Responses of transgenic Arabidopsis plants and recombinant yeast cells expressing a novel durum wheat manganese superoxide dismutase TdMnSOD to various abiotic stresses

Abstract

In plant cells, the manganese superoxide dismutase (Mn-SOD) plays an elusive role in the response to oxidative stress. In this study, we describe the isolation and functional characterization of a novel Mn-SOD from durum wheat (Triticum turgidum L. subsp. Durum), named TdMnSOD. Molecular phylogeny analysis showed that the durum TdMnSOD exhibited high amino acids sequence identity with other Mn-SOD plants. The three-dimensional structure showed that TdMnSOD forms a homotetramer and each subunit is composed of a predominantly-helical N-terminal domain and a mixed / C-terminal domain. TdMn-SOD gene expression analysis showed that this gene was induced by various abiotic stresses in durum wheat. The expression of TdMnSOD enhances tolerance of the transformed yeast cells to salt, osmotic, cold and H 2 O 2-induced oxidative stresses. Moreover, the analysis of TdMnSOD transgenic Arabidopsis plants subjected to different environmental stresses revealed low H 2 O 2 and high proline levels as compared to the wild-type plants. Compared with the non-transformed plants, an increase in the total SOD and two other antioxidant enzyme activities including catalase (CAT) and peroxidases (POD) was observed in the three transgenic lines subjected to abiotic stress. Taken together, these data provide evidence for the involvement of durum wheat TdMnSOD in tolerance to multiple abiotic stresses in crop plants.

Full text

Journal
of
Plant
Physiology
198
(2016)
56–68
Contents
lists
available
at
ScienceDirect
Journal
of
Plant
Physiology
journa
l
h
om
epage:
www.elsevier.com/locate/jplph
Molecular
biology
Responses
of
transgenic
Arabidopsis
plants
and
recombinant
yeast
cells
expressing
a
novel
durum
wheat
manganese
superoxide
dismutase
TdMnSOD
to
various
abiotic
stresses
Feki
Kaouthara,
Farhat-Khemakhem
Amenyb,
Kamoun
Yosrac,
Saibi
Walida,
Gargouri
Alic,
Brini
Faic¸
ala,∗
aBiotechnology
and
Plant
Improvement
Laboratory,
Centre
of
Biotechnology
of
Sfax,
Tunisia
bLaboratory
of
Microorganisms
and
Biomolecules,
Centre
of
Biotechnology
of
Sfax,
Tunisia
cLaboratory
of
Molecular
Biotechnology
of
Eukaryotes,
Centre
of
Biotechnology
of
Sfax,
Tunisia
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
7
December
2015
Received
in
revised
form
23
March
2016
Accepted
23
March
2016
Available
online
28
April
2016
Keywords:
Triticum
durum
Oxidative
stress
Abiotic
stress
Superoxide
dismutase
Reactive
oxygen
species
Antioxidant
enzymes
a
b
s
t
r
a
c
t
In
plant
cells,
the
manganese
superoxide
dismutase
(Mn-SOD)
plays
an
elusive
role
in
the
response
to
oxidative
stress.
In
this
study,
we
describe
the
isolation
and
functional
characterization
of
a
novel
Mn-SOD
from
durum
wheat
(Triticum
turgidum
L.
subsp.
Durum),
named
TdMnSOD.
Molecular
phylogeny
analy-
sis
showed
that
the
durum
TdMnSOD
exhibited
high
amino
acids
sequence
identity
with
other
Mn-SOD
plants.
The
three-dimensional
structure
showed
that
TdMnSOD
forms
a
homotetramer
and
each
subunit
is
composed
of
a
predominantly
␣-helical
N-terminal
domain
and
a
mixed
␣/␤
C-terminal
domain.
TdMn-
SOD
gene
expression
analysis
showed
that
this
gene
was
induced
by
various
abiotic
stresses
in
durum
wheat.
The
expression
of
TdMnSOD
enhances
tolerance
of
the
transformed
yeast
cells
to
salt,
osmotic,
cold
and
H2O2-induced
oxidative
stresses.
Moreover,
the
analysis
of
TdMnSOD
transgenic
Arabidopsis
plants
subjected
to
different
environmental
stresses
revealed
low
H2O2and
high
proline
levels
as
compared
to
the
wild-type
plants.
Compared
with
the
non-transformed
plants,
an
increase
in
the
total
SOD
and
two
other
antioxidant
enzyme
activities
including
catalase
(CAT)
and
peroxidases
(POD)
was
observed
in
the
three
transgenic
lines
subjected
to
abiotic
stress.
Taken
together,
these
data
provide
evidence
for
the
involvement
of
durum
wheat
TdMnSOD
in
tolerance
to
multiple
abiotic
stresses
in
crop
plants.
©
2016
Elsevier
GmbH.
All
rights
reserved.
1.
Introduction
It
is
known
that
drought
and
salt
are
among
the
most
limit-
ing
abiotic
stresses
to
crop
productivity
and
yield.
Variability
of
salt
resistance
within
wheat
plants
was
explained
by
the
presence
of
Kna1
locus
on
chromosome
4DL
of
bread
wheat
(Triticum
aes-
tivum),
which
has
been
linked
to
sodium
exclusion
from
the
leaves
(Dvorák
et
al.,
1994;
Dubcovsky
et
al.,
1996).
High
salinity
causes
a
primary
effect
like
hyperosmotic
stress
and
ion
disequilibrium
producing
secondary
effects,
such
as
ion
toxicity,
oxidative
stress,
hormonal
imbalances
and
nutrient
disturbances
(Deinlein
et
al.,
2014;
Ismail
et
al.,
2014).
The
oxidative
damage
is
a
consequence
of
various
abiotic
environmental
stresses,
and
occurs
through
the
accumulation
of
reactive
oxygen
species
(ROS).
In
plant
cells,
ROS
are
generated
at
low
levels
during
different
metabolic
processes.
∗Corresponding
author
at:
Biotechnology
and
Plant
Improvement
Laboratory,
Centre
of
Biotechnology
of
Sfax:
CBS,
Route
Sidi
Mansour
Km
6,
B.P’1177’,
3018
Sfax,
Tunisia.
E-mail
address:
faical.brini@cbs.rnrt.tn
(B.
Faic¸
al).
Nevertheless,
at
high
levels
ROS
become
toxic
and
affect
nega-
tively
many
normal
cellular
functions,
damaging
DNA,
proteins
and
causing
lipid
peroxidation
(Choudhury
et
al.,
2013;
Miller
et
al.,
2008;
Mittler,
2011).
The
superoxide
radical
(O2
•−)
and
hydrogen
peroxide
(H2O2)
are
included
among
the
ROS.
The
first
is
quickly
converted
to
H2O2and
molecular
oxygen
through
the
involvement
of
the
superoxide
dismutase
(SOD).
H2O2is
then
scavenged
by
cata-
lase
(CAT)
and
different
classes
of
peroxidase
(POD).
In
addition
to
the
enzymatic
components,
plants
produce
non-enzymatic
compo-
nents
such
as
ascorbate,
tocopherols,
carotenoids,
and
glutathione
in
order
to
maintain
redox
homeostasis
(Bowler
et
al.,
1992;
Gill
and
Tuteja,
2010;
Mhamdi
et
al.,
2010;
Suzuki
et
al.,
2012).
Known
as
an
osmoprotectant,
proline
can
act
as
a
potent
non-enzymatic
antioxidant
(Rejeb
et
al.,
2014).
Despite
the
destructive
role
in
cells,
at
moderate
concentrations
ROS
can
act
as
signaling
molecules
in
many
biological
processes,
and
can
also
modulate
the
activi-
ties
of
many
components
in
signaling,
such
as
protein
kinase
and
phosphatase
(Demiral
et
al.,
2011).
Thus,
plants
adjust
their
con-
centrations
between
certain
thresholds
by
means
of
production
and
scavenging
mechanisms.
The
antioxidant
defense
system
produced
http://dx.doi.org/10.1016/j.jplph.2016.03.019
0176-1617/©
2016
Elsevier
GmbH.
All
rights
reserved.

F.
Kaouthar
et
al.
/
Journal
of
Plant
Physiology
198
(2016)
56–68
57
to
regulate
ROS
in
cells
differs
from
one
plant
to
another,
for
exam-
ple,
between
C3and
C4species
under
osmotic
stress
(Uzilday
et
al.,
2014).
In
response
to
abiotic
stress,
it
has
been
reported
that
salt
stress
and
drought
primarily
increase
the
activity
of
some
antioxi-
dant
enzymes
in
numerous
tolerant
plants
like
sesame
(Koca
et
al.,
2007),
barley
(Khosravinejad
et
al.,
2008),
tomato
(Gapinska
et
al.,
2008),
cotton
(Sekmen
et
al.,
2014)
and
pea
(Hernández
et
al.,
1993,
2000).
Moreover,
the
activation
of
SOD
activity
is
observed
in
vari-
ous
plants
exposed
to
short-
or
long-term
Mn-toxicity,
suggesting
that
SOD
is
involved
in
the
detoxification
mechanism
against
oxida-
tive
stress
caused
by
manganese
excess
(Del
Rio
et
al.,
1985;
Shi
et
al.,
2006;
Shenker
et
al.,
2004;
Fonseca
et
al.,
2013).
In
aerobic
organisms,
SOD
plays
a
crucial
role
in
the
defense
against
oxidative
stress.
Based
on
the
metal
co-factor
used
by
SOD,
there
are
usually
Cu/Zn-SOD
and
Fe-SOD
isoenzymes
which
are
associated
with
the
chloroplasts
and
cytoplasm,
and
an
Mn-SOD
isoenzyme
associated
with
mitochondria
(Bowler
et
al.,
1994;
Mittler,
2002).
It
was
sug-
gested
that
Mn
and
Fe-SOD
proteins
are
the
ancient
types
of
SODs,
which
probably
arose
from
the
same
ancestral
enzyme.
The
amino
acid
sequences
of
Cu/Zn-SOD
are
different
from
the
other
isoen-
zymes
and
probably
have
evolved
separately
in
eukaryotes
cells
(Grene,
2002).
Mn-SOD
is
a
metalloenzyme
essential
for
the
sur-
vival
of
all
aerobic
organisms
and
can
act
as
a
tumor
suppressor
by
modulating
ROS
levels
in
cancer
cells
(Holley
et
al.,
2012;
Miriyala
et
al.,
2012).
The
enhancement
of
SOD
activity
has
been
reported
in
vari-
ous
tolerant
plants
against
various
abiotic
stresses.
Furthermore,
the
introduction
of
SOD
genes
in
several
transgenic
plants
results
in
more
efficient
elimination
of
ROS
under
stress
conditions,
sug-
gesting
the
roles
of
SODs
in
plants’
tolerance
to
environmental
stress.
Drought
tolerance
was
enhanced
in
rice
and
Arabidopsis
transgenic
plants
overexpressing
pea
and
jojoba
MnSOD
genes,
respectively
(Liu
et
al.,
2013;
Wang
et
al.,
2005).
Salt
tolerance
was
also
observed
in
transgenic
Arabidopsis
and
poplar
plants
over-
expressing
MnSOD
genes
derived
from
Arabidopsis
and
Tamarix
androssowii
(Wang
et
al.,
2004;
Wang
et
al.,
2010).
The
expression
of
other
SOD
isoenzymes,
Cu/Zn-SOD
and
Fe-SOD,
enhances
oxida-
tive
stress
tolerance
of
the
transgenic
plants
(McKersie
et
al.,
2000;
Prashanth
et
al.,
2008;
Sen
Gupta
et
al.,
1993).
FeSOD
expression
in
transgenic
alfalfa
increases
winter
survival
(McKersie
et
al.,
2000).
However,
it
has
been
reported
in
others
studies
that
the
improve-
ment
of
several
transgenic
plants
with
extragenetic
SODs
was
not
achieved
(Tepperman
and
Dunsmuir,
1990;
Pitcher
et
al.,
1991;
Payon
et
al.,
1997).
This
may
be
attributed
to
the
complexity
of
the
ROS
detoxification
system
and
the
differences
between
SOD
isoen-
zymes.
Despite
the
identification
of
Mn-SOD
genes
responsible
for
abiotic
stress
tolerance
in
many
plants
species,
little
is
known
about
the
Mn-SOD
gene
from
durum
wheat.
To
the
best
of
our
knowledge,
this
is
the
first
study
on
the
function
of
durum
wheat
Mn-SOD
in
response
to
multiple
abiotic
stresses
of
both
the
yeast
system
and
Arabidopsis
plants.
Here,
we
report
on
the
isolation
and
the
functional
characterization
of
a
novel
durum
wheat
superoxide
dis-
mutase
Mn-SOD,
which
was
named
TdMnSOD.
The
results
showed
that
the
TdMnSOD
protein
was
functional
in
the
yeast
system
and
conferred
tolerance
of
both
yeast
cells
and
transgenic
Arabidopsis
plants
to
various
abiotic
stresses.
2.
Materials
and
methods
2.1.
Plant
material,
growth
conditions
and
stress
treatments
Seeds
of
durum
wheat
(Triticum
turgidum
L.
subsp.
durum)
cul-
tivar
Om
Rabia3
(OR3)
were
provided
by
the
Tunisian
Agronomic
Research
Institute.
They
were
sterilized
and
then
germinated
on
Petri
dishes
as
described
by
Brini
et
al.
(2009).
After
the
germina-
tion
in
sterile
water
solution,
seedlings
were
placed
in
containers
containing
modified
half-strength
Hoagland’s
solution
(Epstein,
1972)
for
one
week,
and
in
the
glasshouse
conditions
(25
±
5◦C,
16
h
photoperiod
and
60
±
10%
relative
humidity).
For
stress
treat-
ments,
seedlings
were
transferred
to
different
solutions
containing
200
mM
NaCl
(salt
stress),
15%
PEG
(6000)
(polyethylene
glycol
with
an
average
molecular
weight
of
6000)
(osmotic
stress)
or
10
mM
H2O2(oxidative
stress).
After
3
days
of
stress
application,
samples
(whole
plants)
were
taken
at
different
times
of
exposure
(0–3
and
6
days),
immersed
in
liquid
nitrogen,
and
then
stored
at
−80 ◦C
for
RNA
isolation
and
expression
analysis.
2.2.
Isolation
and
cloning
of
durum
wheat
TdMnSOD
cDNA
Total
RNA
was
extracted
from
whole
10-day-old
durum
wheat
seedlings
(cv.
OR3)
treated
with
salt
stress
(200
mM
NaCl)
for
3
days,
using
the
TRIZOL
reagent
(Invitrogen).
To
remove
the
contaminating
genomic
DNA,
total
RNA
(10
␮g)
was
treated
with
DNase
(Promega).
The
cDNA
was
synthesized
from
DNase-
treated
RNA
samples
(0.5
␮g)
using
M-MLV
reverse
transcriptase
(Invitrogen)
at
37 ◦C
for
1
h
and
using
the
oligo-dT
(18
mer)
primer.
Specific
primers
designed
from
the
nucleotide
sequence
of
Mn-SOD
bread
wheat
(accession
no.
AF092524.1),
which
are
S1
(5-
GCTCTAGAATGGCGCTCCGCACGTTGGCCGCG-3,
XbaI
site
under-
lined)
and
S2
(5-AGGGATCCTCACGCAAGCACTTTTTCATACTCTT-3,
BamHI
site
underlined),
were
chosen
to
amplify
by
PCR
the
open
reading
sequence
of
durum
wheat
manganese
superox-
ide
dismutase
(TdMnSOD).
The
resulting
fragment
was
cloned
in
the
downstream
of
the
constitutive
cauliflower
mosaic
virus
(CaMV)
35S
promoter
between
the
XbaI
and
BamHI
sites
of
the
pBI321
binary
vector
containing
the
hygromycin-resistant
selectable
marker
(Atienza
et
al.,
2007).
The
resultant
plas-
mid,
called
BSOD,
was
sequenced
and
used
as
a
template
to
amplify
the
open
reading
sequence
of
TdMnSOD
using
the
primers
S3
(5-TAGGATCCATGGCGCTCCGCACGTTGGCCGCG-3BamHI
site
underlined)
and
S4
(5-AGGAATTCCGCAAGCACTTTTTCATACTCTT-
3,
EcoRI
site
underlined).
The
resulting
fragment
was
cloned
in
the
expression
vector
pET28a
into
BamHI
and
EcoRI
restriction
sites,
generating
the
recombinant
vector
pSOD.
2.3.
Sequence
analysis
and
three-dimensional
structure
determination
Sequence
analysis
and
multiple
sequence
alignments
were
per-
formed
using
the
biological
sequence
editor
software
BioEdit
7.0.0.
To
predict
the
phylo-genomic
relationships
between
TdMnSOD
gene
and
others
MnSOD
genes,
we
used
the
Molecular
Evolutionary
Genetics
Analysis
5
(MEGA
5)
program
(Tamura
et
al.,
2011).
The
prediction
of
the
protein
secondary
structure
was
per-
formed
using
the
DSSP
program
(Kabsch
and
Sander
1983).
The
automated
comparative
protein
structure
homology
model-
ing
server,
Protein
Homology/analogy
Recognition
Engine
V2.0
(http://www.sbg.bio.ic.ac.uk/phyre2)
was
used
to
generate
the
three-dimensional
structural
model
of
TdMnSOD
using
the
crystal
structure
of
manganese
superoxide
dismutase
from
Arabidop-
sis
thaliana
as
template
(PDB-code
4C7U)
(Marques
et
al.,
2014).
PyMOL
(http://www.pymol.org)
was
used
to
visualize
and
analyze
the
generated
three-dimensional
structural
model
and
to
construct
illustrative
figures.
2.4.
Expression
and
purification
of
TdMnSOD
protein
After
verification
of
the
recombinant
vector
pSOD
by
PCR
analy-
sis,
restriction
analysis
and
DNA
sequencing,
it
was
transformed
into
Escherichia
coli
strain
BL21
cells
(DE3)
for
expression.
The
transformants
were
grown
at
37 ◦C
until
A600 reached
0.6.
The

58
F.
Kaouthar
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al.
/
Journal
of
Plant
Physiology
198
(2016)
56–68
expression
was
induced
by
adding
1
mM
IPTG
(isopropyl
␤-d-1-
thiogalactopyranoside),
and
the
culture
was
kept
for
2
h.
An
aliquot
of
culture
was
removed
and
the
expression
of
the
His-tagged
TdMn-
SOD
was
detected
by
SDS-PAGE.
The
purification
was
done
with
Ni-NTA
column
(QIAGEN).
2.5.
In
vitro
TdMnSOD
activity
and
enzyme
activity
staining
Different
concentrations
of
the
His-tagged
TdMnSOD
protein
were
added
to
the
reaction
mixture
containing
50
mM
potassium
phosphate
buffer
(pH
7.8),
0.1
mM
EDTA,
13
mM
methionine,
2
␮M
riboflavin
and
75
␮M
NBT
(Nitroblue
tetrazolium).
The
mixture
was
exposed
to
cool
white
fluorescent
light
at
a
photosynthetic
pho-
ton
flux
of
50
␮mol
m−2s−1for
15
min.
The
developed
blue
color
in
the
reaction
was
measured
spectrophotometrically
at
560
nm
(McCord
and
Fridovich,
1969).
TdMnSOD
activity
was
expressed
as
units.
One
unit
of
TdMnSOD
was
defined
as
the
volume
of
sample
causing
50%
inhibition
in
color
development.
Staining
for
SOD
activity
was
carried
out
as
described
by
Beauchamp
and
Fridovich
(1971).
TdMnSOD
was
visualized
in
gel
electrophoresis
in
10%
(w/v)
polyacrylamide
slab
gel,
under
native
conditions.
This
gel
was
soaked
in
50
mM
sodium
phosphate
(pH
7.5)
containing
4.8
mM
NBT
in
darkness
for
20
min,
followed
by
soaking
in
50
mM
sodium
phosphate
(pH
7.5)
containing
0.4%
(v/v)
N,N,N’,N’-tetramethylethylenediamine
(TEMED)
and
26
mM
riboflavin,
and
subsequently
illuminated
for
10
min.
Mn-SOD
was
visualized
by
its
insensitivity
to
5
mM
H2O2and
2
mM
KCN.
2.6.
Expression
of
TdMnSOD
in
saccharomyces
cerevisiae
and
stress
tolerance
assays
The
yeast
strain
W303-1B
(MAT˛
ade2
ura3
leu2
his3
trp1)
was
used
to
determine
the
role
of
durum
wheat
TdMnSOD
in
yeast
stress
tolerance.
Before
yeast
transformation,
the
cDNA
fragment
was
released
from
the
recombinant
plasmid
pSOD
by
digestion
with
BamHI
and
EcoRI,
and
then
inserted
into
the
BamHI/EcoRI
sites
of
pYES2
vector
(Invitrogen),
generating
the
recombinant
yeast
vec-
tor
pYES2-TdMnSOD.
The
used
vector
is
a
2-␮m-based
multi-copy
yeast
plasmid
containing
the
URA3
gene
and
the
Gal1
promoter
for
selection
and
expression
in
yeast,
respectively.
Yeast
cells
were
transformed
as
described
by
Ito
et
al.
(1983),
with
the
pYES2
empty
vector
or
with
pYES2-TdMnSOD
construct.
After
the
selection
of
the
recombinant
colonies
on
Yeast
Nitrogen
Base
plates
containing
2%
glucose
and
lacking
uracil
(YNB
Ura−),
stress
tolerance
assays
were
performed
using
the
same
medium
with
2%
galactose
containing
or
not
containing
4
␮M
H2O2,
NaCl
(0.5
and
0.75
M)
or
1.2
M
mannitol.
For
cold
treatment,
the
same
yeast
cells
were
placed
in
a
refriger-
ator
at
−20 ◦C
alcohol
bath
for
24
h.
Aliquots
from
saturated
yeast
cultures
(5
␮l)
and
tenfold
serial
dilutions
(10−1,
10−2and
10−3)
were
spotted
onto
these
media
and
incubated
at
30 ◦C
for
5–6
days.
2.7.
Semi-quantitative
RT-PCR
analysis
Total
RNA
was
isolated
using
the
TRIZOL
reagent
(Invitrogen)
from
frozen
10-day-old
durum
wheat
seedlings
treated
or
not
with
200
mM
NaCl,
15%
(w/v)
PEG
(6000)
and
10
mM
H2O2.
To
remove
the
DNA,
total
RNA
(10
␮g)
was
treated
with
DNaseI
(Promega)
at
37 ◦C
for
15
min
and
further
incubated
at
65 ◦C
for
10
min.
The
reverse
transcription
reaction
was
performed
at
37 ◦C
for
1
h,
and
using
2
␮g
of
the
DNaseI
treated-RNA,
the
oligo-dT
(18mer)
primer
and
M-MLV
reverse
transcriptase
(Invitrogen).
An
aliquot
(2
␮l)
of
the
synthesized
cDNA
was
utilized
as
a
template
for
the
PCR
amplifications
with
the
specific
TdMnSOD
gene
primers
S1
and
S2.
The
wheat’s
actin
gene
(Accession
No.
AY663392)
was
used
as
an
internal
control
for
gene
expression.
The
actin
primers
were
AF
(5-CTGACGGTGAGGACATCCAGCCCCTTG-3)
and
AR
(5-
GCACGGCCTGAATTGCGACGTACATGG-3).
The
PCR
amplifications
were
performed
in
a
final
volume
of
50
␮l
and
following
these
PCR
conditions:
2
min
at
96 ◦C
followed
by
30
cycles
for
30
s
at
96 ◦C,
45
s
at
55 ◦C
and
1
min
at
72 ◦C,
followed
by
a
final
extension
of
5
min
at
72 ◦C.
The
amplified
products
were
resolved
in
1.5%
agarose
gel
and
quantified
using
the
Gel
DocXR
Gel
Documentation
System
(Bio-Rad).
This
software
was
used
to
calculate
average
band
den-
sity,
which
was
recorded
and
used
in
graphic
analyses.
Band
density
was
determined
by
this
software
and
was
given
in
arbitrary
units
and
graphed
using
Microsoft
Excel.
This
semi-quantitative
analysis
was
repeated
three
times
to
validate
the
results.
2.8.
Generation
and
identification
of
transgenic
TdMnSOD
arabidopsis
plants
The
recombinant
plasmid
BSOD
was
introduced
into
Agrobac-
terium
tumefaciens
strain
GV3101
(Konez
and
Schell,
1986).
The
Arabidopsis
thaliana
transformation
was
performed
using
the
floral
dipping
technique
(Clough
and
Bent,
1998),
and
the
transgenic
plants
were
selected
on
Murashige
and
Skoog
(MS)
agar
medium
(Murashige
and
Skoog,
1962)
supplemented
with
20
mg
L−1hygromycin.
To
detect
positive
lines,
genomic
DNA
was
isolated
from
the
leaves
of
different
transformed
plants
and
used
as
a
template
for
PCR
amplifications
with
the
two
TdMn-
SOD
specific
primers
S1
and
S2.
We
used
three
homozygous
lines
in
T3
generation.
The
expression
of
the
transgene
in
these
lines
was
analyzed
by
RT-PCR
using
the
primers
S1
and
S2,
and
the
Arabidopsis
thaliana
␤-tubulin
gene
fragment
as
an
internal
control.
This
constitutive
gene
was
amplified
using
the
specific
primers
TubF
(5-GTCCAGTGTCTGTGATATTGCACC-3)
and
TubR
(5-GCTTACGAATCCGAGGGTGCC-3).
2.9.
Analysis
of
stress
tolerance
of
transgenic
arabidopsis
plants
After
the
sterilization
and
the
stratification
of
wild-type
and
T3
homozygous
seeds
of
the
transgenic
Arabidopsis
lines,
they
were
grown
in
MS
agar
medium
for
one
week
under
a
light/dark
cycle
condition
of
16
h
light/8
h
dark
cycle
at
22 ◦C,
and
then
trans-
ferred
to
MS
medium
supplemented
or
not
with
100
mM
NaCl,
150
mM
NaCl,
150
mM
mannitol,
3
mM
H2O2or
50
␮M
CdCl2and
kept
grown
for
12
days.
For
the
control
condition,
these
seedlings
were
grown
in
MS
agar
medium
and
kept
under
normal
growth
conditions.
2.10.
Leaf
surface
determination
Total
leaf
area
of
transgenic
Arabidopsis
and
wild-type
seedlings
subjected
or
not
to
salt,
osmotic,
oxidative
stresses
and
also
to
cad-
mium
toxicity
was
calculated
using
UTHSCA
image
tool
program
(http://compdent.uthscsa.edu/dig/itdesc.html).
2.11.
Protein
extraction
and
antioxidant
enzyme
activities
Aliquots
of
frozen
fresh
shoot
material
(0.5
g)
were
ground
to
a
fine
powder
with
liquid
nitrogen
and
homogenized
in
a
cold
solution
containing
100
mM
Tris-HCl
buffer
(pH
8.0),
10
mM
EDTA
(Ethylenediaminetetraacetic
acid),
50
mM
KCl,
20
mM
MgCl2,
0.5
mM
PMSF
(Phenylmethylsulfonyl
fluoride),
and
2%
(w/v)
PVP
(Polyvinylpyrolidone).
The
homogenate
was
centrifuged
at
14,000g
for
30
min
at
4◦C,
and
the
supernatant
was
used
for
determination
of
the
antioxidative
enzyme
activities.
Protein
concentration
was
determined
according
to
Bradford
(1976).
Catalase
activity
was
determined
according
to
Aebi
(1984),
by
monitoring
the
disappearance
of
H2O2.
An
aliquot
of
crude
enzyme
extract
was
added
to
the
reaction
mixtures
containing
50
mM
phos-
phate
buffer
(pH
7),
30
mM
H2O2.
Changes
in
optical
density
(OD)
of

F.
Kaouthar
et
al.
/
Journal
of
Plant
Physiology
198
(2016)
56–68
59
the
reaction
solution
at
240
nm
were
recorded
every
20
s.
The
activ-
ity
of
POD
was
determined
according
to
Maehly
and
Chance
(1954)
by
the
guaiacol
oxidation
method.
An
aliquot
of
crude
enzyme
extract
was
added
to
50
mM
phosphate
buffer
(pH
7),
20
mM
gua-
iacol,
and
40
mM
H2O2.
Changes
in
OD
of
the
reacted
samples
at
470
nm
were
recorded
every
20
s.
SOD
activity
was
determined
by
measuring
the
percentage
of
inhibition
of
the
pyrogallol
autoxidation
(Marklund
and
Marklund,
1974).
An
aliquot
of
crude
enzyme
extract
was
added
to
10
mM
pyrogallol
in
Tris-cacodylic
acide-diethylene
triamine
penta
acetic
acid
buffer
(pH
8.2).
The
rate
of
autoxidation
was
taken
from
the
increase
in
OD
of
the
reaction
solution
per
min.
For
Mn-SOD
activ-
ity
assay,
H2O2was
added
to
final
concentration
of
5
mM
in
the
reaction
solution.
The
specific
activities
of
SOD,
CAT
and
POD
were
expressed
as
units
mg−1protein.
One
unit
of
SOD
was
defined
as
the
enzyme
quantity
required
causing
50%
inhibition
of
the
rate
of
the
pyrogallol
autoxidation
in
comparison
with
tubes
lacking
the
plant
extract.
One
unit
of
CAT
was
defined
as
␮mol
ml−1H2O2decomposed
per
minute.
One
enzyme
unit
of
POD
is
defined
as
change
in
one
unit
of
absorbance
min−1.
2.12.
Lipid
peroxidation
The
extent
of
lipid
peroxidation
was
estimated
by
determining
the
amount
of
MDA
(malondialdehyde)
in
the
leaves
by
the
method
of
Draper
et
al.
(1993).
Fresh
shoots
(0.15
g)
were
homogenized
in
0.1%
(w/v)
trichloroacetic
acid
(TCA)
solution.
After
centrifugation
at
15,000g
for
30
min,
an
aliquot
of
the
supernatant
was
added
to
0.5%
(w/v)
Thiobarbituric
acid
(TBA)
in
20%
TCA
solution.
The
mix-
ture
was
heated
at
90 ◦C
for
30
min,
and
then
cooled
on
ice.
The
MDA
equivalent
was
calculated
by
measuring
absorbance
at
532
and
600
nm
in
reference
to
a
MDA
standard
curve.
2.13.
Quantitative
H2O2measurement
and
O2
•−detection
The
quantification
of
H2O2levels
was
determined
following
the
method
described
by
Velikova
et
al.
(2000).
Fresh
leaves
tis-
sues
of
the
non-transformed
and
the
transgenic
Arabidopsis
plants
subjected
or
not
to
different
abiotic
stresses
were
homogenized
with
0.1%
(w/v)
trichloroacetic
acid
(TCA),
and
then
centrifuged
at
12,000g
for
15
min.
An
aliquot
of
the
supernatant
was
added
to
10
mM
phosphate
buffer
(pH
7.0)
and
1
M
potassium
iodide
(KI),
and
the
absorbance
of
this
mixture
was
read
at
390
nm.
Then,
the
H2O2content
was
calculated
using
a
standard
curve
with
concen-
tration
ranging
from
0.05
to
0.1
mM.
In
situ
accumulation
of
superoxide
anion
was
examined
by
his-
tochemical
staining
with
nitro
blue
tetrazolium
(NBT)
according
to
Brini
et
al.
(2011).
The
samples
were
placed
in
the
NBT
solution
(0.1
mM
NBT,
25
mM
HEPES
pH
7.6)
and
subjected
to
vacuum
infil-
tration
for
5
min.
After
incubation
in
the
dark
for
2
h,
the
samples
were
treated
with
80%
ethanol
and
then
observed
under
binocular
loupe
and
photographed
using
an
Olympus
W120
digital
still
cam-
era.
This
staining
assay
was
repeated
three
times
using
three
to
six
different
plants
from
each
stress
treatment.
2.14.
Quantification
of
the
free
proline
content
The
amount
of
free
proline
was
determined
following
the
method
described
by
Bates
et
al.
(1973).
Aliquots
of
fresh
leaf
tissue
(100
mg)
were
placed
in
40%
methanol
solution
and
then
heated
in
a
water
bath
at
85 ◦C
for
60
min.
After
cooling,
an
aliquot
of
this
solution
was
added
to
the
solution
containing
acetic
acid,
orthophosphoric
acid
and
ninhydrin
and
the
mixture
was
placed
at
100 ◦C
for
30
min.
The
addition
of
5
ml
of
toluene
produced
the
apparition
of
two
distinct
phases.
The
upper
phase
that
could
con-
tain
proline
was
collected
in
tubes
and
a
pinch
of
Na2SO4was
added
to
each
tubes.
The
absorbance
of
the
organic
phase
was
determined
at
520
nm.
The
resulting
values
were
compared
with
a
standard
curve
constructed
using
known
amounts
of
proline.
2.15.
Statistical
analysis
All
data
were
subjected
to
one-way
ANOVA
implemented
in
the
SPSS
software
14,
and
treatment
means
separations
were
performed
using
the
Student’s
t-test.
Comparisons
with
P-values
of
<0.05
were
considered
significantly
different.
3.
Results
3.1.
Sequence
characterization
of
the
durum
wheat
TdMnSOD
The
full-length
cDNA
of
TdMnSOD
was
isolated
from
durum
wheat
(Triticum
turgidum
L.
subsp.
durum)
cv.
OR3,
cloned
and
sequenced
as
described
(see
Section
2).
The
TdMnSOD
sequence
was
deposited
to
GenBank
with
the
accession
number
KP696754.
The
open
reading
frame,
678
bp
long,
encodes
for
a
protein
of
225
amino
acids.
Blast
analyses
showed
that
TdMnSOD
shares
94%
identity
with
bread
wheat
TaMnSOD
(AAX68501.1),
82%
iden-
tity
with
Hevea
brasiliensis
HbMnSOD
(CAB53458.1),
74%
identity
with
Arabidopsis
thaliana
AtMnSOD
(AAC24832.1).
According
to
this
analysis,
the
four
residues
His49,
His97,
Asp186
and
His190
implicated
in
active
site
formation
are
identified
(Fig.
1A).
To
detect
the
evolutionary
relationships
of
durum
wheat
TdMnSOD
protein
with
other
plant
Mn-SOD
proteins,
eleven
plant
Mn-SOD
pro-
teins
were
selected
and
compared
using
the
MEGA
5
program.
The
phylogenic
tree
showed
that
the
TdMnSOD
protein
belongs
to
the
subgroups
of
Mn-SOD
proteins,
and
shares
high
sequence
homology
with
Triticum
aestivum
TaMnSOD,
Arabidopsis
thaliana
AtMnSOD
and
Hevea
brasiliensis
HbMnSOD
(Fig.
1B).
The
putative
three-dimensional
structure
model
of
durum
wheat
TdMnSOD
was
proposed
using
the
crystal
structure
of
Arabidopsis
thaliana
AtMn-
SOD
as
a
template
(Marques
et
al.,
2014).
In
silico
analyses
showed
that
TdMnSOD
forms
a
homotetramer
and
each
subunit
is
com-
posed
of
a
predominantly
␣-helical
N-terminal
domain
and
a
mixed
␣/␤
C-terminal
domain.
The
N-terminal
domain
has
only
two
long
helices
␣1
and
␣3,
with
the
extra
␣2
helix
being
absent
in
wheat
TdMnSOD
protein.
The
C-terminal
domain
contains
six
␣-helices
(␣4–␣9)
and
three
␤-strands
(␤1–␤3).
The
residues
His49,
His97
in
the
N-terminal
domain
and
Asp186
and
His190
in
the
C-terminal
domain
are
implicated
in
metal
ion
(Mn3+)
interaction
and
form
the
active
site
(Fig.
1C).
It
is
worth
noting
that
the
valence
of
man-
ganese
in
the
Mn-SOD
resting
state
is
Mn(III),
as
demonstrated
in
the
pea
leaf
Mn-SOD
(Fernández
et
al.,
1982).
All
of
these
results
suggest
that
the
isolated
TdMnSOD
encodes
for
a
manganese
super-
oxide
dismutase,
which
may
have
a
physiological
function
similar
to
other
plants’
Mn-SOD
proteins.
3.2.
Expression
analysis
of
TdMnSOD
in
durum
wheat
exposed
to
different
stress
treatments
Plants’
responses
to
a
series
of
oxidative
stresses
include
changes
at
both
the
mRNA
and
protein
levels
of
the
three
SOD
isoenzymes
(Alscher
et
al.,
2002).
Little
is
known
about
the
effect
of
oxidative
stress
generated
by
various
abiotic
stresses
on
the
expression
of
durum
wheat
TdMnSOD
gene.
Thus,
we
analyzed
the
transcript
abundance
of
TdMnSOD
gene
on
the
cultivar
Om
Rabia3
treated
or
not
with
either
200
mM
NaCl
(salt
stress),
15%
PEG
(6000)
(osmotic
stress),
10
mM
H2O2(H2O2-
induced
oxidative
stress)
or
100
␮M
CdCl2(heavy
metal
stress).
RT-PCR
analysis
showed
a
basal
TdMnSOD
expression
levels
in
the
non-treated
plants.
Moreover,

60
F.
Kaouthar
et
al.
/
Journal
of
Plant
Physiology
198
(2016)
56–68
Fig.
1.
Sequence
and
structure
analysis
of
TdMnSOD
protein.
(A)
Presentation
of
three
part
of
the
amino
acids
alignments
of
TdMnSOD
(AKC00865.1)
with
Triticum
aestivum
TaMnSOD
(AAX68501.1),
Arabidopsis
thaliana
AtMnSOD
(AAC24832.1),
Prunus
persica
PpMnSOD
(CAB56851.1),
Hevea
brasiliensis
HbMnSOD
(CAB53458.1),
Pistacia
vera
PvMnSOD
(ABR29644.1)
and
Avicennia
marina
AmMnSOD
(AAN15216.1),
to
show
the
four
conserved
amino
acids
implicated
in
the
interaction
with
Mn3+ and
active
site
formation.
These
amino
acids
(His49,
His97,
Asp186
and
His190)
are
presented
with
black
triangles.
The
gray
color
corresponds
to
identical
amino
acids.
The
numbers
correspond
to
the
position
of
amino
acids
at
the
ends
of
each
presented
sequences.
(B)
Phylogenetic
tree
of
plants
Mn-SOD
proteins
constructed
by
the
MEGA
program.
The
accession
numbers
of
the
others
Mn-SOD
proteins
are
as
follows;
Citrullus
lanatus
ClMnSOD
(AAS48178.1),
Avicennia
marina
AmMnSOD
(AAN15216.1),Olea
europaea
OeMnSOD
(AAL24044.1),
Nicotiana
plumbaginifolia
NpMnSOD
(CAA32643.1),
Gossypium
hirsutum
GhMnSOD
(ABA00455.1)
and
Nicotiana
tabacum
NtMnSOD
(BAC75399.1).
(C)
Ribbon
diagram
of
TdMnSOD
subunit.
TdMnSOD
protein
has
two
putative
domains;
the
N-terminal
domain
contains
the
two
helices
␣1
and
␣3,
and
the
C-terminal
contains
six
␣-helices
(␣4–␣9)
and
three
␤-strands
(␤1–␤3).
The
residues
His49,
His97
in
the
N-terminal
domain
and
Asp186
and
His190
in
the
C-terminal
domain
are
implicated
in
metal
ion
(Mn3+)
interaction
and
form
the
active
site.

F.
Kaouthar
et
al.
/
Journal
of
Plant
Physiology
198
(2016)
56–68
61
Fig.
2.
(A)
Expression
patterns
of
TdMnSOD
gene
in
response
various
oxidative
stresses.
RT-PCR
analysis
was
performed
using
TdMnSOD
specific
primers
and
total
RNA
isolated
from
durum
wheat
cv.
OR3
treated
or
not
(NT)
with
200
mM
NaCl,
15%
PEG
(6000)
or
10
mM
H2O2during
1–3
and
6
days.
Actin
amplification
was
used
as
internal
control.
(−)
Amplification
in
the
absence
of
templates.
(B)
Densitometric
analysis
of
the
images
of
RT-PCR
products,
using
the
Gel
DocXR
software.
Asterisks
indicate
statistically
significant
differences
(P
<
0.05)
in
expression
level
between
the
NT
plants
and
the
corresponding
treatment.
the
level
of
TdMnSOD
transcripts
increased
and
reached
the
max-
imum
after
3
or
6
days
of
the
exposition
to
salt
or
PEG-induced
osmotic
stresses,
respectively.
Similar
to
osmotic
stress,
TdMn-
SOD
transcript
abundance
was
stable
during
the
first
3
days,
and
increased
significantly
after
6
days
of
H2O2application
(Fig.
2).
3.3.
SOD
activity
of
the
purified
durum
wheat
TdMnSOD
To
analyze
TdMnSOD
activity
in
vitro,
we
expressed
TdMnSOD
in
E.
coli
and
purified
the
His-tagged
protein.
As
shown
in
Fig.
3A,
an
intense
protein
band
was
observed
on
SDS-PAGE
with
a
molecu-
lar
weight
(MW)
of
about
30
KDa.
The
estimated
MW
of
TdMnSOD
is
24
KDa,
and
the
MW
of
His6
is
about
1
KDa.
Thus,
this
intense
band
could
correspond
to
the
purified
His-tagged
TdMnSOD
pro-
tein
(Fig.
3A).
The
native
electrophoresis
gel
stained
for
SOD
activity
in
the
absence
and
presence
of
different
specific
inhibitors
showed
an
intense
band
corresponding
to
the
purified
protein
(Fig.
3B).
The
activity
of
the
purified
TdMnSOD
protein
was
determined
as
described
by
McCord
and
Fridovich
(1969).
The
activity
was
expressed
in
units
and
it
increased
with
increasing
the
amount
of
the
purified
enzyme
(Fig.
3C).
The
specific
activity
of
the
puri-
fied
TdMnSOD
was
about
400
U/mg
protein.
All
of
these
results
suggested
that
TdMnSOD
was
active
and
had
Mn-SOD
activity.
3.4.
TdMnSOD
imparts
various
abiotic
stresses
tolerance
to
transgenic
yeast
cells
To
determine
whether
the
TdMnSOD
protein
can
protect
cells
against
various
environmental
stresses,
we
used
yeast
(Saccha-
romyces
cerevisiae)
as
a
fast
heterologous
model
system.
The
yeast
W303
cells
transformed
with
the
empty
pYES2
(control
cells)
or
with
the
recombinant
vector
pYES2-TdMnSOD
(recombinant
cells)
were
subjected
or
not
to
salt
(0.5
and
0.75
M
NaCl),
oxidative
(4
␮M
H2O2),
osmotic
(1.2
M
mannitol)
and
cold
stresses
on
minimal
galactose
medium.
As
shown
in
Fig.
4,
under
non-stressed
condi-
tions,
the
growth
rate
of
the
recombinant
cells
was
similar
to
the
growth
of
the
control
cells.
Nevertheless,
the
expression
of
TdMn-
SOD
ameliorated
the
growth
of
the
recombinant
cells
compared
to
control
cells
under
these
stress
treatments.
These
results
sug-
gested
that
durum
wheat
TdMnSOD
was
functional
in
yeast
system
and
conferred
abiotic
stress
tolerance
to
yeast
cells.
Based
on
these
results,
we
were
interested
in
investigating
the
role
of
TdMnSOD
in
transgenic
Arabidopsis
plants
under
various
abiotic
stresses.
3.5.
Heterologous
expression
of
TdMnSOD
in
transgenic
arabidopsis
plants
In
plant
cells,
SOD
proteins
play
an
essential
role
in
oxidative
stress
responses.
To
investigate
the
implication
of
durum
wheat
TdMnSOD
in
abiotic
stress
responses
in
planta,
the
corresponding
open
reading
frame
was
placed
under
the
control
of
the
dupli-
cated
cauliflower
mosaic
virus
35S
promoter
and
NOS
terminator
(TNOS)
(Fig.
5A).
After
Agrobacterium-mediated
transformation
of
Arabidopsis
plants
and
selection
with
hygromycin,
five
transgenic
Arabidopsis
plants
were
produced.
The
integrity
and
the
expression
of
the
transgene
were
verified
by
PCR
amplifications
(Fig.
5B)
and
RT-PCR
(Fig.
5C),
respectively.
As
expected,
PCR
products
of
a
frag-
ment
about
670
bp
were
detected
in
the
five
putative
transgenic
lines
(Fig.
5B).
The
expression
of
TdMnSOD
gene
was
performed
in
the
three
lines
(L2,
L7
and
L8)
using
the
constitutively
expressed
ˇ-tubulin
gene
as
control
for
cDNA
amplifications.
The
TdMnSOD
expression
level
was
similar
in
these
selected
lines,
but
absent
in
the
non-transformed
(Wt)
plants
(Fig.
5C).
For
these
lines,
genetic
segregation
data
was
performed
using
the
hptII
gene
and
gave
rise
to
3:1
ratio,
confirming
that
this
marker
segregates
as
a
single
copy
gene.
Thus,
these
three
lines
were
selected
for
further
physiological
analyses.
3.6.
TdMnSOD
confers
tolerance
of
the
transgenic
arabidopsis
plants
to
multiple
abiotic
stresses
Abiotic
stress
tolerance
of
the
three
homozygous
T3
seedlings
lines
(L2,
L7
and
L8)
was
evaluated
in
vitro
by
transferring
one-
week-old
seedlings
to
MS
agar
medium
containing
different
salt
concentrations
(100
and
150
mM
NaCl)
(salt
stress),
150
mM
man-
nitol
(osmotic
stress),
3
mM
H2O2(oxidative
stress)
or
50
␮M
CdCl2
(heavy
metal
stress).
Under
standard
growth
conditions,
the
three
lines
were
phenotypically
indistinguishable
from
Wt
plants.
How-
ever,
these
abiotic
stresses
dramatically
affected
the
growth
of
Wt
plants
compared
to
the
transgenic
Arabidopsis
lines
(Fig.
6A).
When
challenged
to
high
salt
concentration
(150
mM
NaCl),
the
non-
transformed
plants
showed
growth
inhibition
with
the
apparition
of
white
leaves
and
less
root
production,
whereas
the
transgenic
lines
survived
and
developed
more
secondary
roots.
The
exposition
to
H2O2-induced
oxidative
stress
blocked
root
elongation
of
both
Wt
and
the
three
lines,
while
the
aerial
part
of
the
transgenic
lines
continued
to
grow.
By
contrast,
osmotic
and
heavy
metal
stresses
induced
leaf
growth
reduction
of
these
plants
compared
to
normal
conditions.
This
increase
was
more
pronounced
in
Wt
plants
than
that
in
transgenic
lines
(Fig.
6A).
The
effect
of
these
abiotic
stresses
was
analyzed
by
measuring
the
total
leaf
areas
(TLA)
on
Wt
and

62
F.
Kaouthar
et
al.
/
Journal
of
Plant
Physiology
198
(2016)
56–68
Fig.
3.
Analysis
of
TdMnSOD
activity
in
vitro
(McCord
and
Fridovich,
1969).
(A)
SDS-
PAGE
of
total
protein
before
(lane
1)
and
after
(lane
2)
induction
with
IPTG.
Lane
3
corresponds
to
the
purified
His-tagged
TdMnSOD
protein
(25
KDa).
Molecular
masses
are
indicated
on
the
left.
(B)
Gels
stained
for
TdMnSOD
activity
in
the
absence
(Lane
1)
and
presence
of
SOD
inhibitors
(5
mM
H2O2and
2
mM
KCN)
(Lane
2).
(C)
in
vitro
activity
of
the
purified
His-tagged
TdMnSOD.
transgenic
plants.
In
the
absence
of
stress,
similar
TLA
values
were
scored
in
transgenic
and
wild-type
plants
(∼65
mm2).
The
TLA
val-
ues
of
Wt
plants
treated
with
salt
stress
(100
mM
NaCl)
decreased
about
50%
and
75%
under
other
abiotic
stress
(osmotic,
oxida-
tive
and
heavy
metal
stresses).
However,
TLA
values
were
slightly
reduced
about
30%
in
the
three
transgenic
lines
exposed
to
these
different
abiotic
stresses
relative
to
control
conditions
(Fig.
6B)
We
also
measured
whole
seedling
weight
of
the
three
trans-
genic
lines
in
comparison
with
Wt
plants.
Without
stress,
the
fresh
weight
(FW)
was
similar
for
all
plants.
However,
a
reduction
of
about
50%
was
registered
in
the
Wt
plants
treated
with
salt
stress,
and
about
75%
was
registered
in
these
plants
treated
with
other
abiotic
stress.
With
respect
to
the
transgenic
lines,
the
reduction
was
less
pronounced
compared
to
the
non-treated
plants,
which
was
about
25%
in
the
presence
of
these
stress
treatments
(Fig.
6C).
We
quantified
the
level
of
proline
on
leaves
of
the
Wt
and
the
three
lines
subjected
or
not
to
different
stress
treatments.
As
shown
in
Fig.
6D,
both
Wt
and
transgenic
plants
shared
practically
the
same
level
of
production
of
proline
under
standard
conditions.
Nevertheless,
the
transgenic
lines
produced
more
proline
under
salt,
osmotic,
heavy
metal
and
H2O2-induced
stresses
than
the
Wt
plants.
Indeed,
the
increase
of
proline
amount
was
about
1.5–2
fold
in
these
lines
treated
with
these
stresses,
but
only
about
1.2-
to
1.5-fold
in
Wt
plants,
relative
to
non-treated
plants.
Taken
together,
these
results
showed
that
the
transgenic
Ara-
bidopsis
plants
expressing
TdMnSOD
respond
differently
to
the
control
plants,
with
a
significant
tolerance
phenotype
to
multiple
environmental
stresses,
suggesting
that
the
durum
wheat
TdMn-
SOD
protein
plays
a
major
role
against
multiple
abiotic
stresses.
3.7.
Maintenance
of
the
redox
homeostasis
in
tdMnSOD-expressing
arabidopsis
plants
To
compare
ROS
accumulation
in
wild-type
and
transgenic
plants,
we
performed
a
NBT
staining
assay
on
leaves
to
detect
the
superoxide
anion
accumulation.
As
shown
in
Fig.
7A,
under
standard
conditions
a
weak
NBT
staining
was
detected
in
Wt
and
transgenic
lines.
When
seedlings
were
submitted
to
salt,
osmotic,
oxidative
and
heavy
metal
stresses,
an
intense
NBT
staining
was
detected
in
Wt
leaves
as
compared
to
L7
line
(Fig.
7A).
The
accumulation
of
MDA
was
monitored
as
an
indicator
of
membrane
damage
and
lipid
peroxidation.
As
shown
in
Fig.
7B,
under
standard
conditions,
the
MDA
content
was
higher
in
the
Wt
plants
than
those
in
the
three
transgenic
lines.
When
grown
under
stressed
conditions,
all
plant
genotypes
exhibited
increased
levels
of
MDA
contents,
but
this
increase
was
less
marked
in
transgenic
plants
than
in
Wt
plants.
For
peroxide
hydrogen
levels,
the
amounts
were
lower
in
the
three
lines
than
those
in
Wt
plants
under
nor-
mal
conditions.
After
10
days
of
stress
treatments,
both
Wt
and
transgenic
lines
showed
a
marked
increase
in
H2O2levels.
Nev-
ertheless,
the
amount
of
H2O2in
the
transgenic
lines
still
lower
in
L2,
L7
and
L8
lines,
compared
to
the
Wt
plants
(Fig.
7C).
All
of
these
results
suggested
that
the
overexpression
of
TdMnSOD
in
Arabidop-
sis
plants
strengthens
the
antioxidant
system
by
enhancing
ROS
decomposition
capability.
3.8.
Analysis
of
the
antioxidant
enzymes
activities
in
the
transgenic
arabidopsis
plants
The
activities
of
total
SOD
and
Mn-SOD
of
transgenic
lines
were
markedly
higher
than
those
of
Wt
plants
under
normal
growth
conditions
and
stress
treatments.
Indeed,
under
standard
condi-
tions,
the
three
transgenic
plants
exhibited
over
1.5
and
2
in
total
SOD
and
Mn-SOD
activities,
respectively,
as
compared
to
Wt
plants.
On
the
other
hand,
the
application
of
salt,
osmotic,
H2O2-induced
oxidative
stresses
and
cadmium
toxicity
increased
total
SOD
and

F.
Kaouthar
et
al.
/
Journal
of
Plant
Physiology
198
(2016)
56–68
63
Fig.
4.
Stress
tolerance
of
yeast
cells
overexpressing
TdMnSOD
gene.
Cells
of
the
W303
strain
transformed
with
the
empty
vector
(pYES)
or
with
the
recombinant
vector
(pYES
+
TdMnSOD)
were
grown
and
then
five
microliters
of
serial
decimal
dilutions
were
spotted
onto
solid
YNB-Ura/Gal2%
plates
(control)
or
supplemented
with
different
concentrations
of
NaCl
(0.5
and
0.75
M),
4
␮M
H2O2or
0.5
M
mannitol.
For
cold
stress,
the
yeast
cells
were
re-suspended
in
YNB-ura
medium
and
placed
in
a
refrigerator
at
−20 ◦C
alcohol
bath
for
24
h.
Colonies
were
photographed
after
5–6
days
of
incubation
at
30 ◦C.
Fig.
5.
Molecular
analysis
of
TdMnSOD
transgenic
plants.
(A)
Physical
map
of
the
T-
DNA
region
in
the
binary
vector.
The
transgene
was
inserted
between
the
CaMV35S
promoter
(P35S)
and
NOS
terminator
(TNOS);
the
HPTII
marker
is
flanked
by
the
NOS
promoter
(PNOS)
and
terminator
(TNOS).
The
presence
(B)
and
the
expression
(C)
of
TdMnSOD
in
the
transgenic
Arabidopsis
plants
were
analyzed
using
specific
TdMnSOD
primers.
Wt:
Non-
transformed
plants.
A
0.5
kb
of
␤-tubulin
TUB4
gene
fragment
was
amplified
by
RT-PCR
as
an
internal
control.
(−)
negative
control
without
cDNA.
M:
Lambda
PstI
molecular
weight
marker.
Mn-SOD
activities
in
both
transgenic
lines
and
Wt
plants.
However,
both
SOD
and
Mn-SOD
activities
were
notably
higher
in
transgenic
plants
than
in
Wt
plants
(Fig.
8A,
B).
Meanwhile,
we
analyzed
the
activities
of
two
other
active
oxy-
gen
scavenging
enzymes
CAT
and
POD
in
both
Wt
and
transgenic
plants
subjected
to
the
same
stress
conditions.
As
shown
in
Fig.
8C
and
D,
both
CAT
and
POD
activities
in
the
transgenic
lines
were
higher
than
those
of
Wt
plants
under
normal
conditions.
Moreover,
the
application
of
stress
dramatically
increased
these
activities
in
all
these
plants,
whereas
they
were
superior
in
the
transgenic
plants
to
those
of
Wt
plants.
4.
Discussion
Salinization
of
cropland
in
the
Mediterranean
region
is
a
major
limitation
to
crop
yields.
The
response
to
salt
stress
implicates
vari-
ous
physiological,
biochemical
and
molecular
mechanisms.
Salinity
is
well
known
to
induce
oxidative
stress,
which
occurs
due
to
the
production
of
ROS
(Hernandez
et
al.,
2010).
Despite
the
potential
of
ROS
for
causing
harmful
oxidations,
it
is
now
well
established
that
they
are
also
implicated
in
the
control
of
plant
growth
and
devel-
opment
as
well
as
priming
acclimatory
responses
to
stress
stimuli
(Choudhury
et
al.,
2013;
Foyer
and
Noctor,
2009).
To
cope
with
ROS,
living
organisms
have
evolved
antioxidant
defense
systems,
comprised
of
enzymatic
and
non-enzymatic
components.
SOD
is
the
first
antioxidant
enzyme
implicated
in
the
elimination
of
O2•−,
followed
by
many
other
enzymes
like
CAT
and
POD.
Plant
Mn-
SOD
proteins
are
similar
to
bacterial
Mn-SOD,
and
they
have
65%
sequence
similarity
to
one
another
(Alscher
et
al.,
2002;
Bowler
et
al.,
1994).
The
first
Mn-SOD
protein
was
purified
and
charac-
terized
from
pea
leaves
(Sevilla
et
al.,
1982;
Wong-Vega
et
al.,
1991),
and
then
from
various
plants
species
like
maize
(White
and

64
F.
Kaouthar
et
al.
/
Journal
of
Plant
Physiology
198
(2016)
56–68
Fig.
6.
Response
of
TdCAT1-expressing
Arabidopsis
plants
to
various
abiotic
stresses.
(A)
Effect
of
salt
(100
and
150
mM
NaCl),
osmotic
(150
mM
Mannitol),
oxidative
(3
mM
H2O2)
and
heavy
metal
(50
␮M
CdCl2)
stresses
on
the
growth
of
wild-type
plants
and
the
three
Arabidopsis
transgenic
lines
(L2,
L7
and
L8).
For
control
conditions,
the
same
seedlings
were
placed
in
normal
MS
medium
(Control).
Photographs
were
taken
after
10
days
of
stress
application.
(B)
Total
leaf
area
(TLA)
of
individual
leaves
from
the
Wt
and
the
three
lines
L2,
L7
and
L8
under
normal
condition
(C)
or
subjected
to
salt
(Na),
osmotic
(OS)
and
oxidative
(OX)
stresses
and
cadmium
toxicity
(Cd).
Values
are
means
of
five
replicates
of
one
fully
expanded
leaf
per
plant.
Comparison
of
whole
plants
fresh
weight
(FW)
(C)
and
proline
content
(D)
in
leaves
of
transgenic
lines
with
wild-type
plants
under
normal
condition
(C)
or
under
these
abiotic
stresses
conditions.
Values
are
means
±
SE
of
three
biological
replicate
samples.
Each
replicate
sample
was
a
composite
of
leaves
from
five
seedlings.
Asterisks
indicate
significant
differences
between
transgenic
lines
and
wild-type
in
each
stress
treatment
(P
<
0.05).
Scandalios,
1988;
Zhu
and
Scandalios,
1993),
tobacco
(Bowler
et
al.,
1994;
Breusegem
et
al.,
1999)
and
Jojoba
(Simmondsia
chinensis)
(Liu
et
al.,
2013).
In
wheat,
the
three
superoxide
dismutase
isoen-
zymes
were
purified
and
biochemically
characterized
(Robinson
et
al.,
1996).
Thus
far,
there
is
no
conclusive
evidence
for
the
impor-
tance
of
Mn-SOD
on
the
stress
tolerance
of
cereal
crops.
To
evaluate
the
role
of
the
durum
wheat
Mn-SOD
in
the
plant
response
to
various
abiotic
stresses,
we
first
isolated
the
corresponding
open
reading
frame
TdMnSOD
from
the
cultivar
Om
Rabia3,
and
then
we
expressed
it
in
yeast
cells
and
in
Arabidopsis
plants.
The
phylogenic
analysis
of
amino
acids
sequences
showed
that
TdMnSOD
belongs
to
Mn-SOD
group
and
that
it
is
closely
related
to
Triticum
aestivum
TaMnSOD,
Arabidopsis
thaliana
AtMnSOD
and
Hevea
brasiliensis
HbMnSOD
proteins.
In
general,
Mn-SOD
proteins
of
various
plant
species
are
present
in
the
mitochondrial
compartment.
However,
Mn-SODs
are
also
localized
in
peroxisomes
from
different
plant
species,
like
pea
and
watermelon,
among
others
(Alscher
et
al.,
2002;
Bowler
et
al.,
1994;
Del
Rio
et
al.,
1983;
Rodríguez-Serrano
et
al.,
2007).
It
has
been
shown
that
the
mitochondrial
and
perox-
isomal
Mn-SOD
expression
is
regulated
differently
during
pea
leaf
senescence
(Del
Rio
et
al.,
2003).
It
was
reported
that
Mn-SOD
and
Fe-SOD
proteins
exhibit
strict
metal
specificity
despite
their
high
sequence
similarity.
Only
a
small
number
of
cambialistic
SODs
utilize
either
Mn
or
Fe
as
the
active
metal
cofactor
(Alscher
et
al.,
2002).
In
Mn-SOD
proteins,
the
presence
of
manganese
and
the
positively
charged
amino
acids
in
the
active
site
of
the
enzyme
promote
the
catalysis
reaction
of
O2•−
molecules
to
H2O2(Alscher
et
al.,
2002;
Bowler
et
al.,
1994).
Accord-
ing
to
the
sequence
analysis,
the
four
amino
acids
implicated
in
the
active
site
formation
and
in
interaction
with
manganese
are
iden-
tified
in
TdMnSOD,
which
are
His49,
His97
that
are
localized
in
the
N-terminal
domain,
and
Asp186
and
His190
in
the
C-terminal
part.
The
three-dimensional
structure
of
wheat
TdMnSOD
was
identi-
fied
using
the
crystal
structure
of
Arabidopsis
thaliana
AtMnSOD
as
a
template
(Marques
et
al.,
2014).
Accordingly,
the
durum
wheat
TdMnSOD
forms
a
homotetramer
and
each
subunit
is
composed
of
a
predominantly
␣-helical
N-terminal
domain
and
a
mixed
␣/␤
C-terminal
domain.
The
N-terminal
domain
has
two
long
helices
␣1
and
␣3,
with
the
absence
of
the
extra
␣2
helix.
It
has
been
sug-
gested
that
the
absence
of
this
additional
helix
allows
favorable
interactions
between
subunits
to
form
a
dimer
of
dimers
(Trinh
et
al.,
2008).
The
C-terminal
domain
contains
six
␣-helices
and
three
␤-strands.
A
similar
structure
to
TdMnSOD
was
obtained
against
a
Mn-SOD
from
the
thermophilic
fungus
Chaetomium
ther-
mophilum
(CtMnSOD)
(Haikarainen
et
al.,
2014).
All
of
these
results
suggest
that
the
isolated
TdMnSOD
from
durum
wheat
encodes

F.
Kaouthar
et
al.
/
Journal
of
Plant
Physiology
198
(2016)
56–68
65
Fig.
7.
Revelation
of
O2−accumulation
by
NBT
staining
(A),
determination
of
MDA
concentrations
(B)
and
H2O2contents
(C)
in
leaves
of
7
days-old
seedlings
of
wild-type
plants
and
the
three
Arabidopsis
transgenic
lines
(L2,
L7
and
L8)
subjected
or
not
to
salt
(Na),
osmotic
(OS)
and
oxidative
(OX)
stresses
and
cadmium
toxicity
(Cd)
for
12
days.
Asterisks
indicate
significant
differences
between
transgenic
lines
and
wild-type
in
each
stress
treatment
(P
<
0.05).
for
a
manganese
superoxide
dismutase,
which
may
have
the
same
physiological
function
as
the
other
Mn-SOD
proteins.
In
response
to
oxidative
stress,
the
expression
levels
of
some
genes
change
in
plant
cells.
Among
them,
there
are
the
genes
that
encode
for
proteins
with
antioxidant
functions.
For
example,
there
are
many
reports
on
the
changes
in
the
activities
of
vari-
ous
SOD
isoenzymes
and
their
corresponding
mRNA
under
osmotic
stresses
(Zhu
and
Scandalios,
1994).
In
durum
wheat,
the
TdMnSOD
gene
showed
transcript
up-regulation
by
salt
stress,
osmotic
and
H2O2-induced
oxidative
stress.
However,
the
timing
of
the
high
accumulation
of
TdMnSOD
transcripts
diverges
from
one
stress
to
another.
In
agreement
with
our
result,
it
has
been
shown
that
the
expression
of
the
seven
SODs
isoenzymes
changes
when
subjected
to
a
series
of
oxidative
stresses
in
Arabidopsis
thaliana
(Kliebenstein
et
al.,
1998).
The
yeast
S.
cerevisiae
is
a
versatile
tool
for
functional
character-
ization
of
genes
from
higher
eukaryotes
including
plants
(Eswaran
et
al.,
2010).
In
addition,
it
is
an
excellent
model
for
the
study
of
the
mechanisms
underlying
abiotic
stress
tolerance
due
to
the
fact
that
a
high
degree
of
evolutionary
conservation
of
stress
pathways
exists
between
higher
eukaryotes
and
yeast
(Dhar
et
al.,
2011;
Posas
et
al.,
2000).
The
overexpression
of
the
durum
wheat
TdMnSOD
in
yeast
improves
tolerance
of
yeast
cells
to
salt,
osmotic,
cold,
and
H2O2-induced
oxidative
stresses.
These
data
suggested
that
TdMnSOD
is
functional
gene
and
this
durum
wheat
manganese
superoxide
dismutase
plays
a
crucial
role
in
response
to
various
abiotic
stresses
in
yeast
cells.
Similar
salt
and
oxidation
resis-
tance
was
also
observed
in
yeast
cells
expressing
Mn-SOD
from
Chaetomium
thermophilum
(Haikarainen
et
al.,
2014).
The
role
of
Mn-SOD
from
different
plants
in
response
to
salinity,
drought
and
oxidative
stress
was
demonstrated
in
many
transgenic
plants
(Liu
et
al.,
2013;
Wang
et
al.,
2004;
Wang
et
al.,
2005;
Wang
et
al.,
2007).
Furthermore,
cold
tolerance
was
enhanced
in
transgenic
maize
overexpressing
tobacco
Mn-SOD
(Breusegem
et
al.,
1999).
To
the
best
of
our
knowledge,
little
is
known
about
the
function
of
the
durum
wheat
Mn-SOD
in
response
to
environmental
stresses.
In

66
F.
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/
Journal
of
Plant
Physiology
198
(2016)
56–68
0
10
20
30
40
C
Na
Ma
H
Cd
POD (U mg-1 proteins)
10
20
30
40
50
60
C
Na
Ma
H
Cd
MnSOD (U m g-1 proteins)
Wt
L2
L7
L8
C Na OS OX Cd
(A) (B)
40
60
80
100
120
140
160
C
Na
Ma
H
Cd
CAT (U mg-1 proteins)
(C)
(D)
*
*
***
*
*
***
*
*
***
30
40
50
60
70
CNaMaHCd
SOD (U mg-1 proteins)
Wt L2 L7 L8
*
****
C Na OS OX Cd
C Na OS OX Cd
C Na OS OX Cd
Fig.
8.
Analysis
of
total
SOD
(A),
Mn-SOD
(B)
(Marklund
and
Marklund,
1974),
CAT
(C)
and
POD
(D)
activities
of
the
wild-type
(Wt)
plants
and
the
three
Arabidopsis
transgenic
lines
(L2,
L7
and
L8)
under
normal
condition
(C)
or
subjected
to
salt
(Na),
osmotic
(OS)
and
oxidative
(OX)
stresses
and
cadmium
toxicity
(Cd).
Values
are
mean
±
SE
(n
=
4).
At
each
condition,
asterisks
indicate
significantly
greater
mean
values
compared
to
Wt
plants.
this
study,
we
showed
that
the
expression
of
TdMnSOD
in
transgenic
Arabidopsis
plants
enhanced
tolerance
to
multiple
abiotic
stresses.
This
tolerance
was
illustrated
by
higher
growth
rate
and
proline
accumulation.
Proline
has
a
multifunctional
role
in
plants
cells
and
enhances
plant
tolerance
to
a
wide
range
of
abiotic
and
biotic
stresses
(Szabados
and
Savouré,
2010).
In
agreement
with
this,
a
higher
amount
of
proline
was
detected
in
TdMnSOD-expressing
plants
under
different
stress
treatments.
Moreover,
compared
to
wild-type
plants,
TdMnSOD
transgenic
plants
accumulated
low
O2
•−,
H2O2and
MDA
contents
in
leaves.
Determining
MDA
con-
centration
has
often
been
used
as
a
tool
to
assess
the
severity
of
the
oxidative
stress
and
the
degree
of
plant
sensibility.
All
these
observations
suggest
that
the
degree
of
oxidative
stress
was
sig-
nificantly
less
extreme
in
transgenic
Arabidopsis
plants.
This
could
be
the
result
of
a
balance
between
the
ROS-detoxifying
and
ROS-
producing
enzymes.
The
activities
of
Mn-SOD
and
total
SOD
of
transgenic
plants
were
higher
than
those
of
the
wild-type
plants
under
normal
and
stressed
conditions,
indicating
that
the
ability
of
O2
•−elimination
in
transgenic
Arabidopsis
increased
significantly.
In
plant
cells,
the
superoxide
is
dismutated
by
SOD
into
H2O2,
which
is
rapidly
decomposed
into
O2and
H2O
by
CAT
and
POD.
In
the
transgenic
Arabidopsis
expressing
TdMnSOD,
high
activities
of
CAT
and
POD
were
registered
under
normal
and
stressed
conditions.
This
would
be
responsible
for
the
high
ability
to
eliminate
the
ROS
accumulated
in
transgenic
Arabidopsis
plants
under
environmental
stresses.
Similar
result
was
observed
in
transgenic
Arabidopsis
over-
expressing
the
endogenous
Mn-SOD,
which
exhibited
higher
total
SOD,
CAT
and
POD
activities
compared
to
non-transformed
plants
under
salt
stress
(Wang
et
al.,
2004).
All
these
results
suggested
that
the
durum
wheat
TdMnSOD
plays
a
pivotal
role
in
preventing
the
over
accumulation
of
ROS,
and
consequently
enhanced
abiotic
stress
tolerance
of
the
transgenic
plants.
In
summary,
the
novel
TdMnSOD
gene
isolated
from
durum
wheat
encodes
for
a
manganese
superoxide
dismutase,
which
exhibited
a
high
similarity
with
other
Mn-SOD
plants,
and
especially
Arabidopsis
AtMnSOD
and
bread
wheat
TaMnSOD.
Furthermore,
the
three-dimensional
structure
of
TdMnSOD
was
similar
to
Mn-SOD
proteins
from
various
organisms.
Transcrip-
tional
analysis
showed
that
TdMnSOD
was
induced
by
salt,
osmotic
and
H2O2-induced
oxidative
stresses
in
durum
wheat.
We
showed
that
the
TdMnSOD
gene
is
functional
in
the
yeast
system,
and
able
to
impart
abiotic
stress
tolerance
in
yeast
and
in
transgenic
Arabidop-
sis.
Thus,
we
conclude
that
the
wheat
TdMnSOD
gene
is
a
promising
candidate
gene
for
the
development
of
crops
with
multiple
stress
tolerances.
Conflict
of
interest
The
authors
declare
that
they
have
no
conflict
of
interest.
Acknowledgment
This
study
was
supported
by
a
grant
from
the
Ministry
of
Higher
Education
and
Scientific
Research
of
Tunisia.

F.
Kaouthar
et
al.
/
Journal
of
Plant
Physiology
198
(2016)
56–68
67
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