Interaction

between

sugar

and

abscisic

acid

signalling

during

early

seedling

development

in

Arabidopsis

Sugars

regulate

important

processes

and

affect

the

expression

of

many

genes

in

plants

Characterization

of

Arabidopsis

-LRB-

Arabidopsis

thaliana

-RRB-

mutants

with

altered

sugar

sensitivity

revealed

the

function

of

abscisic

acid

-LRB-

ABA

-RRB-

signalling

in

sugar

responses

However

the

exact

interaction

between

sugar

signalling

and

ABA

is

obscure

Therefore

ABA

deficient

plants

with

constitutive

expression

-LRB-

-RRB-

were

generated

Enhanced

expression

did

not

rescue

the

glucose

insensitive

-LRB-

gin

-RRB-

phenotype

of

seedlings

indicating

that

other

ABA

regulated

factors

are

essential

as

well

Interestingly

both

glucose

and

ABA

treatment

of

Arabidopsis

seeds

trigger

a

post-germination

seedling

developmental

arrest

The

glucose-arrested

seedlings

had

a

drought

tolerant

phenotype

and

showed

glucose-induced

expression

of

ABSCISIC

ACID

-LRB-

-RRB-

and

LATE

EMBRYOGENESIS

ABUNDANT

-LRB-

LEA

-RRB-

genes

reminiscent

of

ABA

signalling

during

early

seedling

development

is

a

key

regulator

of

the

ABA-induced

arrest

and

it

is

shown

here

that

functions

in

glucose

signalling

as

well

Multiple

alleles

have

a

glucose

insensitive

-LRB-

gin

-RRB-

phenotype

comparable

to

that

of

other

known

gin

mutants

Importantly

glucose-regulated

gene

expression

is

disturbed

in

the

background

Moreover

was

insensitive

to

sugars

during

germination

and

showed

sugar

insensitive

-LRB-

sis

-RRB-

and

sucrose

uncoupled

-LRB-

sun

-RRB-

phenotypes

Mutant

analysis

further

identified

the

ABA

response

pathway

genes

ENHANCED

RESPONSE

TO

-LRB-

-RRB-

and

as

intermediates

in

glucose

signalling

Hence

three

previously

unidentified

sugar

signalling

genes

have

been

identified

showing

that

ABA

and

glucose

signalling

overlap

to

a

larger

extend

than

originally

thought

Introduction

Photosynthesis

provides

plants

with

sugars

that

play

a

central

role

in

the

plant

life

cycle

as

energy

sources

storage

molecules

structural

components

or

intermediates

for

the

synthesis

of

other

organic

molecules

Next

to

these

metabolic

functions

sugars

act

as

signalling

molecules

with

hormone-like

properties

Both

hexoses

and

disaccharides

are

able

to

induce

signalling

via

different

pathways

-LRB-

Jang

and

Sheen

Loreti

et

al.

Rolland

et

al.

-RRB-

In

plants

glucose

has

been

shown

to

affect

many

processes

including

germination

early

seedling

growth

flowering

and

senescence

-LRB-

Gibson

Smeekens

Rolland

et

al.

-RRB-

Moreover

glucose

feeding

of

Arabidopsis

seedlings

affected

the

expression

of

many

genes

as

shown

by

micro

array

studies

-LRB-

Price

et

al.

Villadsen

and

Smith

Li

et

al.

-RRB-

Sugar-induced

signal

transduction

has

been

shown

to

control

gene

expression

via

diverse

mechanisms

that

include

transcription

translation

and

modification

of

mRNA

and

protein

stability

-LRB-

Rolland

et

al.

-RRB-

Genetic

analysis

showed

that

sugar

signalling

in

plants

is

closely

associated

with

plant

hormone

biosynthesis

and

signalling

in

particular

with

that

of

abscisic

acid

-LRB-

ABA

for

review

see

Finkelstein

and

Gibson

Gazzarini

and

McCourt

León

and

Sheen

Rook

et

al.

Dekkers

and

Smeekens

-RRB-

Four

screens

for

sugar

response

mutants

i.e.

sucrose

uncoupled

-LRB-

sun

-RRB-

impaired

sucrose

induction

-LRB-

isi

-RRB-

glucose

insensitive

-LRB-

gin

-RRB-

and

sugar

insensitive

-LRB-

sis

-RRB-

identified

ABA

deficient

mutants

-LRB-

i.e.

and

-RRB-

and

ABA

-LRB-

-RRB-

as

sugar

insensitive

-LRB-

Arenas-Huertero

et

al.

Huijser

et

al.

Laby

et

al.

Rook

et

al.

-RRB-

encodes

an

domain

containing

transcription

factor

that

binds

a

element

present

in

many

ABA

and

sugar

regulated

promoters

-LRB-

Finkelstein

et

al.

Niu

et

al.

Acevedo-Hernandez

et

al.

-RRB-

These

observations

linked

sugar

regulation

to

ABA

signalling

However

there

are

many

more

examples

of

co-regulation

of

sugar

and

ABA

in

plants

The

expression

of

many

genes

is

co-regulated

by

sugar

and

ABA

A

study

of

Li

et

al.

-LRB-

-RRB-

showed

that

of

the

ABA

upregulated

genes

are

induced

in

response

to

glucose

as

well

in

total

nearly

genes

These

included

genes

in

stress

responses

carbohydrate

and

nitrogen

metabolism

and

signal

transduction

Another

group

of

nearly

genes

was

repressed

by

both

ABA

and

glucose

In

addition

the

authors

identified

a

group

of

genes

that

showed

a

synergistic

upregulation

when

treated

with

both

ABA

and

glucose

including

the

-LRB-

ADP

pyrophosphorylase

large

subunit

-RRB-

gene

which

is

involved

in

starch

biosynthesis

An

earlier

study

showed

that

ABA

was

not

able

to

induce

the

gene

but

that

ABA

in

combination

with

sugar

boosted

transcription

levels

in

comparison

to

sugar

treatment

alone

-LRB-

Rook

et

al.

-RRB-

Interestingly

Akihiro

et

al.

-LRB-

-RRB-

found

a

similar

ABA/sugar

interaction

for

the

regulation

of

expression

and

starch

biosynthesis

in

rice

-LRB-

Oryza

sativa

-RRB-

There

are

many

other

examples

of

processes

and

genes

that

are

regulated

by

sugar

and

ABA

E.g.

the

ABA

stress

and

ripening-induced

-LRB-

ASR

-RRB-

protein

in

grape

-LRB-

Vitis

vinifera

-RRB-

that

binds

to

the

sugar

response

boxes

in

the

promoter

of

the

monosaccharide

transporter

ASR

mRNA

expression

is

responsive

to

sugar

and

is

modulated

by

ABA

-LRB-

Cakir

et

al.

-RRB-

Both

sugar

and

ABA

signals

also

regulate

the

maize

-LRB-

Zea

mays

-RRB-

invertase

gene

-LRB-

Trouverie

et

al.

-RRB-

Thus

ABA/sugar

interactions

were

reported

for

a

multitude

of

processes

and

in

different

plant

species

indicating

that

these

interactions

are

physiologically

relevant

Therefore

a

thorough

understanding

of

these

sugar

and

ABA

interactions

is

of

importance

An

important

strategy

for

the

isolation

of

sugar

response

mutants

makes

use

of

the

effect

of

exogenous

supplied

sugars

on

early

seedling

development

in

Arabidopsis

Although

the

use

of

high

sugar

concentrations

-LRB-

mM

-RRB-

possibly

are

above

the

physiological

range

it

provided

an

efficient

screening

method

for

mutants

-LRB-

reviewed

by

Rognoni

et

al.

-RRB-

Such

elevated

sugar

concentrations

arrest

early

seedling

development

which

is

characterized

by

the

absence

of

cotyledon

greening

and

leaf

formation

The

difference

in

phenotype

between

arrested

and

non-arrested

seedlings

was

exploited

to

isolate

sugar

insensitive

mutants

that

showed

proper

seedling

development

on

high

glucose

-LRB-

gin

-RRB-

and

sucrose

-LRB-

sis

-RRB-

media

In

addition

to

the

aforementioned

role

of

ABA

biosynthesis

and

signalling

such

sugar

response

mutants

revealed

a

role

for

-LRB-

-RRB-

and

ethylene

signalling

in

the

GIN

response

pathway

-LRB-

Zhou

et

al.

Gibson

et

al.

Cheng

et

al.

Moore

et

al.

-RRB-

ABA

deficiency

caused

by

either

mutations

in

or

-LRB-

Koornneef

et

al.

-RRB-

resulted

in

a

clear

gin

phenotype

but

analysis

of

ABA

insensitive

mutants

resulted

in

more

ambiguous

phenotypes

In

addition

to

only

and

showed

a

sugar

insensitive

phenotype

while

for

and

no

or

only

a

weak

phenotype

was

reported

-LRB-

Arenas-Huertero

et

al.

Huijser

et

al.

Laby

et

al.

Brocard

et

al.

Brocard-Gifford

et

al.

-RRB-

Nevertheless

a

role

for

in

sugar

signalling

during

early

seedling

development

was

hinted

too

by

later

studies

E.g.

over

expression

lines

were

sugar

hypersensitive

-LRB-

Finkelstein

et

al.

Zeng

and

Kermode

-RRB-

and

mutants

were

insensitive

to

glucose

in

combination

with

ABA

-LRB-

Nambara

et

al.

-RRB-

This

insensitivity

appeared

to

be

allele

specific

and

the

assay

used

was

different

from

other

sugar

sensitivity

assays

However

in

a

more

recent

report

WT

glucose

sensitivity

during

early

seedling

development

for

the

mutant

was

observed

-LRB-

Yuan

and

Wysocka-Diller

-RRB-

Thus

it

is

unclear

how

and

to

what

extend

sugar

and

ABA

signalling

overlap

ABA

by

itself

blocked

post-germination

early

seedling

development

in

addition

to

the

role

of

ABA

in

the

glucose-induced

seedling

arrest

-LRB-

Lopez-Molina

et

al.

-RRB-

Similar

to

glucose-arrested

seedlings

ABA-arrested

seedlings

lacked

greening

and

leaf

formation

The

ABA-induced

seedling

arrest

is

dependent

on

and

activity

and

both

and

mutant

do

not

arrest

early

seedling

growth

in

response

to

ABA

-LRB-

Lopez-Molina

et

al.

-RRB-

This

ABA

sensitivity

is

confined

to

a

limited

time

window

after

the

start

of

germination

-LRB-

Lopez-Molina

et

al.

Lopez-Molina

et

al.

-RRB-

Adding

ABA

after

this

sensitive

phase

to

seedlings

no

longer

affects

vegetative

development

Similarly

the

glucose-induced

seedling

arrest

could

only

be

triggered

in

a

similar

limited

time

frame

after

sowing

-LRB-

Gibson

et

al.

-RRB-

Here

a

study

of

the

role

of

ABA

biosynthesis

and

ABA

signalling

in

sugar

response

pathways

is

presented

In

particular

this

study

was

focussed

on

the

glucose-induced

early

seedling

developmental

arrest

-LRB-

the

GIN

pathway

-RRB-

Analysis

of

transgenic

ABA

deficient

lines

with

ectopic

expression

-LRB-

-RRB-

suggested

that

additional

ABA-controlled

factors

are

necessary

to

arrest

early

seedling

development

in

response

to

glucose

expression

and

protein

stability

are

controlled

by

ABA

and

is

a

key

component

in

the

ABA

mediated

pathway

that

blocks

early

development

-LRB-

Lopez-Molina

et

al.

Zhang

et

al.

-RRB-

Therefore

we

investigated

the

role

of

in

the

GIN

pathway

encodes

a

domain

transcription

factor

that

contains

four

conserved

domains

one

acidic

activation

domain

-LRB-

-RRB-

and

three

basic

domains

-LRB-

and

Giraudat

et

al.

Finkelstein

et

al.

-RRB-

directs

seed

specific

gene

expression

and

is

required

for

induction

of

desiccation

tolerance

and

seed

dormancy

and

represses

genes

related

to

seed

germination

-LRB-

Ooms

et

al.

Hoecker

et

al.

Nambara

et

al.

Suzuki

et

al.

Finkelstein

et

al.

-RRB-

Other

functions

for

include

regulation

of

flowering

resource

allocation

lateral

root

growth

in

response

to

auxin

and

serve

as

a

signal

transduction

component

downstream

of

PHYB

signalling

in

seeds

-LRB-

Robinson

and

Hill

Suzuki

et

al.

Brady

et

al.

Mazzella

et

al.

-RRB-

Here

we

show

that

multiple

mutants

display

a

gin

phenotype

The

glucose

insensitive

phenotype

observed

for

mutants

was

comparable

to

that

of

other

known

gin

mutants

like

and

Moreover

mutants

showed

a

reduced

expression

level

of

glucose-regulated

genes

In

addition

physiological

and

molecular

experiments

led

to

the

conclusion

that

the

glucose-arrested

seedlings

mimic

ABA-arrested

seedlings

This

was

supported

by

the

observations

that

two

other

ABA

response

mutants

i.e.

and

showed

altered

glucose

responsiveness

as

well

The

identification

of

and

two

other

ABA

response

loci

as

components

in

GIN

signalling

suggests

that

glucose

and

ABA

signalling

share

more

components

than

originally

anticipated

Material

and

methods

Plant

material

Arabidopsis

plants

were

grown

in

a

climate

chamber

at

°C

with

humidity

and

a

h

light/dark

cycle

-LRB-

Sylvania

GRO-LUX

fluorescent

lamps

Technische

Unie

Utrecht

-RRB-

for

seed

production

WT

and

mutant

seeds

that

were

compared

in

all

experiments

presented

were

produced

together

and

stored

under

identical

conditions

Seeds

were

dry-stored

in

paper

bags

at

room

temperature

for

at

least

a

month

before

use

in

our

experiments

The

transgenic

lines

were

constructed

as

follows

cDNA

sequence

was

PCR

amplified

and

cloned

in

pDONR

vector

and

sequenced

Using

Gateway

cloning

was

cloned

behind

the

Cauliflower

Mosaic

Virus

promoter

in

the

vector

The

vector

containing

was

introduced

in

Agrobacterium

which

were

used

to

transform

plants

by

the

floral

dip

method

-LRB-

Clough

and

Bent

-RRB-

Germination

assays

All

germination

assays

were

performed

on

MS

half-strength

Murashige

and

Skoog

medium

-LRB-

pH

-RRB-

including

vitamins

solidified

with

plant

agar

-LRB-

Duchefa

Haarlem

The

Netherlands

-RRB-

Before

plating

seeds

were

surface-sterilized

in

-LRB-

v/v

-RRB-

commercial

bleach

-LRB-

Glorix

-RRB-

for

min

and

rinsed

four

to

five

times

with

sterile

water

After

a

three

days

stratification

period

at

°C

in

the

dark

plates

-LRB-

with

or

without

sugars

present

as

indicated

-RRB-

were

incubated

in

the

growth

chamber

-LRB-

°C

and

h

light/dark

cycle

-RRB-

To

investigate

the

effect

of

stratification

on

sugar-free

-LRB-

MS

-RRB-

media

seeds

were

transferred

to

sugar

media

after

this

stratification

period

Control

experiments

showed

that

transferring

of

seeds

did

not

affect

seed

germination

Germination

defined

as

radicle

emergence

from

the

seed

coat

was

scored

daily

for

days

Experiments

were

performed

in

duplo

each

plate

containing

seeds

and

every

experiment

was

repeated

one

to

three

times

Sugar

response

assays

For

gin

assay

experiments

seeds

were

sterilized

sown

on

MS

media

and

stratified

for

three

days

at

°C

in

the

dark

Hereafter

seeds

were

collected

and

sown

on

MS

media

supplemented

with

sucrose

-LRB-

control

-RRB-

or

on

control

media

with

the

indicated

amount

of

sugar

unless

otherwise

indicated

Different

seed

batches

show

slightly

different

responses

and

therefore

both

and

glucose

media

were

used

Plates

were

incubated

in

the

growth

chamber

-LRB-

°C

and

h

light/dark

cycle

-RRB-

for

up

to

two

weeks

Seedlings

were

scored

gin

when

green

cotyledons

emerged

To

determine

the

isi

phenotype

of

WT

Col

and

the

seeds

were

stratified

first

for

three

days

on

sugar-free

MS

media

After

stratification

the

seeds

were

sown

on

mM

sucrose

media

After

four

days

plant

material

was

harvested

and

expression

levels

determined

For

the

sun

phenotype

analysis

seeds

were

sterilized

sown

on

MS

and

stratified

for

three

days

at

°C

in

darkness

After

stratification

seeds

were

transferred

to

plates

containing

MS

or

MS

with

the

indicated

amount

of

sugar

Seeds

were

light-treated

for

at

least

one

hour

before

they

were

incubated

in

the

dark

-LRB-

wrapped

in

three

layers

of

aluminium

foil

-RRB-

at

°C

After

two

days

plant

material

was

harvested

for

PC

expression

analysis

by

qRT-PCR

Gene

expression

analysis

WT

and

seeds

were

stratified

for

three

days

and

germinated

on

MS

media

Seed

batches

were

germinated

to

approximately

-LRB-

t

-RRB-

before

the

start

of

the

different

treatments

At

this

stage

all

WT

seedlings

were

developmentally

arrested

in

response

to

elevated

glucose

concentrations

and

ABA

treatment

For

RNA

isolation

and

qRT-PCR

analysis

plant

material

was

ground

using

mortar

and

pestle

in

liquid

nitrogen

and

additionally

bead-beaten

in

a

mikro-dismembrator

S

-LRB-

B.

Braun

Biotech

International

Germany

-RRB-

RNA

was

isolated

according

to

Schuurmans

et

al.

-LRB-

-RRB-

Additionally

RNA

was

purified

using

RNeasy

columns

-LRB-

Qiagen

USA

Valencia

CA

-RRB-

RNA

samples

were

DNase

treated

checked

for

the

absence

of

DNA

by

PCR

and

used

for

cDNA

synthesis

and

qRT-PCR

analysis

as

described

before

by

van

Dijken

et

al.

-LRB-

-RRB-

For

expression

analysis

by

qRT-PCR

either

a

TAMRA

labelled

probe

-LRB-

with

TaqMan

Universal

PCR

Master

Mix

Applied

Biosystems

Foster

City

CA

-RRB-

or

SYBR

Green

technology

-LRB-

Power

SYBR

Green

PCR

Master

Mix

Applied

Biosystems

Foster

City

CA

-RRB-

was

used

Relative

quantitation

of

gene

expression

is

based

on

the

comparative

CT

method

-LRB-

User

Bulletin

No.

ABI

PRISM

Sequence

Detection

System

-RRB-

using

the

-LRB-

-RRB-

as

reference

gene

The

primers

were

obtained

for

the

CATMA

database

-LRB-

forward

primer

ID

reverse

primer

ID

-RRB-

The

sequences

of

primers

and

probes

that

were

used

for

gene

expression

analysis

are

listed

in

Table

The

qRT-PCRs

were

run

on

a

ABI-prism

Sequence

Detection

System

-LRB-

Applied

Biosystems

Foster

City

CA

-RRB-

Table

and

probes

used

for

gene

expression

analysisGeneForward

primerReverse

Results

Transgenic

ABA

deficient

lines

with

constitutive

expression

of

are

glucose

insensitive

Screens

for

sugar

insensitive

mutants

revealed

the

importance

of

ABA

and

ABA

signal

transduction

in

particular

-LRB-

Arenas-Huertero

et

al.

Huijser

et

al.

Laby

et

al.

Rook

et

al.

-RRB-

To

study

the

interaction

between

ABA

biosynthesis

and

in

sugar

signalling

we

generated

transgenic

lines

with

constitutive

expression

driven

by

a

CaMV

promoter

in

an

ABA

deficient

-LRB-

-RRB-

background

expression

in

these

transgenic

plants

is

independent

from

plant

sugar

status

Two

lines

homozygous

for

the

construct

were

selected

i.e.

lines

and

Both

lines

germinated

normally

but

showed

a

stunted

growth

phenotype

-LRB-

Fig.

-RRB-

similar

to

what

has

been

reported

previously

for

severe

overexpression

lines

-LRB-

Söderman

et

al.

-RRB-

Expression

analysis

on

control

media

showed

that

expression

is

high

in

and

and

exceeded

glucose-induced

expression

in

developmentally

arrested

WT

Col

seedlings

by

approximately

fold

-LRB-

Fig.

-RRB-

Fig.

of

transgenic

ABA

deficient

lines

with

constitutive

expression

Plant

phenotypes

of

WT

Col

and

two

transgenic

lines

with

constitutive

expression

in

-LRB-

and

-RRB-

after

weeks

of

growth

on

soil

-LRB-

a

-RRB-

expression

was

detected

by

qRT-PCR

in

Col

and

seedlings

grown

for

two

weeks

on

control

or

glucose

media

Values

are

averages

of

two

experiments

SD

-LRB-

b

-RRB-

Seedling

phenotypes

after

two

weeks

of

growth

on

control

sorbitol

-LRB-

sorb

-RRB-

glucose

-LRB-

glc

-RRB-

or

glucose

μM

ABA

-LRB-

glc

ABA

-RRB-

Similar

results

were

obtained

in

three

experiments

-LRB-

c

-RRB-

expression

was

detected

by

qRT-PCR

in

Col

and

seedlings

grown

for

four

days

on

control

half-strength

MS

-LRB-

con

-RRB-

or

half-strength

MS

mM

sucrose

-LRB-

suc

-RRB-

media

Values

are

averages

of

three

experiments

SD

-LRB-

d

-RRB-

The

gin

phenotype

of

Col

and

was

tested

by

growing

the

lines

for

two

weeks

on

control

sorbitol

and

glucose

media

All

lines

showed

greening

on

control

-LRB-

con

-RRB-

and

sorbitol

-LRB-

sorb

osmotic

control

-RRB-

media

as

expected

-LRB-

Fig.

-RRB-

On

glucose

the

Col

seedlings

showed

an

arrested

phenotype

whereas

showed

a

gin

phenotype

as

reported

before

-LRB-

Fig.

León

and

Sheen

-RRB-

Also

expression

levels

in

glucose-arrested

Col

seedlings

were

enhanced

whereas

in

expression

was

very

low

The

and

lines

have

very

high

levels

but

importantly

display

a

gin

phenotype

-LRB-

Fig.

-RRB-

The

gin

phenotype

of

and

the

transgenic

lines

could

be

reverted

to

WT

by

the

addition

of

nM

ABA

as

was

reported

before

for

aba

mutants

-LRB-

Fig.

Arenas-Huertero

et

al.

-RRB-

Thus

in

an

ABA

deficient

background

overexpression

does

not

restore

wild

type

sensitivity

to

glucose

Sucrose

feeding

induces

gene

expression

in

Arabidopsis

seedlings

This

sugar

regulation

of

the

promoter

has

been

used

as

basis

for

a

screen

for

sugar

response

mutants

Rook

et

al.

-LRB-

-RRB-

reported

on

isi

sugar

response

mutants

that

were

unable

to

fully

activate

expression

in

response

to

sucrose

The

identification

of

and

as

mutants

with

an

impaired

sucrose

induction

phenotype

revealed

a

role

for

ABA

biosynthesis

and

signalling

in

control

of

expression

-LRB-

Rook

et

al.

-RRB-

To

study

the

sugar-induced

expression

in

WT

Col

and

stratified

seeds

were

sown

on

mM

sucrose

media

After

four

days

plant

material

was

harvested

and

expression

levels

determined

Sucrose

feeding

resulted

in

an

approximate

six

fold

induction

of

in

Col

seedlings

-LRB-

Fig.

-RRB-

The

induction

of

is

lower

in

the

seedlings

which

is

in

agreement

with

the

isi

phenotype

of

-LRB-

Rook

et

al.

-RRB-

In

the

and

lines

the

expression

level

on

sugar-free

control

media

already

exceeds

that

of

sugar-treated

WT

seedlings

Sugar

feeding

of

and

seedlings

further

boosted

expression

fold

higher

than

sugar-treated

WT

seedlings

-LRB-

Fig.

-RRB-

Glucose

and

ABA

trigger

a

similar

developmental

early

seedling

arrest

High

sugar

concentrations

arrest

early

seedling

development

in

Arabidopsis

After

germination

vegetative

seedling

growth

is

blocked

characterized

by

the

absence

of

cotyledon

greening

and

leaf

formation

Equimolar

concentrations

of

an

osmotic

control

did

not

block

development

in

this

early

seedling

stage

-LRB-

Fig.

Zhou

et

al.

Laby

et

al.

Rognoni

et

al.

-RRB-

Previously

Lopez-Molina

et

al.

-LRB-

-RRB-

showed

that

treatment

with

ABA

arrested

this

post-germination

switch

from

embryonic

to

vegetative

growth

as

well

ABA-arrested

seedlings

showed

neither

greening

nor

leaf

formation

similar

to

glucose-arrested

seedlings

-LRB-

Fig.

-RRB-

In

comparison

to

ABA-arrested

seedlings

glucose-arrested

seedlings

were

larger

due

to

the

longer

hypocotyl

and

larger

cotyledons

-LRB-

Fig.

-RRB-

Moreover

significant

root

growth

was

observed

in

glucose-arrested

seedlings

which

is

absent

in

ABA-arrested

seedlings

-LRB-

Fig.

b

-RRB-

Further

in

glucose-arrested

seedlings

often

a

pink

colouration

was

observed

indicative

for

anthocyanin

accumulation

that

was

absent

in

ABA-arrested

seedlings

-LRB-

Fig.

b

-RRB-

Fig.

and

ABA

signalling

arrest

early

seedling

development

and

induce

a

drought

tolerant

phenotype

seedling

phenotypes

are

shown

after

growth

for

twelve

days

on

control

-LRB-

con

half-strength

MS

sucrose

-RRB-

or

on

con

medium

supplemented

with

either

sorbitol

-LRB-

sorb

-RRB-

glucose

-LRB-

glc

-RRB-

or

μM

ABA

-LRB-

ABA

-RRB-

-LRB-

a

-RRB-

A

close-up

image

of

a

glc-arrested

seedling

-LRB-

left

-RRB-

and

an

ABA-arrested

seedling

-LRB-

right

-RRB-

is

shown

-LRB-

b

-RRB-

Developmentally

arrested

seedlings

have

a

drought

tolerant

phenotype

Seeds

were

grown

on

control

-LRB-

con

-RRB-

or

on

con

media

containing

either

μM

ABA

-LRB-

ABA

-RRB-

or

glucose

-LRB-

glc

-RRB-

for

nine

days

At

day

nine

the

seedlings

were

subjected

to

a

drought

treatment

as

indicated

-LRB-

c

-RRB-

Hereafter

seedlings

were

allowed

to

recover

on

control

media

for

five

days

after

which

survival

was

scored

The

survival

percentage

following

drought

treatment

is

presented

-LRB-

c

-RRB-

ABA-arrested

seedlings

possess

an

enhanced

resistance

to

drought

stress

compared

to

non-arrested

seedlings

-LRB-

Lopez-Molina

et

al.

-RRB-

A

comparison

was

made

between

the

drought

resistance

phenotype

of

glucose

and

ABA-arrested

seedlings

Control

-LRB-

non-arrested

-RRB-

seedlings

did

not

survive

a

drought

treatment

of

h

-LRB-

Fig.

-RRB-

ABA-treated

seedlings

showed

a

near

survival

rate

after

h

of

drought

the

longest

treatment

tested

in

our

experiments

Glucose-arrested

seedlings

showed

an

intermediate

drought

resistance

After

h

of

drought

stress

glucose-treated

seedlings

showed

a

nearly

survival

rate

and

after

h

of

drought

the

survival

rate

was

compared

to

survival

observed

for

control

seedlings

Thus

a

clear

drought

resistant

phenotype

was

observed

for

glucose-arrested

seedlings

The

ABA-induced

early

seedling

developmental

arrest

depends

on

functional

and

genes

and

mutants

did

not

block

early

seedling

development

in

response

to

ABA

Moreover

WT

seedlings

exposed

to

ABA

showed

strongly

enhanced

and

protein

levels

-LRB-

Lopez-Molina

et

al.

-RRB-

Both

genes

encode

transcriptional

regulators

important

for

seed

development

and

LEA

gene

expression

-LRB-

Koornneef

and

Karssen

Parcy

et

al.

Finkelstein

and

Lynch

Carles

et

al.

-RRB-

Lopez-Molina

et

al.

-LRB-

-RRB-

showed

that

during

the

early

seedling

developmental

arrest

several

LEA

genes

were

re-induced

as

well

This

re-induction

of

seed

transcriptional

regulators

and

LEA

gene

expression

suggested

that

a

late

embryogenesis

program

is

re-initiated

by

ABA

treatment

inducing

a

drought

tolerant

phenotype

in

such

arrested

seedlings

-LRB-

Lopez-Molina

et

al.

-RRB-

It

was

investigated

whether

the

same

transcriptional

regulators

and

LEA

genes

were

expressed

by

glucose

signalling

during

early

seedling

development

A

role

for

has

been

proposed

in

GIN

signalling

and

expression

is

sugar

responsive

-LRB-

Arenas-Huertero

et

al.

Laby

et

al.

Brocard

et

al.

Arroyo

et

al.

-RRB-

expression

thus

is

a

good

positive

control

in

this

experiment

Gene

expression

upon

glucose

feeding

was

studied

by

germinating

seeds

on

control

medium

until

the

batch

reached

germination

-LRB-

time

point

zero

t

-RRB-

At

this

time

point

a

sample

was

collected

for

RNA

isolation

while

the

rest

of

the

seeds

were

transferred

to

control

sorbitol

glucose

or

ABA

containing

media

for

an

additional

h

After

h

samples

were

collected

for

gene

expression

analysis

Using

quantitative

RT-PCR

-LRB-

qRT-PCR

-RRB-

the

expression

of

and

was

studied

in

response

to

the

different

treatments

Gene

expression

was

studied

in

three

independent

experiments

In

each

experiment

the

gene

expression

level

at

t

was

set

to

one

-LRB-

Table

-RRB-

Some

variation

in

gene

expression

levels

between

the

three

experiments

was

observed

likely

due

to

the

fact

that

for

each

experiment

independently

generated

seed

batches

were

used

Generally

continued

growth

on

control

media

after

t

resulted

in

a

decreased

expression

of

all

five

genes

-LRB-

Table

-RRB-

In

contrast

treatments

with

sorbitol

glucose

and

ABA

enhanced

the

expression

of

all

genes

studied

except

for

the

sorbitol

treatment

in

the

second

experiment

In

this

particular

experiment

gene

expression

decreased

on

sorbitol

however

this

decrease

was

less

pronounced

compared

to

the

control

treatment

ABA

treatment

resulted

in

the

re-induction

of

and

the

LEA

genes

which

is

in

line

with

results

reported

by

Lopez-Molina

et

al.

-LRB-

-RRB-

Also

glucose

exposure

significantly

induced

the

expression

levels

of

all

five

genes

although

the

expression

levels

are

in

general

somewhat

lower

than

in

response

to

ABA

Thus

is

upregulated

by

glucose

as

well

which

indicated

a

possible

role

for

in

glucose

responsiveness

The

observation

that

the

expression

levels

of

the

genes

studied

are

in

general

lower

after

sorbitol

treatment

compared

to

glucose

treatment

indicates

that

the

glucose-induced

expression

is

not

an

osmotic

response

Table

expression

levels

of

and

after

glucose

treatmentGeneTreatmentExp

relative

expression

levels

of

five

genes

were

determined

using

quantitative

RT-PCR

The

gene

expression

levels

were

measured

at

t

and

after

a

h

treatment

on

control

media

-LRB-

con

half-strength

MS

sucrose

-RRB-

and

on

con

media

supplemented

with

either

sorbitol

-LRB-

sorb

-RRB-

glucose

-LRB-

glc

-RRB-

or

μM

ABA

-LRB-

ABA

-RRB-

media

The

expression

level

at

t

was

set

to

The

experiment

was

performed

three

times

and

the

results

for

each

experiment

are

indicated

Expression

studies

using

seedlings

grown

for

five

days

on

control

sorbitol

-LRB-

-RRB-

or

glucose

-LRB-

-RRB-

media

confirmed

the

induction

of

these

genes

-LRB-

Fig.

-RRB-

In

five

days

old

glucose-arrested

seedlings

enhanced

expression

levels

were

detected

for

and

as

well

as

the

LEA

genes

and

The

induction

of

these

genes

by

sorbitol

was

roughly

five

fold

lower

-LRB-

Fig.

-RRB-

After

h

of

growth

on

ABA-free

media

seedlings

lose

the

capacity

to

re-induce

and

LEA

expression

and

to

arrest

early

seedling

development

in

response

to

ABA

-LRB-

Lopez-Molina

et

al.

-RRB-

To

study

whether

the

induction

by

glucose

is

restricted

to

a

similar

time

frame

seeds

were

germinated

and

grown

for

three

days

on

half-strength

MS

and

subsequently

switched

for

two

additional

days

to

elevated

sorbitol

and

glucose

media

The

re-induction

by

glucose

of

and

was

largely

lost

-LRB-

Fig.

glc

h

-RRB-

The

weaker

induction

by

sorbitol

is

essentially

lost

after

three

days

-LRB-

Fig.

sorb

h

-RRB-

Thus

the

glucose-response

that

induced

and

LEA

expression

is

confined

to

a

similar

time

frame

as

observed

for

ABA

This

is

in

agreement

with

the

finding

that

glucose

regulation

of

and

expression

was

dependent

on

developmental

stage

-LRB-

Arroyo

et

al.

-RRB-

and

that

sugar

arrests

early

seedling

development

only

in

a

small

day

time

frame

after

sowing

-LRB-

Gibson

et

al.

-RRB-

Fig.

glucose-regulated

expression

of

and

LEA

genes

is

restricted

to

a

limited

time

frame

after

the

start

of

germination

The

expression

of

and

genes

was

quantified

by

qRT-PCR

after

germination

and

growth

for

five

days

on

different

media

seeds

were

plated

for

five

days

on

control

media

-LRB-

con

half-strength

MS

sucrose

-RRB-

or

on

con

medium

supplemented

with

either

sorbitol

-LRB-

sorb

-RRB-

or

glucose

-LRB-

glc

-RRB-

The

values

are

an

average

of

three

experiments

and

in

each

experiment

the

expression

on

glc

was

set

to

one

It

was

investigated

whether

this

gene

expression

regulation

is

limited

to

a

small

time

window

after

the

start

of

germination

as

has

been

shown

for

ABA

regulation

-LRB-

Lopez-Molina

et

al.

-RRB-

Therefore

seeds

were

germinated

on

con

media

for

three

days

and

thereafter

switched

to

either

sorb

-LRB-

sorb

h

-RRB-

or

glc

media

-LRB-

glc

h

-RRB-

In

conclusion

the

glucose-induced

block

of

early

seedling

development

mimics

the

ABA-induced

arrest

of

vegetative

development

in

young

seedlings

Both

glucose

and

ABA-arrested

seedlings

have

a

drought

resistant

phenotype

and

show

enhanced

expression

levels

of

seed

expressed

genes

The

observation

that

overexpression

is

unable

to

restore

WT

glucose

sensitivity

in

the

mutant

shows

that

WT

ABA

biosynthesis

is

essential

for

the

GIN

response

Apparently

next

to

additional

factors

which

are

under

control

of

ABA

are

important

for

GIN

signalling

Two

candidates

are

and

Both

play

a

role

in

the

ABA-induced

early

seedling

developmental

arrest

and

gene

expression

and

protein

stability

of

both

transcription

factors

is

affected

by

ABA

-LRB-

Lopez-Molina

et

al.

Lopez-Molina

et

al.

Lopez-Molina

et

al.

Zhang

et

al.

this

study

-RRB-

A

role

for

in

glucose

signalling

has

been

established

has

a

gin

phenotype

-LRB-

although

somewhat

weaker

compared

to

-RRB-

and

expression

is

regulated

by

sugar

-LRB-

Arenas-Huertero

et

al.

Laby

et

al.

Brocard

et

al.

Arroyo

et

al.

this

study

see

Table

-RRB-

However

a

role

for

is

controversial

-LRB-

for

review

Finkelstein

and

Gibson

Leon

and

Sheen

Gibson

Rognoni

et

al.

-RRB-

Since

is

important

for

the

ABA-induced

developmental

arrest

and

is

induced

by

glucose

our

hypothesis

is

that

is

involved

in

GIN

signalling

as

well

Therefore

the

gin

phenotype

and

glucose-regulated

gene

expression

were

studied

in

mutants

mutants

are

glucose

insensitive

The

glucose

insensitive

phenotype

of

WT

and

the

mutant

was

investigated

by

plating

seeds

of

both

genotypes

on

media

with

elevated

glucose

concentrations

-LRB-

glucose

-RRB-

Initially

two

mutant

alleles

were

tested

i.e.

the

weak

allele

-LRB-

producing

normal

drought

tolerant

seeds

-RRB-

and

the

strong

allele

-LRB-

producing

green

drought

intolerant

seeds

Ooms

et

al.

-RRB-

As

expected

WT

early

seedling

development

was

arrested

in

response

to

high

glucose

concentrations

In

contrast

approximately

-LRB-

differed

between

experiments

-RRB-

of

both

and

seedlings

showed

greening

on

high

glucose

media

suggesting

that

is

a

gin

mutant

-LRB-

Fig.

-RRB-

All

genotypes

showed

greening

on

control

and

osmotic

control

media

-LRB-

Fig.

control

and

sorbitol

respectively

-RRB-

The

gin

phenotype

was

observed

for

mutants

in

several

experiments

using

independently

grown

and

harvested

seed

batches

-LRB-

data

not

shown

-RRB-

Next

the

strength

of

the

gin

phenotype

of

was

assessed

in

comparison

to

that

of

the

ABA

deficient

mutant

which

is

in

the

background

as

well

The

mutant

like

other

ABA

deficient

mutants

tested

was

shown

to

possess

a

gin

phenotype

-LRB-

Arenas-Huertero

et

al.

Huijser

et

al.

Laby

et

al.

-RRB-

A

similar

percentage

of

seedling

greening

was

observed

for

and

when

grown

on

glucose

media

-LRB-

Fig.

-RRB-

suggesting

that

both

mutants

possess

a

comparable

gin

phenotype

Fig.

mutants

have

a

gin

phenotype

Representative

and

seedlings

are

shown

that

were

grown

for

days

on

control

media

-LRB-

half-strength

MS

sucrose

-RRB-

or

on

control

medium

supplemented

with

either

sorbitol

or

glucose

-LRB-

a

-RRB-

The

gin

phenotype

of

was

compared

to

that

of

the

ABA

deficient

mutant

Seedlings

were

grown

for

days

on

the

indicated

media

before

seed

germination

and

seedling

greening

were

scored

-LRB-

b

-RRB-

Six

additional

mutant

alleles

-LRB-

and

-RRB-

were

obtained

-LRB-

a

kind

gift

of

Dr.

E.

Nambara

-RRB-

and

tested

for

their

gin

phenotype

These

mutants

were

tested

along

with

two

known

gin

mutants

and

in

the

gin

assay

All

these

mutants

were

retrieved

from

a

screen

for

mutants

insensitive

to

the

unnatural

-LRB-

-RRB-

ABA

-LRB-

Nambara

et

al.

-RRB-

The

ABA

insensitivity

of

the

mutants

was

tested

by

plating

the

mutants

on

half-strength

MS

media

containing

sucrose

μM

ABA

The

ABA

insensitivity

observed

was

in

agreement

with

that

reported

by

Nambara

et

al.

-LRB-

-RRB-

-LRB-

see

Table

-RRB-

The

and

mutants

had

a

strong

ABA

insensitivity

the

and

showed

a

weaker

phenotype

We

did

observe

greening

for

on

ABA

media

while

originally

no

abi

phenotype

was

observed

for

this

allele

-LRB-

Nambara

et

al.

-RRB-

These

mutants

were

isolated

on

-LRB-

-RRB-

ABA

and

the

-LRB-

-RRB-

ABA

insensitivity

does

not

necessarily

parallel

-LRB-

-RRB-

ABA

insensitivity

-LRB-

Nambara

et

al.

-RRB-

Next

these

mutants

were

plated

on

control

sorbitol

and

glucose-containing

media

respectively

On

glucose

media

strong

gin

phenotypes

were

observed

for

and

mutants

as

shown

by

the

high

percentages

of

greening

in

agreement

with

earlier

reports

-LRB-

Arenas-Huertero

et

al.

Huijser

et

al.

Laby

et

al.

-RRB-

Again

a

strong

gin

phenotype

was

observed

for

in

agreement

with

our

observation

using

and

mutants

Five

out

of

the

six

additional

mutants

tested

show

a

gin

phenotype

For

four

mutants

the

gin

phenotype

was

comparable

to

that

of

and

-LRB-

Table

-RRB-

The

strong

ABA

insensitive

mutants

are

strong

gin

mutants

as

well

whereas

the

mutants

with

a

weaker

phenotype

-LRB-

and

-RRB-

show

a

weaker

phenotype

on

glucose

media

However

exceptionally

shows

a

weak

abi

phenotype

but

a

strong

gin

phenotype

The

mutant

lacks

an

ABI

insensitive

phenotype

and

did

not

show

a

gin

phenotype

either

In

conclusion

gin

phenotype

analysis

of

eight

alleles

confirms

the

glucose

insensitive

phenotype

for

Moreover

the

gin

phenotype

of

is

comparable

to

that

of

other

known

gin

mutants

like

and

Table

insensitive

phenotype

of

six

μM

μM

ABAaWT

of

WT

Col

and

six

mutants

were

stratified

on

half-strength

MS

and

after

stratification

switched

to

control

medium

-LRB-

con

MS

sucrose

-RRB-

or

con

medium

containing

sorbitol

glucose

or

μM

ABA

Values

are

the

percentage

of

cotyledon

greening

scored

after

days

The

experiment

was

repeated

once

with

similar

resultsaThe

ABA

insensitivity

scored

for

the

different

mutant

alleles

based

on

cotyledon

greening

by

Nambara

et

al.

-LRB-

-RRB-

Glucose-induced

expression

of

and

is

reduced

in

Glucose-induced

early

seedling

developmental

arrest

is

associated

with

expression

of

and

a

subset

of

LEA

genes

-LRB-

Arenas-Huertero

et

al.

Cheng

et

al.

Arroyo

et

al.

this

study

-RRB-

The

involvement

of

in

glucose-regulated

gene

expression

was

tested

for

three

genes

and

as

a

representative

of

the

LEA

genes

For

expression

analysis

the

weak

and

the

strong

mutants

were

used

Seedlings

of

all

three

genotypes

were

grown

for

h

on

control

media

or

on

control

media

containing

glucose

Both

and

expression

were

sensitive

to

glucose

addition

In

WT

seedlings

glucose

treatment

induced

these

two

genes

on

average

times

in

comparison

to

t

-LRB-

Fig.

c

-RRB-

However

on

glucose

the

expression

levels

of

both

genes

were

much

reduced

in

and

compared

to

WT

-LRB-

Fig.

c

-RRB-

Glucose

treatment

did

not

result

in

a

clear

induction

of

especially

when

compared

to

and

Instead

expression

was

roughly

maintained

to

the

same

level

observed

for

t

but

was

enhanced

when

compared

to

the

control

treatment

which

displayed

a

strong

reduction

of

expression

-LRB-

Fig.

-RRB-

transcripts

were

fold

lower

in

the

mutant

backgrounds

upon

glucose

treatment

Thus

the

mutation

did

not

allow

full

glucose-induced

expression

of

and

Interestingly

the

reduced

expression

is

most

pronounced

in

the

strong

mutant

background

Fig.

glucose-regulated

gene

expression

in

mutants

Glucose-regulated

gene

expression

was

studied

in

WT

and

and

mutants

Gene

expression

was

quantified

by

qRT-PCR

at

t

the

time

of

glucose

application

and

after

h

on

control

medium

-LRB-

con

half-strength

MS

sucrose

-RRB-

or

con

medium

supplemented

with

glucose

At

the

t

time

point

approximately

of

the

seeds

had

germinated

The

values

indicate

an

average

of

three

independent

experiments

-LRB-

two

experiments

for

-RRB-

The

WT

expression

on

glucose

was

set

to

one

in

each

experiment

The

glucose-regulated

expression

in

and

the

two

mutants

was

studied

for

-LRB-

a

-RRB-

-LRB-

b

-RRB-

and

-LRB-

c

-RRB-

The

gene

expression

ratio

of

over

was

calculated

because

all

three

genes

were

already

under

expressed

in

and

at

t

This

ratio

was

calculated

by

dividing

the

average

gene

expression

in

by

the

expression

in

-LRB-

for

both

mutants

-RRB-

at

t

and

after

glucose

treatment

for

all

three

genes

-LRB-

d

-RRB-

Already

at

t

reduced

expression

levels

for

all

three

genes

were

observed

in

the

mutant

backgrounds

This

was

expected

since

the

ABI

transcription

factors

were

found

to

affect

each

others

expression

in

seeds

-LRB-

Söderman

et

al.

-RRB-

Moreover

is

an

important

regulator

of

LEA

gene

expression

-LRB-

Parcy

et

al.

Kermode

-RRB-

To

rule

out

that

this

reduced

expression

is

responsible

for

the

phenotype

observed

after

glucose

treatment

the

gene

expression

ratios

of

WT

over

mutant

were

determined

for

t

and

after

glucose

feeding

For

each

gene

the

expression

level

of

WT

is

divided

by

the

expression

level

of

each

mutant

These

values

show

the

fold

difference

of

expression

between

WT

and

both

mutants

-LRB-

Fig.

-RRB-

E.g.

the

level

of

expression

is

fold

higher

in

WT

compared

to

at

t

After

glucose

treatment

the

fold

difference

for

expression

was

Except

for

the

expression

ratio

of

over

all

WT/mutant

expression

ratios

showed

increased

values

after

glucose

exposure

This

confirms

that

both

mutants

have

a

reduced

ability

to

control

glucose-regulated

gene

expression

Other

sugar

insensitive

phenotypes

of

The

sugar

response

phenotype

of

the

mutant

was

investigated

in

the

sis

sun

and

glucose-induced

delay

of

germination

assays

High

sucrose

concentrations

block

early

seedling

development

similar

to

high

glucose

concentrations

High

sucrose

resistant

mutants

are

known

as

sis

mutants

-LRB-

Laby

et

al.

-RRB-

Several

mutants

affected

in

the

same

genes

were

retrieved

from

gin

and

sis

screens

e.g.

and

The

sis

phenotype

of

WT

and

was

analysed

by

plating

these

genotypes

on

sucrose

media

Both

mutants

show

a

sucrose

insensitive

phenotype

-LRB-

Fig.

b

-RRB-

and

can

thus

be

considered

as

sis

as

well

as

gin

mutants

Fig.

mutant

shows

additional

sugar

signalling

defects

The

sugar

response

phenotype

of

was

assessed

in

three

additional

assays

The

sugar

insensitive

-LRB-

sis

-RRB-

phenotype

was

assessed

by

scoring

the

early

seedling

developmental

phenotype

-LRB-

by

seedling

greening

-RRB-

after

growth

on

elevated

sucrose

concentration

Representative

and

seedling

phenotypes

are

shown

after

growth

on

sucrose

media

for

days

-LRB-

a

-RRB-

The

percentage

of

germination

and

seedling

greening

of

and

was

determined

after

growth

for

days

on

control

-LRB-

half-strength

MS

sucrose

-RRB-

or

on

control

medium

supplemented

with

either

sorbitol

or

sucrose

media

-LRB-

b

-RRB-

The

sucrose

uncoupled

-LRB-

sun

-RRB-

phenotype

was

assessed

by

measuring

plastocyanin

-LRB-

PC

-RRB-

expression

levels

by

qRT-PCR

in

two

days

old

seedlings

grown

on

sucrose

containing

media

in

darkness

Sucrose

repressed

the

PC

transcript

level

in

dark-grown

two

days

old

WT

seedlings

in

comparison

to

WT

seedlings

grown

on

sugar-free

media

however

in

sun

mutants

PC

repression

by

sucrose

is

much

less

pronounced

-LRB-

Dijkwel

et

al.

-RRB-

Indicated

are

the

relative

PC

expression

on

sucrose

containing

media

for

WT

PC-LUC

and

-LRB-

assayed

on

sucrose

-RRB-

and

and

-LRB-

assayed

on

sucrose

-RRB-

Values

indicate

an

average

of

at

least

three

experiments

PC

expression

of

two

days

old

seedlings

grown

on

sugar-free

half-strength

MS

was

set

to

for

each

genotype

in

every

experiment

-LRB-

c

-RRB-

Germination

is

delayed

by

glucose

addition

was

tested

for

glucose-induced

delay

of

germination

by

plating

seeds

on

half-strength

MS

-LRB-

MS

-RRB-

MS

sorbitol

-LRB-

sorb

-RRB-

and

MS

glucose

-LRB-

glc

-RRB-

Sugars

were

already

present

during

stratification

-LRB-

solid

lines

-RRB-

In

addition

seeds

were

stratified

on

sugar-free

MS

media

and

directly

after

stratification

transferred

to

MS

sorbitol

-LRB-

MS

sorb

-RRB-

and

MS

glucose

-LRB-

MS

glc

dashed

lines

-RRB-

Seed

germination

after

the

different

treatments

was

scored

daily

for

eight

days

for

-LRB-

d

-RRB-

and

-LRB-

e

-RRB-

Dijkwel

et

al.

-LRB-

-RRB-

previously

reported

on

sugar

insensitive

mutants

that

have

been

isolated

using

the

sun

screen

Dark-grown

seedlings

transiently

express

photosynthesis

genes

including

plastocyanin

-LRB-

PC

Dijkwel

et

al.

-RRB-

This

transient

increase

of

photosynthesis

gene

expression

is

repressed

on

sucrose

media

In

sun

mutants

sucrose

did

not

repress

this

transient

PC

gene

expression

in

dark

grown

seedlings

-LRB-

Dijkwel

et

al.

-RRB-

is

such

a

mutant

which

was

shown

to

be

allelic

to

-LRB-

Huijser

et

al.

-RRB-

A

possible

sun

phenotype

of

was

investigated

by

quantifying

PC

mRNA

levels

in

two

days

old

dark-grown

seedlings

using

qRT-PCR

Elevated

PC

expression

was

observed

in

sucrose-treated

seedlings

of

compared

to

its

WT

PC-LUC

parent

confirming

its

sun

phenotype

-LRB-

Fig.

-RRB-

Interestingly

the

mutant

showed

enhanced

PC

expression

as

well

and

therefore

has

a

sun

phenotype

-LRB-

Fig.

-RRB-

mutants

displayed

a

nine

fold

increased

PC

expression

level

in

sugar-treated

seedlings

compared

to

WT

In

the

mutants

the

PC

expression

level

is

only

two

fold

increased

indicating

that

is

a

weak

sun

mutant

Sugars

delay

seed

germination

in

Arabidopsis

ABA

plays

a

role

in

this

glucose-induced

delay

of

germination

-LRB-

Ullah

et

al.

Price

et

al.

Dekkers

et

al.

Chen

et

al.

-RRB-

Despite

the

involvement

of

ABA

several

abi

mutants

-LRB-

and

-RRB-

are

normally

sensitive

to

glucose

during

germination

-LRB-

Price

et

al.

Dekkers

et

al.

-RRB-

Sugar

insensitive

mutants

like

and

that

are

insensitive

to

the

glucose-induced

early

seedling

developmental

arrest

are

sensitive

to

glucose

during

germination

-LRB-

defined

as

radicle

emergence

-RRB-

This

indicates

that

the

glucose

response

during

germination

-LRB-

which

delays

radicle

protrusion

-RRB-

and

early

seedling

growth

-LRB-

which

arrests

seedling

development

-RRB-

are

two

distinct

processes

-LRB-

Price

et

al.

Dekkers

et

al.

-RRB-

The

and

alleles

were

plated

on

glucose

The

presence

of

glucose

clearly

delayed

germination

of

WT

seeds

The

osmotic

control

of

sorbitol

only

slightly

affected

germination

compared

to

control

media

Both

mutants

showed

a

clear

resistance

against

glucose

as

displayed

by

their

reduced

germination

delay

-LRB-

data

not

shown

-RRB-

which

is

in

agreement

with

observations

of

Yuan

and

Wysocka-Diller

-LRB-

-RRB-

who

reported

on

the

glucose

insensitivity

of

during

germination

In

our

experiments

seeds

were

sterilized

plated

on

sugar

media

and

after

a

three

day

stratification

period

moved

to

the

growth

chamber

and

germination

was

scored

the

following

days

Interestingly

when

WT

seeds

were

stratified

on

sugar-free

media

-LRB-

half-strength

MS

-RRB-

and

after

stratification

transferred

to

sugar-containing

plates

the

delay

of

germination

by

glucose

was

completely

lost

-LRB-

data

not

shown

-RRB-

Thus

stratification

on

sugar-free

media

suppressed

the

inhibitory

effect

of

glucose

on

germination

This

effect

of

stratification

was

further

investigated

using

higher

-LRB-

-RRB-

glucose

and

sorbitol

concentrations

and

seeds

were

plated

either

on

control

sorbitol

or

glucose

media

stratified

for

three

days

and

moved

to

the

growth

chamber

Alternatively

seeds

were

stratified

on

sugar-free

control

media

and

shifted

to

media

containing

sorbitol

or

glucose

and

then

placed

in

the

growth

chamber

Continuous

treatment

with

glucose

severely

delayed

germination

of

WT

seeds

but

affected

significantly

less

as

expected

-LRB-

Fig.

e

-RRB-

Also

in

this

experiment

stratification

on

sugar-free

media

strongly

suppressed

the

germination

response

to

glucose

in

WT

Even

germination

of

the

mutant

which

already

showed

a

reduced

sensitivity

to

glucose

during

germination

could

be

improved

by

stratification

on

sugar-free

media

The

germination

speed

of

the

mutant

after

stratification

on

sugar-free

media

was

similar

to

the

osmotic

control

indicating

that

this

combination

completely

suppressed

the

germination

delay

by

glucose

The

glucose

response

during

germination

was

affected

by

stratification

but

not

the

osmotic

response

-LRB-

sorbitol

treatment

-RRB-

This

was

observed

for

WT

as

well

as

the

mutant

thereby

differentiating

glucose

from

osmotic

signalling

and

mutant

are

glucose

response

mutants

Our

results

on

the

physiological

and

molecular

level

indicate

that

glucose-arrested

seedlings

mimic

ABA-arrested

seedlings

The

finding

that

is

important

for

glucose

signalling

in

addition

to

and

led

us

to

test

the

glucose

insensitive

phenotype

of

two

other

ABA

signalling

mutants

i.e.

and

Brady

et

al.

-LRB-

-RRB-

proposed

a

genetic

pathway

for

and

the

genes

in

seed

ABA

responsiveness

based

on

double

mutant

analysis

between

the

mutant

and

the

abi

mutants

This

analysis

suggested

that

and

act

at

or

upstream

of

while

acts

at

or

upstream

of

and

-LRB-

Brady

et

al.

-RRB-

encodes

a

phosphatase

protein

that

acts

as

a

negative

regulator

of

ABA

signalling

and

the

mutation

results

in

an

ABA

insensitive

phenotype

-LRB-

Koornneef

et

al.

Leung

et

al.

-RRB-

WT

and

seeds

were

plated

along

with

on

control

sorbitol

and

glucose

media

In

addition

to

the

mutant

displayed

a

gin

phenotype

as

well

although

its

phenotype

is

not

as

strong

as

that

of

-LRB-

Fig.

-RRB-

On

control

and

sorbitol

media

all

seedlings

showed

greening

Fig.

ABA

signalling

mutants

have

sugar

signalling

defects

as

well

Putative

glucose

signalling

defects

were

determined

for

the

ABA

insensitive

mutant

and

the

ABA

oversensitive

mutant

and

were

grown

on

control

media

-LRB-

half-strength

MS

sucrose

-RRB-

or

on

control

medium

supplemented

with

either

sorbitol

or

glucose

After

days

germination

and

seedling

greening

percentage

were

scored

-LRB-

a

-RRB-

To

investigate

whether

has

a

glucose

oversensitive

phenotype

both

WT

Col

and

mutant

were

grown

on

a

sugar

concentration

which

did

not

arrest

early

development

of

WT

Col.

Therefore

seedling

greening

of

Col

and

the

mutant

was

scored

daily

on

half-strength

MS

-LRB-

MS

-RRB-

and

on

MS

glucose

The

seedling

phenotype

was

scored

for

days

-LRB-

b

-RRB-

The

gene

encodes

a

β

subunit

of

a

protein

farnesyl

transferase

-LRB-

Cutler

et

al.

-RRB-

and

acts

as

a

negative

regulator

of

ABA

signalling

The

mutant

has

an

ABA

hypersensitive

phenotype

If

exerts

a

similar

function

in

glucose

signalling

a

glucose

oversensitive

-LRB-

glo

-RRB-

phenotype

is

expected

for

the

mutant

Seeds

of

WT

Col

and

were

plated

on

half-strength

MS

glucose

media

This

glucose

concentration

allowed

seedling

greening

of

the

WT

Col

seedlings

Greening

started

for

WT

seedlings

from

day

four

onwards

However

this

glucose

concentration

arrested

early

seedling

development

of

seedlings

Even

after

eleven

days

no

greening

was

observed

for

seedlings

-LRB-

Fig.

-RRB-

which

indicates

that

indeed

has

a

glo

phenotype

The

mutant

has

a

more

dormant

phenotype

compared

to

WT

-LRB-

Cutler

et

al.

-RRB-

Therefore

seed

germination

was

synchronized

as

much

as

possible

by

using

after-ripened

seeds

and

employing

a

three

day

stratification

period

on

sugar-free

media

On

control

media

-LRB-

half-strength

MS

-RRB-

germination

was

somewhat

delayed

but

that

did

not

affect

cotyledon

greening

On

glucose

germination

reached

similar

germination

levels

as

WT

seeds

despite

that

germination

was

days

delayed

-LRB-

data

not

shown

-RRB-

This

slower

germination

of

does

not

explain

the

much

larger

difference

observed

for

cotyledon

greening

on

glucose

media

Thus

the

small

difference

in

germination

timing

between

and

WT

is

not

responsible

for

the

seedling

greening

phenotype

Discussion

GLUCOSE

INSENSITIVE

signalling

depends

on

a

functional

gene

Our

results

show

that

is

an

essential

regulator

of

GIN

signalling

This

conclusion

is

based

on

three

observations

Firstly

transcript

levels

accumulate

in

response

to

glucose

treatment

In

our

study

seedlings

were

treated

for

a

relatively

short

period

of

h

and

showed

a

fold

enhanced

levels

while

expression

levels

dropped

after

h

growth

on

control

media

-LRB-

Table

-RRB-

The

osmotic

control

conditions

induced

expression

at

most

two

fold

showing

that

the

strong

induction

of

by

glucose

is

not

solely

due

to

osmotic

stress

Secondly

glucose-regulated

gene

expression

was

altered

in

the

mutant

background

Glucose

enhanced

the

expression

of

LEA

genes

and

two

important

regulators

of

GIN

signalling

and

In

WT

glucose

treatment

led

to

a

fold

induction

of

and

and

a

fold

induction

of

The

mutation

caused

fold

under

expression

of

these

glucose-regulated

genes

-LRB-

Fig.

-RRB-

Thirdly

we

observed

a

gin

phenotype

for

seven

out

of

eight

alleles

in

both

and

Col

accessions

The

majority

of

the

alleles

tested

showed

a

gin

phenotype

that

was

comparable

to

well-known

gin

mutants

like

and

-LRB-

Fig.

and

Table

-RRB-

This

indicates

that

is

a

key

regulator

of

GIN

signalling

which

is

in

agreement

with

previous

reports

showing

that

overexpression

-LRB-

either

or

-RRB-

in

Arabidopsis

results

in

a

glucose

oversensitive

phenotype

-LRB-

Finkelstein

et

al.

Zeng

and

Kermode

-RRB-

Kermode

-LRB-

-RRB-

distinguished

at

least

three

functions

for

in

seeds

-LRB-

i

-RRB-

dormancy

induction

and

maintenance

-LRB-

ii

-RRB-

induction

of

storage

proteins

and

desiccation/stress

protectants

and

-LRB-

iii

-RRB-

repression

of

post-germinative

gene

expression

The

observations

that

mutants

showed

a

disturbed

glucose-regulated

induction

of

and

a

gin

phenotype

-LRB-

lack

of

post-germinative

gene

repression

-RRB-

are

in

agreement

with

the

functions

defined

for

Although

a

role

for

in

sugar

signalling

during

early

seedling

development

was

hinted

by

over

expression

lines

-LRB-

Finkelstein

et

al.

Zeng

and

Kermode

-RRB-

and

the

observation

that

mutants

were

insensitive

to

glucose

in

combination

with

ABA

-LRB-

Nambara

et

al.

-RRB-

its

role

remained

controversial

This

because

studies

that

investigated

the

gin

phenotype

of

in

the

background

observed

at

most

a

weak

phenotype

Importantly

however

is

a

sugar

oversensitive

accession

compared

to

the

Col

accession

-LRB-

Laby

et

al.

-RRB-

and

likely

the

sugar

insensitive

phenotype

of

has

been

underestimated

in

comparison

with

mutants

in

genetic

backgrounds

that

show

more

rapid

greening

Further

the

absence

of

a

clear

gin

phenotype

for

in

earlier

studies

could

be

due

to

the

seed

material

used

We

observed

that

freshly

harvested

seeds

were

particularly

sensitive

to

sugars

during

germination

-LRB-

data

not

shown

-RRB-

Low

sugar

concentrations

delay

germination

while

higher

levels

resulted

in

severely

reduced

germination

rates

Therefore

we

routinely

after-ripened

seeds

for

at

least

a

month

before

these

were

used

in

our

sugar

signalling

experiments

Seed

quality

changes

per

batch

produced

which

affects

germination

and

most

likely

responses

to

internal

and

external

stimuli

as

well

Hence

WT

and

mutant

seeds

used

in

the

sugar

response

assays

were

produced

simultaneously

under

the

same

growth

conditions

and

seeds

were

stored

under

identical

conditions

Moreover

we

routinely

stratified

the

seeds

on

sugar-free

half-strength

MS

media

before

the

start

of

the

gin

assays

since

this

procedure

dramatically

improves

germination

rates

following

transfer

to

glucose-containing

media

-LRB-

Fig.

Dekkers

and

Smeekens

-RRB-

Under

these

conditions

we

were

able

to

obtain

clear

gin

phenotypes

within

days

for

in

the

background

In

addition

this

study

identified

both

and

as

sis

mutants

-LRB-

which

are

insensitive

for

the

sucrose-induced

early

seedling

developmental

arrest

Laby

et

al.

-RRB-

as

well

In

addition

mutants

are

insensitive

to

the

glucose-induced

delay

of

germination

-LRB-

Yuan

and

Wysocka-Diller

Fig.

e

-RRB-

and

is

a

weak

sun

mutant

These

observations

imply

a

broader

role

for

in

mediating

sugar

responsiveness

Interestingly

another

domain

transcription

factor

was

found

to

regulate

the

sugar-inducible

sporamin

promoter

-LRB-

Tsukagoshi

et

al.

-RRB-

is

an

active

transcriptional

repressor

A

mutation

in

this

gene

causes

high

sporamin

promoter

activity

in

both

low

and

high

sugar

conditions

Glucose

and

ABA

induce

a

similar

early

seedling

arrest

in

Arabidopsis

Glucose

treatment

of

germinated

seeds

resulted

in

an

enhanced

expression

of

several

transcriptional

regulators

of

late

seed

maturation

-LRB-

and

-RRB-

GIN

signalling

depends

on

these

glucose-induced

factors

as

shown

by

mutant

analysis

-LRB-

León

and

Sheen

this

study

-RRB-

LEA

genes

-LRB-

and

-RRB-

were

also

induced

by

glucose

treatment

The

ABI

transcription

factors

are

known

to

regulate

the

expression

of

LEA

genes

-LRB-

Parcy

et

al.

Finkelstein

et

al.

Finkelstein

and

Lynch

Carles

et

al.

Kermode

-RRB-

LEA

gene

expression

is

correlated

with

drought

and

desiccation

tolerance

-LRB-

Hoekstra

et

al.

-RRB-

and

its

enhanced

expression

is

in

agreement

with

the

drought

tolerant

phenotype

observed

for

glucose-arrested

seedlings

Interestingly

the

ABA

response

during

the

post-germination

developmental

arrest

depends

on

the

same

and

transcription

factors

ABA

similarly

induces

and

a

subset

a

LEA

genes

Lopez-Molina

et

al.

-LRB-

-RRB-

suggested

that

a

late

embryogenesis

program

was

re-induced

by

ABA

which

resulted

in

developmentally

arrested

drought

tolerant

seedlings

and

that

this

may

constitute

an

important

checkpoint

during

germination

and

seedling

development

The

developmentally

arrested

state

could

only

be

induced

within

a

limited

time

frame

and

after

h

ABA

sensitivity

was

lost

Beyond

this

limited

time

window

ABA

did

not

induce

and

protein

levels

nor

re-induce

LEA

gene

transcription

and

did

not

block

early

seedling

development

The

sugar-induced

seedling

arrest

is

restricted

to

a

similar

time

window

-LRB-

Gibson

et

al.

-RRB-

Following

this

time

window

glucose

failed

to

re-induce

and

LEA

genes

Thus

far

it

is

not

clear

whether

this

time

frame

specifies

a

sugar

sensitive

window

Since

ABA

insensitivity

results

in

a

gin

phenotype

the

loss

of

ABA

sensitivity

beyond

this

time

frame

may

account

for

the

glucose

insensitivity

observed

In

conclusion

both

glucose

and

ABA

signalling

depend

on

the

same

set

of

genes

induce

similar

transcription

factors

and

LEA

genes

result

both

in

drought

tolerant

phenotypes

and

are

sensitive

for

a

similar

time

window

This

indicates

that

both

trigger

a

similar

seedling

developmental

arrest

Interestingly

the

induction

of

desiccation

tolerance

in

germinated

radicles

is

limited

to

a

small

time

window

after

germination

as

well

In

germinated

seeds

with

short

radicles

-LRB-

mm

-RRB-

desiccation

tolerance

can

be

induced

while

in

seeds

with

longer

radicles

-LRB-

mm

-RRB-

this

capacity

is

lost

-LRB-

Buitink

et

al.

-RRB-

Possibly

this

phenomenon

depends

on

the

same

genetic

components

and

is

sensitive

in

a

similar

time

window

observed

for

glucose

and

ABA-induced

post-germination

developmental

block

A

possible

role

was

suggested

for

sugar

signalling

in

desiccation

tolerance

induction

in

barrel

medic

-LRB-

Medicago

truncatula

-RRB-

and

cucumber

-LRB-

Cucumis

sativa

-RRB-

radicles

-LRB-

Leprince

et

al.

-RRB-

Non-reducing

di

and

trisaccharides

e.g.

sucrose

protect

the

structure

of

membranes

and

proteins

by

the

formation

of

a

glassy

state

-LRB-

Koster

Hoekstra

et

al.

-RRB-

E.g.

elevated

sucrose

concentrations

were

measured

in

desiccation

tolerant

radicles

-LRB-

Koster

and

Leopold

Bruggink

and

van

der

Toorn

-RRB-

Our

study

suggests

that

such

elevated

sugar

concentrations

may

serve

a

double

role

by

protecting

cellular

structures

and

by

acting

as

a

signal

for

the

induction

of

protectants

such

as

LEA

protein

Interactions

between

sugar

and

ABA

signalling

The

early

seedling

developmental

arrest

phenotype

has

been

extensively

exploited

to

isolate

mutants

with

sugar

signalling

defects

like

gin

mutants

-LRB-

Rognoni

et

al.

-RRB-

The

mutant

revealed

a

role

for

in

glucose

signalling

Such

signalling

does

not

depend

on

its

metabolic

function

-LRB-

Moore

et

al.

-RRB-

and

was

shown

to

interact

with

unusual

partners

-LRB-

a

vacualor

H

ATPase

and

a

proteasome

subunit

-RRB-

to

regulate

glucose

signalling

-LRB-

Cho

et

al.

-RRB-

Other

gin

mutants

suggested

a

link

between

glucose

signalling

and

plant

hormone

biosynthesis

and

signalling

The

lack

of

ABA

biosynthesis

and

signalling

-LRB-

and

-RRB-

resulted

in

glucose

insensitivity

To

study

the

interaction

between

ABA

biosynthesis

and

sugar

signalling

we

used

ABA

deficient

lines

with

constitutive

expression

-LRB-

-RRB-

These

strong

overexpression

lines

affected

expression

-LRB-

ISI

pathway

-RRB-

and

showed

a

stunted

growth

phenotype

similar

to

what

has

been

reported

previously

for

severe

overexpression

lines

-LRB-

Söderman

et

al.

-RRB-

indicating

that

is

active

in

an

ABA

deficient

background

Nevertheless

the

gin

phenotype

of

could

not

be

restored

by

high

expression

levels

-LRB-

Fig.

-RRB-

Thus

is

necessary

but

not

sufficient

for

GIN

signalling

in

an

ABA

deficient

background

indicating

that

other

ABA

regulated

factors

are

essential

for

proper

GIN

signalling

as

well

Both

ISI

and

GIN

signalling

rely

on

ABA

biosynthesis

and

but

surprisingly

overexpression

only

affected

the

isi

phenotype

of

Thus

gin

and

isi

sugar

signalling

depend

on

the

same

components

but

this

study

shows

that

the

relationship

between

sugar

ABA

and

is

different

for

both

sugar

signalling

pathways

ABA

biosynthesis

and

ABA

signalling

are

required

for

the

sugar

induced

seedling

arrest

Genetic

studies

showed

that

two

transcription

factors

which

are

involved

in

ABA

signalling

i.e.

and

function

in

the

sugar-induced

seedling

arrest

-LRB-

Arenas-Huertero

et

al.

Huijser

et

al.

Laby

et

al.

Brocard

et

al.

-RRB-

Here

we

show

that

is

also

essential

for

GIN

signalling

Glucose

induces

ABA

biosynthesis

genes

-LRB-

Cheng

et

al.

-RRB-

and

increased

ABA

levels

were

measured

in

developmentally

arrested

seedlings

ABA

is

able

to

induce

transcription

-LRB-

Table

-RRB-

and

affects

protein

stability

-LRB-

Zhang

et

al.

-RRB-

Thus

glucose

may

impinge

on

via

activation

of

ABA

biosynthesis

also

functions

in

seed

development

and

developmental

timing

-LRB-

Rohde

et

al.

-RRB-

Severe

mutant

embryos

remain

green

and

are

unable

to

acquire

desiccation

tolerance

and

dormancy

during

seed

development

-LRB-

Ooms

et

al.

-RRB-

Such

mature

embryos

resemble

a

developing

seedling

rather

than

a

dormant

embryo

-LRB-

Nambara

et

al.

-RRB-

The

seedling

developmental

arrest

as

induced

by

ABA

and

glucose

is

sensitive

to

developmental

signals

as

well

Arrest

of

seedling

development

by

ABA

and

sugars

only

occurs

within

a

two

to

three

days

time

window

after

imbibition

-LRB-

Gibson

et

al.

Lopez-Molina

et

al.

-RRB-

Therefore

the

phenotype

of

the

severe

mutants

is

probably

not

solely

due

to

ABA

insensitivity

but

to

seed

developmental

defects

and

the

heterochronic

nature

of

this

mutation

as

well

Two

additional

ABA

response

mutants

with

altered

glucose

responsiveness

and

strengthen

the

link

between

glucose

and

ABA

signalling

The

mutant

showed

a

gin

phenotype

that

is

weaker

compared

to

Possibly

additional

seed

developmental

phenotypes

of

may

explain

the

stronger

phenotype

of

the

mutant

compared

to

However

the

ABA

insensitive

phenotype

of

is

somewhat

weaker

compared

to

-LRB-

Koornneef

et

al.

Finkelstein

and

Somerville

-RRB-

which

could

explain

the

weaker

gin

phenotype

as

well

The

mutant

is

hypersensitive

to

ABA

and

shows

a

glucose

oversensitive

phenotype

Interestingly

another

ABA

hypersensitive

mutant

shows

a

similar

glo

phenotype

is

allelic

to

ethylene

-LRB-

Ghassemian

et

al.

-RRB-

and

also

other

ethylene

related

mutants

show

sugar

signalling

defects

-LRB-

Zhou

et

al.

Gibson

et

al.

León

and

Sheen

-RRB-

The

role

of

ABA

and

ABA

signalling

in

sugar

signalling

is

well

established

Thus

far

sugar

signalling

was

thought

to

rely

on

only

a

part

of

the

ABA

signalling

cascade

The

analysis

of

transgenic

lines

showed

that

ABA

related

factors

other

than

are

involved

in

glucose

signalling

Furthermore

the

glucose-induced

early

seedling

arrest

mimics

the

ABA-induced

seedling

arrest

Our

study

indicates

that

all

response

loci

tested

that

act

in

the

genetic

pathway

regulating

ABA

responsiveness

as

proposed

by

Brady

et

al.

-LRB-

-RRB-

affect

glucose

signalling

These

observations

suggest

that

the

genetic

pathways

regulating

glucose

and

ABA

signalling

are

much

more

closely

linked

than

assumed

thus

far

Obviously

both

pathways

are

different

as

well

Genetic

analysis

suggested

that

acts

upstream

of

ABA

signalling

in

glucose

signalling

Moreover

the

mutant

has

a

gin

phenotype

but

is

not

affected

in

ABA

signalling

-LRB-

Chen

et

al.

-RRB-

Such

mutants

may

provide

insight

in

the

mechanism

by

which

glucose

affects

ABA

signalling

Interestingly

glucose

delays

germination

in

a

process

that

depends

on

but

that

does

not

require

and

-LRB-

Gibson

Yuan

and

Wysocka-Diller

Fig.

e

-RRB-

showing

that

the

tight

interaction

between

glucose

and

ABA

is

not

present

in

all

sugar

response

pathways

abscisic acid
aba insensitive
glucose insensitive
seedling development
sugar signalling