Promiscuous

non-catalytic

tandem

carbohydrate-binding

modules

modulate

the

cell-wall

structure

and

development

of

transgenic

tobacco

-LRB-

Nicotiana

tabacum

-RRB-

plants

We

have

compared

heterologous

expression

of

two

types

of

carbohydrate

binding

module

-LRB-

CBM

-RRB-

in

tobacco

cell

walls

These

are

the

promiscuous

modules

-LRB-

a

tandem

and

its

single

derivative

-RRB-

derived

from

a

non-catalytic

of

the

Piromyces

equi

cellulase/hemicellulase

complex

and

the

less

promiscuous

tandem

from

the

Cellulomonas

fimi

xylanase

CBM-labelling

studies

revealed

that

binds

indiscriminately

to

every

tissue

of

the

wild-type

tobacco

stem

whereas

binding

of

was

restricted

to

vascular

tissue

The

promiscuous

had

much

more

pronounced

effects

on

transgenic

tobacco

plants

than

the

less

promiscuous

Reduced

stem

elongation

and

prolonged

juvenility

resulting

in

delayed

flower

development

were

observed

in

transformants

expressing

whereas

such

growth

phenotypes

were

not

observed

for

plants

Histological

examination

and

electron

microscopy

revealed

layers

of

collapsed

cortical

cells

in

the

stems

of

plants

whereas

cellular

deformation

in

the

stem

cortical

cells

of

transformants

was

less

severe

Altered

cell

expansion

was

also

observed

in

most

parts

of

the

stem

whereas

for

the

stem

this

was

observed

in

the

xylem

cells

only

The

cellulose

content

of

the

transgenic

plants

was

not

altered

These

results

support

the

hypothesis

that

CBMs

can

modify

cell

wall

structure

leading

to

modulation

of

wall

loosening

and

plant

growth

Introduction

Plant

cell

expansion

depends

on

the

interplay

between

internal

driving

forces

and

the

controlled

yielding

of

the

cell

wall

The

ability

of

the

wall

to

extend

under

this

internal

pressure

is

largely

determined

by

wall

loosening

processes

that

modify

the

interaction

of

cellulose

microfibrils

with

one

another

and/or

with

hemicelluloses

-LRB-

Darley

et

al.

-RRB-

Likewise

organ

growth

is

brought

about

by

differential

rates

of

wall

loosening

which

causes

differential

tissue

growth

and

consequently

tissue

tension

within

the

organ

-LRB-

Peters

and

Tomos

-RRB-

Thus

progressive

modification

of

the

cellulose

hemicellulose

-LRB-

mostly

cellulose

xyloglucan

-RRB-

networks

is

important

in

plant

development

In

addition

to

their

effect

on

plant

growth

there

are

indications

that

interactions

of

hemicelluloses

with

cellulose

can

affect

cellulose

fibre

properties

Using

the

Acetobacter

model

system

it

was

shown

that

cellulose

glucomannan

and

cellulose

galactomannan

composites

cause

and

loss

of

the

crystallinity

respectively

of

the

native

bacterial

cellulose

-LRB-

Whitney

et

al.

-RRB-

For

cellulose

xyloglucan

composites

loss

of

cellulose

crystallinity

was

observed

-LRB-

Whitney

et

al.

-RRB-

It

was

also

shown

that

the

composites

were

less

stiff

leading

to

a

dramatic

reduction

in

mechanical

strength

for

example

reduction

in

composites

with

xyloglucan

-LRB-

Whitney

et

al.

-RRB-

There

are

also

reports

which

indicate

that

fibre

properties

depend

on

the

side-chain

substitution

of

the

hemicelluloses

Studies

on

Arabidopsis

thaliana

mutants

with

mutations

of

the

and

genes

which

encode

xyloglucan-specific

fucosyl

and

galactosyl

transferases

respectively

revealed

that

the

tensile

strength

of

the

fibre

was

enhanced

by

galactosylation

of

the

xyloglucan

-LRB-

Pena

et

al.

Ryden

et

al.

-RRB-

There

are

indications

that

the

interactions

between

mannan-based

polysaccharides

and

cellulose

can

affect

the

structural

properties

of

the

cell

wall

-LRB-

Carpita

et

al.

Hosoo

et

al.

-RRB-

An

immuno-localisation

study

of

A.

thaliana

revealed

that

mannans

were

present

in

all

thickened

cell

walls

of

stems

and

leaves

including

those

of

the

xylem

parenchyma

and

epidermis

-LRB-

Handford

et

al.

-RRB-

Mannan

transglycosylase

was

recently

identified

and

characterized

as

cell

wall

enzyme

acting

on

mannan-based

plant

polysaccharides

in

primary

cell

walls

of

higher

plants

-LRB-

Schroder

et

al.

-RRB-

This

body

of

evidence

thus

supports

an

emerging

idea

that

mannan-based

polysaccharides

in

cell

walls

may

have

a

role

analogous

to

that

of

xyloglucans

introducing

flexibility

and

forming

a

growth-restraining

network

with

cellulose

Proteins

for

example

expansins

have

been

shown

to

be

involved

in

modifying

cellulose

hemicellulose

interaction

thereby

loosening

the

cell

wall

and

ultimately

regulating

growth

and

developmental

processes

in

which

rearrangements

in

the

cell

wall

are

thought

to

be

important

-LRB-

Cho

and

Cosgrove

Choi

et

al.

Pien

et

al.

Zenoni

et

al.

-RRB-

There

is

evidence

that

-LRB-

heterologous

-RRB-

carbohydrate-binding

modules

-LRB-

CBMs

-RRB-

could

also

affect

these

interactions

-LRB-

Levy

et

al.

Shpigel

et

al.

-RRB-

leading

to

altered

structural

morphology

in

transgenic

plants

-LRB-

Kilburn

et

al.

Quentin

Shoseyov

et

al.

-RRB-

or

plant

growth

-LRB-

Safra-Dassa

et

al.

-RRB-

CBMs

are

non-catalytic

polysaccharide-recognizing

modules

appended

to

glycoside

hydrolases

that

often

degrade

water-insoluble

polysaccharides

and

concentrate

the

enzymes

on

their

target

polysaccharides

CBMs

are

grouped

into

families

based

on

amino

acid

sequence

similarities

there

are

currently

families

of

CBMs

in

the

database

-LRB-

-RRB-

The

biochemical

characteristics

of

members

of

most

of

the

CBM

families

have

been

established

including

those

that

target

non-cell

wall

polysaccharides

such

as

starch

and

glycogen

-LRB-

reviewed

by

Boraston

et

al.

-RRB-

Although

most

CBMs

are

linked

to

the

catalytic

modules

of

carbohydrate-active

enzymes

some

non-catalytic

carbohydrate-binding

proteins

are

not

appended

to

any

catalytic

module

Examples

of

such

proteins

include

putative

CBMs

of

the

A.

thaliana

former

family

now

-LRB-

GenBank

accession

no

-RRB-

-LRB-

Henrissat

et

al.

-RRB-

with

olive

pollen

allergen

Ole

e

-LRB-

GenBank

accession

no

-RRB-

being

the

first

characterized

member

of

this

family

-LRB-

Barral

et

al.

-RRB-

Another

example

is

the

non-catalytic

protein

-LRB-

-RRB-

-LRB-

GenBank

accession

no

-RRB-

which

is

a

component

of

the

anaerobic

fungus

Piromyces

equi

cellulase

hemicellulose

complex

-LRB-

Freelove

et

al.

-RRB-

Because

of

the

heterogeneous

nature

of

the

cell

wall

polysaccharides

several

CBMs

have

evolved

structures

which

enable

them

to

recognize

more

than

one

wall

polysaccharide

-LRB-

Boraston

et

al.

-RRB-

Promiscuous

recognition

of

carbohydrates

by

CBMs

enables

the

associated

enzyme

-LRB-

complex

-RRB-

to

be

more

efficient

in

cell

wall

degradation

McCartney

et

al.

-LRB-

-RRB-

showed

that

different

xylan-specific

CBM

families

can

vary

substantially

in

their

binding

to

the

cell

wall

This

indicates

that

the

specificity

of

CBMs

is

so

intricate

that

after

expression

in

planta

their

effects

on

cell

wall

modification

might

be

very

local

To

really

affect

wall

architecture

it

might

be

important

to

select

CBMs

that

bind

to

different

kinds

of

polysaccharide

-LRB-

promiscuous

-RRB-

so

that

the

effect

is

not

confined

to

one

location

or

cell

type

Most

research

activity

relating

to

the

potential

use

of

CBMs

for

plant

cell

wall

modification

has

been

on

cellulose-specific

CBMs

In

this

investigation

we

have

used

different

CBMs

which

not

only

recognise

hemicellulose

as

opposed

to

but

are

also

to

some

extent

promiscuous

The

modules

are

an

excellent

model

of

promiscuity

in

protein

carbohydrate

recognition

The

modules

bind

soluble

glucomannan

galactomannan

β-glucan

and

hydroxyethylcellulose

-LRB-

HEC

-RRB-

and

insoluble

forms

of

cellulose

and

mannan

-LRB-

Charnock

et

al.

Freelove

et

al.

-RRB-

For

all

the

ligands

tested

two

appended

copies

of

-LRB-

-RRB-

had

affinity

substantially

higher

than

the

additive

value

of

the

individual

modules

This

indicates

the

two

CBMs

act

in

synergy

to

bind

all

their

target

ligands

with

having

more

affinity

than

The

tandem

was

additionally

able

to

interact

with

xyloglucan

and

different

forms

of

xylans

whereas

the

single

CBM

had

little

affinity

for

these

polysaccharides

-LRB-

Freelove

et

al.

McCartney

et

al.

-RRB-

An

ex-situ

labelling

study

has

revealed

that

the

tandem

modules

bind

strongly

to

the

cell

walls

of

the

maize

coleoptile

sheath

and

the

enclosed

developing

leaves

-LRB-

McCartney

et

al.

-RRB-

This

is

indicative

of

the

great

abundance

of

their

interacting

ligands

in

the

primary

cell

walls

of

grasses

It

was

therefore

of

interest

to

investigate

the

heterologous

expression

of

these

promiscuous

CBMs

in

plants

and

more

importantly

to

compare

their

effects

with

those

of

less

promiscuous

CBMs

whose

characteristics

are

described

below

Some

ligand

promiscuity

is

also

observed

for

two

consecutive

family

CBMs

-LRB-

and

-RRB-

from

Cellulomonas

fimi

xylanase

The

tandem

-LRB-

note

the

underlining

used

throughout

this

report

to

distinguish

it

from

-RRB-

has

very

strong

affinity

for

xylan

and

much

lower

affinity

for

insoluble

acid-swollen

cellulose

-LRB-

Bolam

et

al.

-RRB-

It

also

has

an

to

greater

affinity

for

xylan

than

the

individual

modules

We

have

expressed

two

constructs

the

tandem

and

the

single

derivative

and

the

tandem

construct

in

tobacco

-LRB-

all

proteins

targeted

the

cell

wall

-RRB-

under

the

control

of

cauliflower

mosaic

virus

-LRB-

CaMV

-RRB-

promoter

It

is

shown

that

cell

wall

structural

alterations

in

transgenic

plants

expressing

are

more

severe

than

those

in

transformants

Our

results

indicate

that

promiscuous

carbohydrate

binding

modules

can

modulate

cell

wall

structure

and

the

development

of

transgenic

tobacco

plants

Materials

and

methods

Preparation

of

constructs

Three

constructs

were

prepared

for

expression

in

tobacco

plants

one

for

single

one

for

tandem

and

one

for

tandem

The

and

constructs

were

prepared

by

amplifying

their

respective

gene

fragments

from

and

recombinant

plasmid

vectors

used

for

cloning

and

expression

in

Escherichia

coli

-LRB-

Bolam

et

al.

Freelove

et

al.

-RRB-

For

the

fragment

the

polymerase

chain

reaction

-LRB-

PCR

-RRB-

was

performed

using

primers

that

included

BamHI

and

SmaI

recognition

sites

-LRB-

and

the

BamHI

and

SmaI

sites

respectively

are

underlined

-RRB-

The

three

bases

highlighted

in

bold

type

are

the

stop

codon

The

amplified

fragment

of

the

tandem

was

digested

with

BamHI

and

SmaI

-LRB-

Invitrogen

The

Netherlands

-RRB-

and

cloned

into

a

similarly

digested

binary

vector

-LRB-

Hellens

et

al.

-RRB-

Similarly

amplification

of

the

single

fragment

were

performed

using

primers

with

the

same

restriction

sites

as

for

the

tandem

-LRB-

and

the

BamHI

and

SmaI

sites

respectively

are

underlined

-RRB-

The

amplified

fragment

was

digested

with

BamHI

and

SmaI

-LRB-

Invitrogen

The

Netherlands

-RRB-

and

cloned

into

binary

vector

Similarly

amplification

and

cloning

of

the

fragment

were

performed

using

primers

that

included

SmaI

and

EcoRI

recognition

sites

at

either

end

-LRB-

and

the

SmaI

and

EcoRI

sites

respectively

are

underlined

-RRB-

The

cloning

of

the

three

fragments

was

in-frame

with

fusion

peptides

of

two

sequences

which

were

cloned

upstream

in

the

binary

vector

The

first

sequence

codes

for

a

tobacco

transit

peptide

for

transporting

a

cellular

glycoprotein

across

the

plasma

membrane

into

the

cell

wall

-LRB-

Wittink

et

al.

-RRB-

whereas

the

second

sequence

encodes

a

hexahistidine

tag

The

sequence

of

the

transit

peptide

was

obtained

as

a

product

of

annealing

two

oligonucleotide

primers

-LRB-

agcttatgggaagtggtaaagtaacatttgtggctttgctactttgcctctccgtaggggtgatagctt-3

-RRB-

and

-LRB-

ctagaagctatcacccctacggagaggcaaagtagcaaagccacaaatgttactttaccacttcccata-3

-RRB-

The

underlined

nucleotides

produced

overhangs

of

HindIII

and

XbaI

respectively

This

fragment

was

then

cloned

into

the

HindIII

and

XbaI

cloning

sites

of

the

vector

Similarly

the

sequence

of

the

hexahistidine

tag

was

obtained

and

ligated

into

XbaI

and

BamHI

sites

as

annealing

product

of

-LRB-

-RRB-

and

-LRB-

-RRB-

with

the

underlined

nucleotides

producing

overhangs

of

XbaI

and

BamHI

respectively

The

purpose

of

using

the

hexahistidine

tag

was

to

facilitate

affinity

purification

of

the

proteins

The

control

construct

did

not

contain

any

of

the

CBMs

the

transit

peptide

or

the

hexahistidine

epitope

tag

All

constructs

were

sequenced

to

verify

their

integrity

Immunofluorescence

detection

of

and

binding

and

microscopy

Wild-type

tobacco

plants

-LRB-

Nicotiana

tabacum

L.

-RRB-

were

grown

at

°C

under

h

light

and

h

dark

for

weeks

Regions

of

stem

were

excised

and

immediately

fixed

in

PEM

buffer

-LRB-

mmol

L

PIPES

-LRB-

piperazine-N

N

bis

-LSB-

acid

-RSB-

-RRB-

mmol

L

EGTA

-LRB-

ethylene

glycol

bis

-LRB-

β-aminoethyl

ether

-RRB-

N

N

N

N

tetraacetic

acid

-RRB-

and

mmol

L

pH

-RRB-

containing

paraformaldehyde

Samples

were

then

dehydrated

in

an

ethanol

series

-LRB-

and

each

for

min

at

°C

ethanol

and

wax

°C

overnight

-RRB-

and

embedded

in

Steedman

's

wax

Sections

were

cut

to

a

thickness

of

μm

and

collected

on

polylysine-coated

microscope

slides

-LRB-

BDH

Laboratory

Supplies

Dorset

UK

-RRB-

de-waxed

and

re-hydrated

through

a

reverse

ethanol

series

into

phosphate-buffered

saline

-LRB-

PBS

-RRB-

-LRB-

for

min

and

water

for

min

each

and

a

final

step

of

water

min

-RRB-

For

CBM

labelling

sections

were

incubated

in

milk

protein/PBS

-LRB-

to

reduce

any

non-specific

binding

of

proteins

-RRB-

and

μg

mL

for

h.

Immunofluorescence

detection

of

binding

of

and

to

sections

and

microscopy

were

performed

as

described

elsewhere

-LRB-

McCartney

et

al.

-RRB-

Tobacco

transformation

and

regeneration

In

vitro

leaf

explants

of

N.

tabacum

cv

Samsun

NN

were

used

for

Agrobacteriumtumefaciens-mediated

transformation

Cloned

binary

vector

was

co-transformed

with

the

helper

plasmid

pSoup

-LRB-

Hellens

et

al.

-RRB-

into

A.tumefaciens

strain

by

electroporation

This

was

plated

out

on

LB-agar

plates

containing

kanamycin

-LRB-

μg

mL

-RRB-

and

rifampicin

-LRB-

μg

mL

-RRB-

and

incubated

for

three

days

at

°C

to

obtain

single

colonies

The

integrity

of

the

binary

plasmids

was

tested

by

restriction

analysis

of

plasmids

isolated

from

A.

tumefaciens

cultures

used

for

plant

transformation

LB

medium

-LRB-

mL

-RRB-

without

selection

was

inoculated

with

a

single

colony

and

incubated

overnight

at

°C

The

grown

culture

-LRB-

μL

-RRB-

was

added

to

a

Petri

dish

containing

mL

Murashige

Skoog

-LRB-

-RRB-

Leaf

explants

without

major

veins

and

edges

from

young

seedlings

were

transferred

upside-down

in

to

the

medium

Agrobacterium

infection

was

performed

in

the

dark

for

days

at

°C

Two

controls

were

used

for

the

procedure

untransformed

wild-type

control

and

empty

control

without

an

insert

Leaf

explants

were

washed

in

three

changes

of

liquid

washing

medium

of

with

mg

L

carbenicillin

The

washed

explants

were

transferred

upside-up

to

an

plate

containing

mg

L

α-naphthalene

acetic

acid

-LRB-

NAA

-RRB-

mg

L

-LRB-

BAP

-RRB-

mg

L

kanamycin

and

mg

L

carbenicillin

and

incubated

overnight

in

the

dark

at

°C

Plants

were

transferred

to

a

growth

chamber

-LRB-

°C

-RRB-

where

they

were

gradually

adapted

to

light

and

incubated

for

callus

induction

After

two

weeks

in

culture

calli

generated

were

transferred

to

shoot-inducing

medium

containing

mg

L

NAA

mg

L

BAP

mg

L

kanamycin

and

mg

L

carbenicillin

Well-formed

shoots

were

harvested

and

transferred

to

root-inducing

medium

plate

containing

mg

L

kanamycin

mg

L

carbenicillin

and

mg

L

vancomycin

Transformed

plantlets

were

transferred

to

the

greenhouse

to

generate

mature

plants

Between

fifteen

to

eighteen

antibiotic-resistant

tobacco

transformants

were

generated

for

each

of

the

three

CBM

transgenes

RNA

gel

blot

analysis

of

transgenic

plants

Total

RNA

was

isolated

from

to

g

transformed

in-vitro

shoots

as

described

elsewhere

-LRB-

Kuipers

et

al.

-RRB-

Aliquots

of

μg

per

lane

were

separated

on

a

formaldehyde

agarose

gel

and

blotted

onto

a

Hybond-N

nylon

membrane

-LRB-

Amersham

-RRB-

by

vacuum

transfer

in

mol

L

sodium

hydroxide

The

membranes

were

hybridized

with

probes

consisting

of

ng

-LSB-

-RSB-

dCTP-labelled

restricted

fragments

of

or

The

radioactive

labelled

blots

were

exposed

to

X-OMAT

S

and

AR

scientific

imaging

films

-LRB-

Kodak

-RRB-

at

°C

The

blots

were

re-hybridized

after

stripping

with

a

bp

-LSB-

-RSB-

dCTP-labelled

fragment

of

a

tobacco

ribosomal

RNA

gene

-LRB-

GenBank

accession

no

-RRB-

as

a

control

The

ribosomal

probe

was

amplified

from

tobacco

genomic

DNA

using

oligonucleotides

and

for

PCR

amplification

Protein

extraction

Protein

extracts

were

prepared

from

transformed

tobacco

stems

using

mmol

L

sodium

phosphate

buffer

-LRB-

pH

-RRB-

mol

L

NaCl

mmol

L

EDTA

-LRB-

v/v

-RRB-

Tween

protease

inhibitor

-LRB-

Complete

Roche

-RRB-

mg

mL

PVPP

-LRB-

adapted

from

Vincken

et

al.

-RRB-

The

protein

samples

were

incubated

for

h

at

°C

with

gentle

shaking

After

incubation

the

samples

were

centrifuged

at

g

and

°C

for

min

The

supernatant

containing

the

soluble

protein

fraction

was

then

collected

The

protein

samples

were

then

concentrated

with

Centricon

devices

-LRB-

Millipore

-RRB-

according

to

supplier

's

procedure

Protein

concentrations

were

determined

with

an

ESL

protein

assay

-LRB-

Roche

-RRB-

using

BSA

as

standard

Incubation

of

RGS

-LRB-

HIS

-RRB-

size

marker

in

protein

extract

of

wild-type

tobacco

Protein

extracts

were

prepared

from

wild-type

tobacco

as

described

above

either

lacking

or

supplemented

with

mmol

L

EDTA

and

protease

inhibitor

-LRB-

Complete

Roche

-RRB-

RGS

-LRB-

HIS

-RRB-

size

marker

was

incubated

with

the

protein

extracts

as

follows

A

batch

of

RGS

-LRB-

HIS

-RRB-

size

marker

-LRB-

Qiagen

-RRB-

was

dissolved

in

μL

mmol

L

Tris

pH

such

that

the

final

concentration

of

the

individual

proteins

varied

from

to

ng

μL

The

epitope-tagged

size

marker

-LRB-

μL

-RRB-

was

added

to

μL

tobacco

extracts

The

mixtures

were

incubated

at

room

temperature

for

approximately

h

As

a

control

RGS

-LRB-

HIS

-RRB-

size

marker

was

incubated

in

mmol

L

Tris

pH

Western

blot

analysis

The

protein

samples

were

separated

with

SDS-PAGE

on

SDS-polyacrylamide

gels

-LRB-

Mini-Protean

II

apparatus

Bio-Rad

-RRB-

The

separated

proteins

were

electroblotted

on

to

a

nitrocellulose

membrane

in

mmol

L

glycine

mmol

L

Tris

base

-LRB-

w/v

-RRB-

SDS

-LRB-

v/v

-RRB-

ethanol

pH

Blocking

against

non-specific

binding

was

performed

for

h

with

-LRB-

w/v

-RRB-

BSA

-LRB-

Sigma

-RRB-

in

TBS

-LRB-

mmol

L

Tris

mmol

L

NaCl

pH

-RRB-

The

membrane

was

then

incubated

with

a

dilution

of

the

primary

anti-RGS

HIS

antibody

-LRB-

Qiagen

-RRB-

in

TBS

solution

containing

-LRB-

w/v

-RRB-

BSA

for

h

The

membrane

was

washed

twice

in

TBS-TT

buffer

-LRB-

mmol

L

Tris

base

mmol

L

NaCl

pH

-LRB-

v/v

-RRB-

Tween

-LRB-

v/v

-RRB-

Triton

-RRB-

and

once

in

TBS

buffer

for

min

each

The

membrane

was

then

washed

with

dilution

horseradish

peroxidase

-LRB-

HRP

-RRB-

conjugated

sheep

anti-mouse

antibody

-LRB-

Amersham

-RRB-

in

TBS

buffer

containing

-LRB-

w/v

-RRB-

non-fat

dried

milk

The

membrane

was

washed

in

TBS-TT

buffer

four

times

min

each

and

subsequently

submerged

in

Supersignal

ULTRA

substrate

working

solution

-LRB-

Pierce

-RRB-

which

is

a

mixture

of

equal

parts

of

Ultra

luminol/enhancer

solution

and

Ultra

stable

peroxide

solution

Finally

the

membrane

was

exposed

to

X-OMAT

S

Scientific

Imaging

films

-LRB-

Kodak

-RRB-

at

room

temperature

Monitoring

the

growth

of

the

transformed

plants

To

monitor

growth

of

the

transformed

plants

with

time

three

replicates

per

line

were

grown

in

the

greenhouse

The

transformants

were

grown

in

two

series

the

and

series

with

each

series

having

its

separate

controls

Height

measurements

of

the

stems

were

taken

weekly

for

nine

weeks

starting

from

week

two

after

transplant

The

height

measurements

of

six

individual

high

expressers

each

from

transgenic

lines

containing

and

constructs

six

transformed

controls

and

six

untransformed

controls

from

week

to

week

were

subjected

to

one-way

analysis

of

variance

-LRB-

ANOVA

-RRB-

using

an

α

of

To

test

whether

the

differential

stem

elongation

was

significant

a

Fisher

's

unprotected

least

significant

difference

test

was

used

The

hypothesis

of

no

difference

between

plant

heights

was

rejected

at

an

error

of

Light

microscopy

Three

individual

plants

per

transgenic

tobacco

line

and

three

wild-type

plants

as

control

were

used

for

microscopic

examination

Three

transgenic

lines

were

used

per

construct

-LRB-

two

high

expressers

and

one

low

expresser

-RRB-

Stem

samples

were

taken

from

the

second

internode

from

the

top

of

the

plant

Stem

sections

mm-thick

were

fixed

in

glutardialdehyde

-LRB-

Merck

-RRB-

and

paraformaldehyde

-LRB-

Merck

-RRB-

in

mol

L

phosphate

buffer

containing

Triton

for

h

The

samples

were

then

washed

and

dehydrated

in

an

ethanol

series

After

dehydration

they

were

embedded

in

Technovit

resin

-LRB-

USA

-RRB-

-LRB-

Kuroiwa

et

al.

-RRB-

Sections

μm-thick

were

stained

with

toluidine

blue

-LRB-

Aldrich

-RRB-

and

examined

under

a

bright

field

microscope

Cryo-scanning

electron

microscopy

and

quantification

of

cell

size

and

cell

wall

diameter

One

high

expresser

from

each

of

the

and

transgenic

lines

and

one

individual

plant

each

from

their

respective

controls

were

used

for

cryo-SEM

examination

The

plants

that

were

analysed

were

chosen

on

the

basis

of

the

results

obtained

from

the

extensive

light

microscopic

analysis

that

showed

a

consistent

pattern

of

alterations

in

high-expressing

transgenic

plants

Stem

sampling

was

the

same

as

for

the

light

microscopy

Stem

samples

mm-thick

were

mounted

in

a

brass

cylindrical

sample

holder

with

TBS

tissue

freezing

medium

-LRB-

EMS

Washington

PA

USA

-RRB-

The

frozen

samples

were

placed

in

a

sample

holder

in

a

cryo-ultra

microtome

-LRB-

Reichert

Ultracut

-RRB-

and

cut

at

a

specimen

temperature

of

°C

These

samples

were

first

planed

with

a

glass

knife

after

which

the

surface

was

planed

with

a

diamond

knife

-LRB-

Histo

no

trough

mm

°C

Drukker

International

The

Netherlands

-RRB-

After

planing

the

samples

were

placed

in

a

dedicated

cryo-preparation

chamber

-LRB-

CT

HF

Oxford

instruments

UK

-RRB-

All

the

samples

in

the

cryo-preparation

chamber

were

freeze

dried

for

min

at

°C

and

Pa

and

subsequently

sputtered

with

a

layer

of

Pt

The

samples

were

cryo-transferred

into

the

field

emission

scanning

microscope

-LRB-

Jeol

Japan

-RRB-

on

the

sample

stage

at

°C

All

images

were

recorded

digitally

-LRB-

Orion

E.L.I.

sprl

Belgium

-RRB-

at

a

scan

rate

of

s

-LRB-

full

frame

-RRB-

at

the

size

of

bit

The

images

were

optimized

and

resized

for

publication

by

use

of

Adobe

Photoshop

CS

To

quantify

the

micrographic

images

files

were

opened

with

Image

J

software

developed

at

the

National

Institute

of

Health

USA

-LRB-

-RRB-

The

surface

area

of

three

hundred

cortical

and

xylem

cells

was

measured

as

shown

by

the

insets

in

Figs.

and

One

hundred

cells

per

field

of

view

were

measured

in

three

replicates

Similarly

cell

wall

thickness

of

cells

was

quantified

as

shown

by

the

inset

in

Fig.

Fifty

cells

per

field

of

view

were

measured

in

three

replicates

Standard

deviations

of

three

measurements

were

determined

Isolation

of

cell

wall

material

and

analysis

of

the

cellulose

content

Stem

samples

from

one

high

expresser

of

the

and

series

one

vector

control

and

one

wild-type

control

plant

were

ground

to

a

fine

powder

in

liquid

nitrogen

For

each

isolation

g

of

this

stem

material

was

extracted

in

a

mmol

L

Tris

-LSB-

HCl

-RSB-

pH

solution

containing

SDS

for

h

at

room

temperature

-LRB-

RT

-RRB-

with

continuous

shaking

The

cell

wall

material

CWM

was

spun

down

by

centrifugation

at

rpm

for

min

Subsequently

the

residue

was

washed

with

water

ethanol

and

acetone

then

air-dried

The

different

cell

wall

materials

-LRB-

mg

-RRB-

were

hydrolysed

in

mL

mol

L

TFA

The

TFA-insoluble

cellulose

was

spun

down

at

rpm

for

min

and

the

pellet

was

suspended

in

sulfuric

acid

The

suspension

was

heated

and

diluted

appropriately

to

determine

the

cellulose

content

colorimetrically

using

anthrone

as

colouring

agent

-LRB-

Updegraff

-RRB-

The

acid

hydrolysis

was

performed

in

quadruplicate

Results

Expression

of

bacterial

and

genes

in

tobacco

To

detect

expression

of

the

introduced

CBM

genes

in

the

tobacco

plant

we

performed

Northern

blot

analysis

with

total

RNA

Figure

shows

representatives

of

three

classes

of

transcript

expression

-LRB-

high

low

and

-RRB-

of

the

three

CBM

modules

as

revealed

by

the

Northern

analysis

Ten

of

the

plants

were

classified

as

high

expressers

five

as

low

expressers

and

two

as

expressers

For

the

plants

ten

lines

were

classified

as

high

expressers

seven

as

low

expressers

and

two

as

expressers

-LRB-

note

that

expressers

may

include

plants

with

very

low

RNA

expression

which

could

not

be

detected

-RRB-

For

the

plants

six

each

were

classified

as

high

and

low

expressers

whereas

four

plants

were

classified

as

expressers

Preliminary

attempts

at

purifying

CBM

proteins

using

affinity

purification

with

the

hexahistidine

tag

were

not

successful

-LRB-

data

not

shown

-RRB-

Similarly

Western

detection

of

a

hexahistidine

epitope-tagged

fungal

elicitor

protein

that

was

infiltrated

into

the

apoplast

of

leaves

did

not

succeed

in

the

tomato

plants

but

succeeded

in

Arabidopsis

thaliana

-LRB-

van

Esse

et

al.

-RRB-

The

results

indicated

that

the

histidine

tag

was

cleaved

in

the

tomato

plants

but

not

in

A.

thaliana

These

observations

have

since

led

to

the

idea

that

the

hexahistidine

epitope

tags

have

cleavage

sites

recognized

by

proteases

which

are

specific

for

the

cell

walls

of

the

solanaceous

species

Fig.

analysis

of

and

genes

in

transgenic

tobacco

leaves

A

differential

transcript

expression

pattern

is

shown

in

the

upper

panel

with

the

representative

of

each

class

in

the

three

transgenic

lines

-LRB-

a

-RRB-

-LRB-

b

-RRB-

and

-LRB-

c

-RRB-

Line

in

aline

in

both

b

and

c

represent

high

expressers

Lines

and

represent

low

expressers

Line

in

a

line

in

b

and

line

in

c

represent

expressers

The

lower

panel

shows

RNA

blots

for

the

ribosomal

RNA

internal

control

for

each

of

the

series

We

corroborated

this

notion

by

incubating

a

protein

ladder

consisting

of

five

different

proteins

of

known

sizes

each

containing

an

N-terminal

RGS

-LRB-

HIS

-RRB-

tag

in

protein

extracts

of

wild-type

tobacco

-LRB-

Compier

-RRB-

The

tagged

proteins

of

the

size

marker

that

were

incubated

in

tobacco

extract

without

EDTA

and

protease

inhibitor

were

not

detectable

on

a

Western

blot

whereas

the

markers

incubated

in

extract

supplemented

with

EDTA

and

protease

inhibitor

were

detected

This

suggested

that

extracts

from

tobacco

contained

proteases

that

could

remove

the

hexahistidine

epitope

tag

Because

grinding

of

the

plant

material

and

protein

extraction

were

performed

at

°C

and

the

protein

extraction

buffer

contained

EDTA

and

protease

inhibitor

removal

of

the

RGS

-LRB-

HIS

-RRB-

from

the

fusion

proteins

had

probably

occurred

already

in

planta

most

probably

extracellularly

It

should

be

noted

that

with

the

presence

of

the

signal

peptide

the

fusion

proteins

would

be

secreted

into

the

apoplast

Cleavage

of

the

RGS

-LRB-

HIS

-RRB-

was

therefore

likely

to

occur

after

translocation

into

the

extracellular

space

and

have

different

binding

patterns

in

the

stem

Immunofluorescence

detection

of

and

binding

to

stems

of

the

wild-type

tobacco

plant

was

performed

order

to

determine

a

priori

the

particular

tissues

of

the

stem

where

the

introduced

module

would

bind

Indirect

immunofluorescence

micrographs

-LRB-

Fig.

-RRB-

revealed

that

binding

is

restricted

to

the

secondary

cell

walls

of

stem

vascular

tissue

xylem

cells

and

phloem

fibres

-LRB-

Fig.

-RRB-

whereas

indiscriminately

binds

to

every

tissue

of

the

stem

from

the

epidermis

through

the

cortex

and

the

vascular

tissue

to

the

pith

parenchyma

in

the

innermost

part

-LRB-

Fig.

-RRB-

In

the

control

-LRB-

no

CBM

-RRB-

weak

autofluorescence

was

observed

in

the

xylem

vessel

cell

walls

-LRB-

Fig.

-RRB-

This

ex-situ

evidence

for

unselective

binding

of

the

to

both

the

primary

and

the

secondary

cell

walls

of

the

mature

stem

of

the

tobacco

plant

thus

complements

earlier

evidence

for

its

binding

to

a

predominantly

primary-walled

maize

coleoptile

-LRB-

McCartney

et

al.

-RRB-

It

should

also

be

noted

that

tobacco

is

a

dicotyledonous

plant

whereas

maize

is

monocotyledonous

and

that

there

are

many

differences

between

the

polysaccharide

composition

of

the

walls

of

these

species

Both

results

thus

confirm

the

binding

promiscuity

of

the

tandem

as

previously

characterized

-LRB-

Charnock

et

al.

Freelove

et

al.

-RRB-

We

also

conclude

that

the

ligands

are

present

in

the

vascular

tissue

of

the

stem

Fig.

obtained

by

indirect

immunofluorescence

microscopy

showing

binding

of

-LRB-

a

-RRB-

-LRB-

b

-RRB-

and

control

-LRB-

no

CBM

-RRB-

-LRB-

c

-RRB-

to

transverse

sections

of

wild-type

tobacco

stem

The

binds

indiscriminately

to

every

tissue

of

the

stem

from

the

epidermis

through

the

cortex

and

the

vascular

tissue

to

the

pith

parenchyma

in

the

innermost

part

whereas

binding

is

restricted

to

the

vascular

tissues

ct

cortical

parenchyma

xy

xylem

cells

ph

pith

Arrow

head

indicates

the

epidermis

Arrow

indicates

the

phloem

fibres

Scale

bar

μm

Reduced

stem

elongation

and

prolonged

juvenility

are

observed

for

transformants

An

indication

of

reduced

stem

elongation

in

the

transgenic

plants

expressing

the

tandem

was

first

apparent

after

the

third

week

of

transfer

to

the

greenhouse

The

different

stem

elongation

of

the

tandem

plants

and

the

control

plants

became

significant

-LRB-

P

-RRB-

at

weeks

seven

and

eight

when

other

plants

had

reached

their

final

height

-LRB-

Table

Fig.

-RRB-

The

tandem

plants

continued

to

increase

in

height

until

the

eleventh

week

however

when

they

reached

their

final

height

an

average

of

cm

There

was

as

a

result

no

significant

difference

between

the

heights

of

the

tandem

lines

at

maturity

and

the

heights

of

the

single

lines

the

pGreen

vector

control

and

the

wild-type

control

plants

There

was

no

significant

difference

at

any

time

between

stem

elongation

of

transgenic

plants

expressing

and

the

control

plants

Table

height

-LRB-

cm

-RRB-

of

transgenic

lines

CBM

CBM

empty

control

and

wild-type

controlPlant

lineWeeks

after

WT

The

values

are

means

and

standard

deviations

for

six

transgenic

lines

-LRB-

high

expressers

-RRB-

and

six

control

plants

Each

line

has

three

individual

plants

-LRB-

three

replicates

-RRB-

aPlant

height

significantly

different

from

that

of

controlsFig

stem

elongation

in

time

and

delayed

development

of

CBM

transgenic

tobacco

plants

The

picture

was

taken

at

week

eight

In

addition

to

reduced

stem

elongation

it

was

also

observed

that

flower

development

was

delayed

in

the

tandem

plants

We

observed

that

at

week

eight

when

most

plants

had

already

flowered

-LRB-

Fig.

-RRB-

and

set

fruit

-LRB-

data

not

shown

-RRB-

the

tandem

plants

were

just

developing

flower

buds

-LRB-

Fig.

-RRB-

The

flower

buds

eventually

developed

into

normal

flowers

and

also

set

fruits

Most

plants

except

the

plants

had

also

lost

many

leaves

because

of

faster

ageing

-LRB-

Fig.

-RRB-

There

was

no

morphological

or

developmental

change

in

the

transgenic

plants

expressing

the

tandem

gene

compared

with

the

vector

control

The

average

plant

height

of

the

tandem

series

at

maturity

was

cm

which

was

comparable

with

the

cm

average

height

for

their

vector

control

plants

We

also

did

not

observe

a

particular

trend

in

plant

development

with

regard

to

stem

elongation

leaf

abscission

or

flower

formation

Cellular

alterations

are

observed

in

transgenic

plants

The

cellular

basis

for

the

plant

phenotypes

was

examined

by

histological

examination

of

the

transgenic

tobacco

stems

The

examination

revealed

a

prominent

layer

of

collapsed

cells

in

the

cortex

of

the

stems

of

the

tandem

plants

-LRB-

Figs.

-RRB-

We

also

observed

that

this

morphological

alteration

is

consistently

more

severe

in

the

high

expressers

than

in

the

low

expressers

This

alteration

was

also

observed

in

the

high

expressers

of

the

single

although

it

was

much

less

severe

-LRB-

Fig.

-RRB-

For

the

stems

irregularly

shaped

cells

were

observed

in

the

cortex

of

the

high

expressers

-LRB-

Fig.

-RRB-

The

micrographs

of

both

the

empty

vector

control

and

untransformed

wild-type

control

looked

the

same

hence

the

micrograph

was

taken

to

represent

the

controls

-LRB-

Fig.

-RRB-

Altered

cell

enlargement

across

the

tissues

in

the

stems

was

also

observed

This

was

particularly

true

for

the

tandem

plants

-LRB-

compare

for

example

Figs.

and

with

and

respectively

-RRB-

The

altered

cell

size

phenotype

is

widespread

in

the

high

and

low

plants

This

is

not

observed

however

in

the

single

stems

in

which

only

the

high

expressers

had

noticeably

larger

cells

in

the

epidermal

cortex

and

xylem

tissues

than

those

of

the

controls

Similarly

there

was

an

indication

of

larger

xylem

cells

in

the

outermost

layer

of

the

stems

than

in

controls

-LRB-

compare

Figs.

and

-RRB-

There

was

however

no

clear

indication

of

altered

enlargement

of

the

stem

pith

parenchyma

cells

of

the

transgenic

plants

Fig.

sections

of

transgenic

and

control

stems

showing

collapsed

cortical

cells

and

enlarged

cortical

and

xylem

cells

of

the

stems

Sections

of

-LRB-

a

-RRB-

empty

control

-LRB-

b

-RRB-

stem

-LRB-

c

-RRB-

and

-LRB-

d

-RRB-

were

stained

with

toluidine

blue

The

boxed

area

indicates

tissues

shown

with

higher

magnification

in

Fig.

ep

epidermis

ct

cortex

xy

xylem

ph

pithFig

Magnification

-LRB-

-RRB-

of

cross

sections

of

stem

-LRB-

a

d

-RRB-

control

stem

-LRB-

b

e

-RRB-

and

stem

-LRB-

c

f

-RRB-

Micrographs

show

collapsed

cortical

cells

-LRB-

a

-RRB-

enlarged

cortical

and

xylem

cells

of

the

stems

-LRB-

a

d

-RRB-

and

irregularly

shaped

stem

cortical

cells

of

the

stems

-LRB-

c

-RRB-

Asterisks

indicate

irregular

cortical

cells

in

the

transformants

in

the

transformants

these

cells

are

more

apparent

and

are

not

highlighted

ep

epidermis

ct

cortex

xy

xylem

pf

phloem

fibre

Cryo-scanning

electron

microscopy

-LRB-

cryo-SEM

-RRB-

of

tobacco

stems

To

establish

that

the

transgenic

stems

indeed

had

larger

cells

than

those

of

the

controls

we

made

cryo-SEM

examinations

of

stem

samples

Figure

shows

the

collapsed

and

enlarged

cells

of

the

cortex

in

the

sections

of

transgenic

line

of

as

previously

observed

by

light

microscopic

examination

The

cortical

cells

of

the

control

plant

were

smaller

and

the

collapsed

phenotype

was

not

apparent

-LRB-

Fig.

-RRB-

The

micrographs

also

showed

that

xylem

cells

were

indeed

larger

in

the

stems

than

in

the

controls

-LRB-

compare

transgenic

line

Fig.

and

the

empty

vector

control

Fig.

-RRB-

The

micrographs

also

indicated

that

the

cell

walls

of

the

xylem

cells

were

thinner

in

the

stems

-LRB-

Fig.

-RRB-

than

in

stems

of

controls

-LRB-

Fig.

-RRB-

Similar

micrographs

were

obtained

for

the

series

which

confirmed

that

the

radial

surface

area

and

cell

wall

diameter

of

their

stem

xylem

cells

were

larger

and

thinner

respectively

than

those

of

the

controls

-LRB-

data

not

shown

-RRB-

These

notable

observations

were

later

substantiated

by

quantification

Figure

shows

that

almost

and

of

the

stem

cortical

cells

of

the

wild-type

and

control

plants

respectively

were

grouped

in

the

small

class

-LRB-

-RRB-

whereas

fewer

than

of

the

stem

cortical

cells

of

the

plants

were

grouped

in

the

small

class

An

opposite

trend

was

observed

in

the

large

class

-LRB-

-RRB-

fewer

than

of

the

cortical

cells

of

the

two

control

stems

were

grouped

compared

with

more

than

and

of

the

stems

expressing

the

single

and

the

tandem

respectively

We

thus

infer

that

the

cortical

cells

of

the

stems

are

larger

than

those

of

the

control

stems

Similar

quantitative

data

were

obtained

from

the

xylem

cells

depicted

in

Fig.

which

shows

that

only

the

xylem

cells

of

the

stems

are

larger

than

those

of

the

control

stems

There

was

no

difference

between

the

size

of

the

xylem

cells

of

the

single

stems

and

those

of

the

controls

Similarly

we

quantified

the

thickness

of

the

cell

walls

of

the

xylem

cells

of

the

stems

and

the

control

stems

as

presented

in

Fig.

Approximately

of

the

cell

walls

of

the

xylem

cells

of

the

stem

were

grouped

in

the

thin

class

-LRB-

μm

-RRB-

whereas

fewer

than

of

the

stem

xylem

cell

walls

of

the

single

the

control

and

the

wild-type

control

plants

were

grouped

in

this

class

More

than

of

the

xylem

cell

walls

of

the

two

control

stems

were

grouped

in

the

thick

class

-LRB-

μm

-RRB-

whereas

approximately

and

of

the

stem

xylem

cell

walls

of

the

single

and

the

tandem

respectively

were

grouped

in

this

class

We

can

also

infer

from

this

result

that

the

thickness

of

the

cell

walls

of

the

xylem

cells

of

the

stems

-LRB-

particularly

of

the

tandem

-RRB-

are

thinner

than

those

of

the

and

the

wild-type

control

stems

Fig.

electron

micrographs

of

transgenic

tobacco

stems

to

show

details

of

larger

cells

and

thinner

cell

walls

of

the

CBM

stems

compared

with

the

control

-LRB-

a

-RRB-

and

-LRB-

c

-RRB-

and

empty

control

-LRB-

b

-RRB-

and

-LRB-

d

-RRB-

a

and

b

show

the

cortex

tissue

whereas

c

and

d

show

the

xylem

tissue

pf

phloem

fibre

xy

xylem

ct

cortex

Arrow

heads

indicate

collapsed

layer

of

cortical

cellsFig

size

distribution

in

the

cortex

-LRB-

a

-RRB-

cell

size

distribution

in

the

xylem

-LRB-

b

-RRB-

and

cell

wall

thickness

of

xylem

cells

-LRB-

c

-RRB-

in

the

stems

of

transgenic

and

the

wild-type

tobacco

plants

Insets

show

how

the

surface

area

and

wall

thickness

measurements

were

taken

Surface

areas

of

cells

were

measured

cells

per

field

of

view

Cell

wall

diameters

of

cells

were

measured

cells

per

field

of

view

Error

bars

represent

standard

deviation

Grey

bars

represent

wild-type

plants

Diagonal

bars

represent

empty

transformants

Open

bars

represent

single

transformants

Horizontal

brick

bars

represent

double

transformants

A

similar

trend

was

observed

for

the

xylem

cells

of

the

stems

which

were

larger

than

those

of

the

control

plants

-LRB-

data

not

shown

-RRB-

Similarly

the

cell

walls

of

the

xylem

cells

of

the

stems

were

thinner

than

those

of

the

control

stems

-LRB-

data

not

shown

-RRB-

Analysis

of

cellulose

content

A

colorimetric

assay

was

performed

on

samples

of

crystalline

cellulose

obtained

from

four

separate

acid

hydrolyses

to

determine

the

cellulose

content

in

the

stem

cell

walls

of

the

and

transformants

and

the

control

plants

There

were

no

substantial

differences

between

levels

of

cellulose

in

the

stem

cell

walls

of

the

transgenic

tobacco

lines

expressing

the

single

the

tandem

the

tandem

the

empty

vector

control

and

the

wild-type

control

plants

-LRB-

data

not

shown

-RRB-

Hence

it

can

be

concluded

that

use

of

these

CBMs

for

cell

wall

modification

did

not

interfere

with

the

extent

of

cellulose

biosynthesis

Discussion

It

has

been

suggested

that

networks

of

plant

cell

wall

polysaccharides

can

affect

the

properties

of

cellulose

fibres

We

hypothesized

that

expression

of

promiscuous

CBMs

in

tobacco

plants

might

lead

to

modification

of

these

networks

although

they

might

also

affect

plant

development

in

more

general

terms

In

this

study

expression

of

two

promiscuous

Piromyces

proteins

was

targeted

at

tobacco

cell

walls

to

provide

baseline

information

on

the

possibility

of

using

CBMs

for

in-planta

modification

of

the

properties

of

cellulose

fibre

for

industrial

applications

In

parallel

we

also

targeted

the

expression

of

the

less

promiscuous

tandem

protein

of

the

C.

fimi

xylanase

with

a

view

to

comparing

its

effect

with

that

of

the

more

promiscuous

Our

results

reveal

that

the

tandem

modified

the

tobacco

cell

wall

structure

with

consequent

altered

cellular

and

organ

morphology

The

tandem

modules

had

more

effect

than

the

single

Expression

of

the

tandem

modules

in

the

tobacco

cell

wall

affected

plant

development

as

reflected

by

reduced

stem

elongation

rate

enlarged

xylem

and

collapsed

cortical

cells

The

stem

phenotype

of

the

plants

was

more

of

delayed

development

of

the

whole

plant

rather

than

mere

elongation

defects

of

the

stem

We

suggest

that

the

delayed

development

-LRB-

prolonged

juvenility

-RRB-

was

as

a

result

of

reduced

rate

of

anabolism

in

the

tandem

plant

It

could

be

that

structural

remodelling

of

the

cell

wall

has

affected

the

normal

developmental

cues

that

signal

the

cells

to

synthesize

new

cell

wall

materials

It

is

conceivable

that

when

the

cell

walls

are

held

loosely

continuously

as

a

result

of

the

continuous

presence

of

the

rate

of

wall

extension

will

outpace

biosynthetic

rate

Consequently

excessive

thinning

of

the

cell

walls

may

occur

-LRB-

Kutschera

-RRB-

which

may

have

led

to

the

eventual

collapse

of

particularly

the

primary

walled

cells

-LRB-

and

not

so

much

of

the

secondary-walled

xylem

cells

-RRB-

Thus

because

of

the

extensive

loosening

of

their

cell

walls

and

consequent

altered

expansion

the

cortical

cells

may

have

yielded

to

tissue

pressure

within

the

stem

The

altered

cell

enlargement

which

led

to

the

collapse

of

the

cortical

cells

of

the

tandem

stems

is

proposed

as

the

cellular

basis

for

the

reduced

stem

elongation

We

have

not

noticed

that

the

increased

size

of

the

xylem

cells

led

to

thicker

stems

It

is

possible

the

gain

in

the

size

of

the

xylem

cells

is

neutralized

by

the

collapse

of

the

cortical

cells

The

explanation

given

above

is

based

on

the

assumption

that

the

CBMs

do

not

bind

to

polymers

until

they

have

reached

the

wall

It

should

be

noted

however

that

by

placing

a

signal

peptide

at

the

amino

terminus

of

the

CBMs

we

have

targeted

them

through

the

secretory

pathway

which

includes

the

site

of

hemicellulose

biosynthesis

-LRB-

the

Golgi

apparatus

-RRB-

-LRB-

Denecke

et

al.

Vitale

and

Denecke

-RRB-

It

is

therefore

possible

the

CBMs

had

already

bound

to

hemicellulose

before

their

deposition

in

the

cell

wall

As

a

result

the

hemicellulose

content

length

and/or

branching

pattern

might

have

changed

this

in

turn

could

affect

their

interaction

with

cellulose

microfibrils

leading

to

structural

alteration

of

the

cell

wall

The

expression

of

the

modules

in

the

tobacco

cell

wall

altered

the

cellular

morphology

of

the

transgenic

plants

as

was

apparent

from

the

irregular

shape

of

the

cortical

cells

and

altered

cell

enlargement

of

the

xylem

cells

of

the

stems

The

deformation

of

the

cortical

cells

of

the

expressing

plants

is

however

less

pronounced

than

that

of

the

cortical

cells

of

the

transformants

The

more

obvious

phenotypic

changes

in

the

plants

is

consistent

with

the

result

of

the

immunolabelling

study

with

the

proteins

which

revealed

that

binds

to

polysaccharides

that

are

integral

to

both

primary

and

secondary

cell

wall

structure

in

contrast

binds

only

to

xylan

and

non-crystalline

cellulose

As

xylan

is

mainly

present

in

the

secondary

cell

wall

it

is

not

strange

to

see

the

effect

of

most

pronounced

in

the

secondary

walled

xylem

The

finding

that

did

not

label

the

cortex

-LRB-

Fig.

-RRB-

suggests

that

the

deformation

of

the

cortical

cells

is

an

indirect

effect

of

The

cortical

parenchyma

has

no

secondary

walls

suggesting

that

the

walls

are

much

weaker

here

consequently

this

tissue

might

collapse

when

challenged

by

excessive

expansion

of

flanking

tissues

The

expression

of

the

only

had

effect

on

cellular

morphology

and

not

on

plant

morphology

whereas

the

expression

of

the

in

tobacco

led

to

altered

structural

morphology

and

altered

development

of

the

transgenic

plants

The

reduced

stem

elongation

phenotype

of

the

tobacco

plants

is

however

in

contrast

with

the

report

by

Safra-Dassa

et

al.

-LRB-

-RRB-

of

enhanced

growth

of

transgenic

potato

plants

expressing

a

bacterial

These

contrasting

results

might

be

because

of

the

different

specificities

of

the

CBMs

because

we

have

used

double

CBMs

in

our

investigation

as

opposed

to

the

single

CBM

they

used

That

the

experiments

were

performed

on

different

plant

species

may

also

have

contributed

to

the

different

observations

There

are

reports

attributing

cell

wall

phenotypes

in

mutant

or

transgenic

plants

to

abnormal

wall

assembly

in

general

or

depletion

of

the

cellulose

content

in

particular

-LRB-

Szyjanowicz

et

al.

Taylor

et

al.

Tsabary

et

al.

Turner

and

Somerville

-RRB-

In

all

these

instances

the

plants

had

deformed

walls

in

vascular

cells

and

reduced

growth

as

a

result

of

cellulose

depletion

Our

results

reveal

that

wall

deformation

-LRB-

cortical

cells

-RRB-

can

also

occur

without

a

marked

reduction

in

cellulose

content

To

summarise

the

results

of

our

investigation

suggest

that

expression

of

the

promiscuous

tandem

can

alter

cell

morphology

to

a

larger

extent

than

that

of

more

specific

CBM

although

the

actual

effect

is

unpredictable

Use

of

CBMs

for

in-planta

cell

wall

modification

thus

still

needs

further

investigation

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

carbohydrate-binding modules
cellulose–hemicellulose networks
plant growth and development
promiscuous polysaccharide recognition