Overexpression. of dehydrin tas14 gene improves the osmotic stress imposed by drought and salinity in tomato

Article (PDF Available)inJournal of plant physiology 169(5):459-68 · March 2012with166 Reads
DOI: 10.1016/j.jplph.2011.11.018 · Source: PubMed
Abstract
One strategy to increase the level of drought and salinity tolerance is the transfer of genes codifying different types of proteins functionally related to macromolecules protection, such as group 2 of late embryogenesis abundant (LEA) proteins or dehydrins. The TAS14 dehydrin was isolated and characterized in tomato and its expression was induced by osmotic stress (NaCl and mannitol) and abscisic acid (ABA) [Godoy et al., Plant Mol Biol 1994;26:1921-1934], yet its function in drought and salinity tolerance of tomato remains elusive. In this study, transgenic tomato plants overexpressing tas14 gene under the control of the 35SCaMV promoter were generated to assess the function of tas14 gene in drought and salinity tolerance. The plants overexpressing tas14 gene achieved improved long-term drought and salinity tolerance without affecting plant growth under non-stress conditions. A mechanism of osmotic stress tolerance via osmotic potential reduction and solutes accumulation, such as sugars and K(+) is operating in tas14 overexpressing plants in drought conditions. A similar mechanism of osmotic stress tolerance was observed under salinity. Moreover, the overexpression of tas14 gene increased Na(+) accumulation only in adult leaves, whereas in young leaves, the accumulated solutes were K(+) and sugars, suggesting that plants overexpressing tas14 gene are able to distribute the Na(+) accumulation between young and adult leaves over a prolonged period in stressful conditions. Measurement of ABA showed that the action mechanism of tas14 gene is associated with an earlier and greater accumulation of ABA in leaves during short-term periods. A good feature for the application of this gene in improving drought and salt stress tolerance is the fact that its constitutive expression does not affect plant growth under non-stress conditions, and tolerance induced by overexpression of tas14 gene was observed at the different stress degrees applied to the long term.
Journal
of
Plant
Physiology
169 (2012) 459–
468
Contents
lists
available
at
SciVerse
ScienceDirect
Journal
of
Plant
Physiology
jou
rn
al
h
o
mepage:
www.elsevier.de/jplph
Overexpression
of
dehydrin
tas14
gene
improves
the
osmotic
stress
imposed
by
drought
and
salinity
in
tomato
Alicia
Mu
˜
noz-Mayor
a,1
,
Benito
Pineda
b,1
,
Jose
O.
Garcia-Abellán
a
,
Teresa
Antón
a
,
Bego
˜
na
Garcia-Sogo
b
,
Paloma
Sanchez-Bel
a
,
Francisco
B.
Flores
a,
,
Alejandro
Atarés
b
,
Trinidad
Angosto
c
,
Jose
A.
Pintor-Toro
d
,
Vicente
Moreno
b
,
Maria
C.
Bolarin
a
a
CEBAS-CSIC,
Department
of
Stress
Biology
and
Plant
Pathology,
Campus
de
Espinardo,
P.O.
Box
164,
30100
Espinardo-Murcia,
Spain
b
IBMCP-UPV/CSIC,
Laboratory
of
Biotechnological
Breeding,
Camino
de
Vera
14,
46022
Valencia,
Spain
c
University
of
Almeria,
Department
of
Applied
Biology,
Carretera
de
Sacramento
s/n,
04120
Almeria,
Spain
d
CABIMER,
Parque
Científico
y
Tecnológico
de
la
Cartuja,
41092
Sevilla,
Spain
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
10
August
2011
Received
in
revised
form
27
October
2011
Accepted
23
November
2011
Keywords:
Drought
tolerance
Salinity
tolerance
Tomato
Solanum
lycopersicum
tas14
gene
Osmotic
stress
a
b
s
t
r
a
c
t
One
strategy
to
increase
the
level
of
drought
and
salinity
tolerance
is
the
transfer
of
genes
codifying
different
types
of
proteins
functionally
related
to
macromolecules
protection,
such
as
group
2
of
late
embryogenesis
abundant
(LEA)
proteins
or
dehydrins.
The
TAS14
dehydrin
was
isolated
and
character-
ized
in
tomato
and
its
expression
was
induced
by
osmotic
stress
(NaCl
and
mannitol)
and
abscisic
acid
(ABA)
[Godoy
et
al.,
Plant
Mol
Biol
1994;26:1921–1934],
yet
its
function
in
drought
and
salinity
tolerance
of
tomato
remains
elusive.
In
this
study,
transgenic
tomato
plants
overexpressing
tas14
gene
under
the
control
of
the
35SCaMV
promoter
were
generated
to
assess
the
function
of
tas14
gene
in
drought
and
salinity
tolerance.
The
plants
overexpressing
tas14
gene
achieved
improved
long-term
drought
and
salin-
ity
tolerance
without
affecting
plant
growth
under
non-stress
conditions.
A
mechanism
of
osmotic
stress
tolerance
via
osmotic
potential
reduction
and
solutes
accumulation,
such
as
sugars
and
K
+
is
operating
in
tas14
overexpressing
plants
in
drought
conditions.
A
similar
mechanism
of
osmotic
stress
tolerance
was
observed
under
salinity.
Moreover,
the
overexpression
of
tas14
gene
increased
Na
+
accumulation
only
in
adult
leaves,
whereas
in
young
leaves,
the
accumulated
solutes
were
K
+
and
sugars,
suggesting
that
plants
overexpressing
tas14
gene
are
able
to
distribute
the
Na
+
accumulation
between
young
and
adult
leaves
over
a
prolonged
period
in
stressful
conditions.
Measurement
of
ABA
showed
that
the
action
mechanism
of
tas14
gene
is
associated
with
an
earlier
and
greater
accumulation
of
ABA
in
leaves
during
short-term
periods.
A
good
feature
for
the
application
of
this
gene
in
improving
drought
and
salt
stress
tolerance
is
the
fact
that
its
constitutive
expression
does
not
affect
plant
growth
under
non-stress
conditions,
and
tolerance
induced
by
overexpression
of
tas14
gene
was
observed
at
the
different
stress
degrees
applied
to
the
long
term.
© 2011 Elsevier GmbH. All rights reserved.
Introduction
Abiotic
stresses
such
as
drought
and
salinity
impose
severe
pro-
duction
constraints
on
food
production.
Drought
is
a
major
abiotic
stress
that
affects
agriculture
in
45%
of
the
world
(Foolad,
2007)
and
the
potential
yield
losses
by
salinity
are
estimated
at
20%
(Ashraf
Abbreviations:
ABA,
abscisic
acid;
DW,
dry
weight;
FW,
fresh
weight;
IAA,
indole
acetic
acid;
LEA,
late
embryogenesis
abundant;
RWC,
relative
water
content;
TW,
turgent
weight;
WT,
wild
type;
35SCaMV,
Promoter
35S
from
Cauliflower
Mosaic
Virus
(35SCaMV);
s
,
osmotic
potential;
w
,
water
potential.
Corresponding
author.
Tel.:
+34
968
39
63
78;
fax:
+34
968
39
62
13.
E-mail
address:
borjaflores@cebas.csic.es
(F.B.
Flores).
1
Both
authors
contributed
equally
to
this
work.
et
al.,
2008).
The
problem
is
growing,
as
apart
from
natural
salinity
a
significant
proportion
of
recently
cultivated
agricultural
land
has
become
saline.
Although
tolerance
to
drought
and
salt
stresses
is
a
very
complex
trait,
development
of
crop
plants
tolerant
to
stress
is
vital
to
meet
the
growing
food
demand
through
sustainable
agricul-
ture
(Cuartero
et
al.,
2010;
Hirayama
and
Shinozaki,
2010).
Drought
and
salt
stresses
share
common
physiological
osmotic
stresses.
Decreased
soil
water
availability
under
drought
or
decreased
water
potential
of
soil
solution
under
salinity
cause
osmotic
stress,
which
leads
to
decreased
water
uptake
and
loss
of
turgor.
The
differen-
tial
effect
induced
by
salinity
is
the
toxic
effect
induced
by
the
root
uptake
and
shoot
transport
of
saline
ions
(Munns
and
Tester,
2008).
Despite
the
economic
relevance
of
tomato,
the
mechanisms
that
govern
responses
to
these
abiotic
stresses
in
this
horticultural
species
are
not
well
characterized,
and
a
very
small
number
of
genes
0176-1617/$
see
front
matter ©
2011 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jplph.2011.11.018
460 A.
Mu
˜
noz-Mayor
et
al.
/
Journal
of
Plant
Physiology
169 (2012) 459–
468
playing
a
role
in
tomato
tolerance
to
salinity
and
drought
have
been
identified
thus
far
(Atares
et
al.,
2011;
Pineda
et
al.,
2012).
However,
in
spite
of
numerous
reports
of
improved
tolerance
by
the
overexpression
of
different
genes,
the
mechanisms
underly-
ing
the
enhancement
of
tolerance
remain
unclear
in
most
cases.
Thus,
in
order
to
elucidate
the
role
of
AtNHX1
antiporter,
Leidi
et
al.
(2010)
carried
out
a
very
important
study,
finally
demon-
strating
that
tomato
plants
overexpressing
AtNHX1
had
larger
K
+
accumulations
in
the
vacuole
in
all
growth
conditions
tested,
but
no
consistent
enhancement
of
Na
+
accumulation,
as
previously
sug-
gested
(Pardo
et
al.,
2006).
A
strategy
to
increase
the
level
of
drought
and
salinity
tolerance
is
the
transfer
of
genes
codifying
different
types
of
proteins
involved
in
the
molecular
responses
to
abiotic
stress,
such
as
osmoprotec-
tants,
chaperones,
detoxification
enzymes,
transcription
factors,
signal
transduction
proteins
(kinases
and
phosphatases),
heat-
shock
proteins
(HSPs),
and
late-embryogenesis-abundant
(LEA)
proteins
(Campalans
et
al.,
1999;
Capiati
et
al.,
2006;
Khurana
et
al.,
2008;
Orsini
et
al.,
2010;
Amudha
and
Balasubramani,
2011).
LEA
proteins
constitute
a
superfamily
of
proteins
that
were
detected
for
the
first
time
during
the
maturation
phase
of
cotton
embryogenesis,
which
is
the
stage
when
acquisition
of
desiccation
tolerance
occurs
in
the
embryo,
when
they
accumulate
in
high
con-
centrations,
a
characteristic
that
gave
rise
to
their
name
(Dure
and
Chlan,
1981;
Dure
and
Galau,
1981).
This
group
of
very
hydrophilic
proteins
markedly
increase
during
water
deficit
and/or
low
tem-
perature
stress
in
vegetative
organs,
suggesting
a
protective
role
during
water
limitation
(Bies-Etheve
et
al.,
2008;
Popelka
et
al.,
2010),
although
their
precise
functions
and
mechanisms
of
action
are
still
hidden
even
after
twenty
years
of
their
discovery
(Battaglia
et
al.,
2008;
Khurana
et
al.,
2008).
Some
of
the
most
studied
LEA
proteins
in
higher
plants
are
the
group
2
or
dehydrins
(Zhang
et
al.,
2007;
Veeranagamallaiah
et
al.,
2011).
There
have
been
several
studies
of
specific
members
of
this
group
2
of
LEA
proteins
that
confirm
their
accumulation
during
seed
desiccation
and
in
response
to
water
deficit
induced
by
drought,
low
temperature,
or
salinity
(Ismail
et
al.,
1999;
Nylander
et
al.,
2001).
Since
the
expression
of
dehydrins
is
significantly
induced
by
abiotic
stresses
such
as
drought,
cold
and
high
salinity,
it
has
been
suggested
that
a
positive
correlation
exists
between
dehydrin
expression
and
abiotic
stress
tolerance
in
plants
(Saavedra
et
al.,
2006;
Brini
et
al.,
2007).
The
TAS14
dehydrin
was
isolated
and
characterized
in
tomato
(Godoy
et
al.,
1990).
This
gene
was
induced
in
tomato
seedlings
and
adult
plants
under
osmotic
stress
(NaCl
and
mannitol)
and
abscisic
acid
(ABA)
(Godoy
et
al.,
1994),
but
the
physiological
role
played
by
this
gene
during
drought
and
salt
stress
in
tomato
still
remains
unknown.
To
study
the
role
of
the
tas14
gene
in
tomato
and
determine
whether
its
overexpression
increases
drought
and
salinity
toler-
ance,
the
tas14
gene
was
introduced
in
tomato
under
the
control
of
the
constitutive
promoter
35S
from
Cauliflower
Mosaic
Virus
(35SCaMV),
and
growth
and
physiological
responses
to
drought
and
salinity
were
studied
in
the
resulting
transgenic
tomato
plants.
Results
from
different
experiments
described
in
this
paper
show
that
the
tas14
gene
plays
an
essential
role
during
drought
and
salt
stress
in
tomato
by
means
of
improving
its
tolerance
towards
the
osmotic
stress
imposed
by
both
abiotic
stresses.
Several
studies
applying
overexpression
and
ectopic
expres-
sion
of
dehydrins
have
been
previously
published.
For
instance,
the
overexpression
of
multiple
Arabidopsis
dehydrins
led
to
plants
showing
increased
freezing
tolerance
and
improved
survival
when
subjected
to
low
temperature
stress
conditions
(Puhakainen
et
al.,
2004).
Also,
the
ectopic
expression
of
a
wheat
dehydrin
(DHN-5)
in
Arabidopsis
plants
improved
their
tolerance
to
high
salinity
and
water
deficit
(Brini
et
al.,
2007).
With
respect
to
tomato
dehydrins,
a
study
of
the
ectopic
expression
in
yeast
of
one
of
them
(Le4)
was
performed
(Zhang
et
al.,
2000).
The
study
showed
that
the
trans-
formed
yeast
partially
overcame
the
detrimental
effects
of
ionic
and
freezing
stress
by
conferring
tolerance
to
high
concentration
of
KCl,
but
not
to
NaCl
or
sorbitol.
To
our
knowledge,
the
research
described
here
is
the
first
study
to
apply
overexpression
of
a
dehy-
drin
in
a
plant
species
of
such
an
agronomic
interest
as
tomato,
and
where
the
effects
of
the
accumulation
of
this
type
of
LEA
protein
in
tomato
plants
when
subjected
to
water
and
salt
stress
conditions,
in
short-
and
long-term
assays,
have
been
investigated.
Material
and
methods
Transformation
and
molecular
characterization
of
the
transgenic
tomato
plants
The
tomato
746-bp
tas14
cDNA
(X51904)
was
introduced
into
a
tomato
cultivar
of
determined
growth
(Solanum
lycopersicum
L.
cv.
UC82B)
by
Agrobacterium-mediated
transformation
using
a
protocol
previously
described
(Gisbert
et
al.,
2000).
Cotyledonary
explants
were
infected
with
A.
tumefaciens
strain
LBA4404
car-
rying
the
tas14
and
kanamycin
resistance
gene
nptII
sequences
in
the
plasmid
pPM7
vector
containing
the
35SCaMV
promoter.
Transformed
shoots
were
transferred
to
a
rooting
culture
medium
consisting
of
Murashige
and
Skoog
medium
(Murashige
and
Skoog,
1962)
supplemented
with
0.1
mg
L
1
IAA
and
50
mg
L
1
kanamycin.
Only
one
regenerated
plant
from
a
single
poke
was
counted
as
an
independent
transgenic
event.
Twenty
independently
regenerated
kanamycin-resistant
plants
(T0
plants)
were
transferred
into
soil
and
grown
under
stan-
dardized
greenhouse
conditions
(Esta
˜
n
et
al.,
2005)
to
generate
T1
seeds,
which
were
a
mixture
of
azygous
(transformed
line
without
transgene),
homozygous
and
hemizygous
lines.
Progenies
were
obtained
from
those
transgenic
plants
by
selfing
in
con-
trolled
conditions.
These
progenies
(T2
plants)
were
analyzed
for
kanamycin
(50
g
mL
1
)
resistance,
and
azygous
and
homozygous
lines
(T3)
were
identified
according
to
their
kanamycin
resistance
(0%
kanamycin
resistance
in
azygous
line
and
100%
kanamycin
resistance
in
homozygous
line).
The
molecular
verification
of
the
transgenic
plants
was
performed
by
PCR
and
the
number
of
inserted
copies
in
transgenic
plants
was
determined
by
DNA
gel
blot
analysis
using
the
methods
described
in
Pineda
et
al.
(2010).
The
expression
of
the
TAS14
protein
was
verified
by
protein
gel
blot
analysis
as
previously
described
(Godoy
et
al.,
1994).
Drought
and
salt
treatments
and
tolerance
assays
Homozygous
plants
from
the
line
with
higher
expression
levels
of
TAS14
protein
(L4),
named
positive
plants,
and
their
controls,
WT
and
azygous
plants
from
line
L6,
named
negative
plants,
were
tested
applying
different
drought
treatments
at
the
7–8th
leaf
stage.
Plant
culture
for
drought
treatments
was
carried
out
in
a
con-
trolled
growth
chamber.
Seeds
were
germinated
in
a
2:1:1
(v/v)
mixture
of
peat:perlite:siliceous
sand
at
28
C
and
90%
relative
humidity
in
darkness.
When
seedlings
had
developed
2
true
leaves
(25
days
after
sowing),
they
were
transferred
to
5-L
plastic
pots
filled
with
peat
and
plants
were
daily
irrigated
with
half-strength
Hoagland
solution
(Hoagland
and
Arnon,
1950).
The
environmen-
tal
conditions
were
optimized
for
the
growth
of
tomato
seedlings,
varying
the
temperature
along
the
day
between
18
and
25
C,
the
relative
humidity
between
50
and
80%
and
with
a
photoperiod
of
16
h
light/8
h
dark
was
imposed.
A
photosynthetic
photon
flux
(400–700
nm)
of
345
mol
m
2
s
1
at
the
plant
level
was
provided
by
fluorescent
tubes
(Osram
Lumilux
daily-light
58
W
and
Fluora
58
W).
A.
Mu
˜
noz-Mayor
et
al.
/
Journal
of
Plant
Physiology
169 (2012) 459–
468 461
The
drought
response
was
determined
by
using
two
different
procedures.
In
the
first,
drought
stress
was
imposed
by
water-
ing
plants
with
30%
nutrient
solution,
compared
with
the
volume
applied
to
well-irrigated
plants,
for
50
days.
The
well-irrigated
plants
were
irrigated
daily
up
to
pot
capacity,
and
the
nutrient
solution
volume
for
irrigation
of
the
drought-stressed
plants
(30%
of
well-irrigated
plants)
was
calculated,
Drought
tolerance
was
evaluated
on
the
basis
of
plant
biomass
after
30
and
50
days
of
treatment,
and
physiological
response
was
analyzed
in
leaves
and
roots
of
the
first
harvest.
In
the
second
procedure,
the
drought
stress
was
applied
by
withholding
irrigation,
so
water
stress
intensity
increased
with
time,
and
two
successive
dehydration–rehydration
cycles
were
applied.
Thus,
the
plants
dehydrated
for
7
days
were
rehydrated
for
1
day
and
after
this
time
another
similar
dehydration
cycle
was
applied.
Leaves
and
roots
were
taken
for
analysis
at
the
beginning
of
the
experiment
(day
0)
and
on
the
2nd,
4th
and
6th
day
of
each
dehydration
cycle.
Experiments
were
repeated
twice
and
eight
plants
per
treatment
were
used.
The
leaf
relative
water
content
(RWC)
was
analyzed
in
all
samples
and
ulterior
physiolog-
ical
analysis
were
undertaken
only
in
certain
samples
depending
on
the
values
of
RWC
that
were
determined.
To
evaluate
salt
tolerance
at
the
whole
plant
level,
a
first
exper-
iment
was
carried
out
in
controlled
conditions,
by
using
negative
and
positive
plants
and
the
same
environmental
and
culture
condi-
tions
as
in
drought
experiments,
although
salt
treatments
(0,
75
and
150
mM
NaCl)
were
applied
at
a
younger
growth
stage,
when
the
plants
had
developed
two
true
leaves.
At
the
end
of
the
experiment
(25
days),
shoot
biomass
was
measured
and
young
(developing
leaves),
adult
(the
third
completely
developed
leaves)
and
roots
were
taken
for
analysis
from
each
of
the
eight
plants
per
treatment.
A
greenhouse
experiment
was
carried
out
until
fruit
yield
by
using
WT,
negative
and
positive
plants.
Plants
were
grown
as
previ-
ously
described
(Mu
˜
noz-Mayor
et
al.,
2008)
and
the
salt
treatments
(0,
75
and
100
mM
NaCl)
were
maintained
throughout
the
exper-
iment.
In
eight
plants
per
treatment,
ripe
fruits
were
collected
weekly
from
one
month
of
cultivation
and
the
weight
recorded.
The
salinity
response
was
also
studied
at
the
cellular
level
by
using
callus
culture.
Calli
were
initiated
from
leaf
explants
of
neg-
ative
and
positive
plants
and
subcultured
as
previously
described
(Rus
et
al.,
2001).
The
salt
treatments
(0,
100
and
150
mM
NaCl)
were
applied
for
30
days.
Thirty
replicates
per
treatment
were
used
(10
Petri
dishes
with
3
calli
each).
At
the
end
of
the
experiment,
fresh
weight
was
scored
and
a
part
of
the
callus
material
was
taken
for
analysis.
Physiological
measurements
Leaf
water
potential
was
measured
by
inserting
the
youngest
fully
expanded
leaf
in
a
Scholander
pressure
chamber
(Model
3000,
Soil
Moisture
Equipment
Corp.,
CA)
and
determining
the
minimum
pressure
needed
to
extract
water
from
the
cut
end.
In
all
experiments
and
harvests,
fresh
material
was
rinsed
in
deionized
water
and
blotted
carefully
with
tissue
paper.
A
part
of
the
plant
material
was
weighed
for
fresh
weight
(FW)
determina-
tion,
oven
dried
for
48
h
at
80
C,
and
weighed
to
determine
the
dry
weight
(DW).
Another
part
of
the
plant
material
was
placed
into
5-ml
pipette
tips
containing
a
glass
wool
filter
in
the
tip,
and
imme-
diately
frozen
with
liquid
nitrogen
until
analysis.
Subsequently,
sap
was
extracted
from
the
thawed
plant
material
samples
by
centrifu-
gation
and
used
for
analysis.
The
leaf
relative
water
content
(RWC)
was
calculated
as
(FW
DW)/(TW
DW)
×
100,
and
expressed
as
a
percentage.
Leaves
were
excised,
the
fresh
weight
(FW)
recorded
and
incu-
bated
in
water
for
24
h
at
4
C
in
the
dark.
The
leaves
were
blotted
and
the
turgid
weight
(TW)
measured.
Osmolality
was
measured
by
the
freezing
point
depression
method
using
an
osmometer
(Osmomat
030,
Gonotec,
Germany).
Osmolalities
(mOsm
kg
1
)
were
converted
to
osmotic
potential
(1
mOsm
=
2.408
kPa).
Pres-
sure
potential
was
estimated
as
the
difference
between
water
potential
and
osmotic
potential.
The
concentrations
of
inorganic
(Na
+
and
K
+
)
and
organic
solutes
(sugars
and
proline)
were
deter-
mined
as
previously
described
(Mu
˜
noz-Mayor
et
al.,
2008).
The
total
ABA
content
from
samples
was
extracted
and
determined
by
indirect
ELISA,
according
to
the
methodology
described
by
Gosalbes
et
al.
(2004).
Statistical
analysis
Data
were
statistically
analyzed
using
the
SPSS
13.0
software
package
by
ANOVA
and
LSD
test
(P
0.05),
using
the
treatments
as
a
statistical
parameter,
to
determine
significant
differences
between
means.
Results
Characterization
of
tas14
overexpressing
tomato
plants
in
control
and
drought
conditions
Twenty
independent
transgenic
plants
were
generated
by
intro-
ducing
the
tomato
tas14
cDNA
into
the
processing
tomato
cultivar
UC82B,
with
determined
growth
habitus.
Most
of
them
were
pos-
itive
transformants
as
confirmed
by
PCR
analysis
for
both
genes
tas14
and
nptII.
Four
transformants
with
only
one
copy
of
the
over-
expressing
tas14
genetic
construction
were
identified
by
DNA
gel
blot
analysis
for
both
tas14
and
nptII
genes
(data
not
shown).
The
insertion
of
one
copy
was
in
concordance
with
the
segregation
observed
in
T2
for
Kanamycin
resistance
(3:1).
Protein
gel
blot
anal-
yses
were
performed
to
test
for
the
presence
of
TAS14
protein
in
these
4
primary
transformants
(Supplementary
Fig.
1a).
A
14-kDa
band
corresponding
to
TAS14
protein
was
detected
in
the
trans-
genic
plants
but
not
in
WT
nor
in
a
negative
transformant
(L6),
indicating
that
it
was
successfully
expressed
in
the
genetically
mod-
ified
plants.
Although
tas14
gene
is
found
in
the
tomato
genome,
the
expression
of
the
endogenous
gene
is
not
induced
in
standard
culture
conditions
(Godoy
et
al.,
1994).
The
phenotypic
analysis
of
the
tas14
overexpressing
tomato
plants
was
first
carried
out
with
homozygous
transgenic
lines
from
the
4
transformants
with
one
copy
of
the
overexpressing
tas14
genetic
construction.
When
these
lines
were
grown
under
well-irrigated
conditions,
the
growth
pat-
terns
of
the
transgenic
plants
were
similar
to
those
of
the
WT
plants
and
the
lines
without
the
overexpressing
transgene
from
the
same
primary
transformant
(azygous
line),
and
identified
at
the
same
time
as
the
homozygous
lines
(Supplementary
Fig.
1b).
A
preliminary
experiment
was
carried
out
with
the
four
homozygous
lines
(positive
plants)
and
both
WT
and
azygous
plants
from
line
6
(negative
plants)
by
water
withholding.
After
6
days,
both
WT
and
negative
plants
showed
a
dehydrated
aspect,
while
plants
overexpressing
tas14
gene
showed
only
slight
dehydration
symptoms,
especially
line
4,
with
the
highest
expression
of
TAS14
protein
(data
not
shown),
which
was
selected
for
further
experi-
ments.
Next,
the
drought
tolerance
of
the
WT,
negative
and
positive
(homozygous
line
4)
plants
was
tested
after
50
days
of
drought
treatment
(by
irrigating
the
plants
with
30%
of
the
volume
applied
to
well-irrigated
plants).
The
positive
effect
of
the
overexpression
of
the
tas14
gene
had
already
been
noted
by
higher
accumulation
of
shoot
biomass
measured
as
plant
fresh
weight
after
30
days
of
drought
treatment
(Fig.
1a).
At
the
end
of
the
experiment
(50
days),
the
vegetative
shoot
biomass
continued
to
be
significantly
higher
in
positive
than
in
WT
and
negative
plants
under
drought
conditions
(Fig.
1b).
The
most
interesting
characteristic
was
that
the
positive
plants
were
able
to
develop
fruits
after
50
days
of
462 A.
Mu
˜
noz-Mayor
et
al.
/
Journal
of
Plant
Physiology
169 (2012) 459–
468
Fig.
1.
Effects
of
overexpression
of
tas14
gene
on
plant
growth
response
to
drought.
Drought
tolerance
of
wild
type
plants
(WT),
azygous
or
negative
plants
()
and
homozygous
or
positive
plants
(+)
was
tested
by
irrigating
the
plants
with
a
30%
of
the
volume
applied
to
well-irrigated
plants.
The
absolute
and
relative
values
of
shoot
biomass
after
30
days
of
drought
treatment
(a)
and
shoot
biomass
(b)
and
fruit
biomass
(c)
at
the
end
of
the
experiment
(50
days)
are
shown.
Data
are
the
mean
±
SE
(n
=
8).
Significant
differences
at
P
<
0.05
between
lines
were
indicated
with
*.
intense
drought
stress,
while
the
WT
and
negative
plants
had
hardly
any
fruit
at
this
time
(Fig.
1c),
as
illustrated
in
the
photograph
taken
at
the
end
of
the
experiment
(Supplementary
Fig.
1d).
Under
well-irrigated
conditions,
no
significant
differences
were
observed
between
the
shoot
and
fruit
biomass
of
WT,
negative
and
positive
plants
(Supplementary
Fig.
1c).
Drought
tolerance
mechanisms
induced
by
the
overexpression
of
tas14
gene
To
examine
the
physiological
changes
induced
by
the
overex-
pression
of
the
tas14
gene,
water
potential
(
w
)
was
measured
in
leaves
and
osmotic
potential
(
s
),
sugars
and
K
+
contents,
which
are
among
the
most
important
solutes
contributing
to
the
osmotic
potential,
and
the
contents
of
the
osmolyte
proline
and
of
ABA
were
analyzed
in
both
leaves
and
roots
after
30
days
of
drought
treatment
(Table
1).
It
is
interesting
to
note
that
the
physiological
responses
of
the
WT
and
negative
plants
were
similar,
as
no
significant
dif-
ferences
between
them
were
observed
in
any
of
the
parameters
analyzed.
Sugars
were
the
only
solutes
that
increased
in
leaves
of
the
positive
plants,
while
K
+
increased
in
roots.
No
significant
dif-
ferences
were
found
in
the
proline
content
in
leaves,
whereas
the
root
proline
content
significantly
increased
in
the
positive
plants
compared
to
the
WT
and
negative
plants.
ABA
contents
were
simi-
lar
in
roots
and
leaves
of
the
different
types
of
plants.
With
respect
to
the
changes
induced
by
tas14
overexpression
in
w
and
s
,
the
positive
plants
showed
a
water
potential
less
reduced
by
drought
and
an
osmotic
potential
that
was
more
reduced,
with
respect
to
the
WT
and
negative
plants,
so
the
leaf
turgor
potential
(the
dif-
ference
between
w
and
s
)
was
higher
in
leaves
of
the
positive
plants
(Table
1).
Because
plants
may
respond
to
drought
by
using
different
toler-
ance
mechanisms,
depending
on
how
the
stress
is
applied
as
well
as
on
the
duration
of
the
treatment,
negative
and
positive
plants
were
submitted
to
two
successive
cycles
of
complete
withholding
of
irri-
gation
solution
for
7
days,
with
1
day
of
rewatering
and
recovery
of
plants
between
the
two
cycles.
The
RWC
was
maintained
relatively
constant
after
2
and
4
days
of
withholding
irrigation
in
both
nega-
tive
and
positive
plants
(around
90%),
while
the
reductions
induced
by
drought
at
the
6th
day
of
each
dehydration
cycle
were
signif-
icantly
lower
in
the
positive
plants
compared
with
the
negative
plants,
reaching
values
between
75–80%
in
positive
and
50–56%
in
the
negative
plants.
Clear
dehydration
visual
symptoms
(leaf
wilt-
ing)
in
negative
plants
were
observed
after
the
6th
day
of
treatment,
which
was
not
the
case
for
positive
plants
(Supplementary
Fig.
1e).
On
the
basis
of
these
results,
a
physiological
analysis
was
carried
out
at
the
beginning
of
the
experiment
and
after
4
days
of
water
withholding
in
the
first
and
second
dehydration
cycles,
as
neither
the
negative
nor
positive
plants
showed
dehydration
symptoms
yet
at
this
time.
Under
well-irrigated
conditions,
the
physiological
responses
of
negative
and
positive
plants
were
quite
similar
(data
not
shown).
Under
drought,
roots
of
plants
overexpressing
tas14
reduced
their
osmotic
potential
(
s
)
more
than
those
of
the
neg-
ative
plants
in
both
dehydration
cycles,
with
similar
negative
s
values
being
achieved
in
the
first
and
second
cycles
(Fig.
2a).
The
stressed-leaf
s
values
were
also
more
negative
in
positive
than
in
negative
plants
in
both
dehydration
cycles,
although
the
s
val-
ues
in
both
positive
and
negative
plants
were
more
reduced
in
the
second
than
in
the
first
dehydration
cycle
(Fig.
2a).
Sugars
were
the
solutes
that
increased
more
rapidly
in
positive
plants
com-
pared
to
the
negative
plants,
as
they
significantly
rose
from
the
first
dehydration
cycle
in
leaves
and
roots
and
the
differences
were
maintained
in
the
second
cycle
(Fig.
2b).
However,
significant
K
+
increases
in
leaves
and
roots
of
the
positive
plants
were
not
found
until
the
second
dehydration
cycle
(Fig.
2c).
With
respect
to
the
osmolite
proline,
increases
were
induced
by
drought
stress
in
roots
and
leaves
of
both
negative
and
positive
plants,
but
these
increases
are
significantly
higher
in
the
positive
plants,
especially
in
the
first
dehydration
cycle
(Fig.
2d).
Leaf
and
root
ABA
concentrations
of
the
negative
and
posi-
tive
plants
were
analyzed
at
the
beginning
of
the
experiment
and
after
4
days
of
water
withholding
in
the
first
and
second
dehydra-
tion
cycles.
Differences
between
negative
and
positive
plants
were
observed
only
in
leaves
(Fig.
3a),
but
not
in
roots
(data
not
shown).
The
leaves
of
the
positive
plants
accumulated
significantly
more
ABA
(2.5
nmol
g
1
FW)
than
those
of
the
negative
ones
in
the
first
dehydration
cycle
(1.4
nmol
g
1
FW).
In
the
second
cycle,
where
the
ABA
concentrations
were
higher
than
in
the
first,
similar
levels
were
achieved
in
the
leaves
of
both
types
of
plants
(Fig.
3a).
A.
Mu
˜
noz-Mayor
et
al.
/
Journal
of
Plant
Physiology
169 (2012) 459–
468 463
Table
1
Effect
of
TAS14
overexpression
on
plant
physiological
response
after
30
days
of
drought
treatment.
Plant
part
Line
Sugars
(mM)
K
+
(mM)
Proline
(mM)
ABA
(nmol
g
1
FW)
Osmotic
potential
(MPa)
Water
potential
(MPa)
Turgor
potential
(MPa)
Leaf WT 71.3 ±
7.7
130.4 ±
2.6
0.93
±
0.12
1.99
±
0.25
1.01
±
0.023
0.76
±
0.031
0.25
±
0.028
()
87.5
±
4.8
136.8
±
4.4
1.03
±
0.11
2.08
±
0.36
0.97
±
0.041
0.79
±
0.029
0.18
±
0.007
(+)
113.2
±
6.7
*
127.7
±
7.3
0.76
±
0.13
1.60
±
0.17
1.11
±
0.044
*
0.69
±
0.020
*
0.42
±
0.039
*
Root WT
61.1
±
6.7
26.7
±
2.6
0.19
±
0.02
0.09
±
0.02
0.46
±
0.032
()
65.6
±
2.8
33.4
±
3.7
0.18
±
0.01
0.08
±
0.01
0.40
±
0.018
(+)
70.4
±
6.7
43.0
±
2.4
*
0.35
±
0.03
*
0.10
±
0.01
0.44
±
0.024
WT,
negative
()
and
positive
(+)
plants
were
subjected
to
drought
stress
by
irrigation
reduction
(30%
of
the
nutrient
solution
volume
added
to
well-irrigated
plants).
Values
are
the
mean
±
SE
of
eight
plants
*
Significant
differences
between
positive
plants
and
their
controls
(WT
and
negative
plants)
at
P
<
0.05
To
confirm
that
the
greater
increase
of
endogenous
ABA
in
leaves
of
the
tas14
overexpressing
plants
was
associated
with
the
action
mechanism
of
the
tas14
gene,
the
time
course
of
ABA
content
from
the
2nd
to
5th
days
of
the
first
dehydration
cycle
was
compared
in
leaves
of
the
positive
plants
(homozygous
line
for
tas14
gene),
and
a
homozygous
line
overexpressing
other
different
tomato
gene,
the
tsw12
gene,
which
is
involved
in
osmotic
stress
(Torres-Schumann
et
al.,
1992).
From
the
2nd
day
onward,
the
ABA
concentration
in
leaves
increased
significantly
with
drought
in
all
plants
(Fig.
3b).
The
time
course
of
the
ABA
accumulation
under
drought
stress
Fig.
2.
Effects
of
tas14
overexpression
on
plant
physiological
response
to
drought
in
plants
subjected
to
dehydration
cycles.
Osmotic
potential
(a),
sugar
(b),
K
+
(c),
and
proline
(d)
concentrations
were
measured
in
roots
(circles)
and
leaves
(squares)
of
negative
(open
symbols)
and
positive
(solid
symbols)
plants
at
the
beginning
of
the
experiment
(day
0)
and
after
4
days
water
withholding
in
the
first
and
second
dehydration
cycles.
Data
are
the
mean
±
SE
(n
=
8).
Significant
differences
at
P
<
0.05
between
lines
were
indicated
with
*.
464 A.
Mu
˜
noz-Mayor
et
al.
/
Journal
of
Plant
Physiology
169 (2012) 459–
468
Fig.
3.
Effect
of
tas14
overexpression
on
leaf
ABA
concentration
in
plants
subjected
to
dehydration
cycles.
(a)
ABA
concentration
was
measured
in
leaves
of
negative
(open
squares)
and
positive
(solid
squares)
plants
at
the
beginning
of
the
experiment
and
after
4
days
water
withholding
in
the
first
and
second
dehydration
cycles
(0,
1
and
2
in
abscises
axis).
(b)
Time-course
of
ABA
concentration
from
2nd
to
5th
day
of
first
cycle
of
dehydration
in
leaves
of
negative
(open
squares),
positive
(solid
squares)
plants
for
tas14
gene
overexpression,
and
a
transgenic
line
overexpressing
the
tomato
tsw12
gene
involved
in
the
osmotic
stress
(solid
triangles).
Data
are
the
mean
±
SE
(n
=
5).
Significant
differences
at
P
<
0.05
between
lines
were
indicated
with
*.
was
similar
in
the
negative
and
the
tsw12-overexpressing
plants,
increasing
linearly
during
the
progress
of
drought
stress
(between
2
and
5
days
of
water
withholding),
but
the
increases
were
sig-
nificantly
lower
than
in
the
tas14
overexpressing
plants
during
the
whole
period
of
analysis.
Interestingly,
the
highest
differences
between
tas14
overexpressing
and
the
other
plants
(negative
and
tsw12
plants)
were
found
at
the
3rd
dehydration
day.
Thus,
the
leaf
ABA
concentration
in
tas14
overexpressing
plants
was
2.4
nmol
g
1
FW,
whereas
in
negative
and
tsw12
overexpressing
plants
was
around
1.4
nmol
g
1
FW.
These
results
indicate
that
the
mechanism
of
action
of
tas14
is
associated
with
a
rapid
ABA
increase
in
leaves.
Phenotypic
characterization
and
salt
tolerance
mechanisms
induced
by
the
overexpression
of
tas14
gene
To
evaluate
the
effects
of
tas14
overexpression
on
plant
response
to
salinity,
the
same
negative
and
positive
plants
were
grown
from
the
2nd-leaf
stage
at
different
salt
stress
levels
(0,
75
and
150
mM
Fig.
4.
Effect
of
tas14
overexpression
on
plant
growth
response
to
salt
stress.
(a)
Shoot
biomass
was
quantified
in
negative
(open
squares)
and
positive
(solid
squares)
plants
grown
at
different
NaCl
levels
(0,
75
and
150
mM)
for
25
days.
(b)
Fruit
yield
was
quantified
in
WT
(open
circles),
negative
(open
squares)
and
positive
(solid
squares)
plants
grown
at
different
NaCl
levels
(0,
75
and
100
mM)
for
75
days.
Data
are
the
mean
±
SE
(n
=
8).
Significant
differences
at
P
<
0.05
between
lines
were
indicated
with
*.
NaCl)
for
25
days.
The
shoot
biomass
of
negative
and
positive
plants
was
similar
under
control
conditions,
while
with
mild
salt
stress
treatment
(75
mM
NaCl),
the
positive
plants
increased
their
growth
significantly
compared
to
the
negative
plants
after
25
days
of
treat-
ment
(Fig.
4a).
Moreover,
the
shoot
biomass
of
the
positive
plants
grown
at
75
mM
NaCl
was
similar
to
that
of
the
positive
plants
grown
without
salt.
However,
at
a
lower
degree,
a
positive
effect
was
also
observed
at
150
mM
NaCl
for
the
same
period
(Fig.
4a).
These
results
show
that
tas14
also
plays
an
essential
role
during
salt
stress
in
tomato.
To
determine
whether
the
higher
salt
tolerance
induced
by
over-
expression
of
tas14
was
prolonged
along
the
growth
cycle,
the
fruit
yield
of
the
WT,
negative
and
positive
plants
was
determined
after
75
days
at
0,
75
and
100
mM
NaCl
(Fig.
4b).
Fruit
yield
increased
in
saline
medium
in
the
positive
plants
compared
with
WT
and
A.
Mu
˜
noz-Mayor
et
al.
/
Journal
of
Plant
Physiology
169 (2012) 459–
468 465
negative
plants,
with
the
greatest
increase
being
observed
at
mild
salt
levels
(75
mM
NaCl).
The
salinity
physiological
response
was
examined
by
separately
analyzing
young
and
adult
leaves
as
well
as
roots
in
the
plants
of
the
first
experiment
(salt
levels
of
75
and
150
mM
NaCl).
As
shown
in
Table
2,
s
of
the
different
plant
parts
analyzed
decreased
up
to
sig-
nificantly
lower
values
in
positive
plants
compared
with
negative
ones
at
mild
salt
stress
treatment
(75
mM
NaCl),
where
the
most
important
mechanism
contributing
to
the
salt
tolerance
may
be
the
osmotic
tolerance
mechanism.
At
high
stress
level
(150
mM
NaCl),
where
the
toxic
effect
may
be
more
important
than
the
osmotic
effect,
there
was
also
significant
differences
between
the
s
of
negative
and
positive
plants
in
young
and
adult
leaves,
but
sim-
ilar
values
were
found
in
roots
(Table
2).
In
young
leaves,
the
K
+
and
sugar
concentrations
were
significantly
higher
in
positive
than
in
negative
plants
for
both
salt
levels,
whereas
similar
increases
of
young
leaf
sap
Na
+
concentrations
were
found
in
both
negative
and
positive
plants.
However,
the
situation
changed
in
adult
leaves,
as
the
Na
+
concentration
increased
in
positive
plants,
with
respect
to
the
negative
plants
at
75
and
150
mM
NaCl,
whereas
there
were
no
differences
in
K
+
and
sugars
concentrations.
In
roots,
significant
Na
+
increases
were
only
found
at
75
mM
NaCl,
which
agrees
with
the
significant
s
reduction
at
this
level.
Proline
was
also
analyzed
in
this
experiment.
The
salinity
treatments
increased
the
proline
levels
in
the
three
plant
parts
studied,
but
the
increases
were
sig-
nificantly
higher
in
positive
plants,
except
for
adult
leaves
of
plants
subjected
to
150
mM
NaCl
(Table
2).
It
is
interesting
to
note
the
very
high
levels
of
this
osmolite
achieved
in
young
leaves
of
positive
plants.
Finally,
we
attempted
to
ascertain
whether
the
overexpression
of
tas14
enhanced
salt
tolerance
not
only
at
the
whole
plant
level
but
also
at
the
cellular
level.
Moreover,
we
tried
to
confirm
the
ability
of
tas14
gene
to
avoid
loss
of
cellular
water
as
well
as
to
determine
whether
the
mechanism
of
tas14
gene
was
or
was
not
associated
to
a
higher
Na
+
accumulation
within
the
cells.
The
calli
regenerated
from
leaves
of
positive
plants
showed
significantly
higher
fresh
weight
and
water
content
gains
than
the
negative
ones
cultured
for
30
days
in
a
medium
containing
100
and
150
mM
NaCl
(Fig.
5a
and
b).
It
is
interesting
to
point
out
the
significant
increases
of
water
contents
in
positive
calli
grown
under
salt
stress,
which
does
not
occur
under
control
conditions
(Fig.
5b).
These
results
indi-
cate
that
the
overexpression
of
tas14
gene
also
increases
the
salt
tolerance
at
the
cellular
level,
similarly
to
the
response
observed
at
the
whole
plant
level.
Na
+
concentration
was
also
measured
at
the
end
of
the
experiment
(Fig.
5c).
The
Na
+
accumulation
was
simi-
lar
at
100
mM
NaCl
for
both
types
of
calli
but
significantly
lower
at
150
mM
NaCl
in
the
positive
calli
compared
with
negative
ones.
Discussion
The
plants
overexpressing
tas14
gene
with
the
constitutive
pro-
moter
35S
did
not
exhibit
morphological
or
significant
growth
differences
under
unstressed
conditions,
compared
to
wild
type
plants
(Supplementary
Fig.
1b).
This
is
a
good
feature
for
the
poten-
tial
use
in
biotechnology
of
this
gene
in
improving
abiotic
stress
resistance,
since
the
constitutive
overexpression
of
most
stress-
related
genes
generally
causes
slower
growth
and,
consequently,
impacts
negatively
on
the
plant
growth
and
yield
under
non-
stressed
conditions
(Mu
˜
noz-Mayor
et
al.,
2008;
Ray
et
al.,
2009).
The
overexpression
of
the
tas14
gene
enhanced
drought
tolerance
on
the
basis
of
shoot
biomass
(Fig.
1).
It
is
interesting
that
the
positive
effect
of
the
tas14
gene
was
shown
in
spite
of
the
severe
drought
stress
level
applied
in
long-term
assays,
according
to
the
important
reduction
induced
by
drought
stress
in
shoot
vegetative
biomass
and,
especially,
in
fruit
biomass,
in
negative
and
WT
plants,
Fig.
5.
Effect
of
tas14
overexpression
on
callus
growth
response
to
salt
stress.
Calli
were
initiated
from
leaf
explants
of
negative
(open
squares)
and
positive
(solid
squares)
seedlings,
and
further
subcultured
in
medium
with
0,
100
and
150
mM
NaCl.
Fresh
weight
(a),
water
content
(b)
and
Na
+
concentration
(c)
were
determined
after
30
days
of
culture.
Data
are
the
mean
±
SE
(n
=
30).
Significant
differences
at
P
<
0.05
between
lines
were
indicated
with
*.
which
suggests
that
this
gene
has
an
important
role
in
drought
tol-
erance
in
tomato.
It
is
especially
relevant
that
overexpression
of
tas14
gene
increases
drought
tolerance,
since
comparatively
much
less
progress
has
been
made
in
genetics
and
breeding
of
tomatoes
for
drought
tolerance
(Foolad,
2007).
466 A.
Mu
˜
noz-Mayor
et
al.
/
Journal
of
Plant
Physiology
169 (2012) 459–
468
Table
2
Effect
of
TAS14
overexpression
on
plant
physiological
response
after
25
days
of
salt
stress.
Plant
part
NaCl
(mM)
Line
Osmotic
potencial
(MPa)
Na
+
(mM)
K
+
(mM)
Sugars
(mM)
Proline
(mM)
Young
leaf
75
()
1.19
±
0.07
37.6
±
5.3
123.3
±
11.3
44.2
±
6.5
3.10
±
0.41
(+) 1.30
±
0.05
*
45.6 ±
2.6
152.2 ±
9.3
*
58.2
±
3.4
*
8.01
±
1.29
*
150 ()
1.40
±
0.04
92.0
±
4.8
100.1
±
4.5
50.1
±
4.4
8.40
±
0.95
(+)
1.55
±
0.07
*
96.0
±
9.8
125.0
±
5.6
*
65.0
±
4.2
*
11.2
±
1.05
*
Adult
leaf 75
()
1.19
±
0.07
45.0
±
2.8
121.1
±
7.9
42.3
±
3.5
1.60
±
0.30
(+)
1.33
±
0.03
*
73.5
±
3.2
*
129.8
±
6.8
37.4
±
4.4
2.97
±
0.70
*
150
()
1.40
±
0.02
105.0
±
8.0
121.0
±
8.3
39.0
±
3.6
4.90
±
0.60
(+)
1.89
±
0.01
*
140.3
±
12.2
*
128.7
±
4.0
45.7
±
5.0
4.60
±
0.31
Root 75 ()
0.90
±
0.10
131.1 ±
4.2
72.4 ±
4.1
23.4
±
3.7
1.39
±
0.16
(+)
1.20
±
0.07
*
176.9
±
17.3
*
67.9
±
5.6
40.8
±
5.3
*
2.08
±
0.19
*
150 ()
1.33
±
0.14
224.0
±
8.4
50.7
±
9.8
38.2
±
5.8
1.90
±
0.04
(+)
1.36
±
0.08
198.5
±
21.8
56.0
±
5.0
41.2
±
5.6
4.80
±
0.50
*
()
and
(+),
negative
and
positive
plants
were
subjected
to
75
and
150
mM
NaCl.
Values
are
the
mean
±
SE
of
eight
plants
*
Significant
differences
between
lines
at
P
<
0.05
The
overexpression
of
the
tas14
gene
also
increased
salt
tol-
erance,
as
the
shoot
biomass
was
significantly
greater
in
positive
than
in
negative
plants,
with
the
positive
plants
achieving
simi-
lar
shoot
growth
in
medium
without
salt
and
with
75
mM
NaCl
(Fig.
4a).
Moreover,
salt
tolerance
induced
by
overexpression
of
tas14
was
enhanced
throughout
the
growth
cycle,
as
fruit
yield
was
greater
in
positive
than
in
negative
plants
grown
in
saline
medium,
especially
at
75
mM
NaCl
(Fig.
4b).
In
this
study,
the
salt
response
was
also
studied
at
the
cellular
level,
as
several
studies
have
shown
the
role
of
dehydrins
in
ameliorating
the
cellular
effects
of
abiotic
stress
(Battaglia
et
al.,
2008;
Bae
et
al.,
2009).
tas14
over-
expressing
calli
significantly
increased
salt
tolerance
compared
with
negative
calli,
and
this
positive
effect
on
the
growth
was
due
mainly
to
the
high
callus
water
content
(Fig.
5).
Consider-
ing
that
tolerance
at
the
cell
level
in
tomato
is
associated
with
the
ability
to
avoid
dehydration
(Rus
et
al.,
1999),
these
results
corroborate
the
idea
that
the
tas14
gene
seems
to
function
by
increasing
cellular
water
content.
Taken
together,
these
results
sug-
gest
that
tas14
overexpressing
plants
improve
drought
and
salinity
tolerance
without
affecting
the
plant
growth
under