integrate two different stress-induced signals leading to a state of enhanced acclimatization of the entire plant196. This finding indicates that the intracellular networks of ROS signalling in plants can extend to become an intercellular cell-to-cell network that integrates ROS signals from different cells or tissues and coordinate whole plant physiological responses that involve different molecular and metabolic mechanisms162–164,169,196,205,206. Conclusions and perspectives The study of ROS biology in plants initiated with a focus on ROS scavenging and production mechanisms in chloroplasts. This emphasis has changed into studying active ROS production, by for example RBOHs, and its regulation by different post-translational modifications. As ROS levels depend on the interplay between production, scavenging and transport (FIG 1), it will be important to determine the mechanisms that regulate ROS transport, for example by aquaporins or other transporters. Furthermore, our perspective on how ROS are produced in cells during stress should be re-evaluated. Whether the findings that the majority of ROS accumulation in plants during excess light stress is dependent on RBOHs152,160, rather than originating from chloroplasts, extends to other stresses and plant species should be determined. Moreover, this finding highlights that ROS might not be as toxic to cells as initially thought6. Further research is also needed to determine how organelle ROS signalling is linked to the cytosol, nucleus and the apoplast, and how information in the form of ROS signatures is transmitted between these different compartments. The roles of the vacuole, plasmodesmata and endoplasmic reticulum, and the underlying mechanisms that connect ROS signalling between these organelles and the rest of the cell, remain to be determined. Better understanding of the mechanisms that mediate auto-propagating ROS signals in plants and their link to stress responses is also needed. Furthermore, it is unknown whether different channels can actively transport (pump) H2O2 against a potential gradient, and whether cells contain different chaperone molecules that can transport ROS, such as H2O2, from one location to another while protecting them from degradation. The identification of new ROS and redox sensors, redox relays and hubs, and the study of ROS-responsive transcriptional networks, will increase our understanding of how ROS signals are integrated in response to stress. However, to fully elucidate ROS cellular networks, it is necessary to accurately determine ROS levels in different compartments using genetically encoded ROS and redox sensors25,28,29,36,38,40-45,207, as well 17 as to study ROS fluxes between different compartments and organelles. Only by obtaining an allencompassing portrait of the stress-induced ROS signalling landscape of the cell and linking it to plant transcriptional, metabolic and proteomic networks, it will be possible to fully understand the functions of ROS in plants in response to stress. 18 Glossary Acclimatization/Acclimation A process by which plants adjust their metabolism, physiology, and biochemistry to become accustomed to changes in their growth conditions or environment. Aquaporin A transmembrane water channel protein that allows the diffusion of H2O2 from one side of the membrane to the other in a regulated manner. Brassinosteroids A class of polyhydroxysteroids that function as plant hormones involved in many developmental processes and responses to stress. Photosystems I and II Multiprotein complexes that reside on the thylakoid membranes inside chloroplasts and participate in the harvesting of light energy for the purpose of CO2 fixation and sugar biosynthesis. Photorespiration A biochemical pathway that results in the accumulation of H2O2 in peroxisomes, triggered when CO2 concentrations are limited in C3 plants. C3 plants A large group of plants in which the initial product of the assimilation of CO2 through photosynthesis is 3-phosphoglycerate, which contains 3 carbon atoms. Nucleophilic attach Attack of an electron-rich species (the nucleophile) on an electron-deficient species (the electrophile), forming a new bond between the nucleophile and the electrophile. YAP1 A redox-regulated transcription factor that is essential for yeast survival under conditions of oxidative stress. 2C-type Ser/Thr protein phosphatase 2A A family of phosphatases that generally function as negative regulators of different stress responses in plants and are inhibited by ROS-induced redox reactions. DICER proteins Endoribonucleases that cleave double-stranded RNA and pre-microRNAs into short double-stranded RNA fragments called small interfering RNA or microRNA. Leucine-rich-repeat receptor kinases A large gene family in plants composed of a leucine-rich-repeat (LRR)-containing extracellular domain, a transmembrane domain, and an intracellular kinase domain, involved in developmental processes and stress responses. Quinones A redox active class of cyclic organic compounds containing two carbonyl groups, involved in many electron transport reactions, and signalling processes. EF hand A helix–loop–helix structural domain, with an E and F structural orientation of the two α-helices, found in many calcium-binding proteins. Stromules, peroxules, and matrixules Dynamic tubular membrane structures extending from the surface of chloroplasts, peroxisomes and mitochondria, respectively, used for the transport of signals between different organelles and the nucleus. Salicylic acid A phytohormone, characterized by an aromatic ring and a hydroxyl group, involved in the response of plant to different biotic and abiotic stresses. Mediator complex An important component of the eukaryotic transcriptional machinery, linking different transcription factors with RNA polymerase II. Unfolded protein response A cellular stress response pathway triggered by the presence of unfolded proteins inside the endoplasmic reticulum. 19 Plasmodesmata Small channels or pores that transverse the plant cell walls connecting the cytoplasm and plasma membrane of neighboring cells with each other, establishing metabolic and signalling bridges between cells. Stomata Specialized pore structures found in the epidermal layer of plants and used for gas exchange with the atmosphere. List of Supplementary Material Supplementary Box 1. ROS production and scavenging pathways of plants. Supplementary Box 2. Evolution of ROS metabolism in cells. Table S1. Biochemical, physical, and molecular properties of different ROS and RNS in cells. Table S2. Plant (Arabidopsis thaliana) proteins and small molecules regulating ROS levels in cells 20 References 1. Bailey-Serres, J., Parker, J. E., Ainsworth, E. A., Oldroyd, G. E. D. & Schroeder, J. I. Genetic strategies for improving crop yields. Nature 575, 109–118 (2019). 2. Lesk, C., Rowhani, P. & Ramankutty, N. Influence of extreme weather disasters on global crop production. Nature 529, 84–87 (2016). 3. IPCC 2021: Climate Change 2021: The Physical Science Basis. Contribution of Working Group I to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change. vol. In Press (Cambridge University Press, 2021). 4. Zandalinas, S. I., Fritschi, F. B. & Mittler, R. Global warming, climate change, and environmental pollution: Recipe for a multifactorial stress combination disaster. Trends Plant Sci. 26, 588–599 (2021). 5. Halliwell, B. & Gutteridge, J. M. C. Free radicals in biology and medicine. (Oxford University Press, 2015). 6. Mittler, R. ROS are good. Trends Plant Sci. 22, 11–19 (2017). 7. Waszczak, C., Carmody, M. & Kangasjärvi, J. Reactive oxygen species in plant signaling. Annu. Rev. Plant Biol. 69, 209–236 (2018). 8. Smirnoff, N. & Arnaud, D. Hydrogen peroxide metabolism and functions in plants. New Phytol. 221, 1197–1214 (2019). 9. Sies, H. & Jones, D. P. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat. Rev. Mol. Cell Biol. 21, 363–383 (2020). 10. Rodrigues, O. et al. Aquaporins facilitate hydrogen peroxide entry into guard cells to mediate ABAand pathogen-triggered stomatal closure. Proc. Natl. Acad. Sci. U. S. A. 114, 9200–9205 (2017). Revealing the physiological significance of aquaporins for H2O2 mobilization in plants 11. Zou, J. J. et al. Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE8 and CATALASE3 Function in Abscisic Acid-Mediated Signaling and H2O2 Homeostasis in Stomatal Guard Cells under Drought Stress. Plant Cell 27, 1445–1460 (2015). 12. Nie, W. F. et al. Silencing of tomato RBOH1 and MPK2 abolishes brassinosteroid-induced H₂O₂ generation and stress tolerance. Plant. Cell Environ. 36, 789–803 (2013). 13. Suzuki, N., Miller, G., Sejima, H., Harper, J. & Mittler, R. Enhanced seed production under prolonged heat stress conditions in Arabidopsis thaliana plants deficient in cytosolic ascorbate peroxidase 2. J. Exp. Bot. 64, 253–263 (2013). 14. Kim, M. J., Ciani, S. & Schachtman, D. P. A Peroxidase Contributes to ROS Production during Arabidopsis Root Response to Potassium Deficiency. Mol. Plant 3, 420–427 (2010). 15. Liu, D. et al. Tobacco transcription factor bHLH123 improves salt tolerance by activating NADPH oxidase NtRbohE expression. Plant Physiol. 186, 1706–1720 (2021). 16. Miura, K. et al. SIZ1 deficiency causes reduced stomatal aperture and enhanced drought tolerance via controlling salicylic acid-induced accumulation of reactive oxygen species in Arabidopsis. Plant J. 73, 91–104 (2013). 17. Hu, Y. et al. Silencing of OsGRXS17 in rice improves drought stress tolerance by modulating ROS 21 accumulation and stomatal closure. Sci. Rep. 7, 15950 (2017). 18. Yin, Y. et al. BZR1 Transcription Factor Regulates Heat Stress Tolerance Through FERONIA Receptor-Like Kinase-Mediated Reactive Oxygen Species Signaling in Tomato. Plant Cell Physiol. 59, 2239–2254 (2018). 19. Shi, Y. et al. OsRbohB-mediated ROS production plays a crucial role in drought stress tolerance of rice. Plant Cell Rep. 39, 1767–1784 (2020). 20. Martins, L. et al. Redox Modification of the Iron-Sulfur Glutaredoxin GRXS17 Activates Holdase Activity and Protects Plants from Heat Stress. Plant Physiol. 184, 676–692 (2020). 21. Chae, H. B. et al. Thioredoxin Reductase Type C (NTRC) Orchestrates Enhanced Thermotolerance to Arabidopsis by Its Redox-Dependent Holdase Chaperone Function. Mol. Plant 6, 323–336 (2013). 22. Wang, L. et al. The Arabidopsis SAFEGUARD1 suppresses singlet oxygen-induced stress responses by protecting grana margins. Proc. Natl. Acad. Sci. U. S. A. 117, 6918–6927 (2020). 23. Galvez-Valdivieso, G. et al. The high light response in Arabidopsis involves ABA signaling between vascular and bundle sheath cells. Plant Cell 21, 2143–2162 (2009). 24. Kerchev, P. et al. Lack of GLYCOLATE OXIDASE1, but not GLYCOLATE OXIDASE2, attenuates the photorespiratory phenotype of CATALASE2-deficient Arabidopsis. Plant Physiol. 171, 1704–1719 (2016). 25. Hipsch, M. et al. Sensing stress responses in potato with whole-plant redox imaging. Plant Physiol. 187, 618-631 (2021). 26. Koffler, B. E., Luschin-Ebengreuth, N., Stabentheiner, E., Müller, M. & Zechmann, B. Compartment specific response of antioxidants to drought stress in Arabidopsis. Plant Sci. 227, 133–144 (2014). 27. Zia, A. et al. Protection of the photosynthetic apparatus against dehydration stress in the resurrection plant Craterostigma pumilum. Plant J. 87, 664–680 (2016). 28. Schwarzländer, M., Fricker, M. D. & Sweetlove, L. J. Monitoring the in vivo redox state of plant mitochondria: Effect of respiratory inhibitors, abiotic stress and assessment of recovery from oxidative challenge. Biochim. Biophys. Acta - Bioenerg. 1787, 468–475 (2009). 29. Babbar, R., Karpinska, B., Grover, A. & Foyer, C. H. Heat-induced oxidation of the nuclei and cytosol. Front. Plant Sci. 11, 617779 (2021). 30. Torres, M. A., Dangl, J. L. & Jones, J. D. G. G. Arabidopsis gp91 phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc. Natl. Acad. Sci. U. S. A. 99, 517–522 (2002). Revealing the role of RBOHs in generating ROS during early responses to pathogens 31. Kámán‐Tóth, E. et al. Contribution of cell wall peroxidase‐ and NADPH oxidase‐derived reactive oxygen species to Alternaria brassicicola‐induced oxidative burst in Arabidopsis. Mol. Plant Pathol. 20, 485–499 (2019). 32. Xu, Q. et al. An effector protein of the wheat stripe rust fungus targets chloroplasts and suppresses chloroplast function. Nat. Commun. 10, 5571 (2019). 33. Liu, R. et al. A Plasmopara viticola RXLR effector targets a chloroplast protein PsbP to inhibit ROS 22 production in grapevine. Plant J. 106, 1557–1570 (2021). 34. Ding, X., Jimenez‐Gongora, T., Krenz, B. & Lozano‐Duran, R. Chloroplast clustering around the nucleus is a general response to pathogen perception in Nicotiana benthamiana. Mol. Plant Pathol. 20, 1298–1306 (2019). 35. Yang, M. et al. Barley Stripe Mosaic Virus γb Interacts with Glycolate Oxidase and Inhibits Peroxisomal ROS Production to Facilitate Virus Infection. Mol. Plant 11, 338–341 (2018). 36. Bratt, A., Rosenwasser, S., Meyer, A. & Fluhr, R. Organelle redox autonomy during environmental stress. Plant. Cell Environ. 39, 1909–1919 (2016). 37. Mor, A. et al. Singlet Oxygen Signatures Are Detected Independent of Light or Chloroplasts in Response to Multiple Stresses. Plant Physiol. 165, 249–261 (2014). 38. Niemeyer, J., Scheuring, D., Oestreicher, J., Morgan, B. & Schroda, M. Real-time monitoring of subcellular H2O2 distribution in Chlamydomonas reinhardtii. Plant Cell 33, 2935–2949 (2021). 39. Exposito-Rodriguez, M., Laissue, P. P., Yvon-Durocher, G., Smirnoff, N. & Mullineaux, P. M. Photosynthesis-dependent H2O2 transfer from chloroplasts to nuclei provides a high-light signalling mechanism. Nat. Commun. 8, 49 (2017). Revealing chloroplast to nucleus signalling during light stress 40. Ugalde, J. M. et al. Chloroplast-derived photo-oxidative stress causes changes in H2O2 and EGSH in other subcellular compartments. Plant Physiol. 186, 125–141 (2021). 41. Mizrachi, A., Creveld, S. G., Shapiro, O. H., Rosenwasser, S. & Vardi, A. Light-dependent singlecell heterogeneity in the chloroplast redox state regulates cell fate in a marine diatom. Elife 8, e47732 (2019). 42. Nietzel, T. et al. The fluorescent protein sensor roGFP2-Orp1 monitors in vivo H2O2 and thiol redox integration and elucidates intracellular H2O2 dynamics during elicitor-induced oxidative burst in Arabidopsis. New Phytol. 221, 1649–1664 (2019). 43. Marty, L. et al. Arabidopsis glutathione reductase 2 is indispensable in plastids, while mitochondrial glutathione is safeguarded by additional reduction and transport systems. New Phytol. 224, 1569– 1584 (2019). 44. Müller‐Schüssele, S. J. et al. Chloroplasts require glutathione reductase to balance reactive oxygen species and maintain efficient photosynthesis. Plant J. 103, 1140–1154 (2020). 45. Haber, Z. et al. Resolving diurnal dynamics of the chloroplastic glutathione redox state in Arabidopsis reveals its photosynthetically derived oxidation. Plant Cell 33, 1828–1844 (2021). 46. Shapiguzov, A. et al. Arabidopsis RCD1 coordinates chloroplast and mitochondrial functions through interaction with ANAC transcription factors. Elife 8, e43284 (2019). Revealing a role for ROS in retrograde signalling 47. Nomura, H. et al. Chloroplast-mediated activation of plant immune signalling in Arabidopsis. Nat. Commun. 3, 926 (2012). 48. Bienert, G. P. & Chaumont, F. Aquaporin-facilitated transmembrane diffusion of hydrogen peroxide. Biochim. Biophys. Acta 1840, 1596–1604 (2014). 49. Estavillo, G. M. et al. Evidence for a SAL1-PAP chloroplast retrograde pathway that functions in 23 drought and high light signaling in Arabidopsis. Plant Cell 23, 3992–4012 (2011). Revealing a role for ROS in retrograde signalling 50. Inupakutika, M. A., Sengupta, S., Devireddy, A. R., Azad, R. K. & Mittler, R. The evolution of reactive oxygen species metabolism. J. Exp. Bot. 67, 5933–5943 (2016). 51. Jabłońska, J. & Tawfik, D. S. The evolution of oxygen-utilizing enzymes suggests early biosphere oxygenation. Nat. Ecol. Evol. 5, 442–448 (2021). 52. Huang, J. et al. Mining for protein S-sulfenylation in Arabidopsis uncovers redox-sensitive sites. Proc. Natl. Acad. Sci. U. S. A. 116, 20256–20261 (2019). 53. Nietzel, T. et al. Redox-mediated kick-start of mitochondrial energy metabolism drives resourceefficient seed germination. Proc. Natl. Acad. Sci. U. S. A. 117, 741–751 (2020). 54. De Smet, B. et al. In vivo detection of protein cysteine sulfenylation in plastids. Plant J. 97, 765– 778 (2019). 55. Leferink, N. G. H., Duijn, E. van, Barendregt, A., Heck, A. J. R. & Berkel, W. J. H. van. Galactonolactone Dehydrogenase Requires a Redox-Sensitive Thiol for Optimal Production of Vitamin C. Plant Physiol. 150, 596–605 (2009). 56. Chan, K. X. et al. Sensing and signaling of oxidative stress in chloroplasts by inactivation of the SAL1 phosphoadenosine phosphatase. Proc. Natl. Acad. Sci. U. S. A. 113, E4567–E4576 (2016). 57. Zaffagnini, M. et al. Tuning Cysteine Reactivity and Sulfenic Acid Stability by Protein Microenvironment in Glyceraldehyde-3-Phosphate Dehydrogenases of Arabidopsis thaliana. Antioxid. Redox Signal. 24, 502–517 (2016). 58. Akter, S. et al. Chemical proteomics reveals new targets of cysteine sulfinic acid reductase. Nat. Chem. Biol. 14, 995–1004 (2018). 59. Puerto-Galán, L., Pérez-Ruiz, J. M., Guinea, M. & Cejudo, F. J. The contribution of NADPH thioredoxin reductase C (NTRC) and sulfiredoxin to 2-Cys peroxiredoxin overoxidation in Arabidopsis thaliana chloroplasts. J. Exp. Bot. 66, 2957–2966 (2015). 60. Iglesias-Baena, I., Barranco-Medina, S., Sevilla, F. & Lázaro, J.-J. The Dual-Targeted Plant Sulfiredoxin Retroreduces the Sulfinic Form of Atypical Mitochondrial Peroxiredoxin. Plant Physiol. 155, 944–955 (2011). 61. Bender, K. W. et al. Glutaredoxin AtGRXC2 catalyses inhibitory glutathionylation of arabidopsis BRI1-associated receptor-like kinase 1 (BAK1) in vitro. Biochem. J. 467, 399–413 (2015). 62. Niazi, A. K. et al. Cytosolic isocitrate dehydrogenase from Arabidopsis thaliana is regulated by glutathionylation. Antioxidants 8, 16 (2019). 63. Jacques, S. et al. Protein methionine sulfoxide dynamics in Arabidopsis thaliana under oxidative stress. Mol. Cell. Proteomics 14, 1217–1229 (2015). 64. Trnka, D. et al. Molecular basis for the distinct functions of redox-active and FeS-transfering glutaredoxins. Nat. Commun. 11, 3445 (2020). 65. Tarrago, L. et al. Regeneration Mechanisms of Arabidopsis thaliana Methionine Sulfoxide Reductases B by Glutaredoxins and Thioredoxins. J. Biol. Chem. 284, 18963–18971 (2009). 66. Tossounian, M. A. et al. Disulfide bond formation protects Arabidopsis thaliana glutathione 24 transferase tau 23 from oxidative damage. Biochim. Biophys. Acta - Gen. Subj. 1862, 775–789 (2018). 67. Daloso, D. M. et al. Thioredoxin, a master regulator of the tricarboxylic acid cycle in plant mitochondria. Proc. Natl. Acad. Sci. U. S. A. 112, E1392–E1400 (2015). 68. Attacha, S. et al. Glutathione peroxidase-like enzymes cover five distinct cell compartments and membrane surfaces in Arabidopsis thaliana. Plant. Cell Environ. 40, 1281–1295 (2017). 69. Liebthal, M., Schuetze, J., Dreyer, A., Mock, H.-P. & Dietz, K.-J. Redox Conformation-Specific Protein–Protein Interactions of the 2-Cysteine Peroxiredoxin in Arabidopsis. Antioxidants 9, 515 (2020). 70. Delaunay, A., Pflieger, D., Barrault, M. B., Vinh, J. & Toledano, M. B. A thiol peroxidase is an H2O2 receptor and redox-transducer in gene activation. Cell 111, 471–481 (2002). Discovery of thiol peroxidases as sensors of H2O2 71. Miao, Y. et al. An Arabidopsis glutathione peroxidase functions as both a redox transducer and a scavenger in abscisic acid and drought stress responses. Plant Cell 18, 2749–2766 (2006). 72. Foyer, C. H. & Noctor, G. Redox signaling in plants. Antioxid. Redox Signal. 18, 2087–2090 (2013). 73. Rattanawong, K. et al. Regulatory functions of ROS dynamics via glutathione metabolism and glutathione peroxidase activity in developing rice zygote. Plant J. 108, 1097-1115 (2021). 74. Han, Y. et al. Functional Analysis of Arabidopsis Mutants Points to Novel Roles for Glutathione in Coupling H2O2 to Activation of Salicylic Acid Accumulation and Signaling. Antioxid. Redox Signal. 18, 2106–2121 (2013). 75. Terai, Y. et al. Dehydroascorbate Reductases and Glutathione Set a Threshold for High-Light– Induced Ascorbate Accumulation. Plant Physiol. 183, 112–122 (2020). 76. Zhang, T. et al. Glutathione-dependent denitrosation of GSNOR1 promotes oxidative signalling downstream of H2O2. Plant. Cell Environ. 43, 1175–1191 (2020). 77. Gupta, R. & Luan, S. Redox Control of Protein Tyrosine Phosphatases and Mitogen-Activated Protein Kinases in Plants. Plant Physiol. 132, 1149–1152 (2003). 78. Murata, Y., Pei, Z. M., Mori, I. C. & Schroeder, J. Abscisic acid activation of plasma membrane Ca(2+) channels in guard cells requires cytosolic NAD(P)H and is differentially disrupted upstream and downstream of reactive oxygen species production in abi1-1 and abi2-1 protein phosphatase 2C mutants. Plant Cell 13, 2513–2523 (2001). 79. Silver, D. M. et al. Insight into the redox regulation of the phosphoglucan phosphatase SEX4 involved in starch degradation. FEBS J. 280, 538–548 (2013). 80. Kovtun, Y., Chiu, W. L., Tena, G. & Sheen, J. Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc. Natl. Acad. Sci. U. S. A. 97, 2940–2945 (2000). 81. Matsushita, M., Nakamura, T., Moriizumi, H., Miki, H. & Takekawa, M. Stress-responsive MTK1 SAPKKK serves as a redox sensor that mediates delayed and sustained activation of SAPKs by oxidative stress. Sci. Adv. 6, eaay9778 (2020). 82. Byrne, D. P. et al. Aurora A regulation by reversible cysteine oxidation reveals evolutionarily conserved redox control of Ser/Thr protein kinase activity. Sci. Signal. 13, eaax2713 (2020). 25 83. Després, C. et al. The Arabidopsis NPR1 Disease Resistance Protein Is a Novel Cofactor That Confers Redox Regulation of DNA Binding Activity to the Basic Domain/Leucine Zipper Transcription Factor TGA1. Plant Cell 15, 2181–2191 (2003). 84. Lindermayr, C., Sell, S., Müller, B., Leister, D. & Durner, J. Redox Regulation of the NPR1-TGA1 System of Arabidopsis thaliana by Nitric Oxide. Plant Cell 22, 2894–2907 (2010). 85. Tavares, C. P. et al. S-nitrosylation influences the structure and DNA binding activity of AtMYB30 transcription factor from Arabidopsis thaliana. Biochim. Biophys. Acta - Proteins Proteomics 1844, 810–817 (2014). 86. Liu, H. & Charng, Y. Common and Distinct Functions of Arabidopsis Class A1 and A2 Heat Shock Factors in Diverse Abiotic Stress Responses and Development. Plant Physiol. 163, 276–290 (2013). 87. Albertos, P. et al. Redox feedback regulation of ANAC089 signaling alters seed germination and stress response. Cell Rep. 35, 109263 (2021). 88. Lee, E. S. et al. Redox-dependent structural switch and CBF activation confer freezing tolerance in plants. Nat. Plants 7, 914–922 (2021). 89. Charbonnel, C. et al. The siRNA suppressor RTL1 is redox-regulated through glutathionylation of a conserved cysteine in the double-stranded-RNA-binding domain. Nucleic Acids Res. 45, 11891– 11907 (2017). 90. Garcia-Mata, C. et al. A Minimal Cysteine Motif Required to Activate the SKOR K+ Channel of Arabidopsis by the Reactive Oxygen Species H2O2. J. Biol. Chem. 285, 29286–29294 (2010). 91. Tian, Y. et al. Hydrogen peroxide positively regulates brassinosteroid signaling through oxidation of the BRASSINAZOLE-RESISTANT1 transcription factor. Nat. Commun. 9, 1063 (2018). 92. Wu, F. et al. Hydrogen peroxide sensor HPCA1 is an LRR receptor kinase in Arabidopsis. Nature 578, 577–581 (2020). Discovery of HPCA1 as an LRR sensor for H2O2 93. Laohavisit, A. et al. Quinone perception in plants via leucine-rich-repeat receptor-like kinases. Nature 587, 92–97 (2020). 94. Chae, H. B. et al. Redox sensor QSOX1 regulates plant immunity by targeting GSNOR to modulate ROS generation. Mol. Plant 14, 1312–1327 (2021). 95. Green, M. N. et al. Structure of the Arabidopsis thaliana glutamate receptor-like channel GLR3.4. Mol. Cell 81, 3216-3226.e8 (2021). 96. Hao, H. et al. Clathrin and membrane microdomains cooperatively regulate RbohD dynamics and activity in Arabidopsis. Plant Cell 26, 1729–1745 (2014). 97. Nagano, M. et al. Plasma membrane microdomains are essential for Rac1-RbohB/H-mediated immunity in rice. Plant Cell 28, 1966–1983 (2016). 98. Martinière, A. et al. Osmotic stress activates two reactive oxygen species pathways with distinct effects on protein nanodomains and diffusion. Plant Physiol. 179, 1581–1593 (2019). 99. Nühse, T. S., Bottrill, A. R., Jones, A. M. E. & Peck, S. C. Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses. Plant J. 51, 931–940 (2007). 32 Discovery of the redox wave 205. Devireddy, A. R., Zandalinas, S. I., Gómez-Cadenas, A., Blumwald, E. & Mittler, R. Coordinating the overall stomatal response of plants: Rapid leaf-to-leaf communication during light stress. Sci. Signal. 11, eaam9514 (2018). 206. Devireddy, A. R., Arbogast, J. & Mittler, R. Coordinated and rapid whole‐plant systemic stomatal responses. New Phytol. 225, 21–25 (2020). 207. Steinbeck, J. et al. In vivo NADH/NAD+ biosensing reveals the dynamics of cytosolic redox metabolism in plants. Plant Cell 32, 3324–3345 (2020). Competing interests The authors declare no competing interests. Acknowledgments We apologize to the many authors whose important work could not be referred to. This work was supported by funding from the National Science Foundation (IOS-2110017, IOS-1353886, MCB-1936590, IOS1932639), National Institute of Health (GM111364), Interdisciplinary Plant Group, University of Missouri, Research Foundation-Flanders (project G0D7914N), and The Excellence of Science Research project 30829584. Author contributions RM, SIZ, YF and FVB wrote the review, designed the figures, and approved the final version of the manuscript. 33 Figures Fig. 1 ׀ Regulation of ROS metabolism and signalling in plants. a ׀ Formation of ROS by excitation or reduction of atmospheric oxygen. b ׀ Cellular ROS concentrations are regulated by three distinct processes: ROS production, scavenging and transport. These processes determine the steady-state levels of ROS; they also generate different ROS signatures and gradients (characterized by different concentrations of the different types of ROS within organelles and cells), that function as signals. In response to external or internal stimuli, ROS levels change. ROS levels in cells are sensed and decoded through changes in the redox state of different proteins that lead to coordinated responses. In addition to their localized function within cells, ROS production, scavenging and transport can propagate, along membranes, between organelles or between cells, altering the steady-state levels of ROS in the entire plant. Dashed arrows indicate that ROS production, scavenging and transport can be regulated depending on the redox state of the cell. AQP, aquaporin; e, electron; ROS, reactive oxygen species; SOD, superoxide dismutase. 34 Fig. 2 ׀ Production and scavenging of ROS in different compartments in plants during stress. The interplay between ROS production and scavenging in each cell compartment, including the cell wall and apoplast, during stress generates compartment-specific ROS signatures (hypothetical signatures are indicated on right). These are integrated with other (non-ROS) retrograde signals that reach the nucleus, alter the nuclear ROS signature and trigger defense and acclimatization responses. Organelle-to-organelle ROS communication is not depicted. A list of all ROS metabolism reactions and enzymes involved is included in Supplementary Table 2. ER, endoplasmic reticulum, ROOH, organic hydroperoxide; ROS, reactive oxygen species; RNS, reactive nitrogen species. 35 Fig. 3 ׀ Mechanisms of ROS and redox sensing in plants. a ׀ H2O2 alters protein structure and function through oxidation of Cys thiols (directly or through the function of GPXs or PrxRs). H2O2 also affects the ratio between oxidized and reduced GSH (directly or through the function of the ASC-GSH cycle), further altering protein structure and function through S-glutathionylation. These oxidative posttranscriptional modifications (oxi-PTMs) can be reversed through the function of GRXs, PrxR and TRXs allowing ROS such as H2O2 to activate or suppress different cellular functions in a reversible fashion. b ׀ Regulation of PP2A (phosphatase) function by protein oxidation, used to control stomatal aperture closing by ABA in response to water deficit stress. c ׀ Regulation of the transcription factor NPR1 translocation into the nucleus by Cys oxidation and S-glutathionylation, used to control gene expression in response to pathogens. d ׀ Regulation of siRNA binding by the plant protein RTL1, used to control the function of the endoribonuclease complex DICER shown to be involved in responses to viral pathogens. e ׀ Regulation of DNA binding by oxidation of the transcriptional switch BZR1, used to control brassinosteroid responses to many different abiotic stresses including heat and drought. f ׀ Regulation of the ROS/redox receptor HPCA1 by protein oxidation during responses to pathogen infection. Dashed arrows indicate regulation by redox changes. ABA, abscisic acid; APX, ascorbate peroxidase; ARF6, auxin response factor 6; ASC, ascorbate; BZR1, brassinazole-resistant 1; CAT, catalase; Cys, cysteine; DHA, dehydroascorbate; GPX, glutathione peroxidases; GR, glutathione reductase; GRX, glutaredoxin; GSH, glutathione; GSNO, S- 36 nitrosoglutathione; GSSG, oxidized glutathione; HPCA1, hydrogen-peroxide-induced calcium increases 1; MDHAR, monodehydroascorbate reductase; NO, nitric oxide; NPR1, nonexpressor of pathogenesis-related genes 1; OST1, open stomata 1; oxi, oxidized; oxi-PTM, oxidative posttranscriptional modifications; P, phosphate; PIF4, phytochrome-interacting factor 4; PP2A, protein phosphatase 2A; PrxR; peroxiredoxin; PYR/PYL/RCAR, pyrabactin resistance/pyr-like/regulatory components of ABA receptors; red, reduced; RBOH, respiratory burst oxidase homolog; ROS, reactive oxygen species; RTL1, RNAse three like 1; SRX, sulfiredoxin; T, target; TRX, thioredoxins. 37 Fig. 4 ׀ Integration of ROS signals in plant cells. a ׀ ROS signalling in plants can be divided into extrinsic-, intrinsicand organellelocalized pathways. These are integrated through the function of RBOHs, AQPs, various Ca2+ channels, receptors and various kinases and phosphatases that link ROS signalling with calcium, phosphorylation, PA and redox signalling, and trigger transcriptional responses to stress. b ׀ The level of ROS in each organelle can be autonomously controlled through pre-existing organellar ROS production, sensing, scavenging and transport mechanisms, regulated by the nucleus through retrograde/anterograde signalling and newly synthesized (inducible) proteins, and/or modulated through ROS export/import from other organelles (Top). Organelles can impact the levels of ROS in each other or the nucleus through complexes, membrane extensions, diffusion, and/or metabolite/protein-derived signalling (Bottom). c ׀ ROS can accumulate to high levels in different compartments of the cell and impact H2O2 levels in the cytosol and nuclei. Because different compartments are linked with each other and the transport of ROS between different compartments is regulated, different stresses can generate stimulispecific ‘maps’ or ‘landscapes’ of ROS concentrations, across the different cellular compartments, that will alter H2O2 levels in the cytosol and nuclei and trigger stress-specific acclimatization and/or defense mechanisms. Dashed arrow indicates retrograde signalling. Question marks indicate that ROS levels are not known yet. AQP, aquaporin; apoROS, apoplastic ROS; CDPK, Ca2+-dependent protein kinases; chlROS, chloroplastic ROS; CIPK, calcineurin B-like-interacting protein kinases; CPK, Ca2+-dependent 38 protein kinases; cwROS, cell wall-associated ROS; cytROS, cytosolic ROS; ER, endoplasmic reticulum; erROS, endoplasmic reticulum-associated ROS; GPX, glutathione peroxidases; GRX, glutaredoxins; HPCA1, hydrogen-peroxide-induced Ca2+ increase 1; M, metabolite; MAPK, mitogen-activated protein kinase; mitROS, mitochondrial ROS; nROS, nuclear ROS; OST1, open stomata 1; OXI1, oxidative signalinducible 1; P, phosphate; PA, phosphatidic acid; pdROS, plasmodesmatal ROS; perROS, peroxisomal ROS; PRX, peroxidases; PrxR, peroxiredoxin; PDK1, 3-phosphoinositide-dependent protein kinase1; PLD, Phospholipase D; PP2s, protein phosphatase 2; RBOH, respiratory burst oxidase homolog; RLK, receptorlike kinases; ROP, Rho of Plants; ROS, reactive oxygen species; SOD, superoxide dismutase; TRX, thioredoxins; vacROS, vacuolar ROS. 39 Fig. 5 ׀ ROS in early and late responses of plants to stress. a ׀ Different plant sensors for changes in environmental conditions such as temperature, light intensity/quality and osmotic potential are directly linked to ROS signalling through the RBOH signalling hub. These links allow ROS signalling to be triggered during early stages of stress sensing in plants. b ׀ During early responses (seconds to minutes of stress initiation; stages 1 and 2), ROS produced ‘actively’ or ‘passively’ in cells are used to sense stress and trigger signal transduction mechanisms, while during late responses (minutes to hours of stress initiation; stage 3), ROS are used to regulate different networks and metabolic responses, balance plant 40 acclimatization and defense, and induce stress memory. Dashed arrows indicate ROS and other stress metabolites used for early stress signalling. Question marks indicate possible links. ANN1, annexin1; CDPK, Ca2+-dependent protein kinases; CNGC, cyclic nucleotide gated channel; Cry, cryptochrome; e, electron; ETC, electron transport chain; MSL10, mechanosensitive channel of small conductance-like 10; NO, nitric oxide; OSCA1, Reduced hyperosmolality, induced Ca2+ increase 1; P, phosphate; PA, phosphatidic acid; PhyB, phytochrome B; PLD, phospholipase D; PPI, protein-protein interactions; RBOH, respiratory burst oxidase homolog; RLK, receptor-like kinases; ROP, Rho of Plants; ROS, reactive oxygen species; Ubi, ubiquitination. 41