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Stress results in the enhanced accumulation of reactive oxygen species (ROS) in plants, altering the redox state of cells and triggering the activation of multiple defense and acclimation mechanisms. In addition to activating ROS and redox responses in tissues that are directly subjected to stress (termed 'local' tissues), the sensing of stress in plants triggers different systemic signals that travel to other parts of the plant (termed 'systemic' tissues) and activate acclimation and defense mechanisms in them; even before they are subjected to stress. Among the different systemic signals triggered by stress in plants are electric, calcium, ROS, and redox waves that are mobilized in a cell-to-cell fashion from local to systemic tissues over long distances, sometimes at speeds of up to several millimeters per second. Here, we discuss new studies that identified various molecular mechanisms and proteins involved in mediating systemic signals in plants. In addition, we highlight recent studies that are beginning to unravel the mode of integration and hierarchy of the different systemic signals and underline open questions that require further attention. Unraveling the role of ROS and redox in plant stress responses is highly important for the development of climate resilient crops.

Cell penetrating peptides (CPPs) facilitate translocation across biological membranes and are of significant biological and medical interest. Several CPPs can permeate into specific cells and organelles. We examine the incorporation and translocation of a novel anti-cancer CPP in a dioleoylphosphatidylcholine (DOPC) lipid bilayer membrane. The peptide, NAF-1(44-67), is a short fragment of a transmembrane protein, consisting of hydrophobic N terminal and charged C terminal segments. Experiments using fluorescently-labeled NAF-1(44-67) in ~100 nm DOPC vesicles and atomically detailed simulations conducted with Milestoning support a model in which a significant barrier for peptide-membrane entry is found at the interface between the aqueous solution and membrane. The initial step is the insertion of the N terminal segment and the hydrophobic helix into the membrane, passing the hydrophilic head groups. Both experiments and simulations suggest that the free energy difference in the first step of the permeation mechanism in which the hydrophobic helix crosses the phospholipid head groups is −0.4 kcal mol(−1) slightly favoring motion into the membrane. Milestoning calculations of the mean first passage time and the committor function underscore the existence of an early polar barrier followed by a diffusive barrierless motion in the lipid tail region. Permeation events are coupled to membrane fluctuations that are examined in detail. Our study opens the way to investigate in atomistic resolution the molecular mechanism, kinetics, and thermodynamics of CPP permeation to diverse membranes.

Systemic acquired acclimation and wound signaling require the transmission of electrical, calcium, and reactive oxygen species (ROS) signals between local and systemic tissues of the same plant. However, whether such signals can be transmitted between two different plants is largely unknown. Here, we reveal a new type of plant-to-plant aboveground direct communication involving electrical signaling detected at the surface of leaves, ROS, and photosystem networks. A foliar electrical signal induced by wounding or high light stress applied to a single dandelion leaf can be transmitted to a neighboring plant that is in direct contact with the stimulated plant, resulting in systemic photosynthetic, oxidative, molecular, and physiological changes in both plants. Furthermore, similar aboveground changes can be induced in a network of plants serially connected via touch. Such signals can also induce responses even if the neighboring plant is from a different plant species. Our study demonstrates that electrical signals can function as a communication link between transmitter and receiver plants that are organized as a network (community) of plants. This process can be described as network-acquired acclimation.

Heat waves occurring during droughts can have a devastating impact on yield, especially if they happen during the flowering and seed set stages of the crop cycle. Global warming and climate change are driving an alarming increase in the frequency and intensity of combined drought and heat stress episodes, critically threatening global food security. Because high temperature is detrimental to reproductive processes, essential for plant yield, we measured the inner temperature, transpiration, sepal stomatal aperture, hormone concentrations and transcriptomic response of closed soybean flowers developing on plants subjected to a combination of drought and heat stress. Here, we report that, during a combination of drought and heat stress, soybean plants prioritize transpiration through flowers over transpiration through leaves by opening their flower stomata, while keeping their leaf stomata closed. This acclimation strategy, termed ‘differential transpiration’, lowers flower inner temperature by about 2–3°C, protecting reproductive processes at the expense of vegetative tissues. Manipulating stomatal regulation, stomatal size and/or stomatal density of flowers could serve as a viable strategy to enhance the yield of different crops and mitigate some of the current and future impacts of global warming and climate change on agriculture.

Waterlogging stress (WLS) negatively impacts the growth and yield of crops resulting in heavy losses to agricultural production. Previous studies revealed that WLS induces a systemic response in shoots that is partially dependent on the plant hormones ethylene and abscisic acid. However, the role of rapid cell-to-cell signaling pathways, such as the reactive oxygen species (ROS) and calcium waves, in systemic responses of plants to WLS is unknown at present. Here we reveal that an abrupt WLS treatment ofArabidopsis thalianaplants growing in peat moss triggers systemic ROS and calcium wave responses, and that the WLS-triggered ROS wave response of Arabidopsis is dependent on the ROS generating RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), calcium-permeable channels GLUTAMATE-LIKE RECEPTOR 3.3 and 3.6 (GLR3.3 and GLR3.6), and aquaporin PLASMA MEMBRANE INTRINSIC PROTEIN 2;1 (PIP2;1) proteins. We further show that WLS is accompanied by a rapid systemic transcriptomic response that is evident as early as 10 min following waterlogging initiation, includes many hypoxia-response transcripts, and is partially dependent on RBOHD. Interestingly, the abrupt WLS of Arabidopsis resulted in the triggering of a rapid hydraulic wave response and the transient opening of stomata on leaves. Taken together, our findings reveal that the initiation of WLS in plants is accompanied by rapid systemic physiological and transcriptomic responses that involve the ROS, calcium, and hydraulic waves. These findings reveal that systemic plant responses to WLS are rapid and at least partially dependent on cell-to-cell signaling mechanisms.

The CISD2 (NAF-1) protein plays a key role in regulating cellular homeostasis, aging, cancer and neurodegenerative diseases. It was found to control different calcium, reactive oxygen species (ROS), and iron signaling mechanisms. However, since most studies of CISD2 to date were conducted with cells that constitutively lack, overexpress, or contain mutations in CISD2, the relationships between these different signaling processes are unclear. To address the hierarchy of signaling events occurring in cells upon CISD2 disruption, we developed an inducible system to express CISD2, or the dominant-negative H114C inhibitor of CISD2, in human breast cancer cells. Here, we report that inducible disruption of CISD2 function causes an immediate disruption in mitochondrial labile iron (mLI), and that this disruption results in enhanced mitochondrial ROS (mROS) levels. We further show that alterations in cytosolic and ER calcium levels occur only after the changes in mLI and mROS levels happen and are unrelated to them. Interestingly, disrupting CISD2 function resulted in the enhanced expression of the tumor suppressor thioredoxin-interacting protein (TXNIP) that was dependent on the accumulation of mLI and associated with ferroptosis activation. CISD2 could therefore regulate the expression of TXNIP in cancer cells, and this regulation is dependent on alterations in mLI levels.

The complexity of environmental factors affecting plants is gradually increasing due to global warming, an increase in the number and intensity of climate change-driven weather events, such as droughts, heat waves, and floods, and the accumulation of different pollutants. The impact of multiple stress conditions on plants was recently termed ‘multifactorial stress combination’ (MFSC) and defined as the occurrence of three or more stressors that impact plants simultaneously or sequentially. We recently reported that with the increased number and complexity of different stressors, the growth and survival ofArabidopsis thalianaseedlings declines; even if the level of each individual stress is low enough to have no significant effect on plants. This finding is alarming since it reveals that MFSCs of different low-level stressors could impact crops and cause a dramatic reduction in overall growth. However, whether MFSC would impact commercial crop cultivars has not been studied. Here, we reveal that a MFSC of 5 different low level abiotic stresses (salinity, heat, the herbicide paraquat, phosphorus deficiency, and the heavy metal cadmium), applied in an increasing level of complexity, has a significant negative impact on the growth and biomass of a commercial rice (Oryza sativa) cultivar and a maize (Zea mays) hybrid. We further report on the first proteomics analysis of MFSC in plants that identified over 300 proteins common to all 4- and 5-MFSCs. Taken together our findings reveal that the impacts of MFSC on two different crop species are severe, and that MFSC may significantly affect agricultural productivity.

Climate change is causing an increase in the frequency and intensity of droughts, heat waves, and their combinations, diminishing agricultural productivity and destabilizing societies worldwide. We recently reported that during a combination of water deficit (WD) and heat stress (HS) stomata on leaves of soybean plants are closed, while stomata on flowers are open. This unique stomatal response was accompanied by differential transpiration (higher in flowers, while lower in leaves) that cooled flowers during a combination of WD+HS. Here we reveal that developing pods of soybean plants subjected to a combination of WD+HS use a similar acclimation strategy of differential transpiration to reduce internal pod temperature by about 4°C. We further show that enhanced expression of transcripts involved in abscisic acid degradation accompanies this response, and that preventing pod transpiration by sealing stomata causes a significant increase in internal pod temperature. Using an RNA-Seq analysis of pods developing on plants subjected to WD+HS, we also show that the response of pods to WD, HS, or WD+HS is distinct from that of leaves or flowers. Interestingly, we report that although flower, pod and seed numbers per plant are decreased under conditions of WD+HS, seed mass of plants subjected to WD+HS is larger than that of plants subjected to HS, and number of seeds with suppressed/aborted development is lower in WD+HS compared to HS. Taken together our findings reveal that differential transpiration occurs in pods of soybean plants subjected to WD+HS and that this process limits heat-induced damage to seed production.One sentence summaryDifferential transpiration between pods and leaves of soybean plants subjected to a combination of water deficit and heat stress buffers internal pod temperature.

Iron (Fe) is an essential micronutrient whose uptake is tightly regulated to prevent either deficiency or toxicity. Cadmium (Cd) is a non-essential element that induces both Fe deficiency and toxicity; however, the mechanisms behind these Fe/Cd-induced responses are still elusive. Here we explored Cd- and Fe-associated responses in wild-type Arabidopsis and in a mutant that overaccumulates Fe (opt3-2). Gene expression profiling revealed a large overlap between transcripts induced by Fe deficiency and Cd exposure. Interestingly, the use of opt3-2 allowed us to identify additional gene clusters originally induced by Cd in the wild type but repressed in the opt3-2 background. Based on the high levels of H2O2 found in opt3-2, we propose a model where reactive oxygen species prevent the induction of genes that are induced in the wild type by either Fe deficiency or Cd. Interestingly, a defined cluster of Fe-responsive genes was found to be insensitive to this negative feedback, suggesting that their induction by Cd is more likely to be the result of an impaired Fe sensing. Overall, our data suggest that Fe deficiency responses are governed by multiple inputs and that a hierarchical regulation of Fe homeostasis prevents the induction of specific networks when Fe and H2O2 levels are elevated.

Reactive oxygen species (ROS) and the photoreceptor protein phytochrome B (phyB) play a key role in plant acclimation to stress. However, how phyB that primarily functions in the nuclei impacts ROS signaling mediated by respiratory burst oxidase homolog (RBOH) proteins that reside on the plasma membrane, during stress, is unknown. Arabidopsis thaliana and Oryza sativa mutants, RNA-Seq, bioinformatics, biochemistry, molecular biology, and whole-plant ROS imaging were used to address this question. Here, we reveal that phyB and RBOHs function as part of a key regulatory module that controls apoplastic ROS production, stress-response transcript expression, and plant acclimation in response to excess light stress. We further show that phyB can regulate ROS production during stress even if it is restricted to the cytosol and that phyB, respiratory burst oxidase protein D (RBOHD), and respiratory burst oxidase protein F (RBOHF) coregulate thousands of transcripts in response to light stress. Surprisingly, we found that phyB is also required for ROS accumulation in response to heat, wounding, cold, and bacterial infection. Our findings reveal that phyB plays a canonical role in plant responses to biotic and abiotic stresses, regulating apoplastic ROS production, possibly while at the cytosol, and that phyB and RBOHD/RBOHF function in the same regulatory pathway.

Cell-to-cell communication is fundamental to multicellular organisms and unicellular organisms living in a microbiome. A unique cell-to-cell communication mechanism that uses reactive oxygen species (ROS) as a signal (termed the ‘ROS wave’) was recently identified in flowering plants. Here we report that a ROS-mediated cell-to-cell signaling process, like the ROS wave, can be found in ferns, mosses, unicellular algae, amoeba, mammalian cells, and isolated hearts. We further show that this process can be triggered by a localized stress treatment or H2O2 application and blocked by inhibition of NADPH oxidases, and that in unicellular algae, it communicates important stress-response signals between cells. Taken together, our findings suggest that cell-to-cell ROS signaling evolved before unicellular and multicellular organisms diverged. The finding of a ROS wave-like signaling process in mammalian cells further contributes to our understanding of different diseases and could impact the development of new drugs that target cancer or heart disease.

SummaryClimate change-driven extreme weather events, combined with increasing temperatures, harsh soil conditions, low water availability and quality, and the introduction of many man-made pollutants, pose a unique challenge to plants. Although our knowledge of the response of plants to each of these individual conditions is vast, we know very little about how a combination of many of these factors, occurring simultaneously, i.e., multifactorial stress combination, impacts plants.Seedlings of wild type and different mutants of Arabidopsis thaliana plants were subjected to a multifactorial stress combination of six different stresses, each applied at a low level, and their survival, physiological and molecular responses determined.Our findings reveal that while each of the different stresses, applied individually, had a negligible effect on plant growth and survival, the accumulated impact of multifactorial stress combination on plants was detrimental. We further show that the response of plants to multifactorial stress combination is unique and that specific pathways and processes play a critical role in the acclimation of plants to multifactorial stress combination.Taken together our findings reveal that further polluting our environment could result in higher complexities of multifactorial stress combinations that in turn could drive a critical decline in plant growth and survival.Plain Language SummaryThe effects of multiple stress conditions occurring simultaneously, i.e., multifactorial stress combination, on plants is currently unknown. Here we show that different co-occurring stresses can interact to negatively impact plant growth and survival, even if the effect of each individual stress is negligible. We further identify several key pathways essential for plant acclimation to multifactorial stress combination.

The wounding-induced reactive oxygen species (ROS) wave is accompanied by a systemic whole-plant redox response in Arabidopsis.

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.

Plants can send long-distance cell-to-cell signals from a single tissue subjected to stress to the entire plant. This ability is termed “systemic signaling” and is essential for plant acclimation to stress and/or defense against pathogens. Several signaling mechanisms are associated with systemic signaling, including the reactive oxygen species (ROS) wave, calcium wave, hydraulic wave, and electric signals. The ROS wave coordinates multiple physiological, molecular, and metabolic responses among different parts of the plant and is essential for systemic acquired acclimation (SAA) to stress. In addition, it is linked with several plant hormones, including jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). However, how these plant hormones modulate the ROS wave and whether they are required for SAA is not clear. Here we report that SA and JA play antagonistic roles in modulating the ROS wave in Arabidopsis (Arabidopsis thaliana). While SA augments the ROS wave, JA suppresses it during responses to local wounding or high light (HL) stress treatments. We further show that ethylene and ABA are essential for regulation of the ROS wave during systemic responses to local wounding treatment. Interestingly, we found that the redox-response protein NONEXPRESSOR OF PATHOGENESIS RELATED PROTEIN 1 is required for systemic ROS accumulation in response to wounding or HL stress, as well as for SAA to HL stress. Taken together, our findings suggest that interplay between JA and SA might regulate systemic signaling and SAA during responses of plants to abiotic stress or wounding.

Plants can send long distance cell-to-cell signals from a single tissue subjected to stress to the entire plant. This ability is termed ‘systemic signaling’ and is essential for plant acclimation to stress and/or defense against pathogens. Several signaling mechanisms are associated with systemic signaling, including the reactive oxygen species (ROS) wave, calcium wave, hydraulic wave, and electric signals. The ROS wave coordinates multiple physiological, molecular, and metabolic responses among different parts of the plant, and is essential for systemic acquired acclimation (SAA) to stress. In addition, it is linked with several plants hormones, including jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). However, how these plant hormones modulate the ROS wave and whether they are required for SAA is not clear. Here we report that SA and JA play antagonistic roles in modulating the ROS wave. While SA augments the ROS wave, JA suppresses it, during responses to a local wounding or high light (HL) stress treatments. We further show that ethylene and ABA are essential for the regulation of the ROS wave during systemic responses to a local wounding treatment. Interestingly, we found that the redox-response protein NONEXPRESSOR OF PATHOGENESIS RELATED PROTEIN 1 (NPR1) is required for systemic ROS accumulation in response to wounding or HL stress, as well as for SAA to HL stress. Taken together, our findings suggests that an interplay between JA and SA could regulate systemic signaling and SAA during responses of plants to abiotic stress or wounding.One sentence summaryAn antagonistic interaction between SA and JA attenuates the accumulation of ROS in local and systemic tissues during responses of plants to light stress or wounding.

An effective anti-cancer therapy should exclusively target cancer cells and trigger in them a broad spectrum of cell death pathways that will prevent avoidance. Here, we present a new approach in cancer therapy that specifically targets the mitochondria and ER of cancer cells. We developed a peptide derived from the flexible and transmembrane domains of the human protein NAF-1/CISD2. This peptide (NAF-1

A combination of drought and heat stress, occurring at the vegetative or reproductive growth phase of many different crops, can have a devastating impact on yield. In soybean (Glycine max), a considerable effort has been made to develop genotypes with enhanced yield production under conditions of drought or heat stress. However, how these genotypes perform in terms of growth, physiological responses and most importantly seed production, under conditions of drought and heat combination is mostly unknown. Here, we studied the impact of water deficit and heat stress combination on the physiology, seed production and yield per plant of two soybean genotypes, Magellan and Plant Introduction (PI) 548313, that differ in their reproductive responses to heat stress. Our findings reveal that although PI 548313 produced more seeds than Magellan under conditions of heat stress, under conditions of water deficit and heat stress combination its seed production decreased. Because number of flowers and pollen germination of PI 548313 remained high under heat or water deficit and heat combination, the reduced seed production exhibited by PI 548313 under the stress combination could be a result of processes that occur at the stigma, ovaries and/or other parts of the flower following pollen germination.HighlightTolerance to heat stress was found not to confer tolerance to a combination of water deficit and heat stress in soybean, highlighting the need for breeding strategies targeting the stress combination.

Systemic signaling and systemic acquired acclimation (SAA) are essential for plant survival during episodes of environmental stress. Recent studies highlighted a key role for reactive oxygen species (ROS) signaling in mediating systemic responses and SAA during light stress in Arabidopsis (Arabidopsis thaliana). These studies further identified the RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) protein as a key player in mediating rapid systemic ROS responses. Here, we report that tissue-specific expression of RBOHD in phloem or xylem parenchyma cells of the rbohD mutant restores systemic ROS signaling, systemic stress-response transcript expression, and SAA to a local treatment of light stress. We further demonstrate that RBOHD and RBOHF are both required for local and systemic ROS signaling at the vascular bundles of Arabidopsis. Taken together, our findings highlight a key role for RBOHD-driven ROS production at the vascular bundles of Arabidopsis in mediating light stress–induced systemic signaling and SAA. In addition, they suggest that the integration of ROS, calcium, electric, and hydraulic signals, during systemic signaling, occurs at the vascular bundles.

Perception of a change in light intensity leads to the activation of multiple physiological, metabolic, and molecular responses in plants. These responses allow acclimation to fluctuating light conditions, e.g. sunflecks in field grown plants, preventing cellular damage associated with excess light stress. Perception of light stress by a single Arabidopsis (Arabidopsis thaliana) leaf was recently shown to activate different local and systemic responses that include rapid changes in stomatal aperture size; these were found to be coordinated by a systemic process of reactive oxygen species (ROS)-derived ROS production (i.e. the ROS wave). How light intensity is perceived, and how long the ROS wave stays “on” during this process are, however, unknown. Here we show that triggering of the ROS wave by a local excess light stress treatment results in the induction and maintenance of high levels of systemic ROS for up to 6 h. Despite these high systemic ROS levels, stomatal aperture size returns to control size within 3 h, and the systemic stomatal response can be retriggered within 6 h. These findings suggest that the ROS wave triggers a systemic stress memory mechanism that lasts for 3 to 6 h, but that within 3 h of its activation, stomata become insensitive to ROS and open. We further show that the excess light stress-triggered ROS wave, as well as the excess light stress-triggered local and systemic stomatal aperture closure responses, are dependent on phytochrome B function. Our findings reveal a delicate interplay between excess light stress, phytochrome B, ROS production, and rapid systemic stomatal responses.

Climate change is altering environments in which plants and different crops grow and survive. We already experienced an increase in worldwide average earth surface temperatures, as well as frequency and extent of damaging heat waves. These conditions collide in the field with other abiotic stresses such as water deficit, high salinity, increased light irradiation, and so on, generating complex harmful conditions that destabilize agricultural systems. The conditions generated during these episodes of stress combination greatly differ from those occurring in the field when different stress factors occur individually; conditions that have been the focus of study for decades. Fortunately, knowledge of physiological and molecular responses to stress combinations and the cost they inflict on plant growth and yield has been exponentially increasing in the past several years. Understanding plant performance under multiple stress combinations will allow breeding crops capable of maintaining yield production under the new climatic conditions. Here, after reviewing recent data on physiological, hormonal and transcriptional responses to different stress combinations, we highlight the importance of photodamage avoidance, abscisic and jasmonic acid signaling, and the upregulation of genes involved in oxidation-reduction processes, photosynthesis and protein metabolism, for plant acclimation to conditions of high temperatures, in combination with other common abiotic stress factors such as drought or salinity. Finally, we propose new approaches to investigate the response of plants to stress combinations and discuss strategies for improving crop resilience to stress combination.

Systemic acquired acclimation (SAA) is a key biological process essential for plant survival under conditions of abiotic stress. SAA was recently shown to be controlled by a rapid systemic signaling mechanism termed the reactive oxygen species (ROS) wave in Arabidopsis (Arabidopsis thaliana). MYB30 is a key transcriptional regulator mediating many different biological processes. MYB30 was found to act downstream of the ROS wave in systemic tissues of Arabidopsis in response to local high light (HL) stress treatment. However, the function of MYB30 in systemic signaling and SAA is unknown. To determine the relationship among MYB30, the ROS wave, and systemic acclimation in Arabidopsis, the SAA response to HL stress of myb30 mutants and wild-type plants was determined. Although myb30 plants were found to display enhanced rates of ROS wave propagation and their local tissues acclimated to the HL stress, they were deficient in SAA to HL stress. Compared to wild type, the systemic transcriptomic response of myb30 plants was also deficient, lacking in the expression of over 3,500 transcripts. A putative set of 150 core transcripts directly associated with MYB30 function during HL stress was determined. Our study identifies MYB30 as a key regulator that links systemic ROS signaling with systemic transcriptomic responses, SAA, and plant acclimation to HL stress. In addition, it demonstrates that plant acclimation and systemic ROS signaling are interlinked and that the lack of systemic acclimation drives systemic ROS signaling to occur at faster rates, suggesting a feedback mechanism (potentially involving MYB30) between these two processes.

Significance Environmental stresses such as heat, drought, and salinity, especially in combination with intense light conditions, can have devastating economical and sociological impacts. Although our knowledge of how each of these stresses affects plants when applied individually is vast, we know very little about how plants acclimate to a combination of different stresses. Here we reveal that plants can integrate different local and systemic signals generated during conditions of stress combination. We further show that the specific part at which plants sense the two co-occurring stresses makes a significant difference in how fast and efficient they acclimate. Our results shed light on how plants acclimate to and survive a combination of different stresses., Extreme environmental conditions, such as heat, salinity, and decreased water availability, can have a devastating impact on plant growth and productivity, potentially resulting in the collapse of entire ecosystems. Stress-induced systemic signaling and systemic acquired acclimation play canonical roles in plant survival during episodes of environmental stress. Recent studies revealed that in response to a single abiotic stress, applied to a single leaf, plants mount a comprehensive stress-specific systemic response that includes the accumulation of many different stress-specific transcripts and metabolites, as well as a coordinated stress-specific whole-plant stomatal response. However, in nature plants are routinely subjected to a combination of two or more different abiotic stresses, each potentially triggering its own stress-specific systemic response, highlighting a new fundamental question in plant biology: are plants capable of integrating two different systemic signals simultaneously generated during conditions of stress combination? Here we show that plants can integrate two different systemic signals simultaneously generated during stress combination, and that the manner in which plants sense the different stresses that trigger these signals (i.e., at the same or different parts of the plant) makes a significant difference in how fast and efficient they induce systemic reactive oxygen species (ROS) signals; transcriptomic, hormonal, and stomatal responses; as well as plant acclimation. Our results shed light on how plants acclimate to their environment and survive a combination of different abiotic stresses. In addition, they highlight a key role for systemic ROS signals in coordinating the response of different leaves to stress.

Mechanical wounding occurs in plants during biotic (e.g., herbivore or pathogen attack) or abiotic (e.g., wind damage or freezing) stresses and is associated with the activation of multiple signaling pathways. These initiate many wound responses at the wounded tissues, as well as trigger long-distance signaling pathways that activate wound responses in tissues that were not affected by the initial wounding event (termed ‘systemic wound response’). Among the different systemic signals activated by wounding are electric signals, calcium and reactive oxygen species (ROS) waves, and different plant hormones such as jasmonic acid. The release of glutamate from cells at the wounded tissues was recently proposed to trigger several different systemic signal transduction pathways via glutamate-like receptors (GLRs). However, the role of another important compound released from cells during wounding (i.e., extracellular ATP; eATP) in triggering systemic responses is not clear. Here we show that eATP that accumulates in wounded leaves and is sensed by the purinoreceptor kinase P2K is required for the activation of the ROS wave during wounding. Application of eATP to unwounded leaves triggered the ROS wave, and the activation of the ROS wave by wounding or eATP application was suppressed in mutants deficient in P2K (i.e., p2k1-3, p2k2, and p2k1-3p2k2). In addition, the expression of several systemic wound response transcripts was suppressed in mutants deficient in P2K during wounding. Our findings reveal that in addition to sensing glutamate via GLRs, eATP sensed by P2Ks is playing a key role in the triggering of systemic wound responses in plants.One sentence summaryExtracellular ATP plays an important role in triggering the ROS wave and systemic transcriptomics responses during wounding.

As multicellular organisms, plants constantly balance and coordinate many metabolic, physiological, and molecular responses between different cell types and tissues. This process is essential for plant development, growth, and response to different environmental cues. Because plants lack a nervous system, they transmit different signals over long distances via cell-to-cell signaling. Recent studies revealed that reactive oxygen species (ROS), produced by respiratory burst oxidase homologs (RBOHs) at the apoplast play a key role in cell-to-cell signaling. A state of enhanced ROS production by one cell is thereby sensed by a neighboring cell, causing it to produce ROS, creating a continuous chain of cell-to-cell ROS accumulation termed the ‘ROS wave’. This process was found to mediate systemic signals throughout the plant and is required for plant acclimation to different stresses. Although RBOHs were found to produce ROS essential for this process, the identity of the receptor(s) perceiving the apoplastic ROS signal is currently unknow. Here we reveal that the leucine-rich-repeat receptor-like kinase HPCA1 (H2O2-induced Ca2+increases 1) acts as a central ROS receptor required for the propagation of cell-to-cell ROS signals, systemic signaling in response to different biotic and abiotic stresses, and plant acclimation to stress. We further report that HPCA1 is required for systemic calcium signals, but not systemic membrane depolarization responses, and identify key calcium-dependent signal transduction proteins involved in this process. Our findings reveal that HPCA1 plays a key role in mediating and coordinating systemic cell-to-cell ROS and calcium signals that are required for plant acclimation to stress.

Plants are frequently subjected to different combinations of abiotic stresses, such as high light (HL) intensity, and elevated temperatures. These environmental conditions pose a threat to agriculture production, affecting photosynthesis, and decreasing yield. Metabolic responses of plants, such as alterations in carbohydrates and amino acid fluxes, play a key role in the successful acclimation of plants to different abiotic stresses, directing resources toward stress responses, and suppressing growth. Here we show that the primary metabolic response of Arabidopsis (Arabidopsis thaliana) plants to HL or heat stress (HS) is different from that of plants subjected to a combination of HL and HS (HL+HS). We further demonstrate that the combined stress results in a unique metabolic response that includes increased accumulation of sugars and amino acids coupled with decreased levels of metabolites participating in the tricarboxylic acid cycle. Among the amino acids exclusively accumulated during HL+HS, we identified the nonproteinogenic amino acid γ-aminobutyric acid (GABA). Analysis of different mutants deficient in GABA biosynthesis (GLUTAMATE DESCARBOXYLASE 3 [gad3]) as well as mutants impaired in autophagy (autophagy-related proteins 5 and 9 [atg5 and atg9]), revealed that GABA plays a key role in the acclimation of plants to HL+HS, potentially by promoting autophagy. Taken together, our findings identify a role for GABA in regulating plant responses to combined stress.

Mitochondrial inner NEET (MiNT) and the outer mitochondrial membrane (OMM) mitoNEET (mNT) proteins belong to the NEET protein family. This family plays a key role in mitochondrial labile iron and reactive oxygen species (ROS) homeostasis. NEET proteins contain labile [2Fe-2S] clusters which can be transferred to apo-acceptor proteins. In eukaryotes, the biogenesis of [2Fe-2S] clusters occurs within the mitochondria by the iron-sulfur cluster (ISC) system; the clusters are then transferred to [2Fe-2S] proteins within the mitochondria or exported to cytosolic proteins and the cytosolic iron-sulfur cluster assembly (CIA) system. The last step of export of the [2Fe-2S] is not yet fully characterized. Here we show that MiNT interacts with voltage-dependent anion channel 1 (VDAC1), a major OMM protein that connects the intermembrane space with the cytosol and participates in regulating the levels of different ions including mitochondrial labile iron (mLI). We further show that VDAC1 is mediating the interaction between MiNT and mNT, in which MiNT transfers its [2Fe-2S] clusters from inside the mitochondria to mNT that is facing the cytosol. This MiNT-VDAC1-mNT interaction is shown both experimentally and by computational calculations. Additionally, we show that modifying MiNT expression in breast cancer cells affects the dynamics of mitochondrial structure and morphology, mitochondrial function, and breast cancer tumor growth. Our findings reveal a pathway for the transfer of [2Fe-2S] clusters, which are assembled inside the mitochondria, to the cytosol.