Reprinted from BOTANICAL GAZETTE Vol.
No.4, December 1969 c 1969 by The University of Chicago. All rights reserved. Printed in U.S.A. 130,
BOT. GAZ.
130(4) :271-275.1969.
GROSS METABOLIC ACTIVITY ACCOMPANYING THE AFTER-RIPENING
OF DORMANT DOUGLAS-FIR SEEDSl
STEPHEN D. ROSS
University of Washington College of Forest Resources
ABSTRACT
C!.laJ es in the lipid and sug8;r reserv s an? respiratory. activity
of dormant Douglas-fir (Pseudotsuga
.
,
1J!ellZ'leS-/.'l- Mlrb.
Fr.anc ) seeds durmg stratIficatIOn and gernunatlOn were followed to provide some infornla­
tIon on the after-npenmg process. The dormant seeds were characterized by a low level of metabolism even
when placed under conditions known to be favorable for germination. One day of stratification removed the
block P!evel;ting lipid breakdown, bu
. respiratory . activity did n?t attai full . capacity until the fifth day
of stratIficatIOn. Nearly 40% of the lIpId reserves dIsappeared durmg stratIficatIOn, over half of which could
!lot be accounted for by respiration or as sugars. The possible significance of this unaccounted-for carbon
m preparing the dormant seeds for germination is discussed.
Introduction
Although numerous works have been published on
breaking dormancy in the seeds of Douglas-fir (Pse'lt­
dots'ltga menziesi'i Mirb. Franco), little is known
about the after-ripening process. CHING (1959) in­
vestigated the mechanism by which presoaking
Douglas-fir seeds in hydrogen peroxide (H202) brings
about their after-ripening. Changes in O2 uptake and
CO2 production by the seeds following treatment
with H202 provided some information on the physio­
logical activity accompanying after-ripening. The
significance of the gas-exchange patterns found was
not clear since, as CHING noted, H202 can affect the
rates of O2 and CO2 exchange via its influence on nu­
merous metabolic processes. Her study did seem to
indicate though that H202 stimulated respiratory
activity and activated mobilization of the lipid re­
serves.
The present study was initiated in an attempt to
provide a more integrated picture of the physiological
changes occurring during after-ripening. The seeds'
changing physiology during stratification and ger­
mination was assessed by means of lipid and sugar
and gas-exchange determinations. These indices of
metabolic activity cannot provide an insight into the
dormancy mechanism, as was emphasized by MAYER
and POLJAKOFF-MAYBER (1963). They should, how­
ever, indicate how removal of the dormancy block(s)
readies the dormant seeds for germination.
Material and methods
Only one seed lot was investigated since CHING
(1963a, 1963b) found little difference in physiological
1 The research reported in this paper is a condensation of
a Master of Science thesis and was supported financially in
part by the Pacific Northwest Forest and Range Experiment
Station of the U.S. Forest Service (P.L. 85-934) and the Co­
operative Forestry Research (McIntire-Stennis) Act (P.L. 87­
788) and administered by the Institute of Forest Products
of the University of Washington.
activity associated with seed source for coastal
Douglas-fir. The seeds were collected in the fall of
1965 near Elbe, -Washington, at an elevation of ap­
proximately 1, 000 ft. They were dried at room tem­
perature for 1t months and then at 50 C to yield a
final moisture content of between 6% and 9%. Prior
to use the seeds were stored at -17 C in air-tight
containers. All seeds were X-rayed, and the radio­
graphs were used to remove the empty, decayed, and
larva-infected seeds.
Stratification was done by the "naked" method of
ALLEN and BIENTJES (1954). The seeds were pre­
soaked in distilled water at room temperature for 12
hI', drained and blotted surface dry (to reduce mold
growth), and stored at 2 C for the specified stratifica­
tion period. Seeds used in the germination studies
were incubated on a Jacobsen-type germinator at
26 ± 1 C under an 8-hr photoperiod of 350 ft-c.
Total lipid and sugar determinations were made
on duplicate samples of 1.5 g oven-dry weight. The
seeds were immersed in liquid nitrogen to arrest
enzymatic activity and then ground to a fine meal
while still frozen. The crude lipids were extracted in
a Soxhlet apparatus with 75 ml of petroleum ether
(bp 30-60 C) for 24 hI', dried in vacuo, and weighed.
Sugars were extracted from the lipid-free seed meal
with 75 ml of 75% ethanol for 24 hr. The sugar ex­
tracts were clarified with lead subacetate (HASSID
1936) and their reducing power determined by
the Modified Harding and Downs copper reagent
(PLANK 1936). Sucros is a major Douglas-fir seed
sugar (REDISKE 1961; CHING 1963a), but treatment
of the extracts with a fresh, 1% invertase solution
did not reveal its presence. It seems that sucrose was
hydrolyzed during extraction, since sucrose added to
the seed meal prior to extraction was recovered quan­
titatively among the reducing sugars. Acid hydroly­
sis due to the incomplete removal of the chromic acid
cleaning solution from the extraction thimbles is
suspected.
271
272
BOTANICAL GAZETTE
The gas-exchange determinations were performed
in a Gilson Differential Respirometer at 26 ± 0.02 C.
Volume changes were measured over a 2-hr period on
triplicate 20-seed samples. Oxygen uptake was deter­
mined by Warburg's direct method (UMBREIT, BUR­
RIS, and STAUFFER 1957) and CO2 production in a
duplicate flask without KOH in the center well. Be­
cause of problems with the respirometer at the 2 C
temperatures used for stratification, the gas-ex­
change determinations during after-ripening were
made at 26 C after temperature equilibration in the
respirometer for 12 hr.
o
50
Z
40
0::
W
(9
30
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W
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CL
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o L-�-L�L-�����--�----�
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2 3 4 5 6
9
7
12
DAYS
INCUBATED
.LSJ' 260 C
FIG. i.-Cumulative germination percentages for Douglas­
fir seeds after 0 (0), 1 (0), 5 (\7), and 10 (.6.) days of strati­
fication at 2 C.
Lipid, sugar, and gas-exchange determinations
were made periodically during stratification and dur­
ing the incubation of 0- (nonstratified), 1-, 5-, and
10-day stratified seeds. Except where noted the new­
ly germinated seedlings were included in the samples
taken during incubation. The study was conducted
twice, but to conserve space only the average values
are reported here. Differences between the two repli­
cations averaged within 10% of the mean for all
analyses.
Results and discussion
GERMINATION.-Nearly 5% of the nonstratified
seeds germinated during the first 9 days of incubation
(fig. 1). The reduced rate of germination with addi­
tional time on the germinator indicates that the re­
[DECEUBER
maining nonstratified seeds were dormant. The first
5 days of stratification were most effective in terms
of increased germination per day of stratification.
Germination tests conducted for 21 days showed that
the seeds were completely after-ripened by the tenth
day of stratification.
LIPID RESERVEs.-Thirty-eight percent of the
seeds' lipid reserves disappeared between the first
and third days of stratification (fig. 2A). This large
lipid loss was somewhat unexpected, since CHING'S
(1963a) data for different Douglas-fir seed lots show
that only 6% of the crude lipids disappeared after 10
days of stratification. This discrepancy might be due
to seed source differences or to differences in the stage
of seed maturity at time of harvest (REDISKE 1961).
The absence of any real change in the lipid content
of the nonstratified seeds incubated at 26 C (fig. 3A),
in contrast to their rapid mobilization during strati­
fication, points to the existence of a block preventing
lipid breakdown in the dormant seeds. This block was
removed within the first 24 hI' of stratification as
evidenced by the rapid rate of lipid breakdown in
the l-day stratified seeds. Since 1 day of stratifica­
tion did not greatly stimulate germination, it must be
concluded that mobilization of the lipid reserves was
only one step in the after-ripening process.
SUGAR RESERVEs.-Changes in seed sugar content
during stratification are shown in figure 2B. The
significance of this pattern cannot be determined as
neither the sugar-turnover rate nor the rate of lipid­
to-sugar conversion is known. The behavior of the
respiratory quotient (RQ) during stratification (fig.
2C), however, is interesting with respect to lipid-to­
sugar conversions. 'While lipid breakdown was most
intense between the first and third days of stratifica­
tion, the minimum RQ of 0.56, suggesting maximum
glyoxylate cycle activity (STILES and LEACH 1960),
did not occur until the sixth day of stratifi­
cation. I regard this interpretation as provisional
pending biochemical studies on the glyoxylate
cycle. BEEVERS (1961b) warns against placing too
much emphasis on the RQ as an index of glyoxylate
cycle activity, noting that other metabolic processes
also affect it. The possibility is presented that the
rate of lipid-to-sugar conversion during stratification
was not governed by the availability of lipid break­
down products. The inference is that the pathways
involved in mobilizing the lipid reserves to sugars for
subsequent use in respiration and synthetic activities
(BEEVERS 1961a) were sluggish in the dormant seeds.
RESPIRATORY ACTIVITy.-The effect of stratifica­
tion on O2 uptake by the seeds incubated at 26 C is
shown in figure 3B. The curve for the nonstratified
seeds shows how even a few (about 5%) nondormant
seeds can affect the pattern of oxygen uptake for the
sample as a whole. ' Then those nondormant seeds
1969)
273
ROSS-AFTER-RIPENING OF DOUGLAS-FIR SEEDS
that germinated during the first 9 days of incubation
were excluded from the gas-exchange determinations,
the rate of O2 uptake fell to a level only slightly
higher than on the first day (dashed lIne). This nearly
constant rate of O2 uptake by water-imbibed seeds is
characteristic of dormant seeds (STILES and LEACH
1960). One day of stratification reduced this "lag
phase of O2 uptake" to 6 days, and it is not evident in
the patterns for the 5- and 1O-day stratified seeds.
CffiNG (1959) reported that presoaking dormant
Douglas-fir seeds in H202 had a similar effect on O2
uptake.
The present findings support those of CHING
(1959) in showing that the seeds' respiratory activity
increases during the process of after-ripening from a
low level in the dormant seeds. The data do not indi­
cate whether this increase was due to a change in
seed coat permeability to oxygen or to a basic change
in the seeds' respiratory metabolism. CHING placed
little emphasis on the seed coat being a barrier to
oxygen but did not substantiate her view. Regardless
of the dormancy block inhibiting O2 uptake, its re­
moval during stratification increased the supply of
respiratory energy and carbon skeletons for synthetic
acti vities.
CARBON BALANCE SHEET.-Since nearly 40% of
the seeds' initial lipid reserves disappeared during
stratification, it was suspected that the breakdown
products formed might play an important role in
preparing the dormant seeds for germination. Some
information on the fate of these breakdown products
was provided by a carbon balance sheet. The net lipid
and sugar changes and the amount of CO2 produced
during the 10 days of stratification were converted to
an equivalent amount of carbon. Lipid losses were
converted to carbon (1 mg lipids
0. 76 mg carbon)
based on the fatty-acid composition of Douglas-fir
seeds (CHING 1963b). For conversion purposes, seed
sugars were assumed to be hexoses (1 mg hexose
sugar
0.40 mg carbon). Carbon dioxide losses are
considered as respiratory in nature, recognizing that
other decarboxylation reactions contributed to this
=
=
300
(f)
Q
'-
a. 0
-l 0
w
w
W en
Q
:::>
0::
()
(!)
250
200
A
150
(f)
0::
<t
(!)
:::>
(f)
-l
<t
I0
I-
3i
ci
0
w
w
en
30
(!)
"(!)
B
20
0.7
d
ci
0.6
C
0.5
0
I
3
6
10
21
DAYS S TRATIFIED AT 2°C
FIG. 2.-Changes in crude lipids (A), total sugars (B), and the respiratory quotient (C) of Douglas-fir seeds during strati­
fication at 2 C.
274
[DECEMBER
BOTANICAL GAZETTE
loss. The CO2 losses were somewhat exaggerated
since the gas-exchange determinations were made at
26 C after a 12-hr temperature equilibration period
and not at the 2 C stratification temperatures.
Of the 77.2 mg of carbon mobilized during strati­
fication, 1.4% was recovered as sugars, 41.7% was
respired, and 56.9% was unaccounted for. Some of the
unaccounted-for carbon was undoubtedly converted
to starch and other temporary reserves not moni­
tored. Although CHING (1963a) found relatively
little starch buildup following stratification in the
seed lots she investigated, the lipid losses in these
(f)
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..J
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300
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w
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::> "
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W
rials required for the seeds to become after-ripened.
CHING (1963a, 1963b) found that phospholipids, re­
quired in the synthesis of cellular organelles, nearly
doubled following stratification. The possibility also
exists that the seeds were immature when harvested.
Thus the unaccounted-for carbon may have been
used in synthetic activity normally occurring while
the seeds are still attached to the cone scales (RE­
DISKE 1961). This might explain the discrepancy
between the present findings and those of CHING
(1963aJ in the amounts of the lipid reserves mobilized
during stratification. However, it is also possible that
w
w
rn A
B
0.4
(!)
(!) ::i
>- " x "i
0
2
3
4
5
6
7
DAYS INCUBATED AT
9
12
26°C
FIG. 3.-Changes in crude lipids (A) and oxygen uptake
(B) during incubation at 26 C of Douglas-fir seeds after
0(0), 1 (0), 5 (\7), and 10(b.) days of stratification at 2 C.
(0 - - - 0) oxygen uptake by nonstratified seeds after re­
moval of the germinated seedlings.
seeds were also small. It is noteworthy that the pro­
portion of the lipid breakdown products stored as
sugars was negligible. If stratification affected the
seeds' food relations, it did so primarily by facilitat­
ing a faster mobilization of the storage reserves
(lipids and starch) during germination rather than by
providing the developing embryo with an abundant
supply of metabolically active sugars. KOBLET (1932)
arrived at a similar conclusion in his study on the af­
ter-ripening of Pinus strobus seeds.
The unaccounted-for carbon was presumably also
available for various synthetic activities. PACK
(1921) concluded that much of the carbon mobilized
during the stratification of Juniperus seeds was
resynthesized to cell-active and cell-building mate­
the physiological differences associated with seed
source are greater than her study indicated. Further
investigation is needed to determine the fate of the
unaccounted-for carbon and to assess its role in pre­
1
paring the seeds for germination.
Conclusions
The nonstratified Douglas-fir seed was charac­
terized by a low level of metabolism even when
placed under conditions known to be favorable for
germination. Evidence was found that considerable
metabolic activity occurred during stratification.
Stratification apparently facilitated a faster mobili­
zation of the lipid reserves during germination. One
day of stratification activated lipid breakdown, and
1969)
ROSS-AFTER-RIPENING OF DOUGLAS-FIR SEEDS
there is some evidence, albeit highly circumstantial,
that the pathways involved in mobilizing the break­
down products did not become fully developed until
the sixth day of stratification. Stratification also in­
creased the seed's respiratory capacity. This coupled
with mobilization of the lipid reserves is believed to
have helped ready the seed for germination by pro­
viding the embryo with respiratory energy and car­
bon skeletons for synthetic activities. Nearly 40% of
the seed's lipid reserves disappeared during stratifica­
tion, of which almost 60% were not respired or stored
275
as sugars. The significance of this unaccounted-for
carbon in readying the seeds for germination is con­
sidered.
Acknowledgment
I wish to
express my gratitude to Professor DAVID
R. M. SCOTT for his advice and guidance through­
out the course of this study, to the U.S. Forest Ser­
vice for financial assistance, and to the State of
Washington Forest Lands Management Center at
Olympia, Washington, for supplying the seeds.
LITERATURE CITED
ALLEN, G. S., and N. BIENTJES. 1954. Studies on coniferous
tree seed at U.B.C. Forest Chron. 38:485-496.
BEEVERS, H. 1961a. Metabolic production of sucrose from fat.
Nature 191 : 433-436.
-. 1961b. Respiratory metabolism in plants. Row, Peter­
son, New York.
CHING, TE MAY . 1959. Activation of germination in Douglas­
fir seed by hydrogen peroxide. Plant Physiol. 34:557-563.
---. 1963a. Change of chemical reserves in germinating
Douglas fir seed. Forest Sci. 9 :226--231.
---. 1963b. Fat utilization in germinating Douglas fir seed.
Plant Physiol. 38: 722 728.
HASSID, W. Z. 1936. Determination of reducing sugars and su­
crose in plant materials. Ind. Eng. Chern. 8:138-140.
--
-
KOBLET , R. 1932. Uber die Keimung von Pinus strobus unter
besonderer Berucksichtigung der Herkunft des Samens.
Ber. Schweiz. Bot. Ges. 41:199-283.
MAYER, A. M., and A. POLJAKOFF-MAYBER. 1963. The germi­
nation of seeds. Macmillan, New York.
PACK, D. A. 1921. After-ripening and germination of J1tnipems
seeds. BOT. GAZ. 71 :32-60.
PLANK, J. E. VAN DER. 1936. The estimation of sugars in the
leaf of the mangold (Beta vulgaris). Biochem. J. 30:457-483.
REDISKE, J. H. 1961. Maturation of Douglas-fir seed. A bio­
chemical studv. Forest Sci. 7:32-60.
STILES, W., and W. LEACH. 1960. Respiration in plants. Wiley,
New York.
UMBREIT, W. W., R. H. BURRIS, and J. F. STAUFFER. 1957.
Manometric techniques. BUl'gess, Minneapolis.
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