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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
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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.
S.B. was suppo ed by a Uni e si y o No ingham PhD ellowship. D.R. was
suppo ed by a BBSRC DTP PhD ellowship. J.L. and M.A.B., D.A., and M.A.
we e suppo ed, espec i ely, h ough g an s om MICINN (Spain) BIO2011-
27526 and BIO2010-15071. We hank Ch is ian Fankhause and F anc¸ ois
Pa cy o commen s on he manusc ip .
Recei ed: Feb ua y 3, 2015
Re ised: Ma ch 11, 2015
Accep ed: Ma ch 31, 2015
Published: May 14, 2015
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6Cu 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