Oxygen Sensing Coordinates Photomorphogenesis to Facilitate Seedling Survival

Seediscussions,s a s,andau ho p o iles o  hispublica iona :h p://www. esea chga e.ne /publica ion/276922638 OxygenSensingCoo dina es Pho omo phogenesis oFacili a eSeedling Su i al. ARTICLEinCURRENTBIOLOGY:CB·MAY2015 Impac Fac o :9.92·DOI:10.1016/j.cub.2015.03.060·Sou ce:PubMed DOWNLOADS 31 VIEWS 83 12AUTHORS,INCLUDING: MohamadAbbas Uni e si a Poli ècnicadeValència 5PUBLICATIONS92CITATIONS SEEPROFILE No aMa ín-delaRosa TechnischeUni e si ä München 6PUBLICATIONS62CITATIONS SEEPROFILE Da idAlabadi Uni e si a Poli ècnicadeValència 36PUBLICATIONS1,842CITATIONS SEEPROFILE MiguelABlázquez SpanishNa ionalResea chCouncil 86PUBLICATIONS4,717CITATIONS SEEPROFILE A ailable om:MiguelABlázquez Re ie edon:04July2015 Cu en Biology Repo Oxygen Sensing Coo dina es Pho omo phogenesis o Facili a e Seedling Su i al Mohamad Abbas, 1 Sophie Be ckhan, 2 Daniel J. Rooney, 2 Daniel J. Gibbs, 2,3 Jo ge Vicen e Conde, 2 C is ina Sousa Co eia, 2 Geo ge W. Bassel, 2,3 No a Ma ı ´n-de la Rosa, 1 Jose ´Leo ´n, 1 Da id Alabadı ´, 1 Miguel A. Bla ´zquez, 1, * and Michael J. Holdswo h 2, * 1 Ins i u o de Biologı ´a Molecula y Celula de Plan as, Consejo Supe io de In es igaciones Cien ı ´ icas, Uni e sidad Poli e ´cnica de Valencia, Ciudad Poli e ´cnica de la Inno acio ´n, 46022 Valencia, Spain 2 Di ision o Plan and C op Sciences, School o Biosciences, Uni e si y o No ingham, Loughbo ough LE12 5RD, UK 3 School o Biosciences, Uni e si y o Bi mingham, Edgbas on B15 2TT, UK *Co espondence: mblazque[email p o ec ed] (M.A.B.), [email p o ec ed].uk (M.J.H.) h p://dx.doi.o g/10.1016/j.cub.2015.03.060 This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/). SUMMARY Success ul eme gence om he soil is essen ial o plan es ablishmen in na u al and a med sys ems. I has been assumed ha he absence o ligh in he soil is he p eeminen signal pe cei ed du ing ea ly seedling de elopmen , leading o a dis inc mo pho- genic plan (sko omo phogenesis) [1], cha ac e ized by ai s p o iding an adap i e ad an age un il eme gence and pho omo phogenesis. These ai s include supp essed chlo ophyll syn hesis, p omo- ion o hypoco yl elonga ion, and o ma ion o a closed apical hook ha p o ec s he s em cell niche om damage [2, 3]. Howe e , absence o ligh by i sel is no a su icien en i onmen al signal o ea ly seedling de elopmen [4, 5]. Reduced oxygen le els (hypoxia) can occu in wa e -logged soils [6–8]. We he e o e hypo hesized ha below-g ound hypoxia may be an impo an , bu hus a undisco e ed, ecological componen egula ing seedling de elop- men . He e, we show ha su i al and es ablishmen o seedlings ollowing da kness depend on hei abil- i y o sense hypoxia, h ough enhanced s abili y o g oup VII E hylene Response Fac o (ERFVII) an- sc ip ion ac o s. Hypoxia is pe cei ed as a posi i e en i onmen al componen in di e se axa o lowe - ing plan s, p omo ing main enance o sko omo pho- genic ai s. Hypoxia g ea ly enhances su i al once ligh is pe cei ed, while oxygen is necessa y o he subsequen e ec i e comple ion o pho omo pho- genesis. Toge he wi h ligh pe cep ion, oxygen sensing he e o e allows an in eg a ed esponse o he complex and changing physical mic oen i on- men encoun e ed du ing ea ly seedling g ow h. We p opose ha plan s moni o he soil’s gaseous en i- onmen a e ge mina ion, using hypoxia as a key ex e nal cue o p o ec he s em cell niche, hus ensu ing success ul apid es ablishmen upon eme - gence abo e g ound. RESULTS AND DISCUSSION We analyzed he e ec o oxygen a ailabili y and ligh on seed- ling g ow h ollowing ge mina ion in species ep esen ing dis inc b anches o eudico phylogeny: Papa e somni e um,Nico iana ben hamiana, and A abidopsis haliana (A abidopsis). Apical hook de elopmen is cha ac e ized by h ee phases: o ma ion, main enance, and opening [5, 9]. Con a y o he obse a ion ha seedlings kep in da kness unde no mal oxygen le els (no - moxia) e en ually open he hook (Figu e 1A), we ound ha hypoxic condi ions (2% oxygen imposed ollowing he hook main enance phase) s ongly inhibi ed opening in all species, which was e e ed ollowing ans e back o no moxia (Figu es 1A and 1B). The esponse o inal hook angle o inc easing oxygen ensions in A abidopsis is linea , sugges ing s ochas ic cumula i e sensing and esponse o oxygen (Figu e 1C) [10]. E iola ed seedlings main ained unde hypoxia we e able o comple e hook opening when exposed o ligh bu had de ec i e un olding and g eening o co yledons, indica ing ha oxygen is equi ed o he comple e esponse o ligh (Figu e 1D). Oxygen sensing in lowe ing plan s is he e o e a majo compo- nen o sko omo phogenic de elopmen and he ansi ion o pho omo phogenesis. Oxygen is sensed in plan s by he Cys eine (Cys) b anch o he A ginine (A g)/N-end ule pa hway o a ge ed p o eolysis, using g oup VII E hylene Response Fac o s (ERFVIIs) as sub- s a es [11, 12](Figu e 2A). The N-end ule pa hway ela es he in i o s abili y o a p o ein o he na u e o i s N e minus, which may be s abilizing o des abilizing ( he N-deg on) [14]. In no moxia, ERFVIIs a e des abilized h ough oxida ion o N- e minal (N )-Cys, which a ge s he p o eins o deg ada ion ia he N-end ule pa hway. Unde hypoxia, N -Cys is no oxidized and subs a es a e s able, enhancing g ow h and de el- opmen [11–13]. This mechanism is also used o sense ni ic oxide (NO), an essen ial componen o N -Cys oxida ion [15, 16]. The e a e i e ERFVIIs in A abidopsis: RELATED TO AP (RAP)2.12, RAP2.2, RAP2.3, HYPOXIA RESPONSIVE (HRE)1, and HRE2 [17]. We in es iga ed whe he ea ly seedling g ow h in A abidopsis is con olled by oxygen sensing h ough he N-end ule pa hway. We analyzed apical hook de elopmen in mu an seedlings lack- ing ei he E3 ligase (PRT6) o A ginyl- ans e ase (ATE) unc ions Cu en Biology 25, 1–6, June 1, 2015 ª2015 The Au ho s 1 Please ci e his a icle in p ess as: Abbas e al., Oxygen Sensing Coo dina es Pho omo phogenesis o Facili a e Seedling Su i al, Cu en Biology (2015), h p://dx.doi.o g/10.1016/j.cub.2015.03.060 (Figu e 2A). In hese mu an s, subs a es o he Cys-A g/N-end ule pa hway a e cons i u i ely s able, bu wi h di e en N e mini. In con as o wild- ype (WT; accession Col-0) e io- la ed seedlings, apical hooks o p 6 and a e1 a e2 did no ully open in he da k unde no moxia (Figu e 2B). Rema kably, he p 6 ap2.12 ap2.2 ap2.3 h e1 h e2 sex uple mu an (p 6 e VII), which lacks he unc ion o all i e ERFVIIs, e e ed he p 6 pheno ype (Figu es 2B and S1), indica ing ha ERFVIIs ac edundan ly o ep ess hook opening. Opening o apical hooks was also inhibi ed in NO-de icien mu an s, a pheno ype e- e ed in he nia1 nia2 e VII mu an o by ea men wi h NO (Figu es 2C and S1). To con i m ha ERFVIIs a e an in eg al componen o oxygen sensing con olling ea ly seedling de el- opmen , we es ed he abili y o indi idual ERFVIIs o ep ess hook opening by exp essing mu an s able e sions (in which Cys-2 was changed o Alanine, a s abilizing esidue; Figu e 2A) d i en by hei endogenous p omo e s (p omERFVII:MA-ERFVII) p o iding MA-ERFVII p o ein. All i e MA-ERFVIIs we e able o inhibi hook opening compa ed o WT, indica ing ha Cys-A g- N-end ule-media ed deg ada ion o all ERFVIIs con ibu es o hook opening (Figu e 2D). In o de o p o ide unequi ocal e i- dence ha oxygen sensing by ERFVIIs con ols hook opening, we analyzed hook de elopmen in WT and ap2.12 ap2.2 ap2.3 h e1 h e2 (e VII) pen uple mu an (which lacks all ERFVII ac i i y) seedlings unde condi ions whe e hypoxia was imposed a he end o he hook main enance phase (Figu e 2E). Unde hypoxia, WT e iola ed seedlings we e comple ely unable o open hei hooks. This, howe e , was no he esul o a loss o espi a o y ene gy bu a speci ic consequence o oxygen sensing, as e VII mu an seedlings comple ed hook opening unde hypoxia wi h simila kine ics o WT seedlings unde no moxia. In addi ion, e u n o no moxia ollowing a p olonged hypoxic ea men allowed hooks o WT seedlings o open (Fig- u e S1), ea i ming a ole o a as - esponding oxygen-sensing mechanism. We obse ed no e ec o hypoxia on hypoco yl elonga ion, demons a ing ha he obse ed pheno ypes a e no a consequence o hypoxia-induced quiescence (Figu e S1). Apical hook de elopmen has been in ima ely linked o he dy- namics o auxin le els [2]. Acco dingly, we obse ed a s ong co ela ion be ween he p esence o a g adien o auxin ac i i y ac oss he apical hook and he main enance o he hook unde hypoxia (Figu e S1), sugges ing oxygen-media ed egula ion o localized auxin esponses. Gi en ha oxygen is equi ed o comple e esponse o ligh (Figu e 1D), we in es iga ed whe he g eening o co yledons, C 2 4 6 8 10 12 14 O2 (%): 200 150 100 50 0144720 No moxia Hypoxia A A abidopsis haliana Papa e somni e um Nico iana ben hamiana Da k + Ligh (24h) No moxia Hypoxia D B No moxia Hypoxia No moxia Hypoxia O2 96h O2 120h O2 120h O2 (%) 0 5 10 15 180 90 0 Time (h) Figu e 1. Sko omo phogenic T ai s A e Regula ed by Oxygen Sensing in Flowe ing Plan s (A) Images o inal apical hook angle o e iola ed seedlings o A abidopsis haliana,Papa e somni e um, and Nico iana ben hamiana in no moxia o hypoxia (168 h , 168 h , and 144 h in he da k, espec i ely). Hypoxia ea - men was con inuously applied ollowing ini ia ion o he hook main enance phase. (B) Response o he apical hook a e ans e om hypoxia (A) o no moxia o he indica ed imes. (C) Response o he apical hook angle o A abidopsis o inc easing oxygen le els. (D) Images o co yledon g eening o seedlings ollowing ans e o ligh unde no moxia o hypoxia (24 h ). E o ba s indica e SD om he mean. 2Cu en Biology 25, 1–6, June 1, 2015 ª2015 The Au ho s Please ci e his a icle in p ess as: Abbas e al., Oxygen Sensing Coo dina es Pho omo phogenesis o Facili a e Seedling Su i al, Cu en Biology (2015), h p://dx.doi.o g/10.1016/j.cub.2015.03.060 ano he key componen o pho omo phogenesis, is also medi- a ed by oxygen-dependen ERFVII deg ada ion. E iola ed seed- lings o WT and N-end ule mu an s we e analyzed ollowing ans e o ligh unde hypoxic o no moxic condi ions. A e ans e o ligh unde no moxia, o al chlo ophyll le els we e much lowe in p 6,a e1 a e2, and nia1 nia2 mu an s compa ed o WT, bu simila in p 6 e VII (Figu e 2F). Chlo ophyll le els we e also educed in WT seedlings unde hypoxia ollowing ans e o ligh , bu no in e VII. Indi idual s able ERVIIs MA- HRE1 and MA-RAP2.3 d i en by hei endogenous p omo e s we e no able o ep ess chlo ophyll accumula ion, indica ing ha hey ac in combina ion, pe haps as pa o he e ome ic complexes, wi h o he ERFVIIs, unlike hei oles in apical hook main enance. Ou esul s indica e ha oxygen a ailabili y is sensed by ERFVIIs du ing ea ly seedling de elopmen and p omp he hy- po hesis ha low oxygen le els would also enhance seedling su i al du ing exposu e o p olonged da kness. As p e iously epo ed [4], we ound ha a e ex ended exposu e o da k unde no moxia, WT seedlings died, whe eas he e we show ha hypoxia allowed comple e eco e y o co yledon g eening and e en he capaci y o p ima y lea g ow h (Figu es 3A, 3B, and 3C). Hypoxia-media ed su i al was comple ely dependen on ERFVIIs (Figu es 3B and 3C), which es ic ed he accumula- Col-0 p 6 a e1a e2 p 6e VII Col-0 nia1nia2 nia1nia2e VII Col-0 e VII MA-HRE1 MA-HRE2 MA-RAP2.12 MA-RAP2.2 MA-RAP2.3 C- PRT6 P o easomeR-C - ATE1 ATE2 MC- C - O2, NO A-MA- MAP S able A DEBC Time (h) 200 150 100 50 0144720 No moxia Hypoxia 144720 144720 144720 ERFVII: RAP2.12 RAP2.2 RAP2.3 HRE1 HRE2 ox ox F To al chlo ophyll (mg/g) 1 0 0.5 Col-0 p 6 e VII a e1a e2 p 6e VII nia1nia2 MA-HRE1 6h ligh : No moxia Hypoxia No moxia MA-RAP2.3 PCO 14 4 72 0 No moxia MA R AP 2. 3 N N 14 4 4 72 0 0 0 4 0 0 0 0 0 0 14 4 72 0 4 0 Figu e 2. Oxygen Sensing du ing Sko omo - phogenesis and Pho omo phogenesis Is Con olled by ERFVII T ansc ip ion Fac o s and he N-End Rule Pa hway (A) Schema ic o he Cys-A g/N-end ule pa hway. Single amino acid abb e ia ions a e used. ox, oxidized; NO, ni ic oxide; PCO, plan -speci ic Plan Cys eine Oxidase [13]; MAP, Me hionine Amino-Pep idase; ATE A ginyl T ans e ase; PRT6, PROTEOLYSIS E3 ligase. P o ein subs a es a e indica ed as blue o als. (B–E) Dynamics o apical hook angle o e iola ed seedlings o WT (Col-0), N-end ule pa hway and NO syn hesis mu an s, and ansgenics, con ain- ing s abilized ERFVIIs exp essed unde endoge- nous p omo e s (p omERFVII:MA-ERFVII). (F) Chlo ophyll con en o 4-day-old seedlings ans e ed o he ligh o 6 h . E o ba s indica e SD om he mean. ion o eac i e oxygen species (ROS) (Figu e S2). As a con i ma ion o he in ol emen o ERFVIIs in su i al a e long exposu e o da kness, only p 6, bu no p 6 e VII, seedlings we e able o su i e long- e m exposu e o da k no moxia (Figu e 3D). Pho o-oxida i e damage caused by high ROS le els has been linked o excessi e accumula ion o p o ochlo ophyllide (PC) in da kness [4]; he e o e, obse ed educed le els o PC in p 6 mu an s compa ed wi h WT, e VII and p 6 e VII (Figu e 3E), a e he likely explana ion o he enhanced su i al. These esul s demons a e ha s abilized ERFVIIs p o ec seedlings om p olonged exposu e o da k condi ions and pe mi subsequen g ow h o he apical me is em ollowing ans e o ligh . In da kness, chlo ophyll biosyn hesis is known o be ep essed by PIF ansc ip ion ac o s [18]. Howe e , ou analysis o PC accumula ion sugges ed ha ERFVIIs ep ess exp ession o chlo ophyll biosyn hesis genes in low-oxygen en i onmen s. We he e o e analyzed he in luence o ligh and hypoxia on mRNA ansc ip accumula ion o enzymes o e a- py ole syn hesis (Figu es 4A–4D). Fo se e al genes, exp ession in e iola ed seedlings was no in luenced by hypoxia (Figu e S3). Howe e , exp ession o he chlo oplas ic o m o heme syn- hase, FC2, and CHLM,PORA,PORB,PORC GUN1, and GUN4 was g ea ly ep essed in WT by hypoxia bo h in he da k and, ollowing ans e , in he ligh , and was also cons i u i ely ep essed in p 6 independen o oxygen a ailabili y (Figu es 4C and 4D). Fu he mo e, his ep ession was no obse ed in e VII o p 6 e VII mu an s, indica ing ha down egula ion is achie ed by s abilized ERFVIIs. In con as , FC1 (encoding chlo oplas ic and mi ochond ial heme syn hase [19]) exp ession was enhanced unde hypoxia, in an ERFVII-dependen manne (Figu e 4D). Ou esul s sugges a homeos a ic mechanism whe eby hypoxia-s abilized ERFVIIs ep ess se e al s eps o he oxygen- equi ing e apy ole pa hway. Toge he , hese Cu en Biology 25, 1–6, June 1, 2015 ª2015 The Au ho s 3 Please ci e his a icle in p ess as: Abbas e al., Oxygen Sensing Coo dina es Pho omo phogenesis o Facili a e Seedling Su i al, Cu en Biology (2015), h p://dx.doi.o g/10.1016/j.cub.2015.03.060 esul s demons a e an impo an ole o oxygen sensing in chlo- ophyll biosyn hesis by N-end ule con ol o ERFVII s abili y. The obse a ion ha in he ligh chlo ophyll biosyn hesis is no comple ely impai ed in hypoxia sugges s ligh ei he compen- sa es o ep ession o chlo ophyll biosyn hesis by ERFVIIs o p omo es inac i a ion o ERFVIIs. I has been shown ha cy o- solic RAP2.12 mo es o he nucleus in esponse o he hypoxic signal [12]. In ag eemen wi h his esul , we ound ha in e io- la ed seedlings unde hypoxia, he subcellula loca ion o cons i u i ely exp essed s abilized RAP2.3 (35S:YFP-RAP2.3) changed om cy oplasm and nucleus o exclusi ely nucleus (Figu es 4E and S3). Howe e , unde no moxia and ollowing ans e o ligh , his s abilized ERFVII is emo ed om he nu- cleus, h ough a mechanism un ela ed o he ERFVII N-deg on, as N -Cys is no p esen in he analyzed p o ein due o he N - YFP usion. Howe e , i is in e es ing o no e ha his deg ada- ion occu s much mo e slowly in hypoxia. These esul s show ha ligh can o e ide ERFVII unc ion e en unde hypoxia, e en- ually becoming he dominan en i onmen al signal. In addi ion o i s me abolic equi emen o ae obic espi a- ion, highe euka yo es use speci ic sensing o molecula oxygen as a mechanism o con ol physiology and de elop- men [20, 21]. Animals use a di e en Hypoxia-Inducible Fac o (HIF) sys em o sense oxygen [20]. I has been shown ha emb yos o Caeno habdi is elegans exhibi diapause (a es ed de elopmen ) in esponse o hypoxia, con olled Col-0 No moxia A C e VII Hypoxia 8 days No moxia Hypoxia Da k 2 days Ligh No moxia B Col-0 e VII 100 50 0 pe cen age G een co yledons P ima y lea es o med Col-0 e VII 100 50 0 pe cen age Col-0 p 6 e VII p 6e VII Col-0 p 6 e VII p 6e VII Col-0 p 6 e VII p 6e VII Da k No moxia: 7D 10D 14D D No moxia Hypoxia E 4 days da k: Blank 640 660 680 700 Wa eleng h (nm) 80 60 40 20 0 Rela i e Flou escence Col-0 p 6 e VII p 6e VII Figu e 3. S abilized ERFVIIs Enhance Long- Te m Su i al in he Da k (A) Schema ic o expe imen al design o analyze he e ec o long- e m de elopmen in he da k ol- lowed by ans e o he ligh . (B and C) Quan i ica ion and images o co yledon g eening and p ima y lea expansion in WT and e VII seedlings in esponse o ligh ollowing exposu e o da k unde hypoxic o no moxic condi ions. (D) Quan i ica ion o co yledon g eening and p i- ma y lea expansion in WT and mu an seedlings in esponse o ligh ollowing exposu e o inc eased pe iods o da k unde no moxic condi ions. (E) Rela i e luo escence o p o ochlo ophyllide in 4-day e iola ed seedlings. E o ba s indica e SD om he mean. by ma e nal nonau onomous exp ession o neu al HIF-1 ac i i y [22]. The ice Subme gence1A (Sub1A) locus (encoding an ERFVII) p o ides ole ance o sub- me gence-induced hypoxia h ough a quiescence s a egy [21], and wo k e- po ed he e demons a es ha e iola ed seedlings o highe plan s es ic pho o- mo phogenic de elopmen unde low- oxygen s ess and su i e ex ended low- oxygen condi ions h ough s abiliza ion o ERFVIIs. Thus, mo phogene ic and biochemical adap a ions o su i e hyp- oxic en i onmen s in animals and plan s may sha e common ea u es. Ou da a demons a e ha oxygen sensing, in addi ion o ligh pe cep ion, is a key componen o ea ly seedling de elopmen , unc ioning o p o ec he apical me is em s em cell niche and p e en pho o-oxida i e damage. We show ha long- e m su i al o seedlings in he da k depends on hei abili y o sense oxygen and ha coun e -in ui i ely hyp- oxic condi ions a e an impo an en i onmen al componen enhancing long- e m seedling su i al. In addi ion o p e ious wo k demons a ing a ole o ERFVIIs in plan esponses o wa e logging and hypoxia, ou cu en wo k highligh s how sensing o he gaseous en i onmen may play a mo e gene al ole in plan g ow h and de elopmen . Low ligh and high e hylene p oduc ion a e simila i ies be ween he phe- nomenologies o looding and sko omo phogenesis [23, 24]. Howe e , he esponse o e iola ed seedlings desc ibed he e is no ela ed di ec ly o escape o quiescence s a egies associa ed wi h long- e m subme gence [21] bu o apid and non-pe manen hypoxic condi ions ha could occu , o example, a e hea y ain in soil wi h good d aining capaci y. In ag eemen wi h his hypo hesis, oxygen sensing does no appea o in luence one sko omo phogenic ai , hypoco yl elonga ion, as e iola ed p 6 seedlings, and WT and e VII seed- lings unde hypoxia, elonga e hypoco yls simila ly o WT unde no moxia [15](Figu e S1), sugges ing ha oxygen sensing is speci ically ela ed o p o ec ion o he apical me is em s em cell niche. 4Cu en Biology 25, 1–6, June 1, 2015 ª2015 The Au ho s Please ci e his a icle in p ess as: Abbas e al., Oxygen Sensing Coo dina es Pho omo phogenesis o Facili a e Seedling Su i al, Cu en Biology (2015), h p://dx.doi.o g/10.1016/j.cub.2015.03.060 Recen ly i was shown ha oxygen ac s as an in e nal de elop- men al posi ional cue in plan s [25]; in his wo k, we ha e shown ha i also ac s as an en i onmen al posi ional cue. The unde - g ound en i onmen combines bo h oxygen and ligh g adien s. We sugges ha whe eas PIF unc ion in eg a es esponses o ligh in ea ly seedling g ow h [26, 27], ERFVII unc ion in eg a es esponses o he gaseous en i onmen , and he ex en o o e lap be ween he wo pa hways emains o be de e mined. Oxygen sensing may he e o e p o ide an adap i e ad an age o seed- lings g owing h ough he soil, allowing changes in he gaseous a mosphe e o be sensed p io o he i e e sible ansi ion o pho omo phogenic g ow h. Ul ima ely, emo al o ERFVII- ep essi e unc ion is assu ed when seedlings each he soil su - ace h ough oxygen and NO-media ed des uc ion. Toge he wi h ligh pe cep ion, his allows an in eg a ed esponse o he complex and changing physical mic oen i onmen encoun e ed by he g owing e iola ed seedling as i s uggles h ough he soil o each he su ace. AB C DE Figu e 4. Con ol o Te apy ole Syn hesis Gene Exp ession by Oxygen and ERFVIIs (A) Schema ic o expe imen al design. E iola ed seedlings we e g own o 4 days in he da k, he inal 2 days being in ei he no moxia o hypoxia. Then, seedlings we e le in he da k o exposed o ligh o 6 h . (B) Diag am o chlo ophyll biosyn hesis pa hway. Enzymes a e shown nex o in e media e com- pounds o he pa hway. Oxygen (O 2 )-dependen sec ions o he pa hway a e indica ed. (C and D) Exp ession o e apy ole and heme syn hesis genes in WT and mu an lines in esponse o hypoxia and ligh . (E) Changes in subcellula loca ion and abundance o cons i u i ely exp essed YFP-RAP2.3 p o ein in esponse o oxygen and ligh . E o ba s indica e SD om he mean. Cu en Biology 25, 1–6, June 1, 2015 ª2015 The Au ho s 5 Please ci e his a icle in p ess as: Abbas e al., Oxygen Sensing Coo dina es Pho omo phogenesis o Facili a e Seedling Su i al, Cu en Biology (2015), h p://dx.doi.o g/10.1016/j.cub.2015.03.060 SUPPLEMENTAL INFORMATION Supplemen al In o ma ion includes Supplemen al Expe imen al P ocedu es, h ee igu es, and one able and can be ound wi h his a icle online a h p://dx.doi.o g/10.1016/j.cub.2015.03.060. AUTHOR CONTRIBUTIONS M.J.H., M.A.B., D.A., J.L., M.A., G.W.B., and S.B. concei ed and designed ex- pe imen s. M.A., S.B., J.V.C., D.J.G., D.R., C.S.C., G.W.B., N.M-d.l.R., J.L., D.A., and M.J.H. pe o med he expe imen s. M.J.H., M.A.B., D.A., J.L., and M.A. analyzed he da a. M.J.H and M.A.B w o e he manusc ip . ACKNOWLEDGMENTS M.J.H., D.J.G., J.V.C., and C.S.C. we e suppo ed by BBSRC g an s BB/ G010595/1 and BB/K000144/1 (including inancial suppo om SABMille ). G.W.B. was suppo ed by a Ma ie Cu ie In e na ional Incoming Fellowship. 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