Genome-wide

transcriptional

analysis

of

salinity

stressed

japonica

and

indica

rice

genotypes

during

panicle

initiation

stage

Rice

yield

is

most

sensitive

to

salinity

stress

imposed

during

the

panicle

initiation

-LRB-

PI

-RRB-

stage

In

this

study

we

have

focused

on

physiological

and

transcriptional

responses

of

four

rice

genotypes

exposed

to

salinity

stress

during

PI

The

genotypes

selected

included

a

pair

of

indicas

-LRB-

and

-RRB-

and

a

pair

of

japonica

-LRB-

Agami

and

-RRB-

rice

subspecies

with

contrasting

salt

tolerance

Physiological

characterization

showed

that

tolerant

genotypes

maintained

a

much

lower

shoot

concentration

relative

to

sensitive

genotypes

under

salinity

stress

Global

gene

expression

analysis

revealed

a

strikingly

large

number

of

genes

which

are

induced

by

salinity

stress

in

sensitive

genotypes

and

relative

to

tolerant

lines

We

found

probe

sets

to

be

commonly

induced

in

all

four

genotypes

We

found

several

salinity

modulated

ion

homeostasis

related

genes

from

our

analysis

We

also

studied

the

expression

of

a

cation

transporter

reported

by

others

as

a

major

source

of

variation

in

salt

tolerance

in

rice

The

transcript

abundance

of

did

not

change

in

response

to

salinity

stress

at

PI

stage

in

the

shoot

tissue

of

all

four

genotypes

However

we

found

the

transcript

abundance

of

to

be

significantly

higher

in

tolerant

japonica

Agami

relative

to

sensitive

japonica

under

control

and

stressed

conditions

during

PI

stage

Introduction

Rice

is

one

of

the

most

economically

important

crops

of

the

world

Rice

yields

can

be

reduced

by

up

to

when

grown

under

moderate

-LRB-

dS

m

-RRB-

salinity

levels

-LRB-

Zeng

et

al.

-RRB-

Soil

salinity

is

a

serious

constraint

to

rice

cultivation

under

irrigated

agriculture

in

countries

like

Pakistan

and

elsewhere

due

to

poor

quality

of

water

-LRB-

Abdullah

and

Ahmad

-RRB-

Rice

is

the

major

crop

of

many

coastal

regions

which

are

prone

to

sea

water

ingress

during

high

tide

and

of

rainfed

systems

which

experience

increasing

salinity

in

the

root

zone

due

to

a

rising

water

table

Despite

significant

yield

losses

under

saline

conditions

rice

is

potentially

a

good

candidate

for

cultivation

in

areas

prone

to

coastal

flooding

due

to

its

ability

to

tolerate

standing

water

in

the

field

for

long

periods

In

California

salinity

is

an

increasing

concern

for

rice

cultivation

due

to

irrigation

practices

that

involve

use

of

recirculating

water

systems

and

the

requirement

of

water

holding

after

pesticide

application

during

early

growth

stages

-LRB-

Scardaci

et

al.

-RRB-

The

susceptibility

of

rice

to

salinity

stress

varies

with

growth

stage

Rice

is

relatively

salt-tolerant

at

germination

and

in

some

cases

is

not

affected

significantly

by

up

to

dS

m

of

salinity

-LRB-

Heenan

et

al.

Khan

et

al.

-RRB-

Rice

becomes

very

sensitive

at

the

young

seedling

stage

which

impacts

the

stand

density

in

salt-affected

fields

-LRB-

Lutts

et

al.

-RRB-

It

is

relatively

less

sensitive

during

late

reproductive

stage

-LRB-

grain

ripening

-RRB-

From

an

agronomic

perspective

sensitivity

of

yield

components

is

of

primary

importance

Salinity

significantly

reduces

the

tiller

number

per

plant

spikelet

number

per

panicle

fertility

panicle

length

and

primary

branches

per

panicle

-LRB-

Heenan

et

al.

Cui

et

al.

Khatun

et

al.

Zeng

et

al.

-RRB-

Reductions

in

tiller

number

per

plant

and

spikelet

number

per

panicle

were

reported

to

be

the

major

causes

of

yield

loss

in

one

cultivar

of

rice

under

salinity

stress

-LRB-

Zeng

and

Shannon

-RRB-

The

number

of

spikelets

per

panicle

was

determined

to

be

the

most

sensitive

yield

component

This

component

is

determined

at

early

reproductive

stage

around

panicle

initiation

-LRB-

PI

-RRB-

-LRB-

Hoshikawa

Counce

et

al.

-RRB-

The

loss

in

spikelet

number

per

plant

was

most

significant

when

the

stress

was

imposed

before

PI

-LRB-

stage

-RRB-

or

between

PI

and

booting

stage

-LRB-

days

after

PI

-RRB-

-LRB-

Zeng

et

al.

-RRB-

This

loss

of

potential

spikelets

is

attributed

to

degeneration

of

primary

and

secondary

branches

and

flower

primordia

-LRB-

Zeng

et

al.

-RRB-

Considerable

genetic

variability

for

this

yield

parameter

was

reported

based

on

evaluation

of

several

diverse

genotypes

for

salinity

stress

response

-LRB-

Zeng

et

al.

-RRB-

Considerable

variation

for

salt

tolerance

at

critical

stages

in

the

cultivated

gene

pool

has

also

been

reported

by

other

workers

-LRB-

Yeo

and

Flowers

Moradi

et

al.

-RRB-

This

genetic

variability

can

be

utilized

for

improvement

of

salt

tolerance

by

focusing

on

specific

yield

components

However

the

underlying

genes

conferring

tolerance

during

PI

are

at

present

unknown

In

the

current

study

we

have

focused

on

the

salinity

stress

imposed

during

the

PI

stage

considering

the

significant

impact

it

has

on

rice

yields

and

a

lack

of

prior

information

on

molecular

components

of

the

response

to

salinity

stress

at

this

critical

stage

The

genotypes

selected

for

this

study

have

contrasting

physiological

responses

to

salinity

stress

at

PI

based

on

several

seasons

of

experimentation

at

the

U.S.

Salinity

Laboratory

Riverside

-LRB-

Zeng

et

al.

-RRB-

One

of

the

most

salt-tolerant

genotypes

identified

by

Zeng

et

al.

-LRB-

-RRB-

was

an

indica

line

developed

from

a

cross

between

tolerant

land

race

Nona

Bokra

and

sensitive

cultivar

This

genotype

had

the

most

favorable

combination

of

salt

tolerance

at

early

vegetative

and

PI

stages

This

genotype

possesses

good

yield

potential

under

salinity

stress

due

to

high

tillering

ability

and

panicle

weight

-LRB-

Zeng

et

al.

-RRB-

Another

genotype

Agami

was

ranked

high

for

its

ability

to

yield

high

under

salinity

stress

Agami

is

a

salt-tolerant

japonica

subspecies

cultivar

grown

in

Egypt

Several

studies

have

reported

the

popular

California

japonica

cultivar

to

be

very

sensitive

at

all

stages

-LRB-

Zeng

et

al.

-RRB-

Therefore

for

the

current

work

we

have

used

and

Agami

as

the

tolerant

genotypes

representing

indica

and

japonica

subspecies

and

and

as

the

sensitive

genotypes

for

each

of

the

subspecies

Previously

we

focused

on

transcript

accumulation

responses

of

rice

to

salinity

stress

imposed

during

the

early

vegetative

stage

in

two

indica

subspecies

genotypes

which

differed

in

salt

tolerance

as

manifested

by

higher

tillering

capacity

and

maintenance

of

favorable

ion

homeostasis

-LRB-

Walia

et

al.

-RRB-

The

sensitive

genotype

used

for

that

study

was

a

widely

used

sensitive

standard

which

is

susceptible

at

the

vegetative

and

reproductive

stages

was

the

sensitive

parent

of

a

recombinant

inbred

population

developed

at

the

International

Rice

Research

Institute

-LRB-

IRRI

-RRB-

for

mapping

salt

tolerance

quantitative

trait

loci

-LRB-

QTL

-RRB-

in

rice

using

Pokkali

as

the

tolerant

parent

A

salt-tolerant

recombinant

inbred

line

-LRB-

RIL

-RRB-

from

this

population

was

used

as

the

tolerant

genotype

in

our

prior

study

-LRB-

Walia

et

al.

-RRB-

The

transcriptome

of

rice

under

salinity

stress

at

PI

has

been

largely

uncharacterized

Considering

the

sensitivity

of

rice

yield

to

salinity

stress

when

imposed

around

PI

it

is

important

to

identify

specific

genes

that

are

involved

in

the

response

to

stress

at

this

stage

Responsive

genes

are

expected

to

include

those

with

a

role

in

salt

adaptation

and

tolerance

and

which

respond

to

salt

injury

or

constitute

non-adaptive

responses

The

use

of

genotypes

with

well-characterized

phenotypic

responses

to

salinity

stress

at

a

particular

growth

stage

can

help

in

identifying

potential

candidate

genes

which

may

be

important

for

heritable

salt

tolerance

Here

we

perform

whole

genome

transcript

profiling

of

genotypes

from

two

major

subspecies

of

cultivated

rice

chosen

for

their

representative

tolerance

or

sensitivity

to

salinity

stress

during

the

early

reproductive

stage

specifically

at

PI

By

including

indica

genotype

in

the

present

study

on

PI

we

now

have

mRNA

expression

level

responses

at

both

of

the

two

most

salt

sensitive

yield-determining

stages

for

this

genotype

-LRB-

early

vegetative

as

per

Walia

et

al.

and

early

reproductive

in

this

study

-RRB-

Physiological

characterization

of

the

lines

was

also

performed

as

part

of

the

present

study

and

gene

expression

results

were

interpreted

in

the

physiological

context

We

also

incorporated

information

from

known

salt

tolerance

QTL

in

rice

with

the

objective

of

correlating

gene

expression

with

gene

location

on

the

rice

genome

Material

and

methods

Plant

materials

for

expression

studies

Seeds

of

rice

-LRB-

Oryza

sativa

L.

-RRB-

genotypes

and

Agami

were

obtained

from

G.

B.

Gregorio

at

IRRI

of

the

Philippines

and

then

propagated

through

quarantine

at

the

George

E.

Brown

Jr.

Salinity

Laboratory

Riverside

CA

USA

Seed

for

was

supplied

by

California

Cooperative

Rice

Research

Foundation

Inc.

at

Biggs

California

These

genotypes

were

selected

from

the

germplasm

collections

at

the

two

sites

based

on

their

differences

in

salt

tolerance

in

terms

of

agronomic

and

physiological

performance

Extensive

characterization

of

these

lines

was

performed

by

Zeng

et

al.

-LRB-

-RRB-

at

George

E.

Brown

Jr.

Salinity

Laboratory

Riverside

CA

USA

Plant

culture

and

salinity

treatments

The

experiment

was

conducted

in

the

greenhouse

at

Riverside

California

-LSB-

N

latitude

W

longitude

-RSB-

between

May

and

July

Plants

were

cultured

in

tanks

-LRB-

cm

-RRB-

filled

with

sand

and

irrigated

with

nutrient

solution

-LRB-

Yoshida

et

al.

-RRB-

The

nutrient

solution

consisted

of

-LRB-

mM

-RRB-

-LRB-

mM

-RRB-

-LRB-

mM

-RRB-

-LRB-

mM

-RRB-

and

-LRB-

mM

-RRB-

Nutrient

solution

pH

was

maintained

between

and

by

adding

sulfuric

acid

as

needed

Irrigation

solutions

were

prepared

in

L

reservoirs

and

pumped

to

provide

irrigation

to

the

sand

tanks

Overflow

irrigation

was

returned

to

the

reservoirs

through

drainage

by

gravity

Each

reservoir

provided

irrigation

water

to

three

sand

tanks

-LRB-

replicates

-RRB-

three

times

daily

for

min

per

irrigation

cycle

Four

rows

of

each

genotype

were

planted

per

tank

for

control

tanks

An

extra

row

at

each

end

of

the

tank

was

used

as

the

border

row

Each

genotype

was

planted

in

separate

tanks

which

were

marked

for

salinization

later

in

the

experiment

The

water

level

was

maintained

at

the

sand

surface

until

emergence

of

the

seedlings

from

the

sand

and

then

increased

to

cm

above

the

sand

surface

Air

temperature

ranged

from

°C

to

°C

during

the

day

and

°C

during

night

Relative

humidity

ranged

from

to

Light

averaged

μmol

m

s

at

noon

Weak

plants

were

removed

from

the

tanks

Plant

growth

stages

were

closely

monitored

Rough

estimates

of

the

days

to

PI

stage

for

each

genotype

were

available

from

growing

the

genotypes

for

several

years

in

the

same

greenhouse

conditions

and

time

of

the

year

during

previous

experiments

-LRB-

Zeng

et

al.

-RRB-

Salinity

treatment

was

applied

by

adding

NaCl

and

-LRB-

molar

concentration

-RRB-

in

two

steps

over

a

period

of

days

to

each

genotype

about

days

before

PI

-LRB-

Fig.

-RRB-

Electrical

conductivities

-LRB-

ECw

-RRB-

of

nutrient

solutions

were

monitored

using

an

EC

meter

daily

-LRB-

Hanna

Instruments

Woonsockett

RI

-RRB-

The

salinity

in

the

treatment

reservoir

was

allowed

to

stabilize

for

days

to

a

final

electrical

conductivity

of

dS

m

The

non-saline

control

reservoir

with

Yoshida

solution

had

an

ECw

of

dS

m

Two

plants

were

randomly

selected

each

day

for

each

genotype

when

the

plants

were

approaching

PI

The

main

culms

were

dissected

under

a

microscope

to

observe

the

development

of

young

panicles

The

day

when

we

observed

any

plant

of

a

genotype

with

panicle

about

mm

long

was

defined

as

the

PI

date

for

that

genotype

under

the

treatment

in

which

it

was

growing

-LRB-

control

or

salt

stressed

-RRB-

Salinity

delayed

PI

in

the

sensitive

genotype

but

not

in

other

three

lines

Fig.

design

of

salinity

stress

treatment

Salinity

stress

was

applied

days

before

panicle

initiation

-LRB-

PI

-RRB-

A

final

salinity

level

of

dS

m

was

reached

by

a

two-step

addition

of

NaCl

and

Conductivity

in

the

control

tanks

is

represented

by

a

dotted

line

and

the

treated

tank

by

a

solid

line

Diamonds

represent

the

PI

and

ovals

represent

the

harvest

time

points

The

crown

and

growing

point

of

the

main

shoot

were

harvested

days

after

reaching

PI

Phenotypic

characterization

and

gene

expression

studies

Plants

were

characterized

for

phenotypic

responses

to

salinity

stress

on

the

day

of

harvest

for

RNA

extraction

For

phenotypic

characterization

the

shoot

tissue

from

six

plants

was

harvested

for

ion

analysis

Plants

were

washed

with

deionized

water

dried

in

a

forced

air

oven

-LRB-

°C

-RRB-

and

then

ground

into

fine

powder

Shoot

and

K

concentrations

were

determined

on

nitric-perchloric

acid

digests

by

inductively

coupled

plasma

optical

emission

spectrometry

-LRB-

ICP

Perkin-Elmer

Co.

Norwalk

CT

USA

-RRB-

Transpiration

rate

of

the

youngest

fully

expanded

leaf

was

measured

between

h

and

h

one

day

before

harvesting

the

tissue

for

RNA

using

a

LI-COR

Photosynthesis

System

-LRB-

LI-COR

Biosciences

Lincoln

NE

-RRB-

The

following

conditions

for

leaf

gas

measurements

were

used

photosynthetic

photon

flux

density

μmol

m

chamber

concentration

μmol

mol

leaf

temperature

°C

and

chamber

vapor

concentration

mmol

mol

The

plants

were

harvested

or

days

after

reaching

PI

The

main

shoot

was

dissected

for

RNA

extraction

to

obtain

the

growing

point

and

crown

tissue

which

was

snap

frozen

Approximately

plants

were

harvested

per

genotype

per

tank

and

tissue

pooled

to

make

each

biological

replicate

for

RNA

extraction

Three

biological

replicates

-LRB-

one

per

array

-RRB-

were

used

for

each

treatment

Two

plants

from

each

tank

were

allowed

to

grow

to

maturity

to

verify

that

plants

survived

the

imposed

salinity

stress

RNA

extraction

and

processing

for

GeneChip

analysis

RNA

samples

were

processed

as

recommended

by

Affymetrix

Inc.

-LRB-

Affymetrix

GeneChip

Expression

Analysis

Technical

Manual

Affymetrix

Inc.

Santa

Clara

CA

-RRB-

at

the

Core

Instrumentation

Facility

at

the

University

of

California

Riverside

by

Barbara

Walter

Total

RNA

was

initially

isolated

from

frozen

shoot

tissue

using

TRIzol

Reagent

The

RNA

was

purified

using

an

RNeasy

spin

column

-LRB-

Qiagen

Chatsworth

CA

-RRB-

and

an

on-column

DNase

treatment

Eluted

total

RNAs

were

quantified

with

a

portion

of

the

recovered

total

RNA

and

adjusted

to

a

final

concentration

of

μg/μl

All

RNA

samples

were

quality

assessed

prior

to

beginning

target

preparation/processing

steps

by

running

out

a

small

amount

of

each

sample

-LRB-

typically

ng/well

-RRB-

onto

a

RNA

Lab-On-A-Chip

-LRB-

Caliper

Technologies

Corp.

Mountain

View

CA

-RRB-

that

was

evaluated

on

an

Agilent

Bioanalyzer

-LRB-

Agilent

Technologies

Palo

Alto

CA

-RRB-

Single-stranded

then

double-stranded

cDNA

was

synthesized

from

the

poly

-LRB-

A

-RRB-

mRNA

present

in

the

isolated

total

RNA

-LRB-

μg

total

RNA

starting

material

each

sample

reaction

-RRB-

using

the

SuperScript

Double-Stranded

cDNA

Synthesis

Kit

-LRB-

Invitrogen

Corp.

Carlsbad

CA

-RRB-

and

poly

-LRB-

T

-RRB-

nucleotide

primers

that

contained

a

sequence

recognized

by

RNA

polymerase

A

portion

of

the

resulting

ds

cDNA

was

used

as

a

template

to

generate

biotin-tagged

cRNA

from

an

in

vitro

transcription

reaction

-LRB-

IVT

-RRB-

using

the

Affymetrix

GeneChip

IVT

Labeling

Kit

Fifteen

micrograms

of

the

resulting

biotin-tagged

cRNA

was

fragmented

to

strands

of

bases

in

length

following

prescribed

protocols

-LRB-

Affymetrix

GeneChip

Expression

Analysis

Technical

Manual

-RRB-

Subsequently

μg

of

this

fragmented

target

cRNA

was

hybridized

at

°C

with

rotation

for

h

-LRB-

Affymetrix

GeneChip

Hybridization

Oven

-RRB-

to

probe

sets

present

on

an

Affymetrix

Rice

Genome

array

The

GeneChip

arrays

were

washed

and

then

stained

-LRB-

SAPE

streptavidin-phycoerythrin

-RRB-

on

an

Affymetrix

Fluidics

Station

followed

by

scanning

on

a

GeneChip

Scanner

Rice

Genome

array

The

Rice

Genome

array

-LRB-

Affymetrix

Santa

Clara

CA

-RRB-

contains

probe

sets

designed

from

japonica

and

indica

sequences

The

sequence

information

for

this

array

was

derived

from

NCBI

UniGene

Build

-LRB-

-RRB-

GenBank

mRNAs

and

gene

predictions

from

TIGR

's

version

Gene

models

which

had

any

indication

of

transposable

elements

were

removed

from

the

list

of

TIGR

predicted

genes

The

array

is

believed

to

represent

about

distinct

rice

genes

About

of

these

are

anchored

Unigene

EST

and

mRNA

clusters

including

known

rice

full

length

cDNA

clones

and

are

solely

from

TIGR

gene

predictions

To

obtain

annotations

for

the

salt-regulated

probe

sets

we

extracted

the

target

sequence

of

identified

probe

sets

from

the

sequence

information

file

-LRB-

sif

-RRB-

for

the

rice

genome

array

The

target

sequence

extends

from

the

end

of

the

most

probe

to

the

end

of

the

most

probe

The

target

sequences

were

then

searched

using

BLASTn

against

the

TIGR

rice

pseudomolecules

release

-LRB-

-RRB-

and

the

TIGR

Arabidopsis

database

version

Statistical

analysis

of

array

data

The

triplicated

array

data

set

was

analyzed

using

GeneChip

Operating

Software

-LRB-

GCOS

-RRB-

and

DChip

-LRB-

Li

and

Wong

-RRB-

software

The

scanned

images

were

examined

for

any

visible

defects

Satisfactory

image

files

were

analyzed

to

generate

raw

data

files

saved

as

CEL

files

using

the

default

settings

of

GCOS

from

Affymetrix

We

used

a

default

target

intensity

value

-LRB-

TGT

-RRB-

setting

of

The

normalization

factor

value

was

set

to

to

extract

data

without

normalization

Default

parameter

settings

for

the

Rice

Genome

array

were

used

The

scaling

factor

for

the

arrays

ranged

from

to

The

detection

calls

-LRB-

present

absent

or

marginal

-RRB-

for

the

probe

sets

were

made

by

GCOS

Further

analysis

was

done

using

DChip

which

incorporates

a

statistical

model

for

expression

array

data

at

the

probe

level

The

DChip

program

was

set

to

import

GCOS

signals

The

normalization

of

all

arrays

was

performed

using

an

invariant

set

approach

For

calculating

the

expression

index

of

probe

sets

we

used

the

PM

model

and

opted

for

truncating

the

low

expression

values

to

percentile

of

the

probe

set

signals

called

absent

The

expression

values

were

transformed

after

calculating

the

expression

index

DChip

was

used

for

comparative

analysis

of

samples

from

salt

stress

and

control

treated

plants

for

both

genotypes

We

identified

the

differentially

expressed

genes

using

the

empirical

criterion

of

more

than

change

-LRB-

E-B

or

B-E

-RRB-

and

significant

t-tests

of

P

based

on

three

biological

replicates

The

baseline

array

which

is

a

control

sample

is

denoted

by

B

and

salinity-treated

experimental

sample

is

denoted

by

E

The

P-values

assigned

to

differentially

expressed

genes

were

used

as

a

ranking

criterion

for

ordering

of

up-regulated

and

down-regulated

genes

We

used

the

DChip

feature

to

assess

the

false

discovery

rate

-LRB-

FDR

-RRB-

in

our

data

set

using

permutations

in

most

comparisons

Empirical

FDR

was

below

when

identifying

differentially

expressed

genes

All

microarray

data

from

this

work

is

available

from

NCBI

GEO

-LRB-

-RRB-

under

the

series

entry

Expression

validation

by

semi-quantitative

RT-PCR

Several

key

expression

profiles

obtained

from

chip

hybridizations

were

further

validated

by

semi-quantitative

RT-PCR

using

first-strand

cDNA

The

japonica

sequence

of

each

gene

was

obtained

from

the

TIGR

rice

database

Exonic

sequences

were

used

for

the

design

of

primers

using

Primer

Express

-LRB-

Perkin-Elmer

Applied

Biosystems

Foster

City

CA

-RRB-

The

primer

sequences

for

rRNA

-LRB-

F-ATGATAACTCGACGGATCGC

R-CTTGGATGTGGTAGCCGTTT

-RRB-

were

used

as

an

internal

control

The

primer

sequences

for

were

obtained

from

Walia

et

al.

-LRB-

-RRB-

A

cDNA

first

strand

was

synthesized

using

Taq-Man

Reverse

Transcription

Reagents

-LRB-

Applied

Biosystems

Forster

City

CA

Ref

-RRB-

following

the

manufacturer

's

instructions

Two

micrograms

of

total

RNA

was

converted

into

cDNA

Each

cDNA

was

diluted

times

and

μL

of

cDNA

was

used

for

three-step

PCR

The

number

of

PCR

cycles

was

optimized

-LRB-

cycles

-RRB-

for

the

genes

selected

for

expression

validated

and

cycles

for

Results

A

moderate

level

of

salinity

stress

was

imposed

gradually

nine

or

ten

days

before

PI

stage

and

plants

were

sampled

for

phenotypic

characterization

and

expression

profiling

five

days

after

PI

-LRB-

Fig.

-RRB-

All

four

genotypes

used

in

this

experiment

were

known

to

reach

PI

at

different

time

points

based

on

prior

characterization

of

these

genotypes

over

several

growing

seasons

under

comparable

growing

conditions

and

time

of

the

year

One

confounding

factor

in

experiment

involving

salinity

stress

is

that

salinity

is

known

to

delay

PI

in

all

genotypes

With

this

in

mind

we

designed

our

experiment

to

harvest

the

control

and

salinity

stressed

tissue

of

each

of

the

genotype

at

similar

developmental

stage

i.e.

days

after

the

plants

reached

PI

This

was

done

because

developmental

equivalence

when

comparing

control

plants

with

stressed

plants

within

each

genotype

is

more

relevant

when

the

focus

is

on

a

specific

sensitive

stage

such

as

PI

Shoot

ion

uptake

of

salt-tolerant

and

sensitive

lines

Shoot

sodium

ion

analysis

when

assayed

under

salinity

stress

clearly

distinguished

tolerant

from

sensitive

genotypes

Salinity

stress

increased

the

concentration

in

all

genotypes

significantly

-LRB-

Table

-RRB-

The

increase

was

nearly

in

all

genotypes

except

Sodium

ion

accumulation

in

the

tolerant

genotypes

and

Agami

was

much

lower

than

in

the

sensitive

genotypes

Agami

the

salt-tolerant

japonica

maintained

a

lower

concentration

even

under

control

conditions

and

had

the

lowest

concentration

under

stressed

conditions

Shoot

K

concentration

decreased

in

Agami

while

the

concentration

in

other

three

genotypes

did

not

change

significantly

under

salinity

stress

Since

K

ratio

under

salinity

is

considered

a

more

important

index

of

salinity

tolerance

than

ion

concentrations

separately

we

ranked

the

four

genotypes

by

K

ratio

in

descending

order

Agami

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Under

control

conditions

indicas

had

a

lower

K

ratio

than

japonica

varieties

In

addition

to

measuring

and

K

levels

in

the

shoot

tissue

we

also

analyzed

the

chloride

levels

Shoot

chloride

concentrations

increased

in

all

genotypes

under

salinity

stress

Sensitive

japonica

however

maintained

a

much

lower

Cl

level

-LRB-

mmol

kg

compared

to

mmol

kg

in

Agami

-RRB-

than

other

genotypes

Table

responses

of

salt-sensitive

and

salt-tolerant

genotypesTreatmentsNa

K+C

l

Agami

Agami

The

shoot

K

and

Cl

concentrations

were

measured

in

mmol

kg

dry

weight

Transpiration

rate

-LRB-

Tr

-RRB-

is

expressed

as

m

s

Ion

concentration

values

were

obtained

from

six

replicates

Tr

values

are

mean

S.E.

from

to

replicate

measurements

To

further

characterize

the

physiological

status

of

the

four

genotypes

we

measured

the

transpiration

rate

of

the

youngest

fully

expanded

leaf

of

the

main

shoot

-LRB-

Table

-RRB-

The

transpiration

rate

decreased

in

all

genotypes

under

stress

A

noteworthy

observation

was

the

significantly

lower

rate

of

transpiration

maintained

by

tolerant

japonica

Agami

under

control

and

stressed

conditions

relative

to

all

other

genotypes

Tolerance

in

Agami

therefore

seems

to

depend

on

an

avoidance

mechanism

We

also

observed

the

plants

for

visual

damage

symptoms

of

salinity

stress

The

sensitive

genotypes

especially

had

necrosis

on

some

leaves

about

the

length

of

the

leaf

from

the

tip

roughly

one

week

after

the

initiation

of

stress

Expression

responses

of

tolerant

and

sensitive

genotypes

to

salinity

stress

We

used

two

japonica

and

indica

rice

genotypes

with

contrasting

salt

tolerance

for

global

gene

expression

under

salinity

stress

The

developmental

stage

targeted

in

this

study

was

PI

Salinity

stress

was

applied

nine

or

days

before

PI

and

the

tissue

-LRB-

crown

and

growing

point

-RRB-

was

harvested

days

after

the

genotypes

reached

PI

stage

-LRB-

Fig.

see

Materials

and

methods

-RRB-

To

define

differentially

expressed

genes

we

used

the

following

criteria

-LRB-

-RRB-

t-test

P-value

-LRB-

-RRB-

change

and

-LRB-

-RRB-

FDR

below

-LRB-

see

Materials

and

methods

-RRB-

Using

these

criteria

we

identified

and

probe

sets

which

were

up

and

down-regulated

in

salt-sensitive

japonica

respectively

-LRB-

Fig.

-RRB-

The

same

selection

criteria

identified

up

and

down-regulated

probe

sets

in

the

salt-tolerant

japonica

Agami

A

comparable

number

of

probe

sets

-LRB-

-RRB-

were

differentially

up-regulated

in

salt-tolerant

indica

under

salinity

stress

A

total

of

probe

sets

were

down-regulated

in

The

expression

response

of

sensitive

indica

was

characterized

by

up-regulation

of

probe

sets

In

contrast

only

probe

sets

were

significantly

down-regulated

in

response

to

salinity

stress

Complete

lists

of

probe

sets

identified

as

differentially

expressed

in

each

of

the

genotypes

are

provided

as

supplemental

data

-LRB-

Datasheet

-RRB-

Five

probe

sets

identified

as

differentially

expressed

from

the

microarray

datasets

were

checked

using

semi-quantitative

RT-PCR

-LRB-

Supplemental

Fig.

-RRB-

The

results

agreed

for

four

of

the

genes

examined

The

primers

for

the

fifth

gene

did

not

amplify

from

the

indica

subspecies

since

they

were

derived

from

japonica

exonic

sequence

Nineteen

probe

sets

were

significantly

up-regulated

in

all

genotypes

in

response

to

salinity

stress

A

selected

list

of

probe

sets

which

were

commonly

induced

is

presented

in

Table

Only

one

probe

set

Os

with

no

hits

to

the

database

was

down-regulated

by

salinity

stress

in

all

genotypes

Fig.

of

up-regulated

probe

sets

between

different

genotypes

-LRB-

A

-RRB-

Overlap

between

the

sensitive

genotypes

and

-LRB-

B

-RRB-

Overlap

between

the

tolerant

genotypes

-LRB-

C

-RRB-

Overlap

among

the

japonica

subspecies

-LRB-

D

-RRB-

overlap

among

the

indica

subspecies

The

up-regulated

probe

sets

were

identified

using

the

combined

criteria

of

a

or

more

fold

change

P-value

threshold

of

and

false

discovery

rate

below

Table

genes

commonly

induced

in

all

four

genotypes

and

their

P-values

during

the

salinity

stress

imposed

during

reproductive

stagePutative

functionProbe

setRice

protein

riceOs

Low-temperature-induced

protein

Myb-like

DNA-binding

domainOs

RNase

S-like

protein

-LRB-

drought

induced

-RRB-

Os

Low

affinity

nitrate

transporterOs

Putative

Myb-like

DNA-binding

ProteinOs

Bowman

Birk

serine

protease

inhibitorOs

Nitrate

transporter

-LRB-

-RRB-

Os

Anthocyanin

Putative

function

is

based

on

BLAST

hits

against

TIGR

rice

TIGR

Arabidopsis

and

NCBI

nr

database

We

made

several

comparisons

to

identify

genes

which

characterize

the

response

of

japonica

and

indica

to

salinity

as

well

as

genes

which

are

expressed

in

tolerant

and

sensitive

genotypes

-LRB-

Fig.

-RRB-

We

identified

probe

sets

which

were

induced

commonly

between

the

two

japonicas

Agami

and

Similar

comparison

between

the

indica

genotypes

identified

probes

sets

as

commonly

induced

In

an

effort

to

search

for

transcripts

which

may

be

associated

with

salt

tolerance

of

Agami

we

identified

probe

sets

which

were

induced

in

Agami

but

not

in

Two

of

these

probe

sets

-LRB-

Os

and

OsAffx

-RRB-

were

induced

in

Agami

but

down-regulated

in

Probe

set

Os

had

no

sequence

match

to

known

genes

Probe

set

OsAffx

represented

a

germin-like

gene

-LRB-

-RRB-

We

also

identified

several

probe

sets

which

were

induced

in

but

not

in

sensitive

indica

These

included

a

jacalin

lectin

protein

and

nitrate

reductase

-LRB-

-RRB-

The

expression

of

is

known

to

be

induced

in

response

to

salt

stress

and

dehydration

-LRB-

Yamaguchi-Shinozaki

and

Shinozaki

-RRB-

Besides

focusing

on

the

genes

which

were

differentially

expressed

in

response

to

salinity

stress

within

each

of

the

genotypes

we

also

performed

genotype

genotype

comparisons

under

control

conditions

This

analysis

was

performed

to

identify

genes

associated

with

salt

tolerance

that

may

be

inherently

transcribed

at

a

higher

level

in

one

genotype

than

in

another

Interpretation

of

such

genotypic

comparisons

using

plants

profiled

under

control

conditions

is

done

in

light

of

the

fact

that

the

compared

genotypes

have

similar

developmental

stage

but

not

same

chronological

age

We

found

probe

sets

which

were

significantly

up-regulated

in

Agami

relative

to

under

control

conditions

-LRB-

Supplemental

Data

Sheet

-RRB-

A

similar

comparison

yielded

probe

sets

expressed

at

a

higher

level

in

than

-LRB-

Supplemental

Data

Sheet

-RRB-

We

BLAST

searched

the

target

sequence

of

all

the

probe

sets

against

the

rice

genomic

sequence

assembled

at

TIGR

-LRB-

-RRB-

to

obtain

the

genomic

position

of

the

best

BLAST

hit

gene

model

We

found

several

probe

sets

which

were

elevated

in

Agami

relative

to

to

be

tightly

clustered

Clusters

on

chromosome

and

were

particularly

large

-LRB-

Fig.

-RRB-

A

dense

set

cluster

occurred

on

the

long

arm

of

chromosome

This

cluster

localized

to

a

segment

of

Mb

and

represented

rice

gene

loci

Another

set

cluster

on

chromosome

extends

from

Mb

to

Mb

-LRB-

Mb

in

length

-RRB-

This

region

is

of

particular

interest

because

two

QTL

for

salt

tolerance

have

been

mapped

to

this

region

-LRB-

Bonilla

et

al.

Lin

et

al.

-RRB-

The

number

of

gene

clusters

obtained

from

the

comparison

of

indica

genotypes

was

relatively

small

and

most

were

found

on

chromosome

-LRB-

Fig.

-RRB-

Two

of

the

clusters

on

chromosome

were

located

in

the

region

of

a

salt

tolerance

QTL

Saltol

The

first

was

an

set

-LRB-

six

gene

loci

-RRB-

cluster

-LRB-

Mb

-RRB-

and

the

second

was

a

set

-LRB-

eight

gene

loci

-RRB-

cluster

extending

from

Mb

to

Mb

The

positions

of

these

two

clusters

may

coincide

with

two

separate

QTL

in

the

Saltol

region

reported

by

Lin

et

al.

-LRB-

-RRB-

and

Bonilla

et

al.

-LRB-

-RRB-

Additionally

a

dense

set

kb

long

cluster

was

located

on

the

distal

end

of

chromosome

Fig.

chromosomes

with

gene

clusters

based

on

genotypic

comparisons

-LRB-

A

-RRB-

Rice

gene

model

clusters

on

chromosomes

and

obtained

from

genotypic

comparison

of

with

Agami

under

control

conditions

-LRB-

B

-RRB-

Rice

gene

model

clusters

on

chromosome

obtained

by

genotypic

comparison

of

with

using

unstressed

samples

The

chromosome

displays

only

up-regulated

rice

genes

in

the

tolerant

lines

under

control

conditions

The

gene

clusters

with

shaded

area

co-localize

to

previously

identified

as

a

salt

tolerance

QTL

on

chromosome

Genes

involved

in

ion

homeostasis

Maintenance

of

low

and

a

favorable

K

ratio

are

desirable

traits

for

rice

growing

under

saline

conditions

and

ultimately

manifest

as

salt

tolerance

Considering

the

importance

of

ion

transporters

in

exclusion

redistribution

and

compartmentalization

of

ions

we

searched

for

and

identified

several

cation

transport-related

genes

which

significantly

responded

to

the

salinity

treatment

-LRB-

Table

-RRB-

One

such

gene

was

a

sodium

transport

protein

-LRB-

-RRB-

induced

-LRB-

P

-RRB-

in

only

In

contrast

a

sodium/calcium

exchanger

was

down-regulated

in

Another

gene

-LRB-

-RRB-

encoding

for

CLC-d

chloride

channel

was

induced

in

and

by

-LRB-

P

-RRB-

and

-LRB-

p

-RRB-

respectively

Several

genes

associated

with

transport

of

K

were

differentially

regulated

in

response

to

salinity

stress

Three

of

these

genes

belong

to

the

KT-HAK-KUP

family

of

transporters

associated

with

high

affinity

K

uptake

from

the

roots

-LRB-

Santa-Maria

et

al.

-RRB-

In

a

transporter

-LRB-

-RRB-

similar

to

was

up-regulated

by

stress

while

-LRB-

-RRB-

was

down-regulated

Another

cation

channel

gene

-LRB-

-RRB-

with

sequence

similarity

to

was

down-regulated

in

A

potassium

transporter

-LRB-

-RRB-

was

induced

in

by

more

than

-LRB-

P

-RRB-

under

salinity

stress

We

did

not

detect

any

cation

transporters

significantly

responding

to

salinity

stress

in

the

tolerant

genotypes

Table

from

selected

categories

which

were

differentially

expressed

under

saline

conditions

in

one

or

more

rice

genotypesPutative

functionProbe

homeostasisK

transporter

-LRB-

-RRB-

Os

CLC-d

chloride

channelOs

Cation

channel

protein

-LRB-

-RRB-

OsAffx

exchanger

proteinOs

K

transporter

-LRB-

-RRB-

Os

Cation

-LRB-

-RRB-

transport

proteinOs

K

transporter

-LRB-

-RRB-

Os

Flowering

and

inflorescenceCentroradialis

protein

-LRB-

CEN

-RRB-

Os

Zinc

finger

protein

-LRB-

-RRB-

Os

Constan-like

protein

-LRB-

-RRB-

Os

CCT

motifOs

receptor

kinaseOs

MADS-box

proteinOs

Gigantea-like

proteinOs

Floral

homeotic

protein

-LRB-

-RRB-

Os

Flowering

locus

TOsAffx

Homeodomain

-LRB-

-RRB-

Os

Sex

determination

tasselseed

No

apical

meristem

-LRB-

NAM

-RRB-

Os

Auxin-repressed

protein

Auxin-responsive

proteinOs

Auxin

transporter

Phototropic-responsive

protein

kinaseOs

Auxin

response

factor

Anthocyanin

pathwayChalcone

stilbene

synthasesOs

Phenylalanine

Isoflavone

reductaseOs

synthase

Flavonoid

hydroxylase

Os

Dihydroflavonol

Chalcone

isomerase

-LRB-

CHI

-RRB-

Os

acidOsAffx

Fold

change

in

each

genotype

is

expressed

as

-LRB-

is

equal

to

change

numerically

-RRB-

The

P-value

threshold

of

was

used

for

significant

differential

expression

A

-LRB-

-RRB-

indicates

no

change

detected

Since

the

shoot

ion

analysis

indicated

a

strong

exclusion

phenotype

in

the

tolerant

lines

we

further

explored

our

data

set

for

any

association

of

cation

transporters

On

comparing

the

tolerant

Agami

with

under

control

and

salinity

stress

conditions

we

found

a

cation

transporter

-LRB-

-RRB-

to

be

expressing

at

a

higher

level

in

Agami

by

-LRB-

P

-RRB-

and

-LRB-

P

-RRB-

compared

to

under

control

and

salt

stressed

conditions

respectively

On

extending

the

genotypic

comparison

to

indica

genotypes

we

found

transcript

to

be

present

in

at

a

higher

level

than

in

-LRB-

P

-RRB-

under

control

conditions

The

cation

transporter

gene

is

located

in

the

vicinity

of

the

Saltol

region

-LRB-

Bonilla

et

al.

-RRB-

and

has

a

perfect

sequence

match

to

the

gene

reported

to

be

the

tolerance

conferring

gene

in

the

salt

tolerance

QTL

studied

by

Ren

et

al.

-LRB-

-RRB-

We

pursued

the

expression

of

this

gene

using

semi-quantitative

RT-PCR

in

roots

and

shoot

tissue

of

several

rice

genotypes

which

were

part

of

the

salinity

stress

experiment

conducted

during

the

vegetative

stage

study

-LRB-

Walia

et

al.

-RRB-

The

expression

of

in

genotypes

Pokkali

and

was

analyzed

in

the

root

and

shoot

tissues

for

vegetative

stage

-LRB-

V

-RRB-

and

shoot

tissue

of

and

at

the

PI

stage

-LRB-

R

-RRB-

under

control

and

stressed

conditions

-LRB-

Fig.

-RRB-

The

expression

of

increased

in

response

to

salinity

in

the

roots

of

tolerant

lines

Pokkali

and

but

not

in

salt-sensitive

The

mRNA

accumulation

of

in

shoot

tissue

increased

in

response

to

salinity

in

all

four

genotypes

analyzed

during

the

vegetative

stage

Fig.

of

in

different

indica

genotypes

Semi-quantitative

RT-PCR

was

performed

to

study

the

expression

of

in

the

root

and

shoot

tissue

of

and

under

control

and

stressed

conditions

during

vegetative

-LRB-

V

-RRB-

and

reproductive

-LRB-

R

-RRB-

stages

Expression

was

analyzed

for

roots

and

shoot

tissue

of

tolerant

lines

Pokkali

and

during

vegetative

stage

of

growth

The

curly

bracket

encloses

a

control

and

the

corresponding

salt

stressed

sample

Differential

expression

of

auxin

associated

genes

in

rice

under

salinity

stress

Phytohormones

including

auxin

indole

acid

-LRB-

IAA

-RRB-

play

a

central

role

in

plant

growth

and

development

Auxin

is

also

known

to

mediate

responses

to

light

and

gravity

In

our

expression

analysis

several

auxin-related

genes

were

differentially

regulated

by

salinity

stress

indicating

that

salinity

stress

is

associated

with

auxin

response

in

rice

Almost

all

of

these

auxin

related

genes

were

differentially

expressed

in

sensitive

genotypes

and

Most

striking

was

the

response

of

to

salinity

in

this

context

The

expression

of

cytochrome

-LRB-

-RRB-

was

significantly

-LRB-

P

-RRB-

down-regulated

under

salt

stress

The

expression

level

of

is

known

to

determine

auxin

levels

in

plants

-LRB-

Barlier

et

al.

-RRB-

Other

auxin

related

genes

down-regulated

in

response

to

salinity

include

a

putative

and

two

auxin

response

factors

In

contrast

a

mitogen

activated

protein

kinase

cascade

gene

which

is

known

to

suppress

early

auxin

response

gene

transcription

is

induced

in

response

to

salinity

in

An

increased

level

of

nonphototropic

hypocotyl

-LRB-

-RRB-

transcript

was

observed

in

under

salt

stress

A

rice

ortholog

of

is

involved

in

phototropic

response

of

coleoptiles

through

lateral

auxin

translocation

-LRB-

Haga

et

al.

-RRB-

Another

gene

involved

in

auxin

transport

was

found

to

be

down-regulated

in

Agami

Several

of

the

auxin

related

genes

and

their

response

to

salinity

are

listed

in

Table

Effect

of

salinity

stress

on

floral

transition

and

development

in

rice

The

transition

from

vegetative

to

reproductive

stage

termed

floral

transition

is

a

dramatic

phase

change

in

plant

development

The

transition

controlled

by

a

complex

genetic

network

that

integrates

environmental

signals

such

as

day

length

and

temperature

-LRB-

Simpson

et

al.

-RRB-

We

found

several

rice

genes

which

are

associated

with

floral

transition

and

morphological

development

to

be

responsive

to

salinity

stress

imposed

at

the

PI

stage

Centroradialis

-LRB-

CEN

-RRB-

a

gene

which

plays

a

role

in

phase

transition

and

panicle

morphology

was

induced

in

both

the

sensitive

genotypes

and

in

response

to

salinity

A

zinc-finger

protein

-LRB-

-RRB-

linked

to

phase

transition

signaling

in

maize

was

induced

at

a

higher

level

-LRB-

P

-RRB-

in

compared

to

-LRB-

P

-RRB-

In

contrast

the

flowering

locus

T

-LRB-

FT

-RRB-

-LRB-

-RRB-

gene

was

differentially

regulated

in

-LRB-

down-regulated

-RRB-

compared

to

-LRB-

up-regulated

-RRB-

under

salinity

stress

FT

is

known

to

be

downstream

of

gigantea

which

represses

flowering

in

rice

A

gigantea-like

gene

-LRB-

-RRB-

was

down-regulated

in

both

japonica

genotypes

and

Agami

Several

other

genes

involved

in

phase

transition

and

organ

identity

and

size

determination

were

differentially

regulated

in

and

Agami

but

not

in

On

comparing

the

expression

patterns

of

with

we

found

several

genes

including

gigantea

FT

terminal

flower

-LRB-

-RRB-

and

embryonic

flower

-LRB-

-RRB-

to

be

expressing

at

a

higher

level

in

under

control

conditions

Some

of

these

differentially

regulated

genes

and

their

expression

levels

in

different

genotypes

are

detailed

in

Table

Discussion

In

this

study

we

applied

global

gene

expression

profiling

analysis

to

study

the

transcriptional

responses

of

four

rice

genotypes

comprised

of

tolerant

and

sensitive

indicas

and

japonicas

For

these

genotypes

each

with

well-characterized

salinity

tolerance

from

prior

studies

we

measured

gene

expression

during

PI

the

most

salt-sensitive

stage

of

development

considered

to

be

the

major

factor

in

yield

loss

in

rice

The

results

from

this

study

which

used

a

moderate

agronomically

relevant

salinity

stress

demonstrate

striking

differences

in

expression

responses

between

tolerant

and

sensitive

genotypes

The

genotypes

were

also

characterized

for

their

physiological

responses

to

obtain

insights

which

enabled

us

to

correlate

some

of

the

phenotypic

observations

with

expression

results

Under

salinity

stress

two

key

features

one

physiological

and

the

other

within

the

transcriptomes

clearly

distinguished

the

tolerant

genotypes

from

the

sensitive

genotypes

First

is

the

ability

of

the

tolerant

lines

and

Agami

to

exclude

ion

from

the

shoot

tissue

compared

to

sensitive

lines

The

second

feature

observed

at

the

gene

expression

level

is

the

strikingly

small

number

of

genes

which

responded

to

salinity

stress

in

the

tolerant

genotypes

This

is

likely

a

manifestation

of

the

low

shoot

trait

in

tolerant

lines

which

experience

significantly

lower

toxicity

at

the

cellular

level

and

therefore

requires

fewer

adjustments

of

cell

metabolism

and

architecture

Among

the

genes

which

were

commonly

induced

by

salinity

in

all

genotypes

were

abiotic

stress

response

related

genes

such

as

water

stress

induced

-LRB-

-RRB-

low

temperature

induced

protein

-LRB-

-RRB-

and

a

drought-induced

RNase

S-like

gene

The

encodes

for

galactinol

synthase

-LRB-

Takahashi

et

al.

-RRB-

and

was

responsive

to

low

temperature

and

osmotic

stress

Galactinol

synthase

is

required

for

the

committed

step

in

raffinose

family

oligonucleotides

-LRB-

RFOs

-RRB-

It

was

also

induced

in

the

tolerant

line

when

salinity

was

imposed

at

early

vegetative

stage

but

not

in

-LRB-

Walia

et

al.

-RRB-

Besides

the

genes

involved

in

response

to

dehydration

we

also

detected

at

least

two

genes

involved

in

nitrate

transport

which

may

be

compensating

for

salinity

induced

nutritional

deficiency

A

nitrate

transporter

was

induced

in

Mesembryanthemum

crystallinum

in

leaf

and

root

tissue

under

salt

stress

-LRB-

Popova

et

al.

-RRB-

A

low-affinity

nitrogen

transporter

and

both

localizing

to

chromosome

of

rice

were

induced

in

all

rice

genotypes

in

response

to

salinity

stress

-LRB-

Table

-RRB-

Ion

homeostasis

in

rice

under

salinity

stress

Our

data

indicated

that

the

tolerant

genotypes

and

Agami

maintained

significantly

lower

concentration

in

the

shoot

tissue

compared

to

sensitive

lines

under

salinity

stress

Sodium

ion

concentration

in

the

plant

is

considered

to

be

a

good

indicator

of

the

ion

toxicity

levels

to

which

the

cells

are

exposed

The

lower

ion

specific

stress

in

tolerant

genotypes

is

reflected

in

the

relatively

small

number

of

genes

which

respond

to

salinity

in

tolerant

lines

when

compared

to

sensitive

and

We

found

several

ion

homeostasis

related

genes

such

as

ion

transporters

which

were

responsive

to

salinity

stress

in

the

sensitive

genotypes

One

such

gene

encoding

for

transporter

-LRB-

-RRB-

was

induced

by

salinity

stress

in

This

gene

has

high

sequence

similarity

to

the

The

barley

ortholog

of

was

up-regulated

under

salt

stress

in

the

shoot

tissue

of

a

barley

genotype

Morex

during

early

seedling

stage

-LRB-

Walia

et

al.

submitted

-RRB-

This

barley

genotype

particularly

and

barley

in

general

is

known

to

accumulate

a

high

level

of

in

the

shoot

tissue

during

salinity

stress

Salt

tolerance

in

barley

is

believed

to

be

mainly

achieved

through

efficient

compartmentalization

of

toxic

ions

besides

other

mechanisms

conferring

tissue

tolerance

High

in

the

shoot

tissue

was

also

documented

for

rice

genotype

at

vegetative

and

reproductive

stage

under

salinity

stress

-LRB-

Walia

et

al.

Table

-RRB-

The

expression

of

did

not

change

significantly

in

the

other

three

genotypes

examined

Based

on

these

pieces

of

information

we

can

speculate

that

may

be

involved

in

redistribution

or

compartmentalization

of

within

the

plant

in

genotypes

which

are

relatively

inefficient

in

exclusion

from

the

plant

Another

rice

gene

recently

proposed

to

confer

salt

tolerance

through

recirculation

of

ions

is

-LRB-

Ren

et

al.

-RRB-

was

identified

as

gene

underlying

a

rice

QTL

for

maintaining

high

shoot

K

in

the

shoot

under

salt

stress

-LRB-

Lin

et

al.

-RRB-

The

gene

also

encodes

a

HKT-type

transporter

and

functions

as

a

selective

transporter

in

rice

Rice

locus

previously

annotated

as

a

cation

transporter

corresponds

to

based

on

sequence

similarity

This

locus

has

been

reported

to

be

induced

in

shoot

tissue

by

salt

stress

in

both

the

sensitive

and

tolerant

genotypes

under

salinity

stress

imposed

during

early

vegetative

stage

-LRB-

Walia

et

al.

-RRB-

However

the

induction

in

salt-tolerant

was

significantly

higher

than

in

under

stress

In

contrast

to

these

prior

results

with

vegetative

stage

stress

we

did

not

detect

a

significant

up-regulation

of

in

the

shoot

tissue

of

any

of

the

four

rice

genotypes

at

PI

profiled

under

salt

stress

in

this

study

However

on

making

genotype

genotype

comparisons

between

and

we

found

to

be

expressing

at

a

higher

level

in

Agami

compared

to

under

both

control

and

saline

conditions

Interestingly

the

shoot

ion

analysis

indicated

that

Agami

maintains

lower

concentration

than

under

both

control

and

saline

conditions

-LRB-

Table

-RRB-

The

expression

of

was

reported

to

be

up-regulated

by

salt

stress

in

root

but

not

shoot

tissue

-LRB-

Ren

et

al.

-RRB-

In

contrast

our

results

with

indica

genotypes

at

vegetative

stage

indicated

an

up-regulation

of

by

salt

stress

in

the

shoot

-LRB-

crown

plus

growing

point

tissues

-RRB-

an

observational

discrepancy

that

may

be

attributed

to

the

very

different

methods

of

applying

salt

stress

between

the

two

studies

We

pursued

the

expression

analysis

of

further

by

analyzing

the

shoot

and

root

tissues

of

tolerant

indicas

Pokkali

and

and

sensitive

line

-LRB-

Fig.

-RRB-

Our

results

indicate

that

is

salt

up-regulated

in

the

shoots

of

all

four

indica

genotypes

under

the

conditions

that

we

used

Under

the

more

gradual

and

moderate

stress

conditions

that

we

applied

was

up-regulated

in

the

root

tissues

only

in

salt-tolerant

lines

not

in

salt-sensitive

Auxin

and

response

of

rice

to

salinity

stress

Salinity

modulates

the

expression

of

several

auxin-associated

genes

in

the

sensitive

japonica

genotype

The

expression

level

of

the

gene

encoding

was

down-regulated

in

but

not

in

other

genotypes

A

loss-of-function

mutation

in

resulted

in

accumulation

of

indole

-LRB-

IAOx

-RRB-

which

is

redirected

to

auxin

biosynthesis

-LRB-

Bak

et

al.

-RRB-

Taken

together

it

is

plausible

that

auxin

homeostasis

in

is

disturbed

in

response

to

salinity

by

virtue

of

a

reduction

of

gene

expression

Another

gene

involved

in

auxin

signal

transduction

was

also

down-regulated

in

-LRB-

Hayashi

et

al.

-RRB-

Although

the

mechanisms

of

auxin

signal

transduction

in

plants

are

still

largely

obscure

there

is

considerable

evidence

linking

auxin

perception

and

various

ion

transporters

located

in

plant

cell

membranes

-LRB-

Blatt

and

Thiel

Barbier-Brygoo

-RRB-

Anion

channels

have

been

indicated

to

be

involved

in

auxin

signaling

-LRB-

Thomine

et

al.

-RRB-

More

specifically

auxin

stimulated

Cl

efflux/influx

has

been

of

interest

for

several

decades

-LRB-

Bentrup

et

al.

Rubinstein

Babourina

et

al.

-RRB-

Bentrup

et

al.

-LRB-

-RRB-

reported

that

IAA

significantly

reduced

the

uptake

of

Cl

by

cells

of

Petroselinum

sativum

while

Babourina

et

al.

-LRB-

-RRB-

reported

a

stimulatory

effect

of

auxin

on

Cl

uptake

by

oat

coleoptiles

The

modulation

of

auxin

related

genes

in

during

salt

stress

is

attractive

in

context

of

the

shoot

Cl

concentration

in

The

sensitive

genotype

maintained

a

significantly

lower

Cl

concentration

under

salt

stress

compared

to

other

genotypes

-LRB-

Table

-RRB-

Interestingly

a

CLC-d

chloride

channel

-LRB-

-RRB-

was

induced

in

by

-LRB-

P

-RRB-

in

response

to

salinity

stress

Three

of

the

genes

induced

in

in

response

to

salinity

were

associated

with

auxin

The

chloride

channel

-LRB-

-RRB-

was

induced

in

by

-LRB-

P

-RRB-

Also

induced

was

a

gene

for

an

kinase-like

protein

under

salinity

stress

Constitutive

expression

of

a

tobacco

MAPKK

is

reported

to

dampen

auxin

responsiveness

of

a

reporter

gene

-LRB-

Kovtun

et

al.

-RRB-

and

confer

salt

tolerance

-LRB-

Kovtun

et

al.

-RRB-

Additionally

expression

profiling

of

the

salt-overly

sensitive

-LRB-

-RRB-

mutant

in

comparison

to

wild

type

Arabidopsis

revealed

differential

induction

of

auxin-related

genes

in

the

mutant

under

salt

stress

-LRB-

Kamei

et

al.

-RRB-

These

reports

and

our

data

indicate

a

significant

role

of

auxin

in

response

of

some

rice

genotypes

to

salinity

stress

Salinity

stress

and

floral

transition

In

the

current

work

we

focused

on

shoot

meristem

tissue

under

salinity

stress

imposed

during

PI

We

detected

several

genes

associated

with

developmental

transitions

and

flowering

to

be

modulated

by

salinity

stress

Salinity

delays

PI

in

rice

-LRB-

Zeng

et

al.

-RRB-

and

in

our

study

this

delay

was

generally

greater

for

sensitive

than

tolerant

genotypes

In

this

context

we

found

the

up-regulation

of

rice

genes

with

sequence

match

to

terminal

flower

-LRB-

-RRB-

centroradialis

-LRB-

CEN

-RRB-

in

the

sensitive

genotypes

to

be

interesting

Over

expression

of

homologs

of

rice

delayed

phase

transition

and

altered

panicle

morphology

-LRB-

Nakagawa

et

al.

-RRB-

The

delay

in

initiation

of

panicle

in

sensitive

genotypes

especially

under

salinity

stress

was

longer

than

tolerant

genotypes

The

expression

of

was

not

significantly

changed

in

the

tolerant

lines

under

stress

Both

and

CEN

belong

to

the

family

of

phosphatidyl-ethanolamine-binding

proteins

-LRB-

PEBPs

-RRB-

-LRB-

Bradley

et

al.

-RRB-

Another

gene

which

is

involved

in

flowering

transition

is

FT

which

is

also

a

member

of

the

PEBP

family

The

role

of

FT

is

however

antagonistic

to

as

FT

induces

floral

transition

The

expression

level

of

FT

was

down-regulated

in

as

expected

due

to

a

delayed

floral

transition

under

salinity

stress

Surprisingly

FT

was

up-regulated

in

Up-regulation

of

FT

in

was

also

observed

during

the

vegetative

stage

-LRB-

Walia

et

al.

-RRB-

The

expression

of

FT

was

not

modulated

by

salinity

stress

in

tolerant

lines

and

Agami

during

PI

and

in

during

vegetative

stage

An

up-stream

member

of

the

floral

induction

pathway

to

which

FT

belongs

is

gigantea

-LRB-

GI

-RRB-

Suppressed

expression

of

the

rice

ortholog

of

gigantea

-LRB-

OsGI

-RRB-

through

RNA-mediated

interference

-LRB-

RNAi

-RRB-

resulted

in

a

late

flowering

phenotype

under

short

day

conditions

relative

to

wild-type

plants

-LRB-

Hayama

et

al.

-RRB-

The

expression

level

of

gigantea-like

gene

was

significantly

reduced

in

and

Agami

under

salinity

stress

-LRB-

Table

-RRB-

of

these

genes

were

differentially

modulated

in

tolerant

indica

In

brief

the

gene

expression

analysis

identified

genes

which

explain

the

observed

delay

in

PI

of

under

salinity

stress

High

salt

tolerance

has

often

been

linked

to

genotypes

which

take

relatively

long

to

reach

maturity

The

genotypes

selected

for

this

experiment

also

differ

in

their

time

to

reach

maturity

Therefore

it

can

be

argued

that

the

late

maturing

genotypes

and

Agami

are

likely

to

be

more

salt

tolerant

merely

as

a

consequence

of

higher

biomass

translating

into

higher

yield

However

Zeng

et

al.

-LRB-

-RRB-

reported

a

lack

of

correlation

between

the

duration

of

reproductive

growth

period

and

salt

tolerance

based

on

seed

yield

This

study

also

included

three

of

the

four

genotypes

used

in

the

current

study

namely

Agami

and

and

employed

a

comparable

level

of

salinity

stress

Since

difference

in

time

to

maturity

for

different

genotypes

can

potentially

lead

to

confounding

effects

on

gene

expression

measured

by

microarrays

we

decided

to

focus

on

the

developmental

stage

just

after

PI

was

observed

for

each

treatment

regardless

of

the

growth

conditions

-LRB-

control

or

stressed

-RRB-

and

genotype

This

involved

staggered

harvesting

of

control

and

stressed

plants

for

each

of

the

genotype

hence

normalizing

for

delay

in

PI

resulting

from

salt

stress

The

effect

of

difference

in

development

among

genotypes

used

for

transcriptome

analysis

can

be

avoided

by

using

nearly

isogenic

lines

which

differ

in

salinity

tolerance

at

PI

but

have

similar

developmental

behavior

However

such

optimal

lines

focusing

on

tolerance

at

PI

are

presently

not

available

Commonalties

in

the

salinity

stress

response

of

at

early

vegetative

versus

reproductive

stage

Sensitive

genotype

responded

to

salinity

stress

during

PI

by

inducing

a

large

number

of

genes

Up-regulation

of

large

number

of

genes

was

also

reported

for

under

salinity

stress

during

early

vegetative

stage

relative

to

tolerant

genotype

-LRB-

Walia

et

al.

-RRB-

We

explored

the

extent

of

commonalities

in

salinity

induced

responses

of

by

comparing

the

induced

genes

during

vegetative

and

reproductive

stage

One

of

the

most

striking

stress

responses

of

during

the

vegetative

stage

experiment

was

the

induction

of

genes

involved

in

the

flavonoid

pathway

In

the

present

study

we

again

observed

that

several

of

the

flavonoid

pathway

related

genes

were

induced

in

under

salt

stress

in

this

case

at

PI

Among

the

genes

induced

include

those

encoding

for

phenylalanine

ammonia

lyase

chalcone

synthase

dihydroflavonol

and

flavonone

Therefore

up-regulation

of

the

flavonoid

pathway

as

a

response

to

salinity

stress

appears

to

be

a

general

characteristic

of

Interestingly

some

of

the

genes

involved

in

the

flavonoid

pathway

were

down-regulated

in

the

sensitive

japonica

So

the

induction

of

flavonoid

pathway

in

response

to

salinity

stress

is

not

a

universal

response

of

sensitive

rice

genotypes

We

further

compared

the

data

from

the

vegetative

stage

study

with

the

current

PI

data

In

general

we

found

that

the

genes

responding

to

salinity

stress

at

vegetative

stage

were

strikingly

different

from

those

which

responded

when

the

stress

was

imposed

during

PI

A

total

of

probe

sets

were

differentially

expressed

in

response

to

salinity

at

reproductive

stage

but

not

during

vegetative

stage

We

were

especially

interested

in

genes

which

mapped

to

regions

of

the

rice

genome

associated

with

salt

tolerance

based

on

QTL

studies

One

such

gene

-LRB-

-RRB-

encoding

for

SALT

protein

was

induced

during

the

reproductive

stage

This

gene

has

a

jacalin-like

lectin

domain

and

physically

maps

to

the

Saltol

region

on

chromosome

-LRB-

Mb

-RRB-

Interestingly

another

gene

-LRB-

-RRB-

also

encoding

a

SALT-related

protein

was

induced

during

the

vegetative

stage

in

-LRB-

Walia

et

al.

-RRB-

This

gene

also

maps

to

the

Saltol

region

-LRB-

Mb

-RRB-

on

chromosome

The

salT

gene

in

rice

is

responsive

to

salt

stress

and

the

phytohormones

abscisic

acid

and

jasmonic

acid

-LRB-

JA

-RRB-

-LRB-

Claes

et

al.

Moons

et

al.

-RRB-

We

found

some

genes

involved

in

JA

biosynthesis

and

JA

responsive

genes

to

be

induced

by

salinity

stress

in

during

reproductive

stage

but

not

during

vegetative

stage

These

include

two

lipoxygenases

-LRB-

and

-RRB-

a

gene

encoding

for

acid

reductase

-LRB-

-RRB-

-LRB-

-RRB-

and

a

jasmonate

induced

protein

-LRB-

JIP

-RRB-

The

induction

of

JA

biosynthesis

and

responsive

genes

during

salinity

stress

is

interesting

in

context

of

the

report

that

post-treatment

of

salt-stressed

rice

with

JA

dramatically

improves

the

physiological

activities

otherwise

inhibited

by

stress

-LRB-

Kang

et

al.

-RRB-

Results

from

our

laboratory

have

also

indicated

an

ameliorating

role

of

JA

pretreatment

in

salt-stressed

barley

on

shoot

concentrations

and

photosynthetic

performance

-LRB-

Walia

et

al.

-RRB-

Besides

salT

we

found

several

other

key

transcripts

related

to

salinity

stress

response

as

differentially

expressed

during

reproductive

stage

A

potassium

uptake

protein

-LRB-

-RRB-

induced

by

salinity

stress

during

the

reproductive

stage

in

was

one

such

transcript

This

gene

localizes

to

a

region

on

chromosome

-LRB-

Mb

-RRB-

which

is

associated

with

a

major

QTL

for

K

uptake

in

rice

identified

by

Koyama

et

al.

-LRB-

-RRB-

This

region

on

chromosome

was

more

recently

linked

to

a

QTL

controlling

root

concentration

-LRB-

Lin

et

al.

-RRB-

Another

gene

-LRB-

Os

-RRB-

encoding

for

a

salt-tolerance

zinc

finger

protein

-LRB-

-RRB-

was

up-regulated

during

reproductive

stage

under

salinity

stress

The

is

a

transcriptional

repressor

which

is

believed

to

regulate

the

COR/RD

gene

expression

-LRB-

Lee

et

al.

-RRB-

The

expression

of

was

not

up-regulated

in

any

other

genotype

under

salt

stress

Based

on

this

comparison

we

can

state

that

very

few

genes

are

commonly

induced

in

response

to

salinity

stress

in

a

given

genotype

when

sampled

at

two

different

developmental

stages

Electronic

supplementary

materials

Below

are

the

links

to

the

electronic

supplementary

materials

ESM

-LRB-

XLS

kb

-RRB-

ESM

-LRB-

XLS

kb

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ESM

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XLS

kb

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ESM

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kb

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XLS

kb

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ESM

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XLS

kb

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kb

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ESM

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XLS

kb

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ESM

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XLS

kb

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ESM

-LRB-

XLS

kb

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ESM

-LRB-

XLS

kb

-RRB-

ESM

-LRB-

XLS

kb

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rice
salt stress
panicle initiation
microarray