Enhancement

of

stress

tolerance

in

transgenic

tobacco

plants

constitutively

expressing

an

inositol

polyphosphate

from

Arabidopsis

thaliana

Inositol

phosphates

-LRB-

IPs

-RRB-

and

their

turnover

products

have

been

implicated

to

play

important

roles

in

stress

signaling

in

eukaryotic

cells

In

higher

plants

genes

encoding

inositol

polyphosphate

kinases

have

been

identified

previously

but

their

physiological

functions

have

not

been

fully

resolved

Here

we

expressed

Arabidopsis

inositol

polyphosphate

-LRB-

-RRB-

in

two

heterologous

systems

i.e.

the

yeast

Saccharomycescerevisiae

and

in

tobacco

-LRB-

Nicotiana

tabacum

-RRB-

and

tested

the

effect

on

abiotic

stress

tolerance

Expression

of

rescued

the

salt

osmotic

and

temperature-sensitive

growth

defects

of

a

yeast

mutant

strain

-LRB-

-RRB-

that

lacks

inositol

polyphosphate

multikinase

activity

encoded

by

the

gene

Transgenic

tobacco

plants

constitutively

expressing

under

the

control

of

the

Cauliflower

Mosaic

Virus

promoter

were

generated

and

found

to

exhibit

improved

tolerance

to

diverse

abiotic

stresses

when

compared

to

wild

type

plants

Expression

patterns

of

various

stress

responsive

genes

were

enhanced

and

the

activities

of

anti-oxidative

enzymes

were

elevated

in

transgenic

plants

suggesting

a

possible

involvement

of

in

plant

stress

responses

Introduction

During

their

life

plants

are

often

challenged

with

various

types

of

abiotic

stresses

Proper

physiological

and

biochemical

responses

to

such

stresses

are

controlled

by

an

array

of

stress-dependent

signal

transduction

pathways

-LRB-

Xiong

et

al.

-RRB-

Several

signaling

molecules

and

second

messengers

including

calcium

ions

-LRB-

Knight

et

al.

Knight

et

al.

-RRB-

and

inositol

-LRB-

Takahashi

et

al.

-RRB-

have

been

demonstrated

to

be

important

elements

of

signaling

pathways

that

link

the

perception

of

stress

to

various

types

of

downstream

responses

As

one

of

the

central

molecules

in

cell

signaling

is

subject

to

phosphorylation

and

dephosphorylation

by

specific

inositol

phosphate

kinases

and

phosphatases

respectively

which

helps

to

control

intracellular

concentration

-LRB-

Pattni

and

Banting

-RRB-

In

higher

plants

over-expression

of

an

inositol

polyphosphate

-LRB-

-RRB-

possibly

leading

to

intracellular

degradation

of

modulated

stress

and

ABA

signal

transduction

-LRB-

Perera

et

al.

Burnette

et

al.

-RRB-

Likewise

mutation

of

an

inositol

polyphosphate

gene

-LRB-

-RRB-

results

in

increased

sensitivity

to

ABA

and

vein

patterning

defects

in

cotyledons

-LRB-

Carland

and

Nelson

-RRB-

can

also

be

hydrolyzed

by

inositol

polyphosphate

-LRB-

Xiong

et

al.

-RRB-

The

mutant

of

Arabidopsis

accumulated

around

ten-fold

more

than

the

corresponding

wild

type

exhibited

hypersensitivity

towards

ABA

and

was

compromised

in

tolerance

to

freezing

drought

and

salt

stresses

-LRB-

Xiong

et

al.

-RRB-

These

data

demonstrate

the

importance

of

inositol

polyphosphate

phosphatases

in

metabolism

and

stress

signal

transduction

However

evidence

for

a

role

of

inositol

polyphosphate

kinases

in

relation

to

stress

tolerance

is

currently

lacking

in

plants

Inositol

-LRB-

-RRB-

and

more

generally

inositol

polyphosphate

kinases

play

an

essential

role

in

cellular

signal

transduction

and

maintenance

of

homeostasis

by

phosphorylating

inositol

-LRB-

-RRB-

to

inositol

-LRB-

Communi

et

al.

-RRB-

Both

and

are

second

messengers

responsible

for

mobilization

from

intracellular

stores

-LRB-

Berridge

-RRB-

itself

is

also

able

to

regulate

cytosolic

concentration

by

promoting

sequestration

-LRB-

Hill

et

al.

-RRB-

In

Arabidopsis

thaliana

is

encoded

by

two

distinct

genes

and

which

are

ubiquitously

expressed

in

mature

tissues

-LRB-

Stevenson-Paulik

et

al.

Xia

et

al.

-RRB-

Both

of

the

Arabidopsis

use

as

a

substrate

and

display

dual-specificity

inositol

polyphosphate

activities

that

successively

phosphorylate

to

generate

inositol

-LRB-

-RRB-

predominantly

via

an

inositol

-LRB-

-RRB-

intermediate

-LRB-

Stevenson-Paulik

et

al.

Xia

et

al.

-RRB-

Recently

it

was

reported

that

plays

a

role

in

pollen

germination

and

root

growth

-LRB-

Xu

et

al.

-RRB-

while

functions

in

axillary

shoot

branching

through

the

auxin

signaling

pathway

-LRB-

Zhang

et

al.

-RRB-

and

phytate

synthesis

-LRB-

Stevenson-Paulik

et

al.

-RRB-

Although

has

well

been

characterized

at

the

biochemical

level

and

has

been

shown

to

participate

in

plant

development

it

remained

unclear

whether

it

is

also

involved

in

stress

signaling

In

this

study

we

demonstrate

that

restores

the

salt

osmotic

and

temperature-sensitive

growth

defects

of

a

yeast

mutant

strain

-LRB-

-RRB-

deficient

for

the

gene

that

encodes

inositol

polyphosphate

multikinase

We

also

show

that

constitutive

expression

of

in

transgenic

tobacco

enhances

its

tolerance

towards

various

abiotic

stresses

Thus

inositol

polyphosphate

kinase

encoded

by

very

probably

plays

an

important

role

in

signaling

pathways

controlling

the

cellular

response

to

abiotic

stresses

Materials

and

methods

Expression

of

in

yeast

mutant

The

S.

deletion

strain

-LRB-

derived

from

Mat

a

-RRB-

was

obtained

from

EUROSCARF

-LRB-

-RRB-

In

this

strain

the

allele

is

disrupted

through

insertion

of

the

kanamycin

resistance

gene

The

open

reading

frame

was

cloned

into

yeast

expression

plasmid

The

resulting

plasmid

was

used

to

transform

the

yeast

mutant

The

wild

type

strain

was

used

as

control

Ten-fold

serial

dilutions

-LRB-

starting

at

-RRB-

of

each

strain

were

plated

on

YPD

medium

-LRB-

yeast

extract

peptone

glucose

-RRB-

supplemented

with

either

NaCl

-LRB-

or

M

-RRB-

or

mannitol

-LRB-

or

M

-RRB-

and

incubated

at

°C

for

h.

For

growth

at

°C

yeast

cells

were

grown

on

media

containing

no

extra

NaCl

or

mannitol

Each

experiment

was

repeated

at

least

three

times

For

growth

in

liquid

YPD

medium

cells

were

cultured

at

°C

for

three

days

to

saturation

-LRB-

-RRB-

Five

μl

of

the

cell

culture

were

used

to

inoculate

ml

of

synthetic

defined

minimal

medium

supplemented

with

different

concentrations

of

NaCl

or

mannitol

Cell

density

was

measured

at

Quantitative

real-time

PCR

analysis

of

expression

in

Arabidopsis

Ten-day-old

Arabidopsis

seedlings

grown

on

MS

medium

were

used

for

different

treatments

as

described

previously

-LRB-

Shi

and

Zhu

-RRB-

RNA

was

extracted

with

TRIZOL

Reagent

-LRB-

Invitrogen

-RRB-

and

reverse

transcription

of

RNA

was

carried

out

using

M-MLV

RTase

Synthesis

Kit

-LRB-

TaKaRa

-RRB-

Quantitative

real-time

PCR

was

performed

using

the

Rotor-Gene

Series

real

time

DNA

amplification

system

under

the

following

conditions

°C

for

s

cycles

of

°C

for

s

°C

for

s.

primers

were

used

-LRB-

Table

-RRB-

Melting

curve

analysis

was

included

to

verify

specificity

of

the

DNA

amplification

Expression

of

the

reference

gene

Actin

which

served

as

quantifying

control

was

monitored

using

gene-specific

forward

and

reverse

primers

-LRB-

Table

-RRB-

Data

analysis

was

performed

with

Rotor-Gene

software

Table

primers

used

in

this

studyPrimer

namePrimer

sequenceFor

yeast

expression

For

real-time

For

RT-PCRNtActin-F

NtActin-R

Vector

construction

and

plant

transformation

To

create

a

Cauliflower

Mosaic

Virus

-LRB-

CaMV

-RRB-

plant

expression

construct

the

bp

long

cDNA

containing

the

complete

open

reading

frame

was

inserted

via

XbaI

SalI

sites

into

the

binary

vector

pBinAR-HPT

The

resulting

construct

was

introduced

into

Agrobacterium

tumefaciens

-LRB-

strain

-RRB-

Tobacco

-LRB-

Nicotiana

tabacum

cv

-RRB-

leaf

disks

were

infected

with

the

transformed

Agrobacteria

After

two

days

of

co-cultivation

the

explants

were

transferred

to

regeneration

medium

containing

BAP

l

NAA

hygromycin

and

ampicillin

Regenerated

shoots

were

separated

from

the

calli

and

transferred

onto

rooting

medium

containing

MS

salts

l

NAA

and

hygromycin

Rooted

shoots

were

transplanted

into

soil

Seeds

were

harvested

and

homozygous

plants

were

screened

on

a

dark

photoperiod

Salt

stress

tolerance

tests

Wild

type

and

homozygous

transgenic

seeds

were

surface

sterilized

with

a

solution

of

commercial

bleach

-LRB-

sodium

hypochlorite

-RRB-

for

min

and

washed

three

times

with

sterile

water

For

germination

assays

seeds

were

plated

on

MS

medium

supplemented

with

different

concentrations

of

NaCl

-LRB-

and

mM

-RRB-

For

root

growth

measurements

seedlings

cultured

on

solid

MS

medium

were

transferred

to

MS

medium

supplemented

with

different

concentrations

of

NaCl

Plates

were

oriented

vertically

with

seedlings

kept

upside

down

Three

replicates

were

performed

for

each

experiment

Root

length

was

recorded

after

seven

days

of

treatment

To

monitor

stress

effects

on

the

growth

of

wild

type

and

expressing

plants

seedlings

were

transferred

to

MS

medium

supplemented

with

or

mM

NaCl

and

cultured

at

°C

with

cool

white

fluorescent

light

-LRB-

μmol

m

s

-RRB-

under

short

day

condition

-LRB-

dark

-RRB-

For

salt

stress

treatments

and

ion

measurements

wild

type

and

transgenic

seedlings

were

transferred

to

pots

containing

soil

Plants

were

watered

bi-weekly

with

concentrated

MS

salt

solution

supplemented

with

or

without

NaCl

and

grown

at

°C

with

cool

white

fluorescent

light

-LRB-

μmol

m

s

-RRB-

humidity

under

long-day

conditions

-LRB-

dark

-RRB-

for

weeks

Mature

plants

treated

in

the

same

way

were

also

used

to

determine

cellular

concentrations

The

third

leaf

from

the

top

of

each

plant

was

collected

and

dried

at

°C

for

h

and

the

dry

weight

of

each

sample

was

measured

The

dried

leaf

material

was

immersed

in

HAc

followed

by

water

bath

incubation

at

°C

for

h

and

analyzed

for

content

using

atomic

absorption

spectrophotometry

For

hydroponic

salt

tolerance

experiments

wild

type

plants

and

two

independent

transgenic

lines

-LRB-

generation

-RRB-

were

grown

hydroponically

in

Hoagland

solution

Plants

were

grown

at

°C

with

cool

white

fluorescent

light

-LRB-

μmol

m

s

-RRB-

humidity

under

long-day

conditions

-LRB-

dark

-RRB-

The

nutrient

solution

was

replaced

weekly

Oxidative

stress

experiments

Malondialdehyde

-LRB-

MDA

-RRB-

concentration

was

determined

as

a

biomarker

for

oxidative

stress

To

measure

MDA

concentration

leaf

samples

from

plants

grown

at

NaCl

were

ground

in

ml

of

trichlor

acetic

acid

-LRB-

TCA

-RRB-

and

mixed

with

ml

of

thiobarbituric

acid

The

samples

were

then

boiled

for

min

cooled

down

to

room

temperature

and

centrifuged

at

g

The

cleared

supernatant

was

analyzed

by

monitoring

the

difference

in

absorbance

at

and

For

oxidative

damage

experiments

leaf

disks

of

diameter

were

excised

from

healthy

and

fully

expanded

leaves

of

two-month-old

tobacco

plants

The

disks

were

floated

in

a

solution

with

different

concentrations

of

or

water

-LRB-

control

-RRB-

for

h.

Chlorophyll

a

and

b

concentrations

were

measured

as

described

previously

-LRB-

Lichtenthaler

-RRB-

The

treatment

was

carried

out

with

continuous

white

light

at

°C

The

experiment

was

repeated

at

least

three

times

Enzyme

assays

and

protein

determination

Enzyme

assays

were

carried

out

spectrophotometrically

using

a

nucleic

acid

and

protein

analyzer

-LRB-

Du

Beckman

Coulter

-RRB-

One-month-old

seedlings

were

treated

with

or

for

h

and

ground

in

extraction

buffer

-LRB-

mM

ascorbate

in

potassium

phosphate

buffer

pH

-RRB-

at

a

ratio

of

g

leaf

ml

ice-cold

extraction

buffer

The

slurry

was

transferred

to

a

centrifuge

tube

and

centrifuged

at

g

at

°C

for

min

The

supernatant

was

used

for

enzyme

activity

assays

and

total

protein

determination

as

described

previously

-LRB-

Badawi

Bradford

-RRB-

Ascorbate

peroxidase

-LRB-

APX

-RRB-

activity

was

determined

by

measuring

the

rate

of

ascorbate

oxidation

at

in

a

mixture

-LRB-

EDTA

in

potassium

phosphate

buffer

pH

ascorbate

and

soluble

protein

extract

-RRB-

APX

activity

is

expressed

as

micromoles

of

ascorbate

oxidized

per

minute

per

milligram

of

protein

Catalase

-LRB-

CAT

-RRB-

activity

was

measured

by

monitoring

the

decrease

in

absorbance

at

caused

by

the

disappearance

of

in

a

mixture

-LRB-

EDTA

in

potassium

phosphate

buffer

pH

and

protein

extract

-RRB-

The

activity

of

superoxide

dismutase

-LRB-

SOD

-RRB-

was

measured

at

in

a

reaction

mixture

-LRB-

dl-methionine

EDTA

Nitro

Blue

tetrazolium

chloride

and

riboflavin

in

phosphate

buffered

saline

pH

and

protein

extract

-RRB-

Whole

plant

drought

tolerance

test

Wild

type

and

transgenic

plant

seeds

were

surface

sterilized

as

described

above

Seedlings

germinated

on

MS

medium

were

transplanted

to

soil

After

two

months

healthy

plants

of

the

same

size

and

age

were

pooled

into

two

groups

For

the

first

group

-LRB-

individual

plants

each

grown

in

a

pot

-RRB-

plants

were

irrigated

with

g/l

to

simulate

drought

stress

After

days

the

plants

were

photographed

Simultaneously

plants

of

the

second

group

were

subjected

to

drought

stress

by

omitting

watering

After

three

weeks

the

plants

were

re-hydrated

and

observed

for

recovery

Photos

were

taken

days

after

initiation

of

the

stress

treatment

The

relative

humidity

was

maintained

at

The

experiments

were

repeated

twice

with

three

replicates

in

each

experiment

Freezing

tolerance

test

Three-week-old

seedlings

of

wild

type

and

transgenic

plants

grown

on

MS

medium

were

cultured

at

°C

for

one

day

under

long

day

condition

-LRB-

dark

-RRB-

After

cold

acclimation

the

plants

were

left

at

°C

for

or

h

transferred

immediately

to

°C

for

another

h

-LRB-

overnight

-RRB-

Subsequently

plants

were

kept

in

the

greenhouse

at

°C

and

observed

further

Photographs

were

taken

seven

days

after

initiation

of

the

recovery

growth

in

the

greenhouse

Determination

of

proline

concentration

Four-week-old

seedlings

grown

on

MS

medium

were

transferred

onto

filter

paper

in

a

Petri

dish

saturated

with

or

NaCl

incubated

at

°C

for

h

under

continuous

white

light

After

the

treatment

proline

content

was

determined

as

described

previously

-LRB-

Bates

and

Waldren

-RRB-

Western

blot

analysis

Leaves

from

either

Arabidopsis

or

tobacco

plants

were

ground

in

liquid

and

suspended

in

ml

of

mM

potassium

phosphate

buffer

-LRB-

pH

-RRB-

containing

NaCl

and

dithiothreitol

-LRB-

Powell

-RRB-

Homogenates

were

centrifuged

for

min

at

g.

Supernatants

were

transferred

to

new

tubes

and

protein

concentrations

were

determined

as

described

previously

-LRB-

Bradford

-RRB-

A

sample

of

total

protein

was

mixed

with

loading

buffer

boiled

for

min

and

size-fractionated

through

electrophoresis

in

a

SDS

polyacrylamide

gel

Separated

proteins

were

transferred

to

a

PVDF

membrane

-LRB-

Millipore

Corporation

-RRB-

according

to

the

manufacturer

's

instructions

A

polyclonal

antibody

against

protein

purified

from

recombinant

E.

coli

was

raised

in

rabbits

Antiserum

was

purified

by

IgG

affinity

purification

The

antiserum

was

used

at

a

dilution

in

TBST

Peroxidase-labeled

affinity

purified

secondary

antibody

isolated

from

a

pool

of

serum

from

goats

immunized

with

purified

rabbit

IgG

was

used

at

a

dilution

in

TBST

-LRB-

KPL

Corporation

-RRB-

Stress

gene

expression

analysis

using

reverse

transcription

PCR

Three-week-old

tobacco

seedlings

grown

on

MS

agar

plates

were

treated

with

NaCl

for

h.

Total

RNA

was

isolated

with

TRIZOL

Reagent

-LRB-

Invitrogen

-RRB-

and

used

for

reverse

transcription

-LRB-

RT

-RRB-

to

generate

first-strand

cDNA

RT

was

performed

on

total

RNA

per

reaction

using

ReverTra

Ace

-LRB-

TOYOBO

-RRB-

A

portion

of

the

resulting

cDNA

was

then

subjected

to

PCR

amplification

using

gene

specific

primers

-LRB-

Table

-RRB-

Expression

level

of

the

Nicotiana

tabacum

ACTIN

gene

which

served

as

reference

was

monitored

with

NtActin

forward

and

reverse

primers

-LRB-

Table

-RRB-

Statistical

analysis

For

statistical

analyses

the

Student

's

t-test

was

used

to

generate

every

P-value

The

alpha

level

was

The

tests

were

one-tailed

The

data

were

normalized

and

all

samples

were

normally

distributed

with

homogeneity

of

variance

Results

Yeast

complementation

with

In

S.

cerevisiae

the

gene

encodes

an

inositol

polyphosphate

kinase

-LRB-

Odom

et

al.

-RRB-

The

S.

-LRB-

-RRB-

mutant

strain

that

lacks

activity

has

slowed

growth

at

°C

and

is

unable

to

grow

at

°C

-LRB-

Odom

et

al.

-RRB-

We

and

others

have

reported

previously

that

rescues

its

growth

defects

at

°C

-LRB-

Stevenson-Paulik

et

al.

Xia

et

al.

-RRB-

This

result

led

us

to

postulate

that

may

also

be

able

to

rescue

its

growth

defects

when

other

abiotic

stresses

were

imposed

We

observed

that

under

adverse

growth

conditions

such

as

high

salt

concentration

or

upon

osmotic

stress

treatment

growth

of

the

mutant

was

severely

impaired

when

compared

to

that

of

the

wild

type

-LRB-

Fig.

-RRB-

To

test

whether

restores

the

growth

defect

of

the

mutant

strain

under

these

conditions

we

transformed

it

with

the

cDNA

Expression

of

almost

completely

restored

growth

of

the

mutant

under

both

kinds

of

stresses

-LRB-

Fig.

-RRB-

It

also

restored

growth

at

°C

-LRB-

Fig.

-RRB-

as

reported

earlier

-LRB-

Stevenson-Paulik

et

al.

Xia

et

al.

-RRB-

We

also

performed

experiments

using

liquid

media

and

observed

again

that

restores

growth

under

conditions

of

abiotic

stress

-LRB-

Fig.

-RRB-

Fig.

of

in

S.

-LRB-

a

-RRB-

rescues

growth

defects

of

the

yeast

mutant

under

high

salt

or

mannitol

stress

and

at

°C

Ten-fold

serial

dilutions

of

the

strain

were

plated

and

incubated

at

°C

for

h

on

YPD

medium

supplemented

with

either

or

NaCl

or

with

or

mannitol

Plates

incubated

at

°C

contained

YPD

medium

without

extra

NaCl

or

mannitol

WT

wild

type

mutant

mutant

harbouring

plasmid

-LRB-

b

-RRB-

Liquid

yeast

cultures

Cells

were

grown

for

three

days

to

saturation

in

YPD

medium

-LRB-

-RRB-

Five

μl

of

cell

culture

were

then

used

to

inoculate

ml

of

defined

synthetic

minimal

medium

supplemented

with

different

concentrations

of

NaCl

or

mannitol

Cell

density

was

determined

at

various

time

points

as

absorbance

at

nm

Cells

were

grown

at

°C

WT

wild

type

mutant

mutant

harbouring

vector

mutant

harbouring

is

abiotic

stress

regulated

in

Arabidopsis

We

were

interested

to

know

whether

different

kinds

of

stresses

such

as

salinity

cold

and

drought

or

treatment

with

ABA

would

affect

transcript

levels

in

Arabidopsis

plants

To

this

end

we

performed

quantitative

real-time

PCR

-LRB-

qRT-PCR

-RRB-

In

ten-day-old

seedlings

grown

on

MS

medium

transcript

level

increased

slightly

upon

exposure

to

cold

or

drought

stress

but

decreased

in

response

to

salt

or

ABA

treatment

-LRB-

Fig

in

Supplementary

Material

-RRB-

These

results

showed

that

is

a

stress

responsive

gene

potentially

involved

in

different

abiotic

stress

and

ABA-triggered

signaling

pathways

in

plants

We

further

tested

protein

level

by

Western

blot

analysis

Fourteen-day-old

Arabidopsis

seedlings

grown

on

MS

medium

were

subjected

to

different

stress

treatments

for

h

In

seedlings

treated

with

mannitol

protein

level

increased

slightly

however

no

significant

changes

in

protein

level

were

detected

in

seedlings

subjected

to

cold

or

NaCl

stress

-LRB-

Fig

in

Supplementary

Material

-RRB-

Constitutive

expression

of

in

transgenic

tobacco

To

test

whether

constitutive

expression

of

affects

abiotic

stress

tolerance

in

plants

we

introduced

its

open

reading

frame

-LRB-

Fig.

-RRB-

into

the

genome

of

tobacco

-LRB-

Nicotiana

tabacum

cv

-RRB-

by

Agrobacterium

tumefaciens-mediated

transformation

Expression

of

in

the

transgenic

plant

was

controlled

by

the

Cauliflower

Mosaic

Virus

-LRB-

CaMV

-RRB-

promoter

Forty-six

independent

transgenic

plants

-LRB-

generation

-RRB-

were

obtained

six

lines

were

grown

to

produce

seeds

Transgenic

plants

homozygous

for

the

transgene

were

obtained

and

three

lines

-LRB-

and

-RRB-

were

chosen

for

further

experiments

Constitutive

expression

of

did

not

affect

overall

plant

morphology

although

the

transgenic

plants

grew

slightly

faster

than

wild

type

plants

under

normal

growth

condition

Western

blot

analyses

were

performed

to

test

for

the

presence

of

protein

A

band

corresponding

to

was

identified

in

Arabidopsis

plants

similarly

protein

was

detected

in

the

transgenic

but

not

the

wild

type

tobacco

lines

-LRB-

Fig.

-RRB-

indicating

that

it

was

successfully

expressed

in

the

genetically

modified

plants

Fig.

used

for

tobacco

transformation

and

Western

blot

analysis

of

in

wild

type

and

transgenic

tobacco

plants

-LRB-

a

-RRB-

Schematic

map

of

the

binary

construct

used

for

tobacco

transformation

Expression

of

is

driven

by

the

Cauliflower

Mosaic

Virus

promoter

Ocs

ocs

terminator

RB

and

LB

right

and

left

border

of

T-DNA

respectively

-LRB-

b

-RRB-

Western

blot

analysis

Lane

wild

type

Arabidopsis

lane

wild

type

tobacco

lanes

transgenic

expressing

tobacco

lines

and

Coomassie

Bright

Blue

stained

gel

is

shown

below

the

Western

blot

to

demonstrate

equal

loading

of

protein

Expression

of

increases

salt

tolerance

of

transgenic

tobacco

plants

We

first

studied

the

salt

tolerance

of

expressing

plants

during

germination

and

early

stages

of

seedling

development

Seeds

of

wild

type

and

the

three

homozygous

transgenic

lines

and

were

germinated

on

MS

medium

containing

different

concentrations

of

NaCl

Germination

rates

of

wild

type

and

transgenic

seeds

were

similar

when

cultured

on

normal

MS

medium

-LRB-

Fig.

-RRB-

However

germination

of

wild

type

seeds

was

significantly

impaired

on

MS

medium

supplemented

with

or

NaCl

whereas

germination

of

transgenic

seeds

was

less

strongly

affected

After

eight

days

on

high

salt

MS

medium

-LRB-

NaCl

-RRB-

only

of

the

wild

type

seeds

but

more

than

of

the

seeds

obtained

from

expressing

plants

germinated

-LRB-

Fig.

-RRB-

When

sown

on

MS

medium

supplemented

with

NaCl

only

of

the

wild

type

seeds

but

of

the

transgenic

seeds

germinated

after

days

-LRB-

Fig.

-RRB-

At

low

salt

concentration

-LRB-

NaCl

-RRB-

wild

type

and

transgenic

seeds

displayed

similar

germination

rates

-LRB-

data

now

shown

-RRB-

At

NaCl

both

wild

type

and

transgenic

seeds

germinated

However

development

of

wild

type

seedlings

was

impaired

plants

produced

smaller

leaves

and

less

root

biomass

than

did

transgenic

seedlings

-LRB-

Fig.

-RRB-

To

further

test

the

effect

of

salt

on

growth

wild

type

and

transgenic

seedlings

were

transferred

to

MS

medium

supplemented

with

NaCl

at

different

concentrations

After

two

additional

months

strong

growth

retardation

was

observed

in

wild

type

seedlings

Inhibition

of

growth

was

less

evident

in

transgenic

lines

-LRB-

Fig.

-RRB-

A

root-bending

assay

has

previously

been

used

to

test

the

effect

of

elevated

salt

concentration

on

root

growth

-LRB-

An

et

al.

Zhu

et

al.

-RRB-

At

NaCl

on

MS

medium

root

growth

of

both

wild

type

and

transgenic

lines

was

accelerated

but

it

was

inhibited

at

increasing

NaCl

concentrations

At

NaCl

inhibition

of

root

growth

was

significantly

more

pronounced

in

wild

type

than

in

transgenic

plants

-LRB-

Fig.

g

-RRB-

Fig.

rate

and

early

seedling

development

of

transgenic

plants

-LRB-

a

c

-RRB-

Percentage

of

germinating

seeds

of

wild

type

-LRB-

WT

-RRB-

and

transgenic

plants

-LRB-

-RRB-

grown

on

MS

medium

supplemented

with

or

NaCl

Results

are

presented

as

means

and

standard

errors

from

three

independent

experiments

-LRB-

seeds

of

each

line

were

sown

for

each

experiment

-RRB-

-LRB-

d

-RRB-

Wild

type

-LRB-

WT

-RRB-

and

transgenic

seeds

-LRB-

-RRB-

geminated

on

MS

medium

supplemented

with

or

NaCl

Photo

was

taken

three

weeks

after

seeds

were

sown

-LRB-

e

-RRB-

Wild

type

-LRB-

WT

-RRB-

and

transgenic

lines

-LRB-

-RRB-

grown

on

MS

medium

supplemented

with

different

concentrations

of

NaCl

for

months

-LRB-

f

-RRB-

Root

bending

assay

of

wild

type

-LRB-

WT

-RRB-

and

transgenic

plants

-LRB-

-RRB-

on

MS

medium

supplemented

with

or

NaCl

Representative

pictures

are

shown

-LRB-

g

-RRB-

Primary

root

lengths

of

wild

type

-LRB-

WT

-RRB-

and

transgenic

plants

-LRB-

-RRB-

in

the

presence

of

different

salt

concentrations

Root

growth

under

control

condition

-LRB-

NaCl

-RRB-

was

set

to

The

results

are

shown

as

means

and

standard

errors

from

three

independent

experiments

-LRB-

seeds

of

each

line

were

sown

for

each

experiment

-RRB-

indicates

significant

differences

in

comparison

to

the

wild

type

at

P

-LRB-

Student

's

t-test

-RRB-

We

also

performed

salt

tolerance

experiments

with

wild

type

and

two

transgenic

lines

grown

in

the

greenhouse

in

soil

The

plants

were

watered

bi-weekly

with

concentrated

MS

salt

solution

supplemented

with

or

without

NaCl

After

six

weeks

distinct

differences

were

observed

between

wild

type

and

transgenic

plants

exposed

to

NaCl

-LRB-

Fig.

-RRB-

Although

both

sets

of

plants

flowered

under

conditions

of

salt

stress

wild

type

plants

developed

much

shorter

stems

-LRB-

Fig.

-RRB-

produced

fewer

pods

-LRB-

Fig.

-RRB-

and

produced

seeds

that

were

not

viable

We

determined

endogenous

levels

in

leaves

and

roots

of

wild

type

and

transgenic

plants

grown

in

the

absence

or

presence

of

NaCl

Both

wild

type

and

transgenic

plants

exposed

to

salt

contained

elevated

levels

of

ions

in

leaves

and

roots

-LRB-

Fig.

-RRB-

however

ion

concentrations

were

not

significantly

different

between

wild

type

and

transgenic

plants

-LRB-

the

same

was

true

for

plants

grown

under

normal

conditions

-RRB-

We

also

grew

tobacco

plants

in

hydroponic

culture

In

the

absence

of

salt

stress

no

overt

morphological

difference

was

observed

between

wild

type

and

transgenic

plants

However

growth

of

wild

type

tobacco

was

severely

inhibited

in

the

presence

of

NaCl

-LRB-

Fig.

-RRB-

leading

to

a

depression

of

shoot

height

by

about

one

third

and

of

plant

fresh

weight

by

about

in

comparison

to

expressing

tobacco

plants

after

a

two-month

stress

treatment

-LRB-

Fig.

-RRB-

All

these

results

indicated

that

alleviated

the

negative

effects

imposed

by

salt

stress

on

plant

growth

Fig.

tolerance

test

to

compare

wild

type

and

expressing

tobacco

plants

For

each

experiment

twelve

plants

of

each

line

were

used

-LRB-

a

-RRB-

Photograph

of

representative

wild

type

-LRB-

WT

-RRB-

and

transgenic

plants

-LRB-

-RRB-

watered

with

NaCl

for

six

weeks

-LRB-

b

-RRB-

-LRB-

c

-RRB-

Shoot

height

and

pod

numbers

of

wild

type

-LRB-

WT

-RRB-

and

transgenic

-LRB-

-RRB-

plants

treated

with

NaCl

and

indicate

significant

differences

in

comparison

to

the

wild

type

at

P

and

P

respectively

-LRB-

Student

's

t-test

-RRB-

-LRB-

d

-RRB-

content

in

leaves

and

roots

of

wild

type

-LRB-

WT

-RRB-

and

transgenic

plants

-LRB-

-RRB-

grown

in

soil

supplied

with

or

NaCl

solution

-LRB-

e

-RRB-

Photograph

of

a

representative

wild

type

-LRB-

WT

-RRB-

and

transgenic

line

hydroponically

grown

in

nutrient

solution

supplemented

with

NaCl

for

two

months

-LRB-

f

-RRB-

Shoot

height

and

-LRB-

g

-RRB-

fresh

weight

of

wild

type

-LRB-

WT

-RRB-

and

transgenic

lines

and

grown

for

two

months

under

control

-LRB-

NaCl

-RRB-

or

high

salt

-LRB-

NaCl

-RRB-

conditions

Results

are

presented

as

means

and

standard

errors

from

three

independent

experiments

indicates

significant

differences

in

comparison

to

the

wild

type

at

P

-LRB-

Student

's

t-test

-RRB-

Transgenic

plants

showed

increased

tolerance

to

osmotic

drought

freezing

temperature

and

oxidative

stress

In

order

to

determine

whether

expression

of

affects

osmotic

stress

tolerance

in

plants

we

watered

wild

type

and

transgenic

tobacco

plants

-LRB-

lines

and

-RRB-

with

-LRB-

g/l

-RRB-

as

described

previously

-LRB-

Eltayeb

et

al.

-RRB-

Under

such

experimental

conditions

wild

type

plants

were

less

vigorous

flowered

earlier

and

produced

smaller

leaves

than

expressing

plants

-LRB-

Fig.

-RRB-

We

also

tested

the

effect

of

water

shortage

on

plant

growth

After

a

drought

period

wild

type

plants

completely

wilted

while

transgenic

plants

were

less

severely

affected

-LRB-

Fig.

-RRB-

At

the

end

of

the

drought

tolerance

test

the

plants

were

re-watered

and

grown

further

to

allow

seed

setting

Transgenic

plants

produced

larger

shoots

than

wild

type

plants

-LRB-

Fig.

-RRB-

Fig.

of

drought

and

osmotic

stress

on

plant

performance

For

each

experiment

twelve

plants

of

wild

type

-LRB-

WT

-RRB-

and

each

transgenic

line

-LRB-

-RRB-

were

used

-LRB-

a

-RRB-

Wild

type

and

transgenic

plants

grown

in

soil

were

watered

with

g/l

Photograph

was

taken

after

days

of

treatment

-LRB-

b

-RRB-

Water

was

withheld

for

days

to

impose

drought

stress

-LRB-

c

-RRB-

Drought

stressed

plants

were

re-watered

and

grown

for

seed

setting

-LRB-

d

-RRB-

Proline

content

of

wild

type

and

transgenic

plants

after

treatment

with

or

mM

NaCl

for

h.

Results

are

presented

as

means

and

standard

errors

from

three

independent

experiments

indicates

significant

differences

in

comparison

to

the

wild

type

at

P

-LRB-

Student

's

t-test

-RRB-

Proline

accumulation

in

response

to

osmotic

or

salinity

stress

has

been

well

documented

in

prokaryotic

and

eukaryotic

organisms

-LRB-

Schobert

-RRB-

Here

we

compared

the

proline

content

of

wild

type

and

transgenic

plants

treated

with

or

NaCl

Although

proline

accumulated

in

a

dose-dependent

manner

in

both

wild

type

and

transgenic

plants

proline

concentration

was

slightly

higher

in

transgenic

plants

in

the

presence

of

NaCl

-LRB-

Fig.

-RRB-

The

results

presented

in

Fig.

show

that

expression

increased

the

salt

tolerance

of

transgenic

tobacco

plants

Since

salt

stress

exerts

osmotic

stress

to

plant

cells

in

addition

to

its

ionic

toxicity

we

further

determined

if

expression

confers

elevated

tolerance

to

oxidative

stress

on

transgenic

plants

Leaf

disks

from

wild

type

and

transgenic

tobacco

plants

were

exposed

to

increasing

concentrations

of

for

h

As

shown

in

Fig.

transgenic

plants

exhibited

a

strong

tolerance

to

oxidative

stress

Leaf

disks

from

wild

type

plants

were

almost

completely

bleached

at

the

end

of

the

stress

treatment

whereas

those

from

transgenic

plants

showed

significant

resistance

to

damage

-LRB-

Fig.

-RRB-

Fig.

stress

and

short

term

freezing

tolerance

of

wild

type

-LRB-

WT

-RRB-

and

transgenic

tobacco

plants

-LRB-

-RRB-

-LRB-

a

-RRB-

Leaf

disks

were

incubated

in

different

concentration

of

-LRB-

and

respectively

-RRB-

under

continuous

white

light

for

h.

induced

leaf

yellowing

is

delayed

in

the

transgenic

plants

-LRB-

b

-RRB-

Three-week-old

seedlings

were

stored

at

°C

for

h

and

then

transferred

to

normal

temperature

for

further

growth

Photograph

was

taken

seven

days

after

the

freezing

treatment

-LRB-

c

-RRB-

Quantification

of

the

seedling

survival

rate

-LRB-

determined

seven

days

after

the

freezing

treatment

-RRB-

Results

are

presented

as

means

and

standard

errors

from

three

independent

experiments

Fore

each

experiment

seedlings

of

each

line

were

used

indicates

significant

difference

in

comparison

to

the

wild

type

at

P

-LRB-

Student

's

t-test

-RRB-

-LRB-

d

-RRB-

Chlorophyll

content

was

determined

in

leaf

disks

of

wild

type

and

transgenic

plants

kept

in

for

h.

Disks

floated

in

water

served

as

control

Results

are

preans

and

standard

errors

from

three

independent

experiments

and

indicate

significant

differences

in

comparison

to

the

wild

type

at

P

and

P

respectively

-LRB-

Student

's

t-test

-RRB-

To

further

examine

the

effect

of

constitutive

expression

on

plant

performance

the

tolerance

towards

freezing

temperature

was

analyzed

To

this

end

three-week-old

seedlings

were

kept

at

°C

for

or

h

respectively

and

subsequently

allowed

to

recover

for

seven

days

in

the

greenhouse

Most

transgenic

plants

resumed

normal

growth

after

the

h

treatment

at

°C

-LRB-

Fig.

-RRB-

but

only

very

few

wild

type

plants

did

so

When

treated

for

h

at

°C

the

damage

was

less

severe

but

again

transgenic

plants

grew

better

than

wild

type

plants

after

transfer

back

to

the

greenhouse

-LRB-

data

not

shown

-RRB-

Transgenic

seedlings

showed

a

superior

survival

rate

after

short-term

°C

treatment

-LRB-

Fig.

-RRB-

In

the

presence

of

oxidative

stress

chlorophyll

loss

was

significantly

delayed

in

expressing

plants

compared

to

that

of

the

wild

type

-LRB-

Fig.

-RRB-

Lipid

hydroperoxidation

is

an

effective

indicator

of

cellular

oxidative

damage

-LRB-

Yoshimura

et

al.

-RRB-

Changes

in

the

rates

of

lipid

hydroperoxide

production

induced

by

oxidative

stress

were

measured

by

determining

MDA

content

in

leaf

disks

At

high

salinity

-LRB-

NaCl

-RRB-

MDA

concentration

markedly

increased

in

wild

type

plants

whereas

only

a

marginal

increase

was

observed

in

the

two

transgenic

lines

-LRB-

Fig.

-RRB-

Although

superoxide

dismutase

-LRB-

SOD

-RRB-

activity

was

largely

unaffected

in

wild

type

and

transgenic

plants

treated

with

-LRB-

Fig.

-RRB-

a

slight

elevation

of

APX

activity

was

observed

in

transgenic

plants

-LRB-

Fig.

-RRB-

Also

catalase

-LRB-

CAT

-RRB-

activity

increased

in

transgenic

plants

-LRB-

Fig.

-RRB-

These

results

indicate

that

constitutive

expression

of

enhanced

tolerance

to

oxidative

stress-induced

membrane

hydroperoxidation

in

transgenic

tobacco

plants

Fig.

level

anti-oxidative

enzyme

activity

and

expression

level

of

stress

regulated

genes

in

transgenic

plants

-LRB-

a

-RRB-

MDA

levels

in

wild

type

and

transgenic

lines

after

treatment

with

NaCl

-LRB-

b

d

-RRB-

One-month-old

seedlings

were

incubated

for

h

in

or

water

-LRB-

control

-RRB-

under

continuous

white

light

SOD

APX

and

CAT

activities

were

determined

Results

are

presented

as

means

and

standard

errors

from

three

independent

experiments

and

indicate

significant

differences

in

comparison

to

the

wild

type

at

P

and

P

respectively

-LRB-

Student

's

t-test

-RRB-

-LRB-

e

-RRB-

Two-week-old

seedlings

of

wild

type

and

transgenic

lines

-LRB-

-RRB-

were

used

for

RNA

extraction

For

the

salt

stress

experiments

seedlings

were

treated

with

NaCl

for

h

before

RNA

isolation

The

transcriptional

levels

of

five

stress

genes

were

determined

by

RT-PCR

analyses

The

stress

genes

used

for

the

tests

are

as

follows

lipid

transfer

protein

-LRB-

-RRB-

fructose-bisphosphate

aldolase

-LRB-

-RRB-

raffinose

synthase

family

protein/seed

imbibition

protein

-LRB-

-RRB-

group

LEA

proteins

NtERD

-LRB-

-RRB-

and

NtERD

-LRB-

-RRB-

Increased

stress

responsive

gene

expression

in

transgenic

tobacco

plants

Many

genes

responding

to

abiotic

stress

were

cloned

in

recent

years

and

used

as

molecular

markers

to

monitor

the

activity

of

stress-signaling

pathways

in

plants

-LRB-

Kasuga

et

al.

-RRB-

The

stress

experiments

described

above

suggested

that

plays

an

important

role

in

regulating

stress

tolerance

in

plants

To

address

whether

or

not

affects

the

expression

of

stress

responsive

genes

we

investigated

the

transcriptional

levels

of

five

marker

genes

in

wild

type

and

transgenic

tobacco

plants

by

RT-PCR

The

marker

genes

chosen

are

identical

or

homologous

to

those

encoding

lipid

transfer

protein

fructose-bisphosphate

aldolase

raffinose

synthase

chloroplast

ATPase

and

group

LEA

proteins

of

tobacco

As

shown

in

Fig.

transcript

levels

of

these

genes

were

higher

in

transgenic

plants

than

in

the

wild

type

under

both

normal

and

salt

stress

conditions

-LRB-

Fig.

-RRB-

Discussion

A

number

of

genes

encoding

Ins

-LRB-

-RRB-

or

Ins

-LRB-

-RRB-

dual

specificity

were

cloned

and

biochemically

characterized

in

the

past

years

Examples

include

genes

from

yeast

-LRB-

Odom

et

al.

-RRB-

animals

-LRB-

Bertsch

et

al.

-RRB-

human

-LRB-

Dewaste

et

al.

-RRB-

and

higher

plants

-LRB-

Stevenson-Paulik

et

al.

Xia

et

al.

Xu

et

al.

-RRB-

It

has

recently

been

reported

that

in

yeast

-LRB-

S.

cerevisiae

-RRB-

inositol

polyphosphate

kinase

-LRB-

-RRB-

is

required

for

resistance

to

salt

stress

cell

wall

integrity

and

vacuolar

morphogenesis

-LRB-

Dubois

et

al.

-RRB-

and

that

overexpression

of

a

Drosophila

gene

confers

resistance

of

flies

to

but

not

to

paraquat-induced

oxidative

stress

-LRB-

Monnier

et

al.

-RRB-

Further

genetic

studies

indicated

that

the

protective

effect

conferred

by

elevated

expression

is

mainly

due

to

a

lowered

level

and

thus

reduced

ion

release

from

internal

stores

rather

than

an

increased

level

Based

on

these

observations

we

were

interested

to

know

whether

constitutive

expression

of

confers

improved

resistance

to

abiotic

stresses

on

higher

plants

Since

the

Arabidopsis

protein

shares

high

amino

acid

sequence

identity

with

the

yeast

enzyme

which

functions

as

a

transcriptional

regulator

-LRB-

also

called

or

ArgRIII

-RRB-

and

displays

dual-specificity

kinase

activity

-LRB-

Odom

et

al.

-RRB-

we

first

produced

transgenic

Arabidopsis

-LRB-

ecotype

-RRB-

plants

overexpressing

Surprisingly

these

plants

did

not

exhibit

a

significant

improvement

to

salt

or

osmotic

stress

in

our

preliminary

studies

-LRB-

data

not

shown

-RRB-

This

may

be

due

to

the

possible

overlapping/redundant

functions

of

the

two

Arabidopsis

inositol

polyphosphate

kinases

-LRB-

and

-RRB-

in

vivo

as

suggested

earlier

by

the

York

group

-LRB-

Stevenson-Paulik

et

al.

-RRB-

To

examine

this

possibility

we

further

compared

the

growth

of

Arabidopsis

wild

type

as

well

as

transgenic

over-expresser

-LRB-

-RRB-

and

knock-out

mutant

-LRB-

ABRC

-RRB-

plants

All

three

types

of

plants

had

similar

growth

phenotypes

when

subjected

to

salt

or

osmotic

stress

-LRB-

data

not

shown

-RRB-

Here

we

introduced

into

tobacco

plants

and

found

that

transgenic

lines

developed

superior

tolerance

to

various

types

of

abiotic

stresses

Previously

a

vacuolar

H

antiporter

gene

from

Arabidopsis

was

over-expressed

in

tomato

and

canola

to

improve

the

salt

tolerance

of

transgenic

plants

-LRB-

Zhang

and

Blumwald

-RRB-

However

a

comparative

analysis

of

and

K

accumulation

in

leaves

and

roots

of

transgenic

over-expressers

and

wild

type

plants

grown

under

high-salt

conditions

was

not

performed

because

the

high

-LRB-

mM

-RRB-

NaCl

concentration

used

in

these

experiments

was

lethal

to

the

wild

type

plants

In

the

study

reported

here

the

relatively

high

salt

tolerance

of

wild

type

tobacco

plants

made

it

possible

to

perform

such

an

analysis

In

the

presence

of

NaCl

despite

a

similar

increase

of

concentration

in

wild

type

and

transgenic

plants

slightly

more

accumulated

in

leaves

after

long-term

salt

stress

-LRB-

Fig.

-RRB-

This

phenomenon

is

different

from

what

has

been

seen

in

transgenic

wheat

overexpressing

-LRB-

Xue

et

al.

-RRB-

Salinity

causes

two

major

damages

to

plant

cells

water

deficit

resulting

from

the

relatively

high

solute

concentrations

in

the

soil

and

ion

toxicity

due

to

altered

K

ratios

as

well

as

excessive

and

Cl

contents

-LRB-

Apse

and

Blumwald

-RRB-

The

H

exchange

activity

of

is

regulated

in

a

and

pH-dependent

manner

as

reported

earlier

-LRB-

Yamaguchi

et

al.

-RRB-

The

increased

accumulation

of

salt

in

both

leaves

and

roots

suggests

that

unlike

which

mainly

works

through

sodium

compartmentation

may

function

through

both

sodium

extrusion

and

compartmentation

controlled

by

dependent

signal

transduction

although

the

precise

mechanism

of

this

still

remains

to

be

elucidated

Proline

contributes

to

osmotic

adjustment

-LRB-

LeRudulier

et

al.

-RRB-

and

the

protection

of

macromolecules

during

dehydration

-LRB-

Yancey

et

al.

-RRB-

and

as

a

hydroxyl

radical

scavenger

-LRB-

Hong

et

al.

-RRB-

After

treatment

with

NaCl

but

not

NaCl

proline

accumulated

to

slightly

higher

levels

in

transgenic

than

in

wild

type

tobacco

plants

-LRB-

Fig.

-RRB-

An

increase

in

proline

content

was

also

observed

in

salt

tolerant

Arabidopsis

with

suppressed

levels

of

proline

degradation

-LRB-

Nanjo

et

al.

-RRB-

transgenic

salt

tolerant

tobacco

plants

with

enhanced

levels

of

proline

biosynthesis

-LRB-

Kishor

et

al.

-RRB-

and

transgenic

salt

tolerant

tomato

plants

overexpressing

-LRB-

Zhang

and

Blumwald

-RRB-

So

it

is

possible

that

the

elevated

concentration

of

proline

in

transgenic

plants

helps

to

protect

anti-oxidative

enzyme

thus

alleviating

the

negative

effects

imposed

by

salt

on

transgenic

plants

Salt-stress-induced

generation

of

reactive

oxygen

species

-LRB-

ROS

-RRB-

has

been

thought

as

one

of

the

major

causes

of

adverse

effects

of

high

salinity

ROS

are

products

of

chloroplast

and

mitochondrial

metabolism

produced

during

stress

they

cause

membrane

damage

resulting

in

electrolyte

leakage

-LRB-

Apse

and

Blumwald

-RRB-

At

NaCl

a

relatively

strong

increase

of

MDA

production

was

observed

in

wild

type

plants

but

only

a

slight

increase

was

seen

in

the

two

transgenic

lines

-LRB-

Fig.

-RRB-

Furthermore

catalase

-LRB-

CAT

-RRB-

activity

increased

significantly

in

transgenic

plants

after

treatment

with

-LRB-

Fig.

-RRB-

These

results

led

us

to

hypothesize

that

constitutive

expression

of

improves

cell

membrane

integrity

Evidence

supporting

this

hypothesis

was

obtained

from

stress

tolerance

experiments

with

transgenic

Arabidopsis

overexpressing

aldehyde

dehydrogenase

or

transgenic

tobacco

overexpressing

Chlamydomonas

glutathione

peroxidase

-LRB-

Yoshimura

et

al.

-RRB-

The

induction

of

stress-related

genes

has

been

taken

as

a

hallmark

of

stress

adaptation

in

plants

-LRB-

Thomashow

-RRB-

Earlier

studies

have

shown

that

lipid

transfer

proteins

are

induced

by

osmotic

stress

-LRB-

Smart

et

al.

-RRB-

Genes

encoding

plastidic

aldolase

are

induced

by

salt

-LRB-

Yamada

et

al.

-RRB-

Transgenic

plants

overexpressing

such

enzymes

exhibit

improved

tolerance

to

the

respective

stresses

Here

we

observed

that

constitutive

expression

of

under

the

control

of

the

CaMV

promoter

triggered

an

increased

expression

of

various

stress

responsive

genes

in

transgenic

tobacco

Already

in

the

absence

of

salinity

stress

transcript

levels

of

genes

encoding

raffinose

synthase

family

protein/seed

imbibition

protein

-LRB-

-RRB-

and

group

LEA

protein

NtERD

-LRB-

-RRB-

were

found

to

be

slightly

elevated

in

the

transgenic

plants

At

high

salinity

the

expression

levels

of

genes

encoding

lipid

transfer

protein

-LRB-

-RRB-

and

raffinose

synthase

family

protein/seed

imbibition

protein

-LRB-

-RRB-

increased

in

the

transgenic

lines

These

results

suggest

that

acts

in

one

or

more

signal

transduction

pathway

-LRB-

s

-RRB-

thereby

affecting

the

activity

of

stress-related

genes

and

stress

tolerance

Many

stresses

evoke

an

elevation

of

the

cytosolic

calcium

ion

concentration

including

salinity

-LRB-

Knight

et

al.

-RRB-

drought

-LRB-

Knight

et

al.

-RRB-

cold

-LRB-

Knight

et

al.

-RRB-

and

oxidative

stress

-LRB-

Sanders

et

al.

-RRB-

Previously

it

has

been

shown

that

salinity

and

osmotic

stress

induce

a

rapid

and

transient

increase

of

levels

-LRB-

DeWald

et

al.

Takahashi

et

al.

-RRB-

and

the

reported

accumulation

of

occurs

during

a

time

frame

similar

to

that

observed

for

stress

induced

mobilization

Therefore

the

possibility

exists

that

enhanced

abiotic

stress

tolerance

of

transgenic

plants

results

from

changes

of

dependent

signaling

Whether

this

is

indeed

the

case

has

to

be

analyzed

in

future

studies

Plant

growth

and

productivity

are

severely

affected

by

abiotic

stress

Augmented

resistance

towards

various

environmental

stresses

has

been

a

desirable

goal

for

genetic

improvement

of

plants

Stress

tolerance

can

for

example

be

improved

by

altering

the

expression

of

stress-responsive

genes

in

transgenic

plants

Representatives

include

genes

encoding

chitinase

and

glucanase

-LRB-

Hong

and

Hwuang

-RRB-

betaine

aldehyde

dehydrogenase

-LRB-

Yang

et

al.

-RRB-

tonoplast

and

plasma

membrane

H

antiporters

-LRB-

Zhang

and

Blumwald

Apse

and

Blumwald

-RRB-

protein

kinases

-LRB-

Xiong

and

Yang

-RRB-

transcription

factors

-LRB-

Waller

et

al.

-RRB-

peroxidases

-LRB-

Yoshimura

et

al.

-RRB-

DNA

helicase

-LRB-

Sanan-Mishra

et

al.

-RRB-

and

DNA

binding

protein

-LRB-

polypeptide

Kalifa

et

al.

-RRB-

Although

the

precise

mode

of

action

of

in

plant

responses

to

stress

remains

elusive

the

results

in

this

work

provide

direct

evidence

that

alteration

of

expression

can

significantly

modify

plant

tolerance

to

various

abiotic

stresses

indicating

a

great

potential

of

this

gene

for

engineering

stress

tolerant

crops

Electronic

supplementary

material

Below

is

the

link

to

the

electronic

supplementary

material

Quantitative

real-time

RT-PCR

and

Western

blot

analyses

of

in

wild

type

Arabidopsis

seedlings

in

response

to

different

stresses

Fourteen-day

old

seedlings

grown

on

MS

agar

plates

were

treated

with

NaCl

or

mannitol

or

kept

at

°C

for

h.

-LRB-

A

-RRB-

Quantitative

real-time

RT-PCR

analysis

of

transcript

level

The

experiments

were

repeated

three

times

independently

and

the

average

calculated

Error

bars

represent

standard

error

of

the

mean

-LRB-

B

-RRB-

Western

blot

analysis

of

expression

level

Coomassie

Bright

Blue

stained

gel

is

shown

below

the

Western

blot

to

demonstrate

equal

protein

loading

-LRB-

TIF

kb

-RRB-

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

arabidopsis thaliana
atipk2β
inositol phosphate
ip3
stress tolerance
transgenic tobacco