RESEARCH ARTICLE

Protein composition of oil bodies from mature Brassica

napus seeds

Pascale Jolivet1,2, Celine Boulard1,2, Annick Bellamy3,4,5�, Colette Larre6, Marion Barre6,Helene Rogniaux6, Sabine d’Andrea1,2, Thierry Chardot1,2 and Nathalie Nesi3,4,5

1 NRA, UMR 206, Chimie Biologique, Thiverval-Grignon, France2 AgroParisTech, UMR 206, Chimie Biologique, Thiverval-Grignon, France3 INRA, UMR 118, Amelioration des Plantes et Biotechnologies Vegetales, Le Rheu, France4 AgroCampus Ouest, UMR 118, Amelioration des Plantes et Biotechnologies Vegetales, Le Rheu, France5 Universite Rennes 1, UMR 118, Amelioration des Plantes et Biotechnologies Vegetales, Le Rheu, France6 INRA, UR 1268 Biopolymeres, Interactions, Assemblages, Nantes, France

Received: May 23, 2008

Revised: February 17, 2009

Accepted: March 6, 2009

Seed oil bodies (OBs) are intracellular particles storing lipids as food or biofuel reserves in

oleaginous plants. Since Brassica napus OBs could be easily contaminated with protein bodies

and/or myrosin cells, they must be purified step by step using floatation technique in order to

remove non-specifically trapped proteins. An exhaustive description of the protein compo-

sition of rapeseed OBs from two double-zero varieties was achieved by a combination of

proteomic and genomic tools. Genomic analysis led to the identification of sequences coding

for major seed oil body proteins, including 19 oleosins, 5 steroleosins and 9 caleosins. Most of

these proteins were also identified through proteomic analysis and displayed a high level of

sequence conservation with their Arabidopsis thaliana counterparts. Two rapeseed oleosin

orthologs appeared acetylated on their N-terminal alanine residue and both caleosins and

steroleosins displayed a low level of phosphorylation.

Keywords:

Brassica napus / Oil bodies / Post-translational modifications / Protein composition /

Seed

1 Introduction

In oleo-proteaginous plants, storage lipids in the form of tri-

acylglycerol (TAG) are accumulated during seed development

and mobilized upon germination to provide carbon and energy

for the developing seedling. TAGs are deposited in the embryo

or endosperm in organelles named oil bodies (OBs), composed

of a core of TAG surrounded by a monolayer of phospholipids

embedded with proteins, which confer them a remarkable

stability. Although the OB is a relatively simple organelle from a

structural point of view, the mechanisms of its biogenesis and

degradation remain largely unknown [1–3]. OBs must withstand

extremes of desiccation, rehydration, heating and cooling for

months or even years [4] before the storage oil can be mobilized

following seed germination. Proteins embedded in the phos-

pholipid membrane form an effective resistant surface for

dormant and germinating seeds during the environmental

extremes [5]. Moreover, the persistence of the OBs as small

entities provides a large surface area per unit TAG and would

facilitate lipase binding and lipolysis during germination [6].

The most abundant proteins of plant seed OBs are

oleosins [6], which can stand for up to 75–80% of the OB

protein content [7, 8]. Oleosins have received considerable

attention in recent years and they have been studied in

numerous plant species such as sesame [9], sunflower [10],

Abbreviations: IEF, isoelectrofocalization; OB, oil body; TAG,

triacylglycerol

�Current address: Dr. Annick Bellamy, VALINOV, 3 rue Fleming,

F-49066 Angers cedex 01, France

Correspondence: Dr. Pascale Jolivet, UMR AgroParisTech/INRA

206, Chimie Biologique, Centre de Biotechnologie Agro-Indus-

trielle, Thiverval-Grignon 78850, France

E-mail: jolivet@grignon.inra.fr

Fax: 133-1-30-81-53-73

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

3268 Proteomics 2009, 9, 3268–3284DOI 10.1002/pmic.200800449

maize [11], soybean [11], almond [12], coffee [13], cacao [14]

or castor bean [15]. They are characterized by a low mole-

cular mass (around 18–25 kDa) and exhibit a triblock

structure with two terminal amphipathic regions and a

central hydrophobic anchoring region with a highly

conserved proline knot [1]. The steric hindrance and elec-

tronegative repulsion provided by oleosins are supposed to

improve the stability of the OBs. It has been suggested that

the entire surface of an OB is covered by oleosins such that

the compressed OBs never coalesce or aggregate in the cells

of a mature seed [16, 17]. Kim et al. [18] reported the iden-

tification of 16 genes coding for oleosins in the arabidopsis

genome and classified them into three groups according to

their tissue-specific expression profiles: five genes, named

S1–S5, were specifically expressed in maturing seeds, three

additional genes (SM1–SM3) were expressed in maturing

seeds as well as in microspores, and the remaining eight

transcripts (T1–T8) were restricted to the floret tapetum.

Oleosins have also been classified as high- or low-Mr

isoforms (H- and L-oleosin, respectively) depending on their

relative molecular masses [11]. Comparison of amino acid

sequences revealed that the main difference between the

H- and L-oleosins was an insertion of 18 amino acid resi-

dues in the C-terminal part of H-oleosins [19]. Tzen et al.[20] showed that both forms can coexist in OBs but they are

immunologically distinct [6]. Both H- and L-oleosins are

present in monocots and dicots, and thus the duplication

giving rise to them must have occurred before the diver-

gence of monocots and dicots. To date, only oleosins of the

L-form have been described in gymnosperm OBs [21].

Exhaustive descriptions of OB-associated proteins from

mature seeds of the cruciferae family, Arabidopsis thaliana and

Brassica napus, have been recently published through

systematic proteomic analyses [7, 8, 22]. In A. thaliana mature

seeds, four oleosins (S1–S4) were first identified by Jolivet et al.[7], whereas eight were expected in seeds from the gene

expression data [18]. A specific enrichment in seed oleosins

obtained by chloroform/methanol extraction has allowed

subsequently the identification of low-abundance S5 oleosin

[23]. In addition to oleosins, one caleosin (embryo-specific

protein ATS1, AtCLO1), one 17-b-hydroxysteroid dehy-

drogenase (AtHSD1, SLO1 or steroleosin), a probable aqua-

porin and a glycosylphosphatidylinositol-anchored protein of

unknown function have also been identified in purified OBs

from A. thaliana mature seeds [7]. In the case of rapeseed,

while Katavic et al. [22] described three oleosin isoforms using

the non-redundant protein database and Murphy’s team four

[24, 25], we reported the identification of eight oleosins with a

strong residue conservation rate among them and with their

A. thaliana counterparts [8]. Besides oleosins, two enzymes

belonging to the short-chain dehydrogenase reductase family

have also been identified in rapeseed [8, 22], as well as an

embryo-specific protein ATS1 [22]. An OB-associated caleosin

isoform has been previously reported in rapeseed [26]. The

presence of some other proteins such as cruciferins, myrosi-

nases, myrosinase-associated proteins and myrosinase-binding

proteins was also reported by Katavic et al. [22], but they are

likely to result from contamination of their OB-purified frac-

tion. Using a different purification protocol including steps

performed using detergents and urea in order to remove non-

specifically trapped proteins, Jolivet et al. [8] decreased signifi-

cantly this contamination.

In the present work, we compared the protein composi-

tion of highly purified OBs from two double-zero (low erucic

acid and reduced glucosinolate contents) B. napus cultivars

(Explus and Darmor). We focused on developing an exten-

sive analysis method to investigate the protein composition

of seed OBs from these two cultivars. Therefore the proteins

were first identified by a conventional proteomic strategy,

combining protein separation by electrophoresis and MS/

MS. A further analysis was performed using a shotgun

proteomic approach, in which whole OB proteins were

hydrolyzed and the separation conducted at the peptide level

by two stages of LC, prior to MS/MS. Finally, coupling

protease protection strategy to the use of the robust non-

specific protease proteinase K facilitated the characterization

of protein topology in OBs [27, 28]. Protein identification

was performed using not only the public NCBI non-

redundant and Uniprot protein databanks but also a rape-

seed cDNA collection generated from developing seeds [29].

In addition, protein post-translational modifications such as

phosphorylation and N-terminus acetylation were investi-

gated. Proteins from B. napus seed OBs were also char-

acterized using specific antibodies raised against oleosins,

steroleosins or caleosins from A. thaliana OBs [23, 30, 31].

2 Materials and methods

2.1 Plant material

Mature seeds from two double-zero winter-type B. napusvarieties were used for OB isolation. Seeds from double-zero

rapeseeds contain not more than 1–2% of erucic acid in total

fatty acids (versus 48–50% in old varieties) and display a

reduced glucosinolate content (�15–20 mmol/g of seed

instead of �100mmol/g of seed in old varieties), so that

contamination with myrosin cells should be limited. More-

over, nowadays, double-zero rapeseed cultivars are mostly

bred all around the world. Darmor seeds were provided by

the Brassica team of the Plant Breeding and Biotechnologies

Laboratory, Rennes, France. Darmor is a French cultivar

obtained after introgression of the reduced glucosinolate

content trait followed by three backcrosses [32]. Seeds from

Explus were kindly provided by the Experimental Farm of

Grignon, Thiverval-Grignon, France.

2.2 Isolation of OBs from mature seeds

OBs were purified according to Jolivet et al. [7] using a

method adapted from Tzen et al. [33]. In a typical OB

Proteomics 2009, 9, 3268–3284 3269

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

preparation, 300 mg of seeds were ground three times for

30 s 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 purified by floatation using six successive centrifugation

steps (10 000 � g, 41C, 30 min) using a Kontron Ultra-

centrifuge equipped with a swinging-bucket rotor. OB frac-

tions could be sampled during the purification process

(F1–F6). Proteins that were non-specifically associated with

OBs were subsequently removed by detergent washing

(0.1% v/v Tween-20), ionic elution (2 M NaCl) and urea

treatment (7 M). Finally, TAGs in defective or broken OBs

were removed by hexane extraction. The final OB fraction

was collected, resuspended in a minimal volume of buffer 1

and stored at 41C till further use.

2.3 SDS-PAGE of OB proteins

Proteins were quantitated with the Folin Cioccalteu reagent

[34] using BSA as the standard. Protein aliquot from OB was

precipitated with three volumes of cold acetone at �201C 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 SDS

and 0.02% w/v bromophenol blue. SDS-PAGE of proteins

was carried out according to Laemmli [35], using 12% ready-

to-use NuPAGE polyacrylamide gels (Novex, San Diego,

CA). Electrophoresis was run under 100 V for 180 min using

50 mM MES NuPAGE buffer (pH 7.3). Gel was stained with

Coomassie blue (G-250) according to Neuhoff et al. [36].

Molecular masses were estimated with Mark 12TM 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).

2.4 2-DE of OB proteins

Aliquots from OB (50–500 mg protein) were precipitated

with acetone at �201C overnight. The protein pellets

obtained by centrifugation (5000 � g, 41C, 20 min) were

resuspended in 100 mL of isoelectrofocalization (IEF) buffer

(8 M urea, 2 M thiourea, 2% w/v CHAPS, 2% v/v Triton

X-100, 50 mM DTT, 0.5% v/v ampholyte 3–10) as in [22].

Linear IPG gel strips (pH 3–10, 18 cm, GE Healthcare) were

passively rehydrated with IEF buffer and proteins were

applied by cup loading. In order to reveal minor proteins, a

high quantity of OB proteins was loaded (500 mg) and

DryStrip was cut at basic end to remove abundant oleosins.

The IEF was performed on an IPGphor 3 system (GE

Healthcare) using the following focusing method: 300 V for

1 h, 1000 V for 1 h, 6000 V for 1 h, 6000 V for 15 000 Vh.

Before the second dimension, IPG strip was reduced and

alkyled with DTT (5 mg/mL) and iodoacetamide (22.5 mg/

mL) in equilibration buffer (50 mM Tris, pH 6.8, 6 M urea,

30% v/v glycerol and 2% w/v SDS) for 10 min, rinsed in SDS

running buffer and placed onto 12% acrylamide vertical gel.

IPG strip was hold down with 0.5% w/v low melting point

agarose in SDS running buffer. SDS-PAGE in the second

dimension was performed at 60 mA/gel for 2 h.

After 2-DE, gels were washed three times in deionized

water before staining with PageBlueTM Protein Staining

Solution (Fermentas, St. Remy les Chevreuse, France) or

electroblotting [37].

2.5 LC-MS/MS identification of proteins isolated

from polyacrylamide gels (conventional

proteomic strategy)

Protein bands or spots stained with Coomassie blue were

excised from the polyacrylamide gel and stored at �201C.

Trypsin digestion was carried out as in [7] after reduction

with 10 mM DTT and alkylation in the dark with 55 mM

iodoacetamide. After digestion, the resulting peptides were

extracted successively with 5% v/v formic acid, ACN/water

(50/50 v/v) and ACN. Combined extracts were dried and

samples were dissolved in 1% v/v formic acid before LC-MS

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 10mL of samples were loaded

onto a reversed-phase BioBasic-18 column (1� 150 mm,

300 A pore size, 5mm film thickness, Thermo Electron). The

column was eluted at a flow-rate of 0.1 mL/min at 201C with

5% of solvent B (ACN 1 0.1% v/v formic acid) in A (water 1

0.1% v/v formic acid) for 2 min and then with a linear

gradient of B in A from 5 to 45% over 40 min and then 45 to

95% over 5 min before re-equilibration. Eluant from the

column was introduced in the electrospray ionization source

of a Thermo Electron LCQ Deca ion-trap mass spectrometer

operating in positive ion mode. Instrumental parameters

were capillary temperature, 2801C; capillary voltage, 30 V;

spray voltage, 4.5 kV; sheath gas flow, 80 a.u. and 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 a.u. 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) and (iii) fragmentation of these ions.

Protein identification was performed with Bioworks

3.1TM software using A. thaliana and B. napus protein

sequence databases extracted from non-redundant database

downloaded from the NCBI FTP site. A collection of

3270 P. Jolivet et al. Proteomics 2009, 9, 3268–3284

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

B. napus ESTs that has been generated from developing

seeds (10, 15, 20, 25, 40, 45 days after pollination) from

JetNeuf and anthers from Samouraı 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 3

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 terms of (i) trypsin nature of peptides, (ii) Xcorr

magnitude up to 1.8, 2.5 and 3.3 for, respectively, mono-, di-

and tri-charged peptides [38, 39] and (iii) DCN greater than

0.1 [40]. The absence of false-positive identification was

verified using a composite database including original

protein database and its reversed version [38].

2.6 In-solution digestion of whole OBs protein

extracts and multidimensional LC-MS/MS

(shotgun proteomics)

For this approach, analyses were performed within the

platform ‘‘Biopolymers-Interaction-Structural Biology’’

located at the INRA Center of Nantes.

The final OB fraction was precipitated in five volumes of

cold acetone at �201C for 1 h before centrifugation at

10 000 � g for 15 min. The pellet was recovered, dried and

resuspended in 6 M urea, 2% w/v CHAPS and 18 mM DTT.

An equivalent of 500 mg of protein was diluted six times

using 25 mM NH4HCO3 with 10% v/v ACN. Modified

trypsin purchased from Promega (Madison, WI, USA) was

added at a substrate-to-enzyme ratio of 30:1 and the proteins

were continuously digested overnight. After incubation, the

sample was acidified by an aqueous solution of formic acid

to reach a final concentration of 0.1% v/v and stored at

�201C until analysis.

Chromatographic separations were performed using an

AKTAexplorer 10 system (GE Healthcare). In the first step,

the peptide mixture was separated by off-line strong cation

exchange chromatography (Polysulfoethyl A column-

PolyLC, 100 mm length, 2.1 mm id, 5 mm and 200 A pore

size) at a flow-rate of 0.3 mL/min. The solvents consisted of

10 mM KH2PO4 adjusted at pH 3 containing 25% v/v ACN

(solvent A) and 10 mM KH2PO4 adjusted at pH 3 and

containing 25% v/v ACN including 350 mM potassium

chloride (solvent B). Elution started with solvent A for 6 min,

then peptides were eluted using a linear gradient of solvent

B in A from 0 to 50% over 26 min, followed by a rapid

gradient from 50 to 100% of B in 10 min. The elution was

monitored at 220 nm, 14 fractions were collected and further

desalted by reversed-phase chromatography using a mini-

RPC Nucleosil C18 column (50 mm� 2 mm). The column

was equilibrated in 0.1% v/v formic acid and elution was

achieved at a flow-rate of 0.2 mL/min using 75% ACN with

0.1% formic acid. Eluted peptides were collected, dried with

a SpeedVac concentrator and stored at �201C.

The dried strong cation exchange fractions were re-

suspended in 30 mL of 5% v/v ACN containing 0.1% v/v

formic acid and further submitted to nano-scale reversed-

phase chromatography using a Switchos-Ultimate II capil-

lary LC system (Dionex, Amsterdam, the Netherlands)

coupled to a hybrid quadrupole orthogonal acceleration

time-of-flight mass spectrometer (Q-TOF Global, Waters,

Manchester, UK). Chromatographic separation was

performed on a C18 capillary column (Pepmap C18, 75 mm

id, 150 mm length, LC Packings) at a flow-rate of 200 nL/

min. A two-step gradient was applied, a first linear increase

from 2 to 40% of ACN containing 0.1% v/v formic acid

during 50 min, followed by a second increase to 50% within

10 min.

Mass data acquisitions were recorded using the Mass

Lynx software (Waters) using a ‘‘data dependent acquisi-

tion’’ method: MS data were recorded for 1 s on the m/zrange of 400–1500, after which the three most intense ions

were selected and fragmented in the collision cell (MS/MS

measurements); MS/MS data were recorded on the m/zrange of 50–1500. Raw mass spectra were processed with

Protein Lynx Global Server software version 2.1 (Waters).

Protein identity was searched by using MASCOT 2.2 (Matrix

Science, London, http://www.matrixscience.com) against

UniProt/Swiss-Prot and UniProt/TrEMBL databanks (28-11-

2006) restricted to Viridiplantae, or against the TIGR Gene

Indices databank (B. napus: release: 16-06-2006). The

MASCOT search parameters were as follows: trypsin

specificity, one missed cleavage, oxidation of Met as variable

modification and carbamidomethyl (Cys) as fixed modifica-

tion. Mass accuracy was set to 150 ppm for parent ions and

0.3 Da for MS/MS fragments. Only protein hits displaying

more than two peptides above the cut-off significance score

(po0.05) computed by MASCOT were validated. Results

from the three databank searches were compiled and

compared with the results obtained with the contigs

described in Section 2.10, in order to achieve final protein

identification.

2.7 Proteinase K digestion of purified OBs

Proteinase K was used to remove and analyze all externally

accessible protein domains [28]. Purified OBs (1 mg protein)

were diluted in 1 mL of 80 mM Na2HPO4/NaHPO4, pH 7.5,

5 mM MgCl2, 250 mM sucrose and incubated at 41C for

30 min after the addition of proteinase K (Roche Diag-

nostics, Meylan, France) in solution in 10 mM Tris-HCl

buffer (pH 7.5) in order to obtain a 1:100 mass-to-mass ratio

of enzyme to substrate. OBs were isolated as a floating

material by microcentrifuging at 10 000 � g at 41C for

Proteomics 2009, 9, 3268–3284 3271

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

10 min. The lower phase contained a mixture of peptides

obtained from the digestion of accessible domains by

proteinase K. OBs were resuspended in 200 mM Na2CO3

(pH 11) containing 5% v/v SDS and incubated at 371C for

3 h in the presence of proteinase K (10 mg) in order to obtain

peptides from transmembrane protein domains. The reac-

tion was quenched with 5% v/v formic acid and the mixture

was microcentrifuged at 18 000 � g at 41C for 15 min to

remove particulate matter. The two peptide mixtures were

cleaned using Vydac BioSelect solid-phase extraction

cartridge filled with C18 reversed-phase material before LC-

MS/MS analyses. The steps of conditioning, sample load-

ing, washing and elution were carried out according to the

manufacturer’s protocol. Fractions eluted with 30% v/v ACN

and 60% v/v ACN were combined, evaporated to dryness

and dissolved in 1% v/v HCOOH and 5% v/v ACN to be

injected and analyzed as described above. The same filtering

criteria as above were chosen for the analysis of tandem

mass spectra [41, 42]. Identification relevance was evaluated

using reversed database searching [42].

2.8 Search of post-translational modifications

From raw mass spectra, some amino acid modifications

were systematically investigated in the set of the observed

peptides such as the phosphorylation of serine, threonine

and tyrosine residues (mass difference of 180 Da) or the

acetylation of alanine residue (mass difference of 142 Da).

Detection of phosphorylated proteins was also carried out by

means of Phos-tagTM 300/460 phosphoprotein gel stain

(Perkin-Elmer) according to the manufacturer’s protocol.

Samples corresponding to either of total proteins from

purified OBs or a mixture of phosphorylated (bovin b-casein

and chicken ovalbumin) and non-phosphorylated (BSA)

proteins were run on SDS-PAGE gel as described above.

Two gels were realized in the same conditions. The first was

stained with Coomassie blue. The second was fixed in

50% v/v methanol and 10% v/v acetic acid, washed in water

and incubated with Phos-tag dye concentrate at a ratio of

1:100 in stain buffer at room temperature for 90 min. The

gel was destained with destain buffer and then rinsed with

water before viewing with a UV transilluminator imaging

system (302 nm, Ultra-Violet Products).

2.9 Immunoblot analyses of OB proteins

Rabbit sera anti-rS1N, anti-rS2N, anti-rS3N and anti-rS5N

were produced as described previously [23]. Rabbit serum

raised against purified recombinant oleosin S4 was obtained

as described by Roux et al. [43]. Production of rabbit anti-

CLO1 and anti-SLO1 sera was reported by Purkrtova et al.[31] and d’Andrea et al. [30], respectively. Rabbit polyclonal

serum against 2S napin was obtained by subcutaneous

immunization with the purified protein obtained according

to Berot et al. [44]. This anti-2S serum lightly cross reacted

with 12S storage proteins (cruciferins). A more specific anti-

2S serum was also produced against one peptide

(CQQWLHKQAMQSG) belonging to the small chain of

B. napus napin.

For immunoblot analysis, proteins resolved by SDS-

PAGE or 2-D PAGE were blotted on to Immobilon-P PVDF

membrane (Millipore, Molsheim, France). The membrane

was probed with rabbit sera at various dilutions: 1:5000

dilution for anti-rS2N, 1:4000 for anti-rS1N and anti-rS3N,

1:2000 for anti-rS4, anti-rS5N, anti-SLO1, anti-CLO1 and

anti-2S serum, and 1:1000 for anti-peptide from napin.

Rabbit antibodies were revealed with peroxidase-conjugated

goat anti-rabbit IgG from Pierce (Perbio Science, Brebieres,

France). Saturation and incubation with antibodies were

carried out according to d’Andrea et al. [23]. Peroxidase

activity was revealed using SuperSignal West Dura Extended

Duration Substrate from Pierce according to the manu-

facturer’s protocol. Luminescence from peroxidase activity

was recorded using LAS-3000 imaging system and analyzed

using MultiGauge software from Fujifilm (St. Quentin en

Yvelines, France). A MagicMarkTM XP Western protein

standard from Invitrogen was used to visualize standard

bands.

2.10 Sequence analysis

Similarity searches of the databases were conducted

according to Altschul et al. [45] through the Genoplante Info

server. BLASTN option was used to recover rapeseed ESTs

starting from arabidopsis sequences. Alignments were

performed with the ClustalW algorithm and visualized

with BOXSHADE (http://www.ch.embnet.org/software/

BOX_form.html). Selected cDNA clones were obtained from

Genoplante rapeseed cDNA libraries BN15 (ID 5 Lib.

13 977), BN20 (ID 5 Lib. 13 978), BN25 (ID 5 Lib. 13 979),

BN40 (ID 5 Lib. 13 980) and BN45 (ID 5 Lib. 13 981). cDNA

inserts were amplified using T7 and SP6 primers and

subsequently used for sequencing with T7. All DNA

sequences were performed by Genome Express (Grenoble,

France).

To conduct distance analysis among plant oleosins, a

partial alignment of the most conserved region in oleosin

amino acid sequences was generated using ClustalW and

manually adjusted. The distance matrix was calculated and

submitted to a neighbor-joining analysis using the Treecon

program [46] to generate a branching pattern. For statistical

analysis, 1000 bootstraps replications were performed. The

consensus tree was drawn using the Treecon package. The

sequences mentioned in this study are given as follows, with

GenBank accession numbers indicated within parentheses:

for A. thaliana, AtS1 (At3g01570), AtS2 (At3g27660,

OLEO4), AtS3 (At4g25140, OLEO1), AtS4 (At5g40420,

OLEO2), AtS5 (At5g51210, OLEO3), AtSLO1 (At5g50600),

AtSLO2 (At4g10020), AtCLO1 (At4g26740), AtCLO2

3272 P. Jolivet et al. Proteomics 2009, 9, 3268–3284

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

(At5g55240), AtCLO3 (At2g33380) and AtT4 (CAB87943

encoding AtGRP-6, [18]); for B. napus, see Table 1; for

Helianthus annuus, HaOLE1 (CAA44224) and HaOLE2

(CAA55348) see [47, 48]; for Glycine max, GmOLEO1

(P29530) and GmOLEO2 (P29531) see [49]; for Oryza sativa,

OsOLE18 (AAC02240) and OsOLEO16 (AAC02239) see [50];

for Sesamum indicum, SiOLE17 (AAG23840), SiOLE15.5

(AAB58402) and SiOLE15 (AAD42942) see [19]; for Zeamays, ZmOLE18 (AAA67699), ZmOLE17 (AAA68066) and

ZmOLE16 (AAA68065) see [51]; for Gossypium hirsutum,

GhOLE-MatP6 (AAA18524) and GhOLE-MatP7 (AAA18523)

see [52]; for Hordeum vulgare, HvOLE1 (CAA57994) and

HvOLE2 (CAA57995) see [53]; for Ricinus communis,RcOLE1 (AAR15171) and RcOLE2 (AAR15172) see [15]; for

Olea europaea, OeOLE (AAL92479) see [54]; for Coffea cane-phora, CcOLE1 (AAX49389), CcOLE2 (AAX49390), CcOLE3

(AAX49391) and CcOLE4 (AAX49392) see [13]; for Theo-broma cacao, TcOLE1 (AAM46777) and TcOLE2

(AAM46778) see [14] and for Prunus dulcis, PdOLEO1

(Q43804) see [55].

Table 1. Identification of rapeseed orthologs from arabidopsis sequences coding for OB proteins

Protein A. thaliana hit Total ESTno.a)

cDNA cloneb) Sequence namec)

(GenBank accession no.)Identity rate(%)d)

MWe) (kDa)/no. ofresiduesf)

S1 At3g01570 44 Bn25064E15 BnS1-1 (EU678256) 83.6 20.8 /193Bn45001L23 BnS1-2 (EU678257) 83 20.7 /193

S2-OLEO4 At3g27660 21 Bn45001C15 BnS2-1 (EU678255) 76 20.0/188Bn40061D10 BnS2-2 (EU678258) 76.6 19.9/188

S3-OLEO1 At4g25140 91 Bn40040A13 BnS3-1 (EU678265) 86.1 19.6/180Bn25045P23 BnS3-2 (EU678266) 86.7 19.5/180Bn40059C14 BnS3-3 (EU678267) 86.1 20.6/187Bn25047B21 BnS3-4 (EU678268) 86.1 20.7/187Bn25048G13 BnS3-5 (EU678269) 84.4 20.0/183Bn25048E17 BnS3-6 (EU678270) 85 20.0/183Bn40042H01 BnS3-7 (EU678271) 86.7 21.5/195Bn40040E13 BnS3-8 (EU678272) 85 20.6/186Bn25047P14 BnS3-9 (EU678273) 84.4 20.5/186

S4-OLEO2 At5g40420 53 Bn25049K19 BnS4-1 (EU678259) 81.9 22.0/210Bn45042K03 BnS4-2 (EU678260) 80.9 22.2/212Bn40045E19 BnS4-3 (EU678261) 80.4 23.1/220Bn20045J08 BnS4-4 (EU678262) 79.4 23.1/220

S5-OLEO3 At5g51210 14 Bn40046E06 BnS5-1 (EU678263) 74.5 15.7/149Bn40047A18 BnS5-2 (EU678264) 74.5 15.6/149

SLO1 At5g50600- 11 Bn40059G20 BnSLO1-1 (EU678274) 87.9 39.1/349At5g50700 Bn45053I05 BnSLO1-2 (EU678275) 89.9 39.1/349

Bn45050C01 BnSLO1-3 (EU678276) 85.9 38.3/341

SLO2 At4g10020 18 Bn40061E12 BnSLO2-1 (EU678277) 89.7 51.2/456Bn45045P07 BnSLO2-2 (EU678278) 90.7 51.6/461

CLO1- At4g26740- 29 Bn40041H16 BnCLO1-1 (EU678281) 94.2 28.0/245

CLO2 At5g55240 Bn40040I21 BnCLO1-2 (EU678282) 91.4 28.1/245Bn40046D07 BnCLO1-3 (EU678285) 91.4 28.1/245Bn25040L10 BnCLO1-4 (EU678288) 91.8 28.1/245Bn40047C23 BnCLO1-5 (EU678289) 92.6 28.1/245Bn25048H20 BnCLO1-6 (EU678290) 91.8 28.1/245Bn40044L03 BnCLO1-7 (EU678283) 92.2 28.1/245

CLO3 At2g33380 5 Bn15024O15 BnCLO3-1 (EU678279) 82.2 27.5/244Bn15019H09 BnCLO3-2 (EU678280) 82.6 26.9/239

a) Number of rapeseed EST found into the corresponding Genoplante contig.b) Accession number of selected clones used for sequencing the corresponding full-length cDNA.c) Sequences were named as BnS1-1 for B. napus oleosin S1 no. 1 since at least two rapeseed orthologs were found for each arabidopsis

hit due to the polyploid nature of rapeseed genome and gene duplications.d) Percentage of amino acid residue identity between rapeseed and arabidopsis orthologous sequences.e) Calculated molecular weight of the deduced amino acid sequence expressed in kDa.f) Number of amino acid residues.

Proteomics 2009, 9, 3268–3284 3273

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

3 Results

3.1 Identification of sequences coding for seed OB

proteins in B. napus

A candidate gene approach based on arabidopsis sequences

coding for oleosins (S1–S5), steroleosins (SLO1 and SLO2)

and caleosins (CLO1–CLO3) was undertaken to identify the

rapeseed orthologs. Each arabidopsis sequence was blasted

against the rapeseed EST library generated by Genoplante

(see Section 2 and Table 1). ESTs were subsequently aligned

together to determine how many putative independent

rapeseed genes they stood for. For each putative hit, a

selected cDNA clone was obtained from the Genoplante

cDNA library and used to sequence the full-length insert

(Table 1). This allowed us to recover 19 expressed sequences

with high similarities with A. thaliana oleosin genes,

including two genes coding for B. napus oleosin S1 (BnS1),

BnS2 or BnS5, four genes for BnS4 and nine genes for BnS3

(Table 1 and Fig. 1). Similar studies led to the identification

of five steroleosin-like cDNA (three coding for BnSLO1 and

two for BnSLO2) as well as nine caleosin-like sequences

(seven coding for BnCLO1 and two for BnCLO3). All the

corresponding rapeseed cDNA sequences were deposited in

GenBank.

Comparison of the deduced amino acid sequences from

the rapeseed orthologs with their arabidopsis counterparts

displayed high level of sequence identity, ranging from 74.5

to 94.2% (Table 1). Sequence alignment of arabidopsis and

rapeseed oleosin isoforms is shown in Fig. 1. The three

classical structural regions found in plant seed oleosins were

retrieved in rapeseed oleosins: a hydrophilic N-terminus, a

central hydrophobic region of 72 amino acid residues

carrying the typical proline knot sequence (-PX5SPX3P-) and

a C-terminal end. The central region was highly conserved,

whereas N- and C-terminal ends display variations even

within a protein family.

On the basis of the alignment of their C-terminal regions

rapeseed oleosins were assigned in two classes, with BnS1,

BnS2 and BnS4 orthologs belonging to the H-form and

BnS3 as well as BnS5 orthologs belonging to the L-form

(Fig. 1). This was confirmed by a phylogenetic analysis of

plant seed oleosins (Fig. 2), which also led to the conclusion

that rapeseed oleosins from the H-class were closer to the

H-oleosins from other plant species including those from

monocots than from L-related rapeseed oleosins, and viceversa. Nevertheless, within a class (H or L), arabidopsis and

rapeseed oleosins from the different families were closer to

each other than from any other plant oleosin from the same

class. For instance, S3 oleosins from both cruciferous plants

are more related to S5 oleosins than to other plant L-oleosins

and similar results were observed for arabidopsis and

rapeseed S1, S2 and S4 proteins (Fig. 2).

For each of the S1-, S2- or S5-oleosin families, only two

rapeseed sequences were retrieved, which can be grouped

with arabidopsis counterparts, suggesting that a common

ancestral sequence existed before the separation of the

Arabidopsis and Brassica lineages. The two B. napus proteins

likely originate from the B. oleracea genome, on the one

hand, and from the B. rapa genome, on the other hand. For

S4 oleosins, four proteins exist in rapeseed, which could

result from gene duplication in the Brassica lineage. The

situation is even more complex in the S3 protein family

where gene duplication likely occurred to give the actual

nine protein sequences.

3.2 Protein composition of purified OBs

The proteins extracted from the OB fraction obtained through

the last step of preparation (see Section 2) were analyzed by

SDS-PAGE in denaturing conditions (Fig. 3) and an approx-

imate quantification based on band intensity was obtained.

Explus or Darmor OB proteins showed simple patterns with

only six or seven different protein bands visible within the

14–100 kDa range after Coomassie blue staining. The two

cultivars also exhibited very similar patterns with at least five

common bands (1/B, 2/C, 3/D, 4/E and 6/F). All the bands

were excised, subjected to trypsin hydrolysis and the resulting

peptides were analyzed by MS. A complementary study was

performed using a non-conventional shotgun proteomic strat-

egy, starting from the whole protein extract, subjected to trypsin

hydrolysis in solution (see Section 2). A total of 25 proteins were

identified from 135 non-redundant peptide sequences (Table 2

and Supporting Information). They were identified either

through homology with A. thaliana proteins or after searching

EST databanks from B. napus. Oleosins are the major proteins

of rapeseed OBs and stand for 90% of total proteins in the case

of Darmor OBs. Fifteen different oleosins were identified in

rapeseed OBs being orthologs to A. thaliana oleosins (S1–S5).

The major band (band 1/B) was composed of two S1 orthologs,

four S2 orthologs and five S3 orthologs. Either BnS3-5 and

BnS3-6 or BnS3-8 and BnS3-9 cannot be distinguished from the

identified trypsin peptides. In contrast to A. thaliana oleosins,

which are resolved by SDS-PAGE [7], these 11 oleosins

presented a similar electrophoretic behavior because they are

characterized by very close molecular masses (�20.1 kDa).

Moreover, three S4 orthologs with a slightly higher molecular

mass (mean of 22.6 kDa) were recovered in band 2/C. The

N-terminal domain of rapeseed S4 oleosins is particularly

enriched in glycine residues, which represent 32% of its resi-

dues and therefore increase its hydrophobic character and flex-

ibility. Finally, a faint protein band near 15 kDa in the

preparation of Darmor OBs (band A) appeared to contain an S5

ortholog.

Besides oleosins, proteins highly similar to the 17-b-

hydroxysteroid dehydrogenase-like protein (steroleosin 1 or

SLO1) and the embryo-specific protein ATS1 (caleosin or

AtCLO1) already described in A. thaliana [7] were identified

in B. napus seeds (bands 3/D and 4/E). Proteomic analyses

revealed the expression of at least three steroleosin isoforms

and only one caleosin. For this last protein, we have to take

3274 P. Jolivet et al. Proteomics 2009, 9, 3268–3284

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

into account the fact that the identified peptides were

consensus for the caleosin family. This work revealed two

new expressed proteins, which have been characterized only

up to now in other plant species with cloning and nucleotide

sequence [18, 56] or by immunodetection [23]. These

proteins corresponded first to an ortholog to A. thaliana S5

oleosin (At5g51210) and second to a new steroleosin that we

called steroleosin 2 or SLO2, ortholog to a putative short-

chain dehydrogenase reductase of A. thaliana (At4g10020,

91% identity and 94% homology) or to steroleosin-B from S.indicum (68% identity and 87% homology). All the proteins

identified during the progress of this work were obtained

after trypsin digestion with a high sequence coverage

(16–50%, Table 2) except for BnSLO2 (10% sequence

**** BnS3-5 1 -----MTDTAR-THHDITTRDQYP--------------------MMGRDRDQYAIIGRD--QYQGYGQDYSKSRQIAKAA BnS3-6 1 -----MTDTAR-THHDITTRDQYP--------------------MMGRDRDQYAIIGRD--QYQGYGQDYSKSRQIAKAABnS3-8 1 -----MTDTAR-THHDITTRDQYP--------------------LISRDRDQYGMIGRD--QYNMSGQNYSKSRQIAKAT BnS3-9 1 -----MTDTAR-THHDITTRDQYP--------------------LISRDRDQYGMIGRD--QYNMSGQNYSKSRQIAKAT BnS3-3 1 -----MTDTAR-THHDITSRDQYPRDRDQY-------------STIGRDRDKYSMIGRDRDQYNMYGRDYSKSRQIAKAV BnS3-4 1 -----MTDTAR-THHDITSRDQYPRDRDQY-------------SMIGRDRDKYSMIGRDRDQYNMYGRDYSKSRQIAKAV BnS3-7 1 -----MTDTAR-THHDITSRDQYPRDRDQY-------------SMIGRDRDQYSMMGRDRDQYNMYGRDYSKSRQIAKAVBnS3-1 1 -----MADTAR-THHDITSRDQYP--------------------ILGRDRDQYPYGRSD---YQTSGQDYSKTRQIAKAA BnS3-2 1 -----MADTAR-THHDITSRDQYP--------------------ILGRDRDQYPYGRSD---YQTSGQDYSKTRQIAKAA AtS3 1 -----MADTARGTHHDIIGRDQYP--------------------MMGRDRDQYQMSGR--------GSDYSKSRQIAKAA BnS5-1 1 -----MANQTR-THQDIIVRDSRI---------------------------------------TLDRDHPKTGAQMVKVA BnS5-2 1 -----MANQTR-THQDIIVRDSRS---------------------------------------TLDRDHPKTGAQMVKVA AtS5 1 -----MADQTR-THHEMISRDS-------------------------------------------TQEAHPKARQMVKAA BnS1-1 1 ------MADVRTHAHQVQVHPLRQQE---------------------------------GGIKVVYPQSGPSSTQVLAVIBnS1-2 1 ------MADVRTHAHQVQVHPLRQHE---------------------------------GGIKVVYPQSGPSSTQVLAVVAtS1 1 ------MADVRTHSHQLQVHPQRQHE---------------------------------GGIKVLYPQSGPSSTQVLAVF BnS2-1 1 --MANVDRRVNVDRTDKGLQLQPQYEDR-----------------------VGYG--YGYGGNTDYKSCGPSTNQIVALIBnS2-2 1 --MATVERRVQVDPTDKRIHLQPQYEGD-----------------------VGYG--YGYGGRADYKSSGPSSNQIVALIAtS2 1 MANVDRDRRVHVDRTDKRVHQP-NYEDD-----------------------VGFGGYGGYGAGSDYKSRGPSTNQILALIBnS4-1 1 -----MADTHRVDRTDRHLQFQSPYEGGRVSIQYE------GGGGAGGYGG-RGGGYGAEGYKSMMPERGPSSTQVLSFL BnS4-2 1 -----MADTHRVDRTDRHLQFQSPYEGGRVNIQYE------GGGGAGGYGGGRGGGYGAGGYKSMMPERGPSNTQVLSFL BnS4-3 1 -----MADTHRVDRTDRHLQFQPPYEGGRVNIQFEGAGEGYGQSGYGGGGGYGQSGYGGGGYKSMMPESGPSSTQVISFL BnS4-4 1 -----MADTHRVDRTDRHLQFQSPYEGGRVNIQFEGAGGGYGQSGYGGGGGYGQSGYGGGGYKSMMPESGPSSTQVISFL AtS4 1 -----MADTHRVDRTDRHFQFQSPYEGGRGQGQYE------GDR-----------GYGGGGYKSMMPESGPSSTQVLSLL

********************************************************************BnS3-5 53 TAVTAGGSLLVLSSLTLVGTVIALIVATPLLVIFSPILVPALITVALLITGFLSSGGFGIAAITVFSWIYKYAT-GEHPK BnS3-6 53 TAVTAGGSLLVLSSLTLVGTVIALIVATPLLVIFSPILVPALITVALLITGFLSSGGFGIAAITVFSWIYKYAT-GEHPK BnS3-8 53 TAVTAGGSLLVLSSLTLVGTVIALIVATPLLVIFSPILVPALITVALLITGFLSSGGFGIAAITVFSWIYKYAT-GEHPQ BnS3-9 53 TAVTAGGSLLVLSSLTLVGTVIASIVATPLLVIFSPILVPALITVALLITGFLSSGGFGIAAITVFSWIYKYAT-GEHPQ BnS3-3 62 TAVTAGGSLLVLSSLTLVGTVIALTVATPLLVIFSPILVPALITVALLITGFLSSGGFGIAAITVFSWIYKYAT-GEHPQ BnS3-4 62 TAVTAGGSLLVLSSLTLVGTVIALTVATPLLVIFSPILVPALITVALLITGFLSSGGFGIAAITVFSWIYKYAT-GEHPQ BnS3-7 62 TAVTAGGSLLVLSSLTLVGTVIALTVATPLLVIFSPILVPALITVAMLITGFLSSGGFGIAAITVFSWIYKYAT-GEHPQ BnS3-1 52 TAVTAGGSLLVLSSLTLVGTVIALTVATTLLVIFSPILVPALITVALLITGFLSSGGFGIADITVFSWIYKYAT-GEHPQ BnS3-2 52 TAVTAGGSLLVLSSLTLVGTVIALTVATPLLVIFSPILVPALITVALLITGSLSSGGFGIAAITVFSWIYKYAT-GEHPQ AtS3 48 TAVTAGGSLLVLSSLTLVGTVIALTVATPLLVIFSPILVPALITVALLITGFLSSGGFGIAAITVFSWIYKYAT-GEHPQ BnS5-1 36 TGVAAGGSLLVLSGLTLAGTVIAFAVATPLLIIFSPVLVPAVITVVLIITGFLASGGFGIAAITAFSWLYRHMT-GSGSD BnS5-2 36 TGVAAGGSLLVLSGLTLAGTVIALAVATPLLIIFSPVLVPAVITVVLIITGFLASGGFGIAAITAFSWLYRHMT-GSGSD AtS5 32 TAVTAGGSLLVLSGLTLAGTVIALTVATPLLVIFSPVLVPAVVTVALIITGFLASGGFGIAAITAFSWLYRHMT-GSGSD BnS1-1 42 AGVPVGGTLLTLAGLTLAGSVIGLMLAFPLFLIFSPVIVPAAFVIGLAMTGFMASGAIGLTGLSSMSWVLNHIRRVRE-R BnS1-2 42 AGVPVGGTLLTLAGLTLAVSVIGLILAFPLFLIFSPVIVPAAFVIGLAMTGFMASGAIGLTGLSSMSWVLNHIRRVRE-R AtS1 42 VGVPIGGTLLTIAGLTLAGSVIGLMLAFPLFLIFSPVIVPAAFVIGLAMTGFLASGAIGLTGLSSMSWVLNYIRRAGQ-H BnS2-1 54 AGVPIGGSLLALAGLTLAGSVIGFMLSIPLFLLFSPVIVPAALTIGLAVTGILASGLFGLTGLSSVSWVLNYIRGRSD-T BnS2-2 54 VGVPVGGSLLALAGLTLAGSVIGLMLSVPLFLLFSPVIVPAAITIGLAVTAILASGLFGLTGLSSVSWVLNYLRGTSD-T AtS2 57 AGVPIGGTLLTLAGLTLAGSVIGLLVSIPLFLLFSPVIVPAALTIGLAVTGILASGLFGLTGLSSVSWVLNYLRGTSD-T BnS4-1 69 VGVPIVGSLLAIAGLLLAGSVIGLLISIPLFLLFSPVIVPAALTIGLAATGFLASGMFGLTGLSSVSWVLNYLRGTRKSS BnS4-2 70 VGVPIVGSLLAIAGLLLAGSVIGLLISIPLFLLFSPVIVPAALTIGLAATGFLASGMFGLTGLSSVSWVMNYLRGTRKSS BnS4-3 76 VGVPIVGSLLAIAGLLLAGSVIGLMISIPLFLLFSPVIVPAAITIGLATTGFLASGMFGLTGLSSISWVMNYLRRTRG-G BnS4-4 76 VGVPLVGSLLAIAGLLLAGSVIGLMISIPHFLLFSPVIVPAAITIGLATTGFLTSGMFGLTGLSSISWVMNYLRRTRG-S AtS4 59 IGVPVVGSLLALAGLLLAGSVIGLMVALPLFLLFSPVIVPAALTIGLAMTGFLASGMFGLTGLSSISWVMNYLRGTRR-T

BnS3-5 132 GSDKLDSARMKPGS------------------KAQDMKDRAHYYGQQHTGGEHVNTDYRNTDRDRTRGTT--- BnS3-6 132 GSDKLDSARMKLGS------------------KAQDMKDRAHYYGQQHTGGEHVNTDYRNTDRDRTRGTT--- BnS3-8 132 GSDKLDSARMKLGS------------------KAQDMKDRAYYYGQQHTGEEHDRDRDHRTDRDRTRGTQHTT BnS3-9 132 GSDKLDSARMKLGS------------------KAQDMKDRAYYYGQQHTGEEHDRDRDHRTDRDRTRGTQHTT BnS3-3 141 GSDKLDSARMKLGG------------------KVQDMKDRAQYYGQQQTG--------GEHDRDRTRGTQHTT BnS3-4 141 GSDKLDSARMKLGG------------------KVQDMKDRAQYYGQQQTG--------GEHDRDRTRGTQHTT BnS3-7 141 GSDKLDSARMKLGS------------------KAQDLKDRAQYYGQQHTGGYGQQHTGGEHDRDRTRGTQHTT BnS3-1 131 GSDKLDSARMKLGT------------------KAQDIKDRAQYYGQQHTG--------GEHDRDRTRGTHHTT BnS3-2 131 GSDKLDSARMKLGT------------------KAQDIKDRAQYYGQQHTG--------GEHDRDRTRGTHHTT AtS3 127 GSDKLDSARMKLGS------------------KAQDLKDRAQYYGQQHTG--------GEHDRDRTRGGQHTT BnS5-1 115 Q--KIESARMKVGS------------------RGYDTKYGQHNIGVHQQHQQAAS------------------ BnS5-2 115 Q--KIESARMKVGS------------------RGYDTKSGQHNIGVHQQHQQAAS------------------ AtS5 111 ---KIENARMKVGS------------------RVQDTKYGQHNIGVQHQQVS--------------------- BnS1-1 121 MPDELEEAKQRLADMAEYVGQRTKDAGQTIEEKAHDVRESKTYDVRDRDTKGHTATGGDRDTKTTREVRVATT BnS1-2 121 IPDELDEAKQRLADMAEYAGQRTKDAGQTIEDKAHDVRESKTYDVRDRDTKGHTASGGDRDTKTTREVRVATT AtS1 121 IPEELEEAKHRLADMAEYVGQRTKDAGQTIEDKAHDVREAKTFDVRDRDTTKGTHNVRDTKTT---------- BnS2-1 133 VPEQLDYAKRRMADAVGYAGQKGKEMGQYVQDKAHEAHDTSLTTETNGKTRRAHIA----------------- BnS2-2 133 VPEQLDYAKRRMADAVGYAGQKGKEMGQYVQDKAHEAHDTSLTTETTEPGKTRRHT----------------- AtS2 136 VPEQLDYAKRRMADAVGYAGMKGKEMGQYVQDKAHEARETEFMTETHEPGKARRGS----------------- BnS4-1 149 VPEQLEYAKKRMADAVGYAGQKGKGMGQHVQNKAQEAKQYDISKTHDTTTKG-HETTQRTAAA---------- BnS4-2 150 VPEQLEYAKKRMADAVGYAGQKGKEMGQHVQNKAHEAKQYDISKTHDTTTTKGHETTQRTAAA---------- BnS4-3 155 VPDQLEYAKRRMADAVGYAGQKGKEMGQFVQDKAHDAKQYDISKPQDTTTTTTTTTKGHETRTAAA------- BnS4-4 155 VPDQLEYAKRRMADAVGYAGQKGKEVGQFVQDKAHDAKQYDISKPHDTTTTTTTTTKGLETRTAAA------- AtS4 138 VPEQLEYAKRRMADAVGYAGQKGKEMGQHVQNKAQDVKQYDISKPHDTTTKGHETQGRTTAA-----------

PXXXXXSPXXXP (KNOT)

H-form insertion

S3 (OLEO1)

S5 (OLEO3)

S1

S2 (OLEO4)

S4 (OLEO2)

Figure 1. Alignment of

complete amino acid

sequences from A. thali-

ana and B. napus oleo-

sins. Sequences are

aligned with respect to

the three structural

domains (N-terminal,

central hydrophobic and

C-terminal regions) of

known oleosins. Resi-

dues are numbered from

the putative translation

initiation start codon.

Identical residues are

boxed in black and

similar residues are

shaded in gray. Dashes

indicate gaps introduced

in the sequences to

allow maximal align-

ment. Oleosin families

(S1–S5) are indicated on

the right side. Sequen-

ces that show the high-

est conservation rate

among the S3 and S4

families are colored two

by two. Stars above the

sequence display the

central hydrophobic

region (72 amino acids).

The four invariable resi-

dues of the proline knot

sequence (-PX5SPX3P-)

are indicated. The 18-

amino acid insertion

present in the C-terminal

end from the high-mole-

cular-mass isoforms (H

oleosin isoforms) is

boxed. Brackets delimit

the region (85 amino

acid positions) used to

build the distance tree

shown in Fig. 2.

Proteomics 2009, 9, 3268–3284 3275

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

coverage, Table 2) probably due to its lower abundance.

Upon scanning of gels and image analysis, an approximate

quantification on the basis of band intensity was obtained

indicating that in Explus and Darmor seeds, oleosins,

caleosins and steroleosins represented together up to 84 and

95% of OB proteins, respectively. As the resolution of the

different oleosins was rather poor, their relative quantifica-

tion was difficult oppositely to arabidopsis oleosins. Rape-

seed oleosins were apparently composed of nearly 13% S4,

85% S1-S2-S3 and 2% S5 orthologs.

Finally, the presence of some other proteins was noted,

indicating a low contamination of OB fraction with protein

bodies. It is the case of storage proteins (napins and cruci-

ferins) and of proteins related to glucosinolate metabolism

(myrosinase-binding proteins, myrosinase-associated

MyAP5). All these proteins have been largely reported

previously [22]. However, in this work, they were identified

only with few trypsin peptides and a low coverage and their

presence was not systematically detected in all experiments

(Table 2). The contamination of OBs with protein bodies

was controlled through the detection of storage proteins

(cruciferins or napins) using immunodetection. The

presence of storage proteins was investigated in OB frac-

tions F1 and F4 obtained after one or four purification steps

(see Section 2). Figure 4 illustrates immunodetection of

either napins (2S) and cruciferins (12S) using anti-2S serum

or specifically napins (2S) using anti-peptide serum. It is

obvious that storage proteins were efficiently discarded

during the course of OB purification.

It is important to keep in mind that only N- and

C-terminal amphipathic oleosin domains possess trypsin clea-

vage sites. To obtain some information about the hydrophobic

part of oleosins, it is necessary to use another digestion strategy.

Proteinase K, an enzyme showing a broad substrate specificity

and able to degrade many proteins in their native state even in

the presence of detergents, was used on purified OB fraction in

a two-step procedure: first, to remove all externally accessible

protein regions and second to digest the transmembrane

protein regions after OBs splitting in the presence of detergent

[28]. Peptides coming from the two amphipathic terminal

domains of oleosins and from the hydrophobic part were

recovered successively. This strategy led to significantly increase

the total protein sequence coverage especially in the case of

oleosins and caleosins (Table 2 and Supporting Information)

0.1

AtT4

CcOLE4 [L]

PdOLEO1TcOLE2 [L]

RcOLE1

AtS4 [H]

HvOLE1 [H]

HvOLE2 [L]

AtS1 [H]

GhOLE-MatP6

SiOLE17 [H]

AtS5 [L]

BnS2-2

CcOLE2 [L]

OsOLE18 [H]

BnS3-4AtS3 [L]

BnS4-2BnS4-1

BnS4-4BnS4-3

AtS2 [H]BnS2-1

BnS1-2BnS1-1

TcOLE1 [H]GhOLE-MatP7

HaOLE2HaOLE1

CcOLE3 [H]OeOLE [H]

GmOLEO2GmOLEO1

ZmOLE17 [H]ZmOLE18 [H]

CcOLE1 [H]SiOLE15.5 [H]

OsOLE16 [L]ZmOLE16 [L]

RcOLE2SiOLE15 [L]

BnS5-2BnS5-1

BnS3-9BnS3-8BnS3-6BnS3-5

BnS3-2BnS3-1

BnS3-7BnS3-3

1000

875

773

611

548

952

1000

809

922

1000

932

533

999

988

794

917

932

861

645

908

919

510

881

604

946

500

990

867

833

1000

726

813

Figure 2. Dendogram of

relationships between

deduced amino acid

sequences from B. napus

seed oleosins and other

plant seed oleosins. A

multiple alignment of

protein sequences covering

85 residues (see Fig. 1) was

generated. The consensus

tree was obtained by

neighbor-joining analysis

using Treecon [46], boot-

strapped with 1000 itera-

tions and rooted by using

the AtT4 sequence which

codes for a tapetum-specific

oleosin [18]. Bootstrap

values are indicated before

nodes when score was over

50%. Accession numbers of

the aligned oleosin sequen-

ces are those given in

Section 2 for most species

and in Table 1 for B. napus.

H- (high molecular weight)

and L- (low molecular

weight) isoforms are indi-

cated when information is

available in the literature.

The group shaded in gray

includes oleosins of the H-

type.

3276 P. Jolivet et al. Proteomics 2009, 9, 3268–3284

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

that could reach up to 60% if both trypsin and proteinase K

digestions were considered. Some peptides from the hydro-

phobic part of the protein were obtained after OB splitting and

proteinase K digestion (Supporting Information). Especially in

the case of BnS2 and BnS3 oleosins, the existence of over-

lapping peptides series identified unambiguously [41] some

parts of their hydrophobic moiety. Interestingly the peptide

YATGEHPQGSDK appeared to belong to the hydrophobic part

of S3 oleosins and was not accessible to trypsin digestion.

3.3 B. napus OB proteins are immunologically

related with A. thaliana OB proteins

Proteins from A. thaliana- or B. napus-purified seed OBs were

separated by SDS-PAGE, transferred onto a PVDF membrane

and submitted to Western blotting using antibodies raised

against oleosins (S1–S5), caleosin (CLO1) and steroleosin

(SLO1) from A. thaliana (Fig. 5). It was observed that all these

specific antibodies cross reacted with rapeseed OB proteins.

Immunoblots indicated that molecular masses of caleosin and

steroleosin from A. thaliana and B. napus were very close. In

contrast to A. thaliana oleosins, S1, S2 and S3 oleosins from

B. napus were not resolved but were all present in the major

protein band, which is consistent with previous results shown

in Fig. 3. Immunoreaction of S5 oleosin with its specific

antibody was very faint. Separations by 2-D PAGE were

assayed taking into account slightly different molecular masses

and pI in protein families. As caleosins and steroleosins are

very minor toward oleosins, they are revealed with difficulty on

the same 2-D gel than oleosins. Therefore, an aliquot of 500mg

of OB protein instead of 50mg was isoelectrofocalized on a

DryStrip cut down on basic end in order to discard oleosins.

Oleosins, caleosins and steroleosins were revealed by immu-

noblotting. Oleosins were rather poorly focalized (Supporting

Information) when two groups of multiple spots were clearly

observed in the case of caleosins and steroleosins (Fig. 6).

These spots could be identified after an LC-MS/MS analysis

and could be assigned to caleosin isoforms (more than seven

spots) and steroleosin isoforms (three spots). In the case of

caleosin spots, all the identified peptides were consensus for

the caleosin family and it was not possible to distinguish

between different isoforms. In the case of steroleosins,

BnSLO1-2, the more acidic protein was identified in the three

spots while BnSLO1-1 was prevailing in the intermediary

spot (spot 2) and BnSLO1-3 appeared very few abundant

(Supporting Information). These results confirmed the exis-

tence of multiple isoforms for these proteins and might also

suggest the occurrence of post-translational modifications.

3.4 Post-translational modifications of OB proteins

Some post-translational modifications such as phosphor-

ylation (M180) of serine, threonine, tyrosine residues or

acetylation (M142) of an N-terminal alanine residue were

systematically investigated in the set of the observed

peptides through MS. In contrast to the previous work,

which has revealed the possible phosphorylation of

A. thaliana CLO1 [31], no phosphorylation was identified

within the peptides recovered after trypsin or proteinase

K digestion protocols. The phosphorylation status of OB

proteins as revealed by a Phos-tag assay is shown in Fig. 7.

Three protein bands (3, 4 and 6 from bottom to top of the

gel) containing caleosins, steroleosins and b-glucosidase

showed phosphorylation. As it can be seen from the mixture

of standard phosphorylated proteins (b-casein and

ovalbumin), the level of phosphorylation of OB proteins

was low.

The examination of protein sequences deduced from the

corresponding cDNA clones revealed that 10 oleosins

among the 15 oleosins identified possessed an N-terminal

alanine after methionine excision. It appeared that

acetylation of N-terminal alanine could not be observed

after trypsin digestion due to the low molecular mass of the

N-terminal trypsin peptides (often lower than 600 Da),

which make them tedious to find by LC-MS analysis.

Hence, we have inquired acetylation of alanine during

the proteinase K digestion progress, which can lead to

larger peptides. We have observed some peptides corre-

sponding to N-terminal peptides with acetylated alanine

(Supporting Information). Hence, it appeared that one

rapeseed S1 oleosin (BnS1-2) and one S2 (T8580) are

acetylated.

3.5

6.0

14.4

21.5

31.036.5

55.466.397.4

116.3

3.5

6.0

14.4

21.5

36.5

66.3

31.0

55.4

7

6

54

3

2

1A

D

E

F

C

B

M OBE OBD M

3.5

6.0

14.4

21.5

31.036.5

55.466.397.4

116.3

3.5

6.0

14.4

21.5

36.5

66.3

31.0

55.4

7

6

54

3

2

1A

D

E

F

C

B

Figure 3. SDS-PAGE of proteins from OB fraction purified from

Explus (E, 10mg) or Darmor (D, 10mg) seeds. Molecular mass

marker was Mark 12 (M) from Novex. Protein bands were

numbered as listed in Table 2 (columns 6 and 8).

Proteomics 2009, 9, 3268–3284 3277

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Tab

le2.

Ass

ign

men

tso

fp

rote

ins

fro

mp

uri

fied

OB

sfr

om

Darm

or

or

Exp

lus

rap

ese

ed

cult

ivars

a)

Pro

tein

nam

eb

)T

IGR

c) /

Sw

iss-

Pro

tc)

Th

eo

rM

Md

) /p

Id)

A.th

ali

an

ah

itId

en

tity

e) /

sim

ilari

ty(%

)

Darm

or

Exp

lus

AA

g) /

tota

lco

vera

ge

g)(%

)

LC

-MS

/MS

f)

Pep

g) /c

ov

g) %

(ban

d)h

)

2-D

LC

-MS

/M

Si)

Pep

g) /c

ov

g) %

LC

-MS

/MS

f)

Pep

g) /c

ov

g) %

(ban

d)h

)

Pro

tK

j)

Pep

g) /

cov

g) %

Bn

S5-1

15.6

/10.0

At5

g51210/S

580/8

43/1

6.9

(A)

––

3/1

5.5

40/2

7.0

Bn

S1-1

TC

5680

20.6

/8.3

At3

g01570/S

184/9

12/1

0.4

(B)

7/1

8.7

6/1

9.3

(1)

3/1

5.1

66/3

4.4

Bn

S1-2

TC

5691

20.6

/8.3

83/9

23/1

1.4

(B)

4/1

4.1

8/2

1.3

(1)

5/2

2.9

52/2

7.1

Bn

S2-1

TC

6327

19.9

/9.2

At3

g27660/S

280/8

66/1

7.6

(B)

11/2

7.8

11/3

1.5

(1)

14/2

5.1

100/5

3.5

Bn

S2-2

TC

14535

19.7

/7.1

80/8

79/3

9.6

(B)

12/4

5.4

13/4

1.2

(1)

8/2

4.6

99/5

2.9

TC

8580

18.9

/9.1

73/8

27/2

5.3

(B)

8/2

7.5

8/2

5.8

(1)

7/2

2.5

72/4

0.4

TC

5946

20.1

/7.8

82/9

09/4

3.7

(B)

13/4

7.4

11/4

0.0

(1)

17/3

3.1

108/5

6.8

Bn

S3-2

TC

14667

19.4

/9.2

At4

g25140/S

388/9

12/1

1.7

(B)

4/2

1.8

6/2

0.7

(1)

21/4

4.1

94/5

2.5

Bn

S3-4

TC

14082

20.5

/9.4

84/8

64/1

0.7

(B)

2/1

0.7

3/1

6.1

(1)

14/3

2.2

73/3

9.2

Bn

S3-5

/Bn

S3-6

TC

6242

19.9

/9.3

86/8

73/2

2.5

(B)

3/2

2.5

2/5

.5(1

)11/2

5.3

76/4

1.7

Bn

S3-7

21.4

/9.3

82/8

43/1

0.3

(B)

2/1

0.3

3/1

5.5

(1)

13/3

9.2

92/4

7.4

Bn

S3-8

/Bn

S3-9

TC

7507

20.4

/9.1

83/8

51/6

.5(B

)4/1

6.2

1/8

.6(1

)12/3

1.9

83/4

4.9

Bn

S4-1

TC

5860

21.9

/9.5

At5

g40420/S

479/8

67/2

4.9

(C)

9/1

9.6

9/1

2.9

(2)

3/1

3.9

69/3

3.0

Bn

S4-3

TC

6891

22.9

/8.8

76/8

15/1

8.7

(C)

10/4

0.6

13/3

7.9

(2)

11/2

9.2

107/4

8.8

Bn

S4-4

TC

5798

22.9

/9.1

75/8

113/4

7.9

(C)

11/3

1.0

19/4

9.8

(2)

9/2

3.3

124/5

6.6

Bn

CLO

128.1

/5.6

-6.2

At4

g26740/

CLO

191/9

54/1

3.1

(D)

–2/7

.7(3

)7/2

4.9

87/3

5.5

Bn

SLO

1-1

38.9

/7.2

At5

g50600/

86/9

43/1

2.9

(E)

1/3

.27/2

6.4

(4)

2/5

.1166/4

7.6

Bn

SLO

1-2

39.0

/6.3

SLO

189/9

53/1

2.9

(E)

2/5

.712/4

1.0

(4)

3/8

.6209/5

9.9

Bn

SLO

1-3

TC

8344

38.1

/7.1

85/9

23/7

.6(E

)4/1

5.2

6/2

3.2

(4)

2/4

.7140/4

1.0

Bn

SLO

2-2

TC

11781

51.7

/6.9

At4

g10020/

SLO

291/9

4–

3/1

0.6

–1/1

.556/1

2.1

Nap

in1.7

Sla

rge

chain

P01090

9.1

/9.1

At4

g27150

––

–2/1

4.8

12/1

4.8

Cru

cife

rin

CR

U4b

ssu

nit

P33522

20.8

/8.6

At1

g03880

–1/6

.9–

–13/6

.9P

ero

xir

ed

oxin

Q9M

7C

123.9

/6.0

At1

g48130

––

–2/6

.013/6

.0m

yr-

ass

oc

pro

tQ

39308

41.8

/8.5

At1

g54020

––

7/2

0.2

(5)

2/4

.692/2

4.8

Cru

cife

rin

CR

UA

P11090

51.3

/6.6

At5

g44120

––

–2/5

.827/5

.8b

Glu

cosi

dase

Q42618

56.2

/6.0

At3

g21370

2/7

.1(F

)–

2/6

.1(6

)–

65/1

3.2

HS

P82

P55737

80.0

/4.9

At5

g56030

–2/3

.9–

–27/3

.9m

yr-

bin

din

gp

rot

Q96340

99.4

/5.5

At1

g52030

––

1/1

.3(7

)–

13/1

.3

a)

Iden

tifi

cati

on

was

carr

ied

ou

to

nLC

-MS

/MS

an

aly

sis

of

pep

tid

es

ob

tain

ed

aft

er

tryp

sin

or

pro

tein

ase

Kd

igest

ion

.b

)P

rote

inn

am

eas

refe

rred

toT

ab

le1.

c)A

ccess

ion

nu

mb

er

as

rep

ort

ed

inT

IGR

or

Sw

iss-

Pro

td

ata

base

s.d

)T

heo

reti

cal

valu

es

for

mo

lecu

lar

mass

an

dis

oele

ctri

cp

oin

tca

lcu

late

dfr

om

their

am

ino

aci

dse

qu

en

ceu

sin

gE

xP

AS

yp

rote

om

ics

too

ls.

e)

Seq

uen

ces

ali

gn

men

tw

ith

A.

thali

an

ase

qu

en

ces

was

carr

ied

ou

tto

calc

ula

teid

en

tity

an

dsi

mil

ari

tyle

vels

.f)

In-g

el

dig

est

ion

of

pro

tein

ban

ds

by

tryp

sin

.g

)N

um

ber

of

pep

tid

es

(pep

)o

ram

ino

aci

ds

(AA

)re

covere

dfo

reach

pro

tein

.S

eq

uen

ceco

vera

ge

(co

v)

was

calc

ula

ted

taki

ng

into

acc

ou

nt

am

ino

aci

ds

iden

tifi

ed

an

dm

atc

hed

toam

ino

aci

dn

um

ber

of

pro

tein

s.h

)B

an

dn

um

ber

as

sho

wn

inFig

.3.

i)T

ryp

sin

dig

est

ion

of

tota

lp

rote

inextr

act

.j)

Dig

est

ion

of

pu

rifi

ed

OB

sw

ith

pro

tein

ase

Kin

two

step

s(s

ee

Sect

ion

2).

3278 P. Jolivet et al. Proteomics 2009, 9, 3268–3284

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

4 Discussion

4.1 Sequences coding for OB proteins are conserved

between arabidopsis and rapeseed

The proximity between the Brassicaceae genomes allowed

us to develop a candidate gene approach starting from

arabidopsis knowledge to identify the rapeseed sequences

coding for OB proteins. Taking advantage of the availability

of a seed-enriched cDNA library that was used to generate

ESTs [29], we were able to identify 19 sequences coding for

oleosins, five for steroleosin-like proteins and nine for

caleosin-like proteins, with high level of amino acid

conservation with their arabidopsis counterparts. In addi-

tion to DNA sequence conservation, the protein 1-D struc-

ture was also conserved as shown for oleosins (Fig. 1).

Compared with other plant proteins, arabidopsis and rape-

seed hits displayed the closest protein sequences (Fig. 2).

Further work will include the functional validation of the

rapeseed sequences in planta to ensure that they represent

true functional orthologs from arabidopsis ones. For each

arabidopsis hit at least two rapeseed sequences were

retrieved, which can be explained not only by the polyploidy

nature of the B. napus genome but also by the presence of

numerous duplications of chromosomal portions into the

rapeseed genome. For instance, nine sequences coding for

S3 oleosins were identified during the progress of the

present study. Therefore, one further question to be

addressed is the contribution of each of the rapeseed S3

protein to the biogenesis and/or the stability of the seed OBs.

In B. napus genome, both H- and L-oleosins were

retrieved, with a relative balance between the two forms

since 11 oleosin genes belong to the L-class and eight to the

H-form. In the same way we have previously observed that

in A. thaliana seeds the most abundant oleosin was the S3

L-oleosin isoform [7], representing 46% of the total oleosin

content (unpublished data). However, Lee et al. [57] have

observed that H- and L-oleosin isoforms accumulate in OBs

regardless of the dosage of functional H- and L-genes. From

experiments using artificial reconstituted OBs, Tai et al. [19]

suggested that H-oleosins were less stabilizing than L-oleo-

sins. These authors therefore speculated that the H-forms

were involved in specialized biological functions such as the

OB mobilization during seed germination.

4.2 Oleosins are the major components of rapeseed

OBs but steroleosins and caleosins are also

associated with OB proteome

Oleaginous seeds contain major storage proteins besides

OBs. Napins and cruciferins from protein storage vesicles

account for �20 and 60% of total protein in mature rape-

seed, respectively [58]. Hence, the purification of OBs to

homogeneity is difficult to achieve [22, 33]. In this work, the

OB preparation used repeated and numerous floatation

steps. Proteins non-specifically associated with OBs were

removed by detergent washing, ionic elution and finally urea

treatment. Triglycerides from defective OBs were removed

by hexane extraction. Integrity of purified OBs was verified

under fluorescence or electronic microscopy [8]. Finally,

only few proteins were visible from OB fraction (Fig. 3) and

the contamination with storage proteins or proteins involved

in the glucosinolate metabolism was relatively low in

contrast to the results of Katavic et al. [22]. The removal of

napins and cruciferins was monitored by Western blotting

using specific antibodies (Fig. 4).

Fifteen different oleosins were identified by the combination

of several proteomic approaches in purified OBs from rapeseed,

showing high sequence conservation (higher than 75% identity)

with A. thaliana oleosins. Among these 15 oleosins, 13 were

predicted from the Genoplante database and two from a cana-

dian database. It was not possible to distinguish BnS3-5 from

BnS3-6 and BnS3-8 from BnS3-9 through the identified

peptides due to the fact that their respective sequences differ

only with one amino acid. On the contrary it was possible to

identify unambiguously the presence of BnS5-1, BnS3-2 and

BnS3-4 from the peptides obtained after trypsin or proteinase K

digestion (Supporting Information). However, the presence of

BnS5-2, BnS3-1 or BnS3-3 cannot be definitely discarded.

Rapeseed oleosins constituted up to 90% of OB protein content.

In contrast to A. thaliana oleosins, they were not finely resolved

under SDS-PAGE due to their very similar molecular mass,

except for S4 and S5 proteins. All rapeseed oleosins were heavier

Figure 4. Immunodetection of storage proteins in OB fractions

obtained after one (F1) or four (F4) steps of purification. Two

concentrations of protein were systematically assayed and

resolved by SDS-PAGE before immunoblot analysis. The blots

were probed (left) with anti-2S serum, which cross reacts with

rapeseed napins (2S) and cruciferins (12S) or (right) with napin

anti-peptide serum, which specifically cross reacts with napins.

Detection was performed using chemiluminescence. Molecular

masses are indicated in kDa (M, MagicMarkTM XP Western

Protein Standard from Invitrogen).

Proteomics 2009, 9, 3268–3284 3279

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

than the A. thaliana oleosins except for S2. Only four oleosins

were described in rapeseed by Murphy’s team [25, 59–61]. BnV,

BnIII and napII were recognized in our experiments among S3

orthologs as BnS3-9, BnS3-7 and BnS3-4, respectively. It

appeared that BnS2-2 corresponded to napI described only

partially by Murphy [25]. Katavic et al. [22] have found only three

different oleosins, two corresponding to BnS3-4 and BnS3-9,

respectively, and one S2 ortholog. Then in this work we iden-

tified 11 supplementary oleosins. No oleosin from the SM

group [18] was retrieved in B. napus seed OBs.

Proteins highly homologous to hydroxysteroid dehy-

drogenase from A. thaliana (85–89% identity, 92–95%

similarity) or S. indicum (steroleosin-A, 59–61% identity,

Figure 5. Specificity of anti-oleosin, anti-caleosin and anti-steroleosin sera toward oleosins, caleosins and steroleosins from A. thaliana or

B. napus seeds. Proteins (0.1–5 mg) from purified OBs were solubilized in Laemmli buffer and resolved by SDS-PAGE before immunoblot

analysis. The blots were probed with anti-rS1N, -rS2N, -rS3N, -rS4, -rS5N, -CLO1 and -SLO1 sera. Detection was performed using

chemiluminescence. Molecular masses are given in kDa.

Figure 6. Immunodetection of caleosins and steroleosins in OB

fraction (500 mg protein) resolved by 2-D PAGE. IEF was carried

out with an 18 cm, pH 3–10, ImmobilineTM DryStrip, which was

slightly cut down at each end to avoid the trouble caused by very

basic and abundant oleosins. The blot was probed with anti-

CLO1 and -SLO1 sera. Detection was performed using chemilu-

minescence. Molecular masses (kDa) and rough pI are indicated.

ββ

Figure 7. SDS-PAGE of proteins purified from Darmor OB frac-

tion (5 mg) and of a mixture of bovin b-casein, chicken ovalbumin

and BSA (S, 10mg). Molecular mass marker was Mark 12 (M)

from Novex. Protein bands were stained either (left) with

Coomassie blue or (right) with Phos-tag 300/460 phosphoprotein

gel stain (Perkin-Elmer) and imaged on a UV transilluminator.

Protein bands were numbered as listed in Table 2 (column 8) and

in Fig. 3.

3280 P. Jolivet et al. Proteomics 2009, 9, 3268–3284

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

76–77% similarity) were identified in OBs from B. napus.Proteomic analyses allowed unambiguous identification of

the three steroleosins SLO1 found in genomic Brassicadatabases. These proteins with close molecular masses

differed slightly in their amino acid composition and

exhibited different pI. As they cross reacted with specific

antibody raised against A. thaliana SLO1, they were detected

by immunoblotting after 2-D electrophoresis (Fig. 6). Spot

distribution excludes the existence of artifacts due to

proteolysis phenomenon. The same behavior in 2-D PAGE

has been reported previously [22]. Another protein ortholog

to a putative dehydrogenase reductase from A. thaliana or to

S. indicum steroleosin-B was also identified. This protein

(SLO2), which has been only characterized with cloning and

nucleotide sequence in A. thaliana and S. indicum [56], has

never been described earlier in rapeseed. BnSLO2 did not

cross react with anti-SLO1 serum.

Despite the presence of numerous genes (nine) encoding

proteins very homologous to A. thaliana embryo-specific

protein ATS1, we could not identify this protein in previous

work [8]. The present work confirms that caleosins are asso-

ciated with rapeseed OBs. Proteomic analysis did not permit

one to distinguish caleosin isoforms because the identified

trypsin peptides were conserved in the caleosin family.

Protein sequences diverge each other by less than ten amino

acids, leading to similar molecular masses and slightly

different pI (5.6–6.2). A combination of 2-D electrophoresis

and immunodetection using anti-CLO1 serum showed several

spots of caleosin (Fig. 6) in contrast to the result of Katavic

et al. [22] identifying rapeseed ATS1 as a single spot.

4.3 Proteomic analysis gives some information

about the structure of OB integral proteins

Oleosins contain three distinct structural domains: a central

hydrophobic anchoring domain, highly conserved and

devoid of cleavage site for trypsin digestion, and two N- and

C-terminal amphipathic domains. Recovered peptides

through LC-MS/MS analysis after trypsin digestion belon-

ged only to the two amphipathic domains of the oleosins.

Thus, in order to obtain some information about boundaries

of the hydrophobic part of oleosins, we used an alternative

strategy via proteinase K digestion [28]. With this method, a

better coverage of oleosins was achieved with the identifi-

cation of peptides originating from the hydrophobic part of

oleosins and the lower accessibility of this domain embed-

ded within the phospholipid monolayer was confirmed.

Surprisingly we observed that the peptide KYAT-

GEHPQGSD belonging to the C domain of S3 oleosin

orthologs was released by proteinase K after OBs splitting in

the presence of detergent, which suggests that this sequence

could be in the hydrophobic part of the protein contrary to

the result given by Kyte–Doolittle hydropathy plot [62] and to

the topology model hypothesized for A. thaliana S3 oleosin

by Abell et al. [63]. The N-terminal domain of rapeseed S4

oleosins is particularly enriched in glycine residues, which

increase its hydrophobic character and flexibility, leading

possibly to a better coating of OBs.

We could observe acetylation of the N-terminal alanine

residue of at least two oleosins (BnS1-2 and T8580).

N-terminal acetylation is a co-translational modification

found in 50–80% of eukaryotic proteins [64]. In plants,

N-terminal methionine excision is essential and carried out

by two types of Met aminopeptidases functionally inter-

changeable [65]. N-terminal acetylation generally enhances

protein stability and impedes protein turnover rate. Lin et al.[66] have speculated that N-terminal acetylation of oleosins,

which contribute to the structural stability of seed OBs,

prevents ubiquitinated degradation of these proteins to fulfill

the biological function of long-term protection of the OBs.

The presence of multiple spots on 2-D electrophoresis

gels in the case of steroleosins and caleosins might reveal

the existence of post-translational modifications. Phosphor-

ylation on serine, threonine or tyrosine residues was

systematically investigated in this work by MS without any

positive result. On the contrary, phosphorylation of caleo-

sins, steroleosins and b-glucosidase was revealed using a

specific Phos-tag staining assay (Fig. 7). Considering, on the

one hand, the low level of phospho staining and, on the

other hand, the absence of phosphopeptides detection with

proteomic strategy, it appears that the level of phosphor-

ylation of rapeseed OB proteins is rather low. In silicosequence analysis revealed the presence of numerous

potential phosphorylation sites: 9–10 for caleosins, 14–19 for

steroleosins and 29 for b-glucosidase, regularly distributed

all over the sequences. In the same way, only a partial

phosphorylation of Ser225, a highly conserved casein kinase

CK2 phosphorylation site in the C-terminal part of A.thaliana CLO1, was previously described [31] when AtCLO1

protein sequence shows six potential phosphorylation sites.

In conclusion, a combination of complementary approa-

ches allowed us to obtain new information about rapeseed

OB proteome, i.e. identification of all the integral proteins

and preliminary information about their insertion in OBs

and their post-translational modifications. The establish-

ment and the use of a specific rapeseed protein database

from cDNA collection generated from developing seeds

contributed powerfully toward this work. Fifteen different

oleosins (among them eleven have never been previously

suspected) were identified and the existence of S5 oleosin

ortholog was clearly established. The existence of three

steroleosin 1 isoforms was established by proteomic analysis

and immunodetection as well as the presence of a new

protein, we named steroleosin 2 or SLO2, ortholog to a

putative dehydrogenase reductase from A. thaliana or to

steroleosin-B from S. indicum. Caleosins exhibiting a high

endogenous heterogeneity were at last identified. Post-

translational modifications of these proteins were detected

(acetylation of some oleosins, low level of phosphorylation of

steroleosins and caleosins) but must be now confirmed. It

would be interesting to characterize the kinetic properties of

Proteomics 2009, 9, 3268–3284 3281

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

B. napus steroleosins, determine their specificity using

labeled steroids as model substrates and classify these

proteins among the steroid dehydrogenases family. Further

work in our labs is now focused on the study of OB proteins

during seed development and their specific role in the

biogenesis of OBs. For polyploidy species such as B. napus,the production of null mutants is challenging. As oleosins,

caleosins and steroleosins belong to multigene families,

RNAi technology could be more powerful and will be

applied to allow gene expression reduction of all genes at the

same time.

The authors thank Genoplante for free access to the rapeseedcDNA clones. This work was carried out with the financialsupport of the ANR ‘‘Agence Nationale de la Recherche-TheFrench National Agency’’ under the ‘‘Programme National derecherche Genoplante’’ project ‘‘ANR-05-GPLA-023-01’’. Theyalso thank Audrey Geairon (INRA, UR 1268, BISB Platform,Nantes) for excellent technical assistance in mass spectrometryanalyses.

The authors have declared no conflict of interest.

5 References

[1] Hsieh, K., Huang, A. H. C., Endoplasmic reticulum, oleosins,

and oils in seeds and tapetum cells. Plant Physiol. 2004, 136,

3427–3434.

[2] Murphy, D. J., The biogenesis and functions of lipid bodies

in animals, plants and microorganisms. Prog. Lipid Res.

2001, 40, 325–438.

[3] Siloto, R. M. P., Findlay, K., Lopez-Villalobos, A., Yeung, E. C.

et al., The accumulation of oleosins determines the

size of seed oilbodies in Arabidopsis. Plant Cell 2006, 18,

1961–1974.

[4] Rajjou, L., Debeaujon, I., Seed longevity: survival and

maintenance of high germination ability of dry seeds. C. R.

Biol. 2008, 331, 796–805.

[5] Murphy, D. J., Cummins, I., Seed oil-bodies: isolation,

composition and role of oil-body apolipoproteins. Phyto-

chemistry 1989, 28, 2063–2069.

[6] Huang, A. H., Oleosins and oil bodies in seeds and other

organs. Plant Physiol. 1996, 110, 1055–1061.

[7] Jolivet, P., Roux, E., D’Andrea, S., Davanture, M. et al.,

Protein composition of oil bodies in Arabidopsis

thaliana ecotype WS. Plant Physiol. Biochem. 2004, 42,

501–509.

[8] Jolivet, P., Tailliart, K., Boulard, C., Nesi, N., Chardot, T.,

Purification and protein composition of oil bodies from

Brassica napus seeds. O.C.L. 2006, 13, 426–430.

[9] Peng, C. -C., Tzen, J., Analysis of the three essential

constituents of oil bodies in developing sesame seeds. Plant

Cell Physiol. 1998, 39, 35–42.

[10] Beisson, F., Ferte, N., Noat, G., Oil-bodies from sunflower

(Helianthus annuus L.) seeds. Biochem. J. 1996, 317, 955–956.

[11] Tzen, J. T., Lai, Y. K., Chan, K. L., Huang, A. H., Oleosin isoforms

of high and low molecular weights are present in the oil bodies

of diverse seed species. Plant Physiol. 1990, 94, 1282–1289.

[12] Beisson, F., Ferte, N., Bruley, S., Voultoury, R. et al., Oil-

bodies as substrates for lipolytic enzymes. Biochim.

Biophys. Acta 2001, 1531, 47–58.

[13] Simkin, A. J., Qian, T., Caillet, V., Michoux, F. et al., Oleosin

gene family of Coffea canephora: quantitative expression

analysis of five oleosin genes in developing and germinat-

ing coffee grain. J. Plant Physiol. 2006, 163, 691–708.

[14] Guilloteau, M., Laloi, M., Blais, D., Crouzillat, D., Mc Carthy,

J., Oil bodies in Theobroma cacao seeds: cloning and

characterization of cDNA encoding the 15.8 and 16.9 kDa

oleosins. Plant Sci. 2003, 164, 597–606.

[15] Eastmond, P. J., Cloning and characterization of the acid lipase

from castor beans. J. Biol. Chem. 2004, 279, 45540–45545.

[16] Tzen, J. T., Huang, A. H., Surface structure and properties of

plant seed oil bodies. J. Cell Biol. 1992, 117, 327–335.

[17] Leprince, O., Van Aelst, A., Pritchard, H., Murphy, D.,

Oleosins prevent oil-body coalescence during seed imbibi-

tion as suggested by a low-temperature scanning electron

microscope study of dessication-tolerant and -sensitive

oilseeds. Planta 1997, 204, 109–119.

[18] Kim, H. U., Hsieh, K., Ratnayake, C., Huang, A. H., A novel

group of oleosins is present inside the pollen of Arabi-

dopsis. J. Biol. Chem. 2002, 277, 22677–22684.

[19] Tai, S. S., Chen, M. C., Peng, C. C., Tzen, J. T., Gene family

of oleosin isoforms and their structural stabilization in

sesame seed oil bodies. Biosci. Biotechnol. Biochem. 2002,

66, 2146–2153.

[20] Tzen, J. T., Chuang, R. L., Chen, J. C., Wu, L. S., Coexistence

of both oleosin isoforms on the surface of seed oil bodies

and their individual stabilization to the organelles.

J. Biochem. 1998, 123, 318–323.

[21] Wu, L. S. H., Hong, G. H. H., Hou, R. F., Tzen, J. T. C.,

Classification of the single oleosin isoform and character-

ization of seed oil bodies in gymnosperms. Plant Cell

Physiol. 1999, 40, 326–334.

[22] Katavic, V., Agrawal, G. K., Hajduch, M., Harris, S. L., Thelen,

J. J., Protein and lipid composition analysis of oil bodies from

two Brassica napus cultivars. Proteomics 2006, 6, 4586–4598.

[23] d’Andrea, S., Jolivet, P., Boulard, C., Larre, C. et al., Selec-

tive one-step extraction of Arabidopsis thaliana seed oleo-

sins using organic solvents. J. Agric. Food Chem. 2007, 55,

10008–10015.

[24] Li, M., Smith, L. J., Clark, D. C., Wilson, R., Murphy, D. J.,

Secondary structures of a new class of lipid body proteins

from oilseeds. J. Biol. Chem. 1992, 267, 8245–8253.

[25] Murphy, D. J., Keen, J. N., O’Sullivan, J. N., Au, D. M. et al., A

class of amphipathic proteins associated with lipid storage

bodies in plants. Possible similarities with animal serum

apolipoproteins. Biochim. Biophys. Acta 1991, 1088, 86–94.

[26] Hernandez-Pinzon, I., Patel, K., Murphy, D. J., The Brassica

napus calcium-binding protein, caleosin, has distinct

endoplasmic reticulum- and lipid body-associated

isoforms. Plant Physiol. Biochem. 2001, 39, 615–622.

3282 P. Jolivet et al. Proteomics 2009, 9, 3268–3284

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

[27] Rabilloud, T., Membrane proteins ride shotgun. Nat.

Biotechnol. 2003, 21, 508–510.

[28] Wu, C. C., MacCoss, M. J., Howell, K. E., Yates

J. R., III, A method for the comprehensive proteomic

analysis of membrane proteins. Nat. Biotechnol. 2003, 21,

532–538.

[29] Rouquie, D., Lancelot, M. -E., Kreboul, G., Chataigner, V.

et al., The Genoplante oilseed rape chip. 7th Int. Cong. Plant

Mol. Biol. Barcelona, Spain 2003.

[30] d’Andrea, S., Canonge, M., Beopoulos, A., Jolivet, P. et al.,

At5g50600 encodes a member of the short-chain dehy-

drogenase reductase superfamily with 11beta- and 17beta-

hydroxysteroid dehydrogenase activities associated with

Arabidopsis thaliana seed oil bodies. Biochimie 2007, 89,

222–229.

[31] Purkrtova, Z., d’Andrea, S., Jolivet, P., Lipovova, P. et al.,

Structural properties of caleosin: a MS and CD study. Arch.

Biochem. Biophys. 2007, 464, 335–343.

[32] Devouge, V., Rogniaux, H., Nesi, N., Tessier, D.

et al., Differential proteomic analysis of four near-isogenic

Brassica napus varieties bred for their erucic acid and

glucosinolate contents. J. Proteome Res. 2007, 6,

1342–1353.

[33] Tzen, J. T., Peng, C. C., Cheng, D. J., Chen, E. C., Chiu, J. M.,

A new method for seed oil body purification and examina-

tion of oil body integrity following germination. J. Biochem.

1997, 121, 762–768.

[34] Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J.,

Protein measurement with the Folin phenol reagent. J. Biol.

Chem. 1951, 193, 265–275.

[35] Laemmli, U. K., Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature 1970,

227, 680–685.

[36] Neuhoff, V., Arold, N., Taube, D., Ehrhardt, W., Improved

staining of proteins in polyacrylamide gels including

isoelectric focusing gels with clear background at nano-

gram sensitivity using Coomassie Brilliant Blue G-250 and

R-250. Electrophoresis 1988, 9, 255–262.

[37] Lauriere, M., A semidry electroblotting system efficiently

transfers both high- and low-molecular-weight proteins sepa-

rated by SDS-PAGE. Anal. Biochem. 1993, 212, 206–211.

[38] Jiang, X., Jiang, X., Han, G., Ye, M., Zou, H., Optimization of

filtering criterion for SEQUEST database searching to

improve proteome coverage in shotgun proteomics. BMC

Bioinformatics 2007, 8, 323.

[39] Strader, M. B., Tabb, D. L., Hervey, W. J., Pan, C.,

Hurst, G. B., Efficient and specific trypsin digestion of

microgram to nanogram quantities of proteins in

organic-aqueous solvent systems. Anal. Chem. 2006, 78,

125–134.

[40] Eng, J. K., McCormack, A. L., Yates, J. R., An approach to

correlate tandem mass spectral data of peptides with amino

acid sequences in a protein database. J. Am. Soc. Mass

Spectrom. 1994, 5, 976–989.

[41] MacCoss, M. J., Wu, C. C., Yates J. R., III, Probability-based

validation of protein identifications using a modified

SEQUEST algorithm. Anal. Chem. 2002, 74, 5593–5599.

[42] Zybailov, B. L., Florens, L., Washburn, M. P., Quantitative

shotgun proteomics using a protease with broad specificity

and normalized spectral abundance factors. Mol. Biosyst.

2007, 3, 354–360.

[43] Roux, E., Baumberger, S., Axelos, M. A., Chardot, T.,

Oleosins of Arabidopsis thaliana: expression in Escherichia

coli, purification, and functional properties. J. Agric. Food

Chem. 2004, 52, 5245–5249.

[44] Berot, S., Compoint, J. P., Larre, C., Malabat, C., Gueguen,

J., Large scale purification of rapeseed proteins (Brassica

napus L.). J. Chromatogr. B Analyt. Technol. Biomed. Life

Sci. 2005, 818, 35–42.

[45] Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J. et al.,

Gapped BLAST and PSI-BLAST: a new generation of protein

database search programs. Nucleic Acids Res. 1997, 25,

3389–3402.

[46] Van de Peer, Y., De Wachter, R., TREECON for Windows: a

software package for the construction and drawing of

evolutionary trees for the Microsoft Windows environment.

Comput. Appl. Biosci. 1994, 10, 569–570.

[47] Cummins, I., Murphy, D. J., cDNA sequence of a sunflower

oleosin and transcript tissue specificity. Plant Mol. Biol.

1992, 19, 873–876.

[48] Thoyts, P. J., Millichip, M. I., Stobart, A. K., Griffiths, W. T.

et al., Expression and in vitro targeting of a sunflower

oleosin. Plant Mol. Biol. 1995, 29, 403–410.

[49] Kalinski, A., Loer, D. S., Weisemann, J. M., Matthews, B. F.,

Herman, E. M., Isoforms of soybean seed oil body

membrane protein 24 kDa oleosin are encoded by closely

related cDNAs. Plant Mol. Biol. 1991, 17, 1095–1098.

[50] Chen, P. -W., Chai, Y. -J., Wang, L. -D., Tzen, J. et al., Two

embryo-specific cDNAs (accession No. U43930 and

U43931) encoding two oleosin isoforms on the surface

of oil bodies from rice (PGR95-143). Plant Physiol. 1996, 110,

714.

[51] Lee, K., Huang, A. H., Genes encoding oleosins in maize kernel

of inbreds Mo17 and B73. Plant Mol. Biol. 1994, 26, 1981–1987.

[52] Hughes, D. W., Wang, H. Y., Galau, G. A., Cotton (Gossy-

pium hirsutum) MatP6 and MatP7 oleosin genes. Plant

Physiol. 1993, 101, 697–698.

[53] Aalen, R. B., The transcripts encoding two oleosin isoforms

are both present in the aleurone and in the embryo of barley

(Hordeum vulgare L.) seeds. Plant Mol. Biol. 1995, 28,

583–588.

[54] Giannoulia, K., Banilas, G., Hatzopoulos, P., Oleosin gene

expression in olive. J. Plant Physiol. 2007, 164, 104–107.

[55] Garcia-Mas, J., Messeguer, R., Arus, P., Puigdomenech, P.,

Molecular characterization of cDNAs corresponding to genes

expressed during almond (Prunus amygdalus Batsch) seed

development. Plant Mol. Biol. 1995, 27, 205–210.

[56] Lin, L. J., Tzen, J. T., Two distinct steroleosins are present in

seed oil bodies. Plant Physiol. Biochem. 2004, 42, 601–608.

[57] Lee, K., Ratnayake, C., Huang, A. H., Genetic dissection of

the co-expression of genes encoding the two isoforms of

oleosins in the oil bodies of maize kernel. Plant J. 1995, 7,

603–611.

Proteomics 2009, 9, 3268–3284 3283

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

[58] Hoglund, A. S., Rodin, J., Larsson, E., Rask, L., Distribution

of napin and cruciferin in developing rape seed embryos.

Plant Physiol. 1992, 98, 509–515.

[59] Murphy, D. J., Storage lipid bodies in plants and other

organisms. Prog. Lipid Res. 1990, 29, 299–324.

[60] Keddie, J. S., Edwards, E. W., Gibbons, T., Shaw, C. H.,

Murphy, D. J., Sequence of an oleosin cDNA from Brassica

napus. Plant Mol. Biol. 1992, 19, 1079–1083.

[61] Keddie, J. S., Hubner, G., Slocombe, S. P., Jarvis, R. P. et al.,

Cloning and characterisation of an oleosin gene from

Brassica napus. Plant Mol. Biol. 1992, 19, 443–453.

[62] Kyte, J., Doolittle, R. F., A simple method for displaying the

hydropathic character of a protein. J. Mol. Biol. 1982, 157,

105–132.

[63] Abell, B. M., High, S., Moloney, M. M., Membrane protein

topology of oleosin is constrained by its long hydrophobic

domain. J. Biol. Chem. 2002, 277, 8602–8610.

[64] Brown, J. L., Roberts, W. K., Evidence that approximately

eighty per cent of the soluble proteins from Ehrlich ascites cells

are Nalpha-acetylated. J. Biol. Chem. 1976, 251, 1009–1014.

[65] Ross, S., Giglione, C., Pierre, M., Espagne, C., Meinnel, T.,

Functional and developmental impact of cytosolic protein

N-terminal methionine excision in Arabidopsis. Plant

Physiol. 2005, 137, 623–637.

[66] Lin, L. J., Liao, P. C., Yang, H. H., Tzen, J. T., Determination

and analyses of the N-termini of oil-body proteins, ster-

oleosin, caleosin and oleosin. Plant Physiol. Biochem. 2005,

43, 770–776.

3284 P. Jolivet et al. Proteomics 2009, 9, 3268–3284

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com