Global

genome

expression

analysis

of

rice

in

response

to

drought

and

high-salinity

stresses

in

shoot

flag

leaf

and

panicle

To

elucidate

genome-level

responses

to

drought

and

high-salinity

stress

in

rice

a

oligomer

microarray

covering

unique

genes

or

gene

models

was

used

to

profile

genome

expression

changes

in

rice

shoot

flag

leaf

and

panicle

under

drought

or

high-salinity

conditions

While

patterns

of

gene

expression

in

response

to

drought

or

high-salinity

stress

within

a

particular

organ

type

showed

significant

overlap

comparison

of

expression

profiles

among

different

organs

showed

largely

organ-specific

patterns

of

regulation

Moreover

both

stresses

appear

to

alter

the

expression

patterns

of

a

significant

number

of

genes

involved

in

transcription

and

cell

signaling

in

a

largely

organ-specific

manner

The

promoter

regions

of

genes

induced

by

both

stresses

or

induced

by

one

stress

in

more

than

one

organ

types

possess

relative

enrichment

of

two

cis-elements

-LRB-

ABRE

core

and

DRE

core

-RRB-

known

to

be

associated

with

water

stress

An

initial

computational

analysis

indicated

that

novel

promoter

motifs

are

present

in

the

promoters

of

genes

involved

in

rehydration

after

drought

This

analysis

suggested

that

rice

might

possess

a

mechanism

that

actively

detects

rehydration

and

facilitates

rapid

recovery

Overall

our

data

supports

a

notion

that

organ-specific

gene

regulation

in

response

to

the

two

abiotic

stresses

may

primarily

be

mediated

by

organ-specific

transcription

responses

Introduction

Drought

and

highly

saline

soils

are

among

the

most

serious

challenges

to

crop

production

in

the

world

today

This

is

particularly

the

case

in

developing

countries

where

these

abiotic

stresses

severely

limit

crop

growth

and

productivity

Both

traditional

breeding

and

genetic

engineering

of

crop

plants

have

been

utilized

to

improve

drought

and

high-salinity

tolerance

or

resistance

with

the

goal

of

increasing

agricultural

productivity

in

affected

regions

Understanding

plant

responses

to

abiotic

stresses

at

the

genomic

level

provides

an

essential

foundation

for

future

breeding

and

genetic

engineering

efforts

Recent

research

on

drought

and

high-salinity

responses

in

Arabidopsis

implied

that

a

large

proportion

of

the

genome

is

involved

in

drought

-LRB-

Shinozaki

et

al.

-RRB-

or

high-salinity

stress

responses

-LRB-

Xiong

et

al.

Zhu

-RRB-

In

several

cases

it

has

been

shown

that

alteration

of

individual

gene

expression

level

can

significantly

impact

responses

to

drought

-LRB-

Garg

et

al.

Haake

et

al.

-RRB-

or

high-salinity

stresses

in

plants

-LRB-

Kasuga

et

al.

Shi

et

al.

Xu

et

al.

Zhang

et

al.

-RRB-

Genome-wide

identification

of

genes

regulated

by

drought

or

high-salinity

conditions

has

manifold

significance

First

it

provides

a

more

comprehensive

understanding

of

the

transcriptional

responses

to

those

stresses

Second

it

provides

a

starting

point

for

further

elucidating

the

role

of

individual

genes

in

stress

responses

which

will

be

of

great

value

in

crop

engineering

Third

it

aids

in

the

identification

of

stress

responsive

promoters

and

responsible

cis-elements

within

them

that

are

important

both

for

basic

study

and

crop

engineering

applications

DNA

microarrays

provide

a

high

throughput

means

of

analyzing

genome

expression

which

has

been

used

to

study

patterns

of

gene

expression

in

response

to

drought

or

high-salinity

stresses

in

several

plant

species

-LRB-

Seki

et

al.

-RRB-

Initially

a

microarray

containing

full-length

cDNA

clones

from

Arabidopsis

was

used

to

study

gene

expression

under

drought

and

cold

stresses

This

study

resulted

in

the

identification

of

and

cDNA

clones

as

drought

and

cold-inducible

genes

respectively

-LRB-

Seki

et

al.

-RRB-

Other

studies

employed

an

improved

microarray

containing

around

Arabidopsis

full-length

cDNA

clones

to

profile

gene

expression

in

response

to

abscisic

acid

-LRB-

ABA

-RRB-

treatment

-LRB-

Seki

et

al.

-RRB-

as

well

as

cold

drought

and

high-salinity

stresses

-LRB-

Seki

et

al.

-RRB-

Another

study

employed

an

Affymetrix

GeneChip

covering

approximately

genes

from

Arabidopsis

to

monitor

changes

in

gene

expression

under

salt

osmotic

and

cold

stresses

This

study

revealed

that

resulting

expression

changes

varied

significantly

between

root

and

leaf

with

only

minor

overlap

-LRB-

Kreps

et

al.

-RRB-

Similar

studies

have

also

been

performed

in

barley

to

assess

the

drought

and

high-salinity

gene

expression

responses

using

a

microarray

containing

DNA

elements

-LRB-

Ozturk

et

al.

-RRB-

Rice

-LRB-

Oryza

sativa

-RRB-

is

a

model

plant

for

cereal

crops

and

has

perhaps

the

richest

set

of

resources

available

for

plant

genomic

studies

-LRB-

Feng

et

al.

Goff

et

al.

RCSC

Sasaki

et

al.

Yu

et

al.

-RRB-

In

rice

the

high-salinity

stress

response

has

been

analyzed

with

a

microarray

containing

cDNA

clones

from

a

root

cDNA

library

of

salt-tolerant

rice

-LRB-

var

Pokkali

-RRB-

-LRB-

Kawasaki

et

al.

-RRB-

Another

study

with

a

microarray

containing

DNA

elements

detected

possible

abscisic

acid

-LRB-

ABA

-RRB-

or

gibberellin-responsive

genes

-LRB-

Yazaki

et

al.

-RRB-

Later

the

same

group

used

a

rice

cDNA

based

oligonucleotide

array

to

identify

ABA

and

GA

responsive

genes

made

comparison

of

ABA-responsive

genes

and

their

putative

responsible

promoter

elements

between

rice

and

Arabidopsis

-LRB-

Yazaki

et

al.

-RRB-

This

analysis

gave

an

initial

global

view

of

the

ABA

and

GA-responsive

genes

in

rice

A

total

of

genes

induced

by

cold

drought

high-salinity

and

ABA

treatment

were

further

identified

using

a

microarray

containing

full-length

cDNA

clones

-LRB-

Rabbani

et

al.

-RRB-

Recently

an

Affymetrix

rice

genome

array

containing

probe

sets

was

used

to

profile

the

transcriptomes

of

rice

strains

with

salt-tolerant

and

salt-sensitive

genotypes

revealing

genome-wide

differential

transcription

under

salinity

treatments

during

vegetative

growth

-LRB-

Walia

et

al.

-RRB-

Taken

together

previous

studies

in

rice

have

identified

various

genes

regulated

by

different

stress

conditions

However

a

systematic

comparison

of

whole-genome

expression

responses

to

drought

and

high-salinity

stresses

in

various

organs

has

not

yet

been

performed

The

availability

of

the

complete

genome

sequences

of

two

rice

sub-species

-LRB-

Yu

et

al.

-RRB-

makes

the

construction

of

a

whole-genome

microarray

possible

A

whole-genome

oligomer

microarray

for

rice

was

developed

and

successfully

employed

to

obtain

genome

expression

profiles

-LRB-

Ma

et

al.

Jiao

et

al.

-RRB-

Here

we

use

this

whole-genome

microarray

to

monitor

expression

changes

for

a

total

of

genes

in

response

to

drought

and

high-salinity

stresses

in

shoots

at

the

four-tiller

stage

and

in

flag

leaves

and

panicles

at

week

prior

to

heading

This

analysis

revealed

the

extent

of

reprograming

and

alteration

of

cellular

pathways

in

response

to

drought

and

high

salinity

stresses

The

possible

underlying

mechanism

for

the

observed

reprograming

of

the

genome

expression

was

examined

Materials

and

methods

Plant

material

and

stress

treatments

Plant

material

used

in

this

study

was

O.

Sativa

L.

ssp

indica

-LRB-

cv

Minghui

-RRB-

Shoot

samples

were

selected

at

the

four-tiller

stage

-LRB-

vegetative

stage

-RRB-

and

flag

leaf

and

panicle

samples

were

selected

at

one-week-before-heading

-LRB-

reproductive

stage

-RRB-

The

plants

used

for

shoot

samples

were

grown

in

hydropolic

half-strength

Hoagland

solution

up

to

the

four-tiller

stage

and

then

plants

allowed

to

reach

the

reproductive

stages

were

grown

in

soil

For

drought

treatment

samples

plants

were

taken

out

from

the

Hoagland

solution

directly

and

their

roots

were

put

on

filter

paper

for

drought

treatment

Under

this

condition

the

phenotype

of

shoot

was

observed

Materials

at

the

following

stages

were

collected

Stage

-LRB-

-RRB-

when

leaves

were

slightly

rolled

and

leaf

relative

water

content

-LRB-

RWC

-RRB-

was

around

Stage

-LRB-

-RRB-

when

leaves

were

half-rolled

and

leaf

RWC

was

around

Stage

-LRB-

-RRB-

when

leaves

were

completely

rolled

and

leaf

RWC

was

around

For

most

of

the

plants

usually

it

took

half

to

h

to

reach

stage

and

then

after

another

h

or

h

they

could

reach

stage

or

stage

respectively

Rehydration

samples

were

prepared

by

first

subjecting

the

rice

plants

to

drought

treatment

until

leaves

were

completely

rolled

-LRB-

-RRB-

then

supplying

enough

water

and

samples

were

collected

after

h.

For

each

treatment

stage

three

independent

replicates

were

collected

For

sample

collection

under

high-salinity

treatment

-LRB-

mM

NaCl

-RRB-

rice

plants

were

collected

when

plants

reached

stages

or

as

outlined

above

for

drought

treatment

Rice

plants

were

taken

out

from

Hoagland

solution

and

were

directly

put

into

Hoagland

solution

with

mM

NaCl

For

most

of

plants

usually

it

took

h

to

reach

stage

and

then

after

another

and

h

they

could

reach

stage

and

stage

respectively

For

each

stage

three

independent

replicates

were

collected

Samples

from

normal

grown

plants

corresponding

to

each

drought

and

high-salinity

stages

and

rehydration

stage

were

also

collected

at

the

same

time

as

respective

controls

Upon

collection

samples

were

frozen

immediately

in

liquid

nitrogen

and

stored

in

the

°C

freezer

The

estimation

of

RWC

in

rice

plants

was

prior

reported

method

-LRB-

Bare

and

Weatherley

-RRB-

RNA

isolation

probe

labeling

and

hybridization

RNA

preparation

fluorescent

labeling

of

probes

slide

hybridization

washing

and

scanning

were

performed

as

described

previously

-LRB-

Ma

et

al.

-RRB-

Total

RNA

was

prepared

from

frozen

samples

using

the

RNAwiz

reagent

-LRB-

Ambion

-RRB-

Each

RNA

preparation

was

used

to

generate

a

labeled

cDNA

probe

for

hybridization

There

were

at

least

three

high-quality

replicate

data

sets

for

each

experiment

with

each

data

set

obtained

from

an

independent

biological

sample

The

cDNA

synthesis

probe

labeling

and

microarray

hybridization

microarray

slide

washing

and

array

scanning

were

performed

following

previously

described

protocols

-LRB-

Jiao

et

al.

-RRB-

Hybridized

microarray

slides

were

scanned

with

an

Axon

GenePix

scanner

and

independent

TIFF

images

for

both

and

channels

were

used

for

subsequent

analysis

Microarray

and

initial

data

analysis

The

microarray

construction

was

described

previously

-LRB-

Ma

et

al.

Jiao

et

al.

-RRB-

Microarray

data

were

preprocessed

with

Limma

R

Package

-LRB-

Smyth

-RRB-

We

first

performed

Loess

normalization

to

balance

the

dye-bias

and

to

remove

the

print-tip

effect

within

each

array

Quantile

normalization

was

performed

to

remove

the

experimental

variances

across

replicate

arrays

Based

on

the

distribution

of

negative

control

oligos

within

each

slide

we

determined

an

experimental

threshold

for

minimum

non-specific

hybridization

intensity

and

the

gene

oligos

above

of

negative

oligos

were

set

as

to

be

detected

in

each

experiment

To

identify

the

genes

that

respond

to

drought

and

salinity

stress

we

used

the

Limma

R

implementation

to

perform

hypothesis

tests

by

fitting

a

linear

model

to

the

expression

data

During

pair-wise

comparison

between

each

treated

sample

versus

control

a

moderated

t-statistics

were

computed

by

using

an

empirical

Bayes

method

to

shrink

the

gene-wise

sample

variances

towards

a

common

value

-LRB-

Smyth

-RRB-

and

the

differential

level

was

represented

by

a

log

-LRB-

-RRB-

of

intensity

ratio

The

P

value

adjustment

used

in

false

discovery

rate

control

for

multiple

testing

is

Benjamini

and

Hochberg

method

-LRB-

Benjamini

and

Hochberg

Reiner

et

al.

-RRB-

The

genes

with

significantly

differential

expression

were

selected

by

the

adjusted

P

values

less

than

RT-PCR

analysis

of

genes

The

expression

profiles

were

further

quantified

by

RT-PCR

and

compared

to

results

obtained

by

chip

hybridization

The

first

strand

of

cDNA

was

generated

from

μg

of

total

RNA

isolated

independently

from

each

sample

in

a

μl

volume

and

μl

was

used

as

template

in

each

PCR

reaction

-LRB-

cycles

of

min

at

°C

min

at

°C

min

at

°C

-RRB-

A

total

of

eight

drought-induced

genes

were

selected

for

RT-PCR

analysis

-LRB-

the

primers

of

these

genes

are

listed

in

Table

-RRB-

The

gene

of

rice

was

used

as

a

control

for

RT-PCR

experiments

-LRB-

forward

primer

reverse

primer

-RRB-

Functional

classification

GO

terms

used

in

rice

gene

functional

annotations

were

downloaded

from

the

BGI-RIS

database

at

-LRB-

Zhao

et

al.

-RRB-

For

biochemical

pathway

analysis

we

classified

genes

by

associated

biochemical

pathway

-LRB-

s

-RRB-

using

the

AraCyc

database

-LRB-

-RRB-

for

Arabidopsis

which

is

based

on

MetaCyc

pathway

collections

-LRB-

Mueller

et

al.

-RRB-

A

rice

gene

was

considered

to

be

associated

with

a

biochemical

pathway

if

it

had

an

Arabidopsis

homolog

in

that

pathway

To

compare

the

expression

profiles

of

conserved

genes

between

rice

and

Arabidopsis

we

selected

reciprocal

best-matched

gene

pairs

between

rice

and

Arabidopsis

which

are

based

on

a

TBLASTN

search

of

both

rice

and

Arabidopsis

gene

sequences

This

method

used

to

identify

highly

homologous

genes

between

rice

and

Arabidopsis

has

been

described

in

previous

reports

-LRB-

Jiao

et

al.

Ma

et

al.

-RRB-

For

the

diagrams

in

Fig

the

expression

ratios

of

the

selected

rice

genes

at

and

stage

only

were

used

Analysis

of

cis-acting

regulatory

elements

Differentially

expressed

genes

supported

by

available

full-length

cDNA

sequences

were

used

to

further

elucidate

regulatory

cis-elements

in

the

putative

promoter

regions

After

mapping

the

FL-cDNAs

on

indica

rice

pseudo-molecules

kb

DNA

sequences

upstream

from

the

end

of

the

available

cDNA

sequences

were

extracted

and

searched

against

a

plant

cis-regulatory

database

-LRB-

Higo

et

al.

-RRB-

-LRB-

-RRB-

The

abundance

of

known

ABRE

core

and

DRE

core

elements

in

each

query

set

were

counted

Identification

of

novel

cis-regulatory

elements

was

performed

using

the

Improbizer

tool

-LRB-

cis-Site

Seeker

-RRB-

by

Jim

Kent

-LRB-

-RRB-

which

is

based

on

ab-initio

prediction

of

consensus

binding

sites

among

each

co-regulated

gene

set

The

upstream

kb

regions

of

co-regulated

FL-cDNA

genes

were

used

for

common

motif

searching

To

evaluate

the

significance

for

each

putative

novel

motif

we

generated

an

equal

number

of

kb

contrast

sequences

in

each

query

set

by

randomly

arranging

the

four

nucleotides

We

performed

the

standard

Student

's

t-test

on

motif

score

tables

comparing

query

set

versus

contrast

set

The

known

ABRE

motif

predicted

by

Improbizer

has

a

pretty

high

confidence

level

-LRB-

above

-RRB-

and

the

other

two

novel

motifs

were

calculated

to

above

a

confidence

level

Motif

consensus

sequences

were

displayed

by

WEBLOGO

program

at

by

Crooks

et

al.

-LRB-

-RRB-

Gel

shift

assays

Nuclear

protein

extracts

and

gel

mobility

shifts

were

performed

as

described

previously

-LRB-

Gupta

et

al.

-RRB-

Oligomers

were

synthesized

using

the

following

sequences

GCAGATACTGCAGCCAACCTCTCT

GCAGATACTGAAGCCAACCTCTCT

The

complement

sequence

for

each

oligo

was

also

synthesized

After

denaturing

and

annealing

double-stranded

probes

were

used

for

labeling

Clustering

and

chromosomal

location

analysis

of

genes

involved

in

stress

responses

Unsupervised

classification

of

stress-response

genes

was

based

on

log-transformed

ratios

of

all

up

or

down-regulated

genes

detected

in

at

least

one

stage

of

drought

treatment

We

performed

hierarchical

clustering

analysis

to

display

the

expression

pattern

and

tree

diagram

at

different

stress

stages

by

employing

Cluster

and

TreeView

-LRB-

Eisen

et

al.

-RRB-

Classification

of

rehydration-induced

genes

was

based

on

the

log-transformed

ratios

of

rehydration

and

three

stages

of

drought

treatment

in

the

three

organs

and

genes

in

flag

leaf

genes

in

shoot

and

genes

in

panicle

were

grouped

with

the

above

method

To

analyze

the

genome-wide

distribution

of

rehydration-induced

genes

in

the

three

organs

and

drought

and

high-salinity

induced

genes

in

shoot

as

well

as

identify

potential

clusters

of

co-regulated

genes

we

mapped

these

genes

to

the

indica

chromosomes

using

the

BLAT

program

The

expression

profiles

in

Fig.

were

drawn

using

a

customized

Perl

script

plus

a

scalable

vector

graphics

-LRB-

SVG

-RRB-

bundle

Results

Identification

of

drought

and

high-salinity

stress

responsive

genes

using

a

rice

whole

genome

oligomer

microarray

A

previously

reported

rice

genome

microarray

containing

oligomer

probes

for

unique

genes

or

gene

models

-LRB-

Ma

et

al.

Jiao

et

al.

-RRB-

was

used

for

genome-wide

profiling

of

gene

expression

responses

to

drought

and

high-salinity

stresses

Total

RNAs

were

isolated

from

four-tiller

stage

shoots

and

from

flag

leaves

and

panicles

-LRB-

as

described

in

Experimental

procedures

-RRB-

at

three

different

time

points

-LRB-

or

sampling

stages

-RRB-

after

initiation

of

drought

or

high-salinity

stress

treatments

-LRB-

Fig

-RRB-

The

sampling

stages

for

both

drought

and

high-salinity

treatments

were

based

on

morphological

phenotypes

-LRB-

leaf

rolling

state

-RRB-

and

physiological

states

-LRB-

relative

water

content

RWC

see

Bare

and

Weatherley

-RRB-

Stage

one

is

defined

as

exhibiting

slightly

rolled

leaves

with

RWC

Stage

two

is

defined

as

exhibiting

halfway

rolled

leaves

with

RWC

Stage

three

is

defined

as

exhibiting

completely

rolled

leaves

and

with

RWC

For

drought

treatment

the

three

stages

were

designated

as

and

while

the

three

stages

for

the

high-salinity

treatment

were

designated

as

and

-LRB-

see

Fig

-RRB-

The

cDNA

probes

produced

from

the

stress-treated

and

corresponding

untreated

control

sample

pairs

were

hybridized

to

microarray

slides

For

each

sample

point

three

replicates

were

performed

with

dye-swap

to

correct

for

uneven

dye

effects

The

microarray

data

processing

includes

LOESS

normalization

within

slide

and

quantile

normalization

between

slides

to

remove

the

systematic

errors

-LRB-

see

Experimental

procedures

for

details

-RRB-

To

identify

the

differentially

expressed

genes

Limma

R

implementation

was

used

to

perform

pair-wise

comparison

and

a

moderated

t-statistics

was

computed

for

each

gene

of

treated

sample

versus

control

sample

Genes

with

FDR-adjusted

P

value

less

than

were

considered

to

indicate

significant

differences

-LRB-

see

Experimental

procedures

for

details

-RRB-

Numbers

of

genes

up

or

down-regulated

by

drought

or

by

high-salinity

stress

at

each

stage

in

each

organ

were

shown

in

Fig.

In

total

and

drought

up-regulated

genes

and

and

drought

down-regulated

genes

were

identified

in

flag

leaf

shoot

and

panicle

respectively

and

and

high-salinity

up-regulated

genes

and

and

high-salinity

down-regulated

genes

were

identified

in

flag

leaf

shoot

and

panicle

respectively

-LRB-

Fig.

B

-RRB-

The

complete

lists

of

these

genes

are

available

in

the

supplemental

data

-LRB-

Tables

-RRB-

Fig.

number

of

differentially

expressed

genes

at

each

stage

of

drought

and

high-salinity

treatments

in

three

organs

Differentially

expressed

genes

-LRB-

both

induced

or

repressed

-RRB-

are

defined

as

those

with

P

and

three

stages

of

drought

stress

and

three

stages

of

high-salinity

stress

y-axis

shows

the

gene

number

-LRB-

A

-RRB-

Number

of

genes

induced

or

repressed

in

flag

leaf

-LRB-

B

-RRB-

Number

of

genes

induced

or

repressed

in

panicle

-LRB-

C

-RRB-

Number

of

genes

induced

or

repressed

in

shoot.Fig

of

drought

and

high-salinity

genome

expression

responses

in

the

three

rice

organs

-LRB-

A

-RRB-

Total

number

of

drought

and

high-salinity

stress

inducible

genes

and

the

number

of

genes

induced

in

response

to

both

stresses

in

each

of

the

three

rice

organs

Genes

induced

at

least

at

one

stage

under

drought

or

high-salinity

treatments

are

included

in

the

analysis

y-axis

shows

the

gene

number

-LRB-

B

-RRB-

The

total

number

of

drought

and

high-salinity

stress

repressed

genes

and

the

number

of

genes

repressed

in

response

to

both

stresses

in

each

of

the

three

rice

organs

Genes

repressed

at

least

at

one

stage

under

drought

or

high-salinity

treatments

are

included

in

the

analysis

y-axis

shows

the

gene

number

-LRB-

C

-RRB-

Clustering

analysis

of

all

drought

and

high

salinity

responsive

genes

in

each

of

the

three

rice

organs

The

differentially

expressed

genes

-LRB-

both

induced

or

repressed

-RRB-

with

P

at

least

at

one

stage

of

drought

treatment

are

included

The

median

ratio

-LRB-

treated/untreated

sample

-RRB-

is

transformed

and

subject

to

complete

linkage

hierarchical

clustering

and

three

stages

of

drought

treatment

water

recovery

after

drought

and

three

stages

of

high-salinity

treatment

In

total

and

genes

from

flag

leaf

shoot

and

panicle

respectively

were

included

in

this

analysis

-LRB-

D

-RRB-

Relatedness

of

the

genome

expression

patterns

across

selected

stress-treated

rice

organs

A

complete-linkage

hierarchical

clustering

analysis

of

overall

relatedness

for

expression

ratios

from

selected

organs

at

the

stage

of

both

abiotic

stress

treatments

The

abbreviations

for

different

sample

types

are

the

same

as

in

panel

C

Most

of

the

previously

known

genes

responsive

to

both

drought

and

high-salinity

stress

have

been

recovered

from

our

microarray

analysis

Those

include

the

LEA

protein

-LRB-

-RRB-

aquaporin

-LRB-

-RRB-

-LRB-

-RRB-

dehydrin

rab

-LRB-

-RRB-

and

-LRB-

-RRB-

Many

other

responsive

genes

have

been

identified

for

the

first

time

in

this

study

-LRB-

Table

-RRB-

Among

those

newly

revealed

genes

some

are

expected

to

be

involved

in

general

cell

function

or

known

to

be

involved

in

other

stress

responses

while

others

have

putative

or

unknown

function

The

annotation

for

some

of

those

genes

suggested

that

they

include

transcription

factors

from

multiple

families

heat

shock

proteins

various

stress

-LRB-

drought

high-salinity

disease

cold

and

ABA

-RRB-

responsive

genes

protein

kinases

transporters

photosynthesis

enzymes

and

other

metabolic

pathways

-LRB-

Table

-RRB-

The

diversity

of

affected

processes

suggests

a

high

level

of

complexity

in

regulation

There

is

a

significant

overlap

between

genome

expression

profiles

in

response

to

drought

and

high-salinity

stress

To

compare

the

gene

expression

profiles

of

the

drought

and

high-salinity

stress

responses

genes

commonly

regulated

by

both

stresses

at

each

organ

type

were

examined

A

total

of

and

genes

were

up-regulated

and

and

genes

were

down-regulated

by

both

stresses

in

flag

leaves

shoots

and

panicles

respectively

-LRB-

Fig.

B

-RRB-

The

common

genes

represent

and

-LRB-

induced

-RRB-

and

and

-LRB-

repressed

-RRB-

of

all

drought-responsive

genes

in

flag

leaf

shoot

and

panicle

respectively

These

results

indicate

that

about

one-third

even

half

in

flag

leaf

of

the

drought

responsive

genes

in

each

organ

are

also

regulated

by

high

salinity

stress

To

further

compare

gene

expression

under

the

two

abiotic

stresses

all

drought

regulated

genes

were

selected

for

cluster

analysis

-LRB-

see

Experimental

procedures

-RRB-

As

shown

in

Fig.

up

to

half

of

the

genes

up

or

down-regulated

by

drought

stress

also

exhibit

a

similar

expression

pattern

under

high-salinity

stress

The

remainder

of

the

drought

responsive

genes

exhibits

minimal

expression

changes

or

distinct

expression

patterns

in

response

to

high-salinity

stress

Among

genes

specifically

induced

by

drought

stress

some

have

known

functions

while

others

were

previously

predicted

to

have

functions

related

to

drought

stress

or

the

ABA

response

For

example

our

experiments

showed

that

genes

with

putative

functions

including

NAM

HLH

G-box

binding

Zn-finger

transcription

factors

and

protein

kinases

-LRB-

including

MAPK

family

genes

-RRB-

were

all

affected

by

drought

stress

Quite

a

few

known

drought-responsive

genes

such

as

LEA

protein

protein

-LRB-

microtubule

associated

protein

-RRB-

and

ubiquitin

were

also

included

These

genes

were

specifically

induced

by

drought

and

may

function

exclusively

in

the

response

to

drought

stress

in

rice

To

gain

an

overall

view

of

the

effects

of

drought

and

high-salinity

stress

on

various

functional

gene

groups

gene

ontology

-LRB-

GO

-RRB-

categories

for

drought

and

high-salinity

stress

induced

genes

in

three

organs

were

examined

-LRB-

Fig

-RRB-

For

the

majority

of

these

functional

categories

the

relative

numbers

of

genes

responsive

to

the

two

stresses

were

similar

We

also

examined

the

effects

of

drought

and

high-salinity

on

rice

genes

coding

enzymes

in

known

metabolic

pathways

using

shoot

as

a

model

-LRB-

Fig

-RRB-

Some

pathways

are

similarly

regulated

by

the

two

stresses

including

the

Calvin

cycle

TCA

cycle

variation

I

brassinosteroid

biosynthesis

II

gibberellin

biosynthesis

and

IAA

biosynthesis

I

and

the

de

novo

biosynthesis

of

pyrimidine

ribnucleotides/pyrimidine

deoxyribonucleotides/purine

nucleotides

-LRB-

Fig

and

data

not

shown

-RRB-

While

other

pathways

respond

differentially

to

drought

or

high-salinity

stresses

the

representative

pathways

include

sterol

biosynthesis

sugars

and

polysaccharides

ABA

biosynthesis

lignin

biosynthesis

octane

oxidation

cutin

biosynthesis

and

starch

degradation

-LRB-

Fig

-RRB-

For

example

some

steps

in

these

pathways

were

repressed

or

invariable

under

high-salinity

stress

but

were

induced

by

drought

stress

It

is

noticeable

that

one

step

in

the

ABA-biosynthesis

pathway

-LRB-

corresponding

to

Arabidopsis

-RRB-

showed

slightly

inhibited

in

gene

expression

under

high-salinity

stress

-LRB-

only

at

stage

-RRB-

but

was

induced

under

drought

treatment

-LRB-

Fig

-RRB-

Our

informatic

analysis

suggested

that

is

the

closest

Arabidopsis

homolog

with

identity

-LRB-

E

E

-RRB-

From

our

analysis

expression

ratios

-LRB-

transformed

-RRB-

were

-LRB-

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

under

drought

treatment

and

-LRB-

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

under

high

salinity

treatment

The

expression

ratios

of

rice

gene

at

and

stage

were

used

for

the

diagram

in

Fig

It

is

interesting

to

note

that

under

high

salinity

this

gene

was

initially

induced

and

then

inhibited

at

stage

-LRB-

ratio

-RRB-

while

under

drought

treatment

this

gene

was

induced

starting

at

stage

and

maintained

after

stage

-LRB-

ratio

-RRB-

This

example

illustrated

a

distinct

response

kinetics

for

different

responsive

genes

in

response

to

drought

and

high-salinity

stresses

By

comparing

transcriptomes

under

drought

and

high-salinity

stresses

in

three

distinct

organs

we

found

that

drought

responsive

genome

expression

in

flag

leaf

is

closer

to

that

for

high

salinity

stress

than

to

the

drought

expression

response

observed

in

any

other

organ

-LRB-

Fig.

D

-RRB-

In

general

there

seems

to

be

a

closer

relationship

between

the

transcriptome-level

responses

to

the

two

abiotic

stresses

in

the

same

organ

than

between

or

among

transcriptomes

in

distinct

organs

in

response

to

the

same

stress

However

the

extent

of

overlap

in

the

responses

to

drought

or

high-salinity

stresses

varies

for

the

different

rice

organs

For

example

it

is

more

divergent

in

the

responses

to

the

two

abiotic

stresses

in

panicle

while

flag

leaves

show

the

greatest

overlap

Distinct

groups

of

genes

are

induced

during

rehydration

after

drought

stress

In

its

natural

environment

a

plant

's

ability

to

respond

to

rehydration

after

drought

stress

is

important

for

its

survival

although

little

is

known

about

the

gene

expression

changes

that

occur

during

rehydration

Our

experiments

showed

that

after

a

period

of

drought

-LRB-

third

stage

-RRB-

all

the

rice

leaves

rolled

and

turned

yellow

and

the

RWC

dropped

to

When

we

applied

water

to

those

severely

stressed

plants

rice

leaves

started

to

unroll

after

h

and

exhibited

normal

flatness

within

hours

of

rehydration

We

collected

tissue

samples

h

after

rehydration

and

extracted

total

RNA

for

gene

expression

analysis

-LRB-

Fig.

-RRB-

Comparing

to

samples

from

normal

grown

rice

plants

in

flag

leaf

shoot

and

panicle

and

genes

were

up-regulated

after

h

of

rehydration

following

drought

stress

respectively

-LRB-

Fig.

Table

-RRB-

We

further

compared

gene

expression

profiles

during

drought

treatment

with

those

after

rehydration

-LRB-

Fig.

C

-RRB-

The

expression

patterns

of

those

genes

seem

to

belong

to

three

groups

with

flag

leaf

exhibiting

the

greatest

number

of

gene

expression

changes

following

rehydration

Group

I

which

included

and

genes

from

flag

leaf

shoot

and

panicle

respectively

was

induced

during

both

drought

treatment

and

rehydration

Group

II

which

included

and

genes

in

flag

leaf

shoot

and

panicle

respectively

was

repressed

during

drought

stress

but

induced

by

rehydration

-LRB-

for

shoot

and

panicle

see

Table

-RRB-

Group

III

which

included

all

remaining

genes

in

flag

leaf

genes

in

shoot

and

genes

in

panicle

showed

no

significant

expression

changes

under

drought

stress

but

were

up-regulated

during

rehydration

We

suspected

that

group

I

genes

must

be

induced

by

drought

stress

but

have

yet

to

return

to

normal

levels

after

rehydration

Those

genes

in

groups

II

and

III

were

specifically

induced

by

rehydration

Fig.

analysis

of

genes

exhibiting

elevated

expression

after

rehydration

following

drought

-LRB-

A

-RRB-

-LRB-

B

-RRB-

and

-LRB-

C

-RRB-

Cluster

analysis

of

the

genes

exhibiting

significant

elevation

of

expression

-LRB-

with

P

as

threshold

-RRB-

after

h

of

rehydration

treatment

for

flag

leaf

shoot

and

panicle

The

median

ratio

is

transformed

and

subject

to

complete

linkage

hierarchical

clustering

A

total

of

genes

in

flag

leaf

-LRB-

A

-RRB-

genes

in

shoot

-LRB-

B

-RRB-

and

genes

in

panicle

-LRB-

C

-RRB-

are

included

in

this

analysis

and

represent

three

stages

of

drought

treatment

and

represents

rehydration

of

the

stage

-LRB-

D

-RRB-

The

histogram

shows

the

number

of

genes

in

each

of

the

three

rice

organs

induced

under

drought

treatment

at

least

at

one

drought

stage

after

rehydration

following

the

stage

and

the

number

of

genes

with

overlapping

expression

in

various

pairs

of

organs

y-axis

shows

the

gene

numberTable

induced

by

rehydration

and

inhibited

by

drought

stress

in

shoot

and

panicleGene

namePutative

hydrolases

family

motif

I

reaction

center

subunit

IV

I

reaction

center

subunit

n

II

polypeptide

related

protein

a/b

binding

protein

II

kDa

polypeptide

PsbR

precursor

I

reaction

center

subunit

VI

a/b-binding

protein

protein

IX

monomethyl

esteraerobic

oxidative

cyclase

inhibitor/seed

storage/LTP

family

protein

precursor

transformed

ratios

of

all

genes

at

three

stages

of

drought

stress

and

rehydration

stage

were

listed

The

down-regulation

of

group

II

genes

in

response

to

drought

led

us

to

speculate

that

they

may

play

an

important

role

in

conferring

drought

stress

tolerance

whereas

the

up-regulation

of

group

II

and

group

III

genes

during

rehydration

could

be

important

for

recovery

In

both

group

II

and

III

genes

we

found

that

two

classes

of

genes

were

over-represented

among

the

rehydration-inducible

genes

in

shoot

and

panicle

transporter

genes

and

photosynthesis-related

genes

These

transporter

genes

included

-LRB-

folate/biopterin

transporter

-RRB-

-LRB-

putative

potassium

transporter

-RRB-

and

-LRB-

ABC

transporter

putative

-RRB-

and

-LRB-

Transmembrane

amino

acid

transporter

protein

-RRB-

-LRB-

H-ATPase

-RRB-

-LRB-

phosphate

H

symporter

-RRB-

-LRB-

Sodium

sulfate

symporter

transmembrane

region

-RRB-

-LRB-

putative

lipid

transfer

protein

-RRB-

and

-LRB-

aquaporin

-RRB-

The

photosynthesis

related

genes

cover

most

of

the

gene

components

of

the

two

photosystems

such

as

genes

for

putative

chlorophyll

a/b-binding

protein

Photosystem

I

or

II

reaction

center

subunits

and

plastocyanin

These

photosystem

genes

and

transporter

genes

represent

a

large

portion

of

all

genes

induced

by

rehydration

and

their

physiological

roles

in

plants

fit

well

with

a

potential

contribution

toward

plant

recovery

from

drought

stress

Limited

overlap

of

stress

responsive

genes

among

rice

organs

We

also

examined

the

degree

of

overlap

in

expression

of

stress

responsive

genes

in

two

or

more

rice

organs

under

drought

or

high-salinity

stress

Venn

diagrams

revealed

that

only

a

small

portion

of

genes

was

shared

between

each

pair

of

organs

-LRB-

Fig.

-RRB-

The

greatest

overlap

occurred

between

shoot

and

flag

leaf

which

shared

more

inducible

genes

than

shoot

and

panicle

or

flag

leaf

and

panicle

under

both

drought

and

high-salinity

conditions

Under

drought

stress

one-third

of

the

genes

up-regulated

in

flag

leaf

-LRB-

-RRB-

were

also

induced

in

shoot

-LRB-

Fig.

-RRB-

while

under

high

salinity

stress

over

half

of

the

induced

genes

-LRB-

-RRB-

in

shoot

-LRB-

mostly

young

leaves

-RRB-

were

also

up-regulated

in

flag

leaf

-LRB-

Fig.

-RRB-

Only

and

of

genes

up-regulated

in

panicle

were

also

induced

in

flag

leaf

under

drought

and

high

salinity

stresses

respectively

The

percentage

of

genes

shared

between

shoot

and

panicle

is

similar

to

that

between

flag

leaf

and

panicle

-LRB-

Fig.

B

-RRB-

Fig.

of

gene

expression

patterns

among

the

three

rice

organs

in

response

to

drought

and

high-salinity

stresses

-LRB-

A

-RRB-

and

-LRB-

B

-RRB-

Venn

diagram

of

drought

-LRB-

A

-RRB-

and

high-salinity

-LRB-

B

-RRB-

induced

genes

among

the

three

rice

organs

-LRB-

C

-RRB-

and

-LRB-

D

-RRB-

Venn

diagram

of

drought

-LRB-

C

-RRB-

and

high-salinity

-LRB-

D

-RRB-

repressed

genes

among

the

three

rice

organs

Interestingly

only

a

small

fraction

of

stress

responsive

genes

were

expressed

in

all

three

organs

examined

For

example

and

genes

were

induced

in

all

three

organs

under

drought

and

high-salinity

stress

respectively

In

general

most

of

those

genes

exhibited

high

levels

of

expression

as

well

as

strong

inducibility

Among

them

genes

were

induced

by

both

drought

and

high-salinity

stress

in

all

three

organs

This

group

of

genes

includes

a

protein

kinase

-LRB-

-RRB-

chlorophyll

a/b

binding

protein

-LRB-

-RRB-

ABA-responsive

protein

-LRB-

-RRB-

LEA

protein

-LRB-

-RRB-

dehydrin

-LRB-

-RRB-

CHY

zinc

finger

protein

-LRB-

-RRB-

and

homeobox

protein

-LRB-

-RRB-

-LRB-

Table

-RRB-

Table

up

or

down-regulated

by

both

drought

and

high-salinity

stresses

in

all

three

organsGene

namePutative

functionsFlag

leafShootPanicleD

-LRB-

h

-RRB-

S

-LRB-

h

-RRB-

D

-LRB-

h

-RRB-

S

-LRB-

h

-RRB-

D

-LRB-

h

-RRB-

S

-LRB-

h

-RRB-

kinase

a/b

binding

acid-induced

zinc

to

Arabidopsis

protein

Uridine

kinase

family

The

highest

ratio

of

each

gene

at

three

stages

under

drought

-LRB-

D

-LRB-

h

-RRB-

-RRB-

or

high-salinity

-LRB-

S

-LRB-

h

-RRB-

-RRB-

were

transformed

and

listed

Analysis

of

stress

down-regulated

genes

-LRB-

Fig.

D

-RRB-

revealed

a

similar

small

overlap

among

the

three

organs

Only

and

out

of

total

genes

down-regulated

in

flag

leaf

under

drought

stress

were

also

inhibited

in

shoot

and

panicle

respectively

For

high-salinity

stress

and

out

of

down-regulated

genes

in

flag

leaf

were

also

repressed

in

shoot

and

panicle

respectively

Only

and

genes

were

down-regulated

in

all

three

organs

under

drought

and

high

salinity

stress

respectively

while

only

two

genes

were

inhibited

in

all

three

organs

under

both

drought

and

high

salinity

stress

-LRB-

Table

-RRB-

The

shoot

samples

we

used

consisted

largely

of

young

leaves

and

were

thus

physiologically

closer

to

flag

leaf

than

panicle

Indeed

shoot

and

flag

leaf

generally

exhibited

similar

patterns

of

gene

up-regulation

in

response

to

drought

and

high-salinity

stress

However

this

does

not

appear

to

be

the

case

for

repressed

genes

where

overlap

between

shoot

and

flag

leaf

gene

expression

is

minimal

To

validate

the

microarray

data

RT-PCR

analysis

was

used

to

verify

the

transcription

response

of

representative

genes

from

microarray

results

For

this

purpose

we

picked

a

group

of

eight

representative

genes

and

designed

primers

for

RT-PCR

analysis

-LRB-

Table

-RRB-

The

cDNA

templates

were

synthesized

from

total

RNA

samples

prepared

from

shoot

flag

leaf

and

panicle

under

drought

and

unstressed

controls

The

semi-quantitative

RT-PCR

results

for

the

eight

representative

genes

were

shown

in

Fig.

The

expression

patterns

of

all

eight

genes

reflected

changes

observed

by

microarray

analysis

fairly

and

accurately

for

all

three

organs

examined

except

in

three

cases

where

RT-PCR

failed

to

confirm

the

microarray

results

-LRB-

Fig.

-RRB-

indicating

that

our

microarray

data

is

reliable

Fig.

analysis

of

the

representative

drought-induced

genes

among

the

three

rice

organs

Total

RNA

samples

were

prepared

from

the

shoot

-LRB-

and

-RRB-

flag

leaf

-LRB-

and

-RRB-

and

panicle

-LRB-

and

-RRB-

and

taken

from

plants

at

three

stages

of

drought

treatment

and

the

untreated

control

plants

-LRB-

and

-RRB-

The

corresponding

transformed

median

ratio

of

microarray

data

is

shown

at

the

bottom

of

each

mRNA

blot

line

no

expression

The

corresponds

to

cases

where

the

microarray

data

was

not

confirmed

by

the

mRNA

blot

results

Enrichment

of

ABRE

and

DRE

cis-regulatory

elements

in

abiotic

stress-induced

gene

promoters

Previous

reports

have

suggested

that

several

well-characterized

drought

high-salinity

and

cold

inducible

gene

promoters

contain

two

common

cis-regulatory

elements

the

ABA-responsive

element

-LRB-

ABRE

-RRB-

and

the

dehydration-responsive

element

-LRB-

DRE

-RRB-

-LRB-

Seki

et

al.

b

Yazaki

et

al.

Rabbani

et

al.

Dubouzet

et

al.

Nakabayashi

et

al.

Maruyama

et

al.

Yamaguchi-Shinozaki

and

Shinozaki

-RRB-

ABRE

and

DRE

confer

ABA-dependent

and

ABA-independent

gene

expression

in

response

to

water

stress

It

has

been

reported

that

a

single

copy

of

ABRE

in

the

promoter

region

only

induces

relatively

minor

elevation

of

expression

level

while

multiple

copies

of

ABRE

strongly

activate

expression

-LRB-

Shen

and

Ho

Nakabayashi

et

al.

-RRB-

To

elucidate

the

relationship

between

copy

numbers

of

DRE

and

ABRE

cis-regulatory

elements

in

gene

promoter

regions

and

abiotic

stress-induced

gene

transcription

we

divided

the

genes

into

groups

based

on

their

expression

patterns

Genes

induced

specifically

by

drought

or

high-salinity

stress

in

each

of

the

three

organs

defined

six

groups

while

genes

induced

by

both

drought

and

high-salinity

stress

in

each

organ

defined

another

three

groups

Common

inducible

genes

shared

by

at

least

two

organs

under

drought

or

high-salinity

stress

defined

another

nine

groups

We

only

selected

those

genes

among

all

those

groups

with

full-length

cDNA

sequences

available

for

further

promoter

analysis

In

brief

the

kb

upstream

regions

-LRB-

to

bp

-RRB-

preceding

the

ATG

start

codon

of

genes

were

selected

for

promoter

motif

analysis

-LRB-

see

Experimental

procedures

-RRB-

A

similar

number

of

rice

genes

with

full

length

cDNA

available

that

lack

stress

responsive

expression

were

used

as

control

Copy

number

of

ABRE

and

DRE

core

motifs

on

each

promoter

region

were

counted

The

percentages

of

genes

in

each

group

with

to

copies

of

ABRE

core

or

DRE

core

elements

were

then

calculated

Compared

to

control

genes

copy

numbers

of

the

ABRE

and

DRE

core

motifs

in

the

promoter

regions

of

genes

with

organ-specific

expression

in

response

to

drought

or

high-salinity

stress

were

not

markedly

different

-LRB-

Fig.

D

-RRB-

The

promoter

regions

of

genes

responsive

to

both

drought

and

high-salinity

stress

in

each

organ

were

enriched

for

ABRE

and

DRE

core

motifs

compared

to

the

promoters

of

control

genes

-LRB-

Fig.

E

-RRB-

For

example

only

of

control

genes

have

over

four

copies

of

ABRE

core

motif

in

their

promoter

regions

but

and

of

genes

induced

by

both

drought

and

high-salinity

stresses

in

flag

leaf

panicle

and

shoot

respectively

have

over

four

copies

of

the

ABRE

core

motif

in

their

promoter

regions

-LRB-

Fig.

-RRB-

Fig.

of

the

ABRE

and

DRE

core

motif

sequences

among

different

groups

of

drought

and

high-salinity

stress

responsive

genes

In

all

panels

the

x-axis

shows

the

copy

number

of

ABRE

or

DRE

core

motifs

and

the

y-axis

shows

the

relative

number

of

genes

containing

different

copy

numbers

of

ABRE

or

DRE

core

motifs

-LRB-

A

-RRB-

ABRE

core

motif

distribution

among

promoters

of

the

genes

induced

only

by

drought

or

only

by

high-salinity

stress

in

each

single

rice

organ

-LRB-

B

-RRB-

ABRE

core

motif

distribution

among

promoters

of

genes

induced

by

both

drought

and

high-salinity

stress

in

each

rice

organ

-LRB-

C

-RRB-

ABRE

core

motif

distribution

among

promoters

of

genes

induced

in

two

or

three

organs

-LRB-

D

-RRB-

DRE

core

motif

distribution

among

promoters

of

genes

induced

only

by

drought

or

only

by

high-salinity

stress

in

each

rice

organ

-LRB-

E

-RRB-

DRE

core

motif

distribution

among

promoters

of

genes

induced

by

both

drought

and

high-salinity

stress

in

each

rice

organ

-LRB-

F

-RRB-

DRE

core

motif

distribution

among

promoters

of

genes

induced

in

two

or

three

organs

or

by

both

stresses

in

more

than

one

organ

The

following

gene

groups

-LRB-

with

number

of

genes

with

full-length

cDNA

gene

number

-RRB-

were

analyzed

-LRB-

-RRB-

Genes

induced

by

drought

stress

only

in

flag

leaf

-LRB-

-RRB-

-LRB-

-RRB-

Genes

induced

by

high-salinity

stress

only

in

flag

leaf

-LRB-

-RRB-

-LRB-

-RRB-

Genes

induced

by

drought

stress

only

in

shoot

-LRB-

-RRB-

-LRB-

-RRB-

Genes

induced

by

high-salinity

stress

only

in

shoot

-LRB-

-RRB-

-LRB-

-RRB-

Genes

induced

by

drought

stress

only

in

panicle

-LRB-

-RRB-

-LRB-

-RRB-

Genes

induced

by

high-salinity

stress

only

in

panicle

-LRB-

-RRB-

-LRB-

-RRB-

Genes

induced

by

both

stresses

in

flag

leaf

-LRB-

-RRB-

-LRB-

-RRB-

Genes

induced

by

both

stresses

in

shoot

-LRB-

-RRB-

-LRB-

-RRB-

Genes

induced

by

both

stresses

in

panicle

-LRB-

-RRB-

-LRB-

-RRB-

Genes

induced

by

drought

stress

in

both

flag

leaf

and

shoot

-LRB-

-RRB-

-LRB-

-RRB-

Genes

induced

by

drought

stress

in

all

three

organs

-LRB-

-RRB-

-LRB-

-RRB-

Genes

induced

by

high-salinity

stress

in

all

three

organs

-LRB-

-RRB-

-LRB-

-RRB-

Genes

induced

by

high-salinity

stress

in

both

flag

leaf

and

shoot

-LRB-

-RRB-

-LRB-

-RRB-

Genes

induced

by

drought

stress

in

both

flag

leaf

and

panicle

-LRB-

-RRB-

-LRB-

-RRB-

Genes

induced

by

high-salinity

stress

in

both

flag

leaf

and

panicle

-LRB-

-RRB-

-LRB-

-RRB-

Genes

induced

by

drought

stress

in

both

shoot

and

panicle

-LRB-

-RRB-

-LRB-

-RRB-

Genes

induced

by

high-salinity

stress

in

both

shoot

and

panicle

-LRB-

-RRB-

-LRB-

-RRB-

Genes

induced

by

both

stresses

in

all

three

organs

-LRB-

-RRB-

When

genes

expressed

in

at

least

two

organs

under

drought

or

high-salinity

stress

were

compared

to

control

genes

the

ABRE

and

DRE

core

motif

copy

numbers

also

showed

significant

differences

-LRB-

Fig.

F

-RRB-

For

example

of

these

genes

contain

over

four

copies

of

the

ABRE

core

motif

in

their

promoter

regions

compared

with

for

the

control

genes

-LRB-

Fig.

-RRB-

A

similar

pattern

was

found

for

the

DRE

core

element

-LRB-

Fig.

-RRB-

Genes

induced

in

an

organ-specific

manner

by

drought

or

high-salinity

stresses

did

not

contain

a

significantly

different

number

of

copies

of

ABRE

or

DRE

core

motifs

compared

to

control

genes

-LRB-

Fig.

D

-RRB-

Thus

it

is

plausible

that

the

stress-induced

expression

in

this

fraction

of

genes

may

rely

less

on

transcriptional

activation

mediated

by

ABRE

and

DRE

motifs

Unidentified

organ-specific

cis-regulatory

elements

may

exist

in

the

promoter

regions

of

these

genes

and

play

a

more

important

role

in

transcription

activation

under

drought

or

high-salinity

stress

Identification

of

two

novel

cis-regulatory

elements

that

respond

to

rehydration

after

drought

stress

As

shown

in

Table

there

are

and

genes

with

full-length

cDNA

sequences

repressed

by

drought

stress

but

induced

by

rehydration

in

shoot

and

panicle

respectively

An

analysis

of

the

upstream

region

of

these

eight

shoot

genes

identified

a

novel

motif

-LRB-

motif-SP

GGCAGCCG

-RRB-

located

near

to

the

translation

start

codon

-LRB-

to

-RRB-

in

five

of

them

-LRB-

Fig.

-RRB-

To

investigate

the

potential

functional

role

of

this

novel

motif

we

performed

a

gel

shift

mobility

assay

which

involved

incubating

a

bp

probe

containing

motif-SP

from

one

of

those

five

genes

-LRB-

-RRB-

and

a

control

containing

a

single

base

mutation

at

an

invariable

position

with

nuclear

extracts

-LRB-

see

Experimental

procedures

-RRB-

Only

nuclear

extracts

from

drought-treated

shoot

-LRB-

at

the

third

stage

-RRB-

but

not

from

unstressed

shoot

or

shoot

under

rehydration

showed

a

sequence

specific

binding

activity

with

the

probe

-LRB-

Fig.

-RRB-

A

single

base

mutation

at

an

invariable

position

in

the

motif-SP

core

of

the

probe

completely

abolished

protein

binding

activity

suggesting

high

specificity

of

interaction

with

the

core

motif

included

in

this

bp

sequence

This

specific

binding

activity

was

only

observed

in

drought-treated

plants

suggesting

that

it

is

involved

in

transcriptional

repression

under

drought

conditions

Fig.

novel

promoter

motif

associated

with

genes

repressed

by

drought

but

induced

after

rehydration

in

rice

shoot

-LRB-

A

-RRB-

The

core

sequence

of

the

motif

and

the

position

of

the

motif

in

each

promoter

-LRB-

B

-RRB-

The

expression

pattern

of

a

representative

gene

containing

motif-SP

The

histogram

shows

the

transformed

ratio

at

three

drought

stress

stages

-LRB-

-RRB-

and

rehydration

-LRB-

-RRB-

-LRB-

C

-RRB-

Gel

shift

assay

of

motif-SP

Nuclear

proteins

were

extracted

from

shoots

of

untreated

plants

-LRB-

C

-RRB-

plants

at

stage

of

drought

treatment

-LRB-

-RRB-

and

plants

after

rehydration

following

stage

-LRB-

-RRB-

Core

sequences

of

the

probe

TGCAGCCA

and

core

sequence

of

the

probe

with

a

single

base

point

mutation

TGAAGCCA

We

also

searched

promoter

regions

of

genes

inhibited

by

drought

stress

but

induced

by

rehydration

in

panicle

Three

conserved

motifs

were

found

through

this

blind

search

in

of

the

genes

ABRE

motif

the

above-mentioned

motif-SP

and

another

novel

element

motif-P

-LRB-

Fig.

-RRB-

The

presence

of

the

ABRE

motif

suggests

that

a

gene

may

respond

to

drought

stress

by

employing

an

ABA-dependent

pathway

while

motif-SP

may

play

an

important

role

in

drought-stress

repression

and

subsequent

activation

during

rehydration

We

failed

to

detect

specific

binding

activity

using

similar

nuclear

extracts

-LRB-

data

not

shown

-RRB-

and

thus

the

role

of

motif-P

remains

to

be

defined.Fig

motifs

associated

with

genes

repressed

during

drought

and

induced

by

rehydration

in

panicle

The

core

sequence

logos

and

the

position

of

the

motifs

in

each

of

the

promoters

are

listed

Transcription

factor

genes

under

drought

and

high

salinity

stress

are

expressed

in

a

largely

organ

specific

manner

We

further

examined

transcription

factor

genes

whose

expression

are

regulated

by

drought

and

high-salinity

stresses

Among

all

genes

induced

by

drought

or

high-salinity

treatment

in

the

three

organs

a

total

of

genes

-LRB-

Table

-RRB-

were

predicted

to

be

transcription

factors

with

a

DNA-binding

domain

These

transcription

factors

belong

to

various

families

including

bHLH

MYB

HB

NAC

zinc

finger

MADS

bZIP

WRKY

and

HSF

families

These

transcription

factors

could

also

be

divided

into

several

groups

depending

on

their

expression

patterns

Among

the

transcription

factor

genes

genes

were

induced

in

all

three

organs

in

at

least

one

stage

following

either

drought

or

high-salinity

stress

whereas

the

remaining

genes

were

mainly

up

regulated

in

one

or

two

organs

Transcription

factors

induced

only

in

one

individual

organ

were

listed

in

Table

It

is

interesting

to

note

that

over

half

of

the

transcription

factors

were

expressed

in

at

least

two

organs

while

other

transcription

factors

were

activated

in

an

organ-specific

manner

This

suggests

that

the

expression

of

different

transcription

factors

may

play

a

key

role

in

common

or

organ-specific

gene

expression

in

response

to

drought

or

high-salinity

conditions

Table

factor

genes

induced

in

each

organ

by

drought

or

high-salinity

stressGene

name

Putative

functionPanicle

induced

by

high-salinity

onlyS

-LRB-

h

-RRB-

D

-LRB-

h

-RRB-

transcription

DNA-binding

apical

meristem

-LRB-

NAM

-RRB-

binding

factor

zinc

finger

finger

type

-LRB-

RING

finger

-RRB-

DNA-binding

zinc

apical

meristem

-LRB-

NAM

-RRB-

domain

zinc

finger

DNA-binding

finger

N/APanicle

induced

by

drought

onlyS

-LRB-

h

-RRB-

D

-LRB-

h

-RRB-

Panicle

induced

by

both

drought

and

high-salinityS

-LRB-

h

-RRB-

D

-LRB-

h

-RRB-

Shoot

induced

by

drought

onlyS

-LRB-

h

-RRB-

D

-LRB-

h

-RRB-

protein

DNA

binding

domain

DNA-binding

domain

DNA-binding

domain

domain

zinc

DNA-binding

DNA

binding

DNA-binding

DNA-binding

to

DNA-binding

protein

finger

transcription

factor

Shoot

induced

by

both

drought

and

high-salinityS

-LRB-

h

-RRB-

D

-LRB-

h

-RRB-

DNA

binding

Shoot

induced

by

high-salinity

onlyS

-LRB-

h

-RRB-

D

-LRB-

h

-RRB-

DNA

binding

zinc

Flag

leaf

induced

by

drought

onlyS

-LRB-

h

-RRB-

D

-LRB-

h

-RRB-

finger

type

Flag

leaf

induced

by

high-salinity

onlyS

-LRB-

h

-RRB-

D

-LRB-

h

-RRB-

finger

The

highest

ratio

of

each

gene

among

three

stages

of

drought

-LRB-

D

-LRB-

h

-RRB-

-RRB-

or

high-salinity

-LRB-

S

-LRB-

h

-RRB-

-RRB-

were

transformed

and

listed

Minimal

co-regulation

of

neighboring

genes

in

drought

or

high-salinity

stress

responses

Co-regulation

of

adjacent

genes

has

been

observed

in

several

organisms

-LRB-

Hurst

et

al.

-RRB-

including

human

-LRB-

Caron

et

al.

Lercher

et

al.

-RRB-

Drosophila

-LRB-

Spellman

and

Rubin

-RRB-

Arabidopsis

-LRB-

Birnbaum

et

al.

Ma

et

al.

-RRB-

and

yeast

-LRB-

Cohen

et

al.

-RRB-

All

these

studies

identified

co-regulation

of

neighboring

gene

clusters

on

chromosomes

-LRB-

chromatin

domains

-RRB-

In

rice

analysis

of

organ-specific

gene

expression

revealed

that

about

of

the

genome

belongs

to

chromatin

domains

that

exhibit

this

co-expression

pattern

-LRB-

Ma

et

al.

-RRB-

However

similar

analysis

of

light-regulated

genes

failed

to

reveal

significant

co-regulation

-LRB-

Jiao

et

al.

-RRB-

To

investigate

possible

co-regulation

of

neighboring

genes

in

the

drought

and

high-salinity

responses

we

mapped

rehydration

induced

genes

in

three

organs

and

drought

and

high-salinity

induced

genes

in

shoot

to

all

indica

chromosomes

-LRB-

Figs.

-RRB-

This

analysis

indicated

that

significant

gene

clustering

among

rehydration

drought

and

high-salinity

induced

genes

did

not

occur

at

the

chromosomal

level

similar

to

the

pattern

characteristic

of

light-regulated

genes

-LRB-

Jiao

et

al.

-RRB-

Among

the

rice

chromosomes

only

three

regions

showed

significant

enrichment

of

or

more

co-regulated

genes

within

any

given

Mb

domain

For

example

the

Mb

region

on

chromosome

the

Mb

region

in

chromosome

and

the

Mb

region

in

chromosome

-LRB-

Fig.

-RRB-

contain

significant

enrichment

of

rehydration

regulated

genes

in

all

three

organs

although

even

in

those

cases

genes

are

not

directly

adjacent

Fig.

of

drought

high

salinity

and

rehydration

regulated

genes

in

a

representative

rice

chromosome

-LRB-

A

-RRB-

Map

of

chromosome

genes

differentially

expressed

at

the

first

stage

of

drought

first

stage

of

high-salinity

stress

and

h

after

rehydration

in

shoot

-LRB-

B

-RRB-

Map

of

chromosome

genes

differentially

expressed

at

h

after

rehydration

in

the

three

rice

organs

Discussion

This

study

provides

new

insight

into

the

rice

response

to

drought

and

high

salinity

stresses

at

the

whole

genome

level

Using

a

whole

genome

microarray

we

monitored

the

expression

of

unique

or

known

rice

genes

or

gene

models

in

three

different

organs

under

drought

and

high

salinity

stress

Our

work

thus

offers

the

first

comprehensive

picture

of

genome

expression

modulation

in

response

to

drought

and

high

salinity

stress

in

three

distinct

rice

organs

Genome

expression

reprograming

showed

significant

overlap

between

drought

and

high

salinity

responsive

genes

Using

the

criteria

outlined

above

a

total

of

and

rice

genes

showed

significant

up

or

down-regulation

in

response

to

high

salinity

stress

and

drought

stress

in

at

least

one

of

the

three

organs

Our

analysis

suggested

that

out

of

-LRB-

-RRB-

genes

induced

by

drought

were

also

up

regulated

by

high-salinity

stress

in

the

same

organs

This

number

is

consistent

with

previous

studies

reported

for

Arabidopsis

-LRB-

Seki

et

al.

b

Shinozaki

et

al.

-RRB-

In

rice

an

even

higher

percentage

of

drought

induced

genes

were

also

up-regulated

under

high-salinity

stress

as

reported

previously

on

a

smaller

scale

-LRB-

Rabbani

et

al.

-RRB-

which

covered

only

cDNAs

from

plants

under

drought

cold

and

high-salinity

treatment

Thus

our

analysis

should

be

more

representative

of

the

overall

rice

genome

response

to

drought

and

high

salinity

stresses

without

such

bias

However

drought

and

salt

stress

conditions

in

the

field

may

be

more

subtle

and

less

regular

as

in

our

experimental

conditions

thus

our

results

are

only

meant

to

illustrate

some

particular

situations

rice

genome

expression

responses

to

stresses

It

is

interesting

to

note

an

overlap

of

up

to

about

half

of

the

genes

induced

or

inhibited

by

drought

and

high-salinity

stress

in

rice

This

observation

is

consistent

with

current

understanding

that

these

two

stresses

affect

plants

in

overlapping

but

not

identical

ways

At

a

physiological

level

both

stresses

cause

water

depletion

in

the

above-ground

portion

of

the

plants

and

induce

similar

morphological

responses

-LRB-

Fig

-RRB-

At

the

molecular

level

half

of

the

transcriptional

factor

genes

identified

in

our

results

were

shared

by

both

stresses

in

each

organ

which

is

consistent

with

the

observation

that

about

half

of

the

genes

that

respond

to

the

two

stresses

were

shared

at

the

whole

genome

level

Reprograming

of

genome

expression

in

response

to

drought

and

high-salinity

stresses

is

largely

organ

specific

In

this

study

we

examined

whole

genome

expression

profiles

under

drought

and

high-salinity

conditions

in

three

organs

four-tiller

stage

shoot

filling

stage

flag

leaf

and

panicle

Rice

plants

at

these

two

growth

stages

particularly

the

late

one

are

sensitive

to

drought

and

high-salinity

stress

It

is

well

known

that

drought

or

high-salinity

stress

at

the

heading

and

early

panicle

stages

can

severely

compromise

rice

growth

and

development

and

reduce

crop

yield

even

with

late

rehydration

It

is

evident

that

the

rice

genome

is

subject

to

significant

reprograming

with

regard

to

which

portion

of

genome

is

expressed

under

drought

or

high

salinity

stress

Our

results

showed

that

only

a

limited

number

of

drought

and

high

salinity

responsive

genes

were

shared

between

any

two

organs

We

found

that

only

and

of

drought-induced

genes

in

panicle

were

shared

with

those

induced

in

shoot

and

flag

leaf

respectively

and

of

drought-induced

genes

in

shoot

were

also

activated

in

flag

leaf

-LRB-

Fig.

-RRB-

The

percentages

of

genes

shared

between

any

two

organs

under

high-salinity

conditions

were

similar

to

those

under

drought

stress

suggesting

that

responses

to

drought

or

high-salinity

stress

in

different

organs

were

independently

regulated

This

observation

is

similar

to

the

observed

small

overlap

of

cold

responsive

gene

expression

between

root

and

leaf

in

rice

-LRB-

Kreps

et

al.

-RRB-

Genome

expression

reprograming

under

either

drought

or

high

salinity

stress

entails

a

large

number

of

genes

involved

in

many

aspects

of

cellular

function

A

GO

analysis

of

stress

responsive

genes

-LRB-

Fig

-RRB-

indicates

that

in

most

categories

three

organs

activated

similar

scale

-LRB-

number

-RRB-

of

genes

in

response

to

drought

or

high

salinity

stress

while

only

small

percentage

of

responsive

genes

were

overlapped

between

or

among

organs

-LRB-

Fig.

-RRB-

This

suggests

that

responsive

genes

under

the

same

category

are

largely

distinct

individuals

under

distinct

organ

types

It

is

thus

possible

that

homologous

or

functionally

similar

gene

family

members

are

responsive

to

the

same

stresses

in

each

organ

This

is

consistent

with

our

observation

of

organ-specific

transcription

factor

gene

expression

in

response

to

both

drought

and

high

salinity

stresses

-LRB-

Table

-RRB-

The

fact

that

genes

specifically

induced

in

each

organ

do

not

exhibit

enrichment

of

DRE

and

ABRE

motifs

-LRB-

Fig.

-RRB-

suggests

that

organ-specific

transcriptional

factor

gene

expression

may

be

responsible

for

activating

organ-specific

downstream

genes

in

a

secondary

transcriptional

response

to

stress

For

these

genes

it

is

likely

that

certain

organ-specific

promoter

elements

mediate

this

response

pathway

According

to

the

GO

analysis

under

high

salinity

stress

about

storage

proteins

were

up-regulated

in

panicle

while

in

flag

leaf

and

shoot

only

less

than

storage

protein

genes

were

induced

-LRB-

Fig

-RRB-

This

kind

of

difference

among

organs

may

play

a

role

in

organ

specific

response

to

two

stresses

further

function

analysis

of

these

differentially

regulated

genes

among

organs

are

needed

Rice

may

possess

specific

mechanisms

to

facilitate

plant

recovery

during

rehydration

after

drought

According

to

our

microarray

analysis

rice

genes

induced

by

rehydration

were

divided

into

three

groups

according

to

their

expression

patterns

-LRB-

Fig.

-RRB-

somewhat

similar

to

a

previous

report

for

Arabidopsis

-LRB-

Oono

et

al.

-RRB-

This

observation

suggested

that

monocotyledonous

and

dicotyledonous

plants

share

similar

gene

expression

responses

to

rehydration

after

drought

Analysis

of

the

genome-wide

distribution

of

rehydration-regulated

genes

in

the

three

organs

showed

no

significant

co-regulation

of

neighboring

genes

at

the

chromosomal

level

This

observation

is

similar

to

the

case

of

light-regulated

genes

-LRB-

Jiao

et

al.

-RRB-

but

different

from

general

gene

transcription

from

organ

samples

-LRB-

Ma

et

al.

-RRB-

This

distinction

in

the

modulation

of

genome

expression

may

reflect

different

mechanisms

employed

in

response

to

different

developmental

or

environmental

signals

Up

to

now

only

one

possible

cis-element

has

been

reported

to

be

involved

in

the

rehydration

process

after

dehydration

in

Arabidopsis

-LRB-

Satoh

et

al.

Oono

et

al.

-RRB-

In

rice

at

the

whole

genome

level

we

identified

and

genes

induced

by

rehydration

in

flag

leaf

shoot

and

panicle

separately

-LRB-

Fig.

-RRB-

These

genes

provided

us

an

important

starting

point

to

study

rehydration

mechanisms

and

to

search

for

novel

cis-regulatory

promoter

elements

associated

with

dehydration

or

rehydration

Two

novel

cis-regulatory

elements

-LRB-

motif-SP

and

motif-P

-RRB-

were

identified

-LRB-

Fig.

-RRB-

Motif-SP

was

also

found

in

shoot-specific

genes

with

similar

expression

patterns

in

response

to

drought

and

rehydration

Gel

shift

assays

provided

evidence

that

motif-SP

may

function

as

a

cis-element

to

mediate

the

drought-induced

repression

and

late

de-repression

-LRB-

activation

-RRB-

during

rehydration

in

rice

Further

functional

analysis

of

the

promoter

elements

may

substantiate

the

role

of

those

novel

promoter

motifs

Previous

studies

reported

that

drought

stress

suppressed

plant

photosynthesis

systems

and

significantly

modulated

the

activity

of

some

membrane

transporters

-LRB-

Rizhsky

et

al.

Ramachandra

et

al.

Johansson

et

al.

Becker

et

al.

-RRB-

Here

microarray

analysis

revealed

that

genes

involved

in

photosynthesis

and

genes

encoding

transporters

were

repressed

or

maintained

at

low

levels

of

expression

under

drought

but

were

then

strongly

activated

after

rehydration

The

repression

of

metabolic

genes

during

drought

stress

allows

the

plant

to

conserve

energy

and

subsist

on

less

water

conferring

better

drought

tolerance

When

supplied

plenty

of

water

upon

rehydration

activation

of

these

genes

could

aid

in

the

recovery

of

full

photosynthesis

activity

and

transmembrane

solute/water

exchange

thus

helping

plant

resume

its

normal

growth

and

development

quickly

Electronic

supplementary

material

Below

are

the

electronic

supplementary

materials

Fig

-LRB-

JPG

kb

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Fig

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JPG

kb

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Fig

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JPG

kb

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Fig

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JPG

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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

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XLS

kb

-RRB-

ESM

-LRB-

XLS

kb

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ESM

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XLS

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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

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XLS

kb

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ESM

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XLS

kb

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ESM

-LRB-

XLS

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ESM

-LRB-

XLS

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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

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XLS

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ESM

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XLS

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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

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XLS

kb

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rice drought salinity rehydration promoter