Jou nal o Expe imen al Bo any, Vol. 63, No. 15, pp. 5475–5485, 2012
doi:10.1093/jxb/e s199 Ad ance Access publica ion 9 Augus , 2012
This pape is a ailable online ee o all access cha ges (see h p://jxb.ox o djou nals.o g/open_access.h ml o u he de ails)
© 2012 The Au ho s.
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion Non-Comme cial License (h p://c ea i ecommons.o g/licenses/
by-nc/2.0/uk/) which pe mi s un es ic ed noncomme cial use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
Jou nal o Expe imen al Bo any, Vol. 63, No. 2, pp. 695–709, 2012
doi:10.1093/jxb/e 313 Ad ance Access publica ion 4 No embe , 2011
This pape is a ailable online ee o all access cha ges (see h p://jxb.ox o djou nals.o g/open_access.h ml o u he de ails)
RESEARCH PAPER
In Posidonia oceanica cadmium induces changes in DNA
me hyla ion and ch oma in pa e ning
Ma ia G eco, Ad iana Chiappe a, Leona do B uno and Ma ia Bea ice Bi on i*
Depa men o Ecology, Uni e si y o Calab ia, Labo a o y o Plan Cy o-physiology, Pon e Pie o Bucci, I-87036 A ca aca a di Rende,
Cosenza, I aly
* To whom co espondence should be add essed. E-mail: [email p o ec ed]
Recei ed 29 May 2011; Re ised 8 July 2011; Accep ed 18 Augus 2011
Abs ac
In mammals, cadmium is widely conside ed as a non-geno oxic ca cinogen ac ing h ough a me hyla ion-dependen
epigene ic mechanism. He e, he e ec s o Cd ea men on he DNA me hyla ion pa en a e examined oge he wi h
i s e ec on ch oma in econ igu a ion in Posidonia oceanica. DNA me hyla ion le el and pa e n we e analysed in
ac i ely g owing o gans, unde sho - (6 h) and long- (2 d o 4 d) e m and low (10 mM) and high (50 mM) doses o Cd,
h ough a Me hyla ion-Sensi i e Ampli ica ion Polymo phism echnique and an immunocy ological app oach,
espec i ely. The exp ession o one membe o he CHROMOMETHYLASE (CMT) amily, a DNA me hyl ans e ase,
was also assessed by qRT-PCR. Nuclea ch oma in ul as uc u e was in es iga ed by ansmission elec on
mic oscopy. Cd ea men induced a DNA hype me hyla ion, as well as an up- egula ion o CMT, indica ing ha de
no o me hyla ion did indeed occu . Mo eo e , a high dose o Cd led o a p og essi e he e och oma iniza ion o
in e phase nuclei and apop o ic igu es we e also obse ed a e long- e m ea men . The da a demons a e ha Cd
pe u bs he DNA me hyla ion s a us h ough he in ol emen o a speci ic me hyl ans e ase. Such changes a e
linked o nuclea ch oma in econ igu a ion likely o es ablish a new balance o exp essed/ ep essed ch oma in.
O e all, he da a show an epigene ic basis o he mechanism unde lying Cd oxici y in plan s.
Key wo ds: 5-Me hylcy osine-an ibody, cadmium-s ess condi ion, ch oma in econ igu a ion, CHROMOMETHYLASE,
DNA-me hyla ion, Me hyla ion- Sensi i e Ampli ica ion Polymo phism (MSAP), Posidonia oceanica (L.) Delile.
In oduc ion
In he Medi e anean coas al ecosys em, he endemic
seag ass Posidonia oceanica (L.) Delile plays a ele an ole
by ensu ing p ima y p oduc ion, wa e oxygena ion and
p o ides niches o some animals, besides coun e ac ing
coas al e osion h ough i s widesp ead meadows (O , 1980;
Piazzi e al., 1999; Alco e o e al., 2001). The e is also
conside able e idence ha P. oceanica plan s a e able o
abso b and accumula e me als om sedimen s (Sanchiz
e al., 1990; Pe gen -Ma ini, 1998; Mase i e al., 2005) hus
in luencing me al bioa ailabili y in he ma ine ecosys em.
Fo his eason, his seag ass is widely conside ed o be
a me al bioindica o species (Mase i e al., 1988; Pe gen
e al., 1995; La ab ie e al., 2007). Cd is one o mos
widesp ead hea y me als in bo h e es ial and ma ine
en i onmen s.
Al hough no essen ial o plan g ow h, in e es ial
plan s, Cd is eadily abso bed by oo s and ansloca ed in o
ae ial o gans while, in acqua ic plan s, i is di ec ly aken up
by lea es. In plan s, Cd abso p ion induces complex changes
a he gene ic, biochemical and physiological le els which
ul ima ely accoun o i s oxici y (Valle and Ulme , 1972;
Sani zdi Toppi and Gab ielli, 1999; Bena ides e al., 2005;
Webe e al., 2006; Liu e al., 2008). The mos ob ious
symp om o Cd oxici y is a educ ion in plan g ow h due o
an inhibi ion o pho osyn hesis, espi a ion, and ni ogen
me abolism, as well as a educ ion in wa e and mine al
up ake (Ouzonidou e al., 1997; Pe us-Ba beoch e al., 2000;
Shukla e al., 2003; Sobkowiak and Decke , 2003).
A he gene ic le el, in bo h animals and plan s, Cd
can induce ch omosomal abe a ions, abno mali ies in
ª2011 The Au ho (s).
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion Non-Comme cial License (h p://c ea i ecommons.o g/licenses/by-
nc/3.0), which pe mi s un es ic ed non-comme cial use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
RESEARCH PAPER
The scu ellum o ge mina ed whea g ains unde goes
p og ammed cell dea h: iden i ica ion o an acidic nuclease
in ol ed in nucleus disman ling
Fe nando Domínguez1, Ja ie Mo eno2 and F ancisco Ja ie Cejudo1,*
1 Ins i u o de Bioquímica Vege al y Fo osín esis, Uni e sidad de Se illa and CSIC, A da Amé ico Vespucio, 49, 41092-Se illa, Spain
2 Depa amen o de Biología Celula , Facul ad de Biología, Uni e sidad de Se illa, A da Reina Me cedes s/n, 41012 -Se illa, Spain
* To whom co espondence should be add essed. E-mail: [email p o ec ed]
Recei ed 00 2012; Re ised 00 2012; Accep ed 19 June, 2012
Abs ac
P og ammed cell dea h (PCD) is a c ucial phenomenon in he li e cycle o ce eal g ains. In ge mina ing g ains, he
scu ellum allows he anspo o nu ien s om he s a chy endospe m o he g owing emb yo, and he e o e i
may be he las g ain issue o unde go PCD. Thus, he aim o his wo k was o analyse whe he he scu ellum o
whea g ains unde goes PCD and o pe o m a mo phological and biochemical analysis o his p ocess. Scu ellum
cells o g ains ollowing ge mina ion showed a p og essi e inc ease o DNA agmen a ion, and he TUNEL assay
showed ha PCD ex ended in an apical- o-basal g adien along he scu ellum a ec ing epide mal and pa enchy-
mal cells. Elec on- ansmission mic oscopy e ealed high cy oplasm acuola ion, al e ed mi ochond ia, and he
p esence o double-memb ane s uc u es, which migh cons i u e symp oms o acuola cell dea h, whe eas he
nucleus appea ed lobed and had an inc eased he e och oma in con en as he mos dis inc i e ea u es. An acid- and
Zn2+-dependen nucleoly ic ac i i y was iden i ied in nuclea ex ac s o scu ellum cells unde going PCD. This nucle-
ase was no de ec ed in g ains imbibed in he p esence o abscisic acid, which inhibi ed ge mina ion. This nucleoly ic
ac i i y p omo ed DNA agmen a ion in i o on nuclei isola ed om heal hy cells, hus sugges ing a main ole in
nucleus disman ling du ingPCD.
Key wo ds: cell dea h, ge mina ion, nuclease, scu ellum, seed, T i icum aes i um (whea ).
In oduc ion
The p og ammed elimina ion o unwan ed cells is an essen-
ial p ocess o de elopmen o animals and plan s. The wo
mos common o ms o cell dea h in animals a e apop osis and
au ophagy, which can be dis inguished by mo phological and
molecula ea u es (Con ad , 2009). Apop osis is cha ac e ized
by a se ies o well-de ined mo phological changes including cell
sh inkage, cy oplasm con ac ion, and ch oma in condensa ion
p io o he inal engul men by phagocy ic cells (Taa jes e al.,
2008). A he molecula le el, apop osis is cha ac e ized by ac i-
a ion o caspases (cys einyl, aspa a e-speci ic p o eases) and
nuclea DNA agmen a ion (Ki azumi and Tsukaha a, 2011). In
con as , au ophagy is cha ac e ized a he mo phological le el
by he p esence o au ophagic esicles (au ophagosomes) wi hin
he dying cells and he absence o engul men by phagocy es
du ing ea ly s ages o he cell-dea h p ocess (He and Klionsky,
2009). Despi e hese di e ences be ween he wo mechanisms
o cell dea h, gene ic s udies ca ied ou in di e en animal
models ha e iden i ied genes in ol ed bo h in au ophagy and
apop osis (Con ad , 2009). Besides caspases, he e a e a a -
ie y o apop ogenic e ec o s suppo ing he cellula suicide p o-
g amme ha leads o in e nucleosomal DNA agmen a ion and
nuclea condensa ion, such as caspase-ac i a ed DNase (CAD),
mi ochond ial endonuclease G (EndoG), DNaseI, DNaseII,
apop osis-inducing ac o (AIF) ( o e iew, see Samejima and
Ea nshaw, 2005), and apop osis ch oma in condensa ion induce
in he nucleus (Acinus) (Saha a e al., 1999).
5476 | Domínguez e al.
In plan s, p og ammed cell dea h (PCD) is bo h an impo -
an p ocess o de elopmen (Ku iyama and Fukuda, 2002) and
a mechanism o de ence agains pa hogens (Lam, 2004). Whils
plan PCD sha es some simila i ies wi h apop osis o animals,
such as in e nucleosomal agmen a ion o DNA, ch oma-
in condensa ion, and ac i a ion o caspase-like p o eases (Bai
e al., 2010), PCD in plan cells also exhibi dis inc i e ea u es.
The p esence o chlo oplas s, a p ominen acuole, and he cell
wall a e unique cha ac e is ics o plan cells, which a ec PCD
(Williams and Dickman, 2008). In he case o he chlo oplas s,
which cons i u e an impo an sou ce o eac i e oxygen species
p oduc ion in plan cells, i was p oposed ha hese o ganelles
may ha e a signalling unc ion o some plan PCD esponses
(Zapa a e al., 2005). Mo eo e , a combina ion o he unc ion o
he acuole du ing cell dea h and au ophagy may ep esen a plan
al e na i e o he phagocy osis sys em o apop osis (Ha sugai
e al., 2006; Bassham, 2007), which has a speci ic mo phology
e med ‘ acuola cell dea h’ ( an Doo n e al., 2011). A he
molecula le el, al hough he e is inc easing e idence which
connec s he pa icipa ion o p o eases and nucleases in plan
PCD, he enzymes di ec ly in ol ed in he execu ion o nucleus
disman ling in plan s (ch oma in condensa ion, in e nucleosomal
agmen a ion o DNA, and nuclea en elope diso ganiza ion)
a e ye poo ly known.
PCD plays an essen ial ole in he p ocesses o de elopmen
and ge mina ion o ce eal g ains and, hus, he ce eal g ain
has become one o he model sys ems o he s udy o PCD in
plan s. A ini ial s ages o g ain de elopmen , ma e nal issues
such as he nucellus and he nucella p ojec ion cells degene a e
by a p ocess o PCD associa ed wi h cha ac e is ic p o eoly ic
and nucleoly ic ac i i ies (Domínguez and Cejudo, 1998, 2006;
Domínguez e al., 2001). Then he s a chy endospe m, he is-
sue specialized in he accumula ion o s o age compounds,
unde goes PCD du ing ma u a ion (Young e al., 1997; Young
and Gallie, 1999, 2000). Ge mina ion and pos ge mina ion o
ce eal g ains occu s by an o de ed sequence o e en s, which
a e subjec ed o ho monal egula ion and may be summa ized
as ollows: gibbe ellins a e syn hesized a he scu ellum and
di use o he s a chy endospe m (Apple o d and Len on, 1997).
The ho mone is pe cei ed by he aleu one cells, which induce
he syn hesis and sec e ion o hyd oly ic enzymes, including
α-amylases, p o eases, and glucanases, and also he acidi i-
ca ion o he s a chy endospe m, a p ocess ha occu s wi h a
well-es ablished spa io empo al pa e n, as desc ibed o he
whea g ain (Domínguez and Cejudo, 1999). Once he aleu one
cells ha e ca ied ou hei essen ial ole, hese cells ini ia e a
p ocess o PCD, which is also unde he con ol o gibbe el-
lins (Fa h e al., 2000; Domínguez e al., 2004). Besides i s
ini ial ole o p oduce gibbe ellins, he majo unc ion o he
scu ellum in he ge mina ed g ain is he ans e o suga s and
amino acids o he g owing seedling (Wes e al., 1998; Aoki
e al., 2006). In addi ion, he scu ellum is i sel a s o age is-
sue, he con en s o which migh be used o eed he seedling
once he ans e unc ion is inished. So a , he analysis o
PCD in he scu ellum has been limi ed o s udies o emb yo-
genesis du ing maize ke nel de elopmen (Giuliani e al., 2002;
Consonni e al., 2003) o di e en ia ing ascula issue o ge -
mina ed g ains (Domínguez e al., 2002). Howe e , i is no
ye known whe he he scu ellum unde goes a massi e p ocess
o PCD du ing g ain ge mina ion. The p esen s udy add essed
whe he scu ella cells su e PCD in ge mina ed whea g ains
and he iden i ica ion o nucleoly ic ac i i ies in ol ed in
nucleus disman ling. The aim was o compa e he mo phologi-
cal and biochemical ea u es o his dea h p ocess wi h hose o
o he issues unde going PCD in ce eal g ains, such as s a chy
endospe m, aleu one, o nucella cells. The ele ance o his
p ocess o PCD o he scu ellum in he con ex o g ain ge mi-
na ion is discussed.
Ma e ials and me hods
Plan ma e ial
Whea (T i icum aes i um c . Chinese Sp ing) g ains we e s e ilized in
2% ( / ) NaOCl o 20 min and washed wice wi h s e ile wa e , once
wi h 0.01 M HCl and hen ho oughly wi h s e ile dis illed wa e . S e ile
g ains we e allowed o ge mina e a oom empe a u e on s e ile il e
pape soaked wi h wa e . T ea men s wi h ho mones and inhibi o s o
ho mone syn hesis we e ca ied ou on il e pape soaked wi h 20 mM
MOPS-KOH pH 7.0 supplemen ed wi h 10 mM CaCl2. Ho mones and
inhibi o s we e added a he ollowing inal concen a ions: gibbe ellic
acid, GA3, 5 µM; abscisic acid (ABA), 25 µM; paclobu azol (PCB),
500 µM; 24-epib assinolide (EBL), 1 nM, and α-(2-aminoe hoxy inyl)
glycine (AVG), 10 µM. GA3, ABA, EBL, and AVG we e pu chased
om Sigma Chemical and PCB om Duche a Biochimie.
Isola ion o DNA and elec opho esis
Scu ellum discs, dissec ed om whea g ains imbibed o up o 7 days,
we e g ound in liquid ni ogen wi h a mo a and pes le o a ine pow-
de and homogenized in 5 ml o ex ac ion bu e [50 mM TRIS-HCl
pH 8.0, 100 mM NaCl, 50 mM EDTA, 1% ( / ) 2-me cap oe hanol, and
2% (w/ ) SDS]. Fo DNA isola ion, ex ac s we e incuba ed a 45 °C
o 15 min, a oom empe a u e o 30 min, and hen mixed wi h 5 ml
o phenol/chlo o o m (1:1, / ). Samples we e cen i uged a 10,000 g
o 10 min and he uppe phase was p ecipi a ed a –20 °C o 30 min
wi h 2 olumes o ice-cold e hanol. A e cen i uga ion, he DNA pelle
was ai d ied, dissol ed in 250 µl TE bu e (10 mM TRIS-HCl pH 8.0,
1 mM EDTA), and quan i ied spec opho ome ically. RNase A (1.5 µl
o a s ock o 10 mg ml–1) was added and incuba ed a 37 °C o 3 h. A e
his ea men , DNA was again p ecipi a ed and dissol ed in TE bu e .
Finally, DNA samples (20 µg) we e analysed on a 2% aga ose gel and
s ained wi h e hidium b omide. DNA ladde s (500 o 100 bp, Gibco)
we e used o es ima e DNA size.
P epa a ion o nuclea and cy oplasmicex ac s
Scu ellum discs dissec ed om g ains imbibed o up o 7 days
we e g ound in a mo a wi h liquid ni ogen and esuspended in
5 ml homogeniza ion bu e [0.25 M suc ose, 10 mM NaCl, 10 mM
MES-NaOH pH 6.0, 5 mM EDTA, 0.15 mM spe mine, 0.5 mM spe -
midine, 0.2 mM PMSF, 20 mM 2-me cap oe hanol, 0.25 % ( / ) T i on
X-100]. The homogena e was cla i ied by cen i uga ion a 100 g o
1 min and il e ed h ough a nylon mesh (60 µm po e-size, Millipo e).
F ac iona ion was pe o med by adding he il e ed supe na an o
homogeniza ion bu e con aining 30% Pe coll and cen i uga ion a
3000 g o 15 min. The uppe phase was collec ed as he cy oplasmic
ex ac , he Pe coll phase was disca ded, and he nuclei-en iched pel-
le was washed in homogeniza ion bu e and esuspended in 100 µl
ex ac ion bu e [25 mM sodium phospha e pH 7.8, 40 mM KCl,
20% glyce ol, 1% plan p o ease inhibi o cock ail (Sigma), 0.4 M
(NH4)2SO4]. A e ex ac ion on ice o 30 min, he supe na an o he
subsequen cen i uga ion (13,000 g, 20 min, 4 °C) was collec ed as
he nuclea ex ac .
A nuclea -localized nuclease in scu ellum PCD | 5477
TUNELassay
Whea g ains ha es ed a di e en days a e imbibi ion (DAI) we e
longi udinally sec ioned a e emo ing shoo s and oo s, immedi-
a ely ixed in FAE ( o maldehyde/ace ic acid/e hanol (3.7:5:50, / ,
and embedded in Pa aplas Plus (Sigma). In si u de ec ion o DNA
agmen a ion was ca ied ou as p e iously desc ibed (Domínguez
e al., 2001). Pa aplas Plus was emo ed om he g ain sec ions by
ea men wi h xylol, and he sec ions we e hen dehyd a ed wi h a
dec easing e hanol se ies, ea ed wi h p o einase K (20 µg ml–1) in
PBS (10 mM sodium phospha e bu e , 130 mM NaCl), and insed
wice wi h PBS. Endogenous pe oxidase ac i i y was hen quenched
by incuba ion in 1% ( / ) H2O2 in me hanol o 30 min and insed
wice wi h PBS. Fo labelling, sec ions we e incuba ed o 60 min a
37 °C in he p esence o e minal deoxynucleo idyl ans e ase (TdT)
wi h he In si u Cell Dea h De ec ion Ki (Roche Applied Sys ems),
acco ding o he manu ac u e ’s ins uc ions. Con ols we e pe o med
in which TdT was omi ed.
Elec on mic oscopy
Fo mo phological analysis, small agmen s o whea g ains ha es ed
a 1 o 5 DAI we e ixed in 4% ( / ) glu a aldehyde p epa ed in 0.1 M
cacodyla e bu e (pH 7.2) o 3 h a 4 °C. The samples we e dehy-
d a ed in an ace one se ies and embedded in Epon (an epoxy embed-
ding medium). Toluidine blue-s ained semi- hin sec ions used as con ol
we e iewed in a Lei z (A is oplan) ligh mic oscope. Thin sec ions
(60–80 nm) we e cu on a Reiche -Jung Ul acu E ul amic o ome,
s ained wi h u anyl ace a e and lead ci a e, and examined in a Philips
CM-10 ansmission elec on mic oscope.
In-gel nuclease ac i i yassay
The in-gel nuclease ac i i y assay was pe o med as epo ed p e iously
(Domínguez e al., 2004) wi h modi ica ions. Cy oplasmic and nuclea
ex ac s (50 µg p o ein) ob ained as desc ibed abo e we e ac ion-
a ed on SDS-PAGE gels con aining 0.3 mg ml–1 salmon spe m DNA
a 4 °C and 20 mA/pla e. A e elec opho esis, he gels we e washed
wice o 15 min in 1% ( / ) T i on X-100 and hen wice o 15 min
in dis illed wa e . The gels we e hen incuba ed o e nigh in 25 mM
sodium ace a e-ace ic acid bu e (pH 5.5, con aining 1 mM ZnSO4
and 0.2 mM DTT) o 100 mM MOPS-KOH (pH 7.0, con aining 5 mM
CaCl2 and 5 mM MgCl2) a 37 °C. False nucleoly ic ac i i ies associ-
a ed wi h DNA-binding p o eins we e disca ded by incuba ing he
gels in 1% (w/ ) SDS o 2 h a oom empe a u e and hen washed in
wa e o 10 min. Finally, gels we e s ained wi h 1 µg ml–1 e hidium
b omide o 10 min. Nuclease ac i i ies we e pho og aphed on a UV
ligh box. Cy oplasmic con amina ion o plan nuclea ex ac s was ou-
inely analysed by Wes e n blo analysis using phosphoenolpy u a e
ca boxylase (PEPC) as a cy oplasmic ma ke (González e al., 1998).
A ini y-pu i ied polyclonal maize PEPC an ibodies we e pu chased
om Rockland.
In i o endonuclease ac i i yassay
In i o endonuclease ac i i y assay was ca ied ou acco ding o
he me hod desc ibed by I o and Fukuda (2002) wi h modi ica ions.
In b ie , isola ed nuclei om scu ella issue we e incuba ed wi h
nuclea o cy oplasmic ex ac s om scu ellum isola ed om g ains a
7 DAI. Incuba ion was pe o med o 2 h a 30 °C in 25 mM sodium
ace a e-ace ic acid bu e (pH 5.5) o 100 mM MOPS-KOH (pH 7.0).
Reac ions we e s opped by adding an equal olume o lysis bu e
(100 mM TRIS-HCl pH 8.0, 200 mM NaCl, 100 mM EDTA, 2% SDS)
and incuba ion o 1 h a 55 °C. A e ex ac ion wi h phenol/chlo o-
o m/isoamylalcohol (25:24:1, / ), DNA was p ecipi a ed wi h wo
olumes o absolu e e hanol, esuspended in TE bu e , p ecipi a ed
again, and inally esuspended in 25 µl TE bu e . Con amina ing RNA
was emo ed by incuba ion o 3 h a 37 °C in he p esence o RNaseA
( inal concen a ion 60 µg ml–1). DNA was hen e hanol-p ecipi a ed,
esuspended in TE bu e , esol ed on 2% (w/ ) aga ose gels, and isu-
alized using e hidium b omide.
Resul s
The scu ellum o whea g ains ollowing ge mina ion
unde goesPCD
Wi h he aim o es ing whe he he scu ellum o ge mina ed
whea g ains unde goes PCD, his s udy analysed he in e nu-
cleosomal agmen a ion o genomic DNA, a hallma k o PCD.
DNA ladde ing was i s obse ed in g ains a e 4 DAI and
inc eased p og essi ely up o 7 days (Fig. 1A). A mo e p ecise
iden i ica ion o scu ella cells unde going PCD was pe o med
wi h he TdT ( e minal deoxynucleo idyl ans e ase)-media ed
dUDP nick-end labelling (TUNEL) assay. No labelling was
obse ed in sec ions o g ains a 1 DAI (Fig. 1B) hus e eal-
ing he absence o PCD in scu ella cells a hese ea ly s ages;
howe e , he TUNEL assay showed labelling o nuclei o he
pa enchymal and epide mal cells o g ains a 7 DAI (Fig. 1C). In
he cen al egion o he scu ellum, TUNEL s aining o he epi-
helial and pa enchymal cells was i s obse ed in g ains a e
4 DAI and inc eased p og essi ely up o 7 DAI (Fig. 1D–H), in
ag eemen wi h he de ec ion o DNA ladde ing (Fig. 1A). No
labelling o e backg ound was obse ed in con ol sec ions in
he absence o TdT (Fig. 1I).
P e ious analysis o pos ge mina i e p ocesses in whea
g ains e ealed impo an spa io empo al g adien s a ec ing
s a chy endospe m acidi ica ion, aleu one gene exp ession,
and PCD (Domínguez and Cejudo, 1999; Domínguez e al.,
2004). Thus, wi h he aim o es ing whe he scu ellum PCD
akes place wi h any spa io empo al pa e n, ul a hin sec ions
o whea g ains a 1 and 5 DAI we e analysed. A mo pho-
logical symp om o cell dea h, he inc ease o acuoliza ion,
p og essed om he uppe pa o he scu ella epi helium in
con ac wi h he aleu one laye o he lowe pa , which is indic-
a i e o a g adien o PCD in his scu ella issue (Fig. 2A, 2B).
I was no iced ha PCD was ini ia ed in scu ella cells once he
aleu one cells close o he scu ellum had comple ed he p ocess
o PCD and we e almos emp y (Fig. 2B). The TUNEL assay
con i med his pa e n o PCD since in whea g ains a 7 DAI
mos cells o he uppe pa o he scu ellum showed an in ense
labelling, whe eas s aining o cells o he lowe pa was less
in ense (Fig. 2C). These esul s sugges ha he spa ial p og es-
sion o PCD in he scu ellum occu s wi h an apical- o-basal
g adien .
Mo phology o scu ellum cells unde goingPCD
To s udy he mo phological ea u es o scu ellum PCD, his s udy
ocused on he analysis o epi helium and pa enchyma cells o
g ains a 5 DAI, conside ing sepa a ely cy oplasmic and nuclea
e en s. Cha ac e is ic ea u es o he cy oplasm o cells unde -
going dea h, as obse ed in he epi helium, a e he o ma ion
o p o acuoles o igina ed om Golgi cis e nae o endoplasmic
e iculum-de i ed bodies (Fig. 3A, 3B), which appea in g ea
numbe and p obably assume he ole o hyd oly ic enzymes
s o age, un il hese p o acuoles use wi h he cen al acuole
5478 | Domínguez e al.
(Fig. 3C). This dea h p ocess was also cha ac e ized by double-
memb ane esicles seques e ing po ions o cy oplasm (Fig. 3D,
3E), which esembled au ophagosomes o animal cells unde go-
ing au ophagy. Mo eo e , se e al al e a ions o mi ochond ia
could be obse ed including i egula shape, enla gemen , and
b oken c is ae (Fig. 3D–F). In he cy oplasm, ano he au oly ic
compa men cha ac e ized by an elec on- anslucen cy oplasm
could be dis inguished: s o age acuoles e ol ing o ly ic acu-
oles (Fig. 3E). In addi ion, cha ac e is ic memb anous s uc u es
could be obse ed in dying scu ellum cells such as mul ilamel-
la s uc u es (Fig. 3G) o he who ls o med om cy oplasmic
memb anes (Fig. 3H).
Conce ning he nucleus, he cha ac e is ics obse ed in pa en-
chymal scu ellum dying cells include high he e och oma in
Fig. 1. Pa e n o DNA agmen a ion in scu ellum cells o whea g ains ollowing ge mina ion. (A) Whea g ains we e imbibed and
a he days indica ed scu ella we e dissec ed and immedia ely ozen in liquid ni ogen. DNA (20µg) isola ed om each sample was
ac iona ed on 2% (w/ ) aga ose gels. The size o he DNA molecula ma ke s a e indica ed on he igh . (B–I) Longi udinal sec ions o
whea g ains a e 1 (B, D), 3 (E), 5 (F), 6 (G), o 7 (C, H, I) days a e imbibi ion we e subjec ed o he TUNEL assay. Con ol sec ions
(I) we e incuba ed in he absence o TdT enzyme. Analyses we e epea ed a leas h ee imes on independen biological samples and
ep esen a i e esul s a e shown. al, Aleu one laye ; en, s a chy endospe m; se, scu ella epi helium; sp, scu ella pa enchema. Ba s,
100µm (B, C) and 50µm (D–I).
Fig. 2. Apical- o-basal g adien o p og ammed cell dea h in he scu ellum o whea g ains ollowing ge mina ion. (A, B) Ligh
mic oscopy o oluidine blue-s ained ul a hin sec ions o whea g ains 1 (A) and 5 (B) days a e imbibi ion. No e he nuclei enla gemen
and he inc easing acuola ion in he epi helium o he scu ellum. (C) TUNEL assay o nuclea DNA agmen a ion in longi udinal sec ions
o whea g ains 7days a e imbibi ion. The TUNEL assay was pe o med a leas h ee imes and a ep esen a i e scu ellum sec ion is
shown. al, Aleu one laye ; en, s a chy endospe m; se, scu ella epi helium; sp, scu ella pa enchema. Ba s, 50µm (A, B) and 100µm (C)
( his igu e is a ailable in colou a JXB online).
A nuclea -localized nuclease in scu ellum PCD | 5479
con en and deep in agina ions (Fig. 4A), so ha na ow laye s
o cy oplasm a e con ined be ween nuclea segmen s (Fig. 4B;
whi e a ows). A clea symp om o nuclea deg ada ion is he
p esence o emnan s o he e och oma in inside p o acuoles
(Fig. 4C, whi e a owheads), leaking o he cen al au oly ic
acuole (Fig. 4C, black a owheads). O e all, he mo phological
Fig. 3. Mo phological analysis o cy oplasm o epi helial
scu ellum cells in ge mina ed whea g ains a 5days a e
imbibi ion. (A, B) Scu ella epi helium cell showing a acuola ed
cy oplasm in he p oximi y o he he e och oma inized nucleus:
nu, nucleus; m , mi ochond ia; p , p o acuole. (C) Acen al
au oly ic acuole (c ) is o med. (D–G) Appea ance o dis u bed
mi ochond ia (dm ) and double-memb ane esicles (dm ) a he
onse o p og ammed cell dea h: in G, no e he mul ilamella body
(mlb) seques e ing pa o he cy oplasm; ps , p o ein s o age
acuole. (H) Cha ac e is ic memb ane s uc u es localized in
dying cells: wh, who l o med by he cy oplasmic memb ane.
Ba s, 1µm (A, B, C, D, H), 2µm (E), and 0.5µm (F, G).
Fig. 4. Mo phological analysis o nuclei o pa enchymal scu ellum
cells in ge mina ed whea g ains a 5days a e imbibi ion. (A, B) De ail
o a nucleus wi h in agina ions (whi e a ows) and p o ein s o age
acuoles in i s p oximi y. (C) Remnan s o condensed ch oma in inside
p o acuoles (whi e a owheads) o cen al acuole (black a owheads).
c , cen al acuole; nu, nucleus; ps , p o ein s o age acuoles; p ,
p o acuole. Ba s, 0.5µm (A), 5µm (B), and 1µm (C).

5480 | Domínguez e al.
ea u es iden i ied sugges ha scu ellum epi helial and pa en-
chymal cells o whea g ains ollowing ge mina ion unde go
acuola cell dea h, as desc ibed in o he plan issues ( an
Doo n e al., 2011).
A nuclea -localized acid endonucleoly ic ac i i y in
scu ellum cells unde goingPCD
As shown abo e, DNA agmen a ion was iden i ied as a hall-
ma k o scu ellum PCD. To cha ac e ize his p ocess a he bio-
chemical le el, he nucleases localized in he nucleus o cells
unde going PCD we e analysed by in-gel ac i i y assays. Fo
ha pu pose, scu ellum cells we e ac iona ed in o nuclea
and cy oplasmic ac ions acco ding o he scheme depic ed in
Supplemen a y Fig. S1A (a ailable in JXB online). Nuclei iso-
la ed om scu ella cells a ea ly s ages (1–4 DAI) appea ed
in ac , whe eas a 7 DAI showed a lobed and agmen ed
appea ance (Supplemen a y Fig. S1B). P o ein ex ac s om
bo h cy osolic and nuclea ac ions we e subjec ed o analysis
o nucleoly ic ac i i y. A band showing endonuclease ac i i y,
wi h a molecula mass o app oxima ely 70 kDa, was de ec ed
in nuclea ex ac s om scu ellum cells o whea g ains a 7 DAI
when assayed a acid pH, bu no a neu al pH (Fig. 5A, 5B). In
con as , cy oplasmic ac ions showed no de ec able nucleoly ic
ac i i y when assayed a acidic pH, bu showed di e en neu-
al nucleases (Fig. 5A, 5B). Possible con amina ion o nuclea
ac ions wi h cy oplasmic p o eins was uled ou by ou inely
es ing o PEPC, a cy oplasmic enzyme, in he Wes e n blo
analysis (Fig. 5C).
To u he cha ac e ize he p ocess o DNA agmen a-
ion, cell- ee assays we e ca ied ou by incuba ing ei he
he nuclea o cy oplasmic ex ac s om scu ellum cells
unde going PCD (a 7 DAI) wi h in ac nuclei isola ed om
heal hy scu ellum cells (a 1 DAI). The nucleoly ic ac i i y
o he nuclea ex ac s igge ed he in e nucleosomal ag-
men a ion o DNA in in ac nuclei a acid pH in con as o
he cy oplasmic ex ac s, which did no p oduce any DNA
agmen a ion o inc ease he ac i i y o he nuclea ex ac
(Fig. 5D). In ag eemen wi h he in-gel ac i i y esul s, he
nuclea -localized nucleoly ic ac i i y is acidic, as shown by
he low ac i i y de ec ed a neu al pH (Fig. 5E). In addi-
ion, he equi emen o ca ions o his nuclea -localized
nucleoly ic ac i i y was analysed using bo h in-gel and in
i o assays. Fig. 6A shows he ac i a ing e ec o Zn2+ on
he nuclease, whe eas Ca2+ and Mg2+ had no e ec . In i o
assays con i med he ac i a ing e ec o Zn2+ (Fig. 6B). The
ca ion equi emen o he nuclea -localized nucleoly ic ac i -
i y was in con as wi h he cy oplasmic ac i i ies, which
equi ed Ca2+ and/o Mg2+ and we e s ongly inhibi ed by
Zn2+ (Supplemen a y Fig. S2A). Al hough he nucleoly ic
ac i i ies o he cy oplasmic ex ac s did no p oduce DNA
agmen a ion in in ac nuclei, as shown in he cell- ee assay
(Fig. 5D, 5E), hese ac i i ies e ec i ely deg aded naked
DNA, p oducing an unspeci ic DNA smea (Supplemen a y
Fig. S2B). The e o e, he nucleoly ic ac i i y o nuclea
ex ac s is associa ed wi h PCD and is able o p oduce DNA
agmen a ion on an in ac ch oma in s uc u e, being Zn2+-
and acid pH-dependen .
Fig. 5. Iden i ica ion o an acid nuclease in he nucleus
o scu ellum cells unde going p og ammed cell dea h.
(A–C)Scu ellum discs dissec ed om whea g ains a 1, 4, o
7days a e imbibi ion (DAI), as indica ed, we e ac iona ed
in o cy oplasmic and nuclea ex ac s, and aliquo s om bo h
ac ions (25µg p o ein) we e analysed by in-gel nuclease assay
a he indica ed pH. C, F ac ions (15µg p o ein) we e subjec ed
o Wes e n blo analysis and p obed wi h polyclonal an i-PEPC
an ibodies. Molecula -mass ma ke , in kDa, is indica ed on he
le . (D, E) In i o analysis o nuclea DNA agmen a ion. P o ein
ex ac s (4µg p o ein) om nuclea (N), cy oplasmic (C), o a
mix u e o bo h (N+C) ac ions ob ained om scu ella cells o
g ains a 7 DAI we e incuba ed wi h in ac nuclei isola ed om
whea g ains a 1 DAI a he indica ed pH. Following incuba ion,
nuclea DNA was isola ed and aliquo s (20µg) we e ac iona ed
on 2% (w/ ) aga ose gels. DNA ma ke , in kbp, is indica ed on he
le . Assays we e epea ed a leas h ee imes wi h simila esul s
and ep esen a i e esul s a e shown.
A nuclea -localized nuclease in scu ellum PCD | 5481
ABA inhibi s ge mina ion and DNA agmen a ion o
scu ellumcells
The inding o a spa io empo al pa e n o PCD in he scu ellum
(Fig. 2C), and he ac ha PCD s a s once he p oximal aleu-
one cells ha e unde gone PCD, sugges ed ha scu ellum PCD
is igh ly egula ed. As ho mones play an impo an ole in he
con ol o g ain ge mina ion and ea ly seedling g ow h, he e ec
o ho mones and inhibi o s o ho mone syn hesis on scu el-
lum PCD was analysed. Fo ha pu pose, whea g ains we e
imbibed in he p esence o di e en ho mones (GA3, ABA, o
he b assinos e oid EBL) and PCB, an inhibi o o GA syn hesis,
o AVG, an inhibi o o e hylene syn hesis. As expec ed, ABA,
Fig. 6. Cha ac e iza ion o nucleoly ic ac i i ies in he nucleus o
scu ellum cells unde going p og ammed cell dea h. (A) E ec o
ca ions on nucleoly ic ac i i ies. P o ein ex ac s (25µg p o ein)
om nuclea ac ions we e subjec ed o in-gel nuclease assay
by o e nigh incuba ion a 37°C in 25 mM sodium ace a e-ace ic
acid bu e (pH 5.5) con aining 0.2 mM DTT and supplemen ed as
indica ed wi h 1 mM CaCl2, 1 mM MgCl2, o 1 mM ZnSO4. EDTA
was added o a inal concen a ion o 0.1 mM. Molecula -mass
ma ke s, in kDa, a e indica ed on he le . (B) In i o analysis
o nuclea DNA agmen a ion. P o ein ex ac s (4µg p o ein)
om nuclea ac ions om scu ella cells o whea g ains a
7days a e imbibi ion (DAI) we e incuba ed wi h in ac nuclei
isola ed om scu ellum cells o g ains a 1 DAI. Incuba ions we e
pe o med o 2 h a 30°C in 25 mM sodium ace a e-ace ic acid
bu e (pH5.5) con aining 0.2 mM DTT. ZnSO4 and EDTA we e
added o he indica ed concen a ion. DNA was ac iona ed on
2% (w/ ) aga ose gels. DNA ma ke s, in kbp, a e indica ed on he
le . Assays we e epea ed a leas h ee imes wi h simila esul s
and ep esen a i e esul s a e shown.
Fig. 7. E ec o ho mones on scu ellum p og ammed cell dea h.
Whea g ains we e soaked o 7days in he absence o p esence
o he ollowing ho mones o ho mone syn hesis inhibi o s:
5µM GA3, 500µM PCB, 25µM ABA, 1 nM EBL, o 10µM AVG
( inal concen a ions). (A) A e imbibi ion, scu ellum discs we e
dissec ed and genomic DNA was isola ed and analysed in 2%
(w/ ) aga ose gel. DNA ma ke s, in kbp, a e indica ed on he
le . (B) Nuclea ex ac s (25µg p o ein) we e analysed by in-gel
nuclease assay by o e nigh incuba ion a 37°C in 25 mM sodium
ace a e-ace ic acid bu e (pH 5.5) con aining 1 mM ZnSO4 and
0.2 mM DTT. Molecula -mass ma ke s, in kDa, a e indica ed on
he le . (C) In i o analysis o nuclea DNA agmen a ion. P o ein
ex ac s (4µg p o ein) om nuclea ac ions om scu ella cells
o whea g ains unde di e en ho monal ea men (7days a e
imbibi ion, DAI) we e incuba ed wi h in ac nuclei isola ed om
scu ellum discs o g ains a 1 DAI. Incuba ions we e pe o med o
2 h a 30°C in 25 mM sodium ace a e-ace ic acid bu e (pH 5.5)
con aining 1 mM ZnSO4 and 0.2 mM DTT. DNA was ac iona ed
on 2% (w/ ) aga ose gels. DNA ma ke s, in kbp, a e indica ed
on he le . Assays we e epea ed a leas h ee imes wi h simila
esul s and ep esen a i e esul s a e shown. AB, ABA; AV, AVG;
E, EBL; G, GA3; P, PCB.
5482 | Domínguez e al.
and o lowe ex en PCB, exe ed an inhibi o e ec on whea
g ain ge mina ion and seedling g ow h, whe eas GA3 and AVG
did no signi ican ly a ec he pos ge mina i e p ocess and EBL
ea men educed oo elonga ion (Supplemen a y Fig. S3). The
analysis o DNA agmen a ion o scu ellum cells showed ha
only ABA ea men exe ed a clea inhibi o y e ec (Fig. 7A).
The o he ea men s, including PCB o EBL, which a ec ed
he pos ge mina i e p ocess, did no show any signi ican e ec
(Fig. 7A). In ag eemen wi h hese esul s, he in-gel nuclease
assay iden i ied he acid nucleoly ic ac i i y in nuclea ex ac s
om scu ellum cells, wi h he excep ion o he ABA- ea ed
g ains (Fig. 7B). Simila ly, ABA caused a signi ican inhibi-
ion o he nuclea -localized nucleoly ic ac i i y, as de ec ed by
in i o assays (Fig. 7C). Thus, only ABA ea men , which had
a s ong e ec on ge mina ion, was e ec i e o inhibi he bio-
chemical symp oms o PCD o he scu ella cells.
Discussion
The success o ce eal g ain ge mina ion and ini ial s ages o
seedling g ow h depends on he p ecise o ganiza ion o e en s
aking place du ing his p ocess. Because he aleu one cells a e
able o pe cei e gibbe ellins and induce he syn hesis and sec e-
ion o hyd oly ic enzymes, hese cells play a cen al ole o
mobilize he s o age ma e ial o he s a chy endospe m and ha e
ecei ed mo e a en ion han any o he g ain issue. In e es ingly,
once he aleu one cells ha e pe o med hei impo an unc ion,
en e in a p ocess o PCD, which is also ac i a ed by gibbe ellins
(Be hke e al., 1999), hus allowing he use o he aleu one cel-
lula con en s o seedling g ow h. Al hough he scu ellum has
ecei ed less a en ion, i is clea ly a issue essen ial o ge mina-
ion. Indeed, gibbe ellins, he ho mones ac i a ing ge mina ion,
a e syn hesized in he scu ellum (Apple o d and Len on, 1997).
Mo eo e , scu ellum epi helium cells pa icipa e a e y ini ial
s eps o s a chy endospe m mobiliza ion by he sec e ion o
hyd oly ic enzymes oge he wi h he aleu one laye (Okamo o
e al., 1980; Cejudo e al., 1995; Domínguez and Cejudo, 1995).
Ne e heless, he majo unc ion o he scu ellum o ge mina ed
g ains is o se e as ans e ou e o pep ides (Wes e al.,
1998) and suga s (Aoki e al., 2006) o he g owing seedling.
The p esen s udy add essed whe he he scu ellum o whea
g ains unde goes PCD ollowing ge mina ion, so ha hei cel-
lula con en s a e also used by he g owing seedling, and how
scu ellum and aleu one cell dea h is coo dina ed in o he o e all
o ganiza ion o ge mina ion and pos ge mina ion. In addi ion,
his s udy cha ac e ized scu ellum cell dea h mo phologically
and biochemically.
Analysis o genomic DNA om scu ellum discs o g ains a
di e en days a e imbibi ion showed a p og essi e appea -
ance o DNA ladde ing (Fig. 1A), which is indica i e o PCD.
The occu ence o PCD was u he con i med wi h he TUNEL
assay, which e ealed ha bo h epi helial and pa enchymal cells
o he scu ellum unde go PCD, based on he in ense labelling o
nuclei o hese issues. Scu ellum PCD ook place wi h a cha -
ac e is ic spa io empo al pa e n, so ha i was ini ia ed a he
apical egion and p og essed owa ds he basal side o he scu el-
lum (Fig. 2C). A ema kable ea u e o scu ellum PCD is ha
i p oceeds when p oximal aleu one cells showed symp oms o
ha e comple ed he dea h p ocess. Based on hese esul s, i may
be concluded ha scu ellum PCD is coo dina ed wi h aleu one
PCD. Mo eo e , he p og essi e ad ance and he spa io empo-
al pa e n o scu ellum PCD may indica e ha he unc ion o
his issue, o ans e nu ien s om he s a chy endospe m o
suppo ini ial seedling g ow h, does no cease ab up ly once he
p ocess o PCD is ini ia ed.
The spa ial pa e n o scu ella PCD sugges s he exis ence
o a signal o con ol he p ocess. I is known ha scu ellum
cells su e oxida i e s ess in ge mina ing g ains (Se a o and
Cejudo, 2003; Bailly, 2004) and ha hese cells possess di e en
de oxi ying sys ems such as ca alase and supe oxide dismu ase
(Mylona e al., 2007) and 1-Cys pe oxi edoxin, a pe oxidase
speci ically and highly exp essed in seeds and localized in nuclei
o scu ellum and aleu one cells (S acy e al., 1999; Pulido e al.,
2009). Indeed, eac i e oxygen species p oduc ion has an ac i e
ole in aleu one cell dea h (Fa h e al., 2001; Beligni e al., 2002;
Wu e al., 2011). Since aleu one cells p oximal o he op egion
o he scu ellum ha e unde gone PCD, a possible explana ion o
he apical- o-basal pa e n o scu ellum PCD is ha i is due o a
signal o igina ed a he apical-p oximal side aleu one cells, bu
much wo k is s ill needed o es his possibili y.
Once es ablished he occu ence o PCD in he scu ellum o
ge mina ed whea g ains, he mo phological ea u es o his p o-
cess we e analysed. Scu ella cells unde going PCD show acu-
oliza ion in he cy oplasm and a p oac i e in amemb ane sys em
(Figs. 2B and 3) linking he in acellula sec e o y pa hway o
a p ocess o acuola cell dea h ( an Doo n e al., 2011). The
p esence o p ecu so p o ease esicles and au oly ic compa -
men s de i ed om he endoplasmic e iculum (Toyooka e al.,
2000; G eenwood e al., 2005) and Golgi cis e nae (Filono a
e al., 2000) a e conside ed as ea u es o plan cell dea h, esem-
bling mo phological ea u es o au ophagy in animal cells.
Al hough he ole o au ophagy in cell dea h is s ill subjec o
discussion (K oeme and Le ine, 2008), bo h mo phological and
biochemical e idence sugges s ha au ophagy has a p o-dea h
unc ion ei he in de elopmen al (Bozhko e al., 2005a) o
pa hogen-induced PCD in plan s (Liu e al., 2005; Ho ius e al.,
2009). A ea u e o scu ellum PCD is he appea ance o di e -
en deg ees o s uc u al al e a ions o mi ochond ia (Fig. 3F).
In o he euka yo ic cells, mi ochond ia memb anes ha e been
desc ibed as o igin o au ophagosomes (He nández e al., 2003;
Ning e al., 2006; Luo e al., 2009; Hailey e al., 2010). This ole
o mi ochond ia in au ophagy- ype PCD is di e en om he
ole ha hese o ganelles ha e in apop osis- ype PCD, in which
he dis up ion o he mi ochond ia p omo es he ansloca ion
o cy och ome c and o he apop ogenic ac o s o he cy oplasm
(Lam, 2004).
Conce ning he nucleus, i adop s a cha ac e is ic lobed mo -
phology and a highe he e och oma in con en (Fig. 4) as he
mos ele an ea u es. Howe e , emnan s o he e och oma in
could be de ec ed in he cen al au oly ic acuoles (Fig. 4C), as
obse ed in dying cells o soma ic emb yos o No way sp uce
(Filono a e al., 2000), which sugges s ha he nucleus is dis-
man led as cell dea h p og esses. The iden i ica ion o biochemi-
cal componen s pa icipa ing in nucleus disman ling was ano he
objec i e add essed in his s udy.
A nuclea -localized nuclease in scu ellum PCD | 5483
A he molecula le el, he knowledge o enzymes in ol ed
in he execu ion o PCD in plan s is much lowe han in animals.
Despi e he absence o genes encoding caspases in plan s, i
appea s ha caspase-like ac i i ies a e impo an (del Pozo and
Lam, 1998). O he di e en p o eases p oposed o pa icipa e in
plan PCD, only some o hem seem o be essen ial componen s
o nucleus disman ling. This is he case o me acaspase mcII-Pa,
which is ansloca ed o he nucleus in cells unde going PCD du -
ing emb yogenesis (Bozhko e al., 2005b) and pa icipa es in
clea age and ac i a ion o TSN, a phylogene ically conse ed
mul i unc ional egula o o gene exp ession in ol ed in PCD
(Sunds öm e al., 2009). The p esen s udy used he whea g ain
as a model sys em o iden i y nuclea -localized ac o s in ol ed in
he inal s eps o PCD execu ion, c i ically on DNA agmen a ion
and nucleus disman ling. Biochemical analysis allowed he iden-
i ica ion o wo Ca2+/Mg2+ endonucleases, which we e localized,
espec i ely, o he nuclei o aleu one cells (Domínguez e al.,
2004) and nucellus cells (Domínguez and Cejudo, 2006) unde -
going PCD. Al hough bo h endonucleases showed he same ca -
ion equi emen s, he di e en elec opho e ic mobili y sugges ed
ha each issue o whea g ains unde goes PCD wi h he pa icipa-
ion o di e en nucleases. The iden i ica ion o a Zn2+-dependen
endonuclease in he nucleus o whea scu ellum cells unde going
PCD, which p oduced in e nucleosomal agmen a ion o DNA
(Figs. 5–7), is in ag eemen wi h he p oposal o he pa icipa-
ion o di e en nucleases in di e en g ain issues. Among he
endonucleases iden i ied in cells su e ing PCD, only some ha e
been di ec ly in ol ed in nuclea disman ling. This is he case o
ZEN1, a Zn2+-dependen nuclease implica ed in he deg ada ion
o nuclea DNA in Zinnia achea y elemen s (I o and Fukuda,
2002). ZEN1 is localized o acuoles which collapse be o e DNA
is deg aded (Oba a e al., 2001). Howe e , ZEN1 ac i i y did
no p oduce he cha ac e is ic DNA ladde ing shown in animal
apop osis. In plan s, i was p oposed ha nucleus-localized nucle-
ases a e neu al whe eas acuola nucleases a e acidic (Sugiyama
e al., 2000). Thus, he iden i ica ion o an acidic Zn2+-dependen
endonuclease in he nucleus o whea scu ellum cells unde going
PCD may be conside ed an excep ion o his ule, o be added o
he p e iously epo ed acidic Zn2+-dependen nuclease esponsi-
ble o DNA ladde ing iden i ied in ice oo ip cells unde going
PCD in esponse o sal s ess (Jiang e al., 2008).
The appea ance o he nuclea -localized nucleoly ic ac i i y
was comple ely inhibi ed in ABA- ea ed g ains (Fig. 7), which
migh sugges an inhibi o y e ec o ABA on nuclease exp ession.
Howe e , i is well known ha he success o ge mina ion depends
o a spa io empo al sequence o e en s. Mos p obably, he s ong
inhibi o y e ec o ABA on ge mina ion (Supplemen a y Fig. S3)
occu s because i coun e ac s he ac i a ing e ec o gibbe ellins,
hus a es ing ge mina ion a ea ly s ages. As a consequence, he
es o e en s aking place he ea e , including scu ellum PCD,
will no ake place in ABA- ea ed g ains.
Supplemen a y ma e ial
Supplemen a y da a a e a ailable a JXB online.
Supplemen a y Fig. S1. Cy oplasm and nuclei ac iona ion
and isualiza ion o isola ed nuclei
Supplemen a y Fig. S2. Cha ac e iza ion o nucleoly ic
ac i i ies in he cy oplasm o scu ellum cells unde going PCD
Supplemen a y Fig. S3. E ec o ho mones on oo and shoo
eme gence and elonga ion
Acknowledgemen s
This wo k was unded by he Eu opean Regional De elopmen
Fund (ERDF) h ough he Minis e io de Ciencia e Inno ación
(g an no. BIO2010-15430) and Jun a de Andalucía (g an nos.
CVI-5919 and BIO-182).
Re e ences
Aoki N, Sco iel GN, Wang X-D, O le CE, Pa ick JW,
Fu bankRT. 2006. Pa hway o suga anspo in ge mina ing
whea seeds. Plan Physiology 141, 1255–1263.
Apple o d NEJ, Len on JR. 1997. Ho monal egula ion o α-amylase
gene exp ession in ge mina ing whea (T i icum aes i um) g ains.
Physiologia Plan a um 100, 534–542.
Bai S, Willa d B, Kin e M, Chapin LJ, F ancis D, S ead A,
Jones ML. 2010. P o eomic analysis o pos -pollina ion senescence.
Jou nal o Expe imen al Bo any 61, 1089–1109.
Bailly C. 2004. Ac i e oxygen species and an ioxidan s in seed
biology. Seed Science Resea ch 14, 93–107.
Bassham DC. 2007. Plan au ophagy – mo e han a s a a ion
esponse. Cu en Opinion in Plan Biology 10, 587–593.
Beligni MV, Fa h A, Be hke PC, Lama ina L, Jones RL. 2002.
Ni ic oxide ac s as an an i-oxidan and delays p og ammed cell dea h
in ba ley aleu one laye s. Plan Physiology 129, 1642–1650.
Be hke PC, Lonsdale JE, Fa h A, Jones RL. 1999. Ho monally
egula ed p og ammed cell dea h in ba ley aleu one cells. The Plan
Cell 11, 1033–1045.
Bozhko PV, Filono a LH, Suá ez MF. 2005a. P og ammed cell
dea h in plan emb yogenesis. Cu en Topics in De elopmen al
Biology 67, 135–179.
Bozhko PV, Suá ez MF, Filono a LH, Daniel G, Zamya nin
AA, Rod íguez-Nie o S, Zhi o o sky B, Sme enko A. 2005b.
Cys eine p o ease mcII-Pa execu es p og ammed cell dea h du ing
plan emb yogenesis. P oceedings o he Na ional Academy o
Sciences, USA 102, 14463–14468.
Cejudo FJ, Cubo MT, Baulcombe DC. 1995. Amy Iexp ession
du ing whea seed ge mina ion. Plan Science 106, 207–213.
Con ad B. 2009. P og ammed cell dea h du ing animal
de elopmen . Annual Re iew o Gene ics 43, 493–523.
Consonni G, Aspesi C, Ba ban e A, Dol ini S, Giuliani C, Giulini
A, Hansen S, B e chsneide R, Pilu R, Ga azzi G. 2003. Analysis
o ou maize mu an s a es ed in ea ly emb yogenesis e eals an
i egula pa e n o cell di ision. Sexual Plan Rep oduc ion 15,
281–290.
del Pozo O, Lam E. 1998. Caspases and p og ammed cell dea h in
he hype sensi i e esponse o plan s o pa hogens. Cu en Biology 8,
1129–1132.