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Biochemical Systematics and Ecology, Vol. 15, No. 5, pp.
551-558, 1987. 0305-1978/87 $3.00+0.00 Printed in Great Britain.
Pergamon Journals Ltd.
Activities and Subcellular Localization of Enzymes Responsible
for Lipolysis and Gluconeogenesis during the Germination of
Brassica campestris cv. 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 mito- chondria, 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 sepa- rate 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 synthe- tase;
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
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552 NIEVES VILLALOBOS, FERNANDO SIMON, LUISA MARTIN, MAITE
HERRERA AND GREGORIO NICOLAS
[
0,8
~ 06
~04
0.2
0
2:
v I
c0 .5
SC s~- e ~''e
" J ' ' " ' , , , . 7 "~'' (a) ~,"
is
Lipase (pH 5)
(b)
A \ Lipase ',pH 7)
(c)
Lipase
20 ;pr ig) / X (d )
/ \
o I-$-- I I l I I I I ] 2 3 4 5 6 7 8 9 I0
Age of 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 ger- mination 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 particu- larly active between days
5 and 8 and shows maximum activity on day 6 of the process. These
facts are in agreement with the results
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 dif- ferent oleaginous species [5, 6]. During the
germination of turnip seeds, catalase activity (Fig. 5A), present
at very low levels in un- germinated 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
c
.," \
I I 4 5
pH
FIG. 2. ACID LIPASE ACTIVITY AS A FUNCTION OF pH. Seeds germi-
nated for one day.
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GERMINATION OF BRASS~CA CAMPESTR/S SEEDS 553
c
20 --
IO --
O
e /'.% "l /. \.
/ \
I I 8 9
pH FIG. 3. ALKALINE LIPASE ACTIVITY AS A FUNCTION OF pH, Seeds
germinated for six days.
c~
0 ,5 - -
6 7 8
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 acon- itase (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 consider- able
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 gluconeo- genesis, such as
glycolate oxidase (Fig. 5b), fumarase and phosphoenol pyruvate
carboxy- kinase (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 mobiliza- tion 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 centri- fugation were the
following: spherosomes, floating on the upper part; a soluble
fraction, immediately under the layer of spherosomes; a membrane
fraction (using cytochrome reduc- tase 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 gly- oxysomes, 8.07% in the mitochondria; 1.8% in
the membrane fraction and 22.22% in the
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554 NIEVES VILLALOBOS, FERNANDO SIMON, LUISA MARTIN, MAITE
HERRERA AND GREGORIO NICOLAS
v / H
4 --
2 o
o L)
O -
v3c cz I --
03 L)
i -
v
"E o 0 .5 - O c (.}
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GERMINATION OF BRASSICA CAMPESTRIS SEEDS 555
soluble fraction. This high percentage of lipase activity in the
soluble fraction may be indica- tive 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 glyoxy- somes (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 pre- senting its highest
activity in the membrane fraction (31.21%) was active in the
glyoxy- somes (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.
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 accom- panied 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 sec- tions
of cotyledons at 7 days of germination, highlights the appearance
of microbodies inti- mately 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 CHCI 3 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 per- formed
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-dilino- lein 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 phenylhydra- zone at 324 nrn was
determined [23].
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556 NIEVES VILLALOBOS, FERNANDO SIMON, LUISA MARTIN, MAITE
HERRERA AND GREGORIO NICOLAS
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 un-
germinated 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 glutaralde- hyde 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 over- night. 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 con- taining 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.
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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.
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558
FIG. 8. MICROPHOTOGRAPH OF A SECTION FROM A COTYLEDON OF A
TURNIP SEED GERMINATED FOR SEVEN DAYS. LB--lipid body; CW-cell
wall; MB-microbody; PL--plastid.