Plant Physiol.

(1974) 54, 560-563

Adenosine Phosphates in Germinating Radish

(Raphanus sativus L.) Seeds'
Received for publication March 22, 1974 and in revised form May 31, 1974

DONALD E. MORELAND, GRISCELDA G. HUSSEY, CAROLE R. SHRINER, AND FRED S. FARMER

Southern Region, Agricultural Research Service, United States Department of Agriculture, Crop Scienice
Department, North Carolina State University, Raleigh, North Carolina 27607

ABSTRACT ing imbibition of water is, in some way, coordinated and con-
trolled metabolically. Conceivably, end product inhibition,
Changes in concentrations of adenosine phosphates (AMP, substrate induction, hormonal stimulation, and energy re-
ADP, and ATP), oxygen utilization, and fresh weights were lations are all involved and interrelated in the modulation of
measured during the first 48 hours after imbibition of water by metabolism (6). The roles played by each of the known types
quiescent radish seeds (Raphanus sativus L.) at 22.5 C. The of control mechanisms, however, remain to be established.
changes in ATP concentrations, oxygen utilization, and fresh The pivotal role that high energy compounds might play in
weights followed a triphasic time course, characterized by a directing the growth and developmental processes that occur
rapid initial increase, which extended from 0 to approximately in germinating seeds has received limited attention. Brown (4)
1.5 hours, a lag phase from 1.5 to 16 hours, and a sharp linear considered the metabolic implications of changes in the free
increase from 16 to 48 hours. In unimbibed seeds, the concen- nucleotide pattern in germinating seeds. Regulation of the
trations of ATP, ADP, and AMP were <0.1, 0.9, and 2.2 levels of nucleotides by adenylate kinase in seeds was explored
nmoles/seed, respectively. After imbibition of water by the by Bomsel and Pradet (3). Control exerted by the adenylate
quiescent seeds, for 1 hour, the ATP concentration had increased phosphates and the applicability of the energy charge concept
to 2.5, and ADP and AMP concentrations had decreased to 0.3 of Atkinson (1,
2), in the regulation of cellular activity in
and 0.1 nmole/seed, respectively. These early changes occurred germinating seeds, was investigated by Pradet et al. (16) and
also in seeds maintained under anaerobic conditions (argon), later by Ching (6, 7). Only limited information for a relatively
or when treated with either 5 mM fluoroacetate, or 5 mM iodo- few plant species is available concerning changes in the con-
acetate. The concentrations of ADP and AMP did not change centrations of adenylates in seeds during the first few hr after
significantly from 1 to 48 hours. The termination of the lag imbibition of water, and little is known about the interrelations
phase at 16 hours correlated with radicle emergence. Cell divi- between energy-generating systems. A critical evaluation of
sion in the radicles was initiated at approximately 28 hours. the modulation of metabolism by energy relations in
ATP concentrations in seeds maintained under argon or treated ing seeds can conceivably be made when more information
germinat-
with fluoroacetate remained relatively constant from approxi- about the behavior of adenylates becomes available. Con-
mately 2 to 48 hours. In contrast, the ATP concentration of sequently, the objectives of the studies reported herein were
iodoacetate-treated seeds decreased curvilinearly from 4 to 48 (a) to measure changes in the adenylate phosphates (AMP,
hours. Oxidative phosphorylation was estimated to have con- ADP, and ATP) and (b) to determine the
tributed 15, 20, and 65% of the pool ATP at 1.5, 16, and 48 of glycolysis and oxidative phosphorylation relative contribution
hours, respectively.
to the ATP pool,
during the first 48 hr following imbibition of water by quiescent
radish seeds.
MATERIALS AND METHODS
Radish seeds (Raphanuiis sativus L. var. Early Scarlet Globe)
of a uniform size were germinated in the dark in 9-cm Petri
In addition to food reserves, quiescent seeds contain en- dishes on filter paper wetted with 5 ml distilled water or with
zymes and organelles held in a metabolically inactive state. aqueous solutions of 5 mm sodium fluoroacetate or 5 mm so-
When water is imbibed by the seeds at a suitable temperature dium iodoacetate. Unless indicated otherwise, the studies were
and under aerobic conditions, the enzymes and organelles that conducted at 22.5 C. In the study involving effects of anaero-
were formed during seed maturation become activated, and the biosis, the seeds were placed in 50-ml Erlenmeyer flasks which
partially differentiated embryos resume their course of develop- had been modified to permit purging with, and maintaining an
ment. The sequential development of events that occurs follow- atmosphere of, argon.
In the 1-, 4-, and 8-hr treatments, 14 seeds were used. At
' Cooperative investigations of the North Carolina Agricultural
the end of the imbibition or treatment period, the two most
fully developed and the two least developed seeds were dis-
Experiment Station and the Southern Region, Agricultural Re- carded. Measurements were made on the remaining 10 seeds.
search Service, United States Department of Agriculture, Raleigh,
N. C. Paper No. 4305 of the Journal Series of the North Carolina For time periods longer than 8 hr, seven seeds were treated,
Agricultural Experiment Station, Raleigh, N. C. Investigations sup- the most advanced and the least developed seeds or seedlings
ported in part by Public Health Service Grant ES 00044, Coopera- were discarded, and five were assayed. At the sampling times
tive State Research Service Grant 116-15, and National Science indicated, the selected seeds or seedlings were rinsed with
Foundation Grant 28951. distilled water, blotted, and weighed. Seeds or seedlings were
560
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Copyright © 1974 American Society of Plant Biologists. All rights reserved.
 -
Plant Physiol. Vol. 54, 1974 ADENOSINE PHOSPHATES IN RADISH SEEDS 561
homogenized immediately in 3 ml of 7% perchloric acid (v/v)
with a motorized tissue grinder. The homogenate was neutral-
ized with 1.2 ml of 3 N NaOH and 2.0 ml of 0.2 M HEPES
buffer (pH 7.4), and centrifuged to remove precipitated ma- _ 30 12 c
terials and cellular debris. The supernatant was brought to a
volume of 10 ml with distilled water. Whenever necessary, Radicle
dilutions of the supernatant were made with 0.04 M HEPES Emergence
buffer (pH 7.4). -20 -
8 E
The concentration of ATP in the extracts was determined 0
with a luciferin-luciferase assay essentially as described by
Gruenhagen and Moreland (12). ADP and AMP concentra-
tions were determined by difference after enzymatic conversion
to ATP with pyruvate kinase and adenylate kinase, essentially
as described by Chapman et al. (5). All calculations were cor-
rected for interferences imposed by the perchloric acid in the
extracts on the ATP measurements as determined by the in- 00 8 16 24 32 40 48
clusion of internal ATP standards. The energy charge of the Germination Time (Hours )
adenylate pool was calculated, from the molar concentrations FIG. 1. Increases in fresh weight (Q) and oxygen utilization (E)
of the adenylates, as [(ATP) + 1/2(ADP]/[(ATP + (ADP) with time after imbibition of water by radish seeds at 22.5 C. Water
(AMP)] (2). uptake is divided into initial physical hydration (phase 1), a stage
Oxygen uptake of radish seeds and seedlings was measured during which little additional imbibition occurs (phase 2), and a
polarographically with a Clark electrode in a 2-ml, water- period of continuous water uptake (phase 3).
thermostated glass reaction cell maintained at 25 C. After
incubation, the intact seeds or seedlings were placed directly
into the reaction cell, which contained 2 ml of reaction medium
[1I% glucose in 0.03 M potassium phosphate buffer (pH 5.5)].
Ten seeds or seedlings were pooled for each time period mea- c

sured, except that only five seedlings were used for the 48-hr
sample. Utilization of oxygen was linear with time until 4n

anaerobiosis occurred. Oxygen content of the air-saturated

medium was calculated to be 236 nmoles/ml.
Data reported herein are arithmetic averages of duplicate 0
determinations obtained from two separate experiments and a

are presented on a per seed or per seedling basis.

'o
E
RESULTS c

Increases in fresh weight after imbibition of water by radish

seeds at 22.5 C are shown in Figure 1, as a function of time. I-0
-A-,&-&-A
a
- --51 ____3__
-
a
a
0
A
Incremental gains in fresh weights following imbibition of %

water appear to have occurred in three phases. When quiescent 0 8 16 24 32 40 48

Germination Time (Hours)
seeds were placed in contact with water, there was a rapid
initial increase in fresh weight during the first 90 min, which FIG. 2. Changes in concentrations of AMP (A), ADP (El), and
may be attributed to physical hydration of the seed coats ATP (0) with time after imbibition of water by radish seeds at 22.5
and outer layers of the seeds (phase 1). Between 1.5 and 16 hr. C. Phases of water uptake are indicated.
the increase in fresh weight slowed down as exhibited by only
a slight incremental gain with time (phase 2). From 16 to 48 an intermediate concentration of ADP (approximately 0.9
hr, a marked and continuous linear increase in fresh weight nmole/seed) (Fig. 2). There was a very rapid increase in ATP
(phase 3) occurred. Initiation of phase 3 at 16 hr coincided concentration and a corresponding decrease in AMP and ADP
with emergence of the radicles and preceded cell division, concentrations associated with the imbibition of water during
which did not occur until 28 hr. phase 1. The increase in ATP concentration at 1 hr approxi-
Respiration measured as oxygen uptake, after imbibition of mated the sum of the decreases in AMP and ADP concentra-
water by quiescent radish seeds at 22.5 C, is also shown in tions. The slight increase in ATP concentration during phase 2
Figure 1. Oxygen uptake by the unimbibed seeds was too low to paralleled the changes in oxygen utilization (Fig. 1). During the
be detected by the procedure used. However, respiration was phase 3 uptake of water, between 16 and 48 hr, there was a very
initiated during phase 1 of water uptake. In phase 2, between rapid and linear increase in ATP concentration. The marked
1.5 and 8 hr, the rate of respiration did not change appreciably. linear increase in concentration of ATP, which began at around
Between 8 and 16 hr a gradual increase in the rate of respiration 16 hr, coincided with emergence of the radicles. The concentra-
occurred. Beginning with the 16-hr sample and continuing tions of ADP remained relatively low, but were slightly higher
through phase 3, there was a very rapid and linear increase in between 1 and 16 hr than from 16 to 48 hr. AMP concentrations
the rate of respiration, which originated with the emergence of reflected no major changes between 1 and 48 hr and remained
the radicles. The changes in rates of respiration paralleled below those of ADP.
closely the increases in fresh weight. Because of the high AMP and low ATP concentrations, the
The ATP concentration in the unimbibed radish seeds was energy charge of the seeds, before imbibition of water, was
below the limits of sensitivity of the procedure used herein 0.13. At the end of the 1st hr, after imbibition of water, the
(<0.1 nmole). However, the dry seeds contained an appreciable energy charge had increased to 0.89. The change reflected the
concentration of AMP (approximately 2.2 nmoles/seed) and large increase in ATP and corresponding decreases in ADP

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Copyright © 1974 American Society of Plant Biologists. All rights reserved.
562 MORELAND, HUSSEY, SHRINER, AND FARMER
ab
_va
4:3
E E
c a
16 24 32
Germinotion Time ( Hours )
FIG. 3. Changes in ATP concentration with time after imbibition
of water by radish seeds at 22.5 C under aerobic (0) and anaerobic
conditions (0), and treatment with 5 mM fluoroacetate (A) and
5 mM iodoacetate (El). Phases of water uptake are indicated.
and AMP concentrations. After 4 hr, the energy charge had
increased slightly to 0.93 and remained around this value
through the 48-hr sample.
Trends in the ATP content of the radish seeds and seedlings
following placement in an anaerobic (argon) atmosphere, and in
contact with 5 mm iodoacetate and 5 mm fluoroacetate are
presented in Figure 3. For comparative purposes, the curve,
which represents the trend in ATP concentrations obtained
after imbibition in contact with water, is repeated from Figure
2. lodoacetate alkylates sulfhydryl groups and inhibits the
activity of many enzymes that have sensitive sulfhydryl groups
associated with the active centers, including the glycolytic
enzyme glyceraldehyde-3-phosphate dehydrogenase (20). To
be effective, fluoroacetate has to be converted enzymatically
to fluoroacetyl CoA, which is condensed with oxaloacetate to
form fluorocitrate. The fluorocitrate acts as a competitive in-
hibitor of aconitase; utilization of citrate by the tricarboxylic
acid cycle is prevented and citrate accumulates (17).
Maintenance of the seeds in an argon atmosphere and treat-
ment with fluoroacetate did not prevent the reciprocal decrease
in AMP concentration and increase in ATP concentration that
occurred in the untreated control seeds during the 1st hr
after imbibition of water. [Data for ATP only are shown (Fig.
3).] At 1 hr, the ATP concentrations were slightly lower in the
treated seeds than in the untreated controls. The ATP concen-
tration did not increase markedly beyond 1 hr, and essentially
remained constant throughout the 48-hr treatment period.
Treatment with iodoacetate also did not prevent the increase
in ATP concentration during the 1 st hr following imbibition of
water, but the concentration was slightly lower than in the
controls. ATP levels decreased after 4 hr and reached a very
low concentration (0.18 nmole) by 48 hr.
If the mitochondrial contribution of ATP was essentially
arrested, under anaerobic conditions or following treatment
with fluoroacetate, the ATP represented by the area between
the abscissa and the treatment curves can be considered to have
had a glycolytic origin (Fig. 3). The area between the argon
curve and the aerobic curve provides an approximation of the
ATP produced by the mitochondrial system. As reflected in
Figure 3, the concentration of ATP postulated to be contrib-
uted by glycolysis remained relatively constant between 4 and
48 hr in the untreated seeds. The contribution of oxidative
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Copyright © 1974 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 54, 1974
/o O
8 ATP A
40 0
0
oe- _ E
o vm E
0._ -o 30 E -
(L Fresh a N - c)
Weight c m
o 4 -,& o6 20 3
LL
2 Radicle 10
O Length
15.0 17.5 20.0 22.5 25.0 27.5 30.0
Temperature (C )
FIG. 4. ATP concentration (0), radicle length (0), and fresh
weight (A) of radish seedlings after germination of the seeds for 48
hr at different temperatures.
phosphorylation to the ATP pool was estimated to be ap-
proximately 15% at the end of the first phase of water uptake
(1.5 hr), 20% at the end of the second phase (16 hr), and dur-
ing the third phase, amounted to approximately 45 and 65 %
at 24 and 48 hr, respectively.
The energy charge of seeds maintained under anaerobic
conditions and seeds treated with fluoroacetate did not deviate
markedly from the untreated control seeds at all time periods
through 48 hr (0.89 to 0.93). In the iodoacetate treatment,
the energy charge values decreased steadily from 0.89 at 1 hr
to 0.48 at 48 hr.
Seeds maintained under anaerobic conditions and those
treated with iodoacetate showed the same increases in fresh
weight as the seeds germinated under aerobic conditions (Fig. 1)
from 1 to 16 hr. Between 16 and 48 hr, seeds held in the
absence of oxygen and treated with iodoacetate did not leave
the second phase of water uptake. Fresh weight did not in-
crease, but remained at approximately 14 mg/seed. Fluoro-
acetate-treated seeds increased slightly in fresh weight from
14.5 mg at 16 hr to 18.8 mg at 48 hr. In comparison, the
aerobically germinated control seeds had increased in weight
from 14.5 mg at 16 hr to 38.5 mg at 48 hr. Radicles did not
emerge from the seeds held under argon or treated with iodo-
acetate. Radicle emergence did occur in the fluoroacetate
treatment, however, emergence was delayed and instead of
occurring uniformly, was staggered over the period from 24 to
36 hr. In addition, the radicles were shorter and thinner in the
fluoroacetate treatment, than the radicles of the untreated con-
trol seeds.
Effects of temperature on ATP concentration, radicle length,
and fresh weight of radish seedlings measured 48 hr after
imbibition of water by quiescent seeds are presented in Figure 4.
All three curves are bell shaped and show a maximal response
in the temperature range of 22.5 to 25.0 C. Trends in ATP
concentration correlated closely with fresh weight and length
of radicle. However, fresh weight did not decrease as markedly
as ATP content and radicle length, at temperatures higher
than the optimum. Decreased values were obtained at temper-
atures below and above the optimum. As indicated earlier, at
22.5 C, radicles emerged at around 16 hr, but emergence was
delayed at temperatures of 15.5 and 17.5 C. However, when
the 48-hr samples were harvested, all radicles had emerged.
At 30 C, radicles were noticeably thicker than those that de-
veloped at lower temperatures.
Plant Physiol. Vol. 54, 1974 ADENOSINE PHOSPHATES IN RADISH SEEDS
DISCUSSION
Changes in the pool of adenosine phosphates (AMP, ADP,
and ATP) were monitored in this study as a function of time
after imbibition of water by quiescent radish seeds. The values
reported represent the net adenylate content of a pool that is
in dynamic equilibrium with the processes that produce ATP
(glycolytic and mitochondrial systems) and the biosynthetic
processes that utilize ATP. During the course of germination,
following imbibition of water, ATP may not be turned over
at a constant rate. Increases or decreases in the turnover rate
would not be reflected in the concentrations of the pool
adenylates. Little attention has been extended to the determina-
tion of ATP turnover rates in plants. The turnover of high
energy phosphate bonds in germinating lettuce (Lactuca sativa
L.) seeds was estimated to be between 1 and 4/min from 0.5
hr after imbibition of water by mature seeds and the emergence
of the radicles at 15 hr (16).
The significance of energy charge to growth and develop-
ment of higher plants has not been evaluated either extensively
or intensively. The energy charge of quiescent seeds is ex-
tremely low (4, 7, 16), but increases rapidly when water is
imbibed. In the study reported herein, under conditions in
which the oxidative production of ATP was suppressed, either
by anaerobiosis or fluoroacetate, the energy charge remained
above 0.8. However, radicles either did not emerge or radicle
emergence was delayed. Apparently, under these conditions,
the ATP produced mainly through glycolysis was sufficient to
meet the energy demands required to sustain the tissue. The
energy charge dropped markedly when the seeds were treated
with iodoacetate, but only after 24 hr.
In unimbibed seeds, the relative concentrations of AMP,
ADP, and ATP vary considerably between species. In the
quiescent radish seeds used herein, ATP could not be detected
(<0.1 nmole), AMP was relatively high, and the ADP con-
centration was considerably lower than that of AMP. AMP
concentrations in unimbibed seeds of other species range from
extremely low levels to concentrations higher than those of
other adenylates. ADP is present uniformly at an intermediate
concentration, and ATP usually is quite low in concentration
(4, 6, 8, 16). In general, within the first few hr after imbibition
of water, the concentration of ATP exceeds that of either AMP
or ADP, the concentration of ADP remains at an intermediate
level, and AMP drops to extremely low levels (4, 6, 8, 16).
Many of the metabolic events activated by the imbibition of
water by quiescent seeds are common to the germination of all
seeds, however, some specific activities may be limited to cer-
tain seeds because of their particular morphology or the types
of stored food reserves. Some of the events follow a triphasic
time course that is characterized by a rapid initial increase
(phase 1), followed by a lag phase (phase 2), and subsequently
a sharp linear increase (phase 3). However, the time scale and
duration of each phase vary considerably with respect to
species, type of tissue, size of seed, and experimental con-
ditions. Triphasic time courses similar to those presented
herein also have been reported by other investigators for in-
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563
creases in fresh weight (9, 11, 13, 14, 16, 19, 21), respiration
(6, 15, 16, 18, 21, 22), and ATP (16). Uridine incorporation
into RNA also followed a similar triphasic response (10).
Acknowledgmiienzt-We are most appreciative for the use of the controlled tem-
perature germination chambers ma(le available ly Dr. R. J. Downs, Southeastern
Plant Environment Laboratory, Nortlh Carolina State University, Raleighl.
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