Making sense of low oxygen sensing

Making

sense

of

low

oxygen

sensing

Julia

Bailey-Serres

,

Takeshi

Fukao

,

Daniel

J.

Gibbs

,

Michael

J.

Holdsworth

,

Seung

Cho

Lee

,

Francesco

Licausi

,

Pierdomenico

Perata

,

Laurentius

A.C.J.

Voesenek

and

Joost

T.

van

Dongen

1_{CenterforPlantCellBiology,DepartmentofBotanyandPlantSciences,UniversityofCalifornia,Riverside,CA92521-0124,USA} 2_{DivisionofPlantandCropSciences,SchoolofBiosciences,UniversityofNottingham,LoughboroughLE125RD,UK}

3

MaxPlanckInstituteofMolecularPlantPhysiology,AmMuehlenberg1,14476Potsdam-Golm,Germany 4_{PlantLab,InstituteofLifeSciences,ScuolaSuperioreSant’Anna,PiazzaMartiridellaLiberta`33,56127Pisa,Italy}

5_{PlantEcophysiology,InstituteofEnvironmentalBiology,UtrechtUniversity,Padualaan8,3584CHUtrecht,theNetherlands} 6

CenterforBiosystemsGenomics,6708PBWageningen,theNetherlands

Plant-specificgroupVIIEthyleneResponseFactor(ERF) transcriptionfactorshaveemergedaspivotalregulators of flooding and low oxygen responses. In rice (Oryza sativa),theseproteinsregulatecontrastingstrategiesof floodingsurvival.RecentstudiesonArabidopsisthaliana groupVIIERFsshowtheyarestabilizedunder hypoxia butdestabilizedunderoxygen-repleteconditionsviathe N-end rule pathway of targeted proteolysis. Oxygen-dependent sequestration at the plasma membrane maintainsatleastoneoftheseproteins,RAP2.12,under normoxia.Remarkably, SUB1A, the rice group VII ERF thatenablesprolongedsubmergencetolerance,appears toevadeoxygen-regulatedN-endruledegradation.We propose that theturnover of group VII ERFsis of eco-logicalrelevance inwetlandspeciesand might be ma-nipulatedtoimprovefloodtoleranceof crops.

Improvedcropsurvivaloffloodsisneeded

Basedonconservativeexpectationsofhumanpopulation growth, the maintenance of international food security willrequireadoublingofagriculturalproductivityinthe next two decades [1]. This challenge is exacerbated by severe weather events associated with climate change suchas floods,whichhave occurredwithincreasing fre-quency across the globe over the past six decades (Figure 1). However, improvement of crop resilience to waterextremescanbeaccomplishedbyharnessing natu-ralgeneticdiversityinbreedingprograms.Anexampleof thisistheuseofthericeSUBMERGENCE1A(SUB1A) gene,which confersprolongedtolerancetosubmergence (seeGlossary)[2].The effectiveSUB1A-1allelewas iso-lated from an eastern Indian landrace and has been returned to farmers in high-yielding varieties [3]. This new‘Sub1rice’promisestohelpstabilizeharvestsin rain-fedfloodplains,whichrepresent33%ofriceacreage world-wide[4].Thetaskremainstoimprovefloodingtoleranceof other crops.Recent comparative studieswithin and be-tweenspecieshavegreatlyenhancedourunderstandingof mechanisms that facilitate survival of distinct flooding regimes (Table 1). With new insights into low oxygen sensingandresponsemechanismsweareoptimisticthat effectivemeanstolessencropdevastationbyfloodingcan beextendedbeyondricepaddyfields.

Oxygendeprivationisafrequentcomponentofflooding stress

Akeyfeatureoffloodingeventsisthechangeinlevelsof three gases, O2, CO2 and ethylene, due to a near 104 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 speciessuchasricethatconstitutivelyformgasconduits (i.e.aerenchyma)betweensubmergedandaerialorgans. However,ifplantslackgasconduitsorloseoxygenfrom roots,waterloggingrapidlyreducestheoxygen concentra-tionwithincells[9–11].Thepresenceofaerobicmicrobes inthesoilcanfurtherexacerbatethestress.Whenboththe rootandaerialportionsofaplantarewhelmedbywater–a conditiontermedsubmergence–cellularoxygenlevelscan alsodeclinefromnormoxia.Thedegreeofoxygen deficien-cy(hypoxia/anoxia)dependsonmultiplefactorsincluding replenishmentofoxygenthroughphotosynthesis,inward diffusionfromthewaterlayerandcellularconsumptionof oxygen through metabolic activity. Severe oxygen defi-ciency compromises mitochondrial respiration [10,12] andleadstoaninsufficiencyinATPforenergydemanding processes [6,13–15]. However, plants can adjust tothis energycrisisthroughincreasedsubstratelevelATP pro-duction(Figure2).Thisisaccomplishedbycatabolismof solublesugarsandinsomespeciesorcelltypesstarch[16]. Typically, the increasein glycolyticflux iscoupledwith regeneration of NAD+ _{by fermentation of pyruvate to}

Glossary

Anoxia:absenceofoxygen.

Directoxygensensing:sensingofoxygenviaitsmolecularinteractionwitha ligand (i.e. enzyme, protein, chemical compound) that results in an effect of cellularconsequence.

Hypoxia:oxygenlevelsbelownormoxia;theterm‘hypoxic’isoftenusedto describeasituationwheremolecularoxygenisstillpresent,butitslevelhas significantly decreased below 20.6%; cellular oxygen status may be hypoxic or anoxicdependentuponduration,locationandmetabolicactivity.

Hypoxia-responsivegenes:geneswithtranscriptsdifferentiallyregulatedin response to conditions with a low oxygen component.

Indirect oxygen sensing: sensing of change in homeostasis that is a consequenceof oxygendeprivation(i.e.change inATP,ADP,AMP,other metabolite, Ca2+_{, 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.

Waterlogging: flooding of root system. Correspondingauthor:Bailey-Serres,J. (serres@ucr.edu)

ethanolviapyruvatedecarboxylaseandalcohol dehydro-genase (ADH).Because ethanoldiffusesout ofcellsinto 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 ofspecies [17,18]. This includes the generation of 2-oxoglutarate as a coproduct, which can be further metabolizedtosuccinate,viatheTCAcycleenzyme succi-nateCoAligase(SCS),therebyprovidingadditionalATP permoleculeofsucrosemetabolized.Tokeepthese reac-tionsrunning,theoxidationofNADHinthemitochondrial matrixisguaranteedbyreductionofoxaloacetateviathe reversedTCAcyclereactioncatalyzedbymalate dehydro-genase [19,20].Themalateproducedisprobablyfurther converted to fumarate and succinate [21], the latter of whichcouldbeexportedfromhypoxictissuetotheaerated partsoftheplant.Atleastintubersofpotato(Solanum tuberosum), hypoxia stimulates a rearrangement of the mitochondrialrespiratorysupercomplexesthatenhances regenerationofNAD+bythealternativeNAD(P)H dehy-drogenases[22].

EventhoughtheefficiencyofhypoxicATPproductionis low compared to aerobic oxidative phosphorylation, it allows cellstosurviveas longascarbohydratesubstrate remains available. Cell death only becomes inevitable when thereisinsufficientenergyforexclusionofprotons totheapoplasttopreventmembranedepolarizationandto maintainanearneutralcytosolicpH[6,23,24].Avoidance of the severe energy crisis associated with low oxygen stressrequireseconomizationofATPconsumption.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],metaboliccompartmentalization[27],reducedprotein

synthesis[28],increasedproductionofheatshockproteins asmolecularchaperones[29]andadoptionoftheK+ -gra-dienttoenergizemembranetransport[30].Plantsurvival of waterlogging or submergence also depends on their ability to limit or endure oxidative stress, which occurs duringthetransitionfromnormoxiatoanoxiaaswellas uponde-submergence[31–33].

Ethyleneinitiatessubmergencesurvivalstrategiesin riceandwetlandspecies

Recentworkhasexposedmechanismsofresponseto sub-mergencethatcenterongrowthmanagement.Notableare twoantitheticalsurvivalstrategiesdisplayedbybothwild and domesticated species. For example, deepwater rice, cultivated tocopewith slowlyadvancing floods,expends energyreservesintheelongationofinternodalregionsthat are underwater tomaintain photosynthetic tissueabove the air–water interface [34,35]. Similarly, the wetland dicot Rumex palustris, which is well adaptedto shallow but prolonged floods, reorients and extends petioles to elevateleavesabovethesurfaceoffloodwaters[6,7]. How-ever,this‘submergenceescape’strategyisunsuccessfulif energyreservesareexhaustedbeforeescapeofthedeluge. In wetland species capable of surviving transient floods (e.g.Rumexacetosa)[36]andsubmergencetolerantSub1 rice[37],a‘quiescence strategy’minimizesenergy expen-dituresforgrowthuntilde-submergence.

Thegeneticdeterminantsandhormonalsignaling 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 moleculardissectionofsubmergenceresponsesinriceand R. palustris has yielded a model in which a buildup of ethyleneinsubmergedorgansinitiatesahormonal signal-ingcascadethatreducestheantagonismbetweenabscisic

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

Figure1.Numbersoffloodshaveincreasedineachofthepastsixdecadesacrosstheglobe.GraphsshowthenumberoffloodsclassifiedasadisasterintheInternational 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).

Table1.Factorsthatcontributetosurvivaloffloodingoroxygendeprivation

Condition Species Comparison Acclimationor survivalresponse

Causalfactors Refs

Submergence (partial)

Rice(Oryzasativassp. indica) Deepwatertonon-deepwater cultivars;SK1andSK2 transgenics Rapidunderwater internodeelongation; escapestrategy SK1,SK2,ethylene,GA, ABA [34] Submergence (complete)

Rice(ssp.indica,aus andjaponica)

Nearisogeniclines;SUB1A-1 transgenics Growthrestriction; quiescencestrategy SUB1A-1,ethylene, reducedGA responsiveness [2,37,73] Submergence (seed)

Rice(ssp.indica) Anaerobicgerminationof toleranttonon-tolerant cultivars;cipk15mutant

Enhancedcoleoptileand shootelongation

Quantitativetraitloci; enhancedstarch degradation;CIPK15

[74,75]

Submergence (underanoxia)

Rice(ssp.indica) Towheat Seedgerminationand

coleoptileelongation Adjustmentof metabolism;reduced oxygenconsumption [76] Wheat(Triticum aestivum)

Torice Nogerminationand

coleoptileelongation inhibited

Limitedadjustmentof metabolismandoxygen consumption

[76]

Submergence Marshdock(Rumex palustris)

Ecotypeswithfastandslow underwaterelongation responses

Petioleelongation Fastelongationassociated withlowerendogenous ABA

[38]

Commonsorrel (Rumexacetosa)

ToRumexpalustris Limitedpetioleelongation MaintenanceofABA undersubmergence

[36]

Arabidopsisthaliana 86accessions Variedsurvivability Unknown [77]

Meionectesbrownii Variationinlight Photosyntheticaquatic

adventitiousroots

Reducedneedforshoot photosynthate

[78]

Anoxia Rice Towheat Sucroseorglucose-fed

wheatseedssurvive longer

a-Amylaseproduced underanoxiainricebut notinwheatseedlings

[79]

Chlamydomonas reinhardtii

Wildtypetomutant Transcriptomicand

metabolicadjustments Versatilemetabolic adjustmentssuchasH2 production [80–82] Pondweed (Potamogeton distinctus)

Topea(Pisumsativum) Turionelongationand survival

EnhancedH+extrusion andstabilizationof cytosolicpH

[83]

Grape(Vitissp.) Anoxiatolerant(Vitisriparia) tointolerant(Vitisrupestris)

Improvedsurvivalof hypoxiapretreatedroots

Fermentationand maintenanceofion homeostasis(e.g.K+₎ [84] Hypoxiato anoxia

Arabidopsisthaliana Wildtypetoloss-of-function orotherinsertionmutants andoverexpression transgenics

Lowoxygenand/or submergencesurvival

HRE1,HRE2,RAP2.2, RAP2.12;

VERNALIZATION INSENSITIVE3; EXORDIUM1;HEAT SHOCKFACTOR2a; HYPOXIA-RESPONSIVE UNKNOWNPROTEIN (HUP)genes [29,41, 47,62,63,65, 66,72,85,86]

Wildtypetoloss-of-function prt6andate1ate2mutants

Lowoxygenand/or submergencesurvival; seedgerminationunder hypoxia

N-endrulepathway componentsPRT6,ATE1, ATE2

[66,72]

Wildtypetoloss-of-function mutantsandoverexpression transgenics

Seedgerminationunder 0.1%oxygen

NACtranscriptionfactor ANAC102

[87]

Waterlogging Lotusjaponicus WildtypetoN-deficient nodularleghemoglobinRNAi transgenics

Alanineandsuccinate accumulation

ModifiedTCAfluxmode [19]

Poplar(Populus canescens)

Roottoshoot Transcriptomicand

metabolicadjustments; limitedshootresponse

Unknown [88]

Cotton(Gossypium hirsutum)

Roottoshoot Transcriptomicand

metabolicadjustments; shootgrowthinhibition

Unknown [89]

Maize(Zeamays) RootcelltypemRNAs Aerenchymaformation Ethylene,Ca2+_,ROS;

cortexmRNAs

[90]

Rice Responsetocompounds Adventitiousroot

development

Epidermalcelldeath mediatedbyethyleneand ROS [91] Tomato(Solanum lycopersicum) Responsetohormone biosynthesisinhibitors Adventitiousroot development

Ethyleneandauxin [92]

UDP SUS Amylases INV H₂O Glucose Glucose Glucose-1-P Glucose-6P Glucose-1-P UDP-glucose Fructose Fructose-6P Fructose-1,6P₂ Phosphoenolpyruvate Pyruvate Acetaldehyde Glutamate Aspartate 2-Oxoglutarate 2-Oxoglutarate Oxaloacetate Isocitrate Malate Fumarate Glutamate 2-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) FADH₂ 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 ATP or orATP 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

Figure2.Metabolicreconfigurationunderlowoxygenstress.ReducedoxygenavailabilityaltersmetabolismtomaximizesubstratelevelATPproduction.Themodel 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)shuntandag-aminobutyricacid(GABA)shunt.Thehypothesisthatoxygenisconservedisunderfurtherinvestigation.Yellowboxessummarize notablemetabolicadjustments.Bluelinesindicatepathwaysenhancedduringthestress,bluedashedlinesindicatepathwaysproposedtobeactiveduringthestress 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, glutamatedehydrogenase,INV,invertase;LDH,lactatedehydrogenase;MDH,malatedehydrogenase;PDC,pyruvatedecarboxylase;SCS,succinylCoAligase;SUS, sucrosesynthase.

acid(ABA) andgibberellins (GA), whichnormallylimits cellelongation.In rice,naturalvariationinthepresence andabsenceoftheunderwaterescape[SNORKEL(SK)1 and2]andsubmergencetolerance(SUB1A)groupVIIERF determinantsunderliesdifferentialregulationofthe hor-monalcascadeandhormonesensitivitiesthatcontrol un-derwatergrowth[8](Box1).TwoR.palustrispopulations distinguishedbyfastandslowunderwaterpetiole elonga-tion were differentiated by the maintenance of higher levelsofABAandreducedGAresponsivenessintheslow elongatingvarietyduringsubmergence[38].Consistently, thelimitedunderwaterpetiolegrowthandprolonged sub-mergencesurvivalofR.acetosawaslinkedtomaintenance ofABAbiosynthesisthattranslatedintolowerGA respon-sivenessduringsubmergence[36].

Photosynthesiscancontinueinsubmergedleavesandis aided by the gas film that often clings to their surface [39,40].Itfollowsthatthedegreeofoxygendeprivationin photosynthetic tissue may be less extreme than tissues distantfromanoxygensource.Nevertheless,the ethylene-drivenunderwaterelongationofshoottissuecandeplete carbohydratesandlead toanenergycrisis.

Similaritiesintranscriptomeresponsetofloodingand oxygendeprivation

Numerousinvestigationshaveassessedchangesin tran-scriptomesinresponsetolowoxygenstressorfloodingin plants including Arabidopsis, rice, poplar (Populus  canescens)andcotton(Gossypiumhirsutum)[41,42]. Stud-ies performedonseedlingsofthe ArabidopsisCol-0 eco-typeincludeevaluationoftheeffectsofdifferentseverity [43]andduration[28,44]ofoxygendepletion,aswellas theimpactofheatstresspriortoanoxia[29].Becausethe majorityofcellularmRNAsarepoorlytranslatedduring oxygendeprivation[45],changesin polyribosome-associ-atedmRNAswereusedtoevaluatedynamicsinthestress andrecoveryresponses[28].In21celltypesorregionsof rootsandshoots,polyribosomeswerecapturedby immu-nopurificationtoidentifytranscriptsregulatedby short-termoxygendeprivation[46].Thisapproachidentified49 corehypoxia-responsivegenesthatwerestronglyinduced 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

Box1.Contrastingsubmergencesurvivalstrategiesofrice

Most accessions respond to submergence through rapid shoot elongation, which allows emergence from a shallow flood [6]. A limited numberofaccessionsdisplay theabilitytosurviveaslow progressiveflood(escape response)oradeeptransientflashflood (quiescenceresponse)(FigureI).(a)Byamplifyingtheelongationof steminternodes,deepwaterricecanoutgrowaprogressivefloodand survivepartialinundationformonths.Thisdeepwaterescapestrategy iscontrolledbytheSNORKEL(SK)locus,whichencodestwogroup VIIERFs,SK1andSK2[34].SKsareabsentfromlowlandvarieties.(b) Themolecular geneticanalysisofthesubmergence-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,whereasSUB1Aislimitedtosome indicaand auslandraces[2,60].TheSUB1A-1alleleissufficienttoconfersurvival of2weeksorlongerofcompletesubmergence.(c)Modelofthecore submergenceresponsenetworkthatisinfluencedbySKsandSUB1A. GenotypespossesseitherSK1/SK2,SUB1Aorneither.BothSK1/SK2 andSUB1A-1mRNAareethyleneinduced.Indeepwaterrice,SK1/ SK2andtwominorQTLsaugmentaccumulationofbioactiveGAin steminternodesduringsubmergence.Insubmergencetolerantrice varieties,thepresenceofSUB1A-1influencessubmergenceand post-submergence responses in aerial tissue. (1) SUB1A-1 mRNA is ethylene-inducedbutultimatelylimitsethylenebiosynthesis[37].(2) SUB1A-1promotesaccumulationoftwonegative regulatorsofGA responses [SLENDER RICE 1 (SLR1) and SLENDER RICE-LIKE 1 (SLRL1)] [73]. (3) SUB1A-1 does not perturb the submergence-induced decline in ABAcontent butheightens sensitivity to ABA [32]. (4)SUB1A-1limits inductionofgenes associatedwith starch breakdown[37,41,94].(5)SUB1A-1enhancesupregulationofgenes associated with reactive oxygen species (ROS) amelioration and survivalofdehydration,therebyimprovingre-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, SUB1Aisremarkablypositionedtosuspendgrowthandmaintaincell viability during submergence and restore homeostasis during a subsequentrecoveryperiod.

Progressive flood – deepwater rice (SK1/2)

Partial submergence Complete submergence Submergence Reoxygenation ROS ROS amelioration SLR1 SLRL1 Dehydration response Drought recovery Ethylene

ABA SUB1A ABA

SK1/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.

hypoxia-responsivegenes.Ofthe49core hypoxia-respon-sivegenes, 24werealsodifferentiallyregulatedinroots androsetteleavesduringsubmergenceincomplete dark-ness [47]. Finally, meta-analyses that compared tran-scriptomic adjustments tolowoxygen or flooding stress identifiedconservationinthecorenetworkofgenes asso-ciatedwithsignaling,transcriptionandefficient anaero-bic ATP production that is modulated by oxygen deprivationinarangeofplants[41,42].

How doplantcellssenselowoxygenstress?

Based onmechanismsinother eukaryotes,bothindirect and directsensing ofcellular oxygen status could be re-sponsibleforacclimationresponsesthatprolongsurvival of oxygen deprivation in plants [48]. Indirect sensing mechanisms might include perception of altered energy statusthroughchangesinlevelsofadenylates(ATP,ADP and/or AMP),consumable carbohydrates,pyruvate, cyto-solicpH,cytosolicCa2+orlocalizedproductionofreactive oxygenspecies(ROS)andnitricoxide(NO).

Animalandyeastcellssenseandadjustenergy homeo-stasis through Sucrose Non-Fermenting 1 (SNF1)/AMP-activatedproteinkinases[49,50].Theplantenergysensors fallwithinonecladeofSNF1relatives,theSnRK1s,some ofwhichhavebeenimplicatedinlowoxygenresponses.For example,ArabidopsisKIN10andKIN11arenecessaryto limitenergyconsumptionduringhypoxia[51].Whereasin riceseedsgerminatedunderoxygenstarvation,the deple-tion of sucrose activates the SnRK1A energy sensor through the activity of a Calcineurin B-like interacting bindingkinase15(CIPK15)[52].Thissignaltransduction upregulates transcription of genes encoding a-amylases, whichdrivecatabolismofstarchintheseedneededtofuel underwatershootgrowth.Logically,areductionofenergy consumption is beneficial when ATP levels decline. A means of energy conservation during low oxygen stress in plants is selective translation and sequestration of mRNAsduringhypoxia[28].Basedonevidencefromother eukaryotes, the sequestration of a subset of cellular mRNAs, suchas the abundantcohort that encodes ribo-somalproteinsandtranslationfactors,couldberegulated throughSnRK1sandtheTargetofRapamycinkinase[53]. Mitochondria are also thought to contribute to oxygen sensing and signaling in plants, through production of NOand/orreleaseofROSandCa2+duringthetransition fromnormoxiatohypoxia[54,55],asconfirmedinanimals [56,57].

In animals,directoxygensensing regulatesthe accu-mulationoftheasubunitofthehypoxiainduciblefactor (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 functionin transcriptionalactivation. Thereis no corol-larydirectoxygensensingmechanisminplants,because although they possess prolyl hydroxylases they lack HIF1a [41].

GroupVIIERFsregulatelow-oxygenacclimation responses

Theplant-specificERFtranscriptionfactorfamilyincludes over 100 members in rice and Arabidopsis, all of which shareanAPETALA2(AP2)DNAbindingdomain[59].The ERFs havebeenphylogenetically parsedintotenclades, withthegroupVIIERFscharacterizedbyaconservedN terminalmotif(NH2-MCGGAI/L)[59](Figure3a).Fifteen rice (japonica cv. Nipponbare) ERFs were designated groupVIIa(OsERF59-72)andVIIb(OsERF73),based on thepresenceorabsenceoftheconservedNterminalmotif, respectively. The single group VIIb ERF corresponds to SUB1C,whichisfoundinallricevarietiessurveyed[60] and acts downstream of SUB1A [37]. Intriguingly, the groupVIIERFsencodedbySUB1A,SK1andSK2possess variant Ntermini relative tothe rice group VIIaERFs. Arabidopsis encodesfive groupVIIERFs (AtERF71–75), two of which are hypoxia-responsive genes {HYPOXIA

RESPONSIVE ERF1 and 2 [HRE1 (AtERF73;

At1g72360) and HRE2 (AtERF71; At2g47520)]}. As ob-servedforSUB1AandtheSKs,HRE1mRNA accumula-tion is promoted by ethylene, which synergistically enhancesitselevationduringhypoxia[61,62](Figure3b). SeveralrecentreportsindicatethatArabidopsisgroup VIIERFs redundantly regulate hypoxia-responsivegene expressionandsurvivaloflowoxygenstress.Forexample, seedling survival of anoxia was more severely compro-misedinhre1hre2doublemutantseedlingsthanineither singlemutant or the wild type [61,63]. By contrast, low oxygensensitivitywaslessenedinseedlingsthat constitu-tively overexpress either HRE1 or HRE2 mRNA. The ectopicexpressionoftheseERFswassufficienttoheighten induction of the core hypoxia-responsive gene ADH1 or ADHenzymeactivityduringthestress[61,63].However, because hre1hre2 seedlings were able to elevate ADH enzyme activity and ethanol production during hypoxia [63],genetic redundancyis likely to extend tothe other groupVIIERFs[RAP2.12(AtERF75;At1g53910),RAP2.2 (AtERF74; At3g14230) and RAP2.3 (AtEBP/AtERF72; At3g16770)]. Indeed, conditional upregulation of RAP2.12wassufficienttoelevateexpressionofa pADH1:-LUCIFERASEtransgene[64]andRAP2.2overexpression improvedsurvivalofhypoxiainseedlings[65],whereasthe inhibition of either RAP2.2 or RAP2.12 expression via miRNA production limited the induction of ADH1 and severalother hypoxia-responsive genes[66]. The impact ofectopicexpressionofthesegeneswasconditionspecific, as HRE1, HRE2, RAP2.2 and RAP2.12 overexpression significantly increased levels of ADH1 mRNA or ADH activityunderlow oxygenstress butnot undernormoxia [61,65,66]. Nonetheless, RAP2.2, RAP2.3 and RAP2.12 mRNAs accumulate under normoxia in association with polyribosomes[46,67], suggestingtheyare constitutively synthesized.Together,thesefindingshintthat post-trans-lationalregulationlimitsthefunctionofgroupVIIERFsto periodsoflowoxygenstress.

ArabidopsisgroupVIIERFsaredegradedviatheN-end rulepathway

TheconservedN-endrulepathwayoftargetedproteolysis regulatesthehalf-lifeofcertaincellularproteinsbasedon

recognitionofNterminalresiduesby specificN-recognin E3 ligases [68]. In plants, 11 amino acids function as destabilizing residueswhenlocated attheNterminusof a protein, which coupled with an optimally positioned downstream lysine can act as a degradation signal (N-degron)[69].Inplantsandanimalsbutnotyeast,a cyste-ine(Cys)residueattheNterminuscanundergotwosteps ofmodificationthatleadtoproteinrecognitionand degra-dation(Figure3b).Basedonmechanisticstudiesin mam-mals,newlysynthesizedproteinswithaCysasthesecond residue(i.e.NH2–Met1–Cys2)areconstitutivelycleavedby aMetaminopeptidase(MAP)toyieldNH2–Cys2.In Ara-bidopsis, asmallfamilyoffunctionallyredundantMAPs 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 byanN-recogninE3ligase,leadingtoubiquitinationand 26S proteasome-mediated degradation. In Arabidopsis, the genes ATE1and ATE2 encode the Arg transferases [71]andatleastoneE3ligase,encodedbyPROTEOLYSIS 6(PRT6),actsas anN-recogninofNH2–Arg1–Cys2oxidised polypeptides[69].

ThedistinctconservationoftheNterminusofgroupVII ERFsandtheserendipitousobservationthatan Arabidop-sis prt6 mutant constitutively accumulates ADH1 and otherhypoxia-responsivemRNAsinseedsledtothe con-firmationthatgroupVIIERFsarebonafidesubstratesof the N-end rule pathway in plants [66,72] (Figure 3b). Additionalsupportofthisconclusionwasobtainedthrough invitroandinplantaanalyses.

Aninvitrosystemderivedfromrabbitreticulocytes[72] was used to confirm that all five Arabidopsis group VII ERFs are N-endrule substrates.It was alsoshown that theirinstabilityrequiredCys2,asmutationofCys2tothe stabilizingresidueAla2(NH2–Met1–Ala2)eliminated sus-ceptibility toN-endrule turnover.It wasfurther demon-strated in planta that low oxygen stress increased the accumulationofgroupVIIERFssynthesizedwithanative N-terminus(NH2–Met1–Cys2),whereasthosesynthesized with an NH2–Met1–Ala2 N terminus were stable under bothnormoxiaandhypoxia[66,72].Basedonthisevidence, thestabilizationofgroupVIIERFsunderhypoxiaismost probablyrelatedtoaninhibitionoftheCys2oxidationthat is required before the protein can be arginylated and degraded. PM Nucleus ACBP Hypoxia responsive RAP2.12 RAP2.12 RAP2.12 RAP2.12 RAP2.12

Normoxia Flooding Hypoxia

Normoxia ERF ERF ERF Hypoxia Flooding -O₂ HRE2 Ethylene low O2 low O2 Constitutive dark

ethylene _ConstitutiveConstitutiveethylene

? Transcription / translation Arabidopsis group VII ERFs

RAP2.3 RAP2.12 RAP2.2 HRE1 Constitutiveee ethylene ? ? ? RAP2.3 +O₂ 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*

Rice

Group VII ERFs

Arabidopsis

(a)

(b)

(c)

TRENDS in Plant Science Figure 3. Oxygen sensing via N-end rule pathway-targeted turnover of group VII ERFs.(a)NterminalalignmentofArabidopsisgroupVIIERFs.Withtheexception ofSUB1C,allbeginwiththeaminoacids‘Met-Cys’(MC).Thehighlyconserved Arabidopsis N terminal motif is boxed in red and is less conserved in the proteins atlociassociatedwithsubmergenceresponsesinrice.(b)Homeostaticresponse tohypoxiaisregulatedbytheN-endrule-mediatedproteolysisofgroupVIIERFsin Arabidopsis. Group VII ERF transcription factors are either constitutively expressed and/ordifferentiallytranscriptionallyregulatedinresponsetovariablesignals, includinglowO2,ethyleneanddarkness.FourofthefiveERFs(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 rulepathway ofproteolysis. Thisinvolves thefollowing steps:(i) theN terminalMet(M)isconstitutivelycleavedbyamethionineaminopeptidase(MAP); (ii) the exposed Cys (C) is converted to an oxidized (C*) form (e.g. Cys-sulfonic acid)byO2,NOorpossiblyROS;(iii)anArg(R)residueisaddedtotheoxidized

CysbyanarginyltRNAtransferase(ATE);and(iv)theargininylatedproteinis recognizedbyPROTEOLYSIS6(PRT6)orotherE3ligases,whichpolyubiquitinate the protein, targeting it for proteasomal degradation (26S proteasome). The outcome is prevention of transcription of hypoxia-responsive genes under normoxia.Whenoxygenbecomeslimiting(hypoxia),degradationoftheERFsby the N-endrule pathway isinhibited due to a lackof 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,theERFsareonceagaindestabilized,providingafeedbackmechanism that allows the plant to return to aerobic metabolism. (c) AtRAP2.12 localization dynamics. At leastone groupVII ERF,RAP2.12, associates with theplasma membrane(PM)viainteractionwithACBP,limitingitsturnoverundernormoxia. During hypoxia RAP2.12 is relocated to the nucleus and activates gene expression. Upon reoxygenation, RAP2.12 is destabilized, presumably as a consequence of Cys2oxidationandN-endrule-mediateddegradation.

EitherthemodificationoftheNterminusofagroupVII ERFordisruptionofanN-endrulepathwaystepcanaffect survival of low oxygen stress orsubmergence in Arabi-dopsis[66].Forexample,stabilizationofHRE1andHRE2 bymodificationoftheNterminustoNH2–Met1–Ala2was sufficienttoimproveseedgerminationandseedling sur-vival under hypoxia[72]. Inaddition, ate1ate2andprt6 seedlingswereless sensitivetohypoxiawhengrownon sucrose-supplemented medium [72]. The same mutants growntotherosettestageweremoresensitiveto submer-gence in complete darkness [66]. This discrepancy in phenotypemightbeexplainedbydistinctionsinthe avail-ablecarbohydratesinthetwosurvivalassays.Inthelow oxygenexperiments,anaerobicmetabolismwasfueledby sucrose in the medium, whereas in the submergence experimentsitwaslimitedtoendogenousenergyreserves of the plant. Therefore, the absence of PRT6 or ATE activity may enhance anaerobic metabolism to prolong survival in sucrose-fed seedlings but may causea more rapid onset of energy deficiency in submerged plants. Thesefindingsarereminiscentoftheearlierproposalthat abalancebetweenenergyconsumptionandconservation iscrucialtosurvivaloflowoxygenstressandsubmergence [5,37].

Itwasalsoobservedthattheonsetofthetranscription of hypoxia-responsive genes occurs concomitantly with relocalization of RAP2.12 to the nucleus under hypoxia (Figure3c)[66].Duringnormoxia,aGFP-taggedversionof RAP2.12wasprotectedagainstproteindegradationbythe N-endrulepathwayofproteolysisandexcludedfromthe nucleus via interaction with a plasma membrane (PM)-associated Acyl-CoAbindingprotein(ACBP1or ACBP2). RAP2.12 migrated tothe nucleusinresponse tohypoxia and disappeared from the nucleus after reoxygenation. Moreover,transientexpressionofRAP2.12-GFPinleaves of ate1ate2 and prt6 mutants resulted in greater GFP signalintensityinthenucleusundernormoxiaand follow-ingreoxygenation.

In summary, the N-end rule pathway of proteolysis regulatestheaccumulationofgroupVIIERFsand conse-quentiallytheaccumulationofgenetranscriptsassociated with lowoxygen responsesinArabidopsis.It isproposed thatconstitutivelysynthesizedgroupVIIERFsareeither degradedorsequesteredundernormoxia,asconfirmedfor RAP2.12.Asoxygenlevelsfalltheirdegradationbecomes limited, PM sequestration is reversed and the ERF is transported to the nucleus and becomes active in gene regulation. Upon reoxygenation, both constitutively expressedandhypoxia-inducedgroupVIIERFsare desta-bilized. Thus, the N-end rule pathway (i) prevents the excessive accumulationofconstitutively expressed ERFs undernormoxia;(ii)allowsforstabilizationofboth consti-tutive and induced ERFs during hypoxia; and(iii) facil-itatesrapidreversalofERF-regulatedtranscriptionupon reoxygenation. Constitutivelyexpressed groupVII ERFs are proposedtoencode oxygensensorsthat conditionally activatetranscriptionofhypoxia-responsivegenes, includ-ingothergroupVIIERFs[66].Theincreasedsynthesisof N-end rule regulated group VII ERFs by ethylene or darknesscouldfurtherprimecellsforacclimationto oxy-gendeprivation.

ManipulationofN-endruleregulationofgroupVIIERFs andotherproteins

The verification that theN-end rule pathway modulates groupVIIERFaccumulationinthenucleusinan oxygen-dependent manner exposes the first examples of N-end rulesubstratesandahomeostaticlowoxygensensor mech-anisminplants.Basedonavailable genesequencedata, groupVIIERFswiththeconservedN-terminusare broad-ly foundin vascular plant species [66].We propose that futureimprovementoffloodingtolerancecouldbeachieved by manipulation ofsynthesis and turnoverof these pro-teins(e.g.byoverexpression,regulatedexpressionand/or mutationofNH2–Met1–Cys2toNH2–Met1–Ala2).

Giventhe crucial importanceofmodulation ofenergy reservesduringflooding,itisnotsurprisingthatvariation of groupVII ERF susceptibility tooxygen-dependent N-endrule turnover existsin nature.The rice Nipponbare genomeencodes15groupVIIERFswiththeconservedN terminus thatisconsistentwith oxygen-regulated N-end rule-targetedproteolysisinArabidopsis.However,neither SUB1AnorSUB1CareN-endrulesubstratesbasedonin 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 escapeofSUB1AfromN-endrulepathwayturnovercould allow the ethylene-mediated regulation of SUB1A-1 to trigger the sequence ofevents that promotes theenergy managementassociatedwithsubmergencetolerancewell beforeoxygenlevelsreachacriticalnadir.

Concludingremarks:directoxygensensingviathe N-endruleregulatestranscription

Alterationsingeneexpressionassociatedwithincreased catabolism and substrate level ATP production are a hallmark ofreduced oxygen availabilityandflooding in plants. Group VII ERFs play a prominent role in this process.The identification ofan oxygen-dependent pro-teinturnovermechanismthatcontrolstheabundanceof somebutnotallgroupVIIERFsraisesseveralpertinent questions(Box2).Weanticipatethatgenetic manipula-tionofthetargetsofoxygen-regulatedN-endrulepathway turnovercanprovideameanstoimprovesurvivalundera varietyoffloodingconditions.

Box2.Keyquestionsforfutureexperimentation

 CanO2,NOorROS-dependentCys2oxidation,arginylationand

ubiquitinationofgroupVIIERFsbeexperimentallyconfirmed?If so,istheoxidationspontaneousorcatalyzed?

 Whatarethekineticsofoxygen-regulatedgroupVIIERFturnover? DoesERF stabilizationoccur before oxygen deficiencyimpairs cytochromecoxidaseactivity?

 DoestheoxygenlevelaffecttheinteractionbetweenACBPsand RAP2.12?DoesdockingofRAP2.12toACBPimpairCys2

oxida-tion,modificationbyATE,orinteractionwithanE3ligase?Are othergroupVIIERFssimilarlysequestered?

 WhatgenesandnetworksarecontrolledbyindividualgroupVII ERFs?

 Istheactivityor turnoverofSUB1A,whichapparentlyescapes oxygen-mediated N-end rule degradation, controlledupon de-submergence?

 CanmanipulationofgroupVIIERFaccumulationand turnover provideaneffectivestrategytomodulatesurvivaloffloodingin crops?

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;DO1298/2-1)andtheFederalMinistryofEducation and Research (BMBF; HydromicPro), in the Perata group by Scuola SuperioreSant’Anna(A6010PP-A6011PP),andintheVoesenekgroupby grantsfrom theNetherlandsOrganizationfor ScientificResearch and CenterforBioSystemsandGenomics(CBSG2012).N-endrulepathway researchintheHoldsworthgroupissupportedbytheBiotechnologyand BiologicalSciencesResearchCouncil(BB/G010595/1).

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