Biochemical Systematics and Ecology, Vol. 15, No. 5, pp. 551-558, 1987.

Printed in Great Britain.

0305-1978/87 $3.00+0.00
PergamonJournals Ltd.

Activities and Subcellular Localization of Enzymes Responsible for

Lipolysis and Gluconeogenesis during the Germination of
Brassica campestriscv. esculenta Seeds
NIEVES VILLALOBOS, FERNANDO SIMON, LUISA MARTIN, MAITE HERRERA and
GREGORIO NICOLAS
Department of Plant Biology, Faculty of Biology, University of Salamanca, 37008 Salamanca, Spain

Key Word Index--Brassica campestris cv. escuienta; turnip seed germination; lipolysis; gluconeogenesis.
Abstract--During the growth of turnip seedlings, two new lipases have been demonstrated, one with a maximum
activity at pH 4.5 (acid lipase) and the other with a maxima at pH 8.6 (alkaline lipase). Many different enzymes are
involved in gluconeogenesis: catalase, isocitrate lyase, malate synthetase, malate dehydrogenase, aconitase, citrate
synthetase, fumarase, glycolate oxidase, phosphoenol-pyruvate carboxykinase. All of these show maximum activity
coinciding with the stage in which lipid hydrolysis is maximal and when the accumulation of soluble carbohydrates has
also reached its peak. The alkaline lipase as found to be located mainly in the spherosomes, whereas the glyoxysomes
contained the following main activities: catalase, isocitrate lyase, malate synthetase, malate dehydrogenase and citrate
synthetase. Aconitase, together with cytochrome oxidase and fumarase showed their highest activity in the mitochondria, and the presence of malate dehydrogenase, citrate synthetase and glycolate oxidase was also observed in
these organelles. In the membrane-bound fraction, the activities of cytochrome reductase, glycolate oxidase and
phosphoenol-pyruvate kinase were marked, although the latter enzyme was even more active in the soluble fraction.

Introduction

During the germination of oleaginous seeds,

fats are rapidly converted into sugars [1]. This
conversion involves many enzymes and
suggestions have been made concerning the
possible existence of a different subcellular
compartmentalization
for
the
enzymes
involved in both lipolysis and gluconeogenesis.
Ultrastructural studies of cells during different
stages of the germination process have
revealed a decrease in the number of lipid
bodies per cell. This fact suggest that some
lipid bodies, mainly those adjacent to the
glyoxysomes, are degraded before others [2].
Gluconeogenesis is divided mainly into separate compartments: (a) lipid bodies; (b)
glyoxysomes; (c) mitochondria; and (d) the
cytosol. The present work describes the results
obtained in the study of some of the enzymes
involved in the transformation of triglycerides
Abbreviations: IL, isocitrate lyase; MS, maiate synthetase; MDH, malatedehydrogenase: CS, citrate synthetase.
(Received 29 March 1987)

into soluble carbohydrates necessary for the

processes of growth and development of the
embryonic axis in turnip seeds, An attempt
was also made by separating the organelles, to
discover the subcellular location of the
different enzymes acting during lipolysis and
gluconeogenesis in the germination of these
seeds.
Results and Discussion

Lipolysis during the germination of turnip seeds

In ungerminated seeds, approximately 50% of
the total dry weight is accounted for by lipids,
whereas the soluble carbohydrates only
formed 22%. During germination, the lipid
content of the seed decreased gradually,
coinciding with an increase in the amount of
soluble carbohydrates (Fig. la). This is in
accordance with findings reported by other
authors [3] for jojoba seed cotyledons.
It has been well documented that oleaginous
seeds contain lipase(s) that are manifested
during germination [4]. Two kinds of lipase
have been described in turnip seeds: an acid
lipase (Fig. l b) present in dry seeds, whose
551

552

NIEVES VILLALOBOS, FERNANDO SIMON, LUISA MARTIN, MAITE HERRERAAND GREGORIO NICOLAS
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found concerning the mobilization of lipids,

since their faster hydrolysis coincides with the
period of activity of the alkaline lipase which,
as shown in Fig. 1 could be considered to be
the main catalysing enzyme in the lipolysis of
reserve triglycerides.
On studying the optimum pH for activity of
these enzymes (Fig. 2), the acid lipase was
seen to show maximum activity at a pH of 4.5,
whereas for the alkaline form it was 8.6 (Fig. 3).
The neutral lipase showed slight activity at pH
6.5 (Fig. 4), though this could be considered
negligible compared with the activities of the
other two enzyme forms (Fig. lc).

Variation during germination of the enzymes

in valved in gluyconeogenesis
The process of lipid mobilization involves
numerous enzymes. Among them catalase, a
glyoxysomal enzyme that participates in the
13-oxidation of fatty acids, has been used as a
marker to indicate the development of
gluconeogenesis from reserve lipids in different oleaginous species [5, 6]. During the
germination of turnip seeds, catalase activity
(Fig. 5A), present at very low levels in ungerminated seeds, increases until a maximum
was reached during days 3-5; after this it
decreased until a very low level was present in
the last three days of germination. This finding

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I I l I I I I
2

3 4 5 6

Age of

7 8 9 I0

seedling (days)

FIG. 1. CHANGES IN COMPOSITION (a) AND LIPASE ACTIVITY (b, c

and d) DURING THE GERMINATION OF TURNIP SEEDS. Enzymatic
values expressed as units/seed. SC-Soluble carbohydrates;
L--Lipids.

activity increases during the first 24 h of germination and then declines throughout the rest
of the germination process, and an alkaline
lipase (Fig. ld) which can be detected after the
third day of germination; this form is particularly active between days 5 and 8 and shows
maximum activity on day 6 of the process.
These facts are in agreement with the results

.,"

5
pH

FIG. 2. ACID LIPASE ACTIVITYAS A FUNCTION OF pH. Seeds germinated for one day.

GERMINATION OF BRASS~CA CAMPESTR/SSEEDS

553

/'.%

"l
e

20 --

IO --

/. \.

pH
FIG. 3. ALKALINE LIPASE ACTIVITY AS A FUNCTION OF pH, Seeds
germinated for six days.

c~
0,5--

pH
FIG. 4. NEUTRAL LIPASE ACTIVITY AS A FUNCTION OF pH, Seeds
germinated for four days.

is very similar to what was reported for cotton

[7] and castor bean seeds [8].
Isocitrate lyase, malate synthetase and aconitase (Fig. 5) developed their activities during
germination in a way similar to that described
in ref. [9], since they were undetectable or
present at very low levels only when the seeds
had not begun the imbibition period. At 24 h of
germination, it was already possible to note a

small increase in isocitrate lyase and malate

synthetase activities; these continued to rise
gradually until maxima were reached during
days 3-5, in the case of IL, and days 4-6 in the
case of MS (Fig. 5b). Later, the activity of these
two enzymes began to fall and continued to do
so until the end of the process, although it
should be noted that there was still considerable activity up until the eighth day. Similar
activity profiles were observed for citrate
synthetase and malate dehydrogenase (Fig. 5)
which had maximum activities over days 3-5
(CS) and 4-6 (MDH).
Other enzymes participating in gluconeogenesis, such as glycolate oxidase (Fig. 5b),
fumarase and phosphoenol pyruvate carboxykinase (Fig. 5c), had maximum activities, like
the above-mentioned enzymes, over days 3-6
of germination.
A relevant aspect of all these findings is that
all the enzymes participating in the mobilization of reserve lipids show considerable
activity during the days when lipid hydrolysis
was most pronounced; coinciding with this an
accumulation
of
soluble
carbohydrates
occurred. These facts demonstrate that in
turnip seeds there is active gluconeogenesis,
as demonstrated by the presence during
germination of all the enzymes participating in
the process.
Subcellular localization of the different
enzymatic activities
The subcellular fractions obtained by centrifugation were the following: spherosomes,
floating on the upper part; a soluble fraction,
immediately under the layer of spherosomes;
a membrane fraction (using cytochrome reductase as marker) in the interphase between
1.10-1.13 g cm-3; a mitochondrial fraction
(using cytochrome oxidase as marker) at a
density of 1.19 g cm -3 and the glyoxysome
fraction (using catalase as marker) at a density
of 1.28 g cm -3.
When the several enzyme activities were
assayed using the different cellular fractions
(Table 1), most of the alkaline lipase activity
was found in the spherosomes (58.61%),
whereas only 8.98% was found in the glyoxysomes, 8.07% in the mitochondria; 1.8% in
the membrane fraction and 22.22% in the

554

NIEVES VILLALOBOS, FERNANDO SIMON, LUISA MARTIN, MAITE HERRERA AND GREGORIO NICOLAS

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(days)

FIG. 5. DIFFERENT ENZYMATIC ACTIVITIES MEASURED IN HOMOGENATES OF TURNIP SEEDS DURING GERMINATION. Values are expressed as
units/seed.

TABLE 1. SUBCELLULAR LOCALIZATION OF THE ENZYMES INVOLVED IN GLUCONEOGENESIS

Enzyme

Alkaline lipase
Catalase
Cytochrome oxidase
Cytochrome reductase
Isocitrate lyase
Malate synthetase
Malate dehydrogenase
Aconitase
Citrate synthetase
Glycolate oxidase
Fumarase
Phosphoenol pyruvate carboxykinase

Glyoxysomes
nKat
(%)
4.42
1640
0.24
0.42
1.17
4.16
1.95
0.24
0.58
0.16
0.04
0.02

(8.98)
(82)
(1.2)
(1.5)
(73.12)
(82.53)
(39.39)
(20.17)
(38.92)
(14.9)
(2.35)
(1.55)

Mitochondria
nKat
(%)
3.97
40
16.36
3.69
0.11
0.67
1.99
0.64
0.41
0.22
1.04
0.31

(8.07)
(2.0)
(81.8)
(13.18)
(6.87)
(13.29)
(40.2)
(53.78)
(27.51)
(20.5)
(61.17)
(24.03)

Enzymatic activities are expressed as Kats/seed on a 'per 100 seed basis'.

Subcellular fraction
Membrane fraction
nKat
(%)
0.89
34
2.26
12.36
0.74
0.11
0.54
0.11
0.19
0.41
0.19
0.41

(1.8)
(1.7)
(11.3)
(45.12)
(8.75)
(2.18)
(10.9)
(9.24)
(12.75)
(38.21)
(11.17)
(31.7)

Soluble fraction
nKat
(%)
10.93
164
1.14
11.25
0.13
0.05
0.47
0.20
0.18
0.27
0.43
0.55

(22.22)
(13.2)
(5.7)
(40.18)
(8.12)
(0.99)
(9.49)
(16.8)
(12.0)
(25.16)
(25.29)
(42.63)

Spherosomes
nKat
(%)
28.82
22
--0.05
0.05
--0.13
0.013
---

(58.61)
(1.1)
--(3.12)
(0.99)
--(8.72)
(1.2)
---

GERMINATIONOF BRASSICACAMPESTRISSEEDS

soluble fraction. This high percentage of lipase

activity in the soluble fraction may be indicative of breakage, during fractionation, of some
spherosomes since when their own lipase was
used as substrate there was clear autolytic
activity. The existence of high activity in lipid
bodies has already been reported in jojoba
seeds [10] and in ref. [11] working with
soybean seeds, who found maximum lipase
activity in the soluble fraction; these authors
postulated that its origin was mainly from the
glyoxysomes.
Catalase was the enzyme showing the
highest activity and almost all of it (82%) was
present in the glyoxysomes. Similar results
concerning
glyoxysomal
activity
were
observed for the isocitrate lyase with 73.12%
and for the MS, with 82.52%. However, the MDH
and CS, typical enzymes of the glyoxylate
cycle, showed similar activity in the glyoxysomes (39.39% for the MDH and 39.92% for
the CS) and in the mitochondria (40.2% and
27.51% for the MDH and CS respectively).
Aconitase, fumarase and cytochrome oxidase
had their highest activities in the mitochondria,
although a fairly significant activity of all these
enzymes was also seen in the soluble fraction.
Outstanding too was the level reached by
aconitase in the glyoxysomes; this was to be
expected in view of the fact that it is one of the
enzymes associated with the glyoxylate cycle.
Glycolate oxidase, although apparently presenting its highest activity in the membrane
fraction (31.21%) was active in the glyoxysomes (14.9%), the mitochondria (20.5% and
the soluble fraction (25.16%).
However, cytochrome reductase, used as
marker of the membrane fraction--since it is
mainly bound to the endoplasmic reticulum-[12] showed a similar activity in the soluble
fraction (40.18%), which seems to suggest that
during fractionation the breakage of some
microbodies occurs. Such findings coincide
with the results of ref. [12] working with jojoba
seeds; these authors reported the existence of
a certain activity associated with lipid body
membranes. Finally, the phosphoenol pyruvate
carboxykinase was located mainly in the
soluble fraction (42.63%) though its activity
was lower in the membrane fraction (31.7%)
and in the mitochondria.

555

Studies with the electron microscope

In resting seeds and during the first days of
germination, the cell were almost completely
occupied by lipid bodies (Fig. 6). From the day
3 of germination onwards, a pronounced
decrease occurred in these lipid bodies accompanied by the appearance of vacuolated zones.
This situation persisted during the remaining
days of germination studied (Fig. 7). These
results seem to confirm the existence of active
lipid metabolism during the germination of
these seeds. Figure 8, corresponding to sections of cotyledons at 7 days of germination,
highlights the appearance of microbodies intimately related to lipid metabolism [13, 14].
During the other days of germination, it cannot
be said that such microbodies do not appear
but rather that they are masked by the large
amount of lipids present.

Experimental
Plant material. The plant material employed for all the
experiments was turnip seeds (Brass/ca campestris cv.
esculenta). The seeds were germinated and grown on a
glass plate covered with filter paper in the darkness at 25
and 80% RH. All seeding operations were carried out in a
sterile chamber after previously sterilizing the materials to
be used with hypochlorite and UV light.
Analysis of tota/ /ip/ds and soluble carbohydrates. Lipids
were extracted according te the method ef ref. [15]. Total
lipids were determined by drying an aliquot of the CHCI3
extract in a vacuum oven overnight and weighing the lipid
residue. Soluble carbohydrates were extracted, after
rerneving the lipids, with 80% EtOH by the anthrone
method [16]. Proteins were assayed by the method of ref.
[17].
Preparation of enzymatic extracts. All stages were performed between O and 5. To obtain the different enzymatic
extracts the method described in ref. [14] was employed,
using cotyledons obtained at different germination times.
Preparation oforganelles. This procedure was carried out
according te the method of ref. [18].
Enzymatic assays. Two different assays were used for the
lipase: the fluorimetric method [19], with the modifications
introduced in ref. [20], using N-methyl-indoxylimyristate as
substrate and a temperature of 24 and the colorimetric
method [21], using tripalmitin, triestearin, triolein, 1,3-dilinolein and monolinolein as substrate; these were emulsified
in 5% gum arabic for 30 sec in a ultrasonic generator. The
effect of pH on lipase activity was studied using the
following as buffers: succinate hydrochloride (pH 4-6),
imidazoI-HCI (pH 6-7), Tris-HCI (pH 7-9) and glycine-NaOH
(pH 9-10).
The isocitrate lyase assay was performed by measuring
the formation of glyoxylate phenylhydrazone [22]. The
molar extinction coefficient of the glyexylate phenylhydrazone at 324 nrn was determined [23].

556

NIEVESVILLALOBOS, FERNANDOSIMON, LUISA MARTIN, MAITE HERRERAAND GREGORIONICOLAS

Malate synthetase activity (EC 4.1.3.2) was measured by the

method of ref. [22]. Catalase activity (EC 1.11.1.6)was assayed
by the method of ref. [24]. Similarly, the following were
assayed by their respective methods: aconitase (EC 4.2.1.3)
[25]; malate dehydrogenase (EC 1.1.1.37) [26]; citrate
synthetase [27]; fumarase (EC 1.1.3.1) [15] and phosphoenol
pyruvate carboxykinase [28]. Cytochrome oxidase was
determined photometrically [29] and cytochrome reductase
by the method of ref. [3].
Preparation of tissue for electron microscopy. Both ungerminated cotyledons and cotyledons germinated for 10
days were used. The organs were cut into small blocks and
fixed: the pieces were submerged in 3% glutaraldehyde in
0.05 M phosphate buffer, pH 6.8, in a test tube. The tubes
were subjected to a vacuum so that after allowing air to
enter, thus raising the pressure, penetration of glutaraldehyde would be facilitated and the substance would replace
the air remaining between the cells. This operation was
repeated several times until the sample remained stationary
at the bottom of the tubes, showing that they were totally
impregnated with glutaraldehyde. Following this, the
samples were washed 3 0.05 M phosphate buffer pH 6.8.
Fixing was performed in 1% osmium tetroxide in phosphate
buffer for 2 h. The samples were then washed again with
phosphate buffer for 10 min and finally with H20 for 20 min
each wash.
All the samples were placed in a 1.5% agar solution,
dehydrated in a graded acetone series and embedded in
Spurr resin [30]. Polymerization was performed at 60 overnight. Thin sections for electron microscopy were cut using
a LKB ultramicrotome. After being sectioned the samples
were mounted on formvar-coated slot grids. The grids containing the thin sections of the samples were first stained
with 2% uranyl acetate for 20 min at 20; they were then
washed with H20 and stained again with lead citrate.
Observations were made with a Philips EM-300 electron
microscope.

References
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Biophys. 194, 422.
4. Opute, F. I. (1975) J. Exp. Botany 26, 379.

5. Beevers, H. (1969) Ann. NYAcad. $cl, 168, 313.

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557

FIG. 6. MICROPHOTOGRAPH OF A SECTION FROM A COTYLEDON OF A TURNIP SEED GERMINATED FOR ONE DAY. LB--lipid body; GIN--cell
wall.

FIG. 7. MICROPHOTOGRAPH OF A SECTION FROM A COTYLEDON OF A TURNIP TOP SEED GERMINATED FOR SIX DAYS. LS--lipid body;
CW--cell wall; V--vacuole.

558

FIG. 8. MICROPHOTOGRAPHOF A SECTION FROM A COTYLEDON OF A TURNIP SEEDGERMINATEDFOR SEVENDAYS. LB--lipid body; CW-cell
wall; MB-microbody; PL--plastid.