Latex and laticifer starch content of developing leaves of Euphorbia pulcherrima


Spilatro, S.R.; Mahlberg, P.G.

American Journal of Botany 73(9): 1312-1318

1986


Laticifer starch accumulation was compared to laticifer growth for developing leaves of Euphorbia pulcherrima Willd. (poinsettia). Measurements of the laticifer-specific triterpenol, cycloartenol, in latex and in whole leaf extracts were used to calculate the total latex volume in leaves of different developmental stages. Latex volume and starch concentration in the latex were used to determine total laticifer starch and to compare laticifer growth and starch synthesis. Young leaves contained the highest latex and laticifer starch contents on dry wt and leaf area bases. In older expanding leaves, laticifer growth produced an increase in total latex volume accompanied by an increase in total laticifer starch. Laticifer growth and starch accumulation stopped upon cessation of leaf expansion. Starch concentration was similar in latex from all leaves, but differed between plant organs. Thus, laticifer starch accumulation correlated with laticifer growth, but mobilization of the starch out of the laticifer was not observed in old or senescent leaves. This is evidence that laticifer starch grains function within the laticifer independently of degradation or export to other cell types.

Amer.
J.
Bot.
73(9):
1312-1318.
1986.
LATEX
AND
LATICIFER
STARCH
CONTENT
OF
DEVELOPING
LEAVES
OF
EUPHORBIA
PULCHERRIMA
STEVEN
R.
SPILATRO
AND
PAUL
G.
MAHLBERG
Department
of
Biochemistry,
Michigan
State
University,
East
Lansing,
Michigan
48824,
Department
of
Biology,
Indiana
University,
Bloomington,
Indiana
47405
ABSTRACT
Laticifer
starch
accumulation
was
compared
to
laticifer
growth
for
developing
leaves
of
Euphorbia
pulcherrima
Willd.
(poinsettia).
Measurements
of
the
laticifer-specific
triterpenol,
cycloartenol,
in
latex
and
in
whole
leaf
extracts
were
used
to
calculate
the
total
latex
volume
in
leaves
of
different
developmental
stages.
Latex
volume
and
starch
concentration
in
the
latex
were
used
to
determine
total
laticifer
starch
and
to
compare
laticifer
growth
and
starch
synthesis.
Young
leaves
contained
the
highest
latex
and
laticifer
starch
contents
on
dry
wt
and
leaf
area
bases.
In
older
expanding
leaves,
laticifer
growth
produced
an
increase
in
total
latex
volume
accompanied
by
an
increase
in
total
laticifer
starch.
Laticifer
growth
and
starch
accumulation
stopped
upon
cessation
of
leaf
expansion.
Starch
concentration
was
similar
in
latex
from
all
leaves,
but
differed
between
plant
organs.
Thus,
laticifer
starch
accumulation
correlated
with
laticifer
growth,
but
mobilization
of
the
starch
out
of
the
laticifer
was
not
observed
in
old
or
senescent
leaves.
This
is
evidence
that
laticifer
starch
grains
function
within
the
laticifer
in-
dependently
of
degradation
or
export
to
other
cell
types.
STARCH
GRAINS
in
latex
of
Euphorbia
differ
in
their
metabolic
processes
and
biophysical-bio-
chemical
properties
from
grains
of
other
plant
cells
(Biesboer
and
Mahlberg,
1978;
Spilatro
and
Mahlberg,
1985).
The
period
of
laticifer
starch
grain
accumulation
in
relation
to
growth
and
development
of
the
laticifer
is
unclear.
If
starch
grain
formation
only
occurs
in
the
la-
ticifer
tip,
then
the total
starch
content
should
be
established
prior
to
the
expansion
phase
of
laticifer
and
organ
growth,
as
for
a
leaf.
In
this
case
starch
concentration
should
be
expected
to
decrease
during
subsequent
organ
growth.
Alternatively,
if
starch
synthesis
is
a
continuing
process,
the
starch
content
of an
organ
should
progressively
increase
during
the
growth
of
both
the
laticifer
and
the
organ.
Analyses
of
laticifer
starch
content
in
an
or-
gan
is
made
difficult
by
the
presence
of
starch
in
non-laticifer
cells
and
potential
changes
in
latex
composition
during
the
elongation
of
this
cell
in
growing
organs.
The
process
of
starch
synthesis
in
latex
can
be
examined
through
determination
of
starch
contents
within
given
volumes
of
latex
analyzed
throughout
organ
growth.
However,
this
type
of
analysis
would
be
facilitated
with
the
aid
of
a
chemical
marker
which
can
serve
to
measure
the
volume
of
latex
as
well
as
measure
any
potential
changes
in
latex
composition
whether
by
dilution
or
con-
1
Received
14
November
1985;
revision
accepted
7
March
1986.
We
thank
Paul
Ecke
Poinsettias,
Encinitas,
CA,
for
gen-
erously
providing
plants
used
in
this
study.
centration.
Warnaar
(1982)
reported
that
the
triterpenyl
fatty
acid
esters
in
Hoya
latices
can
be
used
to
determine
the
latex
content
of
tissues
and
thus
serve
as
such
a
chemical
marker.
In
this
study
we
describe
cycloartenol
from
the
latex
ofpoinsettia
as
such
a
marker
and
employ
it
to
evaluate
the
process
of
starch
production
in
the
laticifer.
MATERIALS
AND
METHODS
—Plant
materi-
al
Plants
were
grown
under
greenhouse
con-
ditions
(Spilatro
and
Mahlberg,
1985)
to
a
ht
of
1.0
to
1.5
m.
Flowering,
when
desired,
was
induced
with
an
8
hr
photoperiod
for
3
months.
Leaves
of
different
physiological
ages
were
des-
ignated
as
follows.
"Young"
leaves,
located
1-
2
nodes
from
the
shoot
tip,
had
a
blade
length
of
5-7
cm,
were
beginning
rapid
blade
expan-
sion
and
were
still
greening.
"Expanding"
leaves
were
located
at
nodes
3-4,
and
had
a
blade
length
of
11.0-11.5
cm.
"Mature"
leaves,
lo-
cated
at
node
10,
were
fully
expanded
and
had
a
14-17
cm
blade
length.
Leaves
designated
as
"old"
were
the
lowest
nonsenescent
leaves
of
the
plants
excluding
any
leaves
with
necrotic
or
chlorotic
regions.
Old
leaves
typically
were
located
25
to
30
nodes
below
the
shoot
tip.
A
few
senescent
leaves
also
were
analyzed
for
latex
starch
content.
These
leaves
typically
were
yellow
and
necrotic
along
the
edges
and
often
did
not
exude
latex.
Leaf
latex
collection
—Leaves
were
cut
trans-
versely
through
the
primary
vein
and
the
first
10-15
Al
of
exuded
latex
were
collected
for
1312
September,
1986]
SPILATRO
AND
MAHLBERG
-
POINSETTIA
LATEX,
LATICIFER
STARCH
1313
study.
For
analysis
of
triterpenoids
the
latex
was
suspended
in
0.25
ml
chloroform/meth-
anol
(2:1)
and
taken
to
dryness
under
a
stream
of
nitrogen.
For
starch
determination,
the
latex
sample
was
applied
to
a
13
mm
glass
fiber
filter
disk
(Schleicher
and
Schuell,
#32)
presoaked
in
potassium
phosphate
buffer
(0.2
M,
pH
5.3).
Latex
starch
determination
—Laticifer
starch
grains
on
filter
disks
were
freed
of
other
latex
components
by
washing
sequentially
with
5
ml
volumes
of
buffer,
hot
80%
methanol,
chlo-
roform/methanol
(2:1),
and
acetone.
Starch
on
filters
was
initially
hydrolyzed
10
min
in
9
M
sulfuric
acid
(2
ml)
in
a
boiling
water
bath
and
then
assayed
for
by
adding
1
ml
anthrone
re-
agent
(0.4%
in
conc.
sulfuric
acid)
and
reheat-
ing
an
additional
10
min.
After
cooling,
ab-
sorbance
was
read
at
620
nm.
Analysis
of
latex
triterpenoids—Triterpe-
noids
extracted
with
acetone
from
stem
latex
were
separated
by
silica-gel
thin-layer
chro-
matography
(TLC).
TLC
plates
were
devel-
oped
in
a
cyclohexane/ethyl
acetate
(5:1)
sol-
vent
system
and
the
triterpenoids
were
visualized
at
100
C
after
spraying
the
chro-
matograms
with
3%
cupric
acetate
in
8%
phos-
phoric
acid.
Triterpenoids
were
isolated
from
preparative
chromatograms
by
extracting
from
the
silica-gel
with
light
petroleum
(BP
60
C)/
acetone/water
(1:1:1).
The
organic
solvent
lay-
er
containing
the
latex
triterpenoids
was
col-
lected
and
taken
to
dryness
under
a
stream
of
nitrogen.
Triterpenyl-esters
collected
from
TLC
bands
were
saponified
in
0.5
ml
of
methanol
con-
taining
benzene
(10%
v/v)
and
potassium
hy-
droxide
(5%
v/v).
After
one
hr
saponification
at
95
C
the
free
triterpenoids
were
extracted
with
three
1
ml
washes
of
petroleum
ether,
which
were
pooled
and
taken
to
dryness
under
a
stream
of
nitrogen.
Triterpenols
were
acetylated
in
0.5
ml
of
pyridine/acetic
anhydride
(1:1)
for
1.5
hr
at
95
C.
The
resultant
triterpenyl-acetates
were
ex-
tracted
into
light
petroleum
(BP
60
C)
follow-
ing
the
addition
of
3
ml
of
deionized
water.
The
light
petroleum
fraction
was
collected
and
applied
to
a
12
x
45
mm
alumina
column,
eluted
with
20
ml
of
10%
diethyl
ether
in
light
petroleum,
and
subsequently
taken
to
dryness
under
a
stream
of
nitrogen.
Latex
3-hydroxy-triterpenoids
were
digito-
nin
precipitated
according
to
Keller,
Bush
and
Grunwald
(1969)
with
modifications.
A
lipid
extract
of
poinsettia
latex
(20
Al)
in
absolute
ethanol
(1
ml)
was
mixed
with
1%
digitonin
in
80%
ethanol
:
water
and
allowed
to
stand
overnight
at
room
temperature.
The
precipi-
tate
was
centrifuged
at
15,000
rpm
and
the
fragile
pellet
was
carefully
washed
three
times
with
5
ml
volumes
of
80%
ethyl
alcohol
and
once
with
7.5
ml
acetone.
The
purified
triter-
penes
were
dissociated
from
the
digitonide
complex
and
collected
according
to
Weete
and
Kelley
(1977).
Digitonin
precipitated
latex
triterpenoids
were
analyzed
with
a
Hewlett-Packard
5992
gas-liquid
chromatograph-mass
spectrograph
equipped
with
a
1
m
OV-25
column.
Analysis
was
carried
out
with
a
thermal
program
from
240-270
C
at
a
rate
of
3
C/min.
Mass
spec-
trometry
was
performed
with
an
EM
voltage
of
1,700.
Leaf
sampling
procedure
—Leaf
area
was
de-
termined
from
the
wt
of
leaf
silhouettes
using
an
appropriate
standard
curve.
Leaf
silhouettes
were
made
using
"Blue
Vapo"
diazo
paper
prior
to
removal
of
leaves
from
plants.
Silhouettes
were
fixed
by
exposing
the
paper
to
ammonia
vapor,
which
produced
a
permanent
blue
leaf
image.
After
preparing
the
leaf
silhouette,
the
leaf
was
collected
by
severing
the
petiole
through
a
small
region
which
had
been
frozen
with
dry
ice.
The
entire
leaf
was
immediately
frozen
over
dry
ice
and
lyophilized.
This
procedure
prevented
any
loss
of
latex
during
the
collection
of
whole
leaves.
In
one
set
of
experiments
leaves
were
excised
without
initial
freezing
of
the
pet-
iole.
The
latex
was
allowed
to
exude
further
by
excising
and
discarding
the
primary
and
sec-
ondary
veins,
and
blotting
latex
exuded
from
the
interveinal
sections.
These
"deveined"
leaf
sections
were
then
frozen
and
lyophilized
as
above.
Triterpenoid
extraction
from
leaves—Ly-
ophilized
leaves
were
powdered
with
a
mortar
and
pestle,
transferred
to
a
Whatman
10
x
55
mm
extraction
thimble
and
extracted
under
reflux
for
3
hr
in
20
ml
of
chloroform/methanol
(2:1).
The
leaf
residue
remaining
in
the
thimble
was
washed
with
three
2
ml
volumes
of
chlo-
roform/methanol
and
the
extract
and
washes
were
pooled.
For
young
and
expanding
leaves,
the
volume
of
the
pooled
extract
was
reduced
to
approximately
10
ml
under
a
stream
of
ni-
trogen
and
subsequently
taken
to
dryness
with
a
vacuum
evaporator
at
40
C.
The
pooled
ex-
tracts
of
mature
and
old
leaves
were
brought
to
25
ml,
and
a
5
ml
aliquot
taken
to
dryness
with
a
vacuum
evaporator
at
40
C.
Extracts
were
taken
up
in
2
ml
of
acetone
and
the
in-
soluble
residue
was
washed
with
three
2
ml
volumes
of
acetone.
The
extract
and
washes
1314
AMERICAN
JOURNAL
OF
BOTANY
[Vol.
73
13
t
NJ
Time
(elm)
Time
(min)
ttt
Is
Time
(min)
Time
(min)
Fig.
1-4.
1.
Gas-liquid
chromatography
of
poinsettia
latex
lipid
extract.
Eight
major
triterpenoid
peaks,
marked
with
letters
a
through
h,
are
present
in
the
latex.
Internal
standard
is
marked
with
an
i.
2-4.
Thin-layer
chroma-
tography
of
poinsettia
latex
lipid
extracts.
Peaks
are
iden-
tified
with
the
same
letters
as
in
Fig.
1.
2.
Free
triterpenol
fraction.
3.
Triterpenyl
fatty
acid
ester
fraction.
4.
Triter-
penyl
acetate
fraction.
were
pooled,
filtered,
and
taken
to
dryness
un-
der
a
stream
of
nitrogen.
Gas-liquid
chromatography
(GLC)—Leaf
and
latex
extracts
were
taken
up
in
1.0
ml
of
absolute
ethanol
containing
delta-4-andro-
stene-3,
17-dione
as
an
internal
standard
in
preparation
for
GLC.
Triterpenoid
GLC
was
performed
on
a
Hewlett-Packard
5710A
chro-
matograph
equipped
with
a
Hewlett-
Packard
3380A
integrator.
Instrument
conditions
were
as
described
(Spilatro
and
Mahlberg,
1985)
ex-
cept
that
analyses
were
programmed
from
240-
310
C,
at
4
C/min.
The
column
packing
ma-
terial
was
3%
OV-1
on
100/120
mesh
Supel-
coport.
RESULTS
Poinsettia
latex
was
found
to
con-
tain
a
mixture
oftriterpenols,
phytosterols,
and
triterpenyl-esters.
Eight
major
triterpenoid
components
(Fig.
1,
peaks
a-h)
were
separated
by
GLC
of
the
latex
extract
and
no
non-tri-
terpenoid
components
appeared
in
the
chro-
matogram.
This
triterpenoid
profile
was
com-
mon
to
latex
from
leaves
and
stems.
The
latex
triterpenoids
were
further
characterized
to
identify
a
potential
chemical
marker
unique
to
the
laticifer
cell.
TLC
fractionation
of
latex
extract
indicated
the
presence
of
three
major
classes
of
triter-
penoids
presented
as
GLC
profiles
(Fig.
2-4).
Two
of
the
classes
were
identified
as
free
tri-
terpenols
(3-hydroxy-4,4-dimethyl-triterpe-
noids)
(Fig.
2)
and
triterpenyl
acetates
(Fig.
4)
by
cochromatography
with
alpha-amyrin
and
alpha-amyrin
acetate,
respectively.
The
other
major
class
was
the
triterpenyl
fatty
acid
esters
(Fig.
3),
previously
identified
in
poinsettia
latex
by
Warnaar
(1977).
In
the
GLC
profiles,
peaks
a
and
b,
c-g,
and
h
comprised
the
TLC
free-
triterpenol,
triterpenol
acetate,
and
triterpenol
fatty
acid
ester
fractions,
respectively.
Free
phytosterols
(3-hydroxy-4-demethyl-triter-
penes)
occurred
only
as
a
minor
latex
com-
ponent.
Digitonin
precipitation
of
acetone
extract
from
the
whole
leaf
produced
a
GLC
fraction
similar
in
composition
to
the
TLC
free-triter-
pene
fraction
(Fig.
4).
The
component
iden-
tified
as
"b"
in
the
GLC
profiles
comprised
78%
of
this
fraction.
This
fraction
when
ana-
lyzed
by
GC-MS
chromatographed
as
a
single
peak.
The
mass
spectrum
from
the
center
of
this
peak
contained
the
following
fragments:
426
(Mt),
411
(11.2),
408
(10.6),
393
(14.4),
365
(10.6),
339
(6.9),
286
(41.5),
271
(31.9),
203
(100).
This
mass
spectrum
is
in
agreement
with
the
published
spectrum
for
cycloartenol
(Heller
and
Milne,
1978).
The
fragment
of
MW
286
is
diagnostic
for
cycloartenol
(Aplin
and
Hornby,
1966).
When
the
free-triterpenol
frac-
tion
was
acetylated,
the
acetate
of
peak
"b"
cochromatographed
by
GLC
with
authentic
cycloartenol
acetate.
These
results
indicated
that
the
major
free-triterpenol
in
poinsettia
la-
tex
is
cycloartenol.
Hydrolysis
of
the
triter-
penyl
ester
fractions
and
subsequent
analysis
by
GLC
indicated
cycloartenol
did
not
occur
in
an
esterified
form.
The
cycloartenol
content
of
non-laticiferous
cells
was
estimated
from
sections
ofleaflamina
obtained
after
removing
the
primary
and
sec-
ondary
veins,
and
allowing
the
latex
to
exude.
The
cycloartenol
content
of
these
deveined
leaf
sections
was
31.7
±
15.1
pg
per
100
mg
dry
wt
as
compared
to
477
±
43.3
pg
per
100
mg
dry
wt
for
whole
leaves.
The
cycloartenol
found
in
deveined
leaves
was
assumed
to
be
residual
amounts
of
this
compound
in
unexuded
latex,
and
indicated
that
any
cycloartenol
present
in
non-laticiferous
cells
would
not
interfere
with
latex
volume
measurements.
iig/
100
mg
leaf
dry
weight
Azg/cm
2
leaf
area
Leaf
stage'
µg/µl
latex
Young
21.6
±
3.1
2
Expanding
19.7
±
3.0
Mature
14.4
±
2.0
Old
15.8
±
0.9
Total
in
leaf,
Ag
373
±
118
490
±
158
818
±
198
769
±
252
Cycloartenol
789
±
206
29.6
±
9.5
311
±
75
9.7
±
3.0
208
±
32
6.0
±
1.4
245
±
88
8.4
±
2.2
September,
1986]
SPILATRO
AND
MAHLBERG-POINSETTIA
LATEX,
LATICIFER
STARCH
1315
TABLE
1.
Cycloartenol
content
of
poinsettia
leaf
latex
and
whole
leaves
of
different
physiological
ages
'
Description
of
leaf
stages
is
provided
in
text.
2
S.D.
Although
the
cycloartenol
content
was
the
same
for
latex
collected
from
different
parts
of
a
given
leaf,
it
varied
with
leaf
age,
time
of
sampling
and
sampling
procedures.
The
initial
2
to
15µl
of
latex
that
exuded
from
a
sampling
site
contained
constant
cycloartenol
content,
but
the
cycloartenol
concentration
decreased
15-25%
during
subsequent
latex
flow
due
to
dilution
of
latex
by
other
plant
fluids.
The
cy-
cloartenol
content
of
leaf
latex
varied
for
dif-
ferent
sampling
times.
Cycloartenol
content
of
young
leaves
increased
at
weekly
sampling
times
15.6
±
1.7
to
25.1
±
3.5
p,g/A1
latex
over
a
one-month
period.
Latex
cycloartenol
con-
tent
of
old
leaves
appeared
to
vary
over
a
broader
time
period.
During
the
same
one
month
period
no
change
in
cycloartenol
con-
tent
was
observed
in
latex
of
old
leaves,
al-
though
a
40%
increase
in
latex
cycloartenol
content
was
observed
4
months
later.
Cycloartenol
concentration
of
latex
from
mature
and
old
leaves
was
approximately
25%
lower
than
that
from
young
and
expanding
leaves
(Table
1).
Whole-leaf
cycloartenol
con-
tent
increased
more
than
two-fold
from
young
to
mature
stages,
but
was
not
significantly
dif-
ferent
between
mature
and
old
leaves.
Ex-
pressed
on
leaf
dry
wt
or
leaf
area
bases,
the
cycloartenol
content
was
greatest
in
young
leaves
and
decreased
during
leaf
maturation.
Values
for
cycloartenol
content
of
latex
and
whole-leaf
for
leaves
of
a
given
age
were
used
to
calculate
leaf
latex
content
(Table
2).
For
leaves
ofa
given
age,
both
values
were
obtained
on
the
same
day
to
avoid
potential
errors
re-
sulting
from
the
temporal
variability
in
cy-
cloartenol
content
described
above.
Total
latex
volume
increased
approximately
3-fold
from
young
to
mature
leaves
as
compared
to
8-
and
10-fold
increases
in
leaf
dry
wt
and
leaf
area,
respectively.
Leaf
latex
volume
expressed
on
either
a
leaf
area
or
leaf
dry
wt
bases
was
great-
est
for
young
leaves
and
decreased
more
than
50%
in
physiologically
older
leaves.
Latex
con-
tent
on
leaf
area
and
dry
wt
bases
was
similar
in
expanding
mature
and
old
leaves.
The
accumulation
of
laticifer
starch
in
the
laticifer
and
whole
leaf
differed
from
that
of
cycloartenol.
Laticifer
starch
represented
ap-
proximately
0.5%
of
the
latex
dry
wt.
Starch
content
of
latex
was
the
same
for
different
aged
leaves
(Table
3).
Even
in
senescent
leaves
which
still
exuded
latex,
the
latex
had
a
starch
content
similar
to
that
in
other
leaves.
Total
leaf
la-
ticifer
starch
calculated
from
leaf
latex
volumes
and
latex
starch
contents
indicated
the
total
amount
of
leaf
laticifer
starch
increased
during
leaf
expansion
and
was
greatest
in
mature
and
old
leaves.
Laticifer
starch
content
on
leaf
dry
wt
or
leaf
area
bases
was
highest
in
young
leaves,
and
decreased
by
50%
in
older
leaves.
Starch
content
of
leaf
latex
was
greater
than
that
for
either
stem
or
bract
latex.
Values
of
0.80
±
0.33
and
0.43
±
0.29
Ag/A1
latex
were
obtained
for
stems
and
bracts,
respectively.
The
differences
in
starch
contents
of
leaves,
stems,
and
bracts
were
statistically
significant
at
the
0.05
level
(Duncan
multiple
range
test).
No
starch
was
detected
in
latex
from
roots.
DISCUSSION-
Triterpenols
(4,4-dimethyl-3-
hydroxy-triterpenoids)
and
phytosterols
(4-de-
methyl-3-hydroxy-triterpenoids)
are
abundant
constituents
of
Euphorbia
latex
(Ponsinet
and
Ourisson,
1968).
In
poinsettia
we
found
tri-
terpenoids
and
their
esters
to
compose
40%
of
the
latex
dry
wt.
Our
studies
confirm
those
of
Baas
(1977)
in
identifying
cycloartenol
as
the
major
free
triterpenol
in
poinsettia
latex:
how-
ever,
it
occurs
only
as
a
minor
triterpenoid
component
of
the
non-laticifer
cells.
Cycloar-
tenol,
generally
a
minor
triterpenoid
compo-
nent
of
other
plants,
has
been
proposed
to
be
an
intermediate-metabolite
in
plant
triterpe-
noid
biosynthesis
(Rees,
Goad
and
Goodwin,
1968).
The
large,
quantitatively
stable
level
of
cycloartenol
in
the
latex
of
poinsettia
leaves
suggests
that
this
compound
has
a
different
role
in
the
laticifer.
The
abundance
of
cycloartenol
makes
it
an
easily
measurable
latex
marker
with
negligible
interference
from
components
of
non-laticifer
cells.
In
poinsettia
leaves
and
stems,
cycloartenol
content
was
affected
by
latex
dilution
during
1316
AMERICAN
JOURNAL
OF
BOTANY
[Vol.
73
TABLE
2.
Latex
volume
in
poinse
a
leaves
of
different
physiological
ages
Latex
volume'
Leaf
stage'
Total
in
leaf,
µI
µ1/100
mg
leaf
dry
weight
µl/cm'
leaf
area
Young
17.3
±
5.7
3
36.5
±
9.7
1.37
±
0.45
Expanding
24.9
±
7.7
15.8
±
3.6
0.49
±
0.15
Mature
56.8
±
13.7
14.4
±
2.2
0.42
±
0.10
Old
48.7
±
15.9
15.5
±
4.0
0.53
±
0.15
Calculated
from
data
in
Table
1.
See
text.
2
Description
of
leaf
stages
provided
in
text.
S.D.
exudation,
the
developmental
age
of
the
leaves
and
temporal
variation.
In
other
plants
latex
composition
has
been
shown
to
be
influenced
by
sampling
time,
site,
procedure
and
envi-
ronmental
conditions
(Curtis
and
Blondeau,
1946;
Gooding,
1952;
Buttery
and
Boatman,
1976).
In
this
study,
small
sample
size
and
controlled
sampling
times
prevented
potential
errors
due
to
latex
dilution
and
variations
in
composition.
Furthermore,
because
variation
in
the
latex
composition
occurred
between
dif-
ferent
aged
tissues
of
plants,
latex
volume
de-
terminations
required
measurements
from
leaves
of
identical
ages.
Changes
in
cycloartenol
content
reflected
changing
levels
of
latex
within
leaves
during
growth.
The
young
leaf
contained
the
highest
latex
content,
but
as
the
leaf
expanded
the
latex
and
its
cycloartenol
content
decreased.
During
subsequent
leaf
expansion
to
the
mature
stage
leaf,
leaf
and
laticifer
growth
were
proportion-
al,
and
the
latex
content
of
leaves
was
main-
tained
without
significant
change
to
the
old
stage.
Thus,
at
a
stage
prior
to
the
mature
leaf,
laticifer
and
leaf
growth
ended
concurrently.
Similar
trends
were
observed
for
latex
accu-
mulation
during
leaf
development
in
Hoya
australis
(Warnaar,
1982)
and
poinsettia.
In
both
plants
most
or
all
of
the
increase
in
latex
volume
occurred
during
the
period
of
leaf
ex-
pansion.
In
H.
australis
thickening
of
fully
ex-
panding
leaves,
a
process
which
does
not
occur
in
poinsettia,
induced
localized
laticifer
growth,
and
a
resultant
increase
in
latex
per
unit
leaf
area.
The
proportionally
high
latex
content
of
young
leaves
may
have
a
specific
purpose
re-
lated
to
plant
protection.
Latex
has
been
sug-
gested
to
serve
an
antiherbivore
function
(Shukla
and
Krishna
Murti,
1972;
Fahn,
1979).
High
latex
content,
with
its
toxic
components
(Kinghorn
and
Evans,
1975;
Swain,
1977),
may
provide
increased
protection
for
young
leaves
otherwise
more
susceptible
than
old
leaves
to
insect
and
animal
predation.
Young
leaves
of
other
plants
also
have
been
shown
to
have
high
levels
of
secondary
compounds
associated
with
plant
protection
(Rhoades
and
Gates,
1976).
The
metabolic
regulation
of
starch
synthesis
in
laticifers
is
uncertain.
Our
quantitative
anal-
yses
of
starch
content
of
latex
show
that
starch
concentration
in
laticifers
differs
between
plant
organs.
The
proximity
of
primary
photosyn-
thate
production
in
leaves
may
be
responsible
for
the
relatively
high
starch
level
in
leaf
latex,
whereas
translocation
of
photosynthate
into
inflorescences
(bracts)
may
only
support
a
low
level
of
starch
synthesis
in
this
organ.
The
ab-
sence
of
starch
in
root
latex
indicates
that
la-
ticifers
do
not
synthesize
starch
in
all
organs.
Further
investigations
are
necessary
to
relate
laticifer
starch
content
of
different
organs
with
specific
metabolic
control
mechanisms.
The
accumulation
of
starch
in
laticifers
dif-
fers
from
the
common
patterns
of
starch
me-
tabolism
observed
in
other
plants
and
de-
scribed
by
Jenner
(1980)
as
1)
transient
synthesis
in
photosynthetic
cells;
2)
consecu-
tive
periods
of
accumulation
and
degradation
extending
over
time
as
in
some
fruits;
and
3)
successive
periods
of
accumulation
and
deg-
radation
with
an
intervening
period
of
dor-
mancy
(e.g.,
seeds)
or
quiescence
(e.g.,
tubers).
Starch
accumulated
in
the
laticifer
throughout
its
period
of
growth,
but
apparent
net
starch
degradation
did
not
subsequently
occur.
In
pre-
vious
studies
of
laticifer
starch
of
other
Eu-
phorbia
species,
no
diurnal
rhythm
or
net
starch
degradation
in
darkness
was
observed
in
con-
trast
with
that
occurring
in
photosynthetic
cells,
nor
was
starch
degraded
during
flowering
or
fruit
formation
(Biesboer
and
Mahlberg,
1978).
Thus,
laticifer
starch
does
not
appear
to
serve
a
storage
function
under
any
of
these
condi-
tions.
Starch
in
laticifers
may
serve
a
function
dif-
ferent
from
that
in
other
cell
types.
It
is
possible
that
the
function
of
laticifer
starch
may
be
de-
pendent
on
the
physical
properties
of
the
starch
grains
such
that
degradation
and
mobilization
out
of
the
laticifer
is
unnecessary.
Laticifer
starch
grains
of
E.
pulcherrima
previously
have
September,
1986]
SPILATRO
AND
MAHLBERG-POINSETTIA
LATEX,
LATICIFER
STARCH
1317
TABLE
3.
Laticifer
starch
content
of
poinsettia
leaves
of
different
physiological
ages
Laticifer
starch
Leaf
stage'
A
g/
A
I
latex
Total
in
leaf,
A
g
l
ig/
100
mg
leaf
dry
weight
/
/g/cm2
leaf
area
Young
1.10
±
0.04
2
19.0
±
6.8
40.2
±
16.6
1.51
±
0.58
Expanding
1.14
±
0.09
28.4
±
9.0
18.0
±
6.4
0.56
±
0.19
Mature
1.13
±
0.16
64.2
±
17.9
16.3
±
5.3
0.47
±
0.18
Old
1.13
±
0.18
55.0
±
20.0
17.5
±
8.6
0.60
±
0.40
Description
of
leaf
stages
is
provided
in
text.
2
S.D.
been
shown
to
possess
an
unusual
array
ofcom-
positional
and
structural
properties
(Spilatro
and
Mahlberg,
1985),
and
these
may
contribute
to
a
function
unique
to
laticifer
starch
grains
They
have
been
reported
as
components
of
the
wound
plugging
mechanism
in
damaged
latic-
ifers
(Biesboer
and
Mahlberg,
1981).
Thus,
la-
ticifer
starch
grains
can
perform
a
secondarily
derived
function
independent
of
catabolism.
Our
results
indicate
that
laticifer
starch
de-
position
is
an
ongoing
process
during
laticifer
elongation,
but
do
not
indicate
whether
this
deposition
occurs
in
newly
formed
or
preex-
isting
amyloplasts.
Deposition
in
preexisting
amyloplasts
might
be
expected
since
organelle
degeneration
has
been
reported
in
the
vacuo-
lated
region
of
nonarticulated
laticifers
(Marty,
1968,
1971a;
Fineran,
1983).
In
other
studies
we
found
that
the
total
number
of
laticifer
starch
grains
increases
during
the
entire
period
of
la-
ticifer
growth
(Spilatro,
1984),
indicating
the
formation
of
new
amyloplasts.
Marty
(197
lb)
described
a
subpopulation
of
plastids
lacking
starch
grains
in
the
peripheral
cytoplasm
sur-
rounding
the
laticifer
central
vacuole
of
E.
characias.
It
is
possible
that
similar
plastids
in
poinsettia
may
serve
as
precursors
for
the
new
amyloplasts.
However,
the
formation
of
new
amyloplasts
is
paradoxical
in
what
has
been
suggested
to
be
a
degenerating
intracellular
en-
vironment
(Marty,
1971a),
and
requires
fur-
ther
study
to
determine
the
origin
of
laticifer
amyloplasts
and
the
mechanism
regulating
starch
deposition
in
these
elongated
grains.
LITERATURE
CITED
APLIN,
R.,
AND
G.
HORNBY.
1966.
Application
of
mass
spectrometry
to
the
structural
investigation
of
9,
19-
cyclosterols
and
triterpenes.
J.
Chem.
Soc.
(B)
1078-
1019.
BAAS,
W.
1977.
Triterpenes
in
latex
of
Euphorbia
pul-
cherrima.
Planta
Medica
32:
1-8.
BIESBOER,
D.,
AND
P.
MAHLBERG.
1978.
Accumulation
of
non-utilizable
starch
in
laticifers
of
Euphorbia
het-
erophylla
and
E.
myrsinites.
Planta
143:
5-10.
-
-,
AND
1981.
A
comparison
of
alpha-amy-
lases
from
the
latex
of
three
selected
species
of
Eu-
phorbia
(Euphorbiaceae).
Amer.
J.
Bot.
68:
498-506.
BUTTERY,
B.,
AND
S.
BOATMAN.
1976.
Water
deficits
and
flow
of
latex.
In
T.
Kozlowski
[ed.],
Water
deficits
and
plant
growth,
Vol.
4,
pp.
233-289.
Academic
Press,
New
York.
CURTIS,
J.,
AND
R.
BLONDEAU.
1946.
Influence
of
time
of
day
on
latex
flow
from
Cryptostegia
grandiflora.
Amer.
J.
Bot.
33:
264-270.
FAHN,
A.
1979.
Secretory
tissues
in
plants,
pp.
207-220.
Academic
Press,
New
York.
FINERAN,
B.
1983.
Differentiation
of
non-articulated
la-
ticifers
in
poinsettia
(Euphorbia
pulcherrima
Willd.).
Ann.
Bot.
52:
279-293.
GOODING,
E.
1952.
Studies
on
the
physiology
of
latex.
III.
Effects
of
various
factors
on
the
concentration
of
latex
of
Hevea
brasiliensis.
New
Phytol.
51:
139-153.
HELLER,
S.,
AND
D.
MILNE.
1978.
EPA/NIH
mass
spec-
tral
data
base
Vol.
4,
(p.
3266.)
U.S.
Government
Printing
Office,
Washington.
JENNER,
C.
1980.
Storage
of
starch.
In
F.
Loewus
and
W.
Tanner
[eds.],
Ency.
of
plant
physiol.,
Vol.
13A,
pp.
700-747.
Springer-Verlag,
New
York.
KELLER,
C.,
L.
BUSH,
AND
C.
GRUNWALD.
1969.
Changes
in
the
content
of
sterols,
alkaloids,
and
phenols
in
flue-cured
tobacco
during
conditions
favoring
infes-
tation
by
molds.
J.
Agr.
Food
Chem.
17:
331-336.
KINGHORN,
A.,
AND
F.
EVANS.
1975.
A
biological
screen
of
selected
species
of
the
genus
Euphorbia
for
skin
irritant
effects.
Planta
28:
325-335.
MARTY,
R.
1968.
Infrastructure
des
laticiferes
differen-
cies
d'Euphorbia
characias.
C.
R.
Acad.
Sci.,
Paris
267:
299-302.
.
1971a.
Vesicules
autophagiques
des
laticiferes
difrerencies
d'Euphorbia
characias
L.
C.
R.
Acad.
Sci.,
Paris,
Ser.
D.,
272:
399-402.
.
197
lb.
Differenciation
des
plastes
dans
les
la-
ticiferes
d'Euphorbia
characias
L.
C.
R.
Acad.
Sci.,
Paris,
Ser.
D.,
272:
223-226.
PONSINET,
G.,
AND
G.
OURISSON.
1968.
Aspects
parti-
culiers
de
la
biosynthese
des
triterpenes
dans
le
latex
d'
Euphorbia.
Phytochemistry
7:
757-764.
REES,
H.,
L.
GOAD,
AND
T.
GOODWIN.
1968.
Cyclization
of
2,3-oxidosqualene
to
cycloartenol
in
a
cell-free
sys-
tem
from
higher
plants.
Tetrahedron
Lett.
6:
723-
725.
RHOADES,
D.,
AND
R.
GATES.
1976.
Toward
a
general
theory
of
plant
antiherbivore
chemistry.
In
J.
Wallace
and
R.
Marnell
[eds.],
Recent
advances
in
phyto-
chemistry,
Vol.
10,
pp.
168-213.
Plenum
Press,
New
York.
SHUKLA,
0.,
AND
C.
KRISHNA
MURTI.
1972.
The
bio-
chemistry
of
plant
latex.
J.
Scient.
Ind.
Res.
30:
640-
662.
SPILATRO,
S.
1984.
Structure
and
composition
of
laticifer
starch
grains
and
their
metabolism
during
growth
of
1318
AMERICAN
JOURNAL
OF
BOTANY
[Vol.
73
Euphorbia
pulcherrima
Willd.
Ph.D.
Dissertation,
In-
diana
University,
Bloomington.
,
AND
P.
MAHLBERG.
1985.
Composition
and
structure
of
laticifer
starch
grains
of
Euphorbia
pul-
cherrima
Willd.
Bot.
Gaz.
146:
26-31.
SWAIN,
R.
1977.
Secondary
compounds
as
protective
agents.
Ann.
Rev.
Plant
Physiol.
28:
479-501.
WARNAAR,
F.
1977.
Deca-2,4,6-trienoic
acid,
a
new
con-
jugated
fatty
acid,
isolated
from
the
latex
of
Euphorbia
pulcherrima
Willd.
Lipids
12:
707-710.
.
1982.
Investigation
of
Hoya
species
V.
Deter-
mination
of
the
amount
of
latex
present
in
Hoya
aus-
tralis
R.
Br.
ex
Fraill.
and
Hoya
bella
Hook.
and
its
relationship
with
shoot
development.
Z.
Pflanzen-
physiol.
105:
307-314.
WEETE,
J.,
AND
W.
KELLEY.
1977.
Fatty
acids
and
sterols
of
Cronartium
fusiformebasidiospores.
Lipids
12:
398-
401.