Making sense of low oxygen sensing Making sense of low oxygen sensing Julia Bailey-Serres1, Takeshi Fukao1, Daniel J. Gibbs2, Michael J. Holdsworth2, Seung Cho Lee1, Francesco Licausi3,4, Pierdomenico Perata4, Laurentius A.C.J. Voesenek5,6 and Joost T. van Dongen3 1 Center for Plant Cell Biology, Department of Botany and Plant Sciences, University of California, Riverside, CA 92521-0124, USA 2 Division of Plant and Crop Sciences, School of Biosciences, University of Nottingham, Loughborough LE12 5RD, UK 3 Max Planck Institute of Molecular Plant Physiology, Am Muehlenberg 1, 14476 Potsdam-Golm, Germany 4 PlantLab, Institute of Life Sciences, Scuola Superiore Sant’Anna, Piazza Martiri della Libertà 33, 56127 Pisa, Italy 5 Plant Ecophysiology, Institute of Environmental Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands 6 Center for Biosystems Genomics, 6708 PB Wageningen, the Netherlands Review Glossary Anoxia: absence of oxygen. Direct oxygen sensing: sensing of oxygen via its molecular interaction with a ligand (i.e. enzyme, protein, chemical compound) that results in an effect of cellular consequence. Hypoxia: oxygen levels below normoxia; the term ‘hypoxic’ is often used to describe a situation where molecular oxygen is still present, but its level has significantly decreased below 20.6%; cellular oxygen status may be hypoxic or anoxic dependent upon duration, location and metabolic activity. Hypoxia-responsive genes: genes with transcripts differentially regulated in response to conditions with a low oxygen component. Indirect oxygen sensing: sensing of change in homeostasis that is a consequence of oxygen deprivation (i.e. change in ATP, ADP, AMP, other metabolite, Ca 2+ , ROS, pH) that results in an effect of cellular consequence. Normoxia: typically 20.6% oxygen at 1 atm and 20 8C. Submergence: waterlogging and partial to complete immersion of aerial system. Plant-specific group VII Ethylene Response Factor (ERF) transcription factors have emerged as pivotal regulators of flooding and low oxygen responses. In rice (Oryza sativa), these proteins regulate contrasting strategies of flooding survival. Recent studies on Arabidopsis thaliana group VII ERFs show they are stabilized under hypoxia but destabilized under oxygen-replete conditions via the N-end rule pathway of targeted proteolysis. Oxygen- dependent sequestration at the plasma membrane maintains at least one of these proteins, RAP2.12, under normoxia. Remarkably, SUB1A, the rice group VII ERF that enables prolonged submergence tolerance, appears to evade oxygen-regulated N-end rule degradation. We propose that the turnover of group VII ERFs is of eco- logical relevance in wetland species and might be ma- nipulated to improve flood tolerance of crops. Improved crop survival of floods is needed Based on conservative expectations of human population growth, the maintenance of international food security will require a doubling of agricultural productivity in the next two decades [1]. This challenge is exacerbated by severe weather events associated with climate change such as floods, which have occurred with increasing fre- quency across the globe over the past six decades (Figure 1). However, improvement of crop resilience to water extremes can be accomplished by harnessing natu- ral genetic diversity in breeding programs. An example of this is the use of the rice SUBMERGENCE 1A (SUB1A) gene, which confers prolonged tolerance to submergence (see Glossary) [2]. The effective SUB1A-1 allele was iso- lated from an eastern Indian landrace and has been returned to farmers in high-yielding varieties [3]. This new ‘Sub1 rice’ promises to help stabilize harvests in rain- fed floodplains, which represent 33% of rice acreage world- wide [4]. The task remains to improve flooding tolerance of other crops. Recent comparative studies within and be- tween species have greatly enhanced our understanding of mechanisms that facilitate survival of distinct flooding regimes (Table 1). With new insights into low oxygen sensing and response mechanisms we are optimistic that effective means to lessen crop devastation by flooding can be extended beyond rice paddy fields. Corresponding author: Bailey-Serres, J. (serres@ucr.edu) 1360-1385/$ – see front matter � 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.20 Oxygen deprivation is a frequent component of flooding stress A key feature of flooding events is the change in levels of three gases, O2, CO2 and ethylene, due to a near 10 4 reduction in their diffusion in water relative to air [5– 8]. The flooding of root systems – a condition termed waterlogging – has little or no impact in semi-aquatic species such as rice that constitutively form gas conduits (i.e. aerenchyma) between submerged and aerial organs. However, if plants lack gas conduits or lose oxygen from roots, waterlogging rapidly reduces the oxygen concentra- tion within cells [9–11]. The presence of aerobic microbes in the soil can further exacerbate the stress. When both the root and aerial portions of a plant are whelmed by water – a condition termed submergence – cellular oxygen levels can also decline from normoxia. The degree of oxygen deficien- cy (hypoxia/anoxia) depends on multiple factors including replenishment of oxygen through photosynthesis, inward diffusion from the water layer and cellular consumption of oxygen through metabolic activity. Severe oxygen defi- ciency compromises mitochondrial respiration [10,12] and leads to an insufficiency in ATP for energy demanding processes [6,13–15]. However, plants can adjust to this energy crisis through increased substrate level ATP pro- duction (Figure 2). This is accomplished by catabolism of soluble sugars and in some species or cell types starch [16]. Typically, the increase in glycolytic flux is coupled with regeneration of NAD+ by fermentation of pyruvate to Waterlogging: flooding of root system. 11.12.004 Trends in Plant Science, March 2012, Vol. 17, No. 3 129 mailto:serres@ucr.edu http://dx.doi.org/10.1016/j.tplants.2011.12.004 1950 2009 1950 2009 1950 2009 1950 2009 1950 2009 America Africa Oceania Europe 0 100 200 300 500 600 700 Number of flood events per decade 400 Asia TRENDS in Plant Science Figure 1. Numbers of floods have increased in each of the past six decades across the globe. Graphs show the number of floods classified as a disaster in the International Disaster Database of the University of Louvain, Belgium for the period from 1950 through 2009 by geographical region [93]. Events include river or coastal floods, rapid snow melts, heavy rainfall and other occurrences that caused significant social or economic hardship. Adapted from a Millennium Ecosystem Assessment map (http:// maps.grida.no/go/graphic/number-of-flood-events-by-continent-and-decade-since-1950). Review Trends in Plant Science March 2012, Vol. 17, No. 3 ethanol via pyruvate decarboxylase and alcohol dehydro- genase (ADH). Because ethanol diffuses out of cells into the external milieu, its production depletes the plant’s carbon reserves. Therefore, metabolism of pyruvate to alanine provides an alternative, non-detrimental end product of anaerobic metabolism that is observed in a number of species [17,18]. This includes the generation of 2-oxoglutarate as a coproduct, which can be further metabolized to succinate, via the TCA cycle enzyme succi- nate CoA ligase (SCS), thereby providing additional ATP per molecule of sucrose metabolized. To keep these reac- tions running, the oxidation of NADH in the mitochondrial matrix is guaranteed by reduction of oxaloacetate via the reversed TCA cycle reaction catalyzed by malate dehydro- genase [19,20]. The malate produced is probably further converted to fumarate and succinate [21], the latter of which could be exported from hypoxic tissue to the aerated parts of the plant. At least in tubers of potato (Solanum tuberosum), hypoxia stimulates a rearrangement of the mitochondrial respiratory supercomplexes that enhances regeneration of NAD+ by the alternative NAD(P)H dehy- drogenases [22]. Even though the efficiency of hypoxic ATP production is low compared to aerobic oxidative phosphorylation, it allows cells to survive as long as carbohydrate substrate remains available. Cell death only becomes inevitable when there is insufficient energy for exclusion of protons to the apoplast to prevent membrane depolarization and to maintain a near neutral cytosolic pH [6,23,24]. Avoidance of the severe energy crisis associated with low oxygen stress requires economization of ATP consumption. Means to this end include energy efficient sucrose catabolism through sucrose synthase [25], the preferential use of PPi-dependent enzymes [26], constrained catabolism of storage compounds such as starch, lipid and protein [13], metabolic compartmentalization [27], reduced protein 130 synthesis [28], increased production of heat shock proteins as molecular chaperones [29] and adoption of the K+-gra- dient to energize membrane transport [30]. Plant survival of waterlogging or submergence also depends on their ability to limit or endure oxidative stress, which occurs during the transition from normoxia to anoxia as well as upon de-submergence [31–33]. Ethylene initiates submergence survival strategies in rice and wetland species Recent work has exposed mechanisms of response to sub- mergence that center on growth management. Notable are two antithetical survival strategies displayed by both wild and domesticated species. For example, deepwater rice, cultivated to cope with slowly advancing floods, expends energy reserves in the elongation of internodal regions that are underwater to maintain photosynthetic tissue above the air–water interface [34,35]. Similarly, the wetland dicot Rumex palustris, which is well adapted to shallow but prolonged floods, reorients and extends petioles to elevate leaves above the surface of floodwaters [6,7]. How- ever, this ‘submergence escape’ strategy is unsuccessful if energy reserves are exhausted before escape of the deluge. In wetland species capable of surviving transient floods (e.g. Rumex acetosa) [36] and submergence tolerant Sub1 rice [37], a ‘quiescence strategy’ minimizes energy expen- ditures for growth until de-submergence. The genetic determinants and hormonal signaling path- ways that underlie the two flooding survival strategies have been identified. In rice, both strategies utilize the phytohormone ethylene and ethylene response factor (ERF) transcription factors. Combined physiological and molecular dissection of submergence responses in rice and R. palustris has yielded a model in which a buildup of ethylene in submerged organs initiates a hormonal signal- ing cascade that reduces the antagonism between abscisic http://maps.grida.no/go/graphic/number-of-flood-events-by-continent-and-decade-since-1950 http://maps.grida.no/go/graphic/number-of-flood-events-by-continent-and-decade-since-1950 Table 1. Factors that contribute to survival of flooding or oxygen deprivation Condition Species Comparison Acclimation or survival response Causal factors Refs Submergence (partial) Rice (Oryza sativa ssp. indica) Deepwater to non-deepwater cultivars; SK1 and SK2 transgenics Rapid underwater internode elongation; escape strategy SK1, SK2, ethylene, GA, ABA [34] Submergence (complete) Rice (ssp. indica, aus and japonica) Near isogenic lines; SUB1A-1 transgenics Growth restriction; quiescence strategy SUB1A-1, ethylene, reduced GA responsiveness [2,37,73] Submergence (seed) Rice (ssp. indica) Anaerobic germination of tolerant to non-tolerant cultivars; cipk15 mutant Enhanced coleoptile and shoot elongation Quantitative trait loci; enhanced starch degradation; CIPK15 [74,75] Submergence (under anoxia) Rice (ssp. indica) To wheat Seed germination and coleoptile elongation Adjustment of metabolism; reduced oxygen consumption [76] Wheat (Triticum aestivum) To rice No germination and coleoptile elongation inhibited Limited adjustment of metabolism and oxygen consumption [76] Submergence Marsh dock (Rumex palustris) Ecotypes with fast and slow underwater elongation responses Petiole elongation Fast elongation associated with lower endogenous ABA [38] Common sorrel (Rumex acetosa) To Rumex palustris Limited petiole elongation Maintenance of ABA under submergence [36] Arabidopsis thaliana 86 accessions Varied survivability Unknown [77] Meionectes brownii Variation in light Photosynthetic aquatic adventitious roots Reduced need for shoot photosynthate [78] Anoxia Rice To wheat Sucrose or glucose-fed wheat seeds survive longer a-Amylase produced under anoxia in rice but not in wheat seedlings [79] Chlamydomonas reinhardtii Wild type to mutant Transcriptomic and metabolic adjustments Versatile metabolic adjustments such as H2 production [80–82] Pondweed (Potamogeton distinctus) To pea (Pisum sativum) Turion elongation and survival Enhanced H + extrusion and stabilization of cytosolic pH [83] Grape (Vitis sp.) Anoxia tolerant (Vitis riparia) to intolerant (Vitis rupestris) Improved survival of hypoxia pretreated roots Fermentation and maintenance of ion homeostasis (e.g. K + ) [84] Hypoxia to anoxia Arabidopsis thaliana Wild type to loss-of-function or other insertion mutants and overexpression transgenics Low oxygen and/or submergence survival HRE1, HRE2, RAP2.2, RAP2.12; VERNALIZATION INSENSITIVE 3; EXORDIUM 1; HEAT SHOCK FACTOR 2a; HYPOXIA-RESPONSIVE UNKNOWN PROTEIN (HUP) genes [29,41, 47,62,63,65, 66,72,85,86] Wild type to loss-of-function prt6 and ate1ate2 mutants Low oxygen and/or submergence survival; seed germination under hypoxia N-end rule pathway components PRT6, ATE1, ATE2 [66,72] Wild type to loss-of-function mutants and overexpression transgenics Seed germination under 0.1% oxygen NAC transcription factor ANAC102 [87] Waterlogging Lotus japonicus Wild type to N-deficient nodular leghemoglobin RNAi transgenics Alanine and succinate accumulation Modified TCA flux mode [19] Poplar (Populus � canescens) Root to shoot Transcriptomic and metabolic adjustments; limited shoot response Unknown [88] Cotton (Gossypium hirsutum) Root to shoot Transcriptomic and metabolic adjustments; shoot growth inhibition Unknown [89] Maize (Zea mays) Root cell type mRNAs Aerenchyma formation Ethylene, Ca 2+ , ROS; cortex mRNAs [90] Rice Response to compounds Adventitious root development Epidermal cell death mediated by ethylene and ROS [91] Tomato (Solanum lycopersicum) Response to hormone biosynthesis inhibitors Adventitious root development Ethylene and auxin [92] Review Trends in Plant Science March 2012, Vol. 17, No. 3 131 UDP SUS AmylasesINV H 2 O Glucose Glucose Glucose-1-P Glucose-6P Glucose-1-PUDP-glucose Fructose Fructose-6P Fructose-1,6P 2 Phosphoenolpyruvate Pyruvate Acetaldehyde Glutamate Aspartate 2-Oxoglutarate 2-Oxoglutarate Oxaloacetate Isocitrate Malate Fumarate Glutamate2-Oxoglutarate Citrate Succinyl CoA Succinic semialdehyde Oxidative phosphorylation +O2 α-ketoacid Fructose ++ PPi PPi Pi Pi Sucrose Starch UTP UTP UTP ATP ATP ATP ATP NADH NADH NADH NADH NAD(H) FADH 2 NADH Non-circular TCA flux NADH Amino acid LDH PDC GDH NADP(H) MDH GAD ADH NAD+ NAD+ NAD+ NAD+ NAD+ NAD(P)+ NAD(P)+ NADP(H) ATP ATP ADP SCS ATP ATP NH4 + NAD+ NAD+ NAD+ H+ NAD+ ATP + Pi AMP + PPi ATPoror ATP ADP ADP ADP ADP NADH Glycolysis Glycolysis Upregulated by low oxygen (Pasteur effect) to increase substrate level ATP production Fermentation Induced by low oxygen; aids redox equilibration and provides NAD+ to maintain glycolysis GABA shunt GAD uses protons as substrate and may help stabilize cytosolic pH Decreased respiration May occur by downregulation of net NADH production via the TCA cycle or reduced mETC activity; conserves oxygen Starch breakdown Amylase levels increase in some species to keep pace with increased carbon demand Alanine and 2OG shunt Prevents loss of carbon via fermentation and routes 2OG into the oxidative TCA branch to yield additional ATP by substrate level phosphorylation Energy efficiency Sucrose degradation shifts from INV to SUS; some species use PPi consuming enzymes in sucrose breakdown and glycolysis, increasing net production of ATP Lactate Alanine Succinate GABA Ethanol ADP ADP UDP UDP TRENDS in Plant Science Acetyl CoA Figure 2. Metabolic reconfiguration under low oxygen stress. Reduced oxygen availability alters metabolism to maximize substrate level ATP production. The model depicts the major known changes that include enhanced sucrose–starch metabolism, glycolysis, fermentation, a modified tricarboxylic acid (TCA) flow, an alanine and 2- oxoglutarate (2OG) shunt and a g-aminobutyric acid (GABA) shunt. The hypothesis that oxygen is conserved is under further investigation. Yellow boxes summarize notable metabolic adjustments. Blue lines indicate pathways enhanced during the stress, blue dashed lines indicate pathways proposed to be active during the stress and gray dashed lines indicate reactions that are inhibited during the stress. Metabolites that increase during the stress are shown in enlarged black font; metabolites that decrease are shown in red font. Abbreviations are as follows: 2OG, 2-oxoglutarate; ADH, alcohol dehydrogenase; GAD, glutamic acid decarboxylase; GDH, glutamate dehydrogenase, INV, invertase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; PDC, pyruvate decarboxylase; SCS, succinyl CoA ligase; SUS, sucrose synthase. Review Trends in Plant Science March 2012, Vol. 17, No. 3 132 Review Trends in Plant Science March 2012, Vol. 17, No. 3 acid (ABA) and gibberellins (GA), which normally limits cell elongation. In rice, natural variation in the presence and absence of the underwater escape [SNORKEL (SK) 1 and 2] and submergence tolerance (SUB1A) group VII ERF determinants underlies differential regulation of the hor- monal cascade and hormone sensitivities that control un- derwater growth [8] (Box 1). Two R. palustris populations distinguished by fast and slow underwater petiole elonga- tion were differentiated by the maintenance of higher levels of ABA and reduced GA responsiveness in the slow elongating variety during submergence [38]. Consistently, the limited underwater petiole growth and prolonged sub- mergence survival of R. acetosa was linked to maintenance of ABA biosynthesis that translated into lower GA respon- siveness during submergence [36]. Photosynthesis can continue in submerged leaves and is aided by the gas film that often clings to their surface [39,40]. It follows that the degree of oxygen deprivation in photosynthetic tissue may be less extreme than tissues distant from an oxygen source. Nevertheless, the ethylene- driven underwater elongation of shoot tissue can deplete carbohydrates and lead to an energy crisis. Box 1. Contrasting submergence survival strategies of rice Most accessions respond to submergence through rapid shoot elongation, which allows emergence from a shallow flood [6]. A limited number of accessions display the ability to survive a slow progressive flood (escape response) or a deep transient flash flood (quiescence response) (Figure I). (a) By amplifying the elongation of stem internodes, deepwater rice can outgrow a progressive flood and survive partial inundation for months. This deepwater escape strategy is controlled by the SNORKEL (SK) locus, which encodes two group VII ERFs, SK1 and SK2 [34]. SKs are absent from lowland varieties. (b) The molecular genetic analysis of the submergence-tolerant acces- sion FR13A revealed that the SUBMERGENCE 1 (SUB1) locus, encoding two or three group VII ERFs, regulates the quiescence response. SUB1B and SUB1C are invariably encoded at SUB1 in lowland accessions, whereas SUB1A is limited to some indica and aus landraces [2,60]. The SUB1A-1 allele is sufficient to confer survival of 2 weeks or longer of complete submergence. (c) Model of the core submergence response network that is influenced by SKs and SUB1A. Genotypes possess either SK1/SK2, SUB1A or neither. Both SK1/SK2 and SUB1A-1 mRNA are ethylene induced. In deepwater rice, SK1/ SK2 and two minor QTLs augment accumulation of bioactive GA in stem internodes during submergence. In submergence tolerant rice varieties, the presence of SUB1A-1 influences submergence and post- submergence responses in aerial tissue. (1) SUB1A-1 mRNA is ethylene-induced but ultimately limits ethylene biosynthesis [37]. (2) SUB1A-1 promotes accumulation of two negative regulators of GA responses [SLENDER RICE 1 (SLR1) and SLENDER RICE-LIKE 1 (SLRL1)] [73]. (3) SUB1A-1 does not perturb the submergence- induced decline in ABA content but heightens sensitivity to ABA [32]. (4) SUB1A-1 limits induction of genes associated with starch breakdown [37,41,94]. (5) SUB1A-1 enhances upregulation of genes associated with reactive oxygen species (ROS) amelioration and survival of dehydration, thereby improving re-establishment follow- ing de-submergence [32]. (6) SUB1A-1 interacts with a complex network of proteins [95]. (7) SUB1A-1 transiently restricts the progression to flowering during submergence [96]. In summary, SUB1A is remarkably positioned to suspend growth and maintain cell viability during submergence and restore homeostasis during a subsequent recovery period. Similarities in transcriptome response to flooding and oxygen deprivation Numerous investigations have assessed changes in tran- scriptomes in response to low oxygen stress or flooding in plants including Arabidopsis, rice, poplar (Populus � canescens) and cotton (Gossypium hirsutum) [41,42]. Stud- ies performed on seedlings of the Arabidopsis Col-0 eco- type include evaluation of the effects of different severity [43] and duration [28,44] of oxygen depletion, as well as the impact of heat stress prior to anoxia [29]. Because the majority of cellular mRNAs are poorly translated during oxygen deprivation [45], changes in polyribosome-associ- ated mRNAs were used to evaluate dynamics in the stress and recovery responses [28]. In 21 cell types or regions of roots and shoots, polyribosomes were captured by immu- nopurification to identify transcripts regulated by short- term oxygen deprivation [46]. This approach identified 49 core hypoxia-responsive genes that were strongly induced by the stress across all samples evaluated. Also distin- guished were cohorts of mRNAs that were hypoxia-re- sponsive at the organ or cell-specific level, although their modulation was less pronounced than the core Progressive flood – deepwater rice (SK1/2 ) Partial submergence Complete submergence Submergence Reoxygenation ROS ROS amelioration SLR1 SLRL1 Dehydration response Drought recovery Ethylene ABA ABASUB1ASK1/SK2 GA Elongation growth Dehydration Reoxygenation and dehydration Flash flood – Sub1 rice (SUB1A) (c) (a) (b) TRENDS in Plant Science Figure I. Group VII ERFs and pathways that regulate growth responses under distinct flooding regimes. 133 Review Trends in Plant Science March 2012, Vol. 17, No. 3 hypoxia-responsive genes. Of the 49 core hypoxia-respon- sive genes, 24 were also differentially regulated in roots and rosette leaves during submergence in complete dark- ness [47]. Finally, meta-analyses that compared tran- scriptomic adjustments to low oxygen or flooding stress identified conservation in the core network of genes asso- ciated with signaling, transcription and efficient anaero- bic ATP production that is modulated by oxygen deprivation in a range of plants [41,42]. How do plant cells sense low oxygen stress? Based on mechanisms in other eukaryotes, both indirect and direct sensing of cellular oxygen status could be re- sponsible for acclimation responses that prolong survival of oxygen deprivation in plants [48]. Indirect sensing mechanisms might include perception of altered energy status through changes in levels of adenylates (ATP, ADP and/or AMP), consumable carbohydrates, pyruvate, cyto- solic pH, cytosolic Ca2+ or localized production of reactive oxygen species (ROS) and nitric oxide (NO). Animal and yeast cells sense and adjust energy homeo- stasis through Sucrose Non-Fermenting 1 (SNF1)/AMP- activated protein kinases [49,50]. The plant energy sensors fall within one clade of SNF1 relatives, the SnRK1s, some of which have been implicated in low oxygen responses. For example, Arabidopsis KIN10 and KIN11 are necessary to limit energy consumption during hypoxia [51]. Whereas in rice seeds germinated under oxygen starvation, the deple- tion of sucrose activates the SnRK1A energy sensor through the activity of a Calcineurin B-like interacting binding kinase 15 (CIPK15) [52]. This signal transduction upregulates transcription of genes encoding a-amylases, which drive catabolism of starch in the seed needed to fuel underwater shoot growth. Logically, a reduction of energy consumption is beneficial when ATP levels decline. A means of energy conservation during low oxygen stress in plants is selective translation and sequestration of mRNAs during hypoxia [28]. Based on evidence from other eukaryotes, the sequestration of a subset of cellular mRNAs, such as the abundant cohort that encodes ribo- somal proteins and translation factors, could be regulated through SnRK1s and the Target of Rapamycin kinase [53]. Mitochondria are also thought to contribute to oxygen sensing and signaling in plants, through production of NO and/or release of ROS and Ca2+ during the transition from normoxia to hypoxia [54,55], as confirmed in animals [56,57]. In animals, direct oxygen sensing regulates the accu- mulation of the a subunit of the hypoxia inducible factor (HIF) 1a/b transcription factor [58]. HIF1a is constitu- tively synthesized but fails to accumulate under nor- moxia because of oxygen-dependent hydroxylation of specific proline residues that trigger its ubiquitination and 26S proteasome-mediated degradation. As oxygen declines, the prolyl hydroxylases that modify HIF1a are less active. Consequentially, HIF1a accumulates and is trafficked to the nucleus where HIF1a/b can function in transcriptional activation. There is no corol- lary direct oxygen sensing mechanism in plants, because although they possess prolyl hydroxylases they lack HIF1a [41]. 134 Group VII ERFs regulate low-oxygen acclimation responses The plant-specific ERF transcription factor family includes over 100 members in rice and Arabidopsis, all of which share an APETALA2 (AP2) DNA binding domain [59]. The ERFs have been phylogenetically parsed into ten clades, with the group VII ERFs characterized by a conserved N terminal motif (NH2-MCGGAI/L) [59] (Figure 3a). Fifteen rice (japonica cv. Nipponbare) ERFs were designated group VIIa (OsERF59-72) and VIIb (OsERF73), based on the presence or absence of the conserved N terminal motif, respectively. The single group VIIb ERF corresponds to SUB1C, which is found in all rice varieties surveyed [60] and acts downstream of SUB1A [37]. Intriguingly, the group VII ERFs encoded by SUB1A, SK1 and SK2 possess variant N termini relative to the rice group VIIa ERFs. Arabidopsis encodes five group VII ERFs (AtERF71–75), two of which are hypoxia-responsive genes {HYPOXIA RESPONSIVE ERF1 and 2 [HRE1 (AtERF73; At1g72360) and HRE2 (AtERF71; At2g47520)]}. As ob- served for SUB1A and the SKs, HRE1 mRNA accumula- tion is promoted by ethylene, which synergistically enhances its elevation during hypoxia [61,62] (Figure 3b). Several recent reports indicate that Arabidopsis group VII ERFs redundantly regulate hypoxia-responsive gene expression and survival of low oxygen stress. For example, seedling survival of anoxia was more severely compro- mised in hre1hre2 double mutant seedlings than in either single mutant or the wild type [61,63]. By contrast, low oxygen sensitivity was lessened in seedlings that constitu- tively overexpress either HRE1 or HRE2 mRNA. The ectopic expression of these ERFs was sufficient to heighten induction of the core hypoxia-responsive gene ADH1 or ADH enzyme activity during the stress [61,63]. However, because hre1hre2 seedlings were able to elevate ADH enzyme activity and ethanol production during hypoxia [63], genetic redundancy is likely to extend to the other group VII ERFs [RAP2.12 (AtERF75; At1g53910), RAP2.2 (AtERF74; At3g14230) and RAP2.3 (AtEBP/AtERF72; At3g16770)]. Indeed, conditional upregulation of RAP2.12 was sufficient to elevate expression of a pADH1:- LUCIFERASE transgene [64] and RAP2.2 overexpression improved survival of hypoxia in seedlings [65], whereas the inhibition of either RAP2.2 or RAP2.12 expression via miRNA production limited the induction of ADH1 and several other hypoxia-responsive genes [66]. The impact of ectopic expression of these genes was condition specific, as HRE1, HRE2, RAP2.2 and RAP2.12 overexpression significantly increased levels of ADH1 mRNA or ADH activity under low oxygen stress but not under normoxia [61,65,66]. Nonetheless, RAP2.2, RAP2.3 and RAP2.12 mRNAs accumulate under normoxia in association with polyribosomes [46,67], suggesting they are constitutively synthesized. Together, these findings hint that post-trans- lational regulation limits the function of group VII ERFs to periods of low oxygen stress. Arabidopsis group VII ERFs are degraded via the N-end rule pathway The conserved N-end rule pathway of targeted proteolysis regulates the half-life of certain cellular proteins based on PM Nucleus A C B P Hypoxia responsive RAP2.12 RAP2.12 RAP2.12 RAP2.12 RAP2.12 Normoxia Hypoxia Flooding Normoxia ERF ERF ERF Hypoxia Flooding -O2 HRE2 Ethylene low O2 low O2 Constitutiv e dark ethylene Constitutive Constitutive ethylene ? Transcription / translation Arabidopsis group VII ERFs RAP2.3RAP2.12RAP2.2HRE1 Constitutiveee ethylene ??? RAP2.3 +O 2 MC ERF C ERF C* ERF Hypoxia responsive ERF PRT6, Other E3s? 26S proteasome O2, NO MAPs ATEs R-C* Ub ERF ERF ERF Anaerobic metabolism and other responses ERF R-C* R ic e Group VII ERFs A ra b id o p si s (a) (b) (c) TRENDS in Plant Science Figure 3. Oxygen sensing via N-end rule pathway-targeted turnover of group VII ERFs. (a) N terminal alignment of Arabidopsis group VII ERFs. With the exception of SUB1C, all begin with the amino acids ‘Met-Cys’ (MC). The highly conserved Arabidopsis N terminal motif is boxed in red and is less conserved in the proteins at loci associated with submergence responses in rice. (b) Homeostatic response to hypoxia is regulated by the N-end rule-mediated proteolysis of group VII ERFs in Arabidopsis. Group VII ERF transcription factors are either constitutively expressed and/or differentially transcriptionally regulated in response to variable signals, including low O2, ethylene and darkness. Four of the five ERFs (HRE1, HRE2, RAP2.2 and RAP2.12) have been implicated in the regulation of hypoxia-responsive genes. Under oxygen-replete conditions (normoxia), ERFs are degraded via the N- end rule pathway of proteolysis. This involves the following steps: (i) the N terminal Met (M) is constitutively cleaved by a methionine aminopeptidase (MAP); (ii) the exposed Cys (C) is converted to an oxidized (C*) form (e.g. Cys-sulfonic acid) by O2, NO or possibly ROS; (iii) an Arg (R) residue is added to the oxidized Review Trends in Plant Science March 2012, Vol. 17, No. 3 recognition of N terminal residues by specific N-recognin E3 ligases [68]. In plants, 11 amino acids function as destabilizing residues when located at the N terminus of a protein, which coupled with an optimally positioned downstream lysine can act as a degradation signal (N- degron) [69]. In plants and animals but not yeast, a cyste- ine (Cys) residue at the N terminus can undergo two steps of modification that lead to protein recognition and degra- dation (Figure 3b). Based on mechanistic studies in mam- mals, newly synthesized proteins with a Cys as the second residue (i.e. NH2–Met1–Cys2) are constitutively cleaved by a Met amino peptidase (MAP) to yield NH2–Cys2. In Ara- bidopsis, a small family of functionally redundant MAPs catalyzes this reaction [70]. The exposed Cys2 can be spontaneously or enzymatically oxidized in an O2- or NO-dependent manner to Cys-sulfinate or further to Cys-sulfonate [68]. As a result of oxidation, an arginine residue is added to the NH2–Cys2 by an arginyl tRNA transferase (ATE), targeting the protein for recognition by an N-recognin E3 ligase, leading to ubiquitination and 26S proteasome-mediated degradation. In Arabidopsis, the genes ATE1 and ATE2 encode the Arg transferases [71] and at least one E3 ligase, encoded by PROTEOLYSIS 6 (PRT6), acts as an N-recognin of NH2–Arg1–Cys2 oxidised polypeptides [69]. The distinct conservation of the N terminus of group VII ERFs and the serendipitous observation that an Arabidop- sis prt6 mutant constitutively accumulates ADH1 and other hypoxia-responsive mRNAs in seeds led to the con- firmation that group VII ERFs are bona fide substrates of the N-end rule pathway in plants [66,72] (Figure 3b). Additional support of this conclusion was obtained through in vitro and in planta analyses. An in vitro system derived from rabbit reticulocytes [72] was used to confirm that all five Arabidopsis group VII ERFs are N-end rule substrates. It was also shown that their instability required Cys2, as mutation of Cys2 to the stabilizing residue Ala2 (NH2–Met1–Ala2) eliminated sus- ceptibility to N-end rule turnover. It was further demon- strated in planta that low oxygen stress increased the accumulation of group VII ERFs synthesized with a native N-terminus (NH2–Met1–Cys2), whereas those synthesized with an NH2–Met1–Ala2 N terminus were stable under both normoxia and hypoxia [66,72]. Based on this evidence, the stabilization of group VII ERFs under hypoxia is most probably related to an inhibition of the Cys2 oxidation that is required before the protein can be arginylated and degraded. Cys by an arginyl tRNA transferase (ATE); and (iv) the argininylated protein is recognized by PROTEOLYSIS 6 (PRT6) or other E3 ligases, which polyubiquitinate the protein, targeting it for proteasomal degradation (26S proteasome). The outcome is prevention of transcription of hypoxia-responsive genes under normoxia. When oxygen becomes limiting (hypoxia), degradation of the ERFs by the N-end rule pathway is inhibited due to a lack of oxygen-mediated Cys2 oxidation. Stabilized ERFs can then drive the transcription of genes that enhance anaerobic metabolism and other survival responses. Upon return to aerobic conditions, the ERFs are once again destabilized, providing a feedback mechanism that allows the plant to return to aerobic metabolism. (c) AtRAP2.12 localization dynamics. At least one group VII ERF, RAP2.12, associates with the plasma membrane (PM) via interaction with ACBP, limiting its turnover under normoxia. During hypoxia RAP2.12 is relocated to the nucleus and activates gene expression. Upon reoxygenation, RAP2.12 is destabilized, presumably as a consequence of Cys2 oxidation and N-end rule-mediated degradation. 135 Box 2. Key questions for future experimentation � Can O2, NO or ROS-dependent Cys2 oxidation, arginylation and ubiquitination of group VII ERFs be experimentally confirmed? If so, is the oxidation spontaneous or catalyzed? � What are the kinetics of oxygen-regulated group VII ERF turnover? Does ERF stabilization occur before oxygen deficiency impairs cytochrome c oxidase activity? � Does the oxygen level affect the interaction between ACBPs and RAP2.12? Does docking of RAP2.12 to ACBP impair Cys2 oxida- tion, modification by ATE, or interaction with an E3 ligase? Are other group VII ERFs similarly sequestered? � What genes and networks are controlled by individual group VII ERFs? � Is the activity or turnover of SUB1A, which apparently escapes oxygen-mediated N-end rule degradation, controlled upon de- submergence? � Can manipulation of group VII ERF accumulation and turnover provide an effective strategy to modulate survival of flooding in crops? Review Trends in Plant Science March 2012, Vol. 17, No. 3 Either the modification of the N terminus of a group VII ERF or disruption of an N-end rule pathway step can affect survival of low oxygen stress or submergence in Arabi- dopsis [66]. For example, stabilization of HRE1 and HRE2 by modification of the N terminus to NH2–Met1–Ala2 was sufficient to improve seed germination and seedling sur- vival under hypoxia [72]. In addition, ate1ate2 and prt6 seedlings were less sensitive to hypoxia when grown on sucrose-supplemented medium [72]. The same mutants grown to the rosette stage were more sensitive to submer- gence in complete darkness [66]. This discrepancy in phenotype might be explained by distinctions in the avail- able carbohydrates in the two survival assays. In the low oxygen experiments, anaerobic metabolism was fueled by sucrose in the medium, whereas in the submergence experiments it was limited to endogenous energy reserves of the plant. Therefore, the absence of PRT6 or ATE activity may enhance anaerobic metabolism to prolong survival in sucrose-fed seedlings but may cause a more rapid onset of energy deficiency in submerged plants. These findings are reminiscent of the earlier proposal that a balance between energy consumption and conservation is crucial to survival of low oxygen stress and submergence [5,37]. It was also observed that the onset of the transcription of hypoxia-responsive genes occurs concomitantly with relocalization of RAP2.12 to the nucleus under hypoxia (Figure 3c) [66]. During normoxia, a GFP-tagged version of RAP2.12 was protected against protein degradation by the N-end rule pathway of proteolysis and excluded from the nucleus via interaction with a plasma membrane (PM)- associated Acyl-CoA binding protein (ACBP1 or ACBP2). RAP2.12 migrated to the nucleus in response to hypoxia and disappeared from the nucleus after reoxygenation. Moreover, transient expression of RAP2.12-GFP in leaves of ate1ate2 and prt6 mutants resulted in greater GFP signal intensity in the nucleus under normoxia and follow- ing reoxygenation. In summary, the N-end rule pathway of proteolysis regulates the accumulation of group VII ERFs and conse- quentially the accumulation of gene transcripts associated with low oxygen responses in Arabidopsis. It is proposed that constitutively synthesized group VII ERFs are either degraded or sequestered under normoxia, as confirmed for RAP2.12. As oxygen levels fall their degradation becomes limited, PM sequestration is reversed and the ERF is transported to the nucleus and becomes active in gene regulation. Upon reoxygenation, both constitutively expressed and hypoxia-induced group VII ERFs are desta- bilized. Thus, the N-end rule pathway (i) prevents the excessive accumulation of constitutively expressed ERFs under normoxia; (ii) allows for stabilization of both consti- tutive and induced ERFs during hypoxia; and (iii) facil- itates rapid reversal of ERF-regulated transcription upon reoxygenation. Constitutively expressed group VII ERFs are proposed to encode oxygen sensors that conditionally activate transcription of hypoxia-responsive genes, includ- ing other group VII ERFs [66]. The increased synthesis of N-end rule regulated group VII ERFs by ethylene or darkness could further prime cells for acclimation to oxy- gen deprivation. 136 Manipulation of N-end rule regulation of group VII ERFs and other proteins The verification that the N-end rule pathway modulates group VII ERF accumulation in the nucleus in an oxygen- dependent manner exposes the first examples of N-end rule substrates and a homeostatic low oxygen sensor mech- anism in plants. Based on available gene sequence data, group VII ERFs with the conserved N-terminus are broad- ly found in vascular plant species [66]. We propose that future improvement of flooding tolerance could be achieved by manipulation of synthesis and turnover of these pro- teins (e.g. by overexpression, regulated expression and/or mutation of NH2–Met1–Cys2 to NH2–Met1–Ala2). Given the crucial importance of modulation of energy reserves during flooding, it is not surprising that variation of group VII ERF susceptibility to oxygen-dependent N- end rule turnover exists in nature. The rice Nipponbare genome encodes 15 group VII ERFs with the conserved N terminus that is consistent with oxygen-regulated N-end rule-targeted proteolysis in Arabidopsis. However, neither SUB1A nor SUB1C are N-end rule substrates based on in vitro data [72] and the N termini of SK1 and SK2 also deviate from the consensus associated with N-end rule- mediated turnover. This leads us to propose that the escape of SUB1A from N-end rule pathway turnover could allow the ethylene-mediated regulation of SUB1A-1 to trigger the sequence of events that promotes the energy management associated with submergence tolerance well before oxygen levels reach a critical nadir. Concluding remarks: direct oxygen sensing via the N- end rule regulates transcription Alterations in gene expression associated with increased catabolism and substrate level ATP production are a hallmark of reduced oxygen availability and flooding in plants. Group VII ERFs play a prominent role in this process. The identification of an oxygen-dependent pro- tein turnover mechanism that controls the abundance of some but not all group VII ERFs raises several pertinent questions (Box 2). We anticipate that genetic manipula- tion of the targets of oxygen-regulated N-end rule pathway turnover can provide a means to improve survival under a variety of flooding conditions. Review Trends in Plant Science March 2012, Vol. 17, No. 3 Acknowledgments Submergence and hypoxia research in the Bailey-Serres group is supported by the National Science Foundation (IOS-0750811; IOS- 1121626) and the United States Department of Agriculture (2008- 35100-04528), in the van Dongen group by the German Research Foundation (DFG; DO 1298/2-1) and the Federal Ministry of Education and Research (BMBF; HydromicPro), in the Perata group by Scuola Superiore Sant’Anna (A6010PP-A6011PP), and in the Voesenek group by grants from the Netherlands Organization for Scientific Research and Center for BioSystems and Genomics (CBSG2012). N-end rule pathway research in the Holdsworth group is supported by the Biotechnology and Biological Sciences Research Council (BB/G010595/1). References 1 Tester, M. and Langridge, P. (2010) Breeding technologies to increase crop production in a changing world. Science 327, 818–822 2 Xu, K. et al. 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Plant J. 67, 434–446 Making sense of low oxygen sensing Improved crop survival of floods is needed Oxygen deprivation is a frequent component of flooding stress Ethylene initiates submergence survival strategies in rice and wetland species Similarities in transcriptome response to flooding and oxygen deprivation How do plant cells sense low oxygen stress? Group VII ERFs regulate low-oxygen acclimation responses Arabidopsis group VII ERFs are degraded via the N-end rule pathway Manipulation of N-end rule regulation of group VII ERFs and other proteins Concluding remarks: direct oxygen sensing via the N-end rule regulates transcription Acknowledgments References