Oxygen Sensing and Integrative Stress Signaling in Plants

Plants grow in a dynamic environment and continuously face numerous stress conditions in parallel. This fluctuating environment pushed the evolution of extensive metabolic flexibility (Sweetlove et al., 2010; van Dongen et al., 2011). Flooding is an example of an environmental condition that combines many different stress factors simultaneously, including limited gas diffusion (e.g. of oxygen, carbon dioxide, and ethylene), temperature stress, light quality and quantity, and osmotic and oxidative stress. Changes in the internal concentration of oxygen, but also e.g. ethylene, are crucial early signals for the initiation of adaptation strategies to flooding (Sasidharan and Voesenek, 2015; Sasidharan et al., 2017). Initially, a plant cell responds to nearly all these different types of stresses by using the same set of second messengers (such as reactive oxygen species [ROS], calcium, nitric oxide [NO], and lipid molecules) and by activating signaling cascades through kinases (Bjornson et al., 2016; Zhu, 2016). This raises the question of how cells detect and differentiate between stresses. One fundamental aim in low-oxygen research is to unravel how organisms detect and react to the lack of oxygen and respond to an impairment of their energy metabolism (Ratcliffe, 2013). In both plants (Licausi et al., 2011a; Gibbs et al., 2011, 2014; Weits et al., 2014; White et al., 2017) and animals (Loenarz and Schofield, 2011; Ratcliffe, 2013; Bishop and Ratcliffe 2014; Hamanaka et al., 2016), key transcriptional regulators have been identified that regulate the hypoxic response via the integration of cellular signals that are required for their activation (Box 1). Here, we discuss the nature and origin of these various signals under hypoxia that initiate adaptation responses at the transcriptional and (post-) translational level and their potential integration points. In addition, we postulate that it is the integration of different signal inputs that defines a response specific to oxygen limitation. Further, we will discuss the state of current methods and technologies to measure plant internal oxygen concentrations. Abiotic stress perception appears not to rely on the detection of only one specific effector or ligand. Despite all efforts to reveal the primary sensors for diverse abiotic stresses, unequivocal identification remains arduous (Zhu, 2016). In Arabidopsis (Arabidopsis thaliana), the N-end rule pathway of targeted proteolysis was shown to act as a mechanism that controls protein levels of subgroup VII ETHYLENE-RESPONSE FACTOR (ERFVII) transcription factors by regulating their protein turnover in an oxygen-concentration-dependent manner (Gibbs et al., 2011; Licausi et al., 2011a; Box 1). Remarkably, protein abundance of RELATED TO APETALA2.12 (RAP2.12), an ERFVII member in Arabidopsis, is also positively regulated by HYDRAULIC CONDUCTIVITY OF ROOT1 (HCR1), a Raf-like MAPKKK, which could phosphorylate RAP2.12 in vitro. This N-end rule-independent stabilization mechanism is proposed to allow the integration of cation (specifically potassium) availability with the oxygen concentration in the soil (Shahzad et al., 2016). Low-oxygen stress impairs diverse plant cellular functions, including those in the cytosol, nucleus, mitochondria, and endoplasmic reticulum (ER). Actually, molecular oxygen is among the most-utilized molecules in eukaryotic cellular metabolism (Raymond and Segrè, 2006) with more than 350 oxygen-dependent reactions. Thus, decreasing oxygen levels might be indirectly sensed due to an impairment of various metabolic pathways when the cellular oxygen concentration changes around or below the Michaelis-Menten constant K M for oxygen of the enzymes involved. As such, any oxygen-dependent enzyme has the potential to act as a kind of sensor, suggesting simultaneous oxygen sensing at multiple sites of the cell. Then again, it might well be that one or a few oxygen-concentration-dependent enzymes evolved to primarily monitor aerobic metabolism. Taken together, three scenarios of cellular oxygen sensing can be discussed: (1) decentralized sensing at many sites of the cell by e.g. oxygen-consuming enzymes, (2) a primary “oxygen sensor” detects a drop in oxygen to initiate all signaling cascades, and (3) a combination of scenario 1 and 2. A primary oxygen sensor (scenario 2) would detect the cellular oxygen concentration but not the oxygen availability. However, differentiation between “concentration” and “availability” is important, since a low oxygen concentration can result in a high flux of oxygen into the tissue and does not necessarily lead to low-oxygen stress. The advantage of a decentralized oxygen-sensing mechanism (scenario 1) would be that it allows for fine-tuning the response to low-oxygen conditions to a particular situation. By adding up various oxygen-concentration-related signals, such integrative signaling would trigger hypoxic responses only when the actual oxygen concentration does indeed disturb cellular homeostasis. The decentralized oxygen-sensing model further suggests that perturbations of processes in various organelles, at the plasma membrane and in the cytosol can initiate low-oxygen-stress signaling and that these various triggers must be integrated to activate appropriate transcriptional reprogramming and cellular adaptation responses. A specific balance between multiple stress-induced signals could provide a mechanism—like a “cellular fingerprint”—to discriminate between different stress types. In this review, we summarize signaling pathways from different cellular compartments that are initiated by low-oxygen stress and discuss potential integration points for the multiple signal inputs resulting in hypoxia-specific adaptive responses. In Arabidopsis, members of the ERFVII transcription factor family (Box 1) contain a conserved N-terminal domain that makes them oxygen- and NO-dependent substrates of the N-end rule pathway of targeted proteolysis (Gibbs et al., 2015). While the N-end rule is acting as a safeguard mechanism to limit ERFVII abundance under aerobic conditions, several observations suggest that its control is limited to certain Met-Cys proteins, environmental conditions, and developmental phases of the plant. Arabidopsis N-end rule mutant seedlings show constitutive expression of about half of the core hypoxia-responsive genes (Gibbs et al., 2011), indicating that the other hypoxia genes are not controlled by this pathway. In addition, introduction of a GUS reporter driven by the ALCOHOL DEHYDROGENASE1 (ADH1) promoter in Arabidopsis wild-type seedlings or a proteolysis6 (prt6) mutant defective in ERFVII turnover revealed constitutive activation of the promoter in certain tissues of the prt6 mutant, but not in all as compared to the wild-type. Interestingly, in both the wild-type and the prt6 mutant an increased GUS signal was observed after 6 h of hypoxia (Gibbs et al., 2011). Thus, in N-end rule mutants, the hypoxic response is not entirely saturated, nor do mutations in this pathway affect all plant tissues in a similar manner. While constitutive expression of a subset of hypoxic genes is observed in N-end rule mutant seedlings, a recent report revealed that this constitutive expression is less pronounced in adult mutant plants (Giuntoli et al., 2017). As submergence of rosette-stage plants just prior to bolting still results in the activation of hypoxic genes, it strongly argues for additional, N-end rule-independent mechanisms that control either ERFVII activity or alternative transcriptional cascades. In addition, at least one ERFVII, SUBMERGENCE1 A (SUB1A) from rice (Oryza sativa), is not a target of the N-end rule pathway, although it possesses the N-terminal consensus sequence as found in Arabidopsis ERFVIIs (Gibbs et al., 2011). This further suggests alternative regulatory pathways. Limited oxygen availability affects respiration, which impacts on a whole palette of mitochondrial parameters and processes (see Box 2), including reactive oxygen and nitrogen species (ROS/RNS) homeostasis, redox status of NAD(P)H and antioxidant pools, ATP/ADP ratio, the proton-motive force, and calcium and metabolite levels, which in principle could all trigger mitochondrial signaling (Schwarzländer and Finkemeier, 2013). Indeed, roles for these diverse signals in mitochondrial retrograde responses have been established and are discussed by Wagner et al. (2018) in this Focus Issue. The role of mitochondrial retrograde signals in hypoxia has been partly investigated; for example, mitochondrial ROS signals were shown to impact on anoxia tolerance (Chang et al., 2012). Likewise, in animals, mitochondrial electron transport chain (mETC)-derived ROS signals appear to provoke adaptation responses upon hypoxia (Hamanaka et al., 2016). Interestingly, complex III inhibition under aerobic conditions by antimycin-A treatment in part causes induction of hypoxia-responsive genes in Arabidopsis (Ng et al., 2013a), which could be due to disruption of the ATP/ADP ratio and/or increased mitochondrial ROS production. Furthermore, metabolic signals derived from mitochondria might be integrated into cellular reprogramming during hypoxia. This is supported by the notion that overexpression of the mitochondrial UNCOUPLING PROTEIN1 (UCP1), which decreases stress-induced ROS production, results in a hypoxic metabolic pattern and transcriptomic signature (Barreto et al., 2016). UCP in animals have been to the induction of hypoxic responses et al., suggesting a role of this protein family in low-oxygen responses. of plant mitochondria is that the and (see Box 2) are of et al., Remarkably, mitochondrial is oxygen as in low oxygen of complex from the is observed under et al., 2011). mitochondria with under a low also of complex as observed for low-oxygen was also found in in which the of the appears to be to et al., 2013). it still to be that also affects in plant Taken together, the of complex from the does not appear to be the trigger for hypoxic responses in plants but is part of the adaptation response that is initiated by a drop in and a in availability. the oxygen concentration is below a certain is and van Dongen 2011). However, it remains to be at oxygen concentration mitochondrial is of the mitochondrial oxygen concentration is important, as the for oxygen of on cellular conditions such as redox and oxygen concentration et al., 2011). it is to be this impairment is specific to hypoxia or other abiotic stresses similar mitochondrial NAD(P)H and as part of the mitochondrial energy pathways (see Box et al., et al., et al., have roles in the response to diverse abiotic and stresses, which in an energy et al., 2015; Zhu, 2016). mitochondrial ROS with signaling can be observed under or stress et al., et al., indicating that stress signals upon mitochondrial are not hypoxia specific A fundamental question that further is how cells between mitochondrial signals and those by other stresses. in the is for the and of many and the different and pathways for this it is that during stress and stress results in the activation of the protein which the of the protein and the protein quality control and for a The pathway for oxidative protein is in the and on protein and and 2013). and is by through the of from to molecular The of the protein from was found to be for oxygen, which is similar to that of in plants et al., plant a similar as their for oxygen, would be under hypoxia and might act as sensors for low-oxygen Interestingly, only the during the in the was shown to be oxygen in and to the were not et al., 2013). As oxygen-dependent is upon low-oxygen stress, it has been that the a cellular oxygen sensor in animals that to signaling and during hypoxic conditions and is required to this pathway also a role in in mitochondria, oxidative protein on a that in an oxygen-dependent manner et al., suggesting that other protein pathways might have a role in low-oxygen stress. Interestingly, the also an for mitochondrial stress of mitochondrial results in a signal to the nucleus, the mitochondrial retrograde and on both ROS and calcium signals, and several of the protein of the pathway were transcription factors were found to act as of the transcriptional response during et al., 2013; et al., and responses but are at the indicating that mitochondrial signals are at the In this the mitochondrial signal is proposed to activate a that the transcription factors from the which to the to of the genes are also hypoxia including the ERFVII transcription factor a of responses in Arabidopsis (Licausi et al., it would be to the of to low-oxygen tolerance in The of low-oxygen stress in its impact on oxidative in the which energy in the of et al., 2011). However, a low oxygen concentration does not necessarily lead to an energy when the flux of oxygen the mitochondria can with oxygen it is of that any changes in the processes mitochondria are to other compartments to appropriate adaptive responses. By differentiation is required between (1) an energy that is by mitochondrial due to and (2) a status that is by high due to metabolic sensing cellular oxygen levels into the actual energy of the cell could provoke adaptation responses in tissues with high of mitochondrial energy we would to to the of Wagner et al. (2018) in this Focus Issue. energy sensing is conserved and the kinases and target of in both plants and animals et al., 2016). is in response to stress conditions to plant of impairs submergence tolerance in plants et al., 2012). is to that appears to the ratio and ratio in its activation to an energy 2011). different stresses can an energy not of the core genes are not hypoxia specific Dongen and 2015). that provoke a cellular translational which has also been observed during hypoxia et al., et al., In eukaryotic in response to energy availability is in part controlled by the protein et al., 2016). controls initiation and has been shown to be in cells upon hypoxia and In a has in which is under energy conditions, is upon energy et al., and appear to act the of under low-oxygen conditions in plants is well a role for is still as a result of abiotic stress a in ROS homeostasis. In addition, during stress a oxidative is which as a signal to initiate adaptive responses and 2016). mitochondrial ROS in retrograde signaling during oxygen to energy in and cells et al., Hamanaka et al., 2016). in a mitochondrial ROS is observed upon a drop in oxygen levels (Chang et al., 2012). As of antimycin-A this it is that derived from complex III might act as a signaling during mitochondrial upon oxygen In to the of high levels of mitochondria it by The during this either mitochondrial or the to signaling responses. mitochondria, would rely on et al., the cytosol, ROS as signaling molecules are due to the high antioxidant to provide mitochondrial to the ROS sensing and of this signal is to the mitochondrial of by mitochondria during oxygen into the cytosol has been to the activation of and in Arabidopsis (Chang et al., 2012). The that these are in a of and abiotic stress pathways et al., that are integrated into to to activate responses. As of either or do not affect the activation of genes (Chang et al., in the mitochondrial pathway to low-oxygen stress to be Here, it is of to that mitochondria are in with the and which might be in or the mitochondrial ROS to the mitochondrial oxidative upon oxygen a plasma through activation of et al., As ROS during nearly stress, it remains how such signals can be specific for low-oxygen stress. Arabidopsis of which and been in low-oxygen responses et al., et al., et al., 2017). genes including have been shown to on the mechanism through which to low-oxygen signaling further activity submergence tolerance could be to signaling et al., 2017). are by OF and kinases et al., et al., 2011). the oxidative during oxygen by activating The is as which signaling et al., Plants show low-oxygen it was found that the ERFVII transcription factor RAP2.12 the expression of PROTEIN1 to activity et al., 2015). and not only limit the of the ROS but might be in the plants from an oxidative upon on cells suggest a between the mitochondrial ROS and the activation of et al., this has not been in plant but it would be to be ROS on calcium, ROS in can calcium into the cytosol et al., 2010; et al., 2011), which both second messengers be part of a signaling an in calcium levels, which diverse that the calcium signal into a cellular and transcriptional response et al., these are enzymes, and transcription The in calcium levels through the of calcium from the and/or cellular et al., 2017). anoxia revealed a in calcium levels by using an reporter in Arabidopsis et al., oxygen a calcium response was with a calcium of by a calcium response that several In addition, the that inhibition of calcium by calcium in a inhibition of expression under in the of such impairs the expression of under low-oxygen stress et al., the to which calcium and signaling expression is in calcium is after a calcium by transport mechanisms et al., In either on calcium by or by by a proton-motive et al., The transport of calcium is to the levels, which calcium to calcium levels upon a stress Thus, after the calcium levels to be to the a in calcium et al., 2011). suggest that calcium signaling might be to the of low-oxygen stress, as and the under stress conditions might calcium The origin of calcium into the cytosol under low-oxygen stress in plants is partly plasma membrane calcium as well as calcium in part hypoxic responses et al., et al., 2011). calcium for the during oxygen might be the the and roles for and mitochondria in calcium have been in cells et al., et al., it is still to which these to calcium signaling under abiotic stresses in In has been shown to be required for the expression of genes enzymes, including et al., et al., is by it to the as an adaptation response to low-oxygen stress can be by controlled of and a that is regulated in a manner. from Arabidopsis as a potential calcium sensor that under low-oxygen stress to including stress The are under hypoxia to as of et al., 2016). Taken together, the role of calcium in the response to low-oxygen stress, the of calcium homeostasis, and the role of specific calcium sensor and signaling in adaptation to hypoxia. A for the research on low-oxygen stress is that of in oxygen in plants are and methods to the dynamic in oxygen levels with and are for a of low-oxygen signaling at the cellular the numerous methods for of oxygen have been (Box oxygen using oxygen and oxygen sensors are and to current on the and of oxygen in the of oxygen methods in plants is as compared to the were research has driven the of a of methods for the detection of hypoxia such as oxygen and et al., 2010; and 2015; et al., 2015). are on the diffusion of oxygen through a membrane to the which a current that is to the oxygen concentration et al., A is that oxygen is at the the of oxygen in this has been by the of which less for which are with a of to are also less compared to Despite the oxygen and of the have been and have been to measure the oxygen concentration in numerous plants such as and in the of several plant species et al., et al., et al., and oxygen sensors are due to their and their lack of oxygen during the The of oxygen are on the of a by oxygen, resulting in increased when the oxygen concentration can be on the of or to measure the oxygen concentration in a the of with an which was to the oxygen concentration in plant et al., 2012). which of an with a by an can be to measure the oxygen concentration plant tissue (Licausi et al., However, of into the tissue is and oxygen diffusion into the which can lead to oxygen provide a potential to the with the of and oxygen sensors et al., 2012). genes have been identified that specific and upon low oxygen et al., The expression of these genes be as an of hypoxic of of genes, including and has been and et al., 2014; Weits et al., 2014; et al., 2016). are to measure the oxygen concentration in different tissues et al., The are either into the tissue or up by the tissue through and 2015). the signal is using a to the oxygen parameters to be can be in e.g. to and is as several are in animals and rely on transport via the to the A was and to hypoxia in et al., The of the is by oxygen, but when the concentration below the signal However, cellular of appears to be and its into plant cells not of these oxygen such as the of or that with transport proteins, by the cell. as oxygen for plants be with the oxygen concentration in a for oxygen was established et al., 2012). This an protein with a protein of the oxygen and from the to the on the oxygen availability. Remarkably, the ratio could be to the oxygen concentration in a from to a of the oxygen has been to hypoxia in detection of oxygen is on the of which in to cells and strongly to of hypoxic cells et al., 2015). This can be with to provide of the of the oxygen concentration the tissue et al., to oxygen in tissues is This is on the of the of which has a to the tissue oxygen to oxygen in tissues is allows of the oxygen concentration in tissue et al., of the oxygen concentration using or is not limited by the of the and it an to the oxygen concentration in a manner of or and oxygen also for their in on the of in to the and their in plant tissue must be affects diverse organelles, which to stress signals that must be and integrated to initiate an appropriate adaptive response of the plant cell. The diverse and of a drop in oxygen for cellular strongly a in which oxygen sensing does not through one primary sensor but decentralized and at multiple sites in the cell 1). of multiple low-oxygen pathways from diverse cellular to this signaling to the through various pathways. to low-oxygen stress results in an oxidative which to activation of diverse transcription factors The of calcium a signaling and and the activation of are in to ROS by with OF The resulting ROS is through the of which as well as by PROTEIN1 which a complex with and Furthermore, oxygen might oxidative protein in the endoplasmic reticulum through the activity of which results in an protein response In addition, low-oxygen stress causes mitochondrial which is to the via mitochondrial retrograde at the are to the signal to the Further, impairment of the mitochondrial electron transport chain is by a mitochondrial oxidative and an of calcium into the The oxidative results in the activation of and which regulate hypoxic expression by of transcriptional of the further to a drop in levels, which is sensed by the kinases OF and is is under limitation. of RELATED TO APETALA2.12 and other subgroup VII through of the N-end rule pathway under hypoxic aerobic conditions, this pathway oxygen-dependent of the that is by RAP2.12 protein abundance is increased by HYDRAULIC CONDUCTIVITY ROOT1 (HCR1), a Raf-like which the under and integration of the diverse signal inputs at the level of which are regulated by and transcriptional reprogramming resulting in hypoxia the transcriptional at least transcription factors are upon low-oxygen stress in Arabidopsis (Licausi et al., the roles for of these transcription factors or the kind of signals upon low-oxygen stress are it is which signals activate ERFVIIs upon low-oxygen stress in The rule ERFVII target of rice was shown to be a and 2016), suggesting signal Interestingly, activity in Arabidopsis is by a mitochondrial signal upon oxygen (Chang et al., 2012). transcription factor of the might a crucial role in oxygen signaling et al., Furthermore, or signaling was shown to rely on transcription factors that to be et al., et al., 2013). One of the in the is to all transcription factors that act as signal integration points during low-oxygen stress, to the signals and the mechanisms through which act in a and manner. a of retrograde signaling molecules have been including calcium, and diverse et al., 2016). it is how these signals act during low-oxygen stress. the oxidative observed in mitochondria during oxygen was shown to activate (Chang et al., 2012). calcium and ROS might act in to an integrative as observed during stress et al., 2016). signaling does not only signaling to the but also between and 2016). Here, the question raises low signaling of one can be Abiotic stresses in energy metabolism and provoke mitochondrial which the activation of and In addition, signaling molecules calcium and ROS are to all different of stresses. Despite this in stress responses at the cellular and molecular cells can differentiate between stresses and respond be to do plants might be of stress signal ROS or calcium and the and In and cell it was shown that cellular stress sensors act in et al., In the of signaling has only to et al., research (see on the multiple signaling pathways that are by oxygen their and to reveal how the adaptation to low-oxygen stress is and to a particular by This will also the of methods to measure molecular oxygen levels with a high and