Ethylene evolution from bracts and leaves of poinsettia (Euphorbia pulcherrima Willd.)


Woodrow, L.; Grodzinski, B.

Journal of Experimental Botany 38(197): 2024-2032

1987


Ethylene release from fully expanded, red and white bracts and leaves of poinsettia, Euphorbia pulcherrima Willd., was compared. On a laminar (area) basis leaves contained about 50 times more chlorophyll and demonstrated 10 times the photosynthetic rate of the bracts. Both tissues contained starch, however, soluble carbohydrate in the bracts consisted primarily of reducing hexoses while the leaves contained mainly sucrose for translocation. The total free alpha-amino nitrogen content of the bract tissue was twice that of the leaf tissue. The leaves contained more ACC (1-aminocyclopropane-1-carboxylic acid) and produced proportionally more endogenous C2H4 than either the red or white bracts. ACC-stimulated C2H4 release was also greatest from the green tissue indicating that the EFE (ethylene forming enzyme) was most active in the leaves. The specific activity of the 14C2H4/12C2H4 released from [2,3-14C]ACC confirmed ACC as the primary precursor of C2H4 in this tissue. Ethylene release from the non-photosynthetic, bract tissue was not markedly affected by alterations in CO2 or light conditions. In green leaf tissue endogenous ethylene release increased from 1.5 to 6.0 pmol C2H4 cm-2 h-1 while ACC-stimulated ethylene release increased from 10 to 35 pmol C2H4 cm-2 h-1 as the CO2 partial pressure increased from 100 to 1200 .mu.bar.

Journal
of
Experimental
Botany,
Vol.
38,
No
197,
pp.
2024-2032,
December
1987
Ethylene
Evolution
from
Bracts
and
Leaves
of
Poinsettia,
Euphorbia
pulcherrima
Willd.
LORNA
WOODROW
AND
BERNARD
GRODZINSKI'
Department
of
Horticultural
Science,
University
of
Guelph, Guelph,
Ontario
N
1G
2W1,
Canada
Received
30
April
1987
ABSTRACT
Woodrow,
L.
and
Grodzinski,
B.
1987.
Ethylene
evolution
from
bracts
and
leaves
of
Poinsettia,
Euphorbia
pulcherrima
Willd.—J.
exp.
Bot.
38:
2024-2032.
Ethylene
release
from
fully
expanded,
red
and
white
bracts
and
leaves
of
poinsettia,
Euphorbia
pulcherrima
Willd.,
was
compared.
On
a
laminar
(area)
basis
leaves
contained
about
50
times
more
chlorophyll
and
demonstrated
10
times
the
photosynthetic
rate
of
the
bracts.
Both
tissues
contained
starch,
however,
soluble
carbohydrate
in
the
bracts
consisted
primarily
of
reducing
hexoses
while
the
leaves
contained
mainly
sucrose
for
translocation.
The
total
free
alpha-amino
nitrogen
content
of
the
bract
tissue
was
twice
that
of
the
leaf
tissue.
The
leaves
contained
more
ACC
(1-aminocyclopropane-
1-carboxylic
acid)
and
produced
proportionally
more
endogenous
C
2
H
4
than
either
the
red
or
white
bracts.
ACC-stimulated
C
2
H
4
release
was
also
greatest
from
the
green
tissue
indicating
that
the
EFE
(ethylene
forming
enzyme)
was
most
active
in
the
leaves.
The
specific
activity
of
the
14
C
2
1
-
1
4
/
12
C
2
H
4
released
from
[2,3-
14
C]ACC
confirmed
ACC
as
the
primary
precursor
of
C
2
H
4
in
this
tissue.
Ethylene
release
from
the
non-photosynthetic,
bract
tissue
was
not
markedly
affected
by
alterations
in
CO
2
or
light
conditions.
In
green
leaf
tissue
endogeneous
ethylene
release
increased
from
1.5
to
6.0
pmol
C
2
H
4
cm'
h
- '
while
ACC-stimulated
ethylene
release
increased
from
10
to
35
pmol
C2H4
cm
-2
h
as
the
CO
2
partial
pressure
increased
from
100
to
1
200
µbar.
Key
words—Poinsettia,
ethylene,
bracts.
Correspondence
to:
Department
of
Horticultural
Science,
University
of
Guelph,
Guelph,
Ontario
N
1G
2W1,
Canada.
INTRODUCTION
The
flux
of
carbon
through
the
major
metabolic
pathways,
photosynthesis
and
respiration
(µmol
cm'
11
-1
),
in
leaves
is
normally
six
to
seven
orders
of
magnitude
greater
than
the
flow
through
the
ethylene
pathway
(pmol
cm'
h
-
').
Since
ethylene
evolution
is
a
CO
2
-sensitive
process,
the
massive
flux
of
CO
2
associated
with
photosynthetic
activity
in
leaves
may
have
a
substantial
impact
on
processes
subject
to
the
regulatory
properties
of
ethylene
(Grodzinski,
Boesel,
and
Horton,
1982a).
When
gas
exchange
is
limited,
the
photosynthetic
activity
of
the
tissue
can
significantly
alter
the
CO
2
levels
within
the
tissue
and
the
concomitant
C
2
H
4
release
rate.
In
leaf
tissue
from
several
C3
plants,
Nicotiana
tabacum
L.
and
Oryza
sativa
L.
(Kao
and
Yang,
1982),
Xanthium
strumarium
L.
(Grodzinski
et
al.,
1982a,
b),
Ranunculus
sceleratus
L.
(Woodrow,
1983),
Lemna
minor
L.
(Fuhrer,
1985),
Salvia
splendens
L.
(Grodzinski,
1984),
Helianthus
annuus
L.
(Dhawan,
Bassi,
and
Spencer,
1981),
and
two
C
4
species
Zea
mays
L.
(Grodzinski
et
al.,
19826)
and
Gomphrena
globosa
L.
(Grodzinski,
Boesel,
and
Horton,
1983),
ethylene
evolution
has
been
shown
to
drop
if
To
whom
correspondence
should
be
addressed.
©
Oxford
University
Press
1987
Woodrow
and
Grodzinski—Ethylene
and
Poinsettia
2025
the
amount
of
CO
2
available
to
the
tissue
is
reduced
and,
conversely,
to
increase
if
the
ambient
CO
2
concentration
is
raised.
Photorespiratory
CO
2
release
and
amino
acid
meta-
bolism
may
also
modulate
C
2
H
4
release
in
the
light
(Grodzinski,
1984).
Although
light
does
not
appear
to
influence
C
2
H
4
release
directly
in
C3
plants,
the
C4
tissues
studied
have
demonstrated
higher
rates
of
C
2
H
4
release
in
response
to
increasing
light
intensity
when
CO
2
is
not
limiting
(Grodzinski
et
al.,
1982a,
b;
1983).
The
differences
we
have
observed
in
different
monocotyledonous
and
dicotyledonous
C3
and
C4
species
with
respect
to
light
and
CO
2
emphasizes
the
impact
that
photosynthetic
activity
has
on
ethylene
metabolism.
Leaf
responses
to
changes
in
the
photosynthetic
environment
are
rapid
because
the
laminar
structure
of
leaves
facilitates
gas
exchange
as
well
as
light
interception.
Poinsettia
(Euphorbia
pulcherrima
Willd.)
leaves
and
bracts
represent
morphologically
similar
tissue
types
on
the
same
plant
which
differ
in
plastid
and
pigment
development
and
associated
photosynthetic
capacity.
The
use
of
different
tissues
from
the
same
plant
provides
an
alternative
to
the
treatment
of
leaf
tissue
with
chemical
inhibitors
of
photosynthesis
such
as
3-(3,4-dichlorophenyl)-1,1-dimethylurea
(Grodzinski
et
al.,
1982a),
or
inhibitors
of
pigment
synthesis
such
as
norflurazon
(de
Laat,
Brandenburg,
and
van
Loon,
1981)
when
studying
ethylene
release
under
varying
photosynthetic
conditions.
In
poinsettia,
ethylene
has
been
associated
with
substantial
reductions
in
crop
quality
(Staby,
Thompson,
and
Kofranek,
1978)
due
to
petiole
epinasty
of
the
bracts
and
leaves
and
to
potential
involvement
in
bract
and
cyathia
abscission.
The
present
study
provides
data
on
the
relative
contribution
of
bract
and
leaf
tissue
to
total
ethylene
production
and
demonstrates
how
ethylene
produced
by
the
tissue
is
affected
by
environmental
CO
2
concentrations.
MATERIALS
AND
METHODS
Plant
material
Poinsettias,
Euphorbia
pulcherrima
Willd.
cv.
'Annette
Hegg
Dark
Red'
and
cv.
'Annette
Hegg
White'
were
grown
in
the
greenhouse
from
rooted
cuttings
in
10
cm
plastic
pots
according
to
standard
commercial
production
procedures
(McDaniel,
1979).
No
growth
retardants
were
applied.
All
leaf
and
bract
tissue
for
extraction
or
experiments
was
harvested
at
approximately
10.00
a.m.
Fully
expanded
leaves
and
bracts
were
used
for
all
studies
prior
to
any
cyathia
(flower)
abscission.
Tissue
was
prepared
by
punching
8.5
cm
discs
with
a
cork
borer
avoiding
all
major
veins.
At
harvest
all
discs
of
each
tissue
type
were
pooled
and
random
subsamples
were
removed
for
extraction
or
experimental
incubation.
Within
an
experiment
each
treatment
or
extraction
was
replicated
three
times;
each
experiment
was
repeated
twice.
Extraction
and
metabolite
analysis
Subsamples
of
ten
discs
were
extracted
in
hot
80%
aqueous
ethanol.
Three
successive
extracts
were
pooled
and
the
chlorophyll
content
determined
according
to
Wintermans
and
de
Mot
(1965).
The
extract
volume
was
then
reduced
to
approximately
1.0
cm
3
in
a
Speedvac
Concentrator
(Savant
Instruments,
Hicksville,
New
York)
to
remove
all
ethanol
and
partitioned
against
chloroform
to
remove
lipid
and
lipid
soluble
pigments.
The
resulting
aqueous
fraction
was
used
for
determination
of
major
metabolite
pools.
Total
soluble
carbohydrate
(hexoses,
sucrose)
was
estimated
using
anthrone
:
sulphuric
acid
(van
Handel,
1968)
and
standardized
as
glucose
equivalents.
The
hexoses,
glucose
and
fructose
were
determined
by
the
Nelson-Somogyi
reducing
sugar
assay
(Somogyi,
1952)
and
standardized
as
glucose
equivalents.
Sucrose
content
was
calculated
as
the
difference
between
total
soluble
carbohydrate
and
hexose
content.
Alpha-amino
nitrogen
was
determined
by
the
ninhydrin
method
(Yemm
and
Cocking,
1955)
and
expressed
as
alanine
equivalents.
The
starch
content
of
the
extracted
discs
was
determined
by
digestion
with
amyloglucosidase
(Rauser,
1978)
followed
by
quantitation
of
the
liberated
glucose
with
anthrone
:
sulphuric
acid.
All
metabolites
were
expressed
on
a
unit
area
basis.
Anthocyanin
content
was
determined
by
extracting
subsamples
of
10
discs
with
cold
80%
(aqueous)
methanol
containing
1%
HCI
(v/v)
and
measuring
the
optical
density
of
the
extract
at
525
nm
(Craker
2026
Woodrow
and
Grodzinski—Ethylene
and
Poinsettia
and
Wetherbee,
1973).
The
anthocyanin
contents
of
the
tissues
were
compared
as
the
optical
density
of
13
cm
3
extracts
prepared
from
each
subsample.
Extracts
for
the
assay
of
endogenous
ACC
(1-aminocyclopropane-
1-carboxylic
acid)
content
were
prepared
as
described
for
total
metabolite
pools
using
20
discs
per
subsample.
The
resulting
extract
was
assayed
following
procedures
described
by
Lizada
and
Yang
(1979).
The
ACC
content
was
quantified
by
the
addition
of
internal
standards
and
the
efficiency
of
conversion
for
the
poinsettia
extracts
was
84%.
Ethylene
release
Ethylene
release
was
studied
by
incubation
of
tissue
as
described
previously
(Grodzinski
et
al.,
1982a,
b;
1983).
Leaf
discs
(10
per
flask;
total
area
of
5.67
cm
2
)
were
incubated
on
1.0
cm
3
50
mmol
dm
-
K-EPPS
buffer,
pH
8.0
in
25
cm'
flat
bottomed
Micro-Fernbach
flasks
sealed
with
rubber
serum
stoppers.
The
flasks
were
suspended
in
a
shaking
water
bath
at
25
°C
and
illuminated
from
below
with
incandescent
flood
lamps.
The
photon
fluence
rate
was
350
itmol
m
-2
s
(PAR,
400-700
nm)
at
disc
level.
Dark
treatments
were
obtained
by
incubating
the
discs
in
flasks
painted
with
flat
black
paint
and
wrapped
in
aluminium
foil.
The
CO
2
concentration
in
the
flasks
was
maintained
by
suspending
a
plastic
centre
well
in
each
flask
containing
a
strip
of
fluted
filter
paper
and
a
solution
of
2.0
mol
dm
- '
K
2
HPO
4
/KH
2
P0,
buffer
and
NaHCO
3
.
In
this
way
the
CO
2
supply
to
the
discs
was
held
constant
for
the
duration
of
the
experiment.
The
CO
2
concentration
was
varied
by
adjusting
the
pH
of
the
buffer
and
the
NaHCO
3
concentration.
This
technique
represents
a
modification
of
a
method
described
by
Tingey,
Raba,
and
Burns
(1980).
The
CO
2
concentrations
were
verified
by
infrared
gas
analysis
of
headspace
samples
using
an
ADC
225
Mk
III
IRGA
(Analytical
Development
Corp.,
Hoddesdon,
U.K.).
The
CO
2
levels
for
individual
experiments
are
indicated
in
the
figures.
Ethylene
release
by
the
plant
tissue
was
determined
by
withdrawing
500
mm
3
headspace
samples
and
assaying
the
ethylene
content
on
a
Varian
1400
gas
chromatograph
equipped
with
an
activated
alumina
column
and
a
flame
ionization
detector
(column
110
°C;
detector
150
°C).
The
detector
response
was
standardized
against
injections
of
known
quantities
of
C
2
H
4
prepared
by
serial
dilution
of
pure
C
2
H
4
.
The
retention
times
for
ethane
and
ethylene
were
1.3
and
1.8
min
respectively.
For
measurements
of
ACC-stimulated
ethylene
release
1.0
mmol
dm
-3
ACC
was
incorporated
into
the
incubation
medium.
This
parameter
is
an
indicator
of
potential
EFE
(ethylene
forming
enzyme)
activity
(Philosoph-Hadas,
Aharoni,
and
Yang,
1986)
and
provides
a
measure
of
the
capacity
of
each
of
the
tissue
types
to
serve
as
sources
of
ethylene.
"C-ACC
accumulation
and
14
C
2
1-1
4
release
The
radiolabelled
ACC
studies
were
conducted
essentially
as
described
previously
(Grodzinski,
1984;
Woodrow,
1983).
Leaf
discs
were
incubated
with
the
addition
of
2,3-[
14
C]ACC
(847
MBq
mmol
New
England
Nuclear,
Mass.)
to
the
incubation
medium
(1.0
mmol
dm'
ACC;
specific
activity
32.2
MBq
mmol
-
').
The
14
C
2
1-1
4
release
was
determined
by
withdrawing
10.0
cm
3
headspace
samples
at
the
end
of
the
incubation
period
and
injecting
them
into
sealed,
partially
evacuated
20
cm
3
glass
scintillation
vials
containing
0-1
mmol
dm
-3
mercuric
acetate
in
methanol
(Abeles
and
Abeles,
1972).
The
vials
were
held
at
4
°C
for
12
h
to
allow
complete
absorption
of
the
14
C
2
H
4
,
then
scintillation
cocktail
(0.5%
w/v
2,5-diphenyloxazole
in
toluene
:
2-methoxyethanol,
5
:
4
v/v)
was
added
and
the
radioactivity
quantified
by
liquid
scintillation
counting
(Beckman
6800
LSC).
The
sampling
method
removed
26%
of
the
flask
headspace
contents
and
the
mercuric
acetate/methanol
solution
absorbed
100%
of
the
"CAI,
from
the
gas
phase.
Accumulation
of
2,3-[
14
C]ACC
was
determined
by
killing
and
extracting
the
leaf
discs
at
the
end
of
the
2
h
incubation
period.
The
incubation
media
was
removed
and
the
discs
were
washed
with
three
changes
of
distilled
water,
and
then
processed
as
described
for
metabolite
extraction.
After
chloroform
partitioning
aliquots
of
the
aqueous
extract
were
assayed
by
liquid
scintillation
counting
for
total
radioactivity
accumulation
as
described
above.
The
leaf
disc
residues
were
counted
in
a
gel-phase
cocktail
prepared
by
combining
equal
volumes
of
distilled
water
and
scintillation
cocktail
(0.5%
2,5-diphenyloxazole
w/v
in
toluene:
Triton
X-100,
2
:
1
v/v).
The
2,3-[
14
C]ACC
content
was
corrected
for
free
space
uptake
by
comparison
with
a
parallel
[
14
C]sorbitol
incubation.
ACC
uptake
represents
all
ethanol
soluble
forms.
Photosynthetic
and
respiratory
activity
Net
photosynthetic
and
respiratory
gas
exchange
capacity
was
determined
by
Warburg
manometry
using
a
Gilson
illuminated
respirometer
(Umbreit,
Burris,
and
Stauffer,
1972).
Conditions
were
as
described
for
the
ethylene
release
incubations
except
that
Warburg
buffer
(100
mmol
dm'
Woodrow
and
Grodzinski—Ethylene
and
Poinsettia
2027
carbonate/bicarbonate,
pH
90)
was
used
to
allow
the
measurement
of
net
0
2
evolution
or
consumption.
Carbon
dioxide
compensation
points
were
determined
by
allowing
tissue
samples
to
deplete
the
CO
2
level
from
330
µbar
partial
pressure
to
a
constant
value
at
a
light
intensity
of
350
µmol
M
-2
s
-1
.
Anthrone,
ACC,
ninhydrin
and
amyloglucosidase
(11800
U
gm
-1
)
were
purchased
from
Sigma
Chemical
Co.,
St.
Louis,
Missouri.
All
other
chemicals
were
reagent
grade
products
purchased
from
Fisher
Scientific,
Don
Mills,
Ontario.
RESULTS
Photosynthesis
and
carbon
metabolism
The
distinction
between
the
green
leaves
and
the
coloured
(white
and
red)
bracts
was
evident
in
a
comparison
of
several
parameters.
Pigmentation
was
the
most
visible
difference
between
leaf
and
bract
tissue
(Table
1).
The
white
bracts
contained
only
trace
quantities
of
chlorophyll
and
anthocyanin
while
the
red
bracts
contained
the
highest
concentration
of
anthocyanin
and
trace
quantities
of
chlorophyll.
The
green
leaf
tissue
contained
about
the
same
amount
of
anthocyanin
as
the
white
tissue.
The
lack
of
chlorophyll
in
both
red
and
white
bracts
is
correlated
with
the
very
low
net
photosynthesis
rates
relative
to
the
green
leaves
(Table
1).
TABLE
1.
Photosynthesis,
C
2
1-1
4
release,
and
metabolite
concentration
in
red,
white,
and
green
poinsettia
tissue
All
parameters
are
expressed
on
a
unit
(cm
2
)
area
basis.
All
values
represent
the
mean
of
three
replicates
±
s.e.
Red
bract
White
bract
Green
leaf
Net
CO
2
uptake
(nmol
h
-
')
120+10
110+10
1040±50
Chlorophyll
a
and
b
(µg)
1.9+0.2
1.3
+
0.3
6745+2.9
Anthocyanin
(0.D.
525
nm)
0.83+005
0.07+0-01
0.06+0-01
Carbohydrate
(µmol
glucose
equiv.)
Total
soluble
sugar
1-74
+
0-06
123
+
0-07
207
+
0-05
Hexose
1.80+0.10
2.65+042
0.28+0-06
Sucrose
1.79
+
0.06
Starch
1.66+0.21
146+009
1.50+0.15
Total
soluble
a-amino
N
0-47+0-02
0.55+0-03
0.23+0-01
(µmol
ala
equiv.)
ACC
(pmol)
39.9
+
3-5
63-7
+
15-5
115-6
+
6-0
C
2
H
4
release
(pmol
h
')
Endogenous
1.9+0-1
2.4+0.5
5.6+0-7
ACC-stimulated
9.2+0.8
6.2
+
0.6
32.4+1.8
In
addition
the
leaf
tissue
had
a
CO
2
compensation
point
of
60
µbar
at
350
µmol
m
-2
5
-1
,
typical
of
C3
plants,
while
the
bract
tissues
did
not
deplete
the
CO
2
levels
over
the
course
of
an
experiment.
These
data
support
the
view
that
the
bracts,
although
photosynthetically
active
to
a
small
degree,
function
heterotrophically.
The
levels
of
major
metabolites
also
reflect
the
roles
of
the
green
leaves
and
the
bracts
as
source
and
sink
tissues
respectively.
Total
pools
of
soluble
carbohydrates,
as
indicated
by
anthrone
reactivity,
were
similar
in
both
the
leaf
and
bract
tissues.
However,
in
the
bract
tissues
the
reducing
hexoses,
glucose
and
fructose,
accounted
for
the
soluble
component
whereas
in
the
leaf
tissue
the
transport
sugar
sucrose
composed
the
largest
fraction
of
the
2028
Woodrow
and
Grodzinski—Ethylene
and
Poinsettia
soluble
carbohydrates.
This
is
consistent
with
their
function
as
the
major
photo-assimilating
organs
in
these
plants.
Significant
amounts
of
starch
were
present
in
both
leaf
and
bract
tissue
as
the
storage
carbohydrate.
The
concentration
of
total
soluble
alpha-amino
nitrogen
in
the
red
and
white
bract
tissue
was
approximately
twice
that
of
the
green
leaf
tissue.
Ethylene
metabolism
Under
the
conditions
of
the
photosynthetic
assay,
the
green
tissue
had
higher
rates
of
both
endogenous
and
ACC-stimulated
ethylene
evolution
than
the
bract
tissue
(Table
1).
The
green
leaf
tissue
also
contained
the
largest
pool
of
endogenous
ACC,
the
precursor
of
C
2
1-1
4
(Table
1).
In
all
three
tissues,
the
ACC
content
was
sufficient
to
account
for
the
measured
rates
of
endogenous
C
2
H
4
release
for
the
duration
of
the
assays
(Table
1).
Leaf
and
bract
tissues
were
incubated
with
exogenous
ACC
in
order
to
assess
the
capacity
of
the
tissue
to
convert
ACC
to
C
2
I-1
4
(Fig.
1).
When
supplied
with
exogenous
ACC,
the
green
leaf
tissue
evolved
significantly
more
C
2
1-1
4
than
the
bract
tissues
under
all
conditions
except
one.
In
the
totally
artificial
condition
in
which
the
green
photosynthetic
tissue
was
incubated
in
the
light
while
the
CO
2
supply
was
withheld
(i.e.
at
the
CO
2
compensation
point)
the
C
2
I-1
4
release
rate
was
as
low
as
that
from
the
bract
tissue.
In
both
the
red
and
white
bract
tissue,
ACC-stimulated
C
2
I-1
4
release
was
relatively
unresponsive
to
the
light
and
CO
2
incubation
conditions,
even
though
there
was
some
measurable
photosynthetic
activity
in
the
bracts
(Table
1).
Although
both
types
of
bract
tissue
contained
EFE
activity
(Fig.
1)
as
well
as
measurable
pools
of
free
ACC
(Table
1),
the
data
(Fig.
1,
Table
1)
clearly
demonstrate
that
the
photosynthetically
active
green
tissue
had
a
greater
capacity
to
metabolize
exogenous
ACC
to
C
2
1-1
4
and
was
more
dramatically
affected
by
changes
in
the
incubation
conditions
(i.e.
light/CO
2
).
In
order
to
assess
the
degree
of
ACC
accumulation
in
the
tissues,
discs
of
leaves
and
bracts
were
supplied
with
1.0
mmol
dm
-3
ACC,
containing
radiolabelled
2,3-[
14
C]ACC,
(specific
20-
light
ri
light+
co
t
1E1
dark
n
dark+
co
2
,
RED
WHITE
GREEN
Fla
1.
The
effect
of
light
and
CO
2
on
ACC-stimulated
C
2
H
4
evolution
from
red
and
white
bract
tissue
and
green
leaf
tissue
of
poinsettia.
Light
incubations
were
at
an
intensity
of
350
limot
m
2
s
at
disc
level.
The
CO
2
concentration
in
the
'
+
CO
2
'
condition
was
maintained
at
approximately
350
µbar
(see
Materials
and
Methods
for
details).
The
discs
were
incubated
with
1.0
mmol
dm
-3
ACC
for
2
h.
Values
represent
the
mean
of
three
replicates+
s.e.
Woodrow
and
Grodzinski—Ethylene
and
Poinsettia
2029
activity
322
Bq
mmol
').
In
2
h,
the
red
and
white
bract
tissue
accumulated
6.2
±
0-7
and
7.2
±
02
nmol
ACC
cm
-2
respectively
while
the
green
tissue
accumulated
2.9
±
0.2
nmol
cm
-2
even
though
the
bracts
evolved
less
ethylene
(Fig.
1).
The
14
C
2
H
4
evolution
was
also
measured
and
compared
to
total
C
2
H
4
as
determined
by
conventional
gas
chromatographic
analysis
(Table
2).
These
data
show
that
the
total
ACC
supply
(nmol
cm
-2
)
was
not
limiting
to
C
2
H
4
release
(pmol
cm
-2
h
-1
).
Accumulation
of
"C-ACC
in
both
the
light
and
the
dark
was
approximately
1
000
times
greater
than
the
concomitant
C
2
H
4
release
(Table
2)
and
100
times
higher
than
the
endogenous
ACC
concentrations
(see
Table
1).
The
production
of
radioactive
14
C
2
11
4
from
"C-ACC
confirms
that
the
exogenously
supplied
ACC
was
available
for
conversion.
Furthermore,
the
specific
activity
of
the
C
2
H
4
released
from
the
green
leaf
tissue
was
the
same
in
both
the
light
and
the
dark
(Table
2).
The
production
of
14c2*
ri[
*-4
from
2,3-[
14
C]ACC
(Table
2)
and
the
presence
of
ACC
in
the
tissue
(Table
1)
supports
the
role
of
ACC
as
the
precursor
of
C
2
H
4
in
these
tissues.
TABLE
2.
2,3-[
14
C]ACC
accumulation
and
ethylene
release
from
poinsettia
leaf
tissue
Tissue
was
incubated
for
2
h.
The
light
incubations
were
conducted
at
350
pmol
111
-2
S
-1
;
the
CO
2
concentration
was
maintained
at
approximately
350
µbar
by
the
buffered
NaHCO
3
solution
(see
Materials
and
Methods
for
details).
The
specific
activity
of
the
ACC
solution
was
322
MBq
mmol
All
values
represent
the
mean
of
three
replicates
+s.e.
Light
Dark
[
14
C]ACC
accumulation
(nmol
cm'
h
-
')
287±0.15
2.76±0.09
C
2
H
4
release
(pmol
cm'
h
-
')
GC
analysis
29.4
+3-3
218
+4-5
14c
2
H
4
30.9
+2-7
21-0+1-8
Specific
activity
(MBq
mmo1
-1
)
27.8
+
3.0
30.0
+
3.7
As
pointed
out
above
the
green
leaf
tissue
appears
to
be
the
major
source
of
endogenous
ethylene.
It
is
also
the
most
responsive
to
changes
in
CO
2
concentration.
The
data
in
Fig.
2A
and
B
clearly
illustrate
the
change
in
endogenous
and
ACC-stimulated
ethylene
release
as
CO
2
concentration
was
varied
over
a
range
(50-2
000
µbar)
frequently
encountered
in
greenhouses
during
crop
production.
DISCUSSION
Poinsettia
bracts,
particularly
the
white
tissues,
contain
little
chlorophyll
and
exhibit
many
characteristics
of
non-photosynthetic
heterotrophic
tissue.
For
example
the
bract
tissues
had
higher
free
amino
acid
contents
than
the
leaf
tissue.
Tanabe
and
Kawashima
(1982)
found
that
the
achlorophyllous
regions
of
variegated
tobacco
leaves
had
much
higher
free
amino
acid
contents
than
the
green
areas.
In
tobacco
not
all
of
the
amino
acids
were
present
in
proportionally
higher
amounts,
glutamine
and
asparagine
were
selectively
enriched.
When
the
ACC
content
is
expressed
as
a
fraction
of
the
total
pool
of
free
alpha-amino
nitrogen,
which
includes
the
amino
acids,
the
content
of
the
bracts
is
four
times
greater
than
that
of
the
green
tissue.
Despite
the
low
chlorophyll
content
of
the
bracts
both
red
and
white
bracts
fix
CO
2
at
about
10%
of
the
rate
of
the
green
leaf
tissue
(Table
1).
This
rate
of
primary
CO
2
flux
is
five
orders
of
magnitude
greater
than
the
maximal
loss
of
carbon
as
C
2
I-1
4
gas
in
these
tissues
(Fig.
1).
Both
endogenous
and
ACC-stimulated
C
2
H
4
release
from
the
bract
tissue
T
A
CC-
STI
MU
LATE
D
30
.3
20
E
0.
0
A
o
0
0
0
0
0
0
0
0
0
B
50
40
Lu
6
CO
4
Lu
—i
w
5
cc
o.—,
x
O
.c
4
CV
(.0
'E
n
U
O
3
Z
r)
La
E
O
Q.
O
....-
2
0
Z
11J
1
0
2030
Woodrow
and
Grodzinski—Ethylene
and
Poinsettia
0
400
800
1200 1600
2000
2200
CO
2
PARTIAL
PRESSURE
(Mbar)
FIG.
2.
The
effect
of
ambient
CO
2
concentration
on
endogenous
and
ACC-stimulated
C
2
H
4
evolution
from
green
poinsettia
leaf
tissue.
The
light
intensity
at
leaf
disc
level
was
350
µmot
m
-2
s'.
Points
represent
actual
C
2
H
4
and
CO
2
determinations
for
individual
flasks.
The
endogenous
experiment
was
incubated
for
20
h
and
the
ACC-stimulated
experiment
for
2
h.
(Fig.
1)
was
two
to
three
times
less
than
that
from
leaves
except
in
the
experimental
situation
when
the
leaf
tissue
was
incubated
at
the
compensation
point
(i.e.
in
the
light
without
CO
2
).
However,
as
shown
in
Fig.
1,
it
was
only
this
extreme
anomalous
experimental
situation,
when
photosynthetic
leaf
tissue
had
been
allowed
to
deplete
the
CO,
content
of
the
incubation
flask
to
the
CO,
compensation
point
(60
µbar),
that
a
low
ethylene
release
rate,
comparable
to
those
observed
with
bract
tissue,
was
obtained.
Incubation
in
the
dark
when
respiratory
decarboxylation
increases
the
internal
CO,
concentration
or
the
inclusion
of
a
CO,
source
in
the
light
resulted
in
ethylene
release
rates
three
to
four
times
higher.
This
pattern
is
representative
of
results
obtained
with
other
C
3
tissues
(Grodzinski
et
al.,
1982a,
b).
Neither
endogenous
nor
ACC-stimulated
ethylene
release
rates
from
red
and
white
bract
tissue
were
affected
by
ambient
CO,
concentration
or
light
conditions,
to
the
same
extent
as
the
green
leaf
tissue,
suggesting
that
ethylene
release
in
these
tissues
was
saturated
with
respect
to
CO,
(Fig.
1).
Respiratory
activity
and
the
absence
of
a
strong
net
CO,
Woodrow
and
Grodzinski—Ethylene
and
Poinsettia
2031
demand
may
allow
the
internal
CO,
concentration
to
remain
high
enough
for
maximal
rates
of
ethylene
release
in
these
tissues.
In
the
closed
flasks
used
for
C
2
H
4
measurements
neither
white
nor
red
bract
tissue
reduced
the
CO,
level
in
the
light
to
the
compensation
point
typical
of
the
green
tissue.
Ethylene
release
by
poinsettia
leaves
may
fluctuate
as
the
plants
are
alternated
between
light
and
dark
particularly
if
there
are
constraints
on
gas
exchange
or
the
CO,
content
of
the
atmosphere
is
intentionally
manipulated.
Endogenously
produced
C
2
H
4
may
be
an
important
factor
in
any
dense
leaf
canopy.
Variations
between
100
and
1
500
µbar
in
the
atmospheric
CO,
concentration
occur
in
commercial
greenhouses
(Porter
and
Grodzinski,
1985
and
references
therein).
Temporal
and
spatial
CO,
gradients
are
also
encountered
in
outdoor
natural
and
crop
canopies
which
may
have
an
impact
on
ethylene
metabolism.
Heilman,
Meredith,
and
Gonzalez
(1971)
detected
ethylene
within
a
mature
cotton
canopy
up
to
18.6
nmol
dm
-3
at
night
under
still
air
conditions
and
concluded
that
these
accumulations
could
stimulate
abscission
of
the
flowers.
As
demonstrated
in
this
paper,
ethylene
production
by
green
leaf
tissue
is
correlated
with
CO,
concentration
(Fig.
2A,
B).
Both
endogenous
and
ACC-stimulated
ethylene
release
from
green
tissue
is
altered
by
incremental
changes
in
CO,
concentration
(Fig.
2A,
B).
ACC-stimulated
ethylene
release,
under
the
light
conditions
of
this
study
(350
pmol
m
-2
s
1
,
PAR),
is
saturated
with
respect
to
CO,
at
approximately
800
µbar
(Fig.
2B).
Interestingly
endogenous
ethylene
release
is
not
saturated
even
at
2
400
µbar
CO,
(Fig.
2B).
The
ACC-stimulated
rates
reflect
the
CO,
dependence
of
ACC
to
C
2
H
4
conversion
and/or
C
2
1-1
4
retention
(Grodzinski
1982a,
b,
1983;
Fuhrer,
1985;
Kao
and
Yang,
1982).
Recently
Philosoph-Hadas
et
al.
(1986)
have
suggested
that
the
synthesis
of
the
ethylene
forming
enzyme
(EFE)
in
long
term
experiments
(48
h)
is
enhanced
at
high
CO,
levels
resulting
in
higher
ethylene
production
rates
in
addition
to
the
direct
effect
of
CO,
on
ACC
to
C
2
H
4
conversion.
The
endogenous
C
2
H
4
release
rates,
however,
reflect
the
net
effects
of
altered
CO,
concentration
on
all
steps
leading
to
endogenous
ACC
production
as
well
as
regulating
ethylene
release.
Kao
and
Yang
(1982)
found
that
endogenous
ACC
pools
were
not
altered
by
CO,
concentration
or
light/dark
treatment,
and
Fuhrer
(1985)
has
shown
that
the
degree
of
ACC
conjugation
is
not
modified
by
these
parameters.
Investigations
into
the
relationships
between
photosynthesis,
CO
2
,
ethylene
metabolism,
and
associated
plant
responses
are
confounded
by
the
different
photosynthetic
and
photorespiratory
types
C
3
,
C
4
,
and
CAM
(Grodzinski
et
al.,
1982a,
1983;
Grodzinski,
1984)
which
moderate
their
internal
CO,
metabolism
in
distinctive
ways.
This
study
presents
the
first
data
describing
the
ethylene
evolution
patterns
from
naturally
occurring
photosynthetic
and
non-photosynthetic
blade
tissue
of
a
greenhouse
crop.
Ethylene
and
the
pools
of
the
immediate
precursors,
like
other
regulatory
compounds,
are
present
in
minute
quantities
in
plant
tissue
relative
to
the
flux
of
metabolites
through
major
pathways.
It
is
evident,
however,
that
the
synthesis
of
ethylene
is
affected
by
these
pathways
via
CO,
(as
a
substrate
or
product)
and
should
be
considered
in
relation
to
the
overall
metabolic
activity
of
the
tissue.
ACKNOWLEDGEMENTS
The
authors
acknowledge
grants
to
B.G.
from
the
Natural
Sciences
and
Engineering
Research
Council
of
Canada
and
the
Ontario
Ministry
of
Agriculture
and
Food.
The
research
was
conducted
while
L.W.
was
on
educational
leave
from
Agriculture
Canada.
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Light
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Early
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