ARTICLE
Auteur(s) : Pascale Jolivet1,
Kathleen Tailliart1, Céline Boulard1,
Nathalie Nesi2, Thierry Chardot1
1UMR Inra Ina-PG Chimie Biologique, Centre de
Biotechnologie Agro-Industrielle, Thiverval-Grignon, 78850,
France
2UMR Inra AgroCampus Rennes Amélioration des Plantes et
de Biotechnologies Végétales, Domaine de la Motte au Vicomte, Le
Rheu cedex, 35653, France
Article reçu le 2 Juin 2006, accepté le 6 Octobre 2006
Introduction
Oleo proteaginous plants store lipids in oil bodies, composed of a
core of triacylglycerols surrounded by a monolayer of phospholipids
in which different proteins are inserted. Although the lipid body
is a relative simple organelle from a structural point of view, the
mechanisms of its biogenesis and degradation remain largely unknown
[2]. The increase of rapeseed economical value relies in improving
oil extraction while preserving the quality of protein by-products.
In fact, efficient oil extraction from rapeseed is rather
difficult, by comparison to other seeds (soybean, sunflower)
despite similar oil content than sunflower. The process involves
high temperature treatments and use of organic solvent. It is
important to identify molecular and cellular factors involved in
biogenesis of storage seed oil bodies to identify key factors for
the stability of these organelles and to develop milder methods for
extraction of rapeseed oil. Oil bodies must withstand extremes of
desiccation, rehydration, heating and cooling for months before the
storage oil can be mobilized following seed germination. Proteins
embedded in the phospholipid membrane form an effective resistant
surface for dormant and germinating seeds during the environmental
extremes [3].
Description of oil body-associated proteins of Arabidopsis
thaliana has been recently published [1]. The most abundant
proteins found in A. thaliana are (i) four oleosins (S1 to S4)
playing a structural role for stabilizing oil bodies, (ii) one
caleosin with an hypothetic role in oil body maturation by calcium
mediated fusion of microbodies, (iii) one sterol dehydrogenase. In
the case of rape, only few data are available. An average diameter
of 654 nm has been reported for oil bodies isolated from the mature
seeds of Brassica napus [4]. Two, then three, then four oleosins
have been described by Murphy’s team showing a higher similarity
each other [5-7]. Their transport from the site of synthesis on
ribosomes via the endoplasmic reticulum prior to their accumulation
on oil bodies has been also reported [8]. More recently, an oil
body-associated caleosin isoform has been reported in rape seeds
[9].
In this work, we describe the purification and characterization
of oil bodies from two double-low varieties of rape seeds, a
standard variety (Explus) and an oleic variety (Cabriolet). Their
protein complement has been analyzed the most exhaustively as
possible.
Material and methods
Seeds
Mature seeds of Brassica napus (hybrid Explus and variety
Cabriolet) were a generous gift of Monsanto (Saint-Louis, Missouri,
USA). They were characterized by their fresh weight, dry weight and
apparent volume estimated by counting the number of seeds filling
the same volume of 15 mL.
Oil body purification and characterization
Purification
Oil bodies were purified as described by Tzen et al. [10] by
floatation using six successive centrifugation steps (10,000 × g,
4 °C, 30 min) in a Kontron Ultracentrifuge equipped with
a swinging-bucket rotor. In a typical oil body preparation, 300 mg
of fresh weight of seeds were ground 3 times for 30 sec in
5 mL of 10 mM sodium phosphate buffer pH 7.5 containing 0.6 M
sucrose (buffer 1) with a glass Potter and a teflon plunger driven
by a Heidolph motor (rate 7). The sample was cooled on ice between
each grinding cycle, and the potter was rinsed by 5 mL of
buffer 1. The homogenate was overlaid by one volume of 10 mM sodium
phosphate buffer pH 7.5 containing 0.4 M sucrose (buffer 2) and
spun. The oil pad on top was collected and dispersed in 10 mL
of 5 mM sodium phosphate buffer pH 7.5 containing 0.2 M sucrose and
0.1% (v/v) Tween 20. The emulsion was overlaid by one volume of 10
mM sodium phosphate buffer pH 7.5 and spun. The oil body fraction
was resuspended in 10 mL of buffer 1 additionally containing 2
M sodium chloride, overlaid by one volume of 10 mM sodium phosphate
buffer containing 0.25 M sucrose and 2 M sodium chloride and spun.
The oil body fraction was resuspended in 5 mL of 7 M urea and
left on a shaker (60 rpm) at 20 °C for 10 min. Then the
suspension was placed in centrifuge tubes, overlaid by one volume
of 10 mM sodium phosphate buffer pH 7.5 and spun. The oil body
fraction was resuspended in 5 mL of buffer 1 and then mixed
for 10 min at 20°C with 5 mL of hexane. After
centrifugation and removal of the upper hexane layer, the oil body
fraction was collected and resuspended in 5 mL of buffer 1. In
a last step, the resuspension was overlaid by one volume of buffer
2 and centrifuged. The oil body fraction on top was collected (OB),
resuspended in a minimal volume of buffer 1 and stored a 4 °C
till further use.
Microscopy
Nile red (Molecular Bioprobe, Montluçon, France, solution at 1mg
mL–1 in acetone) was added to an aliquot of oil body
suspension (1/10, v/v). After 1 h incubation at 20 °C,
oil bodies were observed at 1000× focus through WIG or WB filters
(for fluorescence) with an Olympus BX 51 light microscope equipped
with 100 X oil immersion objective. Images were recorded using the
Photometrics Cool SNAP software. Oil bodies were also dropped on a
copper grid covered with a carbon film after negative staining
using 3% phosphotungstic acid and observed by electronic microscopy
(JEOL JEM-100S equipment operating at 80 kV under low
illumination).
Light scattering
Oil bodies were diluted in water (generally 10 μL of purified
fraction in 500 μL), and their hydrodynamic diameter was determined
using a Malvern HPPS particle sizer. We used a lipid particle
refraction index equal to 1.46, as determined by Michalski et al.
[11] for milk fat globules. Experiments were performed at
20 °C in a quartz cuvette. Measurements were repeated 3 to 10
times. Standard latex particles (200 nm, Nanospheres, Duke
Scientific, Palo Alto, USA) were dissolved in Milli Q grade
ultrapure water (Millipore Corp, Molsheim, France) and used to
check measurement accuracy.
Lipid extraction and quantification of seeds and oil
bodies
Overall fatty acid composition and quantity were determined by gas
chromatography. The method is based on the transmethylation of
acylated and free fatty acids [12].
One seed (near 5 mg) was ground in 1 mL of 2.5% (v/v)
sulfuric acid in methanol containing 100 μg of heptadecanoic acid.
The sample was heated for 90 min at 80 °C making sure
that no evaporation of solvent occurs. After the addition of 450 μL
of hexane and 1.5 mL of water, fatty acid methyl esters were
extracted into the organic phase by vigorous shaking. The tubes
were centrifuged at low speed (500 g for 5 min) to help the
two-phases separation. The organic upper phase was diluted (1/10)
and 1 μL was analyzed by gas chromatography using a Girdel 30
chromatograph with a moving needle-type injector at 250°C and a
bonded silica capillary column with a stationary phase of 90%
biscyanopropyl – 10% cyanopropyl siloxane (30 m x 0.25 mm
I.D., 0.25 μm film thickness, SP-2380 from Supelco,
Saint-Quentin Fallavier, France). The carrier gas was helium at an
inlet pressure of 1 bar. The column temperature program started at
120 °C, ramping to 210 °C at 3 °C/min. The flame
ionization detector was at 270 °C. Identification of fatty
acid methyl ester (FAME) peaks was based upon retention times
obtained for standards (Supelco). The total amount of fatty acids
was calculated from the ratio between the sum of FAME peak areas
and the heptadecanoic acid methyl ester peak area. Lipids from oil
bodies fraction were extracted according to Folch et al. [13],
after addition of heptadecanoic acid as internal standard and
subjected to methanolysis.
Protein analysis
Electrophoresis
Proteins were quantitated with the Folin Cioccalteu reagent [14]
using bovine serum albumin as standard. Protein aliquots from oil
body fraction were precipitated with 3 volumes of cold acetone at
-20°C for 2 h or overnight. The pellet was dried and
resuspended in a dissociation buffer consisting of 62.5 mM
Tris-HCl (pH 6.8), 10% (v/v) glycerol, 5% (v/v) 2-mercaptoethanol,
2% (w/v) sodium dodecyl sulfate (SDS) and 0.02% (w/v) bromophenol
blue. SDS-PAGE (polyacrylamide gel electrophoresis) of proteins was
carried out according to Laemmli [15], using 12% ready to use Nu
PAGE polyacrylamide gels (Novex, San Diego). Electrophoresis was
run under 100 V for 180 min using 50 mM MES
(2-[N-morpholino]ethane sulfonic acid) NuPAGE buffer (pH 7.3). Gel
was stained with Coomassie blue (G-250) according to Neuhoff and
Harold [16]. Molecular weights were estimated with Mark 12™
standard from Novex.
Gels were scanned (300 dpi) using an EPSON Perfection 1200 PHOTO
scanner, and the TIFF resulting gels were analyzed using the Image
Quant (version 4.2a) software (Molecular Dynamics).
Identification of oil bodies proteins
Protein bands stained with Coomassie blue were excised from the
polyacrylamide gel and stored at – 20 °C. Before trypsin
digestion, gel slices were washed for 5 min with water,
dehydrated for 15 min with acetonitrile and dried by vacuum
centrifugation. Proteins were reduced for 30 min with
150 μL of 10 mM dithiothreitol – 0.1 M ammonium
bicarbonate at 56 °C and alkylated in the dark with
100 μL of 55 mM iodoacetamide – 0.1 M ammonium
bicarbonate for 20 min at 20 °C. The gel pieces were
washed in 0.1 M ammonium bicarbonate, dehydrated with
acetonitrile and vacuum dried. Then proteins were digested
overnight at 37 °C with sequencing grade trypsin (EC 3.4.21.4,
Roche Diagnostics, Meylan, France) at the concentration of
12.5 mg L-1 in the presence of 25 mM ammonium
bicarbonate and 5 mM calcium chloride. The resulting peptides
were extracted successively with 5% formic acid (v/v),
acetonitrile/water (50/50, v/v) and acetonitrile. Combined extracts
were dried and samples were dissolved in 1% formic acid before
liquid chromatography-mass spectrometry analysis.
High performance liquid chromatography was carried out with a
Spectra System equipment (Thermo Separation Products, Riviera
Beach, USA) comprising a SCM1000 vacuum membrane degasser, P4000
gradient pumps and a manual injector. Volumes of 10 μL of samples
were loaded onto a reversed-phase BioBasic-18 column
(1 × 150 mm, 300 Å pore size, 5 μm film thickness,
Thermo Electron Corporation). The column was eluted at a flow-rate
of 0.1 mL min–1 at 20 °C with 5% of solvent B
(acetonitrile + 0.1% formic acid) in A (water + 0.1% formic acid)
for 2 min and then with a linear gradient of B in A from
5 to 45% over 40 min then 45 to 95% over 5 min before
re-equilibration. Eluant from the column was introduced in the
electrospray ionisation source of a Thermo Electron LCQ Deca
ion-trap mass spectrometer operating in positive ion mode.
Instrumental parameters were capillary temperature 280 °C,
capillary voltage 30 V, spray voltage 4.5 kV, sheath gas
flow 80 a.u., auxiliary gas flow 5 a.u. Mass spectra were
acquired scanning from m/z 200 to 2000. Ion fragmentation was
carried out using a normalized collision energy of
35 arbitrary unit. Peptide ions were analyzed using the
data-dependent “triple-play” method as follows: (i) full mass
spectrum scan, (ii) ZoomScan (scan of the major ions with higher
resolution to determine their charge), (iii) fragmentation of these
ions.
Protein identification was performed with Bioworks
3.1TM software using Arabidopsis thaliana and Brassica
napus protein sequence databases extracted from non-redundant
database downloaded from the National Center for Biotechnology
Information (NCBI) FTP site. A collection of Brassica napus ESTs
that has been generated from developing seeds (10, 15, 20, 25, 40,
45 days after pollination) and anthers in the framework of the
French plant genomics programme “Genoplante”
(http://www.genoplante.com) was also used during the progress of
this work. The collection consists of 39 373 ESTs that have been
arranged into 13 083 unique sequences after clustering and
contiging [17]. The unigene set obtained after version 1 of
contiging was used for similarity searches. No enzyme specificity
was set for the query. The database-searching algorithm
SequestTM uses a cross-correlation (Xcorr) and delta
correlation (dCN) functions to assess the quality of the match
between a tandem mass spectrum and amino acid sequence information
in a database. The output data were evaluated in term of (i)
trypsin nature of peptides, (ii) Xcorr magnitude up to 1.7, 2.2 and
3.3 for respectively mono-, di- and tri-charged peptides to
minimize false positives, (iii) dCN higher than 0.1.
Results
Characterization of rape seeds and purified oil bodies
Explus and Cabriolet seeds were characterized only by little
differences (table 1) in weight and
size. On the contrary, water content of Cabriolet seeds was
slightly higher. Oil bodies were purified from mature seeds and
observed under fluorescence or electronic microscopy (figure 1). They proved to
be constituted of spheres able to emit fluorescence light upon
incubation in the presence of Nile red. The size of oil bodies was
estimated using dynamic light scattering or electronic microscopy.
Oil bodies from Cabriolet seeds appeared systematically smaller.
Protein and fatty acid contents were measured showing a ratio fatty
acid/total protein near 1 in the mature seeds and a little
enrichment of oil bodies in fatty acid (× 2 to 3). Protein and fat
contents of seeds and oil bodies were slightly higher in the case
of the standard Explus variety.
Fatty acid composition was determined in seeds and in purified
oil bodies by gas chromatography of fatty acid methyl esters using
heptadecanoic acid methyl ester as a standard (table 2). It was verified that the seeds of
Cabriolet variety were enriched in oleic acid and diminished in
linoleic acid in regards to standard Explus seeds. The same fatty
acid composition was recovered in seeds and oil bodies.
Table 1 Characteristics of rape seeds and purified oil
bodies.
|
|
Explus rape
|
Cabriolet rape
|
|
Seed
|
Fresh weight (mg)
|
5.03
|
5.10
|
|
Estimated volume (mm3)
|
7.9
|
8.9
|
|
Water content (%)
|
8.7
|
12.0
|
|
Protein content (mg/mg)
|
0.45 ± 0.04
|
0.41 ± 0.02
|
|
Fat content (mg/mg)
|
0.43 ± 0.05
|
0.39 ± 0.02
|
|
Oil body
|
Hydrodynamic diametera (nm)
|
1095 ± 212
|
938 ± 119
|
|
Microscopy diameterb (nm)
|
1328 ± 404
|
812 ± 322
|
|
Protein content (μg/mg seed)
|
38.8 ± 3.5
|
33.8 ± 7.4
|
|
Fat content (μg/mg seed)
|
100.3 ± 2.3
|
95.7 ± 2.5
|
ahydrodynamic diameter was estimated using laser dynamic
light scattering.
baverage size was estimated using electronic
microscopy by measurements on near 60 oil bodies.
Table 2 Fatty acid composition of rape seeds and
purified oil bodies.
|
Explus
|
Cabriolet
|
|
Fatty acid
|
Seed
|
Oil body
|
Seed
|
Oil body
|
|
C16:0
|
4.87
|
4.62
|
3.83
|
3.80
|
|
C16:1 (n-7)
|
0.29
|
0.20
|
0.18
|
0.18
|
|
C18:0
|
1.82
|
1.60
|
1.79
|
1.58
|
|
C18:1 (n-9)
|
61.47
|
61.82
|
73.48
|
73.87
|
|
C18:2 (n-6)
|
20.52
|
20.44
|
9.28
|
9.12
|
|
C18:3 (n-3)
|
9.61
|
9.79
|
9.91
|
9.97
|
|
C20:0
|
0.40
|
0.46
|
0.41
|
0.40
|
|
C20:1 (n-9)
|
1.02
|
1.07
|
1.12
|
1.08
|
Protein composition of purified oil bodies
The proteins contained in the oil body fraction obtained through
the last step of preparation (see Material and Methods) were
analyzed by denaturing polyacrylamide gel electrophoresis (figure 2). The two
protein patterns were similar for Explus or Cabriolet seeds. Only
six different protein bands were clearly visible within the 15-70
kDa range after Coomassie blue staining. Upon scanning of gels and
image analysis, an approximate quantification on the basis of band
intensity was obtained (table 3). The
protein bands were identified through the analysis of their trypsin
peptides with liquid chromatography-tandem mass spectrometry. As
Brassica napus genome was not completely sequenced and accessible
as public databases, they were identified in part through homology
with Arabidopsis thaliana proteins. All the proteins were
identified without ambiguity and with a high coverage of the
protein sequence. The major proteins present in rape oil bodies
were very homologous to integral proteins previously described in
A. thaliana oil bodies [1]. Interrogation of ESTs database from B.
napus immature seeds (Genoplante cDNA library) gave the possibility
to determine the real protein sequences in B. napus. Eight oleosins
were identified in rape oil bodies. One could be distinguished from
the others due to its slightly higher apparent molecular mass of 22
kDa (band 5). This oleosin (contig accession number 155968 in the
Genoplante database) corresponded only to a partial protein
sequence which presented 61% identity and 67% similarity with the
N-terminal part of oleosin S4 from A. thaliana and possessed 17
supplementary amino acids. Protein band 6 showing an apparent
molecular mass of 17 kDa was the major band and appeared to contain
7 oleosins the molecular mass of which was comprised between 19.3
and 21.5 kDa. Four oleosins (BnIII, BnV, napII and contig 155846)
presented 82.5 to 86.7% identity with A. thaliana oleosin S3, one
oleosin (contig 156118) presented 80.8% identity with A. thaliana
oleosin S1 and two oleosins (contig 156532 and contig 156533)
presented 77.1 to 80.3% identity with A. thaliana oleosin S2.
Besides oleosins, a protein highly similar to the
11-β-hydroxysteroid dehydrogenase-like protein described in A.
thaliana was identified. However, only a partial sequence was
recovered from ESTs database (235 amino acids against 349 amino
acids for A. thaliana protein). This partial sequence corresponding
to the N-terminal part of the protein presented 89.8% identity and
96.2% similarity with A. thaliana protein.
Finally, the presence of myrosinase-binding protein,
myrosinase-associated MyAP5 and β-glucosidase indicated a low
contamination of oil body fraction with protein body. However, no
storage protein (cruciferins) was identified.
Table 3 Identified proteins in oil bodies purified from
Brassica napus mature seeds.
|
Band
|
Relative intensity (%)
|
Identified protein
|
Apparent molecular weight (kDa)
|
Sequence molecular weight (kDa)
|
Sequence coverage (%)
|
pI
|
A. thaliana orthologues
|
|
1
|
2.6
|
Myrosinase-binding protein
|
88
|
99.404
|
6.8
|
5.5
|
|
|
2
|
6.4
|
β-glucosidase
|
62
|
56.283
|
5.8
|
6.2
|
At3g03640
|
|
3
|
4.4
|
Myrosinase-associated MyAP5
|
46
|
41.797
|
31.3
|
8.5
|
|
|
4
|
9.4
|
11-β-hydroxysteroid dehydrogenase-like
|
40
|
40
|
22.1
|
6.1
|
At5g50600
|
|
5
|
10.7
|
contig 155968
|
22
|
23.024
|
28.6
|
9.1
|
At5g40420/S4
|
|
6
|
64.0
|
BnV
|
17
|
20.286
|
49.7
|
9.2
|
At4g25140/S3
|
|
|
BnIII
|
|
21.540
|
19.0
|
9.3
|
At4g25140/S3
|
|
|
NapII
|
|
19.349
|
53.7
|
9.6
|
At4g25140/S3
|
|
|
contig 155846
|
|
19.515
|
47.2
|
9.2
|
At4g25140/S3
|
|
|
contig 156118
|
|
20.777
|
23.8
|
8.1
|
At3g01570/S1
|
|
|
contig 156532
|
|
20.002
|
46.3
|
9.1
|
At3g27670/S2
|
|
|
contig 156533
|
|
19.880
|
25.5
|
6.9
|
At3g27670/S2
|
Discussion
The oil body preparation used repeated and numerous floatation
steps. Proteins non-specifically associated with oil bodies were
removed by detergent washing, ionic elution and finally urea
treatment. Triglycerides from defective oil bodies were removed by
hexane extraction. So, only few proteins were visible from oil body
fraction and the contamination with proteins involved in the
glucosinolate metabolism was relatively low. Seeds and oil body
fraction had relatively high protein contents compared to other
results. Murphy and Cummins [3] have reported a ratio fatty
acid/total protein near 5.7 for oil bodies but they have noticed
that total protein was assayed by three different methods and that
protein recoveries were relatively low and variable. In fact, they
have carried out protein determination on delipidated oil bodies
and we have verified in that case a poor protein recovery.
Eight oleosins were identified in purified oil bodies from rape.
These oleosins show a high similarity with A. thaliana oleosins.
However, oppositely to A. thaliana oleosins, they were not resolved
under SDS-PAGE due to their very similar molecular mass, except for
rape oleosin S4. Rape oleosins were always heavier than the A.
thaliana corresponding ones except for oleosin S2 the molecular
mass of which was similar for B. napus and A. thaliana. Only four
oleosins were described in rape by Murphy’s team [5, 18-20]. BnV,
BnIII and napII were recovered in our experiments and it appeared
that EST 156533 corresponded to napI described only partially by
Murphy [18]. Then we observed four supplementary oleosins. Brassica
napus is a natural hybrid between B. oleracea and B. campestris and
in consequence, it is not surprising that B. napus contains many
more oleosins than A. thaliana the genome of which is the simplest
in the cruciferae family.
Oleosins contain three distinct structural domains, a central
hydrophobic anchoring domain, highly conserved and containing no
cleavage site for trypsin digestion, and two N- and C- terminal
amphipathic domains. Recovered peptides through liquid
chromatography-tandem mass spectrometry analysis belonged to the
two amphipathic domains of the oleosins. The N-terminal part of B.
napus oleosin S4 was enriched in glycine: 20 glycine residues,
among 64 amino acids for N-terminal part, instead of 11 glycine
residues, among 47 amino acids for N-terminal part in A. thaliana
oleosin S4. This fact increases the hydrophobic character of
N-terminal domain of rape oleosin S4.
A protein highly homologous to sterol dehydrogenase from A.
thaliana was identified. It is also highly similar to Sesamum
indicum sterol dehydrogenase sop2 (65% identity and 83% similarity)
and at a minor degree with sop3 (46% identity and 71% similarity)
[21]. This protein was never described in rape. It will be
interesting to characterize the kinetic properties of B. napus
sterol dehydrogenase, determine its specificity using labeled
steroids as model substrates and classify this protein among the
steroid dehydrogenases family.
Oleosins, the major proteins of oil bodies (75%), were poor
candidates for bidimensional (2D) electrophoresis due to their very
alkaline pI and their low solubility in aqueous medium. However,
oleosin corresponding to contig 156533 has a lower pI. So, it will
be interesting to attempt to separate oleosins using 2D
electrophoresis. To date, no caleosin was identified in our
experiments using proteomic technique. However, ESTs database
reveals the presence of one gene encoding at least one protein very
homologous to A. thaliana caleosin. The use of specific antibody
could be considered to verify this hypothesis.
Acknowledgments
Authors wish to thank C. Gaillard (UR, Biopolymères, Interactions
et Assemblages, INRA, Nantes) for electronic microscopy
measurements, Genoplante for free access to the rapeseed ESTs
database and O. Lapierre and D. Tristant (Centre d’Etude et de
Recherche sur l’Economie et l’Organisation des Productions
Animales) for the gift of rape seeds and financial support of a
part of this work through the project « Produire du lait
autrement ».
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