Ou e e ence: JPLPH 51807 P-au ho que y- 9
AUTHOR QUERY FORM
Jou nal: JPLPH Please e-mail o ax you esponses and any co ec ions o:
E-mail: co ec ions.esch@else ie . homsondigi al.com
A icle Numbe : 51807 Fax: +353 6170 9272
Dea Au ho ,
Please check you p oo ca e ully and ma k all co ec ions a he app op ia e place in he p oo (e.g., by using on-sc een
anno a ion in he PDF ile) o compile hem in a sepa a e lis . No e: i you op o anno a e he ile wi h so wa e o he han
Adobe Reade hen please also highligh he app op ia e place in he PDF ile. To ensu e as publica ion o you pape please
e u n you co ec ions wi hin 48 hou s.
Fo co ec ion o e ision o any a wo k, please consul h p://www.else ie .com/a wo kins uc ions.
Any que ies o ema ks ha ha e a isen du ing he p ocessing o you manusc ip a e lis ed below and highligh ed by lags in
he p oo . Click on he ‘Q’ link o go o he loca ion in he p oo .
Loca ion in Que y / Rema k: click on he Q link o go
a icle Please inse you eply o co ec ion a he co esponding line in he p oo
The e e ence gi en he e is ci ed in he ex bu is missing om he e e ence lis – please make he lis
comple e o emo e he e e ence om he ex : ‘Johnson e al. (2002)’.
Q1 Please con i m ha gi en names and su names ha e been iden i ied co ec ly.
Q2 Re e ence‘Johnsone al.(2002)’isci edin he ex bu no p o ided in he e e ence lis . Please p o ide
i in he e e ence lis o dele e he ci a ion om he ex .
Please check his box o indica e you app o al i
you ha e no co ec ions o make o he PDF ile
Thank you o you assis ance.
Please
ci e
his
a icle
in
p ess
as:
Deme e
Z,
e
al.
Osmo ic
s ess
esponses
o
indi idual
whi e
oak
(Que cus
sec ion,
Que cus
subgenus)
geno ypes
cul u ed
in
i o.
J
Plan
Physiol
(2013),
h p://dx.doi.o g/10.1016/j.jplph.2013.09.013
ARTICLE IN PRESS
G
Model
JPLPH
51807
1–9
Jou nal
o
Plan
Physiology
xxx (2013) xxx–
xxx
Con en s
lis s
a ailable
a
ScienceDi ec
Jou nal
o
Plan
Physiology
j
o
u
nal
homepage:
www.else ie .com/loca e/jplph
Physiology1
Osmo ic
s ess
esponses
o
indi idual
whi e
oak
(Que cus
sec ion,
Que cus
subgenus)
geno ypes
cul u ed
in
i o
2
3
Zi a Deme e a,
Pé e
Kanalasa,
Csaba
Má héa,∗,
Klá a
Csekeb,
E zsébe
Sz˝
oll˝
osia,
Q1
Má a
M-Ham asa,
Ka alin
Jámb ika,
Zol án
Kissa,
Ilona
Mészá osa,∗
4
5
aUni e si y
o
Deb ecen,
Facul y
o
Science
and
Technology,
Depa men
o
Bo any,
PO
Box
14,
H-4010
Deb ecen,
Hunga y6
bHunga ian
Fo es
Resea ch
Ins i u e,
PO
Box
30/A,
H-9600
Sá á ,
Hunga y7
8
a
i
c
l
e
i
n
o
9
10
A icle
his o y:11
Recei ed
25
June
201312
Recei ed
in
e ised
o m
11
Sep embe
2013
13
14
Accep ed
22
Sep embe
201315
A ailable online xxx
16
Keywo ds:17
Que cus
issue
cul u e18
Osmo ic
s ess19
Reco e y20
Guaiacol pe oxidase
21
Single-s and
p e e ing
nuclease22
a
b
s
a
c
Whi e
oaks
(Que cus
sec ion,
Que cus
subgenus)
a e
widely
dis ibu ed
in
Eu ope.
Que cus
pe aea
(ses-
sile
oak),
an
economically
impo an
species
is
p edic ed
o
be
a ec ed
by
clima e
change.
Q.
pubescens
(pubescen
oak)
and
Q.
i giliana
(I alian
pubescen
oak)
a e
economically
less
impo an ,
d ough
ol-
e an
species.
F equen
hyb idiza ion
o
whi e
oaks
was
obse ed
and
cu en ly
he
in og ession
o
Q.
pubescens
and
Q.
i giliana
in
non-medi e anean
egions
o
Eu ope
has
been
epo ed.
Ou
goal
was
o
use
issue
cul u es
es ablished
om
indi idual
ees
o
he
abo e
axa
and
hei
pu a i e
hyb ids,
all
p esen
in
he
o es
s and
o
Sík ˝
okú
LTER
Resea ch
A ea
(NE
Hunga y)
as
simple
expe imen al
model
sys ems
o
s udying
d ough /osmo ic
s ess
ole ance.
Tissue
cul u es
a e
mo e
sui able
models
o
such
s udies,
han
seedlings,
because
hey
a e
gene ically
iden ical
o
he
pa en
plan s.
Polye hylene
glycol
(PEG6000)
ea men s
we e
used
o
his
pu pose.
The
iden ifica ion
o
axa
was
based
on
lea
mo phological
ai s
and
mic osa elli e
analysis
and
showed
ha
Q.
pe aea
is
gene ically
dis inc
o
all
o he
axa
examined.
We
es ablished
six
callus
lines
o
Que cus.
As
expec ed,
in
Q.
pe aea
cul u es
PEG6000
induced
se e e
loss
o
esh
weigh
and
he
abili y
o
eco e
a e
emo al
o
he
osmo icum,
which
was
no
cha ac e is ic
o
Q.
pubescens
and
Q.
i giliana.
Pu a i e
hyb ids
exhibi ed
an
in e media e
esponse
o
osmo ic
s ess.
Ac i i y
gels
showed
he
inc ease
o
single-s and
p e e ing
(SSP)
nuclease
and
no
significan
change
o
guaiacol-pe oxidase
ac i i ies
in
d ough -sensi i e
geno ypes/cul u es
and
no
significan
inc ease
o
SSP
nuclease
ac i i ies
accompanied
wi h
inc eases
o
guaiacol-pe oxidase
ac i i ies
in
d ough - ole an
ones.
This
indica es
ha
d ough /osmo ic
s ess
ole ance
is
associa ed
o
inc eased
capaci y
o
sca eng-
ing
eac i e
oxygen
species
and
hence
less
suscep ibili y
o
DNA
damage.
Ou
esul s
confi m
ha
issue
cul u es
o
oak
a e
sui able
model
sys ems
o
s udying
d ough /osmo ic
s ess
esponses.
© 2013 Published by Else ie GmbH.
In oduc ion
23
Oaks
(Que cus
spp.)
a e
widesp ead
in
Eu ope
and
play
an24
impo an
ecological
and
sil icul u al
ole.
High
gene ic
and
mo -25
phological
a iabili y
has
been
epo ed
o
he
Que cus
sec ion
26
(=Lepidobalanus;
o
whi e
oaks
sensu
Nixon,
1993)
o
he
Que -27
cus
subgenus
(He zog,
1996;
Gailing
e
al.,
2007).
The
h ee
main28
whi e
oak
species
a e
Que cus
obu
L.,
Q.
pe aea
(Ma .)
Liebl
and29
Q.
pubescens
Willd.
Se e al
o he
oak
axa
ha e
also
been
dis-
30
inguished
om
he
Eu opean
b oadlea ed
o es s,
o
which
Q.31
dalechampii
Ten.,
Que cus
polyca pa
Schu ,
and
Q.
i giliana
Ten.32
a e
impo an .
The
wo
o me
ones
esemble
o
Q.
pe aea
sensu33
s ic o
and
a e
usually
included
in
he
agg ega e
o
Q.
pe aea
sensu34
∗Co esponding
au ho .
Tel.:
+36
52512900;
ax:
+36
52512943.
E-mail
add esses:
[email p o ec ed]
(C.
Má hé),
[email p o ec ed] (I.
Mészá os).
la o,
while
Q.
i giliana
belongs
o
he
agg ega e
o
Q.
pubescens 35
sensu
la o
(Schwa z,
1936a,b;
Bo dács
e
al.,
2002).
In e specific 36
hyb idiza ion
is
e y
common
be ween
whi e
oak
species
(Cu u 37
e
al.,
2007;
Lepais
e
al.,
2009;
Sal ini
e
al.,
2009;
Lepais
and 38
Ge be ,
2011)
which
inc eases
he
gene ic
di e si y
in
na u al
pop- 39
ula ions
(Bo o ics
e
al.,
1998;
Gömö y
and
Schmid o á,
2007; 40
Kanalas
e
al.,
2008). 41
Eu opean
whi e
oaks
di e
in
he
p e e ence
o
ecological
con- 42
di ions
and
g ow
in
a ious
habi a s.
The
dis ibu ion
o
oaks
is 43
mos ly
dependen
on
hei
capaci y
o
esis
d ough
o
excess
o 44
wa e
in
he
soil
o
e en
he
wo
phenomena
successi ely
(Jones, 45
1959;
Johnson
e
al.,
2002).
Among
he
h ee
main
whi e
oak
species
Q246
Q.
pubescens
is
he
mos
d ough
ole an
one
and
occupies
wa m 47
and
xe ic
si es
in
Eu ope
(Bo o ics
e
al.,
1998;
Yu uko
and
Zhele , 48
2001;
Thomas
e
al.,
2002;
Gallé
e
al.,
2007;
Siam
e
al.,
2009).
Q. 49
pe aea
g ows
p edominan ly
on
mesic
o
ela i ely
d y
si es
on 50
lowe
al i ude
slopes
and
idges,
whe eas
Q.
obu
can
popula e 51
lowland
si es
wi h
we
and
empo a ily
wa e logged
soils
(Jones, 52
0176-1617/$
–
see
on
ma e ©
2013 Published by Else ie GmbH.
h p://dx.doi.o g/10.1016/j.jplph.2013.09.013
Please
ci e
his
a icle
in
p ess
as:
Deme e
Z,
e
al.
Osmo ic
s ess
esponses
o
indi idual
whi e
oak
(Que cus
sec ion,
Que cus
subgenus)
geno ypes
cul u ed
in
i o.
J
Plan
Physiol
(2013),
h p://dx.doi.o g/10.1016/j.jplph.2013.09.013
ARTICLE IN PRESS
G
Model
JPLPH
51807
1–9
2Z.
Deme e
e
al.
/
Jou nal
o
Plan
Physiology
xxx (2013) xxx–
xxx
Table
1
Iden i y
o
Que cus
axa
used
in
his
s udy,
based
on
16
lea
quan i a i e
ai s
measu ed
acco ding
o
Bo o ics
(2000)
and
Kanalas
e
al.
(2008,
2009).
Explan
code
numbe Iden i y
A149
Q.
pe aea
D137 Q.
pe aea
×
Q.
dalechampii
D89
Q.
pe aea
×
Q.
pubescens
A211
Q.
i giliana
×
Q.
polyca pa
B50
Q.
i giliana
A75
Q.
pubescens
1959;
Aas,
1998).
Q.
dalechampii
Ten.
(Theodo opoulos
e
al.,
1995)53
and
Que cus
polyca pa
Schu
(Ma ula,
2009)
a e
dis inc
om
Q.54
pe aea
sensu
s ic o
in
ecological
equi emen s.
Bo h
a e
mo e55
d ough
ole an
and
g ow
in
wa me
si es.
Q.
dalechampii
Ten.56
and
Q.
i giliana
ha e
been
desc ibed
om
a id
si es
o
sou he n57
Eu ope
(Ofle ea
e
al.,
2011).
Those
oak
species
o
hei
hyb ids58
ha
a e
mo e
capable
o
s and
d y
and
ho
summe
pe iods
could59
be
impo an
ools
o
he
u u e
o es y
as
global
wa ming
and60
equen
d ough
e en s
a e
p edic ed
o
inc ease
he
p essu es
o61
Eu opean
oak
o es s
(IPCC,
2007).
62
In
Hunga y
mixed
o es s
o
sessile
oak
and
Tu key
oak
(Que ce-63
um
pe aeae-ce is)
co e
he
la ges
pa
o
o es ed
a ea.
The64
Sík ˝
okú
Long- e m
Ecological
Resea ch
Si e
(LTER)
(Bükk
Moun-65
ains,
no h-eas e n
Hunga y)
ep esen s
his
o es
ype
(Jakucs,66
1985)
and
has
in e na ional
epu a ion
as
a
o me
IBP
and
MAB67
a ea
and
cu en
LTER
Eu ope
ne wo k
membe .
The
a ea
is
si ua ed68
in
he
ansi ion
be ween
o es
and
o es -s eppe
zone
(47◦55N,69
20◦26E,
320–340
m
a.s.l.)
and
is
ulne able
o
he
clima e
change70
(Mészá os
e
al.,
2007).
In ense
o es
moni o ing
esea ch
has
been71
unning
o
40
yea s
in
he
si e.
The
o es
s and
is
cu en ly
cha ac-
72
e ized
by
he
dominance
o
axa
om
Que cus
sec ion
and
Q.
ce is73
(Mészá os
e
al.,
2007;
Kanalas
e
al.,
2008,
2009).
Based
on
lea 74
mo phological
ai s
he
assignmen
o
ees
o
whi e
oak
species75
showed
ha
his
o es
s and
is
mos ly
composed
o
Q.
pe aea76
senso
s ic o
(70.2%).
1%
o
sampled
ees
was
assigned
o
Q.
poly-77
ca pa,
2.5%
was
assigned
o
Q.
i giliana
and
4%
was
assigned
o78
Q.
pubescens.
22,
3%
o
ees
we e
conside ed
as
pu a i e
hyb ids:79
Q.
pe aea
×
Q.
dalechampii
(5.1%),
Q.
pe aea
×
polyca pa
(2.5%),
Q.80
pe aea
×
Q.
pubescens
(5.6%),
Q.
pe aea
×
Q.
i giliana
(7.1%)
and81
Q.
i giliana
×
Q.
pubescens
(2%)
(Kanalas
e
al.,
2008,
2009).
The82
pe cen age
o
hyb ids
is
ela i ely
high
as
compa ed
o
oak
commu-83
ni ies
om
o he
Eu opean
egions
(see
Cu u
e
al.,
2007;
Guge li84
e
al.,
2007
o
examples).
The
oak
decline
obse ed
in
he
1980s85
in
Eu ope
app oached
his
si e
oo,
he
die-back
o
ees
occu ed86
p ima ily
in
popula ion
o
Que cus
pe aea
sensu
la o.
87
Plan
issue
cul u e
echniques
including
callus
and
cell
sus-88
pension
cul u es
o e
many
ad an ages
–
e.g.
hey
p o ide
ully89
con ollable
sys ems
–
o
physiological/biochemical
s udies.
Con-90
sequen ly
hey
we e
applied
o
oak
species
as
well,
e.g.
o
s udying91
he
ole
o
ABA
in
he
ma u a ion
o
Q.
ilex
emb yos
(Mau i
and92
Manzane a,
2004)
o
o
dehyd in
p o eins
in
Q.
obu
soma ic93
emb yos
(ˇ
Sunde líko á
e
al.,
2009).
E en
hough
in
i o
cul u es94
ep esen
di e en
de elopmen al
s ages
and
gene
exp ession
pa -95
e ns
han
ma u e
plan s,
cell
and
callus
cul u es
o
sessile
oak
and96
peduncula e
oak
p o ed
o
be
excellen
models
and
a e
cu en ly97
used
o
he
s udy
o
osmo ic
s ess- ela ed
ansc ip ional
changes
98
and
o he
physiological
esponses.
They
a e
hough
o
be
sui able99
o
physiological
s udies
a
issue,
cell
and
molecula
le el
(Gleeson100
e
al.,
2004;
Po h
e
al.,
2005; ˇ
Sunde líko á
e
al.,
2009).101
In
ou
s udy
we
ha e
selec ed
ees
o
se e al
pu a i e
102
d ough -sensi i e and
- ole an
axa
belonging
o
Que cus
sec ion103
co-occu ing
in
he
o es
s and
o
Sík ˝
okú
P ojec
LTER
si e
(Jakucs,104
1985)
o
es ablishing
in
i o
cul u es
(Table
1).
Wha
could
be
he105
impo ance
o
issue
cul u es
in
his
espec ?
Ins ead
o
s udying
106
he
e ec
o
en i onmen al
s ess
in
ma u e
ees
we
can
examine107
physiological
esponses
in
in
i o
cul u es
unde
con olled
condi- 108
ions.
In
i o
cul u es
es ablished
om
ege a i e
issues
a e
mo e 109
likely
o
eflec
he
gene ic
backg ound
o
o iginal
plan s/explan s, 110
han
e.g.
seedlings
wi h
unce ain
gene ic
o igin.
Ou
p incipal
aim 111
was
o
s udy
d ough /osmo ic
s ess
ole ance
o
di e en
oak 112
geno ypes
in
a
model
–
in
i o
cul u e
sys em.
Fo
his
pu pose, 113
we
needed
o
es ablish
a
p ocedu e
o
c ea ing
s able
issue
cul- 114
u es
since
he e
we e
no
li e a u e
da a
o
cul u e
eady- o-use 115
o
hose
oak
geno ypes.
The
use
o
axenic
cul u es
allowed
us
o 116
a oid
po en ial
supe imposing
e ec s
o
mul iple
en i onmen al 117
condi ions. 118
Pe oxidases
(E.C.
1.11.1.7)
play
a
ole
in
he
sca enging
o
eac- 119
i e
oxygen
species
(ROS)
known
o
ele a ed
le els
du ing
d ough . 120
The
p esence
o
sca enging
sys ems
is
a
good
indica o
o
d ough 121
ole ance
(Reddy
e
al.,
2004).
In
he
absence
o
p o ec ion
agains 122
oxida i e
s ess,
DNA
and
RNA
damage
occu s.
This
may
be
accom- 123
panied
by
inc eases
in
he
ac i i y
o
nucleases,
among
hem,
single 124
s and
p e e ing
(SSP)
nucleases
(EC
3.1.30.1)
(Reddy
e
al.,
2004; 125
Roldán-A jona
and
A iza,
2009).
The e o e,
we
s udied
PEG6000 126
induced
changes
in
s ess
enzyme
– pe oxidase,
nuclease
–
ac i - 127
i ies
ha
accompanied
al e a ions
o
esh
weigh
and
eco e y
o 128
oak
issue
cul u es.
PEG6000
is
widely
used
o
modeling
osmo ic 129
s ess
in
highe
plan s,
due
o
minimal
side-e ec s
and
he
incapa- 130
bili y
o
plan
cells
o
me abolize
i
(see
Hohl
and
Schop e ,
1991; 131
Guó h
e
al.,
2010
o
examples).
Ou
basic
hypo hesis
was
ha 132
osmo ic
s ess
esponses
will
be
di e en
o
in
i o
cul u es
es ab- 133
lished
om
explan s
de i ed
om
indi iduals
o
d ough
sensi i e 134
(Q.
pe aea
sensu
s ic o)
om
hose
o
d ough
ole an
species
(Q. 135
pubescens,
Q.
i giliana).
Since
Q.
dalechampii
is
closely
ela ed
o
Q. 136
pe aea
(Bo o ics
e
al.,
1998),
hus
he
sensi i i y
o
d ough
o
is- 137
sue
cul u es
o
indi iduals
ep esen ing
hyb ids
be ween
hem
(Q. 138
pe aea
×
Q.
dalechampii)
was
expec ed
o
be
close
o
d ough
sen- 139
si i e
Q.
pe aea.
In
con as ,
cul u es
o
Q.
i giliana
×
Q.
polyca pa 140
hyb ids
we e
expec ed
o
be
mo e
ole an
o
osmo ic
s ess
han
Q. 141
polyca pa,
due
o
he
highe
d ough
ole ance
o
Q.
i giliana
pa - 142
en .
Cul u es
o
Q.
pe aea
×
Q.
pubescens
hyb id
we e
p esumably 143
in e media y
be ween
he
wo
pa en s
wi h
espec
o
d ough
ol- 144
e ance.
I
in
i o
expe imen s
confi m
he
expec ed
esponses
o 145
indi iduals
o
non-hyb id
axa
(i.e.
d ough
sensi i i y
o
Q.
pe aea 146
and
ole ance
o
Q.
pubescens
and
Q.
i giliana),
hey
can
be
used
o 147
es ing
o
cul u es
de i ed
om
Que cus
geno ypes
(e.g.
pu a i e 148
hyb ids)
wi h
unknown
d ough
sensi i i y. 149
The
main
goal
o
his
s udy
was
o
o e
a
model
sys em
based 150
on
in
i o
cul u es
o
selec ed
indi idual
whi e
oak
ees
o
he 151
es ima ion
o
hei
geno ype-dependen
d ough
ole ance. 152
Ma e ials
and
me hods 153
Plan
ma e ial
and
issue
cul u e 154
Plan
ma e ial
was
collec ed
in
he
oak
o es
o
Sík ˝
okú 155
Resea ch
A ea,
No h-Eas e n
Hunga y
(47◦55N,
20◦26E, 156
320–340
m
a.s.l.)
in
ea ly
sp ing
be o e
lea
flush.
Young
(2–4 157
yea s
old)
shoo s
we e
cu
om
he
lowe
canopy
o
105–110 158
yea s
old
ma u e
ees
o
Que cus
pe aea
(Ma uschka)
Lieblein, 159
Q.
pubescens
Willd.,
Q.
i giliana
Ten.
and
o
pu a i e
hyb ids 160
and
ans e ed
o
labo a o y.
Each
explan
was
collec ed
om 161
a
selec ed
single
ee
pe
geno ype
lis ed
in
Table
1
and
used 162
o
es ablishmen
o
issue
cul u e
and
subsequen
physiological 163
expe imen s.
All
explan s
o igina ed
om
he
same
ecological 164
condi ions.
A e
collec ion
shoo s
we e
kep
unde
condi ions 165
o
22
±
2◦C,
20
␮mol
m−2s−1PFD
and
12/12
h
pho ope iod
un il 166
he
de elopmen
o
young
lea es
(12
±
4
mm
leng h).
These 167
young
lea es
we e
used
as
explan s
o
he
induc ion
o
issue 168
cul u es.
The
induc ion
and
main enance
o
callus
p oduc ion 169
Please
ci e
his
a icle
in
p ess
as:
Deme e
Z,
e
al.
Osmo ic
s ess
esponses
o
indi idual
whi e
oak
(Que cus
sec ion,
Que cus
subgenus)
geno ypes
cul u ed
in
i o.
J
Plan
Physiol
(2013),
h p://dx.doi.o g/10.1016/j.jplph.2013.09.013
ARTICLE IN PRESS
G
Model
JPLPH
51807
1–9
Z.
Deme e
e
al.
/
Jou nal
o
Plan
Physiology
xxx (2013) xxx–
xxx 3
Table
2
Key
pa ame e s
o
he
SSR
ma ke s
used
o
mic osa elli e
analysis
o
whi e
oak
ees
used
in
his
s udy.
SSR
p ime
pai
Repea
mo i
P ime
sequence
No.
o
alleles/
no.
o
ees
Obse ed
he e ozygosi y
Re e ence
o
he
me hod
o
mic osa elli e
analysis
ZAG1/5
F (GT)5(GA)9 GCTTGAGAGTTGAGATTTGT
7/6
0.500
S einkellne
e
al.
(1997)
ZAG1/5
R
GCAACACCCTTTAACTACCA
ZAG
9
F
(AG)12
GCAATTACAGGCTAGGCTGG
7/6
0.833
S einkellne
e
al.
(1997)
ZAG
9
R
GTCTGGACCTAGCCCTCATG
ZAG
110
F
(AG)15
GGAGGCTTCCTTCAACCTACT
9/6
1.000
S einkellne
e
al.
(1997)
ZAG
110
R
GATCTCTTGTGTGCTGTATTT
ZAG
11
F (TC)22 CCTTGAACTCGAAGGTGTCCTT 4/6 0.833 Kamp e e
al.
(1998)
ZAG 11 R GTAGGTC
A
AAACCATTGGTTGACT
ZAG
96
F (TC)20
CCCAGTCACATCCACTACTGTCC
7/6
1.000
Kamp e
e
al.
(1998)
ZAG
96
R
GGTTGGGAAAAGGAGATCAGA
ZAG
112
F
(GA)32
TTCTTGCTTTGGTGCGCG
2/6
0.167
Kamp e
e
al.
(1998)
ZAG
112
R
GTGGTCAGAG
ACTCGGTAAGTATTC
was
achie ed
on
WPM
medium
(Woody
Plan
Medium,
Lloyd
170
and
McCown,
1980)
solidified
wi h
0.8%
(w/ )
aga
(Di co,171
Law ence,
KS,
USA).
The
plan
g ow h
egula o s
(PGRs)
used
we e
172
0.05–4
mg
L−1(0.53–21.5
␮M)
␣-naph haleneace ic
acid
(NAA)173
and
0.05–1
mg
L−1(0.25–0.5
␮M)
indole-3-bu y ic
acid
(IBA)
as174
auxins
and
0.1–4
mg
L−1(0.44–17.7
␮M)
N6-benzyladenine
(BA)
as175
a
cy okinin.
All
PGRs
we e
om
Sigma–Ald ich,
Budapes ,
Hunga y.176
The
design
o
PGR
con en
o
issue
cul u e
media
was
based
on177
he
me hods
o
Seckinge
e
al.
(1979),
Tanaka
e
al.
(1995),
Cuenca178
e
al.
(1999)
and
To ibio
e
al.
(2004).
G ow h
condi ions
o
in
i o
179
cul u e
we e:
14/10
h
pho ope iod
wi h
a
pho on
fluence
a e180
o
10
␮mol
m−2s−1du ing
he
ligh
pe iod
and
empe a u es
o 181
22
±
2◦C/18
±
2◦C.182
De e mina ion
o
axonomical
s a us
o
whi e
oak
indi iduals
by
183
lea
mo phological
ai s184
Lea
mo phological
ai s
we e
assessed
in
all
ees
selec ed
o 185
es ablishmen
o
in
i o
cul u es
in
a
o me
comp ehensi e
ax-186
onomic
su ey
o
198
whi e
oak
ees
in
he
o es
s and
(Kanalas187
e
al.,
2008,
2009).
B iefly,
in
his
su ey
fi e
lea es
we e
collec ed188
om
he
lowe
canopy
o
ees.
Al oge he
means
o
16
lea
quan-
189
i a i e
ai s
we e
used
in
nume ic
classifica ion
analysis
wo ked190
ou
p e iously
o
each
axon
(Bo o ics,
2000;
Kanalas
e
al.,
2008,191
2009).
This
analysis
allowed
o
classi y
he
indi idual
ees
as
oak
192
species
sensu
s ic o
and
hyb ids.193
Mic osa elli e
(SSR)
analysis
194
Fo
DNA
ex ac ion
win e
bud
samples
o
he
indi idual
ees195
lis ed
in
Table
1
we e
used.
A e
g inding
wi h
liquid
ni ogen
he196
ex ac ion
was
ca ied
ou
by
he
Qiagen
Plan
Mini
Ki
(BioMa ke ,
197
Gödöll˝
o,
Hunga y).
DNA
concen a ion
o
ex ac s
was
checked
by198
gel
elec opho esis
on
a
0.5%
aga ose
(Ro h
Ro i®ga ose
NEEO,
RK199
Tech,
Budapes ,
Hunga y)
gel.
Polyme ase
chain
eac ions
we e
pe -200
o med
wi h
he
ollowing
SSR
ma ke s
(fluo escen
dyes
a
he
201
5-ends
a e
indica ed
in
b acke s):
ZAG
1/5
(6-FAM),
ZAG
9
(6-FAM),202
ZAG
110
(HEX)
(S einkellne
e
al.,
1997),
ZAG
11
(TET),
ZAG
96203
(TET),
ZAG
112
(HEX)
(Kamp e
e
al.,
1998).
Key
pa ame e s
o 204
SSR
ma ke s
a e
p esen ed
in
Table
2.
PCR
mas e mixes
and
op i-
205
malisa ion
p ocedu es
we e
made
up
acco ding
o
S einkellne
e
al.206
(1997)
and
Kamp e
e
al.
(1998)
and
comp ised
he
ollowing
com-207
ponen s
o
15
␮L
final
eac ion
olume:
5×
bu e
(P omegaGoTaq208
Flexi)
4
␮L;
MgCl21
mM
(0.6
␮L)
in
case
o
ma ke s
ZAG
1/5
and209
ZAG
9,
2
mM
(1.2␮L)
in
case
o
he
o he
ma ke s;
P ime
F
and210
R
(Ro h
Ro i®ga ose
NEEO,
RK
Tech,
Budapes ,
Hunga y):
0.25
pM211
(0.375
␮L)
each
o
ZAG
9,
0.75
pM
(1.125
␮L)
o
ZAG
1/5
and212
0.34pM
(0.5
␮L)
o
he
emaining
ma ke s;
dNTPmix
(P omega213
10
mM)
0.4
␮L;
polyme ase
enzyme
(P omegaGoTaq
Flexi)
0.4
U;214
DNA
sample
1
␮L
(app ox.
10
ng/␮L).
Fo
he
PCRs
an
Eppendo 215
Mas e cycle
G adien
he mocycle
was
used
wi h
he
ollowing 216
p og am:
in
case
o
ZAG
1/5
and
ZAG
9
ini ial
dena u a ion
95◦C217
15
min,
dena u a ion
95◦C
50
s,
p ime
annealing
55◦C/ZAG
9
and 218
65◦C/ZAG
1/5
50
s,
elonga ion
72◦C
1
min
45
s,
epe i ion
o
las 219
h ee
s eps
in
35
cycles,
final
elonga ion
72◦C
10
min.
In
case
o
he 220
o he
ou
ma ke s:
ini ial
dena u a ion
95◦C
15
min,
dena u a ion 221
95◦C
30
s,
p ime
annealing
50◦C
30
s,
elonga ion
65◦C
1
min
30
s, 222
epe i ion
o
las
h ee
s eps
in
35
cycles,
final
elonga ion
65◦C223
15
min.
F agmen
analyses
we e
ca ied
ou
by
an
ABI
P ism
310 224
gene ic
analyse
(Applied
Biosys ems
Li e
Technologies,
Budapes , 225
Hunga y)
in
mul iplexed
uns.
C
ma ix
se
and
TAMRA
500
size 226
s anda d
we e
applied
(Applied
Biosys ems
Li e
Technologies).
The 227
e alua ion
o
agmen
sizes
was
done
by
he
GeneMappe
so - 228
wa e. 229
The
aw
geno ype
da a
se
wi h
he
SSR
agmen
leng h
sizes 230
was
analyzed
by
he
GenAlEx
6.4
(Peakall
and
Smouse,
2006)
pop- 231
ula ion
gene ic
so wa e.
The
gene ic
dis ances
be ween
pai s
o 232
indi iduals
we e
calcula ed
based
on
he
sha ed
alelle
equen- 233
cies.
The
gene ic
ela ionship
among
samples
was
ep esen ed
on
a234
dend og am
cons uc ed
wi h
he
unweigh ed
pai -g oup
a e age 235
amalgama ion
me hod
(UPGMA)
(Snea h
and
Sokal,
1973).
Fo
his 236
pu pose
he
Clus e
Analyses
op ion
o
S a is ica
6.0
so wa e
was 237
used. 238
PEG
ea men s
and
eco e y
expe imen s 239
Callus
cul u es
we e
g own
on
solidified
WPM
medium
(see 240
Resul s
sec ion
o
PGR
con en ).
They
we e
ans e ed
on
liq- 241
uid
medium
o
he
same
composi ion
(2
mL
cul u e
medium
in 242
10
mL
s e ile
plas ic
flasks,
Labsys em,
Budapes ,
Hunga y)
and 243
ea ed
o
24
h
wi h
0,
5,
10,
20
and
40%
(w/ )
polye hylene
gly- 244
col
6000
(PEG6000,
VWR,
Leu en,
Belgium).
PEG6000
solu ions 245
co esponded
o
osmo ic
po en ials
o
−0.05,
−0.15,
−0.49
and 246
−1.75
MPa.
Du ing
ea men s
wi h
he
osmo icum,
cul u es
we e 247
gen ly
shaken
(100
pm)
on
a
o a o y
shake
(E.
Bühle
KS-15,
E. 248
Bühle
GmbH,
Hechingen,
Ge many).
F esh
weigh
(FW)
o
calli 249
was
measu ed
a
he
s a
and
he
end
o
PEG6000
ea men s 250
by
means
o
an
analy ical
balance
(Model
AA
200
DS,
accu acy 251
±10
␮g,
Den e
Ins umen
Co.,
A ada).
FW
was
in
he
ange
o 252
20–50
mg.
Be o e
FW
measu emen
a
he
s a
o
expe imen s, 253
excess
liquid
medium
was
emo ed
by
gen le
cen i uga ion
wi h- 254
ou
a ec ing
callus
g ow h
and
iabili y
(1000
pm,
5
s
on
a
He aeus 255
Bio uge,
Kend o
Labo a o y
P oduc s,
Ha au,
Ge many).
Enzyme 256
ac i i ies
(see
below)
we e
measu ed
di ec ly
a e
ea men s 257
wi h
he
osmo icum.
Pe cen age
o
FW
inc ease
was
calcula ed 258
on
he
basis
o
he
di e ence
be ween
FW
a
he
end
and
a
he 259
s a
o
expe imen s.
Reco e y
expe imen s
we e
pe o med
as
ol- 260
lows:
ollowing
PEG6000
ea men s,
cul u es
om
PEG
con aining 261
Please
ci e
his
a icle
in
p ess
as:
Deme e
Z,
e
al.
Osmo ic
s ess
esponses
o
indi idual
whi e
oak
(Que cus
sec ion,
Que cus
subgenus)
geno ypes
cul u ed
in
i o.
J
Plan
Physiol
(2013),
h p://dx.doi.o g/10.1016/j.jplph.2013.09.013
ARTICLE IN PRESS
G
Model
JPLPH
51807
1–9
4Z.
Deme e
e
al.
/
Jou nal
o
Plan
Physiology
xxx (2013) xxx–
xxx
medium
we e
washed
ou
wo
imes
by
gen le
shaking
(100
pm)262
in
he
p esence
o
liquid
cul u e
medium
lacking
he
osmo icum263
ollowed
by
u he
cul u e
on
aga -solidified
medium
o
30
days.264
F esh
weigh
o
eco e ed
calli
was
also
measu ed
as
desc ibed
ea -265
lie .
Beside
FW
measu emen s
a
he
s a
and
end
o
cul u e
pe iod,266
he
p esence
o
iable,
g een
callus
issue
a
he
end
o
cul u e
was267
moni o ed
as
well.268
SSP
nuclease
ac i i y
gels269
The
ac i i y
o
SSP
nucleases
(EC
3.1.30.1)
was
assayed
on270
polyac ylamide
gels,
basically
as
desc ibed
be o e
(Jámb ik
e
al.,271
2011).
In
b ie ,
PEG6000
ea ed
calli
we e
ex ac ed
wi h
10
mM272
T is–HCl
(Sigma–Ald ich,
Budapes ,
Hunga y),
pH
8.0,
150
mM273
NaCl
(Reanal,
Budapes ,
Hunga y),
14.6
mM
2-me cap oe hanol274
(Sigma–Ald ich)
and
2%
(w/ )
poly inyl-py olidone
(PVP,
Me ck,275
Da ms ad ,
Ge many).
P o ein
ex ac s
(20
␮g/well)
we e
loaded276
on
SDS-
and
single-s anded
DNA
con aining
polyac ylamide
gels277
along
wi h
a
molecula
weigh
ma ke
(Sigma–Ald ich).
P o ein278
con en
o
ex ac s
was
de e mined
acco ding
o
B ad o d
(1976).
279
A e
ena u a ion
o
enzymes,
gels
we e
incuba ed
in
T is–HCl,
pH280
6.8
(14
h,
39◦C)
o
assaying
hei
ac i i y,
s ained
wi h
0.5
␮g
mL−1
281
e hidium
b omide
(Sigma–Ald ich,
Budapes ,
Hunga y)
and
exam-282
ined
wi h
an
UV
ansillumina o .
SSP
nuclease
ac i i ies
appea ed283
as
clea
bands,
no
s ained
wi h
e hidium–b omide.
To al
nuclease284
ac i i ies
on
gels
we e
quan ified
wi h
he
aid
o
CpA las®so wa e285
and
exp essed
as
ela i e
band
in ensi ies,
whe e
he
alue
o
con-286
ol
ac i i ies
was
1.
The
molecula
weigh
o
ssDNase
isoenzymes287
was
es ima ed
wi h
he
UVI-TEC®so wa e.288
Pe oxidase
ac i i y
gels289
PEG6000
ea ed
calli
we e
ex ac ed
a
4◦C
wi h
a
bu e 290
con aining
100
mM
KH2PO4/K2HPO4(VWR
In e na ional
L d.,291
Deb ecen,
Hunga y),
pH
7.2,
8
mM
MgCl2(Reanal,
Budapes ,292
Hunga y),
4
mM
di hio h ei ol
(DTT,
Sigma–Ald ich),
1%
( / )
T i-293
on
X-100
(Reanal),
2%
(w/ )
PVP
(Me ck).
A e
cen i uga ion
( wo
294
imes
o
30
min)
a
15,000
×
g
wi h
a
He aeus
Bio uge,
p o ein295
con en
o
supe na an s
was
assayed
by
he
me hod
o
B ad o d296
(1976).
10
␮g
p o ein
was
loaded
on o
each
well
o
na i e
7.5%297
(w/ )
polyac ylamide
gels.
Elec opho esis
was
pe o med
a
4◦C,298
ollowed
by
gel
s aining
o
30–60
min
in
a
bu e
con aining299
100
mM
sodium
ace a e
(VWR),
10%
( / )
hyd ogen
pe oxide
and300
1
mM
guaiacol.
Pe oxidase
(E.C.
1.11.1.7)
ac i i y
was
isible
due301
o
da k-colo ed
e aguaiacol
bands
(Dixi
e
al.,
2011).
Guaiacol-302
pe oxidase
ac i i ies
we e
quan ified
wi h
he
aid
o
CpA las®
303
so wa e,
and
exp essed
as
o
SSP
nucleases.304
Da a
analysis305
All
expe imen s
we e
pe o med
a
leas
ou
imes
wi h
six306
pa allel
callus
samples
pe
expe imen
o
each
geno ype
and
ep-307
esen a i e
da a
a e
p esen ed
in
he
Resul s
sec ion.
The
mean
±
SE308
o
quan i a i e
da a
was
calcula ed
and
plo ed
wi h
he
aid
o
309
Sigma
Plo
10.0
so wa e,
whe e
i
was
app op ia e.
Plo s
ep e-310
sen
mean
±
SE
alues
o
di e en
calli/ he
espec i e
indi idual311
geno ype.
Quan i a i e
da a
we e
subjec ed
o
s a is ical
analysis
312
by
wo-way
ANOVA.
This
in ol ed
All
Pai wise
Mul iple
Compa i-
313
son
P ocedu es
(Holm-Sidak
me hod)
wi h
an
o e all
significance314
le el
o
0.05.
This
me hod
allowed
o
analyze
he
e ec s
o
PEG
con-315
cen a ions
wi hin
a
single
geno ype
as
well
as
di e ences
be ween316
geno ypes
wi hin
a
single
PEG
concen a ion.
Di e ences
we e
con-
317
side ed
significan
a
P
<
0.05.
Fig.
1.
Clus e
dend og am
showing
gene ic
dis ances
be ween
Que cus
axa
in ol ed
in
his
s udy.
Resul s 318
Gene ic
ela ionships
be ween
Que cus
explan s
s udied 319
Explan s
used
in
his
s udy
o igina ed
om
six
indi idual 320
ma u e
ees
om
he
Que cus
(=Lepidobalanus)
sec ion
co- 321
occu ing
a
he
Sík ˝
okú
Resea ch
Si e
(Bükk
M s.,
Hunga y).
Lea 322
mo phological
ai s
o
he
indi idual
ees
we e
s udied
in
o de 323
o
es ima e
hei
axonomical
iden i y.
Based
on
his,
we
iden i- 324
fied
h ee
non-hyb id
and
h ee
hyb id
axa
(Table
1).
In
o de 325
o
es ima e
he
gene ic
ela edness
o
selec ed
ees
a
mic osa el- 326
li e
analysis
was
applied.
Then
gene ic
dis ances
we e
calcula ed 327
be ween
indi iduals
based
on
he
sha ed
allele
con en
o
mul ilo- 328
cus
geno ypes
de i ed
om
he
six
SSR
loci
analyzed
(Fig.
1).
This 329
e ealed
ha
A149
(Q.
pe aea)
is
gene ically
dis inc
o
all
o he 330
indi iduals
examined,
including
A75
(Q.
pubescens)
and
B50
(Q.
i - 331
giliana).
D89,
a
pu a i e
Q.
pe aea
×
Q.
pubescens
hyb id
and
A211, 332
a
pu a i e
hyb id
be ween
Q.
i giliana
and
Q.
polyca pa,
appea ed 333
o
be
in
he
same
clus e
wi h
B50
and
A75.
D137,
a
pu a i e
hyb id 334
be ween
Q.
pe aea
and
Q.
dalechampii
was
gene ically
dis an
o 335
A149
and
appea ed
o
be
in
he
same
clus e ,
bu
a
a
ela i ely 336
high
dis ance
o
all
o he
indi iduals
(Fig.
1). 337
The
es ablishmen
and
main enance
o
issue
cul u es
om
whi e 338
oak
explan s 339
We
ha e
induced
and
s abilized
callus
cul u es
o
he
fi s
ime 340
om
six
Que cus
geno ypes
o igina ing
om
Sík ˝
okú
Resea ch 341
A ea
(Table
1).
A
wide
ange
o
g ow h
egula o
concen a ions 342
was
es ed
(see
Ma e ials
and
me hods
sec ion).
Calli
appea ed
a e 343
30
±
3
days
o
cul u e
o
young
lea
explan s.
PGR
combina ions
o 344
callus
induc ion
and
main enance
as
well
as
emb yogenesis
and 345
o ganogenesis
we e
geno ype
dependen
(da a
no
shown).
This
is 346
a
gene al
ule
o
issue
cul u es
o
ela ed,
bu
gene ically
di e en 347
plan
axa
(see
Duncan
e
al.,
1985;
Má hé
e
al.,
2012
o
exam- 348
ples).
Howe e ,
we
ha e
ound
a
PGR
combina ion,
whe e
all
callus 349
samples
o igina ing
om
di e en
oak
geno ypes
we e
o
simila 350
mo phology
(undi e en ia ed
s a e),
g ow h
a e
and
iabili y.
This 351
was
WPM
medium
con aining
4
mg
L−1NAA
and
0.5
mg
L−1BA.
A 352
his
PGR
con en ,
g ow h
o
all
s able
cul u es
p o ed
o
be
con- 353
s an
since
hey
we e
ini ia ed
(a
ime
pe iod
o
a
leas
one
yea ), 354
he e o e
hey
we e
sui able
o
osmo ic
s ess
expe imen s. 355
I
is
wo h
men ioning,
ha
we
could
no
es ablish
an
e ficien 356
mic op opaga ion
sys em
(i.e.
mass
plan
egene a ion
wi hou
an 357
in e media y
callus
s age)
om
he
a o emen ioned
oak
geno ypes. 358
Howe e
he
choose
o
PGRs
(NAA
and
BA
ins ead
o
2,4-d
and359
kine in)
excluded
somaclonal
a iabili y
du ing
callus
induc ion 360

Please
ci e
his
a icle
in
p ess
as:
Deme e
Z,
e
al.
Osmo ic
s ess
esponses
o
indi idual
whi e
oak
(Que cus
sec ion,
Que cus
subgenus)
geno ypes
cul u ed
in
i o.
J
Plan
Physiol
(2013),
h p://dx.doi.o g/10.1016/j.jplph.2013.09.013
ARTICLE IN PRESS
G
Model
JPLPH
51807
1–9
Z.
Deme e
e
al.
/
Jou nal
o
Plan
Physiology
xxx (2013) xxx–
xxx 5
Fig.
2.
The
e ec
o
PEG
concen a ion
on
esh
weigh
changes
o
calli
wi h
di e -
en
axonomic/gene ic
o igin.
Cul u es
we e
g own
on
WPM
medium
con aining
4
mg
L−1NAA
and
0.5
mg
L−1BA.
(A)
FW
changes
a e
PEG
ea men s;
(B)
FW
changes
du ing
eco e y
o
PEG
ea ed
calli.
Mean
±
SE
alues
o
di e en
calli/ he
espec i e
indi idual
geno ype
a e
plo ed.
As e iscs
ep esen
significan
di e -
ences
be ween
PEG
ea men s
o
a
single
geno ype,
while
le e codes
ep esen
di e ences
be ween
callus
lines
wi hin
a
single
PEG
ea men .
Di e ences
we e
conside ed
o
be
significan
a
P
<
0.05.
and
main enance,
hus
callus
cul u es
p o ed
o
be
sui able
o
361
osmo ic
s ess
expe imen s.
362
The
e ec s
o
PEG6000
on
wa e
loss
and
eco e y
o
Que cus363
callus
cul u es364
Two-way
ANOVA
e ealed
ha
he e
was
a
significan
ela ion-365
ship
(P
<
0.05)
be ween
he
geno ype
o
indi idual
oak
ees
and
he366
e ec s
o
PEG
ea men s
conce ning
all
physiological
pa ame e s367
(g ow h,
eco e y
and
enzyme
ac i i ies)
s udied
(Figs.
2–5).368
The
measu emen
o
FW
a e
ea men
wi h
he
osmo icum369
e ealed
ha
o
A149,
significan
wa e
loss
occu ed
a
10%
370
PEG6000
and
inc eased
p og essi ely
and
significan ly
as
PEG6000371
concen a ions
inc eased.
In
con as ,
A75
and
B50
we e
ole an 372
o
osmo ic
ea men
and
significan
wa e
loss
occu ed
only
a 373
40%
PEG6000
(Fig.
2A).
Pu a i e
hyb ids
we e
in e media e
wi h
374
espec
o
he
e ec s
o
he
osmo icum:
wa e
loss
occu ed
a
20%375
PEG6000
and
inc eased
a
40%
PEG6000
(Fig.
2A).
A
eco e y
a e 376
osmo ic
s ess,
changes
o
FW
showed
ha
o
A149,
callus
g ow h377
was
sligh ly
s imula ed
by
10%
PEG6000,
bu
inhibi ed
significan ly
378
a
highe
concen a ions,
while
o
A211
and
B50
he e
was
a
an-379
sien
s imula ion
a
5–10%
PEG6000
(Fig.
2B).
A e
washou
o
he380
osmo icum
he e
was
a
significan
inhibi ion
in
he
g ow h
o
A211
381
calli
p e ea ed
wi h
40%
PEG6000
and
p e ea men
wi h
20–40%
382
PEG6000
inhibi ed
he
g ow h
o
B50
calli.
Thus
he e
was
no
eco -
383
e y
in
hese
cases.
In
case
o
cul u e
lines
D137,
D89
and
A75,
he e384
was
a
gene al
inhibi ion
o
callus
g ow h
by
PEG6000
(Fig.
2B).385
S a is ical
analysis
o
da a
e ealed
ha
PEG6000
induced
wa e
386
loss
o
A75
calli
was
significan ly
di e en
o
all
o he
callus
lines.387
Pai wise
compa ison
showed
significan
di e ences
in
PEG6000 388
induced
wa e
loss
be ween
A149,
B50
and
A211
only
a
highe 389
(20–40%)
concen a ions
(Fig.
2A).
Conce ning
callus
g ow h
du - 390
ing
eco e y
expe imen s,
al hough
significan
di e ences
we e 391
obse ed
be ween
callus
lines
a
ce ain
PEG6000
concen a ions 392
(Fig.
2B),
significan ly
highe
g ow h
a e
o
A75
and
B50
calli
as 393
compa ed
o
lines
A149,
D137,
D89
and
A211
was
no
de ec ed. 394
Conce ning
he
p esence
o
iable,
compac ,
g een
callus
issue 395
du ing
eco e y
expe imen s,
we
ha e
made
he
ollowing
obse - 396
a ions.
Fo
A149
(Q.
pe aea),
his
ype
o
issue
was
no
p esen
a 397
p e ea men
wi h
10–40%
PEG6000:
i
was
eplaced
by
nec o ic- 398
like,
b owning
issue
ha
sugges ed
he
accumula ion
o
phenolic 399
compounds
as
a
s ess
eac ion
(Fig.
3).
In
con as ,
o
A75
(Q. 400
pubescens)
and
B50
(Q.
i giliana),
issue
iabili y
pe sis ed
e en 401
a
40%
PEG
(Fig.
3).
D89
as
a
pu a i e
hyb id
be ween
Q.
pe aea 402
and
Q.
pubescens,
was
in e media e
in
his
espec :
callus
iabili y 403
was
los
only
a
20–40%
PEG.
The
pu a i e
hyb ids
D137
and
A211 404
beha ed
simila ly
o
A75
and
B50
(Fig.
3). 405
Osmo ic
s ess- ela ed
enzyme
ac i i ies 406
Ac i i y
gels
e ealed
ha
SSP
nuclease
isoenzyme(s)
wi h 407
molecula
weigh (s)
in
he
ange
o
45–55
kDa
we e
p esen
and 408
ac i e
in
all
cul u e
lines.
One
dominan
band
o
50
kDa
was 409
de ec ed
(Fig.
4A).
In
con ol
cul u es,
his
ac i i y
was
weake 410
in
lines
A149,
D137
and
D89
as
compa ed
o
A211,
B50
and
A75 411
(Fig.
4A).
The
e ec s
o
PEG6000
ea men s
we e
dependen
on 412
cul u e
line/geno ype.
Excep
A75
(Q.
pubescens)
cul u es,
osmo ic 413
s ess
induced
ansien
inc eases
in
nuclease
ac i i ies
as
com- 414
pa ed
o
con ols,
wi h
maximal
ac i i ies
a
5–20%
PEG6000. 415
Significan
inc eases
we e
de ec ed
o
D89
(5–10%
PEG6000),
A149 416
(10–20%
PEG6000)
and
A211
(20%
PEG6000).
In
case
o
B50
(Q.
i - 417
giliana)
he e
was
only
a
sligh
s imula ion
o
SSP
nuclease
ac i i y 418
by
PEG
(Fig.
4A
and
B).
In
case
o
A75,
PEG6000
did
no
inc ease 419
no ably
he
enzyme
ac i i y,
bu
a
5–10%
PEG6000,
wo
bands
wi h 420
s ong
ac i i ies
we e
de ec able
in
he
molecula
weigh
ange
o 421
50
kDa
(Fig.
4A
and
B).
5–20%
PEG6000
induced
he
appea ance
o 422
one
addi ional
band
wi h
ela i ely
weak
ac i i y
in
case
o
B50
and 423
A211
(Fig.
4A). 424
Two-way
ANOVA
e ealed
significan
di e ences
be ween
SSP 425
nuclease
ac i i ies
o
PEG6000
ea ed
A149
and
D137,
B50,
A75, 426
espec i ely.
A
20%
PEG,
A149
was
cha ac e ized
by
significan ly 427
highe
SSP
nuclease
ac i i y
han
he
es
o
callus
lines.
Besides 428
A149,
a
10%
PEG6000,
enzyme
ac i i y
o
A211
was
significan ly 429
highe
as
compa ed
o
A75
(Fig.
4B). 430
Conce ning
guaiacol
pe oxidase
ac i i ies,
one
band
wi h
s ong 431
ac i i y
appea ed
in
all
cul u es.
PEG6000
dec eased
his
enzyme 432
ac i i y
in
A149
(Q.
pe aea),
al hough
his
dec ease
was
no
sig- 433
nifican
(Fig.
5A
and
B).
A
non-significan
dec easing
e ec
was 434
obse ed
in
he
case
o
pu a i e
hyb ids
D89
and
A211
as
well, 435
bu
ansien
inc eases
we e
de ec able
a
10%
and
5–10%
PEG6000, 436
espec i ely
(Fig.
5A
and
B).
PEG
ea men s
inc eased
guaiacol
pe - 437
oxidase
ac i i ies
in
he
pu a i e
hyb id
D137,
Q.
i giliana
(B50) 438
and
Q.
pubescens
(A75)
wi h
peaks
a
5%
(D137,
B50)
and
20% 439
PEG6000
(A75)
(Fig.
5A
and
B).
An
addi ional
band
o
weak
pe - 440
oxidase
ac i i y
was
obse ed
in
case
o
D89,
A211
and
A75.
Fo 441
A75,
his
band
was
p esen
only
a e
ea men s
wi h
20%
PEG6000 442
(Fig.
5A). 443
Two-way
ANOVA
e ealed
significan
di e ences
be ween
pe - 444
oxidase
ac i i ies
o
PEG6000
ea ed
A149
and
D137,
B50,
A75, 445
espec i ely
(Fig.
5B).
A
5%
PEG6000,
he
enzyme
ac i i y
o
B50 446
was
significan ly
highe ,
han
A75.
Conce ning
o e all
e ec s
o
he 447
osmo icum,
pe oxidase
ac i i ies
o
he
pu a i e
hyb id
D89
did
no 448
di e
significan ly
o
A149,
bu
i
had
significan ly
lowe
ac i i ies, 449
Please
ci e
his
a icle
in
p ess
as:
Deme e
Z,
e
al.
Osmo ic
s ess
esponses
o
indi idual
whi e
oak
(Que cus
sec ion,
Que cus
subgenus)
geno ypes
cul u ed
in
i o.
J
Plan
Physiol
(2013),
h p://dx.doi.o g/10.1016/j.jplph.2013.09.013
ARTICLE IN PRESS
G
Model
JPLPH
51807
1–9
6Z.
Deme e
e
al.
/
Jou nal
o
Plan
Physiology
xxx (2013) xxx–
xxx
Fig.
3.
Rep esen a i e
eco e y
expe imen s
wi h
Que cus
issue
cul u es
o
di e en
axonomic/gene ic
o igin.
PEG
ea men s
we e
ollowed
by
washou
o
he
osmo icum
and
u he
cul u e
on
a
medium
supplemen ed
wi h
4
mg
L−1NAA
and
0.5
mg
L−1BA.
Scale
ba :
5
mm.
han
D137
and
B50.
A211
had
significan ly
lowe
ac i i ies,
han450
B50
and
D137
(Fig.
5B).
451
Discussion452
We
ha e
es ablished
six
no el
s able
callus
lines
om
oak
geno-453
ypes
belonging
o
he
Que cus
sec ion
(=Lepidobalanus)o Que cus
454
subgenus.
These
cul u es
we e
sui able
o
compa ing
di e ences
455
in
osmo ic
s ess
esponses
among
geno ypes.
Based
on
p e ious456
wo k
on
he
p oduc ion
o
s able
issue
cul u es
o
Q.
obu
(Cuenca457
e
al.,
1999;
To ibio
e
al.,
2004),
we
es ablished
he
p ope
cul u e458
media
sui able
o
d ough
s ess
expe imen s
wi h
PGR
con en
o 459
4
mg
L−1NAA
and
0.5
mg
L−1BA.460
Clea
dis inc ion
om
molecula
ma ke s
be ween
Q.
pe aea461
and
Q.
pubescens
is
o en
di ficul
(Sal ini
e
al.,
2009).
Howe e ,462
mic osa elli e
and
isoenzyme
da a
ha e
p e iously
demons a ed463
ha
Q.
pe aea
and
Q.
pubescens
a e
dis inc
species,
bu
c oss-464
ing
be ween
hem
is
possible
(Samuel
e
al.,
1995;
B uschi
e
al.,465
2000).
By
applying
6
mic osa elli e
loci,
in
his
s udy
he
selec ed466
Q.
pe aea
indi idual
ee
could
be
clea ly
dis inguished
om
all
467
o he
Que cus
(=Lepidobalanus)
indi iduals,
including
Q.
pubescens468
and
pu a i e
hyb ids
(Fig.
1).
Thus,
significan
gene ic
dis ances469
could
be
obse ed
be ween
indi iduals
cha ac e ized
by
di e en 470
lea
mo phological
ai s.
This
is
o
pa icula
impo ance,
since
di -
471
e en
lea
mo phologies
o
oaks
do
no
necessa ily
eflec
no able472
gene ic
di e ences
(Cu u
e
al.,
2007).
The
clea
dis inc ion
o
Q.473
pe aea
o
all
o he
indi iduals
s udied
was
confi med
by
in
i o
474
mo phogenesis
expe imen s
as
well:
his
indi idual
explan
could
475
no
egene a e
oo s,
in
con as
o
all
o he
explan s
s udied,
whe e
476
e ficien
oo
p oduc ion
was
egula ly
obse able
( o
be
published477
elsewhe e).
Na i e
gels
e ealed
a
single
main
pe oxidase
ac i i y478
band
o
all
cul u e
lines.
In
case
o
D89,
A211
and
A75
a
mino
479
addi ional
band
appea ed.
In e es ingly,
his
band
was
inducible480
by
PEG
in
case
o
A75,
ha
is,
i
was
de ec able
only
du ing
osmo ic 481
s ess
(Fig.
5A,
a owheads).
This
addi ional
band
u he
suppo ed 482
mic osa elli e
da a:
in
case
o
he
gene ically
mo e
dis inc
lines 483
A149
(Q.
pe aea)
and
D137
i
was
no
p esen ,
while
i
appea ed
in 484
gene ically
ela ed
cul u e
lines
men ioned.
In
case
o
SSP
nuclease 485
ac i i y
pa e ns,
in
he
gene ically
ela ed
lines
A211,
B50
and
A75, 486
wo
bands
appea ed
a
PEG6000
ea men s,
bo h
in
he
molecu- 487
la
weigh
ange
o
45–55
kDa
(Fig.
4).
I
should
be
no ed
howe e , 488
ha
many
nucleases
a e
glycop o eins
and
he
p esence
o
absence 489
o
glycoside
esidues
depends
on
he
physiological
s a e
o
cells 490
(Desai
and
Shanka ,
2003).
The e o e
i
is
possible
ha
double 491
bands
de ec ed
eflec
a
single
p o ein
wi h
di e en
glycosyla ion 492
s a es. 493
D ough /osmo ic
s ess,
as
a
significan
numbe
o
abio ic 494
s esses,
leads
o
he
inc ease
o
ROS
in
plan
cells
(Mi le ,
2002). 495
Resis ance
o
d ough
in ol es
an
inc eased
capaci y
o
sca - 496
enging
ROS
h ough
supe oxide
dismu ases
(SOD),
ca alases
and 497
pe oxidases
(Wang
e
al.,
2003).
Non-enzyma ic
and
enzyma ic 498
sca enging
mechanisms
a e
s imula ed
du ing
summe
midday 499
cha ac e ized
by
high
ligh
exposu e,
empe a u e
and
wa e
defi- 500
ciency
in
Q.
sube
(Fa ia
e
al.,
1996).
In
con as ,
o
d ough
and
sal 501
sensi i e
Q.
obu ,
among
ROS
sca enging
sys ems,
only
supe ox- 502
ide
dismu ase
(SOD)
ac i i y
and
isoenzyme
pa e n
was
modified 503
a
exposu e
o
NaCl
(Sehme
e
al.,
1995).
D ying
o
ecalci an 504
Q.
obu
aco ns
is
leading
o
he
loss
o
emb yo
iabili y,
associ- 505
a ed
wi h
he
accumula ion
o
ROS
and
low
le els
o
sca enging 506
enzymes
(Hend y
e
al.,
1992).
In
case
o
a
d ough
ole an
Q.
obu 507
geno ype,
ele a ed
le els
o
ROS
sca enging
enzyme
(SOD,
asco - 508
ba e
pe oxidase,
ca alase,
dehyd oasco ba e
educ ase,
glu a hione 509
educ ase)
ac i i ies
we e
de ec ed
(Schwanz
and
Polle,
2001).
In 510
he
absence
o
a
p ope
sca enging
capaci y,
cellula
s uc u es
and 511
mac omolecules
including
DNA
(single
s and
b eaks)
a e
signifi- 512
can ly
damaged
(Reddy
e
al.,
2004).
Following
oxida i e
damage 513
Please
ci e
his
a icle
in
p ess
as:
Deme e
Z,
e
al.
Osmo ic
s ess
esponses
o
indi idual
whi e
oak
(Que cus
sec ion,
Que cus
subgenus)
geno ypes
cul u ed
in
i o.
J
Plan
Physiol
(2013),
h p://dx.doi.o g/10.1016/j.jplph.2013.09.013
ARTICLE IN PRESS
G
Model
JPLPH
51807
1–9
Z.
Deme e
e
al.
/
Jou nal
o
Plan
Physiology
xxx (2013) xxx–
xxx 7
Fig.
4.
The
e ec
o
PEG
on
SSP
DNase
ac i i ies
o
Que cus
calli
o
di e en
gene ic
o igin,
g own
on
a
medium
supplemen ed
wi h
4
mg
L−1NAA
and
0.5
mg
L−1BA.
(A)
Rep esen a i e
gels.
A ows
indica e
he
p esence
o
addi ional
bands;
(B)
enzyme
ac i i ies
ep esen ed
by
ela i e
band
in ensi ies
as
measu ed
wi h
CP
A las
so -
wa e.
Mean
±
SE
alues
o
di e en
calli/ he
espec i e
indi idual
geno ype
a e
plo ed.
As e iscs
ep esen
significan
di e ences
be ween
PEG
ea men s
o
a
single
geno ype,
while
le e codes
ep esen
di e ences
be ween
callus
lines
wi hin
a
single
PEG
ea men .
Di e ences
we e
conside ed
o
be
significan
a
P
<
0.05.
o
DNA,
cells
a e
cha ac e ized
by
he
ac i i y
o
nucleases
in ol ed514
in
epai
(Roldán-A jona
and
A iza,
2009).515
Se e al
SSP
nucleases
inducible
by
d ough /osmo ic/sal
s ess516
ha e
been
iden ified.
Fo
example,
ba ley
BnucI
is
a
sal
s ess517
inducible
ype
I
nuclease,
a
pu a i e
glycop o ein
sligh ly
smalle 518
han
36
kDa
om
ba ley
(Mu amo o
e
al.,
1999).
Hyd ogen
pe -519
oxide
and
d ough
s ess
induces
he
ac i i y
o
se e al
nuclease520
Fig.
5.
The
e ec
o
PEG
on
guaiacol
pe oxidase
ac i i ies
o
Que cus
calli
o
di e en
gene ic
o igin,
g own
on
a
medium
supplemen ed
wi h
4
mg
L−1NAA
and
0.5
mg
L−1
BA.
(A)
Rep esen a i e
gels.
A owheads
indica e
he
p esence
o
addi ional
bands.
(B) Enzyme ac i i ies
ep esen ed
by
ela i e
band
in ensi ies
as
measu ed
wi h
CP
A las
so wa e.
Mean
±
SE
alues
o
di e en
calli/ he
espec i e
indi idual
geno-
ype
a e
plo ed.
As e iscs
ep esen
significan
di e ences
be ween
PEG
ea men s
o
a
single
geno ype,
while
le e codes
ep esen
di e ences
be ween
callus
lines
wi hin
a
single
PEG
ea men .
Di e ences
we e
conside ed
o
be
significan
a
P
<
0.05.
isoenzymes
using
ssDNA
as
subs a e
o
molecula
weigh s
ang- 521
ing
be ween
26
and
38
kDa
in
cauliflowe
seedlings.
Two
o
hem 522
appea ed
be
he
same
enzyme,
inducible
by
bo h
s ess
ac o s 523
(Le´
sniewicz
e
al.,
2010).
The
main
nuclease
isoenzyme
p o ed
o 524
be
modula ed
by
osmo ic
s ess
in
his
s udy
appea ed
o
ha e
a525
molecula
weigh
o
50
kDa
(Fig.
4).
To
ou
bes
knowledge,
nucle- 526
ases
o
simila
molecula
weigh ,
wi h
changing
ac i i y
a
abio ic 527
s esses
a e
unusual
in
plan s.
I
should
be
no ed
howe e ,
ha
a528
significan
numbe
o
nucleases
consis
o
subuni s
o
lowe
size 529
(see
Desai
and
Shanka ,
2003
o
a
e iew).
Thus,
he
na u e
o 530
Que cus
nuclease
o
50
kDa
needs
u he
in es iga ion. 531
In
case
o
d ough
sensi i e
indi iduals
(A149,
D89),
PEG- 532
induced
inc ease
o
SSP
nuclease
ac i i y
was
accompanied
by, 533
no
significan
changes
o
dec eases
o
guaiacol-pe oxidase
ac i - 534
i ies
in
callus.
In
con as ,
o
d ough - ole an
axa
(A75,
B50), 535
PEG
ea men s
led
o
unchanged
o
sligh ly
inc easing
SSP
nucle- 536
ase
ac i i ies
oge he
wi h
inc eases
o
pe oxidase
ac i i ies 537
(Figs.
4
and
5,
see
de ails
below).
This
means
ha
ROS
canno 538
Please
ci e
his
a icle
in
p ess
as:
Deme e
Z,
e
al.
Osmo ic
s ess
esponses
o
indi idual
whi e
oak
(Que cus
sec ion,
Que cus
subgenus)
geno ypes
cul u ed
in
i o.
J
Plan
Physiol
(2013),
h p://dx.doi.o g/10.1016/j.jplph.2013.09.013
ARTICLE IN PRESS
G
Model
JPLPH
51807
1–9
8Z.
Deme e
e
al.
/
Jou nal
o
Plan
Physiology
xxx (2013) xxx–
xxx
Table
3
O e iew
o
po en ial
osmo ic
s ess/d ough
ole ance
o
PEG6000
ea ed
oak
callus
lines
as
shown
by
di e en
physiological
pa ame e s.
This
classifica ion
was
based
on
significan
di e ences
in
physiological
esponses
as
e ealed
by
wo-way
ANOVA.
Physiological
pa ame e s FW G ow h
( eco e y)
Viabili y
( eco e y)
ssDNase
ac i i y
Guaiacol-pe oxidase
ac i i y
Po en ially
d ough
ole an
B50,
A75
n.d.
A211,
B50,
A75
B50,
A75
D137,
B50,
A75
Po en ially
d ough
sensi i e
A149,
D89
n.d.
A149,
D89
A149,
D89,
A211
A149,
D89
Is
he
di e ence
be ween
A75
and
B50
significan ?
Only
a
40%
PEG6000
Yes
No
No
Yes
n.d., no
de ec able
on
he
basis
o
a ailable
da a.
be
sca enged
in
d ough -sensi i e
axa,
ollowed
by
DNA
s and539
b eaks
leading
o
ac i i y
inc eases
o
nucleases
p obably
in ol ed540
in
epai .
D ough - ole an
axa
a e
expec ed
o
be
able
o
sca -541
enging
ROS
e ficien ly,
hus
DNA
s and
b eaks
occu
p obably542
wi h
less
equency.
Howe e ,
changes
(inc eases
o
dec eases)
543
o
SSP
nuclease
ac i i ies
we e
no
p opo ional
wi h
changes
o 544
pe oxidase
ac i i ies
o
all
geno ypes
and/o
PEG
concen a ions545
(Figs.
4
and
5).
This
indica es
ha
besides
ROS
le els,
o he
mech-
546
anisms
could
modula e
nuclease
ac i i ies.547
In
case
o
callus
line
A149
(Q.
pe aea),
ea men s
wi h
PEG6000548
induced
concen a ion-dependen
wa e
loss,
as
seen
by
he549
dec ease
o
callus
esh
weigh .
The
capaci y
o
calli
o
g ow
and
550
o
p oduce
g een,
iable
issues
dec eased
a e
emo al
o
20–40%551
and
10–40%
PEG6000
espec i ely,
as
shown
by
eco e y
expe -552
imen s
(Figs.
2
and
3).
Meanwhile,
pe oxidase
ac i i y
dec eased
553
and
SSP
nuclease
ac i i y
inc eased
(Figs.
4
and
5).
All
hese
e ec s554
o
osmo ic
s ess
in
a
model
expe imen
(Table
3)
confi m
p e-555
ious
findings
(Thomas
e
al.,
2002)
and
ou
hypo hesis,
ha
is,556
sessile
oak
is
gene ally
a
d ough /osmo ic
s ess
sensi i e
species.
557
In
con as ,
cul u es
o
A75
(Q.
pubescens)
and
B50
(Q.
i giliana)558
a e
esis an
o
PEG6000
induced
wa e
loss
and
hey
a e
able559
o
eco e
e en
a e
ea men s
wi h
high
concen a ions
o
he560
osmo icum
(e en
hough
hei
inc ease
in
esh
weigh
is
inhib-
561
i ed
by
PEG
in
eco e y
expe imen s,
Figs.
2
and
3).
Osmo ic
s ess562
inc eases
pe oxidase
ac i i ies
and
induces
only
sligh
inc eases
o
563
SSP
nuclease
ac i i ies
in
hese
cul u es.
O e all,
hese
esul s
con-564
fi m
p e ious
findings
and
ou
hypo hesis,
ha
is,
issue
cul u es565
o
Q.
pubescens
and
Q.
i giliana
o igin
a e
d ough /osmo ic
s ess
566
ole an
(Table
3).
Mo eo e ,
mic osa elli e
da a
show
a
close
ela -
567
edness
be ween
A75
and
B50
(Fig.
1).
Fo
D89,
PEG6000
induced568
wa e
loss
was
in e media y
as
compa ed
o
A149
and
A75,
as569
shown
by
changes
o
callus
esh
weigh
and
he
pe sis ence
o
570
callus
eco e y
a e
ea men
wi h
10%
PEG
(Figs.
2A
and
3).
On571
he
o he
hand,
osmo ic
s ess
induced
he
inc ease
o
ssDNase572
ac i i y
and
excep
ea men s
wi h
10%
PEG,
he
dec ease
o
pe -
573
oxidase
ac i i y
(Figs.
4
and
5).
O e all,
hese
pa ame e s
sugges
574
ha
d ough
esponse
o
D89
is
in e media y
be ween
A149
and575
A75,
adding
u he
p oo
o
he
idea
ha
D89
is
a
hyb id
be ween576
Q.
pe aea
and
Q.
pubescens.
Howe e ,
all
physiological
pa ame e s
577
sugges
ha
cul u es
o
D89
a e
mo e
ole an
o
d ough ,
han578
A149,
bu
s ill
can
be
conside ed
as
d ough
sensi i e
(Table
3).579
Pe oxidase
ac i i ies
o
PEG6000
ea ed
D137
calli
inc eased
as
o
580
d ough
esis an
geno ypes
(Fig.
5
and
Table
3),
e en
hough
we581
ha e
ini ially
hypo hesized
ha
as
a
hyb id
be ween
Q.
pe aea
and582
Q.
dalechampii,
his
geno ype
is
ela i ely
d ough
sensi i e.
Indeed,583
mic osa elli e
da a
confi med
ha
D137
is
gene ically
close
o
584
he
clus e
con aining
Q.
pubescens,
han
o
Q.
pe aea
(Fig.
1).
On585
he
o he
hand,
Q.
dalechampii
lineage
could
also
explain
a
highe 586
d ough
ole ance.
Conce ning
A211,
i s
wa e
loss
and
abili y
o587
eco e
was
in e media y
be ween
d ough
sensi i e
A149
and
588
ole an
A75
(Figs.
2A,
3
and
Table
3),
bu
PEG6000
induced
SSP589
nuclease
and
pe oxidase
ac i i y
changes
we e
simila
o
A149
590
(Figs.
4
and
5).
Mo phological
and
mic osa elli e
da a
we e
no
com-
591
pa able
o
his
cul u e
line.
Lea
mo phology
ai s
da a
sugges ed
592
ha
A211
is
a
hyb id
be ween
Q.
i giliana
and
Q.
polyca pa,
bu 593
he
analysis
o
SSR
ma ke s
sugges ed
i s
close
ela edness
o
he 594
analyzed Q.
pubescens
ee
(Table
1
and
Fig.
1).
Thus,
e en
hough 595
his
indi idual
ee
is
pu a i e
hyb id
be ween
a
d ough
sensi i e 596
and
a
d ough
esis an
oak
axon,
genes/p o eins
esponsible
o 597
d ough
ole ance
seem
o
be
weakly
exp essed. 598
I
should
be
no ed
ha
a
gi en
oak
species
can
be
cha ac e - 599
ized
by
high
in a-popula ional
gene ic
a iabili y
(He zog,
1996)600
and
as
a
consequence,
di e ences
be ween
physiological
esponses 601
o
indi iduals
may
occu .
Thus,
in a-popula ional
spec um
o 602
d ough
ole ance
o
oaks
in
he
Sík ˝
okú
LTER
a ea
needs
u he 603
s udies. 604
Table
3
shows
he
es ima ed
osmo ic
s ess/d ough
ole ance
o 605
he
six
oak
cul u e
lines
s udied
as
shown
by
significan
physiologi- 606
cal
esponses
o
PEG
ea men s.
All
physiological
changes
induced 607
by
osmo ic
s ess
indica ed
ha
d ough
sensi i i y
o
Q.
pe aea,
Q. 608
pubescens,
and
Q.
i giliana
cul u es
confi med
p e ious
labo a o y 609
and
field
s udies
including
ecological
equi emen s
o
hei
pop- 610
ula ions
(Cocha d
e
al.,
1992;
Damesin
and
Rambal,
1995;
Gallé 611
e
al.,
2007;
Siam
e
al.,
2009;
Rod íguez-Calce ada
e
al.,
2010; 612
Ofle ea
e
al.,
2011)
and
p o ed
ou
hypo hesis:
such
in
i o
cul- 613
u es
can
be
addi ional
model
sys ems
o
s udying
s ess
esponses 614
o
axa
wi h
unknown
d ough
esponses
(D89,
D137
and
A211 615
in
he
p esen
s udy).
In e es ingly,
B50
(Q.
i giliana)
and
A75
(Q. 616
pubescens)
had
significan ly
di e en
esponses
in
eco e y
expe - 617
imen s
and
conce ning
pe oxidase
ac i i ies
(Figs.
2,
3
and
5
and 618
Table
3),
e en
hough
ou
da a
and
p e ious
findings
sugges
ha 619
bo h
Q.
pubescens
and
Q.
i giliana
a e
d ough
ole an
species
(see 620
Table
3;
Siam
e
al.,
2009;
Ofle ea
e
al.,
2011
o
example).
In
gen- 621
e al,
mic osa elli e
da a
confi med
ela i ely
high
gene ic
dis ances 622
be ween
d ough
sensi i e
and
d ough
ole an
oak
indi iduals. 623
The
p esen
wo k
o e s
an
in
i o
model
o
s udying
physiologi- 624
cal
esponses
o
oak
o
d ough ,
by
using
issue
cul u es
es ablished 625
om
selec ed
indi idual
ees
wi h
di e en
axonomic
iden i y
co- 626
occu ing
in
he
same
o es
s and.
Fu he
s udies
bo h
in
he
field 627
and
labo a o y,
he
la e
conce ning
issue
cul u es
om
di e en 628
indi iduals
om
he
same
popula ion
will
e eal
na u al
a iabili y 629
in
d ough
ole ance
o
whi e
oaks.
We
sugges
ha
in
i o
callus 630
cul u es
as
simplified
expe imen al
sys ems
a e
help ul
ools
con- 631
ibu ing
o
he
modeling
o
d ough
ole ance
o
field
g own
oak 632
plan s
wi h
di e en
gene ic
backg ound
and
could
be
o
gene al 633
applicabili y
o
plan
s ess
biology
esea ch. 634
Acknowledgemen s 635
The
s udy
was
suppo ed
by
Na ional
Resea ch
Founda ion 636
(OTKA
No.
K68397
and
K101552)
and
Eu opean
Union
and
he 637
Eu opean
Social
Fund
co-financed
p ojec
TÁMOP-4.2.2/B-10/1- 638
2010-0024. 639
Re e ences 640
Aas
G.
Mo phologische
und
ökologische
Va ia ion
mi eleu opäische
Que cus- 641
A en:
Ein
Bei ag
zum
Ve s ändnis
de
Biodi e si ä .
Lib i
Bo anici:
Band
19. 642
Eching:
IHW-Ve lag;
1998.
p.
213. 643