Differential Proteomic Analysis of Four Near-Isogenic Brassica napus Varieties Bred for their Erucic Acid and Glucosinolate Contents Vanessa Devouge,† He´ le` ne Rogniaux,† Nathalie Ne´ si,‡ Dominique Tessier,† Jacques Gue´ guen,† and Colette Larre´ *,† INRA Centre de Nantes, BIA, Rue de la Ge´raudie`re, BP 71627, 44316 Nantes, France, and INRA Centre de Rennes, APBV, UMR118, INRA-AgroCampus Rennes, BP 35327, 35653 Le Rheu, France Received September 4, 2006
Four near-isogenic B. napus varieties, with decreasing amounts of erucic acid and glucosinolates reflecting the actual breeding process, were used to characterize the proteins affected during this process. Following improvement of 2-DE conditions, proteins differentially accumulated were identified by mass spectrometry analysis. Accumulation of cruciferins was found to be only slightly affected, whereas significant quantitative differences were mainly found for proteins involved in defense system and carbohydrate metabolism. Keywords: Brassica napus • seed • proteome • erucic acid • glucosinolates • 2-D-electrophoresis • mass spectrometry
Introduction Oleaginous seeds such as soybean, rape, and sunflower are of importance for both human food and animal feed. Rapeseed (Brassica napus) is the major oilseed produced in the European Union with a seed production of about 15 Mtonnes in 2004/ 2005. This production is expected to expand, thanks, in large part, to the EU’s nascent biodiesel production industry. Rapeseed was subjected to intensive selective breeding in the 1970s to improve seed quality. These efforts resulted in obtaining a canola-quality rapeseed (double-low “00”, “zero erucic acid, zero glucosinolates”) with the reduction in two toxic compounds: the erucic acid in the edible oil was eliminated, and the glucosinolate level was substantially decreased in the meal for feedstock uses.1,2 These improvements were primarily made through breeding research programs that paid little attention to the protein composition of meals, which is nevertheless responsible for amino acid intake.3 Currently, the remaining content of glucosinolates in seeds of rapeseed commercial varieties is about 12 µmol/g of seed,4 and the reduction of glucosinolate contents or their complete elimination remains a breeding goal.5 More recently, because of the low cost of vegetable oil production, interest in engineering enhanced seed oil quantity and quality for industrial uses has prompted new breeding projects.6 With the exception of those concerning glucosinolates, most breeding programs are focused on oil quantity or composition. Concerning the protein content trait, it is generally considered to be negatively correlated with oil content.7,8 Despite the increasing production of meals from various oleaginous crops, only little information is available concerning the impact of * To whom correspondence should be addressed. Dr Colette Larre´. E-mail:
[email protected]. Fax: 33(0)2 40 67 50 25. † INRA Centre de Nantes. ‡ INRA Centre de Rennes.
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breeding on seed protein composition. Most of the studies on rapeseed proteins were dealing with their uses as meal for animal feeding or with their potential uses for food and nonfood applications. Sixty percent of seed proteins are storage proteins of the 12S globulin- and 2S albumin-type (12S also called cruciferin and 2S also called napin, respectively). Their structure and physicochemical properties, as well as their chemical or enzymatic modifications,9 have been widely investigated, leading to a better understanding of the molecular bases of their functionalities.10-13 Surprisingly, only two studies deal with the variability of the protein composition in rapeseed. They reported that the cruciferin/napin ratio (12S/2S) increased in modern double low varieties (“00”) than in old varieties rich in both erucic acid and glucosinolates.14,15 This lack of information prompted us to evaluate in the present study the effects of the elimination of erucic acid on one hand and of the decrease in glucosinolates on the other hand on the wholeseed proteome of Brassica napus. The characterization of whole-plant protein composition was made possible with proteomics during this past decade16 and, more recently, characterizations of seed tissues. Seed-specific proteomic studies have provided reference maps on maize, medicago, and barley,17-19 as well as information on seed filling20 and Arabidopsis germination process.21,22 In the case of Brassica napus, a complete proteomic analysis of wholeseed protein expressed at five developmental stages revealed the metabolic pathways operating during seed filling.23 Whereas structural and biologically active proteins remain minor, 12S globulins accumulate in large amounts from mid-maturation and are highly represented in mature seed proteins. Although total seed proteome comparison between genotypes has never been investigated within Brassica, the capability of two-dimensional electrophoresis (2-DE) to distinguish between genotypes has been extensively demonstrated.24-29 A study performed on 21 maize (Zea mays L.) inbred lines 10.1021/pr060450b CCC: $37.00
2007 American Chemical Society
Effect of Breeding on B. napus Seed Proteome
Figure 1. Pedigree of the four varieties studied. Varieties that contain both erucic acid and glucosinolates are noted (++), those selected for elimination of erucic acid are quoted (0+), and varieties without erucic acid and lower amount of glucosinolates are noted (00).
revealed 70 markers, based on protein shifts revealed by 2-D PAGE, that were used to describe the pedigree relationships between lines.30 In the case of Arabidopsis, the comparison of 2-D protein maps from eight ecotypes revealed only 25% of common spots from an average of 250 detected spots and up to 10% of specific spots for one ecotype.31 A proteomic analysis comparing 32 potato genotypes revealed that most of the detected proteins showed significant qualitative and quantitative differences between genotypes.32 On the other hand, in the same study, very few protein variations were found between genetically modified lines of potato and their parental controls.32 Taken together, these results suggest that genotypic variations could be extensively and well reflected by proteome analyses. In the present study, we used an improved 2-DE approach to compare the seed proteome of four Brassica napus varieties that differ in their oil erucic acid content and/or their glucosinolates level. These genotypes were derived from a nearisogenic family to minimize the protein variations due to genotypic factors other than those induced by breeding for low erucic acid and glucosinolate contents. In a first step, we optimized the protein extraction procedures and quality of 2-DE for proteomic analysis. In a second step, a quantitative comparison of the four Brassica napus genotypes was performed and the differentially accumulated proteins were identified by mass spectrometry. The results are then discussed with regard to the breeding process.
Materials and Methods Plant Material and Growth Conditions. Four winter-type Brassica napus varieties, namely Gaspard, JetNeuf, Darmor, and Darmor-bzh, that belong to a near-isogenic family, generously provided by the Joint Research Unit, “Plant Improvement and Biotechnologies”, of the INRA Research Centre in Rennes, France, were used in this study. The pedigree of the 4 varieties is summarized in Figure 1. Briefly, Gaspard, that contains high levels of both erucic acid and glucosinolates (noted “++”) was developed from a cross between Matador and Hambourg. A cross between Gaspard and Primor, that carries a low erucic allele, followed by 3 backcrosses with Gaspard leads up to
research articles JetNeuf (noted “0+”). A similar strategy was undertaken to reduce the glucosinolate content in JetNeuf, using a Polish spring genotype (Bronowski), and leads up to Darmor (noted “00”). In a final step, a dwarf Darmor line (referred to as Darmor-bzh) was obtained after a cross between Darmor and a dwarf Primor, followed by three backcrosses with Darmor. Darmor-bzh displays a mutation in the BZH gene that encodes a member of the GRAS gene family.33 Plants were grown under controlled and reproducible conditions (16-hours photoperiod; 18-20 °C night/20-22 °C day temperature). Flowers were pollinated manually and tagged using the day of pollination. Selfing bags were put on the inflorescences to avoid cross pollination. Dry mature seeds were harvested at 60 days after pollination. Seeds were removed from the siliques and immediately frozen in liquid nitrogen. Samples were stored at -80 °C before analyses. All samples were produced in triplicate. Preparation of Protein Extract and Protein Quantification. Total protein extracts were prepared from dry mature Brassica napus seeds. The three methods (A, B, C) described below were used to extract proteins from total seed or after delipidation. For delipidation, seeds were ground in dichloromethane at 4 °C and centrifuged (7000 g for 10 min). (A) Direct extraction in an aqueous solution: seeds or defatted pellets were carefully ground in 20 µL per mg of seeds of an extraction buffer using a mortar and a pestle, and then centrifuged. Extraction buffers containing various chaotropic agents and detergents were used: UC (8 M Urea, 2% Chaps, 18 mM DTT), UCT (8 M Urea, 2% Chaps, 2 M Thiourea, 18 mM DTT) and UCT-C80 (8 M Urea, 2% Chaps, 2 M Thiourea, 18 mM DTT, 2% ASB C8Φ). After 2 h shaking at room temperature, the extracts were centrifuged (13 000 g for 30 min). Supernatants were removed and centrifuged a second time for 30 min. (B) Extraction with aqueous-phenol followed by protein precipitation from the phenol phase according to Hurkman and Tanaka.34 (C) Extraction of non-proteic material prior to protein solubilization: seeds (or defatted pellets) were ground at 4 °C in a buffer containing 10% TCA and 0.07% β-mercaptoethanol in 100% acetone. The pellet was treated according to Chan et al.35 and resuspended in a UCT or UCT-C80 protein extraction medium. All chemicals were of pure grade and purchased from Amersham Pharmacia Biotech (Uppsala, Sweden) and Sigma Chemical (St. Louis, MO), except ASB C8Φ, which was purchased from VWR international (Strasbourg, France). Extractions were performed at least in triplicate, and protein quantification of each extract was carried out with the Non Interfering Protein Assay (Geno Technology, St Louis, MO), according to the supplier’s recommendations. 2-D Electrophoresis. Two-dimensional polyacrylamide gel electrophoresis (2-DE) were performed using the IPGphor Isoelectric Focusing System (Amersham Pharmacia Biotech, Uppsala, Sweden) for the first dimension, and the Hoefer DALT System (Amersham Pharmacia) for the second dimension. Prior to IEF, protein extracts were adjusted to a volume of 350 µL by the addition of Destreak Rehydration Solution (Amersham Pharmacia) containing 40 mM 2-hydroxyethyl disulfide (SigmaAldrich) and 0.5% v/v IPG Buffer (Amersham Pharmacia). Proteins were then loaded on immobilized pH gradient gel strips (18-cm Immobiline Dry Strip, Amersham Pharmacia) and IEF was performed at 20 °C. Prior to the second dimension, Journal of Proteome Research • Vol. 6, No. 4, 2007 1343
research articles each gel strip was incubated, first with 6 mL equilibration buffer (50 mM Tris HCl, pH 8.8, 6 M urea, 30% v/v glycerol, 2% SDS) containing 64 mM DTT for 10 min and, subsequently, in the same buffer containing 135 mM iodoacetamide. Proteins were separated in the second dimension either on 10% or on 15% polyacrylamide gels. Protein Staining and Analysis of 2-Dimensional Gel Electrophoresis. For qualitative evaluation of the extraction methods, gels were stained either by Coomassie Blue or by silver nitrate according to Blum et al.36 The four varieties were quantitatively compared using gels stained with Coomassie Brilliant Blue G250 (Sigma-Aldrich). Briefly, proteins were first fixed 2 × 1 h in 50% ethanol, 2% phosphoric acid. Gels were then washed 1 h in 2% phosphoric acid. Gels were submitted to a sensitization step for 20 min in 17% ethanol, 15% ammonium sulfate, and 2% phosphoric acid, and 0.1% Coomassie Brilliant blue was then added to this buffer. After 3 days of staining, gels were washed 10 min in deionized water, 10 min in 20% ethanol, and finally, 10 min in deionized water. For each variety, at least four replicated 2-D gels were made. The relationship between the amount of loaded proteins and the number of detected spots was examined by increasing the amounts of protein loaded from 50 to 800 µg. Up to 300 µg of proteins, the number of spots increased almost linearly with the amount of loaded proteins, from 56 ( 7 to 286 ( 20. For higher loadings (500 and 800 µg), the gain in the number of detected spots was weaker and induced losses of 20% and 40% of expected spots from the linear relationship obtained from lower loadings. Moreover, streakings appeared in the gel and numerous spots overlapped, especially for the most abundant spots inducing a lower spot resolution. The gels were all characterized by the presence of six to ten very intense spots compared to the others. The linear range of spot quantification was examined for these spots in order to define the optimal amount of proteins to load to perform a differential quantitative analysis. For all major spots, a linear relationship between spot intensity and protein load was obtained up to 300 µg loaded with R values varying in the range of 0.88 to 0.99. It was not possible to avoid a saturating effect of these proteins while maintaining effective detection of others. Therefore, the whole quantitative differential study was performed using two electrophoretic conditions, one to focus on major proteins the other to focus on minor proteins. Image acquisition was performed using a GS-710 Imaging Densitometer (BioRad) with a resolution of 300 micron pixel-1. The normalization step, spot detection, and spot quantification were performed using ImageMaster 2-D Platinum software (Amersham Biosciences). For all varieties, relative quantification of the detected spots was made in percent of total spot volume for each gel (% vol) that allowed normalization. Replicated gels for each extraction or for each genotype were matched to each other and only spots detected in at least three replicates were used to create master gels. For quantification of minor proteins, some spots were detected as non Gaussian peak and therefore considered as saturated spots, they correspond to the major seed storage proteins and were removed in silico as described by Salekdeh et al., 2002.37 ANOVAs were performed to assess significant differences between varieties. A multiple Range Test using the method of Least Significant Differences at 99% confidence level was used to determine which pair of factor level was significant. Statistical analysis were performed using ImageMaster 2-D Platinum software or StatGraphics (STATGRAPHICS Plus 5.1, Statistical Graphics Corp). 1344
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Protein Identification by Mass Spectrometry. Protein spots whose expression profiles were significantly different between the varieties were picked up manually and prepared for mass spectrometry. Protein Hydrolysis. Protein spots of approximately 1 mm3 were picked up manually and in gel digested prior to mass spectrometry. Briefly, gel spots were washed with 100 µL of 25 mM NH4HCO3, followed by 100 µL of 50% acetonitrile in 25 mM NH4HCO3. Proteins were then reduced by incubation with 10 mM DTT (1 h, 57 °C), and alkylated with 100 µL of 55 mM iodoacetamide (45 min at room temperature). Gel spots were further washed as described above and were finally dried in a vacuum centrifuge. The proteins were digested overnight at 37 °C by the addition of 10-20 µL of Trypsin (12.5 ng/µL in 25 mM NH4HCO3; modified trypsin purchased from Promega, Madison, WI). The resulting peptide mixture was acidified by the addition of 1 µL of an aqueous solution of formic acid (1%, vol.), and stored at -20 °C until analysis. Peptide Mass Fingerprinting by MALDI MS Analysis. MALDITOF mass spectrometry experiments were performed on a M@LDI-LR instrument (Micromass/Waters, Manchester, UK), equipped with a conventional laser at 337 nm. One microlitre of the peptide mixture was mixed with 1 µL of the matrix preparation (5 g/L R-cyano-4-hydroxy-cinnamic acid, 5 g/L 2,5dihydroxybenzoic acid, 70% [v/v] acetonitrile and 0.1% [v/v] trifluoroacetic acid), and spotted onto the MALDI sample probe. Mass data acquisitions were processed with Masslynx software (Micromass/Waters). LC-MS/MS Analysis. Nanoscale capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the digested proteins were performed using a SwitchosUltimate capillary LC system (LC Packings/Dionex, Amsterdam, The Netherlands), coupled with a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-TOF Global, Micromass/Waters, Manchester, UK). Chromatographic separation was conducted on a reverse-phase capillary column (Pepmap C18, 75-µm i.d., 15-cm length, LC Packings) at a flow rate of 200 nL min-1. The gradient consisted of a linear increase from 2 to 50% of acetonitrile in 50 min, followed by a rapid increase to 60% of acetonitrile within 10 min. Mass data acquisitions were processed with Masslynx software (Micromass/Waters) using the so-called “data dependent acquisition” mode: MS data were recorded for 1 s on the massto-charge (m/z) range of 400-1500, after which the most intense ions were selected and fragmented in the collision cell (MS/MS measurements) for 8 s; MS/MS data were recorded on the m/z range of 50-1500. Databank Searches. Mass data were processed with Protein Lynx Global Server software (Micromass/Waters). Protein identification was achieved by searching mass data in the UniProt/Swiss-Prot and UniProt/TrEMBL databanks (10-012006), or in the TIGR Gene Indices databank (Brassica napus: release: 29-09-2004). Results from the three databank searches were compared in order to achieve final protein identification. For MALDI-MS data, a peptide mass tolerance of 100 ppm was used, and a minimum of seven peptides of the spectrum matched to the protein was required for validation. Unidentified proteins or ambiguous identifications at this stage were submitted to further LC-MS/MS analysis. In the case of LCMS/MS data, a mass tolerance of 100 ppm was applied to the parent ion and 0.2 Da to the fragment ions. Each identification was manually inspected and validated. Known amino acid
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Effect of Breeding on B. napus Seed Proteome
Table 1. Seed Characteristics: Seed Weight as Well as Their Contents in Proteins, Lipids, Erucic Acid, and Glucosinolates Are Given for Each of the Four Varietiesa
Gaspard JetNeuf Darmor Darmor-bzh a
seed weight
lipid content (% weight of seed)
protein content (% weight of seed)
glucosinolates (µmol/g)
erucic acid (% of total fatty acids)
5.42a ( 0.23 4.85b ( 0.31 5.60a ( 0.57 4.57b ( 0.68
45.80 ( 2.31 44.22 ( 3.72 46.32 ( 3.08 44.07 ( 2.40
20.48 ( 0.55a 22.04 ( 0.51b 19.14 ( 0.31c 19.08 ( 0.62c
83.0 ( 6.3a 72.2 ( 11.8a 22.4 ( 1.8b 21.2 ( 2.3b
51.1 ( 0.4a 1.5 ( 1.0b 0.2 ( 0.1b 0.6 ( 0.6b
Means with the same letter in the same column are not statistically different; a, b, and c pointed statistically different valor for Student test P < 0.01.
modifications were eventually allowed in a second phase, to increase the sequence coverage of identified proteins. Western Blot Analysis. Rabbit polyclonal serum against 12S cruciferin was obtained by sub Cutaneous (S.C.) immunization with the purified protein obtained according to Be´rot et al. 2005.38 One mg of 12S in PBS (0.010 mol/L Na2HPO4, 0.15 mol/L NaCl, pH7.2), emulsified with complete (first injection) or incomplete (boost injections) Freund’s adjuvant, was injected every 15 days. The serum was collected after the fourth injection. Proteins (500 µg) were separated on 15% acrylamide gels as described in the “2-D procedure” and then incubated 10 min in 20% ethanol containing 25 mM Tris base, 192 mM Glycine, and 0.1% (w/v) SDS. Proteins were transferred (1 h, 500 mA) on nitrocellulose membrane using a Dalt Blotting Kit (Amersham Biosciences) and in the same buffer. Membranes were saturated for 1 h in 150 mL of Phosphate Buffer Salt (PBS, 0.15 mM NaCl, 2.68 mM KCl, 1.46 mM KH2PO4, 8.10 mM Na2HPO4) containing 5% defatted milk. Membranes were then washed 2 × 10 min in PBS containing 0.05% (v/v) Tween 20, prior to a 1 h incubation in PBS-Tween containing a 1/5000e anti-12S Rabbit antibody. Membranes were then washed for a second time, 2 × 10 min, in PBS containing 0.05% (v/v) Tween 20 prior to a 1 h incubation in PBS containing a 1/3000e anti-Rabbit antibody/HRP. Finally, membranes were incubated 2 × 10 min in PBS-Tween and one time for 10 min in PBS prior to HRP 4CN detection.
Results The four varieties were characterized by their seed weights and by their total contents in erucic acid, glucosinolates, lipids and proteins (Table 1). The reduction in erucic acid levels, followed by the decrease in glucosinolate levels, was effective across the four near-isogenic varieties used in this study. The total lipid content, when expressed in % of seed weight, did not significantly vary across the four varieties. Concerning the protein content JetNeuf exhibited the highest value, followed by Gaspard, and then by Darmor and Darmor-bzh which were not statistically different from each other. Optimization of Extraction Procedures for Proteomic Analysis. For optimizing proteomic analysis, five different extraction procedures (Methods: A using three buffers, B, and C) were checked on seeds of the Gaspard genotype; the protein recovery yields and the quality of the 2-D electrophoretic profiles, evaluated by the number of detected spots and resolution, were compared on total or defatted seeds. The extraction yield of the different methods is given in Table 2. It varies significantly depending on the method, with extraction yields ranging from 37 to more than 90% of the total protein content of the seed. The best results were obtained with UCT and UCT-C80 extraction methods. Since lipids represent close to 50% of the seed weight, the influence of a delipidation step was investigated. Regardless
Table 2. Comparison of Extracted Proteins in Relation with the Extraction Procedure Using Gaspard Dry Mature Seedsa method of extraction
delipidation step
extraction yield (% of total proteins)
UC UC UCT UCT UCT-C80 UCT-C80 TCA TCA PHENOL PHENOL
no yes no yes no yes no yes no yes
67.11 ( 10.81 58.28 ( 1.44 90.81 ( 4.05 77.02 ( 1.44 99.54 ( 6.84 76.12 ( 3.06 68.11 ( 2.16 57.83 ( 0.36 52.52 ( 0.54 37.20 ( 6.66
a The delipidation was performed with dichloromethane. Quantification of the corresponding amounts of extracted proteins was performed with NI Protein Assay. UC: 8M Urea-2% Chaps; UCT: 8M Urea-2% Chaps-2M thiourea; UCT-C80: 8M Urea-2% Chaps-2M thiourea-2% sulfobetain ASBC80; TCA: trichloracetic acid precipitation; PHENOL: phenol separation.
of the extraction procedure, the delipidation resulted in a loss of the extracted proteins that could be as high as 29% of the extracted proteins without delipidation. The delipidation step resulting in a considerable loss of extractable proteins, the extraction of proteins will be consequently made on non defatted seeds. The 2-D electrophoretic patterns were compared for the nondefatted extracts obtained by the various extraction procedures described above, except for the UC method, which gave us the poorest results for protein recovery when compared with UCT and UCT C80 methods. All gels were loaded with the same amount of proteins, i.e., 300 µg for Coomassie blue staining and 100 µg for silver staining respectively. The silver staining was only performed to validate the results obtained from the Coomassie blue staining. The total number of detected spots slightly varied depending on the extraction method: 332 ( 11 for UCT, 324 ( 13 for UCT-C80, 309 ( 9 for TCA and 324 ( 14 for the phenol extraction method; the percentage of matched spots between replicates varied from 83 to 94%. The comparison of the master gels by pair did not show any unique spots except for the TCA extraction method. Although this extraction method led to less detected spots than the other methods, four unique spots were observed (Figure 2). The sum of their volumes represented 2.06 ( 0.09% of the total volume of spots. All four were identified as beta subunits of cruciferin. The other modes of extraction did not induce qualitative differences of the spot patterns, but quantitative variations in the spots intensity could be observed. This is particularly striking in the case of high molecular weight basic proteins, which were extracted more efficiently by the phenol method. As the UCT-C80 method gave the higher protein extraction yield and a number of spots similar to phenol method, it was chosen for further analysis. Since the seed proteins were almost exhaustively extracted with UCT-C80 (Table 2), the relative Journal of Proteome Research • Vol. 6, No. 4, 2007 1345
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Figure 2. Electrophoretic profiles of total protein extract of Gaspard dry mature seeds obtained after different extraction methods. (A) Electrophoretic profile of spots on 15% acrylamide gel showing the four differential extracted spots. (B, C, D, and E) Enlargement of the area of interest in relation with extraction procedure with (B) UCT extraction, (C) UCT-C80, (D) TCA, (E) Phenol. Molecular markers are in kDa. Black arrow pointed on spots which corresponded to differentially extracted proteins. Proteins were separated on IPG strip pH 3-10NL followed by a 15% acrylamide SDS-PAGE.
intensity of spots in two-dimensional gels is expected to reflect the relative amount of the corresponding proteins in the seed. Optimization of 2-DE for Quantitative Differential Analysis. Electrophoretic conditions were optimized, on the one hand, to increase the number of detected proteins in a wide pI and molecular weight range and, on the other hand, to make possible the further quantitative comparison of spots between the varieties. Therefore, the optimization was done on the protein loading and on the electrophoretic conditions. Gels that were loaded with protein amounts until 200 µg displayed very faint spots and no saturated spot. At higher loadings, some saturated spots occurred. Therefore a loading of 200 µg of proteins was chosen for differential quantitative analysis of the most intense spots. However, using this loading, most of the minor spots, especially present in the top part of the gel, were very weak or undetectable. The use of 10% acrylamide gels added to a higher load (500 µg) made it possible to improve the resolution of these minor spots and also to increase the total number of spots to 495 ( 1346
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40. Under these particular conditions, the saturated spots were found at the bottom, on the basic side of the gels. In order to be able to perform differential quantitative analysis, these saturated spots were manually removed from the whole set of images and the remaining spots re-normalized as described in Materials and Methods. Differential Analysis of Four Brassica napus Varieties Revealed Only Quantitative Differences. Seed protein extracts were prepared using the UCT-C80 method for the four varieties. The extraction yields varied between 82 and almost 99% (data not shown); the best yields were obtained for Gaspard and Darmor, which display the heaviest seeds and also the higher amount of lipids (Table 1). Seed Storage Protein Analysis. Differential analysis was carried out on 15% acrylamide gels using 200 µg protein loads, in order to focus on major spots mainly represented by the seed storage proteins. First of all, Western blot analyses were performed to localize the 12S globulin subunits on 2-D-gels using specific anti-12S antibodies. Western blot analysis revealed more than 20 spots corresponding to cruciferin after protein separation on pH 3-10 NL gradient gels (Figure 4). Some of them were picked on gels and submitted to mass spectrometry analysis for identification in order to validate the antibody specificity. The alpha polypeptides were identified in fourteen spots corresponding to seven isoforms, while beta polypeptides were found in nine spots. Two additional spots were identified as seed storage protein-like. Several other proteins detected as cruciferin subunits by Western blotting were confirmed to belong to this globulin family on the basis of consensus peptides determined by mass spectrometry but unfortunately could not be undoubtedly identified as a specific isoform due to the lack of discriminating peptides (data not shown). These results confirm the specificity of the antibody used here, it can be noted that this antibody raised against native 12S forms appeared to better recognize the alpha subunits of the 12S. These 12S globular proteins are hexameric and it is well-known that the alpha polypeptides are located in the periphery of the structure while the beta polypeptides are inside and also more hydrophobic. These elements can explain a better recognization of the alpha polypeptides. As breeding for erucic acid and glucosinolates seed content was shown to affect the 12S/2S ratio in the seed,14,15 we were interested to know whether it would also affect the differential expression of cruciferin isoforms. For the four varieties, sets of gels were submitted to image analysis. Statistical analysis revealed that two spots were differentially expressed between the varieties (spots 686, 688). They were both identified as cruciferin beta polypeptides (Table 3). Their percentage of volume range from 0.005 to 1.82% of the total spots volume but it is worth noting that the sums of the volume of these spots were not statistically different between the four varieties; they represented less than 3% of the total volume of spots detected in these conditions. By comparison, the whole of the spots identified as cruciferin accounted for 32.2 ( 2.9, 25.2 ( 5.6, 27.18 ( 4.3, and 30.08 ( 1.6% of the total spots volume on gels for Gaspard, JetNeuf, Darmor, and Darmor-bzh, respectively. Nonstorage Protein Analysis. For each variety, differential analysis was carried out with an equivalent of 500 µg of proteins on a 10% acrylamide set of gels, in order to focus on the high molecular weight proteins. A slightly but significantly different number of spots was detected between Gaspard, JetNeuf, Darmor, and Darmor-bzh with 495 ( 40, 456 ( 29, 562 ( 8,
Effect of Breeding on B. napus Seed Proteome
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Figure 3. Immunolocalization of 12S globulins by Western blot. (A) Western blot was performed with anti 12S globulin antibodies. (B) Proteins were separated on IPG strips 3-10 NL, in denaturating conditions and submitted to a second migration step on 15% acrylamide gels prior Coomassie Blue staining. Molecular weight marker are shown next to the gels. Spots identified as 12S globulins alpha subunit by mass spectrometry analysis are pointed in yellow and beta subunit in pink. The two differentially detected spot of cruciferin (spot 686 and 688) are pointed by black lanes and their spot ID.
Figure 4. Map of protein extract of Brassica napus mature seed (variety JetNeuf)- Localization of identified spots of protein with a differentially expression profile between the four varieties studied. IEF was performed on pH 3-10NL strips. Proteins were separated in denaturizing conditions on 10% acrylamide gels prior Coomassie Blue staining. Molecular weight marker are shown next to the gels. Spots are pointed with black lanes and their group ID.
and 604 ( 32 spots, respectively. The matching was performed between Gaspard and JetNeuf, JetNeuf and Darmor and at last between Darmor and Darmor-bzh. The number of common spots was respectively : 442, 453, 552. The nonmatched spots after a visual control being discarded because they were either incompletely focused or in streakings. Nevertheless, among the common spots, the ANOVA pointed out 69 spots having different average % of volume among the studied varieties. A further multiple range test at 0.01 confidence level revealed that
the larger number of different spots (55) was found between Gaspard (++) and JetNeuf (0+), that differ in their acid erucic content while JetNeuf (0+) and Darmor (00) that differ in their glucosinolate contents presented only 25 different spots. On the other hand, the two “00” varieties, Darmor and Darmorbzh, differed by only 11 spots. Out of the 69 spots, we identified 36 proteins (Figure S5, Supporting Information) listed in Table 3. Only one part of the results of the multiple range analysis is shown, the nonidentified spots are not presented and we chose to focus on the significant differences observed between Gaspard (++) and JetNeuf (0+) then on JetNeuf (0+) and Darmor (00) (Table 4). As expected, Darmor-bzh was shown to be very close to Darmor in terms of protein composition. In fact, only seven differentially accumulated proteins were identified, three of them are myrosinase binding protein (spots 2907, 3166, 3175), one myrosinase precursor (spot 2802), one beta glucosidase (spot 2759), one glyceraldehyde 3-phosphate dehydrogenase (spot 3065), plus one of unknown function (spot 3201). Thirty-two differentially expressed proteins were identified between Gaspard and JetNeuf. Thirty of them were significantly overexpressed in JetNeuf. The two others (spots 688 and 3166), identified as 12S seed storage protein and myrosinase binding protein, respectively, were detected at higher level in the ancestor variety. In Gaspard (++), the beta-glucosidase class 1 (TC 358 in the TIGR gene indices) was less accumulated. This protein was detected in seven very faint spots in Gaspard, whereas many of these spots were very intense spots in JetNeuf, Darmor, and Darmor-bzh (spots 2759, 2760, 2762, 2763, 2764, 2774, and 2781). Other spots belonging to proteins implicated in carbohydrate pathways such as glyceraldehyde 3-phosphate dehydrogenase (spots 3049, 3065 and 3067), and alcohol dehydrogenase (spot 3014), were detected at lower levels in Gaspard. That was also the case for thirteen proteins that participated in the plant detoxification/defense system such as myrosinases (spots 2795, 2802), myrosinase binding protein (spots 2565, 2575, 2579, 2580, 2905, 2906, 2907), epithiospecifier protein (spot 3032), peroxiredoxin antioxidant protein (spot Journal of Proteome Research • Vol. 6, No. 4, 2007 1347
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Table 3. Identified Spots with a Differential Expression Pattern between the Four Brassica napus Varietiesa no. of spots
2759 2760 2762 2763 2764 2774 2781 3014 3049 3065 3067
identification
TC358 homologue to beta glucosidase TC358 homologue to beta glucosidase TC358 homologue to beta glucosidase TC358 homologue to beta glucosidase TC358 homologue to beta glucosidase TC358 homologue to beta glucosidase TC358 homologue to beta glucosidase alcohol dehydrogenase G3PC_ARATH glyceraldehyde 3P dehydrogenase cytosolic TC 121 - G3PC_SINAL glyceraldehyde 3P dehydrogenase cytosolic TC 121 - G3PC_SINAL glyceraldehyde 3P dehydrogenase cytosolic
2625 2626 2738
elongation factor 2 elongation factor 2 TC 505, TC348, TC 783 HSP 70
686
cruciferin beta subunit (CRU1_BRANA group) TC1082 similar to cruciferin BNC2 beta subunit, complete
688
2565 2575 2579 2580 2795 2802 2904 2905 2906 2907 3032 3166 3175 3258 3279 3283 3289
myrosinase binding protein myrosinase binding protein myrosinase binding protein myrosinase binding protein TC114 - MYRO_BRANA myrosinase precursor TC114 - MYRO_BRANA myrosinase precursor TC713 - similar to myrosinase binding protein_ incomplete TC713 - similar to myrosinase binding protein_ incomplete TC713 - similar to myrosinase binding protein_ incomplete TC713 - similar to myrosinase binding protein_ incomplete TC 1614 similar to epithiospecifier TC731 - similar to myrosinase binding protein TC731 - similar to myrosinase binding protein TC 563 peroxiredoxin antioxidant TC551 similar to GSH-dependent dehydroascorbate reductase 1,putative TC625 - similar to Q9ZRW8 gluthatione transferase glutathione S transferase PM24
2982 3183
TC 293 Actin7 (Actin 2) TC883 - homologue to T24C10.1 protein (At1g54870/F14C21_16)
3201
TC 215 - similar to unknown protein MRA 19
a
matching protein
% cov
carbohydrate pathway Q42618 38.97 Q42618 37.5 Q42618 39.71 Q42618 35.66 Q42618 43.01 Q42618 16.72 Q42618 37.54 Q9SEQ0 7.95 Q42352 18.04
matched peptides
theor. pI
theor. MW
exp. pI
exp. MW
20 18 18 18 20 10 16 4 5
6.4 6.4 6.4 6.4 6.4 6.4 6.4 5.8 6.6
62140 62140 62140 62140 62140 62140 62140 41279 36914
7.4 7.7 7.2 6.7 6.9 7.9 8.1 6.1 7.2
61267 61104 61022 61762 61350 61185 60940 38062 36893
P47096
40.98
13
6.3
39931
7.7
36778
P47096
48.36
15
6.3
39931
8.2
36770
18 24 8 9 8
5.8 5.8 -
94185 94185 -
6.4 6.6 4.5
95050 94916 68563
protein biosynthesis Q9SGT4 20.57 Q9SGT4 28.25 Q40980, 35.77 O48563, 14.05 Q9HLA8 storage functions 27.89 P33524
20.52
defense/detoxification Q96340 30.12 Q96340 39.90 Q96340 21.13 Q96340 35.01 Q00326 47.58
6
-
-
5
9.2
20959
20 24 13 22 26
5.5 5.5 5.5 5.5 6.6
99463 99463 99463 99463 62695
4.9 5.3 5.2 5.1 6.3
110425 111157 112389 110182 57607
Q00326
35.06
20
6.6
62695
6.1
57453
Q9LIF8
23.79
6
6.0
49724
6.1
47566
Q9LIF8
21.37
11
6.0
49724
6.8
47607
Q9LIF8
42.74
10
6.0
49724
6.2
47443
Q9LIF8
29.43
8
6.0
49724
6.5
47443
Q93VB6 Q9LIF8
13.67 54
5 13
5.7 5.8
38890 31484
5.4 6.4
37092 31547
Q9LIF8
45.33
10
5.8
31484
6.0
31434
Q9M7C1 Q9RWR4
43.45 68.20
8 14
6.0 5.9
26378 23956
5.4 5.5
24683 23641
Q9ZRW8
45.29
12
5.8
25650
6.4
23441
P46422 19.9 other proteins P53492 42.85 Q9FZ42 22.06
7
5.9
23997
7.2
23271
13 8
5.2 6.1
41793 31522
4.5 6.2
40356 31182
unknown proteins Q941A4 87.35
31
5.9
28544
5.9
29558
Identifications were performed either by MALDI MS or LC MS/MS analysis.
3258), and GSH-dependent dehydroascorbate reductase (spot 3279) or glutathione S-transferase (spot 3289). Out of the six remaining spots, three corresponded to proteins implicated in protein biosynthesis (sp. 2625, 2626, and 2738) and were identified as elongation factors or heat shock proteins. The 1348
Journal of Proteome Research • Vol. 6, No. 4, 2007
spots 2982 (actin), 3183 (TC883) and 3201 (unknown protein) were equally less detected in Gaspard. To access the potential effect of breeding for low glucosinolate content, the comparative analysis of the electrophoretic profiles between JetNeuf and Darmor was performed and led
research articles
Effect of Breeding on B. napus Seed Proteome
Table 4. List of Differentially Detected Spots between Gaspard (G) and JetNeuf (JN) and JetNeuf (JN) Minus Darmor (D)a spot id
protein identification
G minus JN
JN/G
JN minus D
D/JN
2580 2625 2626 2738 2759 2762 2763 2764 2781 2795 2802 2905 2982 3014 3032 3049 3065 3067 3201 3258 3279 3289 688 2565 2575 2579 2760 2774 2906 2907 3166 3183 686 2904 3283
myrosinase binding protein elongation factor 2 elongation factor 2 TC 505, TC348, TC 783 HSP 70 TC358 homologue to beta glucosidase TC358 homologue to beta glucosidase TC358 homologue to beta glucosidase TC358 homologue to beta glucosidase TC358 homologue to beta glucosidase TC114 - MYRO_BRANA -myrosinase precursor TC114 - MYRO_BRANA -myrosinase precursor TC713 - similar to myrosinase binding protein_ incomplete TC 293 Actin7 (Actin 2) alcohol dehydrogenase TC 1614 similar to epithiospecifier G3PC_ARATH glyceraldehyde 3P dehydrogenase cytosolic TC 121 - G3PC_SINAL - glyceraldehyde 3P dehydrogenase cytosolic TC 121 - G3PC_SINAL - glyceraldehyde 3P dehydrogenase cytosolic TC 215 - similar to unknown protein MRA 19 TC 563 peroxiredoxin antioxidant TC551 similar to GSH-dependent dehydroascorbate reductase 1,putative glutathione S transferase PM24 TC1082 similar to cruciferin BNC2 beta subunit, complete myrosinase binding protein myrosinase binding protein myrosinase binding protein TC358 homologue to beta glucosidase TC358 homologue to beta glucosidase TC713 - similar to myrosinase binding protein_ incomplete TC713 - similar to myrosinase binding protein_ incomplete TC731 - similar to myrosinase binding protein TC883 - homologue to T24C10.1 protein (At1g54870/F14C21_16) cruciferin beta subunit (CRU1-BRANA group) TC713 - similar to myrosinase binding protein_ incomplete TC625 - similar to Q9ZRW8 gluthatione transferase
-0.202 -0.202 -0.064 -0.081 -1.255 -1.024 -0.135 -0.436 -0.14 -0.144 -0.085 -0.324 -0.075 -0.109 -0.126 -0.091 -0.197 -0.173 -0.312 -0.214 -0.271 -0.156 0.302 -0.565 -0.146 -0.24 -0.348 -0.194 -0.255 -0.459 0.393 -0.247 -0.29 0.006 -0.11
1.9 1.4 1.6 1.5 14.4 14.7 5.3 5.4 28.0 1.7 1.3 4.0 1.9 1.7 1.7 2.2 1.9 2.2 1.4 1.5 2.1 1.6 0.0 3.9 2.0 2.4 10.2 5.9 2.0 3.6 0.4 1.3 1.4 1.0 1.3
0.198 0.198 -0.021 0.052 -0.134 0.011 0.01 0.04 -0.048 0.058 -0.029 0.019 0.05 -0.013 0.055 -0.002 -0.042 -0.005 0.064 0.152 0.105 0.013 -0.358 0.552 0.154 0.191 -0.177 -0.138 0.145 0.096 -0.583 0.292 0.474 0.067 0.181
0.6 0.9 1.1 0.8 1.1 1.0 0.9 0.9 1.3 0.8 1.1 1.0 0.7 1.1 0.8 1.0 1.1 1.0 1.0 1.0 0.8 1.0 72.6 0.3 0.3 0.5 1.5 1.6 0.7 0.9 2.9 0.7 0.6 0.7 0.7
a Values corresponded to the absolute differences of mean spot volume between Gaspard (G) and JetNeuf (JN) and JetNeuf (JN) minus Darmor (D). They were obtained from the multiple range test at 1% significant level. Significative differences are in italics. Negative values are indicative of an overexpression either in JN (first row) or in Darmor (second row) and inversely for positive values.
to the identification of 13 proteins differentially expressed out of which 7 were identified as myrosinase binding proteins (Table 4). Furthermore, 9 spots of protein (spots 686, 2565, 2575, 2579, 2904, 2906, 2907, 3183, and 3283) were found at higher levels in JetNeuf, whereas 4 spots (spots 688, 2760, 2774, and 3166) were less detected in this genotype. Out of the 13 spots differentially expressed between JetNeuf and Darmor, 3 (spots 686, 2904 and 3283) were found to be exclusively different between JetNeuf and Darmor whereas 10 were also variable between Gaspard and JetNeuf either more accumulated in Gaspard (spots 688, 3166) or in JetNeuf (spots 2565, 2575, 2579, 2760, 2774, 2906, 2907, 3183). It is interesting to note that only 2 spots out of the 35 showed an increased expression from Gaspard to JetNeuf; these 2 spots were identified as myrosinase binding protein (spot 3166) and cruciferin subunit (spot 688).
Discussion Mature seeds of four near-isogenic varieties of Brassica napus were characterized at the proteome level. Darmor and Darmorbzh, selected for their low contents in both erucic acid and glucosinolates, contained less total proteins as expressed in % of seed weight. Although a negative correlation between proteins and lipids contents in seeds has often been highlighted, this was not observed for these four varieties. On the other hand, our results point out an effect of breeding on the total protein content of these varieties whereas no difference
was observed on the total lipid content. These results rose the question of a potential effect of breeding on the protein composition of the seed. Improvement of Extraction and Electrophoresis Conditions. Two-dimensional gel electrophoresis has become a classical tool to achieve the separation and quantification of proteins at the proteome scale. However, the detection and, even more, the relative quantification of a large number of proteins strongly depend on the quality of sample preparation. We thus compared five different extraction methods, which were inspired from the existing literature and were adapted to B. napus seeds in the present study.39-44 These methods can be classified into two categories: on one hand, those based on the direct solubilization of proteins (UC, UCT, UCT-C80) and, on another hand, those involving a precipitation step of either the proteins (TCA) or of the non-proteic material (phenol extraction method). One of the main specificity of cruciferous seeds compared to some other plant materials is their high lipid content. Therefore, the benefit of a delipidation step before protein extraction was tested. We found that this delipidation procedure induced a significant loss of proteins, that could be caused by two phenomena: first, some proteins might be co-extracted in the dichloromethane phase and, second, some of the pelleted proteins might be less prone to further solubilization. It is interesting to note that the expected benefits of delipidation regarding electrophoretic quality, i.e., clearer background, Journal of Proteome Research • Vol. 6, No. 4, 2007 1349
research articles sharper and better defined spots,45 were also obtained while using the UCT-C80 extraction method directly on non-defatted seeds. In this last method, the addition of ASB-C8Φ as a supplementary detergent to the extraction buffer makes it possible to separate the lipids from the protein aqueous extract in a creamed upper phase, without the need of a precipitation step. Our comparison of the extraction methods based on protein recovery and electrophoretic criteria led to the poorest performance of phenol solubilization, followed by urea extraction. These results agree with those of Natarajan et al.46 on soybean seeds. However, they diverge from those obtained by Saravanan and Rose47 who found that the phenol method gave the best extraction yield on various tomato tissues, on banana and on avocado. These discrepancies confirm that extraction should be adapted to the type of organ or tissue studied and also to the plant species. If one considers extraction methods based on direct solubilization of proteins, the extraction yield significantly increased from 67% with only urea and Chaps to 91% with the use of thiourea, which is an effective chaotrope.48 These results are similar to those of Me´chin et al.17 who concluded that the solubilization of maize endosperm proteins was greatly improved by the addition of thiourea. Similarly, the addition of ASB-C8Φ as a supplementary detergent to the extraction buffer in the UCT-C80 method made it possible to extract as much as 99% of the total proteins of the seed, compared to extraction without this molecule. This is mainly resulting from an improved solubilization of membrane proteins, as was already reported by Chevallet et al.49 and Henningsen et al.50 Moreover, the addition of this highly efficient detergent (ASB-C8Φ) by inducing a separation of lipids not only significantly improved the extraction yield but also the electrophoresis quality. The comparison of the 2-D gels loaded with the different extracts revealed a majority of matched or redundant spots, but it is important to note that only TCA extracts revealed few unique spots. These results are not consistent with those of Carpentier et al.,51 who show that 21% of the spots differed qualitatively between the TCA and phenol methods, whereas the efficiency of extractions was not statistically different. Regarding the composition of the extracts by 2-DE, they mentioned that some spots, especially those located in the basic region (7
