Biochemistry of Oleoresinosis Monoterpene and Diterpene Biosynthesis in Lodgepole Pine Saplings Infected with Ceratocystis clavigera or Treated with Carbohydrate Elicitors

Abstract

Elevated levels of monoterpenes and diterpene resin acids are produced in the stems of lodgepole pine (Pinus contorta var latifolia) saplings when wounded and inoculated with the blue-stain fungus Ceratocystis clavigera or when wounded and treated with a pectic fragment from tomato leaves (PIIF) or a fungal cell wall fragment (chitosan). This induced defensive response (hyperoleoresinosis) is the result of a transient rise in the ability to biosynthesize cyclic monoterpenes and diterpene resin acids as measured by the in vivo incorporation of label from [U-(14)C]sucrose relative to untreated controls, and is accompanied by a corresponding rise in the levels or activities of the relevant terpene cyclases as determined by in vitro assay using labeled acyclic precursors. The results indicate that juvenile P. contorta responds to infection and biotic elicitors much like the mature tree, and they suggest that the Pinaceae possess a mechanism for elicitor recognition and induced defense similar to that of other higher plants.

Full text

Plant
Physiol.
(1987)
85,
1123-1128
0032-0889/87/85/1123/06/$0
1.00/0
Biochemistry
of
Oleoresinosis'
MONOTERPENE
AND
DITERPENE
BIOSYNTHESIS
IN
LODGEPOLE
PINE
SAPLINGS
INFECTED
WITH
CERA
TOCYSTIS
CLA
VIGERA
OR
TREATED
WITH
CARBOHYDRATE
ELICITORS
Received
for
publication
July
2,
1987
and
in
revised
form
September
8,
1987
RODNEY
CROTEAU*,
SANDRA
GURKEWITZ,
MARK
A.
JOHNSON,
AND
HENRY
J.
FISK
Institute
of
Biological
Chemistry,
Washington
State
University,
Pullman,
Washington
99164-6340
ABSTRACI
Elevated
levels
of
monoterpenes
and
diterpene
resin
acids
are
produced
in
the
stems
of
lodgepole
pine
(Pinus
contorta
var
latifolia)
saplings
when
wounded
and
inoculated
with
the
blue-stain
fungus
Ceratocystis
clavigera
or
when
wounded
and
treated
with
a
pectic
fragment
from
tomato
leaves
(PIIF)
or
a
fungal
cell
wall
fragment
(chitosan).
This
induced
defensive
response
(hyperoleoresinosis)
is
the
result
of
a
transient
rise
in
the
ability
to
biosynthesize
cyclic
monoterpenes
and
diterpene
resin
acids
as
measured
by
the
in
vivo
incorporation
of
label
from
[U-
'4Clsucrose
relative
to
untreated
controls,
and
is
accompanied
by
a
corresponding
rise
in
the
levels
or
activities
of
the
relevant
terpene
cyclases
as
determined
by
in
vitro
assay
using
labeled
acycic
precursors.
The
results
indicate
that
juvenile
P.
contorta
responds
to
infection
and
biotic
elicitors
much
like
the
mature
tree,
and
they
suggest
that
the
Pinaceae
possess
a
mechanism
for
elicitor
recognition
and
induced
de-
fense
similar
to
that
of
other
higher
plants.
25,
26)
and
the
broad
outlines
of
pine
defense
biology
have
been
delineated
(2,
15).
The
biosynthesis
of
resin
terpenoids,
however,
is
poorly
understood,
as
is
the
biochemistry
of
the
defense
reaction;
present
concepts
are
based
largely
on
analogy
to
terpen-
oid
metabolism
and
phytoalexin
production
in
herbaceous
spe-
cies
(7,
15,
17).
The
lack
of
fundamental
information
about
this
defense
response
is,
in
part,
a
result
of
the
difficulties
encountered
in
experimenting
with
mature
trees
in
the
forest
setting,
the
system
in
which
the
phenomenon
was
first
discovered
and
with
which
most
early
observations
were
made
(17,
19,
21,
23,
24).
In
this
communication
we
provide
an
account
of
induced
oleo-
resinosis
in
2-year-old
lodgepole
pine
saplings
(maintained
in
a
greenhouse)
in
response
to
inoculation
with
'blue
staining'
fungus
(C.
clavigera)
and
with
carbohydrate
elicitors,
we
report
in
vivo
experiments-
which
demonstrate
the
elevated
formation
of
mon-
oterpenes
and
diterpene
resin
acids,
and
we
describe
the
isolation
and
enhanced
activity
of
the
terpenoid
cyclases
which
catalyze
the
committed
steps
of
monoterpene
and
diterpene
biosynthesis
in
this
species.
The
terpenoid
fraction
of
pine
resin
contains
20
to
50%
volatile
monoterpenes
(C10),
minor
levels
of
sesquiterpenoids
(C5),
and
50
to
80%
nonvolatile
diterpene
acids
(C20)
(8,
18),
and
the
induced
secretion
of
these
materials
constitutes
an
important
component
of
the
defense
response of
pines
to
attack
by
bark
beetles
and
their
associated
pathogenic
fungi
(2,
13,
26).
Resistant
lodgepole
pine
(Pinus
contorta
Douglas)
produces
high
levels
of
a-pinene
and
limonene
(Fig.
1)
in
the
secondary
resin
elicited
by
attack
(relative
to
the
constitutive
[preformed]
resin
contained
in
the
resin
ducts)
(19);
however,
the
major
factor
in
resistance
to
the
mountain
pine
beetle
(Dendroctonus
ponderosae
Hopkins)
and
associated
fungus
(Ceratocystis
clavigera
Robinson
et
Dav-
idson)
appears
to
be
a
rapid
and
vigorous
secondary
resinosis
resulting
from
the
increased
production
of
all
constitutive
ter-
penoids
(21,
23).
Mature
lodgepole
pine
inoculated
with
C.
clavigera,
a
pectic
fragment
from
plant
cell
walls,
or
a
fungal
cell
wall
fragment
(chitosan)
respond
by
producing
resin
monoter-
penes
to
the
same
or
greater
extent
as
control
trees
in
which
the
response
is
elicited
by
living
mountain
pine
beetle,
suggesting
that
pines,
like
other
higher
plants,
possess
a
common
recogni-
tion-defense
mechanism
(17).
The
chemistry
of
pine
resin
and
the
role
of
resin
components
in
resistance
to
infestation
and
infection
have
been
discussed
(20,
'
Supported
in
part
by
United
States
Department
of
Agriculture
Grant
86-CRCR-1-2052
and
by
a
grant-in-aid
from
Hercules
Incorporated.
Scientific
Paper
No.
7810,
Project
0268,
from
the
Research
Center,
College
of
Agriculture
and
Home
Economics,
Washington
State
Univer-
sity,
Pullman,
WA
99164.
MATERIALS
AND
METHODS
Plant
Materials,
Substrates,
and
Reagents.
Two-year-old
lodgepole
pine
saplings
(Pinus
contorta
Douglas
var
latifolia
Engelmann)
of
22
to
28
cm
in
height
were
obtained
from
the
U.S.
Forest
Service
Pine
Nursery
(Bend,
OR;
courtesy
of
J.
Wojtowych)
and
the
Potlatch
Corp.
(Lewiston,
ID;
courtesy
of
Dr.
R.
Blair)
and,
following
transfer
to
10-in
deep
pots
(620
cc)
in
Grace
Forestry
Mix
(pH
4.5-5.0)
(W.
R.
Grace
&
Co.,
Memphis,
TN)
and
cold
conditioning
at
0
to
4°C
for
1
month,
they
were
maintained
in
a
greenhouse
(30C
day/18°C
night,
fertilized
weekly
with
a
complete
fertilizer
of
N:P:K
=
15:30:15
with
trace
nutrients)
for
a
minimum
of
6
weeks
to
break
dor-
mancy
before
use.
The
'blue
stain'
fungus
Ceratocystis
clavigera
(Robinson
et
Davidson)
was
obtained
from
ATCC
(No.
24286)
and
was
main-
tained
on
malt
agar
at
24C.
Two
weeks
after
transfer
the
fungal
mats
were
harvested,
washed
by
repeated
suspension
in
25
mM
sodium
phosphate
(pH
7.0)
and
centrifugation,
and
the
wet
paste
was
used
as
the
inoculum.
[l
-3H]Geranyl
pyrophosphate
(100
Ci/mol)
and
[l
-3H]farnesyl
pyrophosphate
(84.8
Ci/mol)
were
prepared
as
described
previ-
ously
(6).
[1-3H]Geranylgeranyl
pyrophosphate
(10.6
Ci/mol)
was
a
gift
from
R.
M.
Coates,
University
of
Illinois
and
abietic
acid
(a
mixture
of
abietic
and
dehydroabietic
acids)
was
from
Aldrich
Chemical
Co.
Methylation
of
the
mixture
with
BF3
in
methanol
(see
below)
gave
largely
methyl
dehydroabietate
which
was
converted
to
the
corresponding
alcohol
by
reduction
with
LiAlH4
in
ether,
and
thence
to
the
abietriene
by
transformation
to
and
reduction
of
the
primary
iodide
with
NaBH3CN
(14).
[U-
'4C]Sucrose
(514
Ci/mol)
was
obtained
from
New
England
Nu-
clear.
The
sources
of
other
reagents
and
standards
have
been
1123

Plant
Physiol.
Vol.
85,
1987
c-Pinene
P-Pinene
3-Carene
0c-
Phellandrene
jB-Phellandrene
Abietic
Acid
Dehydroabietic
FIG.
1.
Lodgepole
pine
oleoresin
componeni
described
(1
1).
Time-Course
of
Induced
Oleoresinosis.
Saplings
M
at
midstem
with
a
single,
3
mm
deep
puncture
1
gauge
needle
and
the
wound
was
either
untreated
with
C.
clavigera
mycelia,
or
injected
with
10
A
aqueous
solution
of
a
pectic
plant
wall
fragment
(proteinase
inhibitor
inducing
factor)
(3)
or
chitose
trol
saplings
were
unwounded.
At
2-d
or
4-d
inter
d,
six
saplings
subjected
to
each
treatment
were
hi
the
stem
segment
from
1
cm
above
to
1
cm
belo'
site
was
excised
with
a
razor
blade.
For
the
corresl
trols,
a
2
cm
segment
from
the
midstem
was
tak(
segments
were
chopped
into
1
mm
sections
on
a
r
num
foil
and
sections
from
like
treatments
were
ir
100-ml
round
bottom
flask,
along
with
the
sticky,
pad
and
razor
blade,
and
the
entire
contents
sub
haustive
simultaneous
solvent
extraction
(diethyl
distillation
(J&W
Scientific,
apparatus
No.
320-100
p-cymene
and
2
mg
octadecanoic
acid
as
internal
st
ethereal
distillate
containing
the
volatiles
(with
t
internal
standard)
was
concentrated
to
small
volu
lyzed
by
capillary
GLC-MS.
The
aqueous
still
pol
adjusted
to
0.1
N
KOH,
the
solids
removed
by
filtra
solids
and
still
pot
washed
with
ether.
The
wash
sol)
utilized
to
extract
nondistillable
neutral
compour
alkaline
aqueous
residue,
which
was
subsequently
E
HCI,
saturated
with
NaCl,
and
extracted
with
CH(
the
resin
acids.
Following
removal
of
solvent,
ti
fraction
(with
the
octadecanoate
internal
standard)
dried
and
weighed,
as
was
the
oven
dried
plant
r
the
resin
acids
of
P.
contorta
are
largely
of
the
abietic
type
(1,
23)
and
are
thermally
transformed
to
dehydroabietic
acid
(9)
N
9
upon
solvent
extraction-steam
distillation,
no
additional
precau-
tions
(10)
were
taken
and
the
conversion
to
methyl
esters
(pri-
marily
methyl
dehydroabietate)
was
carried
out
by
1
h
reflux
in
excess
15%
BF3
in
methanol.
The
methyl
esters
formed
were
recovered
by
pouring
the
reaction
mixture
into
15
ml
of
brine
followed
by
ether
extraction
(2
x
10
ml),
and
the
pooled
ether
Limonene
phases
were
dried
over
anhydrous
Na2SO4,
concentrated
to
small
volume,
and
the
contents
examined
by
combined
capillary
GLC-
MS.
Calibration
of
the
method
with
a-pinene
and
abietic
acid
indicated
that
volatile
monoterpenes
were
recovered
in
about
85%
yield,
whereas
resin
acids
were
recovered
(as
methyl
dehy-
droabietate)
in
an
overall
yield
of
approximately
80%.
In
vivo
Tracer
Studies.
The
protocol
for
these
experiments
was
essentially
identical
to
that
described
above.
In
this
instance,
wounded
saplings
and
wounded
saplings
treated
with
PIIF
or
chitosan
were
employed,
along
with
unwounded
controls.
Three
saplings
subjected
to
each
treatment
were
harvested
at
4-d-
intervals,
and
each
sapling
was
injected
24
h
before
harvest
with
15
,l
of
an
aqueous
solution
containing
5
,uCi
of
[U-14C]sucrose
(5
,ul
injections
at
three
sites
radial
from
the
wound
site
at
900
intervals).
Isolation
of
volatile
terpenoids
and
diterpene
resin
acids
(as
methyl
esters)
was
as
before;
however,
in
this
case,
products
were
analyzed
by
radio-GLC
following
the
addition
of
appropriate
monoterpene
and
resin
acid
carrier
standards,
which
were
obtained
from
the
oleoresin.
Isolation
and
Assay
of
Terpene
Cyclases.
A
similar
protocol
was
employed
for
these
studies
carried
out
with
wounded
sa-
plings,
and
wounded
saplings
treated
with
PIIF
or
chitosan,
as
well
as
with
unwounded
controls.
Harvest
was
again
at
4-d
intervals;
however,
in
this
instance
any
exuded
oleoresin
was
wiped
from
the
stem
with
a
pentane
soaked
tissue
before
removal
Acid
of
the
2
cm
stem
segment.
A
dozen
such
segments
for
each
treatment
at
each
time
point
were
obtained
and
the
outer
tissue
ts.
isolated
by
scraping
down
to
the
secondary
xylem
with
a
razor
blade.
The
scrapings
were
frozen
in
liquid
N2
and
ground
to
a
fine
powder
with
a
mortar
and
pestle.
The
powder
was
then
vere
wounded
transferred
to
15
ml
of
chilled
100
mM
Na-phosphate
buffer
(pH
by
sterile
18-
6.4)
containing
20%
glycerol,
5
mM
DTE,
10
mM
Na2S205,
10
1,
or
smeared
mM
Na-ascorbate,
15
mM
MgCl2,
and
1
g
each
of
insoluble
PVP
J
of
a
0.1%
(GAF
Corp.)
and
powdered
polystyrene
resin
(methanol
washed
termed
PIIF
XAD-4,
Rohm
and
Haas,
Inc.),
and
thoroughly
homogenized
in
an
(12).
Con-
a
loose-fitting
Ten-Broeck
homogenizer.
The
homogenate
was
vals,
up
to
12
filtered
through
several
layers
of
cheesecloth
and
the
resulting
arvested,
and
filtrate
was
centrifuged
at
105,000g
to
provide
the
soluble
super-
w
the
wound
natant
used
as
the
enzyme
source.
This
preparation
was
concen-
ponding
con-
trated
to
about
3
ml
by
ultrafiltration
(Amicon
PM-30)
and
En.
The
stem
passed
through
a
2
x
10
cm
column
of
Sephadex
G-25
equili-
)ad
of
alumi-
brated
with 25
mM
Mes-5
mM
Na-phosphate
buffer
(pH
6.5)
iserted
into
a
containing
10%
glycerol,
0.5
mM
DTE,
15
mM
MgCl2
and
1.5
resin
covered
mM
MnCl2
to
accomplish
adjustment
to
assay
conditions
(6).
jected
to ex-
The
assay
for
terpene
cyclase
activity
was
initiated
by
the
ether)-steam
addition
of
a
saturating
concentration
(20
gM)
of
the
appropriate
0)
using
3
mg
1-3H-labeled
acyclic
precursor
for
monoterpenes
(geranyl
pyro-
andards.
The
phosphate),
sesquiterpenes
(farnesyl
pyrophosphate)
and
diter-
he
p-cymene
penes
(geranylgeranyl
pyrophosphate)
to
1
ml
of
the
enzyme
me
and
ana-
solution
(40-80
Ag
protein
by
the
Bio-Rad
dye-binding
assay),
t
residue
was
and
the
mixture
was
incubated
for
1
h
at
30°C
in
a
Teflon-
tion,
and
the
capped
vial.
The
reaction
was
terminated
by
chilling
the
vial
in
vent
was
then
ice
and
extraction
of
the
contents
with
pentane
(2
x
1.5
ml).
ids
from
the
The
pooled
pentane
extract,
brought
to
1%
ether,
was
passed
acidified
with
through
a
1
x
5
cm
column
of
silicic
acid
to
isolate
the
terpene
-13
to
recover
olefins
(6).
The
tritium
content
of
this
fraction
was
determined
he
resin
acid
by
scintillation
counting
of
an
aliquot
and
the
material
analyzed
was
vacuum
by
radio-GLC
following
the
addition
of
appropriate
internal
*esidue.
Since
standards.
The
original
reaction
mixture
was
next
extracted
with
1124
CROTEAU
ET
AL.

OLEORESIN
BIOSYNTHESIS
IN
PINE
DEFENSE
ether
(2
x
1.5
ml)
and
the
combined
extracts
passed
through
the
same
silicic
acid
column
to
provide
the
oxygenated
terpenoids
(6),
which
were
analyzed
as
before
by
aliquot
counting
and
radio-
GLC
following
the
addition
of
internal
standards.
Analytical
Procedures.
The
general
procedures
for
radio-TLC
on
silica
gel
G
(with
or
without
12%
AgNO3)
and
for
radio-GLC
have
been
described
(6,
1
1).
The
developing
solvent
for
analyzing
diterpene
olefins
was
hexane:diethyl
ether
(99:1,
v/v).
The
col-
umn
used
for
radio-GLC
was
stainless
steel
(10
ft
x
l/8
in
o.d.)
packed
with
10%
SE-30
on
80/100
mesh
Gas-Chrome
Q
and
operated
at
a
He
flow
of
45
cm3/min
(80°C
for
monoterpene
olefins,
220°C
for
diterpene
resin
acid
methyl
esters).
Analytical
capillary
GLC
analysis
(FID)
and
combined
GLC-MS
analysis
(70
eV)
were
performed
on
a
25
m
fused
silica
column
coated
with
SE-30
and
programmed
from
80
to
240C
at
5°C/min
with
He
as
carrier
gas.
Quantitation
was
by
electronic
integration
of
detector
output
with
respect
to
the
internal
standard,
and
by
assuming
a
response
factor
of
one.
Procedures
for
liquid
scintillation
spectrometry
have
been
described
(6,
11).
Samples
were
quench-corrected
by
internal
standardization
([3H]-
or
['4C]toluene)
and
counted
to
<1%
probable
error
(efficiency
for
3H
=
30%;
for
'4C
=
72%).
RESULTS
Time-Course
of
Induced
Oleoresinosis.
Lodgepole
pine
sa-
plings
were
examined
for
alterations
in
oleoresin
content
in
response
to
fungal
infection
and
to
carbohydrate
elicitors
of
plant
cell
wall
and
fungal
wall
origin.
Two-year-old
lodgepole
pines
were
wounded
and
inoculated
with
C.
clavigera,
a
symbiont
of
the
mountain
pine
beetle,
or
treated
with
the
biotic
elicitors
PIIF
(3)
or
chitosan
(12),
and
the
total
monoterpene
content
of
the
reaction
zone
was
monitored
over
a
12-d
period
by
combination
of
steam
distillation
and
gas
chromatographic
analysis.
Fungus-
infected
stems
accumulated
3
times
the
monoterpene
level
of
untreated
controls
after
12
d,
whereas
the
PIIF
and
chitosan
treatments
resulted
in
nearly
a
5-fold
increase
in
monoterpene
content
with
a
perceptibly
more
rapid
rate
of
accumulation
(Fig.
2a).
Wounding
alone
led
to
a
lesser
increase
(1.5-fold)
in
mono-
terpene
level,
which
is
a
characteristic
wound
response
in
conifers
(2,
19,
21,
23)
while
inoculation
with
spores
rather
than
mycelia
led
to
a
relatively
slow
increase
in
monoterpene
content
to
slightly
over
twice
the
control
level
in
12
d
(data
not
shown).
Monoterpene
content
of
the
pine
stem
oleoresin
was
examined
by
GLC-MS
at
the
10-d
period
following
wounding
and
fungal
infection
(Table
I).
Significant
increases
in
the
levels
of
the
monoterpene
olefins
a-pinene,
f,-pinene,
3-carene,
and
,3-phel-
landrene
(for
structures
see
Fig.
1),
which
are
thought
to
be
toxic
to
this
fungus
(19,
23),
were
found
in
infected
stem
tissue
compared
to
controls.
The
levels
of
a-phellandrene
and
limonene
decreased,
probably
because
of
volatility
losses
with
little
com-
pensating
production
of
these
compounds.
Examination
of
the
monoterpene
content
of
the
oleoresin
elicited
by
PIIF
and
chi-
tosan
treatment
(at
12
d)
revealed
a
composition
similar
to
that
of
the
infected
tissue,
but
with
slightly
higher
proportions
of
,l-
pinene
and
3-carene,
as
well
as
limonene.
The
alteration
in
diterpene
resin
acid
content
of
the
oleoresin
was
also
measured
(by
gravimetric
determination
of
free
organic
acids)
in
response
to
the various
treatments,
and
was
shown
to
roughly
double
upon
infection
and
to
increase
nearly
3-fold
upon
treatment
with
PIIF
or
chitosan
(Fig.
2b).
GLC-MS
analysis
of
the
methyl
esters
verified
the
presence
of
dehydroabietate
(major)
and
chromatographically
similar
resin
acids
(minor
components)
of
mol
wt
=
316
(30),
and
incidently
indicated
a
slight
decrease
in
the
levels
of
the
fatty
acids
C,8g:
and
C182
which
comprised
about
10%
of
the
oleoresin
of
untreated
controls.
Little
can
be
said
about
the
composition
of
the
native
diterpene
resin
acid
fraction
since
the
oleoresin
isolation
procedure
(steam
distillation
Co
a)
c
0)
0)
0
C
0
Q-
L-
I0
a)
co
~o
C.)
C
01)
Time
(day)
FIG.
2.
Effect
of
time
after
treatment
on
the
accumulation
of
mono-
terpenes
(panel
a)
and
diterpene
resin
acids
(panel
b)
in
lodgepole
pine
stems
aseptically
wounded
(w),
infected
with
C.
clavigera
(i)
or
treated
with
PIIF
(p)
or
chitosan
(c).
Control
stems
were
unwounded
(u).
For
experimental
conditions
and
analytical
procedures
see
"Materials
and
Methods."
Table
I.
Effect
of
Wounding
and
Infection
on
the
Monoterpene
Composition
of
Lodgepole
Pine
Stems
Two-year-old
lodgepole
pine
saplings
were
infected
with
Ceratocystis
clavigera
or
wounded
aseptically
and
the
monoterpene
content
of
the
reaction
zone
was
determined
10
d
after
treatment.
The
controls
were
intact
saplings.
The
monoterpenes
were
isolated
by
solvent
extraction-
steam
distillation,
identified
by
GLC-MS,
and
quantitated
by
integration
of
chromatographic
peak
areas
with
reference
to
the
internal
standard
as
described
in
"Materials
and
Methods."
Monoterpene
Control
AsepWic
Infected
mg-g-'
tissue
dry
weight
a-Pinene
0.44
0.69
1.68
Camphene
trace
trace
0.09
,B-Pinene
1.55
2.79
5.97
3-Carene
0.27
0.34
1.33
a-Phellandrene
0.14
0.14
0.08
Limonene
0.85
0.70
0.22
f,-Phellandrene
1.22
2.14
3.14
Total
monoterpenes
4.47
6.79
12.51
1125

Plant
Physiol.
Vol.
85,
1987
in
air,
exposure
to
Lewis
acid)
resulted
in
the
isomerization/
oxidation
of
the
acids
present
with
the
resulting
production
of
dehydroabietate
(9).
The
mass
spectral
identification
of
the
latter,
as
the
methyl
ester
(30),
did
however
confirm
the
presence
of
abietic-type
resin
acids
in
P.
contorta
saplings,
as
anticipated
from
studies
on
the
resin
acid
content
of
mature
trees
(1,
23).
The
nonvolatile
neutral
components
of
the
oleoresin
were
also
subjected
to
preliminary
(gravimetric)
analysis,
and
were
shown
to
increase
roughly
2.5-fold
in
response
to
infection.
This
mate-
rial
comprised
about
15%
of
the
oleoresin
produced
in
response
to
infection
(volatile
monoterpenes
about
40%,
and
free
acids
about
40%)
and
GLC
analysis
of
this
fraction
revealed
a
very
complex
mixture
of
more
than
twenty
components
(at
>2%),
as
expected
for
this
class
of
wood
extractives
(22).
In
Vivo
Tracer
Studies.
In
order
to
determine
if
the
induced
accumulation
of
oleoresin
was
due
to
de
novo
synthesis,
a
prelim-
inary
attempt
was
made
to
assess
oleoresin
biosynthesis
by
application
of
labeled
acetate,
mevalonate,
or
sucrose
to
the
wound
site.
These
efforts
were
thwarted
by
the
presence
of
oleoresin
which
prevented
either
adequate
application
of
sub-
strate
to,
or
uptake
by,
the
relevant
tissue.
An
alternate
approach,
whereby
tracer
was
injected
at
sites
distributed
radially
on
the
stem
from
the
wound
site,
was
taken;
yet,
even
under
these
circumstances
the
incorporation
of
acetate
and
mevalonate
was
quite
poor
presumably
due
to
compartmentation
barriers.
[U-
14C]Sucrose
proved
to
be
the
most
suitable
precursor
of
oleoresin
terpenoids
and
was
used
to
examine
biosynthetic
capability
over
a
12-d
time
course
by
radial
injection
of
this
substrate
24
h
prior
to
harvest
and
product
analysis.
Incorporation
of
label
from
sucrose
into
monoterpenes
of
the
untreated
controls
was
quite
low
(<0.1
%),
whereas
wounding
produced
a
notable
(5-fold
at
maximum),
but
transient,
increase
in
the
rate
of
incorporation
into
these
materials
(Fig.
3a).
Wounded
saplings
that
had
been
treated
with
either
PIIF
or
chitosan
exhibited
the
most
rapid
rate
of
[U-'4C]sucrose
labeling
(after
24
h),
reaching
a
maximum
4
or
8
d
following
elicitor
treatment
and
declining
to
about
50%
of
maximum
at
12
d
(Fig.
3a).
The
rates
of
de
novo
resin
synthesis
observed
following
elicitor
treatment
were
some
15
to
20
times
higher
at
maximum
than
were
those
of
untreated
controls,
a
rather
higher
ratio
than
the
3-
to
5-fold
difference
in
oleoresin
content
observed
analyti-
cally
between
treated
and
control
saplings
in
the
earlier
time
course
experiments
(Fig.
2).
It
should
be
noted,
however,
that
the
oleoresin
content
of
the
untreated
controls
represents
primary
(preformed)
resin
which
is
stored
in
the
resin
ducts
and
not
resin
produced
in
response
to
external
stimulus.
Radio-GLC
analysis
of
the
labeled
monoterpenes
generated
from
[U-14C]sucrose
in
response
to
chitosan
treatment
after
8
d
indicated
the
presence
of
a-pinene,
13-pinene,
3-carene,
and
f,-phellandrene,
with
lesser
quantities
of
camphene,
a-phellandrene,
and
limonene
(Fig.
4a),
the
de
novo
biosynthesis
of
which
could
be
anticipated
from
the
earlier
analytical
studies
(Table
I).
Incorporation
of
label
from
sucrose
into
diterpene
resin
acids
(measured
as
'4C-methyl
dehydroabietate)
was
low
in
untreated
controls
(<0.1
%),
whereas
wounding
resulted
in
a
transient
increase
in
incorporation
to
about
twice
that
of
control
levels,
and
both
PIIF
and
chitosan
treatment
resulted
in
an
increase
of
incorporation
rate
(at
maximum)
to
over
10
times
that
of
the
untreated
control
(Fig.
3b).
For
the
reasons
noted
earlier,
precise
definition
of
resin
acid
composition
was
not
possible;
however,
the
presence
of
methyl
dehydroabietate
as
the
major
component
of
the
labeled
resin
acid
fraction
at
each
time
point
was
verified
by
radio-GLC,
confirming
that
a
transient
increase
in
the
biosyn-
thesis
of
abietane-type
resin
acids
had
occurred.
A
roughly
10-fold
increase
in
the
incorporation
of
label
into
neutral,
nonvolatile
substances
was
also
observed
in
response
to
wounding
and
treatment
with
PIIF
or
chitosan
(data
not
shown)
to
1.6
-
0).
0)c
0.
0.
-
0
CI
0
L.
00
o
.~
b
0.8-
C
p
0)
0.4
w
U
0
I
I
0
4
8
12
Time
(day)
FIG.
3.
Effect
of
time
after
treatment
on
the
incorporation
of
[U-'4C]
sucrose
(in
24
h)
into
monoterpenes
(panel
a)
and
diterpene
resin
acids
(panel
b)
of
lodgepole
pine
stems
aseptically
wounded
(w)
or
wounded
and
treated
with
PIIF
(p)
or
chitosan
(c).
Control
stems
were
unwounded
(u).
For
experimental
conditions
and
analytical
procedures
see
"Materials
and
Methods."
but
the
absolute
incorporation
rate
was
too
low
(i.e.
about
0.2%
incorporation
at
maximum)
to
permit
detailed
radiochemical
analysis
of
this
fraction.
Measurement
of
Terpene
Cyclase
Activity.
To
confirm
the
transient
increase
in
the
biosynthesis
of
oleoresin
terpenoids
in
P.
contorta
saplings
in
response
to
treatment
with
PIIF
and
chitosan,
an
effort
was
made
to
measure
alteration
in
the
activity
of
the
cyclase
enzymes
responsible
for
generating
parent
CO0,
C15,
and
C20
terpenoid
compounds
from
the
universal
acyclic
branch-
point
metabolites
geranyl,
farnesyl,
and
geranylgeranyl
pyro-
phosphate.
Cell-free
extracts
(105,OOOg
supernatants)
from
con-
trol
and
treated
tissue
were
prepared
at
4-d
intervals
following
initiation
of
the
experiment
and
they
were
assayed
under
general
conditions
developed
for
comparable
enzymes
from
herbaceous
species
(6).
Assay
of
the
105,000g
supernatant
prepared
from
treated
tissue
with
[
1-3H]geranyl
pyrophosphate
as
substrate
gave
rise
to
readily
detectable
levels
of
monoterpene
olefins,
well
above
the
levels
observed
in
controls
(Fig.
5a).
Maximum
activity
was
observed
between
4
and
8
d
following
treatment,
with
a
diminution
of
activity
to
roughly
50%
of
maximum
at
12
d.
Radio-GLC
analysis
of
the
olefin
fraction
(obtained
by
assay
at
d
4
following
chitosan
treatment)
revealed
the
presence
of
labeled
a-pinene,
(l-
pinene,
3-carene,
and
,3-phellandrene,
with
lesser
quantities
of
1126
CROTEAU
ET
AL.

OLEORESIN
BIOSYNTHESIS
IN
PINE
DEFENSE
0
0.
a1)
0
0
48
Time
(min)
FIG.
4.
Radio
gas-liquid
chromatograms
of
the
monoterpenes
isolated
from
lodgepole
pine
stems
(8
d
after
wounding
and
treatment
with
chitosan)
that
had
been
administered
5
ACi
of
[U-'4C]sucrose
for
24
h
(panel
a),
and
of
the
monoterpenes
isolated
from
a
cell-free
extract
of
lodgepole
pine
stems
(4
d
after
wounding
and
treatment
with
chitosan)
that
had
been
incubated
with
20
gM
[1-3H]geranyl
pyrophosphate
for
I
h
(panel
b).
The
smooth
lower
tracing
(panel
c)
is
the
detector
response
obtained
from
coinjected
authentic
standards
of
a-pinene
(1),
camphene
(2),
,B-pinene
(3),
3-carene
(4),
a-phellandrene
(5),
limonene
(6),
and
j3-
phellandrene
(7).
For
experimental
conditions
and
analytical
procedures
see
Materials
and
Methods."
camphene,
a-phellandrene,
and
limonene
as
expected
(Fig.
4b).
The
oxygenated
monoterpene
fraction
was
comprised
largely
of
geraniol,
released
by
phosphatases
in
the
preparation,
and
the
level
of
this
activity
was
similar
to
that
of
the
control
and
changed
little
over
the
time-course
(i.e.
15
±
3%
conversion
of
substrate).
Assay
with
[1-3H]famesyl
pyrophosphate
as
substrate
of
the
soluble
enzyme
system
prepared
from
treated
tissue
gave
rise
to
detectable
levels
of
sesquiterpene
olefins
which
were
well
above
those
of
controls.
The
time-course
of
activity
paralleled
that
of
the
monoterpene
cyclase
activity
from
tissue
treated
with
PIIF
or
chitosan,
but
the
overall
level
of
activity
was
about
8-fold
less,
precluding
radio-GLC
analysis.
Radio-TLC
analysis,
by
scraping
and
counting
regions
of
the
plate,
indicated
that
the
labeled
compounds
in
this
fraction
chromatographed
in
a
broad
range
of
RF
values
in
the
region
of
the
sesquiterpene
olefins
caryophyl-
lene
and
humulene.
Analysis
of
the
oxygenated
sesquiterpene
fraction
indicated
the
presence
of
labeled
farnesol
(by
radio-
TLC),
and
the
putative
phosphatase
activity
changed
little
over
the
time-course.
Although
little
is
known
about
the
biosynthesis
of
diterpene
resin
acids,
a
sufficient
theoretical
framework
exists
to
suggest
that
the
initial
cyclic
products
of
the
pathway
are
olefins
of
the
pimaradiene
and/or
abietadiene
type
(7,
29).
Assay
with
[1-3H]
geranylgeranyl
pyrophosphate
as
substrate
of
the
soluble
enzyme
system
prepared
from
treated
tissue
gave
rise
to
diterpene
olefins
80
V)
a)
c
a2)
a)
0
0
40
40-'
0
L-
0.
cm
E
-C
E
C
CO)
.5
a)
cr
40
20
0
4
8
12
Time
(day)
FIG.
5.
Effect
of
time
after
treatment
on
the
rate
of
conversion
of
[
1-
3H]geranyl
pyrophosphate
to
monoterpene
olefins
(panel
a)
and
of
[1-
3H]geranylgeranyl
pyrophosphate
to
diterpene
olefins
(panel
b)
in
cell-
free
extracts
of
lodgepole
pine
stems
aseptically
wounded
(w)
or
wounded
and
treated
with
PIIF
(p)
or
chitosan
(c).
Control
stems
were
unwounded
(u).
For
details
of
the
assay
and
product
analyses
see
"Materials
and
Methods."
at
overall
rates
comparable
to
those
observed
for
cyclization
of
geranyl
pyrophosphate
to
cyclic
monoterpene
olefins,
and
the
time-course
was
also
similar
with
respect
to
the
controls
(Fig.
Sb).
Radio-GLC
evaluation
of
this
material
suggested
the
pres-
ence
of
at
least
two
components;
however,
the
lack
of
suitable
standards
and
the
low
specific
activity
of
the
precursor
(and
thus
low
count
levels)
prevented
adequate
analysis
of
these
products.
Radio-TLC
separation
indicated
the
labeled
material
to
chro-
matograph
with
an
RF
value
above
that
of
abietriene
(aromatic
C-ring).
The
oxygenated
diterpene
fraction
was
shown
to
contain
primarily
geranylgeraniol
by
radio-TLC
analysis.
DISCUSSION
The
results
presented
here
indicate
that
lodgepole
pine
saplings
respond
to
fungal
infection
and
challenge
with
biotic
elicitors
by
the
production
of
oleoresin,
much
like
their
mature
counterparts
(17,
19,
21,
23,
24).
The
chemical
composition
of
the
'induced'
oleoresin
differs
somewhat
between
saplings
and
mature
trees,
most
notably
in
the
minor
contribution
of
limonene
to
the
sapling
resin
and
in
the
greater
proportion
of
diterpene
resin
acid
(19,
23).
Similar
differences
were
observed
between
the
primary
(constitutive)
resin
of
saplings
and
mature
trees,
and
such
devel-
opmental
variation
is
not
unexpected
(27).
1127

Plant
Physiol.
Vol.
85,
1987
The
observation
that
lodgepole
pine
responds
to
the
presence
of
fungus
and
to
the
same
carbohydrate
fragments
of
plant
and
fungal
cell
walls
that
elicit
defensive
reactions
in
the
Solanaceae
and
Leguminosae
(28)
suggests
that
the
Pinaceae
possess
a
rec-
ognition
mechanism
for
induced
defense
similar
to
that
of
other
higher
plants
(2,
17).
Although
the
role
of
blue
stain
fungi
in
pathogenesis
has
been
questioned
(4)
and
the
detailed
influence
of
various
oleoresin
components
is
still
controversial
(5),
there
can
be
little
doubt
that
C.
clavigera,
as
well
as
biotic
elicitors
which
might
be
expected
to
arise
in
the
process
of
infection,
do
elicit
the
production
of
a
complex
oleoresin
which
in
turn
is
detrimental
to
the
growth
and
development
of
both
the
fungus
and
the
bark
beetle
which
serves as
the
vector
(19-21,
25).
By
these
criteria,
induced
oleoresinosis
can
be
seen
to
resemble
phytoalexin
production
(16);
however,
it
need be
emphasized
that
the
process
represents
greatly
enhanced,
although
relatively
slow,
production
of
constitutive
metabolites,
which
is
quite
un-
like
the
typical
phytoalexic
response.
Finally,
evidence
based
on
in
vivo
tracer
studies
is
presented
for
a
transient
increase
in
the
de
novo
biosynthesis
of
relevant
monoterpene
and
diterpene
oleoresin
components
in
the
tissue
of
the
reaction
zone
in
response
to
challenge.
Furthermore,
this
transient
increase
in
biosynthetic
capability
is
manifested
at
the
level
of
the
cyclases
which
catalyze
the
committed
steps
of
terpenoid
biosynthesis
from
branch-point
acyclic
precursors.
Whether
the
levels
or
activities
of
other
enzymes
of
the
terpenoid
pathway
are
similarly
enhanced
in
response
to
challenge
is
pres-
ently
uncertain.
This
is
a
question
which
can
be
readily
addressed
with
pine
saplings;
a
model
system
for
which
the
induction
and
analytical
procedures
are
now
being
optimized.
Acknowledgments-We
thank
Greg
Wichelns
for
raising
the
plants,
Nancy
Madsen
for
typing
the
manuscript,
and
C.
A.
Ryan
and
A.
A.
Berryman
for
helpful
discussion.
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