The

microRNA

regulated

SBP-box

genes

and

control

shoot

maturation

in

Arabidopsis

Throughout

development

the

Arabidopsis

shoot

apical

meristem

successively

undergoes

several

major

phase

transitions

such

as

the

juvenile-to-adult

and

floral

transitions

until

finally

it

will

produce

flowers

instead

of

leaves

and

shoots

Members

of

the

Arabidopsis

SBP-box

gene

family

of

transcription

factors

have

been

implicated

in

promoting

the

floral

transition

in

dependence

of

and

accordingly

transgenics

constitutively

over-expressing

this

microRNA

are

delayed

in

flowering

To

elaborate

their

roles

in

Arabidopsis

shoot

development

we

analysed

two

of

the

regulated

Arabidopsis

SBP-box

genes

i.e.

the

likely

paralogous

genes

and

Single

and

double

mutant

phenotype

analysis

showed

these

genes

to

act

redundantly

in

controlling

the

juvenile-to-adult

phase

transition

In

addition

their

loss-of-function

results

in

a

shortened

plastochron

during

vegetative

growth

altered

inflorescence

architecture

and

enhanced

branching

In

these

aspects

the

double

mutant

partly

phenocopies

constitutive

over-expressing

transgenic

plants

and

thus

a

major

contribution

to

the

phenotype

of

these

transgenics

as

a

result

of

the

repression

of

and

is

strongly

suggested

Introduction

During

maturation

plants

pass

through

several

developmentally

distinct

growth

phases

in

which

the

shoot

gradually

gains

reproductive

competence

-LRB-

Poethig

-RRB-

After

the

transition

from

embryonic

to

postembryonic

growth

plants

undergo

at

least

two

further

phase

transitions

the

vegetative

as

well

as

the

reproductive

phase

change

During

vegetative

growth

rosette

leaves

are

initiated

at

the

flanks

of

the

shoot

apical

meristem

-LRB-

SAM

-RRB-

with

a

certain

frequency

referred

to

as

plastochron

-LRB-

Erickson

and

Michelini

-RRB-

After

going

through

the

reproductive

phase

transition

also

known

as

the

floral

transition

the

SAM

starts

to

initiate

floral

buds

instead

of

leaves

In

Arabidopsis

as

in

many

other

plants

showing

day

length

dependent

flowering

the

floral

transition

is

preceded

by

a

transition

from

juvenile

to

adult

growth

This

switch

known

as

the

vegetative

phase

change

is

physiologically

defined

as

achieving

competence

to

respond

to

photoperiodic

induction

of

flowering

-LRB-

Poethig

-RRB-

The

transition

from

juvenile

to

adult

growth

is

gradual

and

rather

subtle

but

generally

can

be

followed

by

several

morphological

markers

In

Arabidopsis

for

example

leaves

produced

in

the

juvenile

phase

have

long

petioles

are

small

round

and

lack

abaxial

trichomes

In

contrast

short

petioles

elliptical

anatomy

and

the

development

of

trichomes

on

the

abaxial

side

represent

adult

traits

-LRB-

Telfer

et

al.

-RRB-

Regulation

of

these

developmental

transitions

is

largely

dependent

on

-LRB-

changes

in

-RRB-

environmental

cues

such

as

day

length

light

intensity

and

temperature

as

well

as

on

endogenous

factors

such

as

the

plant

hormone

gibberellin

-LRB-

Telfer

et

al.

-RRB-

Whereas

the

molecular

genetic

mechanisms

underlying

the

floral

transition

are

already

worked

out

in

increasing

detail

-LRB-

Komeda

-RRB-

it

has

only

been

recently

that

we

begin

to

understand

the

molecular

genetic

basis

of

the

vegetative

phase

change

Most

genes

suggested

to

play

a

role

in

promoting

the

latter

phase

change

have

been

identified

by

the

analysis

of

mutants

showing

a

precocious

onset

of

adult

traits

and

intriguingly

link

vegetative

phase

change

to

RNA

silencing

pathways

These

genes

include

the

Arabidopsis

ortholog

of

exportin

HASTY

-LRB-

HST

Telfer

and

Poethig

Bollman

et

al.

-RRB-

the

zinc-finger-domain

protein

encoding

locus

SERRATE

-LRB-

SE

Clarke

et

al.

-RRB-

and

ZIPPY

-LRB-

ZIP

-RRB-

an

AGO-family

member

-LRB-

Hunter

et

al.

-RRB-

More

recently

screens

for

mutations

with

zip-like

phenotypes

resulted

in

alleles

of

SUPRESSOR

OF

GENE

-LRB-

-RRB-

and

RNA-DEPENDENT

-LRB-

-RRB-

both

genes

required

for

posttranscriptional

gene

silencing

-LRB-

PTGS

-RRB-

and

acting

in

the

same

pathways

as

ZIP

and

HST

-LRB-

Peragine

et

al.

-RRB-

Furthermore

a

precocious

vegetative

phase

change

has

also

been

found

in

dicer-like

-LRB-

-RRB-

mutants

-LRB-

Gasciolli

et

al.

Xie

et

al.

Yoshikawa

et

al.

-RRB-

One

explanation

for

the

observed

effects

could

be

that

target

genes

of

this

silencing

pathway

play

a

positive

role

on

the

vegetative

phase

change

and

their

down-regulation

consequently

promotes

juvenility

Hence

mutations

in

genes

involved

in

this

silencing

pathway

as

the

ones

described

above

cause

an

accelerated

vegetative

development

In

line

with

this

idea

members

of

the

plant

specific

SBP-box

gene

transcription

factor

family

have

been

implicated

in

promoting

vegetative

and

floral

phase

transitions

In

particular

overexpression

of

the

Arabidopsis

SBP-box

gene

leads

to

early

flowering

and

a

significant

earlier

appearance

of

abaxial

trichomes

on

the

rosette

leaves

-LRB-

Cardon

et

al.

Wu

and

Poethig

-RRB-

Interestingly

together

with

of

other

family

members

expression

is

post-transcriptionally

controlled

by

and

probably

also

by

the

very

closely

related

-LRB-

Rhoades

et

al.

Schwab

et

al.

Wu

and

Poethig

Gandikota

et

al.

-RRB-

Consistent

with

its

role

of

down-regulating

and

related

SPL

target-genes

constitutive

overexpression

of

encoding

loci

has

been

shown

to

cause

the

production

of

a

significantly

larger

number

of

leaves

with

juvenile

characteristics

and

a

delay

in

flowering

-LRB-

Schwab

et

al.

Wu

and

Poethig

-RRB-

Although

the

available

data

clearly

point

to

a

regulatory

role

for

the

miRNA

regulated

SPL

genes

in

the

temporal

development

of

the

Arabidopsis

shoot

the

contribution

of

the

single

genes

to

the

described

phenotypes

remains

to

be

determined

Therefore

we

identified

and

isolated

mutant

alleles

for

single

SPL

genes

In

comparison

to

other

targeted

SPL

genes

available

expression

data

-LRB-

AtGenExpress

Schmid

et

al.

-RRB-

show

and

to

be

already

quite

active

in

the

vegetative

shoot

apex

Accordingly

their

mutant

phenotypes

were

found

to

affect

vegetative

development

Here

we

report

the

mutant

analysis

of

and

two

likely

paralogous

members

of

the

SQUAMOSA

PROMOTER

BINDING

PROTEIN-LIKE

-LRB-

SPL

-RRB-

transcription

factor

family

-LRB-

Cardon

et

al.

-RRB-

and

discuss

their

redundant

regulatory

role

on

the

vegetative

phase

change

and

the

temporal

initiation

of

rosette

leaves

Materials

and

methods

Plant

material

and

plant

growth

conditions

All

of

the

genetic

stocks

described

in

the

paper

were

in

Columbia

background

The

T-DNA

insertion

lines

-LRB-

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

were

obtained

at

the

Nottingham

Arabidopsis

Stock

Centre

-LRB-

NASC

-RRB-

The

T-DNA

insertion

lines

GABI-Kat

-LRB-

-RRB-

and

WiscDsLox

-LRB-

-RRB-

were

obtained

from

GABI-Kat

and

the

Arabidopsis

Biological

Research

Centre

-LRB-

ABRC

-RRB-

respectively

Insertion

mutant

information

for

NASC

or

ARBC-lines

was

obtained

from

the

SIGNAL

website

at

Plants

homozygous

for

the

T-DNA

insertions

were

identified

by

PCR

using

T-DNA

left

border

and

gene-specific

primers

T-DNA

specific

left

border

primers

for

SALK

GABI-Kat

and

WiscDsLox

T-DNAs

were

and

respectively

In

combination

with

the

respective

left

border

primer

we

used

the

following

gene-specific

primers

for

for

and

for

GABI-Kat

and

for

WiscDsLox

The

stable

insertion

mutant

-LRB-

-RRB-

was

obtained

from

the

ZIGIA-population

-LRB-

Unte

-RRB-

Plants

homozygous

for

a

four

base

pair

insertion

in

the

first

exon

of

caused

by

the

excision

of

the

transposon

were

backcrossed

with

wild

type

twice

to

obtain

plants

exclusively

containing

the

four

base-pair

insertion

without

any

further

transposon

contamination

In

order

to

identify

plants

containing

the

mutation

we

used

the

following

primer

combination

-LRB-

forward

-RRB-

and

-LRB-

reverse

recognises

the

insertion

-RRB-

All

plants

were

grown

in

plastic

trays

or

pots

filled

with

ready-to-use

commercial

pre-fertilized

soil

mixture

-LRB-

Type

Werkverband

eV

Sinntal-Jossa

Germany

-RRB-

For

stratification

seeds

were

kept

on

moist

paper

at

°C

in

the

dark

for

days

before

transferring

to

soil

-LRB-

i.e.

sowing

-RRB-

in

growth

chambers

at

°C

relative

humidity

Germination

and

cultivation

of

the

plants

in

long-day

conditions

-LRB-

h

light

h

dark

-RRB-

were

either

under

approx.

-LRB-

-RRB-

or

-LRB-

-RRB-

light

provided

by

fluorescent

tubes

-LRB-

and

Osram

Munich

Germany

-RRB-

Plants

in

short-day

-LRB-

SD

-RRB-

conditions

-LRB-

h

light

h

dark

-RRB-

were

cultivated

under

approx.

light

To

determine

sensitivity

to

photoperiodic

induction

of

flowering

stratified

seeds

were

germinated

in

a

modified

SD

with

a

h

light

period

The

developing

plants

were

kept

in

these

conditions

for

days

before

they

were

transferred

to

similar

growth

chambers

with

continuous

light

provided

by

Osram

HQIT

W

lamps

Batches

of

plants

were

returned

to

the

modified

SD

conditions

after

and

days

Phenotypic

analysis

Flowering

time

was

measured

as

the

time

between

sowing

and

anthesis

-LRB-

opening

of

first

flower

-RRB-

Bolting

time

was

recorded

when

the

main

inflorescence

had

reached

a

height

of

cm

Inflorescence

height

was

measured

between

the

rosette

and

the

first

flower

of

the

main

inflorescence

of

plants

with

their

first

siliques

fully

ripened

In

order

to

count

the

number

of

site

shoots

all

site

shoots

longer

than

cm

were

scored

Abaxial

trichomes

were

scored

using

a

Leica

MZFLIII

stereomicroscope

-LRB-

Wetzlar

Germany

-RRB-

An

estimation

of

the

rosette

leaf

initiation

rate

-LRB-

L/D

day

-RRB-

was

obtained

by

dividing

the

number

-LRB-

L

-RRB-

of

rosette

leaves

having

reached

at

least

cm

in

length

through

the

number

of

days

-LRB-

D

-RRB-

between

sowing

and

determination

Note

that

this

value

reflects

but

does

not

equalize

-LRB-

average

-RRB-

plastochron

as

it

should

be

corrected

for

the

true

start

of

initiation

of

the

first

leaf

as

well

as

the

time

needed

for

the

first

adult

leaf

to

reach

a

size

of

cm

Histological

analysis

Apical

regions

were

isolated

from

plants

grown

in

SD

for

days

by

trimming

with

a

razor

blade

The

tissue

was

fixed

in

M

pH

for

h

and

embedded

in

paraffin

using

a

Leica

tissue

processor

-LRB-

Wetzlar

Germany

-RRB-

Embedded

apices

were

cut

into

μm

thin

cross

sections

using

a

Jung

Autocut

and

photographed

using

a

Zeiss

Axiophot

microscope

-LRB-

Göttingen

Germany

-RRB-

equipped

with

a

camera

-LRB-

JVC

Yokohama

Japan

-RRB-

The

first

cross

section

in

which

the

apex

was

visible

plus

two

successive

sections

were

used

to

determine

the

diameter

of

the

apical

region

From

these

three

measured

values

the

average

was

taken

for

comparison

In

addition

cross

sectional

area

and

circularity

factor

-LRB-

4π

A

P

A

is

area

P

is

perimeter

-RRB-

of

the

leaf

primordia

outlined

by

hand

on

the

photographs

were

determined

as

well

The

measurements

were

performed

with

help

of

the

program

ImageJ

s

-LRB-

Wayne

Rasband

National

Institutes

of

Health

USA

-RRB-

treatments

the

double

mutant

and

the

overexpressor

were

grown

in

conditions

Immediately

after

germination

half

of

the

plants

were

treated

by

spraying

μM

Tween

and

this

was

repeated

twice

per

week

until

they

started

flowering

The

other

half

of

the

plants

was

similarly

treated

with

Tween

Phylogenetic

comparison

Multiple

alignments

of

amino

acid

sequences

were

generated

by

the

program

ClustalW

of

the

MacVector

software

package

-LRB-

Accelrys

Ltd.

Cambridge

UK

-RRB-

using

the

BLOSUM

matrix

with

an

open

gap

penalty

of

and

an

extend

gap

penalty

of

Only

the

SBP-domain

was

used

for

the

phylogenetic

reconstruction

The

tree

was

constructed

using

the

neighbour-joining

algorithm

of

the

MacVector

software

package

Quantitative

real-time

PCR

analysis

To

perform

quantitative

RT-PCR

-LRB-

using

the

real-time

PCR

detection

system

Bio-Rad

Munich

Germany

-RRB-

apical

regions

were

collected

-LRB-

roots

and

as

much

of

the

leaves

as

possible

were

removed

using

tweezers

-RRB-

of

plants

cultivated

and

days

after

sowing

in

conditions

Total

RNA

was

extracted

using

the

RNeasy

plant

mini

kit

-LRB-

Qiagen

Hilden

Germany

-RRB-

including

an

on-column

DNase

digestion

First-strand

cDNA

was

synthesized

using

SuperScript

III

RNase

H

reverse

transcriptase

-LRB-

Invitrogen

-RRB-

starting

with

μg

of

total

RNA

primed

with

an

oligo

-LRB-

T

-RRB-

primer

-LRB-

Gibco

BRL

Karlsruhe

Germany

-RRB-

primers

-LRB-

forward

-RRB-

and

-LRB-

reverse

-RRB-

as

well

as

primers

-LRB-

forward

-RRB-

and

-LRB-

reverse

-RRB-

were

designed

to

generate

a

PCR

product

of

and

bp

respectively

Based

on

the

analysis

of

Czechowski

et

al.

-LRB-

-RRB-

expression

was

used

as

reference

for

transcript

normalization

with

the

primer

pair

-LRB-

forward

-RRB-

and

-LRB-

reverse

-RRB-

The

PCR

efficiencies

for

and

primers

were

determined

to

be

and

respectively

Quantifications

in

triplicate

were

performed

using

the

Brilliant

SYBRGreen

QPCR

kit

-LRB-

Stratagene

La

Jolla

CA

USA

-RRB-

according

to

the

manufacturer

's

protocol

in

a

final

volume

of

μl

PCR

was

carried

out

in

μl

optical

reaction

vials

-LRB-

Stratagene

-RRB-

heated

for

min

at

°C

to

hot-start

the

Taq

polymerase

followed

by

cycles

of

denaturation

-LRB-

s

at

°C

-RRB-

annealing

-LRB-

s

at

°C

-RRB-

and

extension

-LRB-

s

at

°C

-RRB-

Semi-quantitative

RT-PCR

analysis

Total

RNA

was

extracted

from

seedlings

of

the

wild-type

mutant

and

transgenic

lines

using

the

RNeasy

plant

mini

kits

-LRB-

Qiagen

-RRB-

RT-PCR

with

equal

amounts

of

RNA

was

performed

using

the

one-step

RT-PCR

kit

-LRB-

Qiagen

-RRB-

knockout

lines

were

identified

using

the

following

primer

pair

-LRB-

forward

-RRB-

and

-LRB-

reverse

-RRB-

mRNA

is

expected

to

yield

a

bp

fragment

whereas

genomic

DNA

is

expected

to

yield

a

bp

fragment

knockout

lines

were

identified

using

the

following

primer

pair

-LRB-

forward

-RRB-

and

-LRB-

reverse

-RRB-

mRNA

is

expected

to

yield

a

bp

fragment

whereas

genomic

DNA

is

expected

to

yield

a

bp

fragment

RT-PCR

of

the

loading

control

-LRB-

-RRB-

was

performed

with

the

primer

pair

-LRB-

forward

-RRB-

and

-LRB-

reverse

-RRB-

to

yield

a

fragment

of

bp

when

derived

from

mRNA

-LRB-

genomic

is

expected

to

yield

a

bp

fragment

-RRB-

Statistics

Graphical

representations

of

numerical

data

were

generated

with

the

Microsoft

Excel

program

-LRB-

Microsoft

Germany

Munich

-RRB-

and

statistical

tests

were

performed

using

the

Student

's

t-Test

within

this

program

P-values

lower

than

were

considered

to

be

statistically

relevant

and

the

data

involved

to

represent

significant

differences

Remaining

techniques

and

methods

Standard

molecular

biology

techniques

were

performed

as

described

by

Sambrook

et

al.

-LRB-

-RRB-

Graphical

plots

and

digital

photographic

images

were

cropped

and

assembled

using

Adobe

Photoshop

-LRB-

Adobe

Systems

San

Jose

CA

USA

-RRB-

Results

Molecular

characterization

of

the

Arabidopsis

SBP-box

genes

and

Mutant

alleles

for

the

Arabidopsis

SBP-box

genes

-LRB-

-RRB-

and

-LRB-

-RRB-

were

obtained

from

screening

publicly

available

electronic

databases

and

seed

stock

centres

for

transposon

or

T-DNA

tagged

SPL

genes

For

we

identified

three

insertion

alleles

designated

as

to

and

confirmed

the

nature

and

position

of

their

mutations

-LRB-

see

Materials

and

methods

Fig.

-RRB-

The

first

allele

was

identified

in

the

En-transposon

mutagenised

ZIGIA

population

-LRB-

Baumann

et

al.

Unte

-RRB-

and

most

likely

resulted

from

the

excision

of

an

inserted

transposon

leaving

behind

a

insertion

footprint

in

the

first

exon

The

result

of

this

is

a

frame

shift

in

the

coding

sequence

and

the

generation

of

a

stop-codon

base

pairs

after

the

insertion

site

Both

and

represent

T-DNA

insertion

mutant

alleles

identified

within

respectively

the

SALK

collection

-LRB-

Alonso

et

al.

-RRB-

and

the

GABI-Kat

collection

-LRB-

Li

et

al.

-RRB-

Fig.

characterization

of

and

-LRB-

a

-RRB-

Schematic

representation

of

the

genomic

loci

of

and

The

positions

of

the

mutations

identified

are

indicated

by

open

triangles

numbered

according

to

the

respective

alleles

Boxes

represent

exons

The

SBP-box

sequences

are

depicted

in

black

the

remaining

coding

sequences

in

grey

and

the

untranslated

and

regions

are

left

blank

-LRB-

b

-RRB-

Changes

in

transcript

levels

of

and

in

the

shoot

apical

region

during

plant

development

in

as

determined

with

qRT-PCR

and

normalized

against

For

comparison

relative

transcript

levels

were

arbitrarily

set

to

one

for

days

after

sowing

Error

bars

indicate

standard

deviation

-LRB-

c

-RRB-

Absence

of

and

transcripts

in

seedlings

of

the

respective

mutants

as

validated

by

RT-PCR

Presence

of

the

respective

transcripts

in

wild

type

seedlings

is

shown

for

comparison

and

the

amplification

of

transcript

as

quality

control

and

reference

for

quantification

Fragment

lengths

are

indicated

on

the

left

in

base

pairs

-LRB-

bp

-RRB-

Also

three

independent

T-DNA

insertion

lines

for

could

be

obtained

and

confirmed

-LRB-

see

Materials

and

methods

Fig.

-RRB-

Two

alleles

designated

as

and

were

identified

within

the

SALK

collection

and

one

within

the

WiscDsLox

T-DNA

collection

According

to

data

available

from

the

AtGenExpress

micro-array

database

-LRB-

Schmid

et

al.

-RRB-

both

and

transcript

levels

increase

during

development

and

are

preferentially

found

in

the

shoot

apical

region

and

in

young

flowers

We

confirmed

this

temporal

expression

pattern

with

the

help

of

qRT-PCR

-LRB-

Fig.

-RRB-

In

growing

conditions

-LRB-

see

Materials

and

methods

-RRB-

and

transcript

levels

remain

comparable

during

the

first

weeks

Thereafter

the

expression

level

of

starts

to

increase

followed

by

that

of

Around

days

after

sowing

-LRB-

DAS

-RRB-

at

about

the

time

plants

have

undergone

their

reproductive

phase

transition

transcript

levels

have

become

approximately

two

and

a

half

times

higher

in

comparison

to

and

six

times

in

comparison

to

day

Arabidopsis

lines

carrying

as

a

transgene

a

genomic

fragment

encompassing

the

locus

for

and

with

a

GUS

reporter

gene

inserted

downstream

of

the

ATG

start

codon

confirmed

the

predominantly

apical

expression

of

-LRB-

Supplementary

Fig.

-RRB-

RT-PCR

performed

on

mRNA

isolated

from

whole

seedling

plants

homozygous

for

any

of

the

three

or

mutant

alleles

-LRB-

see

Materials

and

methods

-RRB-

did

not

result

in

the

detection

of

RNA

derived

of

the

respective

genes

-LRB-

Fig.

-RRB-

This

strongly

suggests

that

all

mutant

alleles

isolated

represent

functional

null-alleles

Accordingly

plants

homozygous

for

any

of

the

three

mutant

alleles

showed

highly

identical

phenotypes

as

did

all

three

homozygous

mutants

-LRB-

see

phenotypic

analysis

below

Supplementary

Fig.

-RRB-

Allelic

tests

confirmed

that

the

observed

phenotypes

are

indeed

due

to

mutation

in

either

or

respectively

-LRB-

data

not

shown

-RRB-

With

over

of

their

amino

acid

residues

identical

and

show

high

similarity

on

the

level

of

their

proteins

Also

a

phylogenetic

comparison

based

on

the

SBP-box

of

all

SPL

genes

in

Arabidopsis

revealed

and

as

most

closely

related

and

most

likely

forming

a

pair

of

paralogous

genes

-LRB-

Fig.

-RRB-

Based

on

this

close

relationship

some

degree

of

functional

redundancy

could

be

expected

and

therefore

we

created

double

mutant

lines

to

uncover

such

redundancy

To

ascertain

that

phenotypic

changes

in

the

mutant

plants

are

solely

due

to

the

loss-of-function

of

and

we

generated

two

different

homozygous

double

mutant

lines

with

the

allelic

combinations

and

respectively

Both

lines

exhibit

the

same

phenotype

as

described

in

the

next

section

For

further

detailed

analysis

the

line

was

chosen

and

in

the

following

referred

to

as

for

simplicity

Fig.

relationship

of

the

Arabidopsis

SBP-box

genes

as

based

on

the

conserved

SBP-domain

The

orthologous

sequence

of

Chlamydomonas

has

been

used

as

outgroup

The

likely

paralogous

pair

and

is

boxed

in

grey

targeted

SPL

genes

are

marked

with

an

asterisk

Only

bootstrap

values

over

are

shown

Phenotypic

analysis

of

and

mutants

For

a

phenotypic

analysis

we

compared

and

single

mutants

double

mutants

to

wild

type

as

well

as

to

an

transgenic

line

-LRB-

kindly

provided

by

D.

Weigel

and

R.

Schwab

-RRB-

An

interesting

aspect

of

the

over-expressing

plants

as

already

noticed

by

Schwab

and

co-workers

-LRB-

-RRB-

is

an

increased

rate

of

rosette

leaf

initiation

which

in

combination

with

a

modest

delayed

flowering

results

in

the

obvious

denser

rosettes

of

fully

developed

plants

-LRB-

Fig.

-RRB-

In

addition

advanced

plants

became

very

bushy

-LRB-

Fig.

-RRB-

We

found

these

phenotypic

aspects

also

displayed

by

the

double

mutant

albeit

less

pronounced

-LRB-

Fig.

b

-RRB-

To

quantify

the

contribution

of

and

to

these

phenomena

we

compared

the

number

of

rosette

and

cauline

leaves

of

the

respective

single

and

double

mutants

and

of

the

overexpressor

to

wild

type

-LRB-

Table

-RRB-

Whereas

in

growing

conditions

the

line

produced

ca

eleven

more

rosette

leaves

in

comparison

to

wild

type

the

single

mutant

lines

produced

on

average

only

rosette

leaves

more

Again

with

ca

six

extra

rosette

leaves

the

double

mutant

differed

more

from

wild

type

than

the

single

mutants

and

showed

a

stronger

tendency

towards

the

phenotype

of

the

overexpressor

The

number

of

cauline

leaves

remained

very

comparable

among

all

mutants

and

wild

type

although

some

reduction

may

be

observed

particularly

in

the

mutants

Fig.

analysis

of

and

mutants

-LRB-

a

-RRB-

Flowering

and

double

mutant

plants

shown

next

to

wild

type

and

the

overexpressor

Plants

shown

next

to

each

other

are

of

the

same

age

and

grown

in

parallel

under

conditions

-LRB-

b

c

-RRB-

wild

type

double

mutant

-LRB-

b

-RRB-

and

overexpressor

-LRB-

c

-RRB-

at

a

more

advanced

stage

of

development

in

comparison

to

the

plants

shown

in

aTable

evaluation

of

and

mutant

alleles

in

comparison

to

Col

wt

and

a

transgene

under

LD

conditionsRosette

leavesCauline

leavesBolting

-LRB-

DAS

-RRB-

Anthesis

-LRB-

DAS

-RRB-

Juvenile

leavesaInfloresc

heightb

-LRB-

cm

-RRB-

wt13.11.13.90.516.31.220.91.55.51.212.11.3spl9-114.31

c0.715.91.419.6c1.78.30.88.01.0spl9-215.61

c0.516.91.220.81.49.20.98.20.8spl9-315.71

c0.617.5c1.521.31.59.60.78.60.8spl15-115.61

c0.416.60.820.91.07.10.710.31.2spl15-214.91

d1.43.40.918.5d1.422.3c1.810.9d0.86.9d1.4spl9-2

S

plants

per

genotype

were

used

for

determinationDAS

days

after

sowing

SD

standard

deviationValues

significantly

different

from

wt

at

confidence

level

are

shown

in

italicsaNumber

of

rosette

leaves

formed

before

the

first

leaf

with

abaxial

trichomesbMeasured

from

rosette

to

first

flowercValues

significantly

different

from

wt

at

but

not

at

confidence

leveldValues

significantly

different

from

single

mutants

at

confidence

leveleValues

significantly

different

from

double

and

single

mutants

at

confidence

level

Also

with

respect

to

the

development

of

side

shoots

the

double

mutant

differed

more

from

wild

type

than

the

single

mutants

In

fact

and

single

mutants

were

found

not

to

differ

significantly

from

plants

that

had

formed

on

average

side

shoots

of

at

least

cm

in

length

by

the

time

that

the

first

siliques

ripened

With

an

average

of

side

shoots

the

double

mutant

did

significantly

differ

from

wild

type

as

did

the

transgenic

line

with

on

average

side

shoots

Taken

together

the

phenotypic

data

of

the

double

mutant

clearly

suggests

a

redundant

function

of

and

in

shoot

development

and

in

the

maintenance

of

apical

dominance

is

assumed

to

target

besides

and

exclusively

other

SPL

genes

-LRB-

Rhoades

et

al.

-RRB-

These

too

were

shown

to

be

down

regulated

in

over-expressing

plants

-LRB-

Schwab

et

al.

-RRB-

As

in

comparison

to

the

double

mutant

the

over-expressor

displays

an

even

more

severe

aberrant

phenotype

it

can

also

be

deduced

that

in

addition

to

and

other

SPL

genes

act

redundantly

to

control

shoot

development

and

apical

dominance

In

addition

to

the

number

of

leaves

formed

before

the

appearance

of

the

first

flowers

we

also

determined

for

the

same

plants

the

time

they

needed

to

bolt

as

well

as

to

anthesis

-LRB-

Table

-RRB-

On

average

the

and

single

mutants

behaved

similar

to

wild

type

but

as

expected

based

on

the

data

of

Schwab

et

al.

-LRB-

-RRB-

the

line

bolted

and

flowered

somewhat

later

The

double

mutants

showed

an

intermediate

behaviour

Whereas

for

the

single

mutants

the

few

more

leaves

formed

may

be

accounted

for

by

the

slight

delay

in

the

transition

to

flowering

this

delay

is

unlikely

to

explain

the

increased

rosette

leaf

number

of

the

double

mutant

In

line

with

the

observation

of

Schwab

and

co-workers

-LRB-

-RRB-

who

reported

a

leaf-initiation

rate

per

day

in

SD

of

vs.

for

the

overexpressor

and

the

wild

type

respectively

this

is

probably

best

explained

by

assuming

a

shortened

plastochron

during

vegetative

growth

To

uncover

a

possible

cause

or

consequence

for

this

increased

rate

of

leaf

initiation

we

microscopically

examined

cross

sections

of

the

vegetative

shoot

apex

to

determine

size

and

phyllotaxy

of

the

double

mutant

and

the

overexpressor

and

compared

these

to

wild

type

To

this

purpose

plants

were

grown

for

days

in

SD

conditions

whereafter

the

number

of

rosette

leaves

having

reached

at

least

cm

in

length

were

recorded

and

their

apices

dissected

fixed

and

embedded

in

paraffin

-LRB-

see

Materials

and

methods

-RRB-

At

this

age

plants

were

found

to

have

formed

on

average

leaves

of

cm

or

more

the

double

mutant

and

the

overexpressor

plants

already

-LRB-

Fig.

-RRB-

As

the

plants

were

of

the

same

age

these

differences

most

likely

reflect

differences

in

plastochron

Alternatively

one

may

assume

large

temporal

differences

per

genotype

concerning

initiation

of

the

first

leaf

and/or

development

of

the

last

leaf

recorded

to

have

reached

cm

in

length

However

we

obtained

no

indications

for

such

discrepancies

and

noted

increasing

differences

in

rosette

density

during

the

entire

vegetative

growth

phase

of

wild

type

and

mutants

From

these

data

a

relative

-LRB-

-RRB-

increase

in

leaf

initiation

rate

of

the

transgenics

over

wild

type

can

be

deduced

a

value

that

quite

well

matches

the

observation

of

Schwab

et

al.

-LRB-

-RRB-

Leaf

initiation

rate

of

the

double

mutant

seem

to

be

increased

by

a

factor

of

-LRB-

-RRB-

in

comparison

to

wild

type

Fig.

formation

of

double

mutant

in

comparison

to

wild

type

and

overexpressor

-LRB-

a

-RRB-

Determination

of

the

average

number

of

rosette

leaves

of

at

least

mm

in

length

formed

by

the

primary

shoot

and

-LRB-

b

-RRB-

of

the

average

diameter

of

the

primary

shoot

apex

of

double

mutants

wild

type

and

overexpressor

plants

after

having

grown

for

days

in

SD

-LRB-

c

d

-RRB-

Average

circularity

-LRB-

c

-RRB-

and

cross

sectional

area

-LRB-

d

-RRB-

of

leaf

primordia

as

determined

from

cross

section

through

primary

shoot

as

shown

in

e

g.

Values

represent

averages

of

subsequent

primordia

as

indicted

in

different

shades

of

grey

according

to

the

legend

shown

in

d.

-LRB-

e

g

-RRB-

Cross

sections

through

primary

shoot

apices

of

a

wild

type

-LRB-

e

-RRB-

a

double

mutant

-LRB-

f

-RRB-

and

a

overexpressor

plant

-LRB-

g

-RRB-

after

having

grown

for

days

in

SD

Error

bars

in

a

d

indicate

standard

deviation

-LRB-

n

-RRB-

Successive

leaf

primordia

in

e

g

are

sequentially

numbered

starting

with

the

youngest

-LRB-

-RRB-

Marginal

meristem

on

one

side

of

leaf

number

-LRB-

counted

from

the

centre

outwards

-RRB-

is

encircled

in

e

and

g.

Scale

bar

in

e

g

represents

μm

After

sectioning

the

paraffin

embedded

material

a

small

but

not

significant

difference

in

average

SAM-diameter

of

the

double

mutant

and

wild

type

could

be

observed

-LRB-

Fig.

e

f

-RRB-

However

with

an

average

diameter

of

μm

the

overexpressor

showed

also

a

slight

but

yet

significant

-LRB-

P

-RRB-

decrease

in

its

SAM

size

compared

to

-LRB-

Fig.

e

g

-RRB-

Furthermore

both

the

double

mutant

and

the

overexpressor

exhibited

the

same

phyllotaxy

as

wild

type

with

rosette

leaves

initiated

either

clock

or

anticlockwise

with

an

angle

of

divergence

of

about

between

successive

leaves

and

forming

a

spiral

lattice

with

a

parastichy

pair

-LRB-

-RRB-

-LRB-

Fig.

g

-RRB-

From

these

observations

it

is

concluded

that

the

observed

shorter

plastochron

is

neither

the

result

nor

the

cause

of

an

altered

phyllotaxy

in

the

double

mutant

or

the

overexpressor

The

shortened

plastochron

however

seems

to

correlate

with

a

reduced

SAM

size

As

obvious

from

cross

sections

shown

in

Fig.

g

the

young

leaves

of

the

overexpressor

appear

more

roundish

in

shape

in

comparison

to

wild

type

leaves

at

similar

positions

In

particular

the

vacuolated

cells

surrounding

their

midveins

seem

larger

and

the

developing

laminas

reduced

i.e.

represented

by

less

small

cytoplasm

rich

cells

along

their

lateral

margins

In

addition

the

stipules

of

the

overexpressor

seem

to

be

more

prominent

In

these

aspects

of

leaf

development

the

double

mutant

seems

to

behave

intermediate

-LRB-

Fig.

-RRB-

To

quantify

the

difference

in

shape

and

size

of

the

leaf

primordia

we

determined

their

circularity

and

cross

sectional

area

-LRB-

see

Materials

and

methods

-RRB-

starting

from

the

first

leaf

cross

section

found

to

be

separated

from

the

apical

meristem

To

reduce

effects

due

to

imperfect

cross

sectioning

i.e.

not

absolute

perpendicular

to

the

longitudinal

axes

as

well

as

in

correlating

sequentially

numbered

primordia

of

different

sections

we

averaged

the

values

obtained

over

successive

primordia

As

shown

in

Fig.

circularity

of

the

youngest

leaf

primordia

was

highly

similar

between

the

different

genotypes

Circularity

of

subsequent

older

primordia

decreased

in

all

however

more

rapidly

in

wild

type

such

that

on

average

leaf

did

differ

significantly

between

the

genotypes

Interestingly

it

can

not

be

excluded

that

this

difference

is

a

direct

consequence

of

a

shortened

plastochron

in

the

mutant

lines

In

particular

as

the

leaf

initiation

rate

of

the

overexpressor

line

lies

roughly

one

and

a

half

times

above

that

of

wild

type

Accordingly

and

with

respect

to

absolute

age

leaf

of

the

overexpressor

may

be

more

comparable

to

leaves

of

wild

type

and

to

which

indeed

no

significant

difference

in

circularity

was

found

However

in

cross

sectional

area

these

young

leaves

differed

significantly

On

average

for

wild-type

leaves

and

for

leaf

of

the

overexpressor

It

is

known

that

the

shape

and

other

characteristics

of

newly

formed

leaves

progressively

change

in

correlation

with

the

vegetative

phase

transition

-LRB-

Telfer

et

al.

-RRB-

Furthermore

likely

due

to

a

changed

plastochron

the

correlation

between

leaf

number

and

flowering

seems

to

differ

for

the

mutants

and

wild

type

Therefore

we

further

investigated

the

possibility

that

the

observed

differences

correlate

with

relative

altered

timing

of

the

vegetative

phase

transition

Functional

analysis

of

and

during

the

vegetative

phase

transition

In

order

to

determine

the

timing

of

the

vegetative

phase

change

we

used

the

absence

or

presence

of

abaxial

trichomes

on

rosette

leaves

as

a

morphological

marker

for

leaves

formed

during

the

juvenile

or

adult

growth

phase

respectively

-LRB-

Telfer

et

al.

-RRB-

On

average

in

our

growing

conditions

the

first

abaxial

trichomes

developed

on

rosette

leaf

number

six

of

wild-type

plants

-LRB-

Table

-RRB-

The

double

mutants

displayed

its

first

abaxial

trichomes

on

leaf

number

twelve

and

the

overexpressor

on

leaf

number

Although

less

than

the

double

mutant

the

respective

single

mutants

also

developed

significantly

more

juvenile

leaves

than

wild

type

-LRB-

Table

-RRB-

We

distinguished

juvenile

and

adult

growth

phase

based

on

a

phase

dimorphism

i.e.

in

abaxial

trichomes

However

on

a

plant-physiological

level

the

juvenile

phase

in

Arabidopsis

is

characterized

as

being

incompetent

to

respond

to

photoperiodic

induction

of

flowering

-LRB-

Poethig

-RRB-

To

determine

if

this

competence

was

indeed

affected

small

populations

of

plants

of

wild

type

the

single

and

double

mutants

and

the

overexpressor

were

germinated

and

cultivated

for

weeks

in

non-inductive

SD

conditions

-LRB-

see

Materials

and

methods

-RRB-

The

plants

were

then

brought

into

continuous

light

and

batchwise

shifted

back

to

SD

after

either

or

days

Their

flowering

response

was

recorded

within

a

period

after

this

inductive

treatment

Plants

not

flowering

within

this

period

also

did

not

flower

after

months

like

plants

of

a

control

group

representing

all

genotypes

that

were

kept

continuously

in

SD

As

shown

in

Table

the

inductive

treatment

caused

a

flowering

response

in

of

the

plants

of

all

genotypes

Three

days

also

sufficed

to

induce

all

or

almost

all

of

the

wild-type

and

single

mutant

plants

whereas

the

response

of

the

double

mutant

and

particular

of

the

overexpressor

already

declined

One

day

of

continuous

light

still

enough

to

induce

half

or

more

of

the

wild-type

and

single

mutant

plants

did

not

induce

flowering

in

any

of

the

overexpressor

plants

and

only

in

one-tenth

of

the

double

mutants

These

results

thus

demonstrate

that

also

according

to

physiological

criteria

and

redundantly

promote

the

juvenile-to-adult

phase

transition

In

addition

other

SPL

genes

are

expected

to

contribute

as

well

based

on

the

behaviour

of

the

overexpressor

Table

floral

induction

in

wild

type

and

spl

mutantsPercentage

of

plantsa

induced

after

treatment

with

continuous

light

daysaTwo

populations

of

plants

per

genotype

were

evaluated

within

a

week

period

following

inductive

treatment

The

role

of

gibberellin

in

the

function

of

and

The

plant

hormone

gibberellin

-LRB-

GA

-RRB-

is

known

to

promote

flowering

in

many

plants

and

in

Arabidopsis

it

is

particularly

required

for

flowering

in

SD

-LRB-

Wilson

et

al.

-RRB-

Exogenous

application

of

GA

will

induce

abaxial

trichomes

on

leaves

where

these

are

normally

not

present

although

they

will

not

appear

earlier

than

leaf

three

-LRB-

Telfer

et

al.

-RRB-

In

order

to

test

whether

the

double

mutant

and

the

overexpressor

are

defective

in

gibberellin

sensitivity

or

biosynthesis

we

exogenously

applied

and

compared

the

onset

of

abaxial

trichome

production

to

mock

treated

and

wild-type

plants

grown

in

-LRB-

Fig.

-RRB-

Like

in

wild

type

the

treatment

strongly

reduced

the

number

of

rosette

leaves

without

abaxial

trichomes

i.e.

juvenile

leaves

with

about

a

factor

of

three

This

result

shows

that

the

double

mutant

and

the

overexpressor

remained

sensitive

to

However

in

both

the

double

mutant

and

the

overexpressor

the

amount

of

applied

could

not

reduce

the

number

of

juvenile

leaves

to

that

of

obtained

in

treated

wild

type

Fig.

of

GA

on

double

mutant

and

overexpressor

plants

in

comparison

to

wild

type

The

number

of

juvenile

rosette

leaves

formed

is

shown

for

plants

that

were

either

regularly

sprayed

with

-LRB-

μM

-RRB-

or

mock

treated

Error

bars

indicate

standard

deviation

-LRB-

n

-RRB-

Discussion

Transgenic

plants

constitutively

over-expressing

the

plant

specific

have

been

described

for

Arabidopsis

-LRB-

Schwab

et

al.

Wu

and

Poethig

-RRB-

and

rice

-LRB-

Xie

et

al.

-RRB-

Recently

overexpression

of

a

encoding

locus

has

also

been

shown

to

be

the

cause

of

the

natural

maize

mutant

phenotype

-LRB-

Chuck

et

al.

-RRB-

Interestingly

in

all

three

different

species

overexpression

of

this

well

conserved

miRNA

-LRB-

Axtell

and

Bartel

Arazi

et

al.

-RRB-

causes

a

similar

phenotype

suggesting

an

evolutionary

conserved

role

for

the

function

of

and

its

SPL

target

genes

Generally

in

comparison

to

the

respective

wild

type

over-expressing

plants

are

smaller

flower

later

tend

to

lose

apical

dominance

and

initiate

more

leaves

with

a

shorter

plastochron

targets

eleven

SBP-box

genes

in

Arabidopsis

but

the

results

presented

here

clearly

show

that

already

simultaneous

silencing

of

the

two

likely

paralogous

target

genes

and

well

approximate

the

over-expressing

phenotype

regarding

the

traits

mentioned

above

and

thus

act

as

important

and

functionally

redundant

transcription

factors

regulating

diverse

processes

in

shoot

maturation

and

most

likely

in

combination

with

other

regulated

SPL

genes

In

agreement

with

this

latter

statement

is

our

observation

that

in

addition

to

and

mutation

of

a

third

controlled

gene

-LRB-

T-DNA

insertion

line

-RRB-

results

in

triple

mutant

plants

showing

an

even

better

approximation

to

the

overexpressor

phenotype

-LRB-

Supplementary

Fig.

despite

the

absence

of

detectable

transcript

single

homozygous

mutant

plants

lack

an

obvious

mutant

phenotype

data

not

shown

-RRB-

In

greenhouse

LD

conditions

we

found

the

triple

mutant

to

have

produced

on

average

-LRB-

n

-RRB-

rosette

leaves

in

comparison

to

for

the

double

mutant

wild-type

and

the

overexpressor

plants

grown

in

parallel

produced

and

rosette

leaves

before

flowering

respectively

and

positively

regulate

the

juvenile-to-adult

growth

phase

transition

It

became

clear

with

the

detailed

analysis

of

Arabidopsis

overexpressors

by

Wu

and

Poethig

-LRB-

-RRB-

as

well

as

with

the

description

of

the

mutant

in

maize

by

Chuck

et

al.

-LRB-

-RRB-

that

one

of

the

major

phenotypic

alterations

in

over-expressing

plants

is

an

extended

juvenile

growth

phase

This

suggests

that

one

of

the

important

functions

of

targeted

SBP-box

genes

is

to

promote

the

vegetative

phase

change

In

agreement

with

this

observation

Wu

and

Poethig

-LRB-

-RRB-

showed

that

overexpression

of

the

regulated

gene

and

its

likely

paralogs

leads

to

a

greatly

shortened

juvenile

phase

in

Arabidopsis

Based

on

morphological

markers

-LRB-

abaxial

trichomes

-RRB-

and

physiological

parameters

-LRB-

response

to

photo-inductive

stimulus

-RRB-

we

found

that

the

double

mutant

exhibit

a

delayed

vegetative

phase

transition

and

therefore

conclude

that

both

genes

are

very

likely

involved

in

the

positive

regulation

of

this

developmental

process

in

a

redundant

fashion

Most

likely

because

of

this

redundancy

photoperiodic

induction

of

the

single

and

mutants

is

not

much

affected

However

the

effect

on

the

appearance

of

abaxial

trichomes

as

a

marker

for

the

juvenile-to-adult

phase

transition

appears

to

be

stronger

in

the

mutant

in

comparison

to

This

may

be

due

to

the

fact

that

in

shoot

apical

development

expression

of

starts

to

increase

before

that

of

-LRB-

Fig.

-RRB-

As

and

promote

the

juvenile-to-adult

growth

phase

transition

and

thus

competence

to

respond

to

photoperiodic

induction

of

flowering

it

is

interesting

to

note

that

both

and

themselves

are

strongly

upregulated

in

the

shoot

apex

upon

such

induction

-LRB-

Schmid

et

al.

-RRB-

An

additional

role

for

these

genes

in

establishing

inflorescence

or

floral

meristem

identity

may

thus

be

suggested

and

negatively

regulate

leaf

initiation

rate

Our

data

on

the

leaf

initiation

rate

suggest

that

and

act

negatively

on

leaf

initiation

Silencing

of

both

genes

leads

to

a

shorter

plastochron

Other

regulated

SPL

genes

may

act

similarly

as

the

plastochron

is

even

further

shortened

in

the

overexpressor

plants

A

few

mutants

are

known

to

cause

a

shortened

plastochron

and

most

of

them

simultaneously

affect

phyllotaxy

However

we

found

that

shortening

of

the

plastochron

due

to

loss

of

SPL

gene

function

is

neither

the

cause

nor

the

result

of

a

changed

spatial

distribution

of

leaf

primordia

at

the

shoot

apex

A

shorter

plastochron

without

an

altered

phyllotaxy

has

also

been

reported

for

two

rice

mutants

and

-LRB-

Itoh

et

al.

Miyoshi

et

al.

Kawakatsu

et

al.

-RRB-

encodes

a

cytochrome

protein

whereas

encodes

a

RNA

binding

protein

In

both

mutants

the

reduction

in

plastochron

is

accompanied

by

an

increase

in

the

size

of

the

SAM

and

a

higher

rate

of

cell

division

However

although

the

SAM

of

is

actually

smaller

than

that

of

it

has

a

shorter

plastochron

Furthermore

higher

cell

division

activity

associated

with

constitutive

overexpression

of

CyclinD

shortened

the

plastochron

in

tobacco

without

altering

SAM

size

-LRB-

Cockcroft

et

al.

-RRB-

These

observations

suggest

as

already

noticed

by

Kawakatsu

et

al.

-LRB-

-RRB-

that

not

SAM

size

but

rather

cell

division

rate

is

decisive

in

plastochron

duration

Also

our

results

may

lend

support

to

this

hypothesis

as

both

the

double

mutant

and

the

overexpressor

exhibit

a

clearly

shorter

plastochron

than

wild-type

plants

but

their

SAM

sizes

differ

only

marginally

Therefore

it

will

be

of

interest

to

determine

if

and

other

regulated

SPL

genes

control

cell

division

rate

in

the

SAM

and

if

so

in

particular

if

their

role

is

mediated

through

the

phytohormone

cytokinin

Not

only

is

cytokinin

a

major

positive

regulator

of

cell

proliferation

and

division

in

plants

-LRB-

Werner

et

al.

-RRB-

it

is

also

in

mutual

dependence

of

auxin

a

major

determinant

in

the

outgrowth

of

lateral

shoots

-LRB-

Sachs

and

Thimann

Chatfield

et

al.

-RRB-

This

latter

aspect

may

explain

the

reduced

apical

dominance

observed

for

the

double

mutant

and

the

overexpressor

Finally

mutants

disrupting

cytokinin

signalling

are

known

to

result

in

reduced

leaf

initiation

rates

in

addition

to

a

smaller

SAM

and

other

effects

-LRB-

Nishimura

et

al.

Higuchi

et

al.

-RRB-

Do

and

negatively

regulate

leaf

maturation

rate

Based

on

their

observations

of

the

pla

mutants

in

correlation

to

the

expression

of

the

respective

genes

in

leaf

primordia

but

not

in

the

SAM

Kawakatsu

et

al.

-LRB-

-RRB-

proposed

that

the

rate

of

leaf

maturation

plays

a

significant

role

in

regulating

the

rate

of

leaf

initiation

In

addition

these

authors

postulated

a

model

in

which

the

inhibitory

effect

of

pre-existing

leaf

primordia

on

the

initiation

of

the

next

leaf

is

lost

as

they

mature

Similarly

a

shortened

plastochron

in

the

mutants

and

the

overexpressor

may

also

be

due

to

precocious

maturation

of

their

leaves

as

suggested

by

our

comparison

of

cross

sections

through

successive

leaf

primordia

of

wild

type

and

the

overexpressor

Even

if

the

shape

i.e.

circularity

of

the

primordia

may

not

significantly

differ

after

correction

for

an

altered

plastochron

by

comparing

primordia

based

on

age

and

not

on

serial

sequence

number

their

cross

sectional

area

seems

to

increase

more

rapidly

in

the

overexpressor

In

particular

the

cells

surrounding

the

midvein

in

overexpressor

leaves

appear

to

enlarge

more

rapidly

and

do

not

modulate

the

role

of

GA

in

the

vegetative

phase

change

Exogenous

application

of

has

been

found

to

accelerate

abaxial

trichome

production

in

Arabidopsis

suggesting

that

gibberellins

function

to

regulate

vegetative

phase

change

-LRB-

Telfer

et

al.

-RRB-

These

findings

are

also

supported

by

mutant

analysis

For

example

in

spindly

-LRB-

spy

-RRB-

mutants

which

undergo

constitutive

GA

response

abaxial

trichomes

occur

on

leaves

initiated

significantly

earlier

than

in

wild

type

-LRB-

Jakobsen

and

Olszewski

Telfer

et

al.

-RRB-

On

the

other

hand

Telfer

et

al.

-LRB-

-RRB-

found

that

mutants

blocked

in

GA

biosynthesis

as

well

as

GA

insensitive

mutants

are

significantly

delayed

in

the

appearance

of

abaxial

trichomes

Loss-of-function

mutants

for

the

here

examined

and

genes

clearly

delay

the

appearance

of

abaxial

trichomes

Our

treatment

of

double

mutant

and

transgenic

plants

with

high

doses

of

showed

that

like

in

wild

type

their

number

of

juvenile

leaves

can

be

reduced

but

not

to

numbers

equal

to

those

found

for

similarly

treated

wild

type

In

fact

the

ratios

of

juvenile

to

adult

leaves

of

wild

type

and

mutant

phenotypes

remain

highly

comparable

to

untreated

plants

From

this

we

conclude

that

the

role

and

other

controlled

SPL

genes

play

in

the

vegetative

phase

change

is

unlikely

to

be

GA

mediated

although

a

minor

contribution

to

GA

sensitivity

can

not

be

excluded

Outlook

targeted

members

of

the

SBP-box

family

of

transcription

factors

in

both

mono

and

dicots

appear

to

play

an

important

role

as

positive

regulators

of

shoot

maturation

and

of

the

vegetative

to

reproductive

phase

transition

in

particular

Both

genetic

factors

i.e.

and

SBP-box

genes

have

also

been

suggested

to

be

major

determinants

in

the

transition

from

undifferentiated

to

differentiated

embryogenic

calli

of

rice

-LRB-

Luo

et

al.

-RRB-

As

the

interaction

between

SBP-box

genes

and

is

of

ancient

origin

in

land

plants

-LRB-

Arazi

et

al.

Riese

et

al.

-RRB-

it

will

be

interesting

to

learn

to

what

extent

their

molecular

interplay

is

of

importance

to

developmental

phase

transitions

in

plants

in

general

Electronic

supplementary

material

Below

is

the

link

to

the

electronic

supplementary

material

Supplementary

Fig.

Expression

of

as

detected

with

a

GUS

reporter

gene

construct

A

GUS

reporter

was

constructed

by

subcloning

a

bp

genomic

fragment

of

beginning

bp

upstream

of

the

start

codon

and

extending

to

bp

downstream

of

the

codon

into

the

binary

vector

-LRB-

kindly

provided

by

Guido

Jach

MPIZ

Cologne

-RRB-

A

ß-glucuronidase

-LRB-

GUS

-RRB-

reporter

was

subsequently

cloned

in

frame

shortly

downstream

of

the

start

codon

and

the

whole

construct

stably

transformed

into

Arabidopsis

background

-LRB-

A

B

-RRB-

GUS

staining

of

mature

embryo

gently

squeezed

out

of

the

imbibed

seed

-LRB-

A

-RRB-

and

of

a

ca.

day

old

seedling

grown

in

LD

-LRB-

B

-RRB-

Bars

in

A

and

B

represent

respectively

μm

and

mm

-LRB-

TIF

KB

-RRB-

Supplementary

Fig.

Phenotypes

of

and

related

mutants

-LRB-

A

-RRB-

Flowering

plants

homozygous

for

and

alleles

in

background

next

to

wild

type

-LRB-

B

-RRB-

Flowering

plants

homozygous

for

and

alleles

in

background

next

to

wild

type

-LRB-

C

-RRB-

Flowering

plants

from

left

to

right

of

wild

type

the

double

mutants

and

the

triple

mutant

and

the

overexpressor

-LRB-

TIF

KB

-RRB-

O O O O O O O O O B I O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

arabidopsis
juvenile phase
mirna
phase change
shoot maturation