Cu en Biology 23, 1094–1100, June 17, 2013 ª2013 Else ie L d All igh s ese ed h p://dx.doi.o g/10.1016/j.cub.2013.04.072
Repo
An hocyanins Double he Shel Li e
o Toma oes by Delaying O e ipening
and Reducing Suscep ibili y o G ay Mold
Yang Zhang,
1
Eugenio Bu elli,
1
Rosalba De S e ano,
2
Henk-jan Schoonbeek,
1
And eas Magusin,
1
Chia a Paglia ani,
3
Nikolaus Wellne ,
4
Lionel Hill,
1
Diego O zaez,
5
An onio G anell,
5
Jona han D.G. Jones,
6
and Ca hie Ma in
1,
*
1
John Innes Cen e, No wich Resea ch Pa k, No wich,
NR4 7UH, UK
2
Depa men o Soil, Plan , En i onmen al, and Animal
Sciences, Uni e si y o Naples Fede ico II, 80055 Po ici, I aly
3
Depa men o Ag icul u al, Fo es y, and Food Sciences,
Uni e si y o Tu in, ia Leona do da Vinci 44,
10095 G ugliasco TO, I aly
4
Ins i u e o Food Resea ch, No wich Resea ch Pa k, Colney,
No wich, NR4 7UA, UK
5
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, 46022 Valencia, Spain
6
The Sainsbu y Labo a o y, No wich Resea ch Pa k, Colney,
No wich, NR4 7UH, UK
Summa y
Shel li e is an impo an quali y ai o many ui , including
oma oes. We epo ha en ichmen o an hocyanin, a
na u al pigmen , in oma oes can signi ican ly ex end shel
li e. P ocesses la e in ipening a e supp essed by an ho-
cyanin accumula ion, and suscep ibili y o Bo y is cine ea,
one o he mos impo an pos ha es pa hogens, is
educed in pu ple oma o ui . We show ha educed sus-
cep ibili y o B. cine ea is dependen speci ically on he
accumula ion o an hocyanins, which al e he sp eading
o he ROS bu s du ing in ec ion. The inc eased an ioxidan
capaci y o pu ple ui likely slows he p ocesses o o e ip-
ening. Enhancing he le els o na u al an ioxidan s in oma o
p o ides a no el s a egy o ex ending shel li e by gene ic
enginee ing o con en ional b eeding.
Resul s and Discussion
Impo an challenges o he cul i a ion o oma oes include
pos ha es losses and educed quali y due o ui senes-
cence and pa hogen in ec ion. Many oma oes g own o esh
consump ion a e picked when s ill i m and g een, s o ed a
low empe a u e, and exposed o exogenous e hylene o
induce colo and ipeness be o e eaching he supe ma ke
shel . Al hough e ec i e in limi ing pos ha es losses, hese
p ocedu es nega i ely a ec oma o la o , a oma, and ex u e
[1]. The common use o mu an s a ec ed in ipening has
simila nega i e impac s on la o . O e he las wo decades,
gene ic enginee ing has been used o ex end oma o shel li e
by educing he ac i i y o cell-wall-deg ading enzymes [2–5]
and enhancing he le els o speci ic me aboli es [6, 7].
An hocyanins a e wa e -soluble pigmen s esponsible o
he ed, pu ple, and blue colo s o many lowe s and ui [8].
They a e p oduced by plan s o a ac pollina o s and seed
dispe se s [9]. An hocyanin p oduc ion is also commonly
induced unde s ess condi ions [10] and in ec ion by pa ho-
gens [11]. Besides physiological oles in plan s, die a y an ho-
cyanins a e associa ed wi h p o ec ion agains ce ain cance s
[12], ca dio ascula diseases [13], and o he ch onic human
diso de s [13].
We ha e shown ha ec opic exp ession o wo genes
encoding ansc ip ion ac o s, Delila (Del) and Rosea1
(Ros1), om snapd agons, unde he con ol o he ui -
speci ic E8 p omo e , esul s in inc eased exp ession o all
he genes commi ed o an hocyanin biosyn hesis o c ea e
in ensely pu ple oma o ui [14]. While g owing he pu ple
oma oes, we obse ed ha hey had imp o ed shel li e
compa ed o wild- ype, ed ui . The shel li e o ood is
de ined as he pe iod du ing which a s o ed p oduc emains
sui able o consump ion and is no mally de e mined by he
deg ee o so ening, sh i eling, and o ing o ui . Conse-
quen ly, bo h ui so ening la e du ing ipening and pa hogen
in ec ion in luence he shel li e o oma oes. Pu ple ui om
Del/Ros1 oma o plan s ha e no mal size, shape, and numbe
o seeds. Howe e , pu ple ui exhibi delayed ipening a e
b eake compa ed o ed ui . This is e iden om he appea -
ance o he pu ple ui bo h on he ine and du ing pos ha es
s o age and om a educed le el o ungal in ec ion unde
ei he condi ion (Figu es 1A and 1B).
Bo h wild- ype (WT) and pu ple oma oes we e ha es ed
when ipe and s o ed unde s e ile condi ions. Fo pu ple ui ,
49 days o s o age a 18C we e equi ed o obse e 50% o he
ui so ened, equi alen o he le el o so ening obse ed in
ed ui a 21 days. Comple e collapse was obse ed in pu ple
ui a e 10 weeks s o age, compa ed o 5 weeks o ed ui .
Wi h a ex u e analyze , he i mness o ed ui was measu ed
as 50% lowe han ha a b eake , a e 2 weeks a 18C,
whe eas he same educ ion in i mness was eached a e
5 weeks s o age o pu ple ui . These esul s indica ed ha
exp ession o Del and Ros1 can mo e han double he shel
li e o oma o ui (Figu e 1C). These di e ences we e accom-
panied by g ea e abili y o esis ensile o ces in pu ple
oma oes compa ed o ed ui o equi alen age (Figu e 1D).
P oduc ion o e hylene, equi ed o ull ipening in climac-
e ic ui such as oma o, inc eased jus a e b eake and
was 2- old g ea e in pu ple ui han in ed ui (Figu e S1A
a ailable online). Measu emen s o cu icle hickness e ealed
no di e ences be ween WT and pu ple oma o (Figu es
S1B–S1D). In addi ion, Fou ie ans o m in a ed (FT-IR) spec-
oscopy indica ed ha he e we e no signi ican cell wall com-
posi ional di e ences be ween pu ple oma o peel and ed
oma o peel 1 week a e b eake (Figu e S2E). These obse a-
ions implied ha he ex ended shel li e o pu ple ui was due
o nei he impai ed e hylene p oduc ion no al e ed cu icle/
peel composi ion.
The suscep ibili y o pu ple ui o pa hogens was in es i-
ga ed by in ec ion o in ac o wounded oma oes wi h
B. cine ea, he causal agen o g ay mold disease, one o he
mos impo an pos ha es pa hogens o oma oes [15].
When in ac ui we e sp ayed wi h a B. cine ea spo e suspen-
sion wi hou wounding, he p opo ion o pu ple ui showing
*Co espondence: ca hi[email p o ec ed]
se e e symp oms o in ec ion was subs an ially lowe han o
ed ui (Figu es S2A and S2B). When wounded ui we e
inocula ed wi h he B. cine ea spo e suspension, he size o
he lesions did no inc ease 1 day pos inocula ion (dpi) in ei he
ui ype, indica ing ha he ungus needs abou 24 h o
es ablish a e inocula ion. F om 2 dpi, howe e , he e was
g ea e sp ead o in ec ion in ed ui han in pu ple ui . A
3 dpi, he a e age size o he lesions in pu ple oma oes was
signi ican ly smalle han in ed ui , indica ing educed sus-
cep ibili y o B. cine ea in ec ion (Figu e 1E). Quan i a i e
PCR wi h DNA ex ac ed om in ec ed oma oes con i med
ha he e was signi ican ly mo e Bo y is g owing on ed ui
han on pu ple ui a 3 dpi (Figu e 1F). Reduced pa hogen
suscep ibili y was also obse ed in pu ple ui in og essed
in o he MoneyMake gene ic backg ound (Figu e S2C), indi-
ca ing ha he lowe suscep ibili y o pu ple oma oes o
B. cine ea is no dependen on a speci ic gene ic backg ound.
The suscep ibili y o oma o ui o nec o ophic pa hogens
inc eases du ing ipening [16, 17]. A co ela ion be ween ui
age la e in ipening and inc eased suscep ibili y was obse ed
in ed ui . Howe e , in pu ple ui , suscep ibili y o B. cine ea
did no inc ease om he b eake s age when an hocyanin
p oduc ion was induced (Figu e 1G). This obse a ion sug-
ges ed a speci ic ole o an hocyanins in limi ing he sp ead
o ungal in ec ion, as suppo ed by he in e media e suscep i-
bili y displayed by wo di e en Del/Ros1 lines (C and Y) ha
p oduce lowe le els o an hocyanins han line N (used o
he ini ial es s) [14](Figu es S2D–S2F).
To ensu e ha he e ec s on delayed ipening and pa hogen
suscep ibili y we e compa ed a exac ly he same de elop-
men al s age, we used i us-induced gene silencing (VIGS)
o silence he exp ession o Del and Ros1 in pu ple ui in
he MoneyMake backg ound (in which la ge ui size allows
dissec ion o issue sec o s ela i ely easily). Ag o-in il a ed
Del/Ros1 ui showed a pheno ype o pu ple and ed sec o s,
he la e de ining hose pa s o he ui whe e Del and Ros1
had been silenced [18] and hyd ophilic an ioxidan capaci y
was educed (Figu es 2A and 2B). In olde ui , he ed sec o s
we e clea ly so e and he issues we e mo e collapsed han in
pu ple sec o s, demons a ing he sho e s o age li e o ed
sec o s compa ed o pu ple sec o s (Figu e 2A). Red sec o s
also showed g ea e suscep ibili y o B. cine ea han pu ple
sec o s (Figu e 2A).
Gene exp ession p o iles o ed and pu ple sec o s o VIGS-
Del/Ros1 ui we e compa ed. Samples we e ha es ed a 8,
30, and 45 days a e b eake . A 3- old di e ence in exp ession
le els (pu ple e sus ed) was se as he h eshold o signi i-
can changes de ec ed using he TOM2 mic oa ay. Two hun-
d ed and o y one genes showed signi ican di e ences in
exp ession be ween pu ple and ed sec o s o e a leas wo
s ages (Figu e S3A). Func ional anno a ion e ealed ha
many o hese genes a e in ol ed in p ima y and seconda y
me abolism, cell wall modi ica ion, oxida i e s ess, and pa h-
ogen esis ance (Figu es S3B and S3C and Da a Se S1).
Reduced exp ession o many genes known o be in ol ed in
o e ipening was obse ed in pu ple sec o s (Figu es S3B
Figu e 1. Accumula ion o An hocyanins in Toma o F ui Delays La e Ripening and Dec eases Pa hogen Suscep ibili y
(A) Wild- ype, ed (i and ii) and ansgenic, pu ple (iii and i ) oma o ui s we e agged du ing he ini ial s ages o de elopmen and ha es ed and pho o-
g aphed a he end o he g een s age (i and iii). The same ui , s o ed a oom empe a u e, was epho og aphed a e 2 mon hs (ii and i ). The scale
ba ep esen s 2 cm.
(B) Se e e symp oms o oppo unis ic in ec ion no mally associa ed wi h o e ipe ed, wild- ype oma o ui on he ine (le ) we e no obse ed in pu ple,
Del/Ros1 oma o ui o he same age g own unde iden ical g eenhouse condi ions ( igh ). The scale ba ep esen s 2 cm.
(C) Pu ple ui showed slowe so ening as de e mined by isual inspec ion compa ed o wild- ype, ed ui . Pe cen ages o ui showing o e ipening
symp oms we e assessed e e y week du ing s o age es s. E o ba s show he SEM (n = 4). F ui s we e ha es ed a 14 days pos b eake (d0 = 14 dpb).
(D) Tex u e s eng h changes in Mic oTom and Del/Ros1 ui s du ing s o age es s. A e age alues we e calcula ed o a leas eigh indi idual ui s, and
e o ba s indica e he SEM.
(E) Symp oms o wounded ed and pu ple ui s a e inocula ion wi h B. cine ea B05.10. Whi e do s ep esen he lesion ma gins.
(F) Quan i a i e PCR e ealed mo e Bo y is g owing on he WT oma oes han on pu ple ui , 3 dpi. Bo y is g ow h was calcula ed by compa ison o he
a io o Bo y is DNA o oma o DNA. E o ba s show he SEM (n = 3). *p < 0.05 compa ed o con ol ed oma o.
(G) The ipening- ela ed inc ease in suscep ibili y o Bo y is did no occu in pu ple ui . Lesion diame e was measu ed 3 dpi. E o ba s show he SEM
(n R3). The scale ba ep esen s 2 cm. *p < 0.05 and **p < 0.01 o alues o pu ple oma oes compa ed o ed oma oes a he same s age.
See also Figu es S1 and S2.
High-An hocyanin Toma oes Ha e Longe Shel Li e
1095
and S3C), indica ing ha he supp ession o exp ession o
hese genes in pu ple oma oes con ibu es o he ex ended
shel li e o he ui .
The supp ession o genes in ol ed in o e ipening in pu ple
ui was con i med by quan i a i e RT-PCR. Genes encoding
polygalac u onase (SlPG2a)[4] and b-galac osidase (SlTBG4)
[5], in ol ed in cell wall so ening, showed subs an ially lowe
exp ession in pu ple ui du ing ipening (Figu es 2C and 2D).
The lowe le els o gene exp ession esul ed in lowe o al
ac i i ies o polygalac u onase and b-galac osidase in pu ple
oma oes compa ed o ed oma oes (Figu es 2E and 2F).
Al hough he silencing o he indi idual genes migh only
ha e mino e ec s on so ening [3, 5], he combined supp es-
sion o a numbe o di e en cell wall modi ica ion enzymes
likely educes signi ican ly he a e o ui so ening.
To iden i y speci ic e ec s o an hocyanins on ex ension o
shel li e, we silenced dihyd o la onol 4- educ ase (SlDFR), a
key gene in an hocyanin biosyn hesis, using VIGS in pu ple
oma oes. On he same ui , VIGS-SlDFR-silenced, o ange
sec o s showed simila exp ession le els o Del and Ros1 o
nonsilenced, pu ple sec o s, whe eas SlDFR exp ession was
subs an ially educed (Figu e S4A). An hocyanin le els we e
educed by 80%, al hough o he la onoids accumula ed in
he silenced sec o s, gi ing hem an o ange colo (Figu es
S4B and S4C). SlDFR-silenced sec o s we e sensi i e o
B. cine ea, whe eas pu ple sec o s on he same ui emained
esis an (Figu e 3A). Compa ed o nonsilenced sec o s,
SlDFR-silenced sec o s had educed hyd ophilic an ioxidan
Figu e 2. Delayed Ripening and Reduced Pa h-
ogen Suscep ibili y A e Associa ed wi h he Accu-
mula ion o An hocyanins, and Exp ession o
Ripening-Rela ed Genes Is Supp essed in Pu ple
Toma oes
(A) VIGS-Del/Ros1 oma o ui s showed educed
accumula ion o an hocyanins in silenced a eas
(i, pic u es aken 14 days a e b eake ). The ed
sec o s showed quicke so ening han pu ple
sec o s (ii, pic u es aken 42 days a e b eake ).
Pu ple sec o s showed educed suscep ibili y o
B. cine ea 3 dpi (iii). Red sec o s o VIGS-silenced
oma oes we e mo e suscep ible o B. cine ea 3
dpi (i ). All scale ba s ep esen 2 cm.
(B) VIGS-Del/Ros1 silenced sec o s had educed
hyd ophilic an ioxidan capaci y compa ed o
pu ple sec o s. E o ba s show he SEM (n = 3).
S, silenced sec o s; NS, nonsilenced sec o s.
*p < 0.05 and **p < 0.01 compa ed o he WT.
(C and D) Quan i a i e RT-PCR analysis o genes
encoding cell-wall-modi ying enzymes in WT and
Del/Ros1 ui s du ing ipening. Polygalac u onase
2a (SlPG2a) (C) and b-galac osidase 4 (SlTBG4) (D)
a e shown. E o ba s show he SEM (n = 3).
(E and F) To al polygalac u onase (E) and b-galac-
osidase (F) ac i i ies in ed and pu ple ui a
di e en s ages du ing ipening. E o ba s show
he SEM (n = 3). *p < 0.05 and **p < 0.01 compa ed
o he WT a he same s age.
See also Figu e S3,Table S1, and Da a Se S1.
capaci y (Figu e 3B), al hough his was
highe han he hyd ophilic an ioxidan
capaci y o WT ed ui , due o he accu-
mula ion o la onols. S o age es s indi-
ca ed ha VIGS-SlDFR-silenced ui
could be kep longe han WT ui
bu no as long as pu ple oma oes
(Figu e 3C). We con i med hese obse a ions by c ossing
Del/Ros1 plan s o he aw mu an o oma o in he Ailsa C aig
gene ic backg ound, which lacks DFR ac i i y and canno
make an hocyanins [19]. In he F2, he plan s ha con ained
Del/Ros1 bu lacked DFR ac i i y (aw
2/2
) p oduced o ange
ui due o high le els o la onols. Like he VIGS-SlDFR-
silenced sec o s, he aw
2/2
, Del/Ros1 ui we e no less sus-
cep ible o B. cine ea han we e ed oma oes (Figu e 3D).
The o ange ui had 2- old highe hyd ophilic an ioxidan ca-
paci y han he pa en al aw
2/2
line (Figu e 3E) and hey could
be kep longe pos ha es , al hough no as long as pu ple
oma oes (Figu e 3F). Consequen ly, he delay in o e ipening
and he enhanced pa hogen esis ance o pu ple oma oes a e
no due o o - a ge s o he Del and Ros1 ansc ip ion ac o s.
Resis ance o B. cine ea is speci ically he esul o he accu-
mula ion o an hocyanins, whe eas he delay in o e ipening
is mos likely associa ed wi h he inc eased hyd ophilic an iox-
idan capaci y o he ui .
Le els o oxida i e s ess inc ease ma kedly in he la e
s ages o ipening and may acili a e many o he me abolic
changes associa ed wi h ma u a ion o oma o ui [20]. Com-
pa ison o a cul i a wi h sho e shel li e o one wi h longe
shel li e showed educed sca enging abili y and inc eased
le els o eac i e oxygen species (ROS) [21]. Acco dingly,
inc ease o an ioxidan capaci y o educ ion o le els o
ROS wi h di e en an ioxidan s can ex end shel li e [6, 22,
23]. Taken oge he , ou da a sugges ha ele a ion o he
le els o an ioxidan s in ui educes he issue-damaging
Cu en Biology Vol 23 No 12
1096
ac i i y o oxida i e s ess and hus is he mos likely cause o
he delay in o e ipening obse ed in pu ple (Del/Ros1) and
o ange (VIGS-SlDFR and Del/Ros1,aw
2/2
) oma oes.
Malondialdehyde (MDA) is a byp oduc o lipid pe oxida ion
and can be used o measu e damage esul ing om oxida i e
s ess du ing issue senescence [21, 24]. MDA le els in ed
Mic oTom ui inc eased la e in ipening. In pu ple oma oes,
howe e , MDA le els did no inc ease signi ican ly up o
4 weeks a e b eake (Figu e 4A). Lowe oxida i e damage in
pu ple oma o was associa ed closely wi h inc eased o al an i-
oxidan capaci y du ing o e ipening, which esul ed p inci-
pally om he accumula ion o an hocyanins (Figu e 4B). Highe
hyd ophilic an ioxidan capaci y/lowe ROS le els we e associ-
a ed wi h supp ession o ipening- ela ed enzyme ac i i ies
such as polygalac u onase andb-galac osidase, an e ec likely
o be o impo ance in ex ending shel li e, since down egula ion
o some o he co esponding genes by an isense has been
shown o esul in ui ha a e i me o longe han con ols
[3, 5] and hei combined supp ession may ex end shel li e
ye u he .One explana ion o he induced exp ession o hese
genes, la e in ipening, is ha i is he esul o inc eased ROS
signaling. Ou da a sugges ha ROS signaling is an impo an
de e minan o he a e o ipening, la e in ui de elopmen .
High hyd ophilic an ioxidan capaci y can supp ess bo h ROS
Figu e 3. Inhibi ion o An hocyanin Biosyn hesis in Pu ple Toma oes Al e s Suscep ibili y o Bo y is and Pos ha es S o age
(A) VIGS-SlDFR silenced sec o s had inc eased suscep ibili y o B. cine ea compa ed o nonsilenced sec o s on he same ui . Pic u es we e aken a 3 dpi.
Whi e do s indica e lesion sizes. Scale ba s ep esen 2 cm.
(B) The hyd ophilic an ioxidan capaci y o VIGS-SlDFR-silenced sec o s was lowe han ha o nonsilenced sec o s, al hough s ill highe han ha o WT
ui due o he accumula ion o la onols. E o ba s show he SEM, n = 3. **p < 0.01 o di e ences in TEAC alues o hyd ophilic ex ac s o silenced and
nonsilenced issues.
(C) S o age es s indica e VIGS-SlDFR-silenced ui can be kep o longe han WT ui bu o less ime han nonsilenced pu ple ui . F ui s we e ha es ed
2 weeks a e b eake , and he imes o show o e ipening symp oms ( isual o ing and collapse o ui ) we e eco ded. E o ba s show he SEM, n = 7.
**p < 0.01 compa ed wi h WT, ed ui .
(D) High le els o la onols accumula e in aw
2/2
Del/Ros1 F2 oma o ui ob ained by c ossing Del/Ros1Mic oTom wi h aw
2/2
(DFR
–
) mu an s. The o ange,
la onols-en iched oma o (le ) was mo e suscep ible o B. cine ea. Pic u es we e aken a 3 dpi. Whi e do s show lesion bound ies. Scale ba s
ep esen 2 cm.
(E) Compa isons o an ioxidan capaci ies o aw
2/2
,aw
2/2
Del/Ros1and Del/Ros1 ui . E o ba s show he SEM, n = 3. Solid ba s show hyd ophilic an iox-
idan capaci y, and open ba s show lipophilic an ioxidan capaci y. **p < 0.01 compa ed wi h pa en al aw
2/2
ui .
(F) aw
2/2
Del/Ros1 ui had a longe shel li e han pa en al aw
2/2
ui bu a sho e shel li e han Del/Ros1 ui . Times (a e b eake ) o ui o show
o e ipening symp oms ( isual o ing and collapse o ui ) we e eco ded. E o ba s show he SEM, n = 10. **p < 0.01 compa ed wi h pa en al aw
2/2
ui .
See also Figu e S4.
High-An hocyanin Toma oes Ha e Longe Shel Li e
1097
ac i i y and signaling and consequen ly may delay he p o-
cesses o o e ipening, bo h di ec ly and indi ec ly.
Reduced suscep ibili y o B. cine ea is associa ed speci ically
wi h an hocyanin accumula ion. An hocyanin le els ha e been
associa ed wi h educed suscep ibili y o Bo y is in g apes
[25] and may educe pos ha es spoilage o ui s in gene al
by Bo y is.Whenweg ewB. cine ea on aga pla es
Figu e 4. Ex ended Shel Li e in Pu ple Toma oes
Is Associa ed wi h Thei High An ioxidan
Capaci y
(A) Malondialdehyde le els in pe ica p o ed and
pu ple Mic o om ui du ing ipening. E o ba s
show he SEM (n = 3). *p < 0.05 compa ed wi h
WT, ed ui a same s age
(B) T olox equi alen o al an ioxidan capaci y
(TEAC) o wa e ex ac s om ed and pu ple
oma oes du ing ipening. E o ba s show he
SEM (n = 3). *p < 0.05 and **p < 0.01 in compa ison
o WT, ed ui a he same s ages.
(C) Addi ion o juice om ei he ed o pu ple
oma oes o he g ow h medium had no e ec
on g ow h o B. cine ea. PDA medium (i), PDA
wi h 15 mg/li e T iademinol (an inhibi o o ungal
g ow h) (ii), PDA supplemen ed wi h 50% ed
juice (iii), and PDA supplemen ed wi h 50%
pu ple juice (i ) a e shown. Pic u es we e aken
3 days a e pla e inocula ion. Scale ba s ep e-
sen 2 cm.
(D) 3,30-diaminobenzidine (DAB) s aining o
hyd ogen pe oxide p oduced 24 h a e inocula-
ion o B. cine ea: ed (i) and pu ple (ii) ui s
s ained wi h DAB, 24 h a e inocula ion, wound
only ed (iii) and pu ple (i ) ui s ained 24 h a e
wounding. Scale ba s ep esen 1 mm.
(E) The le els o ROS in ed and pu ple oma oes
we e al e ed by in il a ion o a wa e con ol,
10 mM diphenyleneiodonium chlo ide (DPI, ROS
inhibi o ), o 50 uni s/ml glucose oxidase plus
1% glucose (G+GO, ROS induce ). F ui s we e
wounded and in il a ed 1 h p io o B. cine ea
inocula ion. Pic u es we e aken 3 dpi. Whi e
do ed lines ep esen lesion ma gin. All scale
ba s ep esen 2 cm.
(F) Model o he mechanism o shel li e ex en-
sion in pu ple, high-an hocyanin oma oes.
supplemen ed wi h ed and pu ple ui
juice, nei he ex ac inhibi ed he g ow h
o he ungus (Figu e 4C). This indica es
ha an hocyanins do no supp ess he
g ow h o B. cine ea di ec ly and ha
he esis ance equi es li ing hos cells.
Be ween 24 and 48 h a e in ec ion
wi h B.cine ea, lesions on ed ui sp ead
quickly, while on pu ple ui hei size e-
mained small (Figu e 1E). 3,30-diamino-
benzidine (DAB) s aining o H
2
O
2
in in-
ec ed ed and pu ple ui s du ing his
pe iod showed ha a ROS bu s was
gene a ed a he in ec ion si e. Howe e ,
he ROS bu s on ed ui sp ead widely,
whe eas on pu ple ui s ong ROS
induc ion was es ic ed o he inocula-
ion si e (Figu e 4D). The oxida i e bu s
is hough o po en ia e in ec ion by ne-
c o ophic pa hogens ha eed on dead
issue, acili a ing he expansion o
disease lesions [26–28]. Vacuum in il a ion o diphenyleneiodo-
nium chlo ide (DPI), an NADPH oxidase inhibi o , in o ed ui
p io o B. cine ea inocula ion es ic ed he sp ead o lesions,
whe eas in il a ion o pu ple oma oeswi h glucose and glucose
oxidase (which induce ROS, h ough he gene a ion o H
2
O
2
)
inc eased lesion g ow h in pu ple ui (Figu e 4E). These da a
sugges ha in pu ple oma oes, an hocyanins al e he
Cu en Biology Vol 23 No 12
1098

dynamics o he ROS bu s gene a ed by B.cine eain ec ion and
limi he induc ion o cell dea h necessa y o g ow h o he
nec o oph.
In addi ion o hei high nu i ional alue [14], an hocyanin-
ich pu ple oma oes ha e 2- old longe shel li e, he
combined esul o inc eased esis ance o oppo unis ic pa h-
ogens and slowe ipening a la e s ages. These ai s a e asso-
cia ed wi h he accumula ion o an hocyanins in oma oes.
An hocyanins speci ically al e he sp ead o he ROS bu s
gene a ed as pa o nec o ophic in ec ion and so educe sus-
cep ibili y o B. cine ea. Accumula ion o an hocyanins esul s
in high hyd ophilic an ioxidan capaci y, which educes he
inc ease in ROS le els, ha occu s la e in ui de elopmen ,
and he educ ion in ROS may supp ess he la e s ages o
ipening (Figu e 4F). The associa ion o slowe ipening wi h
ele a ed hyd ophilic an ioxidan capaci y o ui o e s new,
ye b oad, a ge s o b eede s o ex end he pos ha es shel
li e o ui . Addi ionally, an hocyanins could be used o educe
he suscep ibili y o ipe ui speci ically o Bo y is cine ea,
he mos impo an ungal pa hogen o so ui .
Accession Numbe s
The GEO accession numbe o he TOM2 mic oa ay da a epo ed in his
pape is GSE46341.
Supplemen al In o ma ion
Supplemen al In o ma ion includes Supplemen al Expe imen al P oce-
du es, ou igu es, one able, and one da a se and can be ound wi h his
a icle online a h p://dx.doi.o g/10.1016/j.cub.2013.04.072.
Licensing In o ma ion
This is an open-access a icle dis ibu ed unde he e ms o he C ea i e
Commons A ibu ion-NonComme cial-No De i a i e Wo ks License, which
pe mi s non-comme cial use, dis ibu ion, and ep oduc ion in any medium,
p o ided he o iginal au ho and sou ce a e c edi ed.
Acknowledgmen s
E.B. and C.M. we e suppo ed by he Eu opean Union FP6 FLORA p ojec
(FOOD-CT-01730), and Y.Z., E.B., and C.M. a e suppo ed by he Eu opean
Union FP7 ATHENA collabo a i e p ojec (g an ag eemen 245121). Y.Z. is
also suppo ed by a Ro a ion S uden ship om he John Innes Founda ion.
C.M. and E.B. we e suppo ed by he co e s a egic g an o he Biological
and Bio echnological Science Resea ch Council (BBSRC) o he John Innes
Cen e and a e cu en ly suppo ed by he Ins i u e S a egic P og am
Unde s anding and Exploi ing Plan and Mic obial Seconda y Me abolism
(BB/J004596/1) om he BBSRC. E.B. was suppo ed by a sho - e m
EMBO ellowship o unde aking pa o he esea ch epo ed in his pape .
H.-J.S. was suppo ed by g an BB/G042960/1 om he BBSRC and he
John Innes Founda ion, and A.G. and D.O. a e suppo ed by he Fundacio
´n
Genoma (Cali om p ojec ) and he Spanish Minis y o Science and Educa-
ion (p ojec BIO2010-15384). This wo k bene i ed om he ne wo king
ac i i ies wi hin he Eu opean- unded COST ACTION FA1106 Quali yF ui .
We hank And ew Da is o pho og aphy. C.M. and J.D.G.J. a e Di ec o s
o No olk Plan Sciences, a company b inging bio echnological imp o e-
men s in c ops o ma ke .
Recei ed: Decembe 28, 2012
Re ised: Ma ch 27, 2013
Accep ed: Ap il 25, 2013
Published: May 23, 2013
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