Increased

Terpenoid

Accumulation

in

Cotton

-LRB-

Gossypium

hirsutum

-RRB-

Foliage

is

a

General

Wound

Response

The

subepidermal

pigment

glands

of

cotton

accumulate

a

variety

of

terpenoid

products

including

monoterpenes

sesquiterpenes

and

terpenoid

aldehydes

that

can

act

as

feeding

deterrents

against

a

number

of

insect

herbivore

species

We

compared

the

effect

of

herbivory

by

Spodoptera

littoralis

caterpillars

mechanical

damage

by

a

fabric

pattern

wheel

and

the

application

of

jasmonic

acid

on

levels

of

the

major

representatives

of

the

three

structural

classes

of

terpenoids

in

the

leaf

foliage

of

Gossypium

hirsutum

plants

Terpenoid

levels

increased

successively

from

control

to

mechanical

damage

herbivory

and

jasmonic

acid

treatments

with

E-β-ocimene

and

heliocide

and

showing

the

highest

increases

up

to

Herbivory

or

mechanical

damage

to

older

leaves

led

to

terpenoid

increases

in

younger

leaves

Leaf-by-leaf

analysis

of

terpenes

and

gland

density

revealed

that

higher

levels

of

terpenoids

were

achieved

by

two

mechanisms

-LRB-

-RRB-

increased

filling

of

existing

glands

with

terpenoids

and

-LRB-

-RRB-

the

production

of

additional

glands

which

were

found

to

be

dependent

on

damage

intensity

As

the

relative

response

of

individual

terpenoids

did

not

differ

substantially

among

herbivore

mechanical

damage

and

jasmonic

acid

treatments

the

induction

of

terpenoids

in

cotton

foliage

appears

to

represent

a

non-specific

wound

response

mediated

by

jasmonic

acid

Introduction

Plants

produce

a

broad

range

of

defense

chemicals

that

act

as

deterrents

or

toxins

against

herbivores

and

pathogens

Several

different

strategies

have

evolved

concerning

the

deployment

of

such

compounds

When

the

synthesis

of

defense

chemicals

occurs

solely

after

initial

attack

this

reduces

metabolic

costs

-LRB-

Gershenzon

-RRB-

as

defenses

are

produced

only

when

needed

Such

induced

defenses

have

ecological

advantages

-LRB-

Agrawal

and

Karban

-RRB-

and

might

be

a

useful

strategy

for

plants

that

are

sporadically

attacked

However

plants

that

risk

frequent

and

heavy

damage

may

be

better

protected

by

investing

in

constitutive

defense

compounds

-LRB-

Wittstock

and

Gershenzon

-RRB-

The

accumulation

of

constitutive

defenses

within

plants

usually

conforms

to

expectations

of

the

optimal

defense

theory

-LRB-

ODT

-RRB-

which

predicts

that

the

highest

protection

level

will

be

found

in

parts

with

the

highest

fitness

value

such

as

young

tissues

and

reproductive

organs

-LRB-

McKey

Rhoades

-RRB-

This

has

been

shown

for

a

variety

of

plant

secondary

metabolites

from

different

classes

such

as

alkaloids

-LRB-

Hartmann

and

Zimmer

Baldwin

-RRB-

glucosinolates

-LRB-

Porter

et

al.

Brown

et

al.

-RRB-

furanocoumarins

-LRB-

Berenbaum

and

Zangerl

-RRB-

phenylpropanoids

-LRB-

Harborne

Opitz

and

Schneider

-RRB-

and

terpenes

-LRB-

Gershenzon

and

Croteau

-RRB-

Under

abiotic

or

biotic

stress

plants

often

exhibit

induced

responses

by

enhancing

the

accumulation

of

constitutive

compounds

in

certain

tissues

that

increases

their

deterrence

or

toxicity

to

enemies

-LRB-

Baldwin

Wittstock

and

Gershenzon

Vazquez-Flota

et

al.

-RRB-

These

changes

also

may

be

rationalized

as

a

way

for

plants

to

reduce

the

costs

of

defense

by

producing

high

concentrations

of

constitutive

defenses

only

when

and

where

these

are

needed

However

more

information

is

required

to

understand

how

the

levels

of

constitutive

defenses

change

under

different

stresses

in

plants

on

an

organ-by-organ

basis

Among

the

vast

number

of

defensive

compounds

in

plants

terpenoids

form

the

largest

group

with

over

known

structures

-LRB-

Buckingham

-RRB-

Some

of

the

best-studied

examples

for

constitutive

plant

defenses

are

terpenoid

volatiles

such

as

monoterpenes

-LRB-

-RRB-

which

are

considered

defensive

against

herbivores

and

pathogens

-LRB-

Gershenzon

and

Dudareva

-RRB-

Monoterpenes

are

frequently

constituents

of

oils

and

resins

that

are

accumulated

in

complex

secretory

structures

such

as

glandular

trichomes

secretory

cavities

or

resin

ducts

-LRB-

Fahn

-RRB-

Among

the

most

comprehensively

studied

systems

are

monoterpenes

in

the

glandular

trichomes

of

peppermint

-LRB-

Gershenzon

et

al.

McConkey

et

al.

-RRB-

However

less

is

known

about

the

accumulation

of

other

classes

of

terpenes

Cotton

-LRB-

Gossypium

hirsutum

-RRB-

is

particularly

suitable

for

the

study

of

terpenoid

accumulation

because

a

large

variety

of

terpenes

are

produced

constitutively

in

all

photosynthetically

active

parts

of

the

plant

and

stored

in

subepidermal

glands

Beside

monoterpenes

G.

hirsutum

produces

sesquiterpenes

-LRB-

Elzen

et

al.

-RRB-

and

terpenoid

aldehydes

such

as

hemigossypolone

-LRB-

-RRB-

and

the

heliocides

to

-LRB-

Fig.

Hedin

et

al.

-RRB-

All

three

classes

of

terpenoids

in

cotton

are

biosynthetically

related

-LRB-

Stipanovic

et

al.

Davis

and

Essenberg

-RRB-

The

precursor

of

sesquiterpenes

farnesyl

diphosphate

is

formed

by

addition

of

a

isopentenyl

diphosphate

unit

to

geranyl

diphosphate

the

precursor

of

all

monoterpenes

The

heliocides

are

formed

from

Diels

Alder-type

reactions

of

the

hemigossypolone

-LRB-

derived

from

δ-cadinene

-RRB-

to

the

monoterpene

myrcene

for

the

formation

of

heliocides

and

or

the

monoterpene

E-β-ocimene

for

the

formation

of

heliocides

and

-LRB-

Fig.

-RRB-

Fig.

products

of

three

structural

classes

which

are

accumulated

in

glands

of

the

foliage

of

Gossypium

hirsutum

α-pinene

-LRB-

E

-RRB-

β-ocimene

myrcene

-LRB-

E

-RRB-

β-caryophyllene

γ-bisabolene

α-humulene

hemigossypolone

heliocide

heliocide

scheme

for

the

biosynthesis

of

heliocide

in

cotton

The

accumulation

of

terpenoid

aldehydes

in

cotton

leaves

was

shown

to

be

increased

after

herbivory

-LRB-

Bezemer

et

al.

-RRB-

and

these

compounds

have

been

considered

to

function

as

feeding

deterrents

on

generalist

herbivores

such

as

Spodoptera

exigua

-LRB-

McAuslane

et

al.

-RRB-

The

heliocides

specifically

have

been

correlated

with

antifeedant

effects

and

are

locally

and

systematically

induced

in

response

to

herbivore

attack

-LRB-

Karban

and

Carey

Croxford

et

al.

Alborn

et

al.

-RRB-

However

besides

the

terpenoid

aldehydes

little

information

is

available

on

the

accumulation

of

other

terpenes

found

in

subepidermal

glands

In

studying

the

control

of

terpenoid

accumulation

in

cotton

it

is

of

interest

to

determine

if

higher

concentrations

arise

from

increases

in

gland

number

or

from

increases

in

the

amount

of

terpenoids

per

gland

-LRB-

McAuslane

et

al.

-RRB-

Increasing

numbers

of

subepidermal

glands

in

cotton

leaves

were

observed

after

attacks

of

spider

mites

and

caterpillars

-LRB-

McAuslane

et

al.

Agrawal

and

Karban

-RRB-

To

learn

more

about

the

control

of

terpenoid

accumulation

in

cotton

we

compared

the

response

of

plants

to

herbivore

damage

mechanical

damage

and

the

application

of

jasmonic

acid

-LRB-

JA

-RRB-

a

ubiquitous

mediator

of

defense

responses

in

the

plant

kingdom

-LRB-

Browse

-RRB-

Differences

in

the

reaction

of

plants

to

these

treatments

were

investigated

with

respect

to

plant

growth

gland

production

and

accumulation

of

major

compounds

from

three

classes

of

terpenoids

terpenoid

aldehydes

monoterpenes

and

sesquiterpenes

at

the

level

of

the

individual

leaf

We

also

examined

how

the

accumulation

was

influenced

by

the

number

of

glands

in

the

total

foliage

or

the

relative

filling

of

glands

with

terpenoids

Methods

and

Material

Plants

G.

hirsutum

L.

of

a

single

variety

Deltapine

acala

-LRB-

Delta

and

Pine

Company

Hollandala

MS

USA

-RRB-

were

reared

in

a

growth

chamber

under

high

light

intensity

-LSB-

mmol

-LRB-

-RRB-

s

of

photosynthetically

active

radiation

-RSB-

a

light

period

°C

°C

-LRB-

day/night

-RRB-

and

relative

humidity

Plants

were

established

in

pots

containing

Seramis

clay

granulate

-LRB-

Masterfood

GmbH

Verden/Germany

-RRB-

which

were

watered

daily

and

fertilized

weekly

with

a

complete

fertilizer

-LSB-

g/l

Flory

-LRB-

N/P/K

-RRB-

Euflor/Germany

-RSB-

Under

these

conditions

optimal

growth

and

production

of

the

various

terpenoids

was

obtained.For

all

experiments

leaves

were

numbered

sequentially

such

that

number

refers

to

the

first

true

leaf

initiated

right

after

the

cotyledons

-LRB-

Fig.

-RRB-

In

this

study

leaves

were

investigated

and

collectively

referred

to

as

total

foliage

Fig.

of

a

Gossypium

hirsutum

plant

at

the

end

of

experiment

approximately

Leaves

are

numbered

according

to

the

order

of

development

Grid

pattern

indicate

leaves

which

were

damaged

either

mechanically

or

by

caterpillars

of

Spodoptera

littoralis

Herbivores

Eggs

of

the

generalist

herbivore

Spodoptera

littoralis

-LRB-

Boisd.

Lepidoptera

Noctuidae

-RRB-

Egyptian

cotton

worm

were

obtained

from

Syngenta

-LRB-

Basel

Switzerland

-RRB-

After

hatching

larvae

were

reared

in

plastic

boxes

containing

artificial

diet

which

consisted

of

g

of

bean

flour

ml

water

g

ascorbic

acid

g

acid

ml

α-tocopherol

ml

Mazola

germ

oil

ml

formaldehyde

and

ml

agar

solution

Fresh

diet

was

applied

every

third

day

Larvae

were

reared

at

constant

°C

relative

humidity

and

ambient

light

conditions

Two

weeks

after

hatching

larvae

reached

the

third

instar

and

were

used

for

the

study

Experiment

Treatment

of

Cotton

Plants

Three

weeks

after

germination

when

plants

had

developed

four

to

five

true

leaves

the

length

of

the

midrib

of

leaf

was

determined

as

a

good

measure

for

growth

stage

Next

plants

at

comparable

growth

stage

were

transferred

into

a

second

growth

chamber

-LRB-

Vötsch

Germany

-RRB-

with

abiotic

conditions

identical

to

the

chamber

where

plants

were

reared

Here

plants

were

maintained

separately

in

plastic

pots

and

watered

twice

a

day

to

ensure

adequate

water

supply

After

h

plants

were

divided

into

four

treatment

groups

of

six

individuals

each

Plants

of

one

group

were

damaged

mechanically

three

times

on

leaf

on

the

first

day

and

three

times

on

leaf

on

the

fourth

day

by

using

a

fabric

pattern

wheel

In

every

case

leaves

were

scratched

four

times

in

parallel

to

the

midrib

-LRB-

two

times

on

each

side

-RRB-

For

a

second

group

three

caterpillars

of

Spodoptera

littoralis

were

caged

on

each

plant

on

leaf

during

the

first

day

and

on

leaf

during

the

fourth

day

until

larvae

consumed

at

least

one

third

of

the

available

leaf

material

Plants

of

the

third

group

were

watered

with

a

JA

solution

in

water

-LRB-

Sigma

-RRB-

during

the

first

day

JA

concentrations

in

this

range

are

commonly

used

to

induce

the

biosynthesis

of

secondary

products

in

plants

-LRB-

Koch

et

al.

van

Poecke

and

Dicke

-RRB-

Afterward

this

solution

was

replaced

by

pure

tap

water

A

control

group

of

plants

was

not

treated

at

all

One

week

after

the

start

of

treatments

all

true

leaves

were

harvested

separately

frozen

in

liquid

nitrogen

and

stored

at

°C

until

further

analyses

Foliar

Glands

Just

after

removal

all

leaves

were

scanned

to

calculate

leaf

areas

by

using

the

program

ImageJ

-LRB-

Wayne

Rasband

National

Institutes

of

Health

USA

-RRB-

In

this

procedure

herbivore-damaged

leaves

were

used

as

templates

to

reconstruct

the

areas

of

undamaged

leaves

In

addition

surface

view

pictures

were

taken

of

two

circular

sections

from

the

center

of

each

leaf

half

with

a

stereomicroscope

connected

to

a

digital

camera

In

doing

so

leaves

were

illuminated

from

below

with

a

lamp

to

visualize

filled

subepidermal

glands

as

pigmented

spots

After

the

number

of

filled

glands

was

counted

gland

densities

for

sections

were

calculated

Based

on

these

sections

gland

numbers

and

gland

densities

per

leaf

and

per

total

foliage

were

extrapolated

These

values

were

slightly

underestimated

because

of

higher

gland

densities

at

leaf

edges

-LRB-

personal

observations

-RRB-

Analysis

of

Monoterpenes

and

Sesquiterpenes

True

leaves

were

separately

analyzed

for

their

content

of

constitutive

terpenes

Depending

on

available

material

mg

of

ground

frozen

leaf

were

extracted

with

μl

pentane/n-hexane

Additionally

μg

of

nonylacetate

were

added

as

internal

standard

before

extracts

were

shaken

for

h

at

room

temperature

After

extracts

were

cooled

to

°C

they

were

filtered

through

cotton

and

to

remove

remaining

water

A

subset

of

extracts

was

analyzed

qualitatively

by

gas

chromatography

mass

spectrometry

-LRB-

GC

MS

-RRB-

on

a

Hewlett-Packard

gas

chromatograph

-LRB-

injector

temperature

°C

μl

splitless

injection

-RRB-

coupled

to

a

Hewlett-Packard

quadrupole

mass

selective

detector

Separations

were

performed

on

a

column

-LRB-

m

mm

μm

film

Agilent

Technology

-RRB-

with

ml

min

helium

as

the

carrier

gas

The

following

temperature

gradient

was

used

°C

for

min

increased

to

°C

at

°C

min

further

increased

to

°C

at

°C

min

followed

by

an

increase

to

°C

at

°C

min

maintained

at

°C

for

min

and

a

final

heating

to

°C

for

min

Mass

spectrometry

was

performed

with

an

ionization

potential

of

electron

volts

and

a

scan

range

of

m/z

from

to

Compound

identification

was

based

on

comparisons

with

mass

spectra

in

the

Wiley

and

National

Institute

of

Standards

and

Technology

-LRB-

NIST

-RRB-

libraries

or

on

direct

comparison

of

mass

spectra

and

retention

times

with

available

standards

For

quantification

all

samples

were

analyzed

on

a

Hewlet-Packard

gas

chromatograph

and

a

flame

ionization

detector

-LRB-

temperature

°C

-RRB-

The

separation

procedure

was

identical

to

the

method

for

GC

MS

analysis

Terpenes

were

quantified

by

using

nonylacetate

and

corrected

with

response

factors

according

to

Scanlon

and

Willis

-LRB-

-RRB-

Concentrations

and

levels

of

terpenes

per

subepidermal

gland

were

calculated

for

single

leaves

and

for

total

foliage

Eight

individual

monoterpenes

and

sesquiterpenes

were

analyzed

which

represent

more

than

of

the

total

terpenes

in

G.

hirsutum

Analysis

of

Terpenoid

Aldehydes

True

leaves

were

separately

analyzed

for

their

content

of

terpenoid

aldehydes

according

to

Stipanovic

et

al.

-LRB-

-RRB-

Depending

on

available

material

mg

of

ground

frozen

material

were

extracted

with

ml

of

ethyl

acetate/n-hexane

-LRB-

-RRB-

and

μl

of

HCL

Extracts

were

shaken

at

°C

for

h.

Afterwards

extracts

were

removed

from

the

leaf

material

evaporated

with

nitrogen

and

redissolved

in

and

solvent

B

-LRB-

see

below

-RRB-

from

the

following

high-performance

liquid

chromatography

-LRB-

HPLC

-RRB-

procedure

Reversed

phase

HPLC

analysis

was

performed

on

a

Chromolith

column

-LRB-

Merck

μm

mm

-RRB-

with

single

wavelength

detection

at

nm

The

column

was

eluted

with

trifluoracetic

acid

-LRB-

solvent

A

-RRB-

and

EtOH/MeOH/MeCN

EtOAc/iPrOH

-LRB-

solvent

B

-RRB-

with

the

following

gradient

B

-LRB-

min

-RRB-

B

-LRB-

min

-RRB-

B

-LRB-

min

-RRB-

B

-LRB-

min

-RRB-

Flow

rate

was

held

constant

at

ml

min

and

temperature

was

kept

at

°C

Five

compounds

the

hemigossypolone

-LRB-

-RRB-

and

the

heliocides

-LRB-

-RRB-

were

identified

by

their

retention

times

according

to

the

literature

-LRB-

Stipanovic

et

al.

-RRB-

and

additionally

by

liquid

chromatography

mass

spectrometry

-LRB-

LC

MS

-RRB-

measurements

Quantification

was

done

by

using

standard

curves

of

purified

compounds

Concentrations

and

levels

per

subepidermal

gland

were

calculated

for

single

leaves

and

for

the

total

foliage

Hemigossypolone

and

the

four

heliocides

represent

approximately

of

the

total

terpenoid

aldehyde

content

As

a

sesquiterpene

aldehyde

hemigossypolone

was

included

in

our

analysis

with

other

terpenoid

aldehydes

rather

than

with

the

sesquiterpenes

In

comparison

to

other

cotton

varieties

-LRB-

Bezemer

et

al.

-RRB-

the

terpenoid

aldehyde

gossypol

-LRB-

-RRB-

was

found

only

in

traces

by

LC

MS

and

was

not

quantified

Experiment

Eighteen

plants

at

the

growth

stage

of

two

true

leaves

were

chosen

and

prepared

for

treatments

in

a

way

comparable

to

the

first

experiment

Plants

were

divided

into

three

groups

Plants

of

one

group

were

damaged

mechanically

nine

times

on

leaves

and

during

six

consecutive

days

by

using

a

mechanical

fabric

pattern

wheel

-LRB-

procedure

see

experiment

-RRB-

For

a

second

group

two

caterpillars

of

Spodoptera

littoralis

were

caged

on

each

plant

on

one

cotyledon

during

day

on

leaf

during

day

and

on

leaf

during

day

until

larvae

consumed

at

least

one

third

of

the

available

leaf

material

A

control

group

was

not

treated

at

all

One

week

after

the

start

of

treatments

leaf

from

each

plant

was

harvested

Leaf

areas

and

gland

number

per

leaf

were

determined

according

to

experiment

Afterwards

leaves

were

frozen

in

liquid

nitrogen

and

stored

at

°C

until

further

analyses

of

mono

and

sesquiterpenes

Levels

of

terpene

classes

were

calculated

based

on

the

levels

of

eight

individual

compounds

that

represent

more

than

of

the

accumulated

monoterpenes

and

sesquiterpenes

in

G.

hirsutum

Statistics

Experiment

Total

Foliage

The

effects

of

mechanical

damage

herbivory

and

JA

application

on

foliage

area

levels

of

total

monoterpenes

sesquiterpenes

and

terpenoid

aldehydes

as

well

as

on

levels

of

individual

major

terpenoids

were

analyzed

with

analyses

of

variance

Data

were

tested

for

normality

and

equal

variances

For

normally

distributed

data

one-way

analyses

of

variance

-LRB-

ANOVAs

-RRB-

were

used

Differences

between

treatments

and

controls

were

tested

for

significance

by

using

Dunn

's

post

hoc

tests

Data

not

normally

distributed

were

analyzed

by

using

Kruskall

Wallis

one-way

ANOVAs

on

ranks

and

Dunn

's

post

hoc

tests

Analyses

were

performed

with

Sigma

Stat

The

effect

of

foliage

area

and

treatment

on

the

total

number

of

glands

was

tested

by

analysis

of

codeviance

Generalized

linear

models

-LRB-

GLM

-RRB-

with

the

log

link

as

link

function

were

performed

To

deal

with

overdispersion

a

quasi-Poisson

distribution

was

used

in

the

models

instead

of

Poisson

distribution

The

models

were

simplified

by

removing

non-significant

terms

and

by

factor-level

reduction

-LRB-

Crawley

-RRB-

These

analyses

were

performed

in

R

version

All

data

are

presented

as

mean

SE

except

when

it

is

mentioned

otherwise

Single

Leaves

The

effects

of

leaf

position

treatment

and

their

interactions

on

levels

of

terpenoids

per

gland

were

tested

by

using

nested

two-way

ANOVA

For

this

purpose

terpenoid

data

were

root

or

log-transformed

to

normalize

them

For

single

leaf

positions

the

effects

of

the

treatments

on

the

leaf

area

were

tested

with

one-way

ANOVAs

The

effect

of

leaf

area

and

treatment

on

the

total

number

of

glands

was

tested

by

analysis

of

codeviance

-LRB-

GLM

see

above

-RRB-

To

analyze

the

effect

of

leaf

area

and

treatment

on

gland

density

levels

of

terpenoids

per

gland

and

terpenoid

concentrations

per

milligram

fresh

leaf

material

ANCOVAs

were

used

These

analyses

were

performed

in

R

version

Changes

in

per

gland

concentrations

of

individual

terpenoids

at

specific

leaf

positions

after

treatments

were

tested

with

Sigma

Stat

For

normally

distributed

data

one-way

ANOVAs

were

used

Differences

between

treatments

and

controls

were

tested

for

significance

by

using

Dunn

's

post

hoc

tests

Data

not

normally

distributed

were

analyzed

using

Kruskall

Wallis

one-way

ANOVAs

on

ranks

and

Dunn

's

post

hoc

tests

Statistics

Experiment

Single

Leaves

The

effect

of

the

treatments

on

the

areas

of

leaf

was

tested

with

one-way

ANOVA

The

effect

of

leaf

area

and

treatment

on

the

total

number

of

glands

was

tested

by

analysis

of

codeviance

-LRB-

GLM

see

above

-RRB-

To

analyze

the

influence

of

leaf

area

and

treatments

on

the

accumulation

of

mono

and

sesquiterpenes

an

ANCOVA

was

achieved

All

analyses

were

performed

in

R

version

Results

Experiment

Terpenoid

Accumulation

in

Total

Cotton

Foliage

after

Induction

The

accumulation

of

terpenoid

classes

among

the

four

different

treatments

was

first

compared

on

the

basis

of

total

foliage

-LRB-

Fig.

-RRB-

For

monoterpenes

sesquiterpenes

and

terpenoid

aldehydes

there

was

a

gradual

increase

in

the

following

order

control

mechanical

damage

herbivory

and

JA

treatment

-LRB-

ANOVA

on

ranks

monoterpenes

H

P

sesquiterpenes

H

P

terpenoid

aldehydes

H

P

-RRB-

Levels

of

all

three

classes

were

elevated

in

herbivore-damaged

-LRB-

Dunn

's

tests

monoterpenes

Q

sesquiterpenes

Q

aldehydes

Q

all

P

-RRB-

and

JA-treated

plants

-LRB-

Dunn

's

tests

monoterpenes

Q

sesquiterpenes

Q

aldehydes

Q

all

P

-RRB-

in

comparison

to

the

controls

Fig.

-LRB-

SE

N

-RRB-

levels

of

terpenoids

in

the

total

foliage

of

cotton

plants

which

were

either

damaged

at

leaves

and

mechanically

-LRB-

Mech

-RRB-

fed

upon

by

larvae

of

Spodoptera

littoralis

-LRB-

Herb

-RRB-

treated

with

jasmonic

acid

-LRB-

JA

-RRB-

or

left

as

untreated

controls

-LRB-

Ctrl

-RRB-

days

previously

Asterisks

indicate

significant

differences

between

treatment

and

control

plants

based

on

Kruskall

Wallis

one-way

ANOVAs

on

ranks

and

Dunn

's

post

hoc

tests

P

All

individual

compounds

showed

the

same

pattern

that

was

seen

for

total

terpenoid

accumulation

a

gradual

increase

from

control

to

mechanical

damage

herbivory

and

JA

treatment

In

out

of

compounds

this

increase

was

significant

-LRB-

Table

-RRB-

for

herbivore

and

JA-treated

plants

compared

to

the

control

plants

while

mechanically

damaged

plants

showed

tendencies

for

increased

accumulation

Among

monoterpenes

considerable

differences

in

accumulation

after

treatment

were

shown

-LRB-

E

-RRB-

β-ocimene

increased

up

to

eightfold

after

both

herbivory

and

JA

treatment

in

comparison

to

controls

whereas

myrcene

levels

slightly

but

significantly

increased

after

herbivory

and

JA

treatment

and

the

amounts

of

α

and

β-pinene

did

not

show

significant

differences

between

treatments

and

control

Little

variation

in

the

induction

pattern

occurred

when

comparing

individual

sesquiterpenes

Levels

of

all

major

sesquiterpenes

including

β-caryophyllene

α-humulene

γ-bisabolene

and

β-bisabolol

doubled

after

herbivory

and

were

higher

after

JA

treatment

in

comparison

to

control

plants

Among

terpenoid

aldehydes

considerable

differences

appeared

Whereas

the

amounts

of

hemigossypolone

were

doubled

levels

of

heliocides

especially

and

increased

much

more

after

herbivory

and

JA

treatment

compared

to

the

controls

Heliocide

for

example

showed

up

to

a

ninefold

increase

after

herbivory

and

up

to

a

increase

after

JA

treatment

in

comparison

to

control

plants

For

these

increases

were

approximately

five

and

tenfold

respectively

Meanwhile

the

highest

levels

of

and

were

found

in

the

JA-treated

plants

reaching

up

to

that

of

control

levels

Table

levels

-LRB-

SD

N

-RRB-

of

terpenoid

products

in

total

foliage

of

G.

hirsutum

plants

that

were

either

mechanically

damaged

with

a

fabric

pattern

wheel

damaged

by

larvae

of

Spodoptera

littoralis

-LRB-

herbivory

-RRB-

treated

with

jasmonic

acid

-LRB-

JA

-RRB-

or

left

as

untreated

controlsCompoundANOVA

-LRB-

df

-RRB-

Levels

of

terpenoids

-LRB-

mg

-RRB-

F/HP

ValueControlMechanical

damageHerbivoryJAMonoterpenesα-Pinene7.2130

-LRB-

E

-RRB-

Sesquiterpenes

-LRB-

E

-RRB-

Terpenoid

Heliocide

Heliocide

Heliocide

Heliocide

The

effect

of

treatment

on

terpenoid

levels

were

tested

using

one-way

ANOVAs

For

normally

distributed

data

one-way

ANOVAs

were

performed

-LRB-

F

values

-RRB-

For

not

normally

distributed

data

Kruskall

Wallis

one-way

ANOVAs

on

ranks

were

performed

-LRB-

H

values

-RRB-

P

significant

differences

from

the

control

according

to

Bonferoni

post

hoc

tests

P

significant

differences

from

the

control

according

to

Dunn

's

post

hoc

tests

Growth

and

Subepidermal

Gland

Production

of

Cotton

Foliage

ANOVA

showed

an

effect

of

the

treatments

on

the

total

leaf

area

-LRB-

F

P

-RRB-

Whereas

controls

mechanically

and

herbivore-damaged

plants

were

highly

comparable

in

size

-LRB-

mean

-RRB-

JA-treated

plants

-LRB-

mean

-RRB-

showed

a

reduction

in

the

total

area

of

foliage

down

to

that

of

the

controls

-LRB-

Bonferoni

post

hoc

test

P

-RRB-

Because

of

significantly

smaller

leaves

in

the

JA-treated

plants

the

effect

of

treatment

on

the

production

of

subepidermal

glands

was

tested

by

using

a

GLM

with

leaf

area

as

a

covariable

For

all

treatments

this

covariable

showed

an

impact

on

the

number

of

glands

-LRB-

t

P

Fig.

-RRB-

In

comparison

to

the

controls

an

increase

in

the

number

of

glands

was

found

for

herbivore-damaged

plants

-LRB-

t

P

-RRB-

and

for

plants

that

were

treated

with

JA

-LRB-

t

P

-RRB-

Because

there

were

no

differences

between

mechanically

damaged

and

non-treated

plants

the

data

of

both

groups

were

combined

in

this

analysis

Fig.

of

leaf

area

and

treatment

on

the

number

of

glands

in

the

total

foliage

of

cotton

plants

which

were

either

damaged

at

leaves

and

mechanically

-LRB-

Mech

-RRB-

fed

upon

by

larvae

of

Spodoptera

littoralis

-LRB-

Herb

-RRB-

treated

with

jasmonic

acid

-LRB-

JA

-RRB-

or

left

as

untreated

controls

-LRB-

Ctrl

-RRB-

days

previously

Regressions

were

drawn

based

on

transformed

data

from

a

generalized

linear

model

-LRB-

GLM

-RRB-

with

a

quasi-Poisson

error

distribution

Data

for

control

and

mechanically

damaged

plants

were

combined

due

to

factor

level

reduction

Single

Leaf

Analysis

To

assess

more

precisely

plant

response

to

the

treatments

analyses

were

carried

out

at

the

level

of

individual

leaves

For

all

treatments

the

distribution

of

leaf

areas

followed

a

consistent

pattern

within

plants

with

the

biggest

leaves

at

intermediate

positions

-LRB-

Fig.

-RRB-

Whereas

no

differences

in

leaf

area

were

evident

among

mechanically

damaged

herbivore-damaged

and

control

plants

plants

treated

with

JA

exhibited

significant

smaller

areas

for

leaves

in

comparison

to

corresponding

control

leaves

-LRB-

ESM

Tables

and

-RRB-

Because

of

the

negative

impact

of

JA

treatment

on

the

leaf

area

the

following

analyses

of

the

effect

of

treatments

on

gland

and

terpenoid

production

were

performed

by

using

leaf

area

as

covariable

-LRB-

statistical

results

see

ESM

Tables

and

-RRB-

Fig.

-LRB-

SE

N

-RRB-

leaf

area

number

of

glands

gland

density

terpenoid

level

per

gland

and

terpenoid

concentration

of

cotton

plants

which

were

either

damaged

at

leaves

and

mechanically

-LRB-

Mech

-RRB-

fed

upon

by

larvae

of

Spodoptera

littoralis

-LRB-

Herb

-RRB-

or

treated

with

jasmonic

acid

-LRB-

JA

-RRB-

days

previously

Areas

of

herbivore-damaged

leaves

and

were

extrapolated

from

remaining

leaf

material

For

each

leaf

position

asterisks

indicate

significant

differences

from

corresponding

control

leaves

based

on

one-way

ANOVAs

-LRB-

a

-RRB-

on

analyses

of

codeviance

with

leaf

area

as

covariable

-LRB-

b

-RRB-

and

ANCOVAs

with

leaf

area

as

covariable

-LRB-

c

e

-RRB-

P

Gland

number

per

leaf

revealed

a

similar

distribution

pattern

among

leaves

for

all

treatments

The

oldest

leaf

contained

the

lowest

gland

number

which

increased

gradually

toward

leaf

and

again

decreased

in

the

expanding

leaves

and

-LRB-

Fig.

-RRB-

Young

leaves

-LRB-

-RRB-

of

herbivore-treated

plants

showed

significantly

elevated

numbers

of

glands

in

comparison

to

corresponding

leaves

of

control

plants

Lower

but

still

significant

increases

in

gland

numbers

were

found

for

leaves

and

-LRB-

youngest

leaf

-RRB-

of

plants

that

were

treated

with

JA

compared

to

control

leaves

Mechanical

damage

showed

nearly

no

effect

on

gland

number

Changes

in

leaf

area

and

gland

numbers

after

treatment

should

have

an

impact

on

gland

densities

Independent

of

treatment

gland

densities

increased

gradually

from

the

oldest

leaf

toward

the

youngest

leaf

-LRB-

Fig.

-RRB-

This

pattern

was

enhanced

in

herbivore-damaged

and

JA-treated

plants

because

of

significantly

higher

gland

densities

in

young

leaves

-LRB-

-RRB-

compared

to

those

leaves

from

the

control

plants

Mechanical

damage

did

not

cause

an

increased

gland

density

in

any

leaf

To

analyze

how

terpenoid

production

was

altered

by

treatment

total

terpenoid

levels

per

gland

were

calculated

Treatments

had

a

considerable

effect

on

the

pattern

of

terpenoids

per

gland

among

leaves

-LRB-

Fig.

-RRB-

Control

plants

showed

highest

levels

in

the

oldest

leaf

and

lowest

levels

in

the

youngest

one

In

contrast

mechanically

and

herbivore-damaged

plants

exhibited

highest

values

in

younger

leaves

having

significantly

elevated

total

terpenoid

levels

in

comparison

to

those

of

control

leaves

Plants

that

were

treated

with

JA

showed

significantly

elevated

levels

of

total

terpenoids

per

gland

in

young

and

intermediate

leaves

in

comparison

to

the

controls

The

increase

in

total

terpenoid

and

gland

production

caused

by

the

treatments

as

well

as

altered

leaf

areas

had

a

considerable

impact

on

the

pattern

of

the

total

terpenoid

concentrations

in

leaves

Total

terpenoid

concentrations

showed

a

consistent

distribution

pattern

among

leaves

for

all

treatments

-LRB-

Fig.

-RRB-

There

was

a

gradual

increase

starting

from

the

oldest

leaf

toward

the

youngest

leaves

and

All

treatments

enhanced

this

pattern

by

causing

significantly

higher

concentrations

in

intermediate

and

young

leaves

in

comparison

to

the

leaves

of

corresponding

control

plants

The

lowest

effect

was

found

for

mechanically

damaged

plants

whereas

plants

treated

with

JA

reached

the

highest

concentration

with

mean

levels

of

approximately

μg

terpenoids

per

milligram

fresh

weight

The

analyses

of

individual

terpenoids

revealed

a

significant

impact

of

treatment

leaf

position

and

interactions

between

these

two

factors

on

levels

of

all

compounds

per

gland

-LRB-

Table

-RRB-

The

lowest

effects

of

treatment

were

found

for

the

monoterpenes

α

and

β-pinene

whereas

the

biggest

changes

appeared

for

-LRB-

E

-RRB-

β-ocimene

and

heliocides

and

Differences

of

single

terpenoid

levels

per

gland

among

leaves

appeared

to

be

biggest

for

-LRB-

E

-RRB-

β-ocimene

and

heliocides

but

in

case

of

the

latter

this

was

due

to

its

unusual

absence

in

leaves

and

-LRB-

Fig.

-RRB-

Significant

interactions

between

treatment

and

leaf

position

revealed

that

changes

in

levels

of

total

and

single

terpenoids

of

induced

plants

were

not

consistent

in

all

leaves

which

is

illustrated

in

Fig.

As

with

total

terpenoid

levels

per

gland

-LRB-

Fig.

-RRB-

mechanical

damage

and

herbivory

caused

significantly

elevated

accumulation

of

individual

compounds

per

gland

in

the

youngest

leaves

in

comparison

to

control

plants

In

contrast

JA

treatment

induced

elevated

levels

in

young

but

also

in

intermediate

leaves

compared

to

control

leaves

JA

treatment

led

to

the

highest

increases

of

most

compounds

especially

for

heliocides

in

comparison

to

those

in

the

corresponding

leaves

of

control

plants

Fig.

-LRB-

SE

N

-RRB-

concentrations

of

terpenoids

per

gland

among

true

leaves

of

Gossypium

hirsutum

plants

which

were

either

damaged

at

leaves

and

mechanically

-LRB-

Mech

-RRB-

fed

upon

by

larvae

of

Spodoptera

littoralis

-LRB-

Herb

-RRB-

treated

with

jasmonic

acid

-LRB-

JA

-RRB-

or

left

as

untreated

controls

-LRB-

Ctrl

-RRB-

days

previously

Asterisks

indicate

significant

differences

-LRB-

P

-RRB-

from

corresponding

control

leaves

based

on

one-way

ANOVA

's

and

Bonferoni

post

hoc

tests

Pound

signs

indicate

significant

differences

-LRB-

P

-RRB-

from

corresponding

control

leaves

based

on

Kruskall

Wallis

one-way

ANOVAs

on

ranks

and

Dunn

's

post

hoc

testsTable

of

nested

two-way

ANOVA

for

the

effects

of

treatment

-LRB-

mechanical

damage

herbivory

jasmonic

acid

application

-RRB-

leaf

position

and

interactions

on

levels

of

accumulated

terpenoids

per

gland

in

plants

of

G.

hirsutumCompoundTreatment

-LRB-

df

-RRB-

Leaf

Number

-LRB-

df

-RRB-

Treatment

Leaf

Number

-LRB-

df

-RRB-

FPFPFPTotal

-LRB-

E

-RRB-

Sesquiterpenes

-LRB-

E

-RRB-

Terpenoid

Heliocide

Heliocide

Heliocide

Heliocide

Experiment

Morphological

and

Chemical

Changes

in

Young

Leaves

after

Induction

In

addition

to

herbivore-treated

plants

and

untreated

controls

this

supplementary

experiment

also

included

mechanically

damaged

plants

that

were

injured

more

frequently

and

over

a

longer

period

than

in

experiment

ANOVA

showed

an

effect

of

the

treatments

on

areas

of

young

leaves

-LRB-

leaf

F

P

Fig.

ESM

Table

-RRB-

Herbivory

reduced

the

areas

of

these

leaves

in

comparison

to

those

of

the

corresponding

leaves

of

the

control

plants

-LRB-

t

P

-RRB-

Therefore

the

effect

of

mechanical

damage

and

herbivory

on

the

production

of

glands

were

tested

by

using

a

GLM

with

leaf

area

as

a

covariable

For

both

treatments

leaf

area

showed

an

impact

on

the

number

of

glands

-LRB-

t

P

-RRB-

In

comparison

to

the

control

plants

a

significant

increase

in

the

number

of

glands

was

found

for

mechanically

damaged

plants

-LRB-

t

P

-RRB-

and

an

even

more

pronounced

increase

for

herbivore-treated

plants

-LRB-

t

P

Fig.

-RRB-

Fig.

-LRB-

SE

N

-RRB-

leaf

area

number

of

glands

and

monoterpene

and

sesquiterpene

levels

of

leaf

from

Gossypium

hirsutum

plants

which

were

either

damaged

mechanically

at

leaves

and

-LRB-

Mech

-RRB-

fed

upon

by

larvae

of

Spodoptera

littoralis

-LRB-

Herb

-RRB-

on

one

cotyledon

leaves

and

or

left

as

untreated

controls

-LRB-

Ctrl

-RRB-

Asterisks

indicate

significant

differences

from

corresponding

control

leaves

based

on

one-way

ANOVAs

-LRB-

a

-RRB-

on

analyses

of

codeviance

with

leaf

area

as

covariable

-LRB-

b

-RRB-

and

ANCOVAs

with

leaf

area

as

covariable

-LRB-

c

d

-RRB-

P

P

P

The

accumulation

of

mono

and

sesquiterpenes

in

leaf

after

treatment

followed

a

comparable

pattern

a

gradual

increase

from

control

to

mechanical

damage

and

herbivory

-LRB-

ANOVA

monoterpenes

F

P

sesquiterpenes

F

P

Fig.

d

-RRB-

Levels

of

monoterpenes

were

elevated

after

herbivory

in

comparison

to

the

controls

-LRB-

t

P

-RRB-

Levels

of

sesquiterpenes

were

elevated

after

mechanical

damage

-LRB-

t

P

-RRB-

and

herbivory

-LRB-

t

P

-RRB-

in

comparison

to

the

controls

Discussion

Cotton

-LRB-

G.

hirsutum

-RRB-

accumulates

a

large

variety

of

terpenes

constitutively

including

monoterpenes

-LRB-

-RRB-

sesquiterpenes

-LRB-

-RRB-

and

terpenoid

aldehydes

-LRB-

and

-RRB-

This

study

is

the

first

to

describe

the

accumulation

of

all

three

of

these

classes

after

actual

or

simulated

herbivory

Increased

levels

of

terpenoids

were

observed

in

the

total

foliage

week

after

mechanical

damage

feeding

by

Spodoptera

littoralis

caterpillars

or

treatment

with

JA

in

comparison

to

the

untreated

controls

A

closer

look

revealed

that

after

damage

to

older

leaves

-LRB-

leaves

and

numbered

from

the

base

-RRB-

the

increase

was

restricted

mainly

to

young

leaves

-LRB-

leaves

-RRB-

Similarly

studies

on

cotton

that

investigate

the

effects

of

herbivory

by

larvae

of

S.

exigua

and

the

wireworm

Agriotes

lineatus

also

showed

elevated

concentrations

of

terpenoid

aldehydes

in

young

cotton

leaves

-LRB-

McAuslane

et

al.

McAuslane

and

Alborn

Bezemer

et

al.

-RRB-

Such

an

induction

pattern

is

in

agreement

with

ODT

which

predicts

that

an

increase

in

the

accumulation

of

defense

chemicals

occurs

preferentially

in

plant

parts

with

the

highest

fitness

value

such

as

young

tissues

or

reproductive

organs

-LRB-

McKey

Frischknecht

et

al.

Ohnmeiss

and

Baldwin

-RRB-

All

three

classes

of

cotton

terpenoids

are

stored

in

subepidermal

pigment

glands

found

in

leaves

and

other

organs

After

treatment

G.

hirsutum

displayed

two

ways

to

achieve

elevated

terpenoid

accumulation

-LRB-

-RRB-

production

of

additional

glands

and

-LRB-

-RRB-

increased

filling

of

existing

glands

-LRB-

Fig.

-RRB-

We

showed

that

production

of

additional

glands

was

restricted

to

leaves

that

were

still

under

development

or

newly

formed

after

treatment

This

trend

also

has

been

observed

after

attack

by

spider

mites

or

larvae

of

S.

exigua

on

cotton

-LRB-

McAuslane

et

al.

Agrawal

and

Karban

-RRB-

In

other

plants

such

as

birch

and

tomato

an

elevated

number

of

defense

structures

like

glandular

trichomes

is

also

produced

in

young

leaves

after

induction

by

herbivores

-LRB-

Boughton

et

al.

Valkama

et

al.

-RRB-

Besides

the

fact

that

according

to

ODT

plants

have

been

selected

to

increase

their

resistance

to

herbivores

especially

in

young

tissue

the

formation

of

subepidermal

pigment

glands

in

cotton

might

be

necessarily

restricted

to

newly

developing

leaves

because

of

developmental

constraints

In

counting

the

number

of

cotton

leaf

glands

under

a

stereomicroscope

we

may

have

overestimated

the

production

of

new

glands

after

treatment

if

herbivory

triggered

the

filling

of

pre-existing

glands

that

were

unpigmented

-LRB-

H.

T.

Alborn

personal

communication

-RRB-

However

by

using

several

microscopic

techniques

we

did

not

find

any

evidence

for

unfilled

glands

in

the

leaves

analyzed

Therefore

we

assume

that

actual

or

simulated

herbivory

does

indeed

induce

increased

numbers

of

glands

in

cotton

The

increased

filling

of

existing

glands

with

terpenoids

was

shown

in

the

youngest

leaves

of

induced

plants

for

all

major

terpenoid

classes

However

considerable

differences

in

the

magnitude

of

this

additional

accumulation

were

observed

among

individual

terpenoid

compounds

Among

terpenoid

aldehydes

levels

of

heliocides

and

showed

the

highest

increase

after

treatments

Other

studies

on

cotton

species

also

have

demonstrated

that

these

two

heliocides

increase

more

than

and

hemigossypolone

-LRB-

HGQ

-RRB-

after

herbivory

by

Spodoptera

species

-LRB-

McAuslane

et

al.

McAuslane

and

Alborn

Agrell

et

al.

Bezemer

et

al.

-RRB-

Here

we

showed

that

levels

of

-LRB-

E

-RRB-

β-ocimene

increased

in

a

magnitude

similar

to

and

and

that

this

compound

increased

most

among

monoterpenes

and

sesquiterpenes

Interestingly

-LRB-

E

-RRB-

β-ocimene

is

a

direct

precursor

in

the

formation

of

and

combining

with

hemigossypolone

in

a

Diels

Alder-type

reaction

-LRB-

Stipanovic

et

al.

Fig.

-RRB-

Therefore

in

non-treated

plants

the

biosynthesis

of

this

monoterpene

could

represent

a

limiting

step

in

the

production

of

the

appropriate

heliocides

This

proposition

is

supported

by

the

fact

that

in

cotyledons

of

G.

hirsutum

which

do

not

contain

any

heliocides

hemigossypolone

is

present

-LRB-

S.

Opitz

unpublished

-RRB-

but

-LRB-

E

-RRB-

β-ocimene

is

absent

A

comparable

regulation

mechanism

that

involves

myrcene

could

determine

the

levels

of

and

-LRB-

Stipanovic

et

al.

-RRB-

Our

study

compared

the

terpenoid

content

of

total

cotton

foliage

after

three

different

treatments

and

showed

a

gradual

increase

in

terpenoid

levels

in

the

following

order

mechanical

damage

with

a

fabric

pattern

wheel

herbivory

by

S.

littoralis

caterpillars

and

application

of

JA

However

a

single

incidence

of

mechanical

injury

is

hard

to

compare

with

continuous

feeding

damage

by

an

insect

Studies

on

lima

bean

have

demonstrated

that

both

the

intensity

and

frequency

of

mechanical

damage

can

alter

the

reaction

of

a

plant

-LRB-

Mithöfer

et

al.

-RRB-

This

is

consistent

with

the

results

of

experiment

where

more

frequent

mechanical

damage

caused

significantly

higher

numbers

of

glands

-LRB-

Fig.

-RRB-

a

response

not

detectable

after

the

less

frequent

mechanical

damage

in

experiment

-LRB-

Fig.

and

-RRB-

However

in

both

experiments

herbivory

induced

stronger

reactions

of

plants

than

mechanical

damage

Elicitors

found

in

the

regurgitate

of

herbivores

are

known

to

trigger

the

biosynthesis

of

defense

metabolites

-LRB-

Alborn

et

al.

-RRB-

If

such

elicitors

play

a

role

in

cotton

defense

reactions

mechanical

injury

may

never

result

in

terpenoid

accumulation

comparable

to

that

caused

by

herbivory

The

strongest

increase

in

both

terpenoid

and

gland

production

appeared

in

plants

that

were

treated

with

JA

a

ubiquitous

plant

hormone

known

to

mediate

defense

responses

to

biotic

and

abiotic

stresses

-LRB-

Browse

-RRB-

A

previous

study

with

cotton

showed

that

the

application

of

the

methylated

derivative

methyl

jasmonate

induced

the

synthesis

and

emission

of

volatile

terpenes

a

response

also

observed

for

herbivore-damaged

plants

-LRB-

Rodriguez-Saona

et

al.

-RRB-

Obviously

JA

plays

an

important

role

in

mediating

damage-induced

signaling

in

G.

hirsutum

Given

the

effects

of

exogenous

application

herbivory

can

be

assumed

to

trigger

elevated

internal

levels

of

JA

as

in

other

species

-LRB-

Baldwin

et

al.

Creelman

and

Mullet

-RRB-

For

example

in

tobacco

lima

bean

or

maize

internal

JA

levels

were

determined

in

ranges

of

single

nanograms

per

gram

fresh

weight

showing

to

increases

within

the

first

hours

after

induction

-LRB-

Baldwin

et

al.

Koch

et

al.

Schmelz

et

al.

-RRB-

However

internal

JA

levels

of

cotton

still

need

to

be

investigated

If

the

irrigation

of

plants

with

μM

JA

in

our

study

led

to

a

non-physiological

high

internal

concentration

of

JA

this

could

explain

why

JA

treatment

induced

terpenoid

accumulation

to

a

greater

extent

than

herbivory

We

observed

additionally

that

JA

inhibited

leaf

development

a

response

also

seen

in

other

plants

-LRB-

Sembdner

and

Parthier

-RRB-

Studies

on

the

induction

of

chemical

defense

in

plants

after

herbivory

always

raise

questions

about

the

specificity

of

such

reactions

to

particular

enemies

Certain

plants

have

been

shown

to

induce

different

spectra

of

defenses

in

response

to

different

herbivores

-LRB-

Turlings

et

al.

Traw

and

Dawson

Delphia

et

al.

-RRB-

However

in

this

study

we

did

not

find

substantial

differences

in

the

pattern

of

induction

among

treatments

for

the

major

terpenoids

measured

-LRB-

see

Table

-RRB-

The

amounts

of

single

compounds

varied

in

magnitude

leading

to

changes

in

the

terpenoid

profile

but

these

profile

changes

tended

to

be

comparable

among

treatments

The

induction

of

elevated

terpenoid

accumulation

in

leaves

of

G.

hirsutum

may

thus

be

a

non-specific

reaction

of

plants

to

damage

Similar

non-specific

reactions

to

damage

are

known

for

terpenes

in

other

species

-LRB-

Banchio

et

al.

-RRB-

as

well

as

for

alkaloids

-LRB-

Frischknecht

et

al.

Baldwin

et

al.

-RRB-

phenolics

-LRB-

Cipollini

-RRB-

and

glucosinolates

-LRB-

Bodnaryk

-RRB-

The

fact

that

cotton

plants

react

to

damage

with

elevated

levels

of

terpenoids

accumulated

in

the

subepidermal

pigment

glands

of

their

foliage

suggests

that

these

compounds

function

in

plant

defense

Caterpillar

species

such

as

S.

exigua

or

Heliothis

virescens

prefer

feeding

on

glandless

instead

of

glanded

cultivars

of

G.

hirsutum

thus

supporting

the

argument

that

the

gland

terpenes

play

a

key

role

in

defense

of

cotton

against

herbivores

-LRB-

Montandon

et

al.

McAuslane

and

Alborn

-RRB-

Leaf

material

from

plants

of

glanded

cultivar

lines

that

had

been

induced

by

herbivory

showed

increased

deterrent

or

toxic

effects

on

Spodoptera

species

compared

to

material

from

uninduced

plants

a

finding

thought

to

be

due

to

their

elevated

contents

of

terpenoid

aldehydes

-LRB-

Alborn

et

al.

McAuslane

et

al.

Anderson

et

al.

-RRB-

Indeed

terpenoid

aldehydes

such

as

gossypol

hemigossypolone

and

the

heliocides

and

exhibit

strong

toxicity

to

caterpillars

of

H.

virescens

and

Pectinophora

gossypiella

after

addition

to

artificial

diet

-LRB-

Elliger

et

al.

-RRB-

While

the

feeding

deterrent

effects

of

induced

cotton

foliage

are

usually

attributed

to

elevated

terpenoid

aldehydes

contents

-LRB-

Hedin

et

al.

McAuslane

et

al.

McAuslane

and

Alborn

-RRB-

we

showed

that

cotton

plants

also

increase

their

levels

of

monoterpenes

and

sesquiterpenes

Thus

these

compounds

may

also

participate

in

defense

against

herbivores

A

sesquiterpene

caryophyllene

oxide

has

been

demonstrated

to

synergize

the

negative

effect

of

gossypol

the

dominant

terpenoid

aldehyde

in

roots

and

seeds

of

cotton

on

larval

development

of

H.

virescens

-LRB-

Gunasena

et

al.

-RRB-

Additionally

caryophyllene

retarded

the

growth

and

delayed

the

time

of

development

of

these

larvae

Other

studies

have

shown

that

single

sesquiterpenes

such

as

β-bisabolene

impair

the

development

of

insect

herbivores

and

deter

them

from

feeding

-LRB-

Bowers

et

al.

Gonzalez-Coloma

et

al.

Zipfel

-RRB-

Beside

their

function

against

herbivores

terpenoids

are

also

considered

to

play

a

defensive

role

against

fungi

or

pathogens

Because

plants

are

exposed

especially

to

infestation

at

wound

sites

the

elevated

terpenoid

levels

in

damaged

leaves

might

have

a

critical

role

in

helping

plants

to

cope

with

pathogens

For

cotton

studies

have

shown

that

certain

terpenoid

aldehydes

possess

antifungal

and

antipathogen

activities

-LRB-

Zhang

et

al.

Abraham

et

al.

-RRB-

A

good

example

for

the

antibiotic

activity

of

terpenoids

in

other

species

comes

from

conifers

where

the

growth

and

germination

of

bark

beetle

associated

pathogens

is

inhibited

-LRB-

Keeling

and

Bohlmann

-RRB-

In

addition

analyses

of

terpene-rich

essential

oils

from

a

variety

of

plant

species

have

shown

antibacterial

and

antifungal

activities

indicating

the

potential

role

of

these

substances

in

plant

defense

-LRB-

Oyedeji

and

Afolayan

Ozer

et

al.

-RRB-

In

summary

our

results

demonstrate

that

elevated

levels

of

terpenoids

in

cotton

leaves

after

real

and

simulated

herbivory

represent

a

general

wound

response

that

is

mediated

by

JA

The

increase

in

terpenoids

is

due

to

the

production

of

additional

glands

in

which

terpenoids

are

stored

as

well

as

the

increased

filling

of

existing

glands

As

all

three

classes

of

terpenoids

-LRB-

monoterpenes

sesquiterpenes

and

terpenoid

aldehydes

-RRB-

were

elevated

after

damage

these

substances

may

act

synergistically

in

defense

against

herbivores

or

pathogens

-LRB-

Stipanovic

et

al.

-RRB-

Electronic

supplementary

material

Below

is

the

link

to

the

electronic

supplementary

material

ESM

-LRB-

DOC

kb

-RRB-

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

gossypium hirsutum
cotton
spodoptera littoralis
terpenoid aldehydes
monoterpenes
sesquiterpenes
pigment glands
jasmonic acid
mechanical damage
induction