Isolation and Characterization of Peanut Spherosomes - Plant ...
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Plant Physiol. (1967) 42, 585-597
Isolation and Characterization of Peanut Spherosomes
T. J. Jacks,' L. Y. Yatsu, and A. M. Altschul
Seed Protein Pioneering Research Laboratory2, P. 0. Box 19687, New Orleans, Louisiana 70119
Received January 10, 1967.
Summary. Spherosomes of cotyledons of germinating peanuts (Arachis hypogea
L.) were examined by electron microscopy and found to be particles about 1.0 to 2.0 ,u
in diameter bounded by a limiting membrane. Isolated spherosomes appear similar
to spherosomes in situ. The isolated spherosomes are composed of 98.1 % total
lipids, 0.77 % phospholipid and 1.27 % protein by dry weight. The amounts of
protein and phospholipid associated with the isolated spherosomes are sufficient to
account for limiting membranes. Spherosomes amply account for the lipid in a peanut
cotyledon. The activity of lipase and fatty acyl-Coenzyme A synthetase is not
associated with the isolated spherosomes. This suggests that peanut spherosomes
are principal sites of lipid storage hut not of lipid degradation.
Tntracellutlar particles, called spherosomes (2), at 1.5,000 X g for 20 minutes to prodtuce a creamy
have been observed in plant cells by light and band or fat pad on the surface of the supernatanit
electron microscopy. The affinity of spherosomes liqluid, the sutpernatant liquid an(d a pellet. The fat
for lipophilic stains (6) and the relative abuindance pa(l was removed with a spatula and resuispende(d
of spherosomes in the storage tissuie of oil seeds in 15 ml of 0.25 M stucrose. This sulspension and
(22) in(licate that spherosomes contain lipid re- the suiperulatant liquiiid were centrifuged againi at
serves in planit cells. This paper presents evidence 1.5,000 X q for 20 minuites. The restultant pellets
that spherosoines in peanuit cotyledons are the were combined with the previouls pellet, resuispen(le(d
principal sites of lipid storage but appear lipoly- in 7 ml of 0.25 M sucrose and( designated the mito-
tically inert during germination. Isolated sphero- chond(rial fraction. The 2 liquid( supernatants were
somes were examined with the electron microscope also combined. The washed spherosomes (fat pad)
and sttudies of the morphology and chemical analvsis were resuspended in 7 ml of 0.25 M sucrose.
of the spherosomes are described. Spherosomal fractions prepared for chemical
analysis were washed with distilled water instead
Materials and Methods of the 0.25 -i sucrose.
Electron Microscopy. Isolated spherosomes were
Secd Germiniation. Peantut seeds (Arachis hy- embedded in an agar medium to facilitate manipu-
pogca L.), Virginia 56R variety, were soaked for lation through the fixation procedures. The spher-
3 houirs in a 0.1 % (w/v) suispension of Orthoside3 osomal fraction was mixed with a 2 % (w/v)
and germinated on wet paper toweling in the dark soltution of agar at 430 and centrifuged at 2600 X g
at 210. until the agar gelled. Half mm3 cuibes of the gel
Fractionation of Cotyledons. Cotyledons were which contained the spherosomes were removed
removed from the seedlings, rinsed with distilled from the centrifuge ttube and taken throuigh the
water and blotted dry. The cotyledons (22 g) were fixation process.
homogenized for 20 seconds with 55 ml of 0.25 M Fixation was accomplished by 1 of 3 procedlures.
sucrose. The mixtuire was strained and centrifuged The material was fixed in: i) aquleouis 2 % potas-
at 1000 X g for 5 minuites. The pellet was dis- siuim permanganate at 00 for 1 hour, ii) 2 % os-
carded an(d the suipernatant liquiid was centrifulged mium tetroxide solution in 0.1 Mi phosphate buiffer,
pH 7.2, overnight, iii) 6 % gluitaraldehyde in 0.3 M
sucrose containing 0.1 M phosphate buffer, pH 7.2,
I
Postdoctoral Research Associate. overnight. It was then serially dehydrated in
2 One of the laboratories of the Southern Utilization
Research and Development Division, Agricultural Re- aqueous acetone. The first 2 preparations were
search Service, United States Department of Agriculture. embedded in Maraglas (5). The dehydrated, glu-
3 Mention of trade names does not imply recommend- taraldehyde-fixed preparation was transferred to
ation by the Department over other equally suitable prod- hexane and rinsed thoroughly. This defatted ma-
ucts.
4The abbreviation used is: FACS, fatty acyl-CoA terial was then serially hydrated, fixed in 2 %
synthetase. Downloaded from on February 15, osmium tetroxide
2020 - Published in 0.1 M phosphate buffer, pH
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Copyright © 1967 American Society of Plant Biologists. All rights reserved.
585586 PLANT PHYSIOLOGY
7.2, for several hours, serially dehydrated again and free fatty acid in the reaction mixtures. The re-
embedded in Maraglas as above. action mixture (final vol 1 ml) contained 40
Thin sections were cut on the Servall Porter- ,Lmoles of acetate buffer, pH 4.6, 0.08 % Poly-
Blulm microtome using a diamond knife. The Tergent J-300 (non-ionic detergent), 2 % winter-
sections were stained in uranyl acetate and lea(l ized cotton seedI oil and enzyme preparation. Oil
citrate (19) an(d observed in a Phillips EM-200 was emulsifiecl with the detergent before addition
microscope. to the reaction mixture. The addition of 5 ml of
Chemical Analysis. The spherosomal and mito- extraction media (isopropanol, heptane, 1 N sulfuric
chondrial fractions were dried to constant weight acid, 4:1:0.2 respectively) to the incubating mix-
over P.05 in vacuio at room temperature. Whole ture stopped the enzymic reaction and the free
tissue was grolln(l with mortar ancl pestle uinder fatty acids were extracted by the double extraction
litquid nitrogenl to a fine powder before drying. procedure of Dole and Meinertz (4). The amount
TIhe drie(d samples (100 mg) were extracte(d 3 of extracte(d fatty acid was determined colorimetri-
times with 5 ml of chloroform-methanol (3:1) for cally (13). Formation of free fatty acids by the
30 minutes at 550 and twice with 3 ml of ether for enzyme preparation was a function of the period
2 minutes at room temperature. The chloroform- of incuibation an(d the amouint of enzyme prepara-
methanol and ether extracts were combined and the tioIn.
solvents were removed by evaporation in vacuo.
The weighits of the residue and lipid were deter-
mined after drying to constant weights in vacuo Results
over P2,05 and paraffin chips at room temperature.
Total lipid-phosphoruis was determined after Figure 1 shows a section of a peanut cotyledon
digestion of the sample by the method of Martin germinated for 1 day. The abundance of particles
and Morton (11). After digesting, 1.0 ml of H9O with diameters of 1.0 to 2.0 ,u is evident. They
was added to the liquid digest and the mixture held appear as electron-transparent bodies surrounded
for 30 minutes in a water bath at 900 to hydrolyze by electron-dense membranes and are identified as
any pyrophosphate formed during the digestion. spherosomes.
The phosphorus content was estimated by the The isolated spherosomal fraction is shown in
method of Allen (1). figure 2. The average diameter and staining char-
Nitrogen was determined by the procedure of acteristics of these spherosomes are similar to the
Minari and Zilversmit (12) ; protein was deter- spherosomes in the intact tissue (cf fig 1). A
mined by the miethod of Lowry et al. (9). small amouint of contaminating material was foutnd
Enzyme AXssays. Activity of FACS4 [acid: associated with the isolated spherosomes but this
CoA ligase (AMP), ECI 6.2.1.2] was determined material is not morphologically recognizable. Fig-
by measuring the formation of fatty acyl-CoA. ure 3 shows isolated spherosomes fixed with OS04.
The reaction mixtuires (final vol 1 mln) contained Since OS04 reacts with the unsaturated fatty acids
enzyme preparation, 60 umoles of tris-HCl buffer, of the lipid, the interiors of the spherosomes appear
pH 7.4, 6 ,lmoles of MgCI2, 6 jumoles of potassium electron-dense. Spherosomes fixed with 0s04 have
buityrate, pH 7, 1.2 ,umoles of CoA, pH 7, and 3 spherical shapes rather than the irregular forms
,umoles of potassium ATP [madle from disodium of spherosomes fixed with KM?1nO4. Figure 4 is
ATP using Dowex 50-X8 (HI) resin and neutral- an electron micrograph of spherosome "ghosts"
izing the effluent with dilute KOH]. The reaction fixed with OS04 afteritreatment of the spherosomal
was stopped by the addition of 3 ml of 95 % fraction with hexane. Only the electronl-dense,
ethanol to the incubating mixture, the mixtture was limiting membranes are evident.
filtered andl 0.5 ml of the filtrate was added to 2.0 The composition of the spherosomal and mito-
ml of 95 % ethanol. Formation of fatty acid-CoA chondrial fractions and of whole cotyledons is
was determined from the absorbance of the etha- shown in table I. The mitochondrial fraction is
nolic soluition at 232 m,u (17). Absorbance in- presented only for comparative puirposes; the data
creased linearly with increasing time of incubation obtained from whole cotyledons were uised to de-
an(l with increasing amounts of enzyme prepara- termine the effect of germination on their lipid
tion. The suibstitution of 6 jumoles of ammonium content and to calculate the amounilt of lipid in the
oleate for potassiutm butyrate gave results similar seed that is spherosomal.
to those obtained with potassium butyrate. The The average (Iry weight of the peanut cotyle-
presence of sodium ions in the reaction mixtuires dons decreases from 345 mg per cotyledonl before
was avoided because these ions may inhibit the germination to 143 mg per cotyledon after germi-
formation of fatty acyl-CoA in a manner similar nating 11 days. Since this corresponds to a 55 %
to their inhibitory effect on acetyl-CoA forma- decrease in lipid content per cotyledon during
tion (20). germination (cf table I), it appeared likely that
Activity of the acid lipase (glycerol-ester hy- spherosomes might be the intracellular site of the
drolase, EC 3.1.1.3) present in germinating peanuts enzymes associated with lipolysis in germinating
(18) was measured by determining
Downloaded the 15,
from on February increase of cotyledons.
2020 - Published Therefore, the activities of lipase and
by www.plantphysiol.org
Copyright © 1967 American Society of Plant Biologists. All rights reserved.JACKS ET AL.-SPHEROSOMES 587
.
.
t.w
'6
if
C.,.
r
L'C
N
\..
_
aw
i s.
'f:
Fic.. 1. Cotyledon of a peanut seed germinated 1 day and fixed in KMnO4. S, spherosome; A, aleurone grain; M
mitoehondria; CW, cell wall. X 22,000. In all figures the bar represents l,u.
Downloaded from on February 15, 2020 - Published by www.plantphysiol.org
Copyright © 1967 American Society of Plant Biologists. All rights reserved.JACKS ET AL.--SPHEROSONMES 589
k
I.1
;f
trIl " -
i
_
_
1 -..
FIG. 2. Spherosomal fraction fixed in KMnO4. X 16,D0.
Downloaded from on February 15, 2020 - Published by www.plantphysiol.org
Copyright © 1967 American Society of Plant Biologists. All rights reserved.JACKS ET AL.-SPHEROSOMES 591
'r
{
.,,
.9
.1
FIG. 3. Spherosomal fraction fixed in OSO4. X 24,000.
Downloaded from on February 15, 2020 - Published by www.plantphysiol.org
Copyright © 1967 American Society of Plant Biologists. All rights reserved.JACKS ET AL.-SPHEROSOMES 593
l4k.
1k' ,a.A.".
... 4.' "'. - -
I.
A
t. .Al
*J .
p .:;4~ il
4,.
C' ;A
'.4
- a.*
it I
_
* ,,
.W,.
a
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s' t_ IN: ..
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ip - t
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it II.i
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4
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FIG. 4. Spherosomal fractioln fixed in OSO4 after treating with hexane. X 23,000.
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Copyright © 1967 American Society of Plant Biologists. All rights reserved.JACKS E-,T AL.-SPHEROSOMNIE S 595
Table I. Cominposition of Sphw-osomal and Mitochlondrial Fractions and Whole Cotyledons of Peanut
Materials were prelpared anid analy zecd as descrihed under Materials and Methods. The l)eriod of germination
was 11 days. Values rep)resent averages of duplicate determiinationis on 2 samples and are given as percent of the
dry %xveight of recoverel mnaterialK'
Total Non-lipid Phospho-
MIa-terial lipidi residue Recoverv** liipid*** Proteint
UIJngermiinated cotyledons 48.3 51.7 97.2 1.07 28.6
Germinated cotyledons 52.5 47.5 103.2 1.54 24.3
Spll)erosomes 98.1 1.9 104.7 0.77 1.27
NI itoclhoindria 35.0 65.0 100.4 6.45 49.5
* Total lipid + noni-lipid residue = 100 %.
** Dry weight of sample (100 mg) = 100 %.
* Phospholipid - lipid phosplhorus X 24.8. Total phospholipid of peanut contains 4.04 % phosphorus (16).
t Protein =- noni-lipid nitrogeni X 5.46. Purified peanuit protein contains 18.3 % nitrogen (8).
Table II. Distribution of Catalytic Activity, Protein and Lipid in Cell Fractions
Fractions were prepared anlxd analyzed as described under Materials and Metlhods. Values represent averages of
2 or more determinations and are given as percent of recovered activity or of recovered protein. Values for the
distribution of lipid in germinating peanuts were calculated from the data of table I and the protein distribution of
this table.
Fattv
Fraction'* Lipase acyl-CoA Protein*" Lipid
Svintlhetase
Supernatant596 PHYSIOLOGY
Discussion lipase and FACS in spherosomes are not sufficient
to account for the decrease in lipid content which
occuirs dturing germination, whereas these activities
Peanutt spherosomes, isolated (figs 2 andl 3) or in tinfractionated homogenates amount to nearly
in situi (fig 1), appear morphologically similar to twice the required theoretical activity. Thtus it is
the corresponding particles in intact planit an(I apparent that spherosomes are the sites of lipi(d
animal cells (10, 14, 22). The compositioni of the storage, but not of lipid degra(lation.
particles isolatedl from peanuits (table I) or ctil- Although lipid and lipolytic activity are spatially
thired animal cells (10) is also similar. These separable by centrifugation in vitro (table II),
similarities suggest that spherosomes are common lipolysis occurs in vivo as shown by the (lecrease
t-o b)oth animal and( planit cells. in the amotunt of lipid per cotyledon during germi-
The magnitud(1e of the lipid content of peantit nation (see Results) as well as the decrease in the
spherosomes imparts a lowv density to these particles. ntumber of spherosomes per cell (14). This poses
In fact, since the bulk of peanut oil consists of the problem of how the lipolytic enzymes and the
oleate residues (7), the density of a spherosome spherosomes interact during germination for the
is estimated as 0.92. Spherosomes, therefore, mi- utilization of lipid.
grate to the axis of rotation dturing centrifugation
and form a distinct layer, or fat pad, above the
isolation mediuim (0.25 M suicrose). Literature Cited
The spherosomal membrane, shown as an elec-
tron dense botundary in electron micrographs (cf 1. ALLEN, R. J. L. 1940. The estimation of phos-
fig 2), accounts for the phospholipid and most of phorus. Biochem. J. 34: 858-65.
the protein associated with the spherosomes. Mac- 2. BUVAT, R. 1963. Electron microscopy of plant
kenzie et al. ( 10) have provided formulae to protoplasm. Intern. Rev. Cytol. 14: 41-155.
3. CHANG, Y. F., T. C. CHENG, AND H. Ho. 1963.
calculate the amounts of monolayered protein and Biochemical studies on fat and carbohydrate metab-
phospholipid needed to cover the suirface of vari-ous- olism during peanut seed germination. Taipei. Nat.
sized spheres. Using these formtulae and assuming Taiwan Univ. Coll. Agr. Mem. 7: 12-25.
a spherosomal density of 0.92, we calcullate that 4. DOLE, V. P. AND H. MEINERTZ. 1960. Microde-
spherosomes with diameters of 1.0 to 2.0 JU would termination of long-chain fatty acids in plasma
contain at least 0.75 to 0.35 % protein by weight, and tissues. J. Biol. Chem. 235: 2595-99.
respectively, and at least 1.15 to 0.58 % phospho- 5. ERLANDSON, R. A. 1964. A new Maraglas, D. E.
lipid by weight, respectively. The amouints of these R. 732, embedment for electron microscopy. J.
Cell Biol. 22: 704-09.
materials (table I) are sufficient to coat sphero- 6. FREY-WYSSLING, A. AND K. MijHLETHALER. 1965.
somes of 1.5 ,u diameter with 1 layer or 3.1 ,u Ultrastructural plant cytology. Elsevier Publish-
diameter with 2 layers of phospholipid. The ing Company, New York. 377 p.
amount of protein could coat spherosomes of 1.0 ,u 7. HOFFPAUIR, C. L. 1953. Peanut composition. Re-
diameter with 2 layers and larger spherosomes with lation to processing and utilization. Agr. Food
more than 2 layers of protein. Chem. 1: 668-71.
The formation of "ghosts" (fig 4) after treat- 8. JONEs, D. B. 1931. (Rev. 1941). Factors for con-
ing the spherosomes with hexane clearly demon- verting percentages of nitrogen in foods and feeds
into percentages of proteins. U. S. Dept. Agr.
strates the spherosomal membranes. The interior Circ. 183. 22 p.
of the spherosomes is electron-transparent after 9. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND
OsO4 fixation (cf fig 3) since the internal lipid of R. J. RANDALL. 1951. Protein measurement with
the spherosomes was extracted in the hexane. The the Folin phenol reagent J. Biol. Chem. 193: 265-
spherosomal membranes remained intact and appear 75.
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Ilt was calcuilated (table IT) that the sphero- AND D. E. PI-IILPOTT. 1966. Regulation of cell
somal fraction prepared from germinating cotyle-
lipid metabolism and accumulation. IV. The iso-
latioII -inld composition of cvtoplasmic lipid-rich
(ions accounts for the lipid present in the cotvledon. particles. Biochemistrv 5: 145461.
Apparently, then, spherosomes represent the prin- 11. MARTIN, E. M. AND R. K. MORTON. 1956. The
cipal site of lipid storage in peantut cotyledons. chemical composition of microsomes and mito-
Since more than half of the lipid content per chondria from silver beet. Biochem. J. 64: 221-
cotyledon is uitilized during germination, it seemed 35.
likely that the lipolvtic enzymes might be associated 12. MINAR1, 0. AND D. B. ZILVERSMIT. 1963. Use of
with the spherosomes. However, most of the lipase KCN for stabilization of color in direct nessleriza-
activity is associated with the mitochondrial frac- tion of Kjeldahl digests. Anal. Biochem. 6: 320-
27.
tion and the FACS activity with the supernatant 13. MOSINGER, F. 1965. Photometric adaption of
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FACS agrees with other findings obtained from Lipid Res. 6: 157-59.
germinating peanuts (15) and castor beans (21). 14. PALEG, L. AND B. HYDE. 1964. Physiological ef-
The relatively Downloaded
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scopy of barley a!eurone cells. Plant Physiol. Lipolysis and the free fatty acid pool in seedlings.
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