Introduction
High oleic acid oil is one of the interestingly functional food, it has more monounsaturated fatty acids (MUFAs), which is helpful to reduce cholesterol in blood plasma, and lower low density lipoprotein (
Robert et al., 2004), thus reduce the risk of coronary heart disease (
Iwona et al., 2006), and diets rich in MUFAs would have favorable outcomes on blood lipids and factor VIIc which can be useful to prevent heart disease (
Allman et al., 2005;
Iwona et al., 2006). In addition, it has lower saturated fat and more heat-stable than commodity-grade oil (
Fuller et al., 1967;
Mark et al., 2001), which is desirable for frying purposes, refining and storage.
As the largest oilseed crop in China, high oleic acid rapeseed will increase considerably in the near future (
Zhang et al., 2011). With Xiangyou 15 variety as raw material treated by
60Co (
Guan et al., 2006), more than 100 high oleic acid rapeseed materials (oleic acid content more than 75%) were obtained after 6 years directional selection. The mutants have higher oleic acid content and the similar agronomic traits with Xiangyou 15 variety (
Guan and Li, 2008). Several studies have been done in mature period of seeds, and some difference were found in FAD2 and FAD3 genes between high oleic acid rapeseed and Xiangyou 15 (
Guan et al., 2006;
Xiao et al., 2009;
Guan et al., 2012).
A comparative proteomic approach based on 2-DE and mass spectrometry was adopted to identify proteins that changed in the 15-day-seedlings of M15 (oleic acid content is 78.9%) compared with Xiangyou 15 variety. And the gene levels of differentially expressed proteins were verified by qPCR analysis. The objective of these studies was to find out whether there has some remarkably differences between Xiangyou 15 variety and M15 in seedling period in seedling period.
Materials and methods
Materials
The samples of Xiangyou 15 variety and M15 in this study were provided by Hunan branch of national oilseed crops improvement center. And the two rapeseeds were planted in the same salver at 25°C for 15 days. The seedling of Xiangyou 15 variety and M15 has little difference. 1 g 15-day-seedlings were mixed together and briefly washed with sterile water thrice, then frozen in liquid nitrogen immediately and stored at - 80°C for protein extraction (Fig. 1).
The determination of fatty acid content
The fatty acid content of rapeseed samples was determined by gas chromatograph. 1 mg rapeseed was dried at 80°C, crushed with high speed disintegrator, and then moved to 10 mL test tube, 1 mL 0.5 M KOH-CH3OH and 2 mL aether and petroleum ether mixture with a volume rate of 1 to 1. And placed at 40°C for 24 h, then 2 mL H2O was add to stratify, 1 μL upper sampling was extracted to determine with methyl heptadecanoate as the internal standard. And the gas chromatograph Agilent-6890A (Amercia), working conditions were: the inlet temperature of 270°C, the column temperature was 190°C with 5°C per min to 230°C, then holding 1 min, detector temperature was 260°C, nitrogen flow rate was 250 mL per min, hydrogen flow rate was 30 mL per min, air flow rate was 300 mL per min, split ratio was 40: 1, and column flow rate was 1 mL per min.
Protein extraction
Frozen seedling sample (1.0 g) was ground to powder in a mortar using liquid nitrogen precooled. The powders were displaced to a 10 mL centrifuge tube and then added with 10 volumes precooled (–20°C) sample buffer (8 M urea, 2% CHAPS). The mixture was vortexed, and then treated with sonicator in a beaker which contained with the mixture of ice and water. The swing was set to 21%. And the procedures were: cumulative ultrasounds for 60 s, ultrasound 0.1 s per 1 s.
The mixture was vortex extracted for 30 min at 25°C, centrifuged under 13 000 ×
g for 15 min at 4°C. The supernatant was transferred carefully to a new centrifugal tube, 4 volumes acetone (–20°C) was added, and then placed at –20°C overnight. The mixture was centrifuged under 13 000 g for 20 min at 4°C, the sediment was washed with –20°C acetone containing 0.07% DTT thrice,then dried using freeze-drying centrifuge (FDU-2100, EYELA, Japan). The powder was dissolved by rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT, 0.2% Bio-Lyte [pH 4–7], and 0.001% bromophenol blue) per 1 mg pellet. The content of protein was quantified using the Bradford method (
Bradford, 1976) with bovine serum albumin as the standard.
Two-dimensional polyacrylamide gel electrophoresis
About 1.0 mg protein was dissolved in 450 μL rehydration buffer and applied to IPG strips (24 cm, pH 4–7, Bio-Rad). The IPG strip was rehydrated for 14 h in 400 μL rehydration buffer containing the protein sample. Then the isoelectric focusing (IEF) was performed at 20°C using a IPGphor II IEF system (Amersham Biosciences, Uppsala, Sweden) under the procedure listed in Table 1.
After IEF separation, the strips were equilibrated twice for 15 min each using equilibration buffer (6 M urea, 37.5 mM pH 8.8 Tris-HCl, 20% glycerol, and 2% DTT) with 1% DTT and 4% iodacetamide (IAA). The second dimension separation was performed by SDS-PAGE on a vertical slab acrylamide gel (12% total monomer, with 2.6% crosslinker) using an Ettan Dalt SIX electrophoresis system (Amersham Biosciences, Uppsala, Sweden). The electrophoresis initiated with a constant electric current of 10 mA for 45 min and was switched to 20–30 mA until the bromophenol blue frontier reached the bottom of the gel. After SDS-PAGE, the gel was fixed for 1 h in a solution containing 40% methanol and 10% acetic acid, washed with deionized water for 15 min, and then stained in a staining buffer (0.12% Coomassie brilliant blue G-250, 10% ammonium sulfate, 10% phosphoric acid, and 20% methanol) for 24 h (
Candiano et al., 2004), and then destaining by ddH
2O until the background was clear. The experiment was repeated in triplicate for each sample.
Gel image analysis
Images of stained gels were acquired by the image scanner Typhoon 9410 (Amersham Biosciences, Uppsala, Sweden) in a transmission mode. Image analysis was performed with PDQuest 8.0 software (Bio-Rad). After background subtraction and spot detection, spots were matched and normalized using the method of total density in the gel image. Only spots with statistically significance (Student’s t-test, p<0.05) and reproducible changes were considered, and the protein spots with an abundance ratio of at least two were selected as differentially expressed proteins.
Proteins identification
To identify proteins in protein spots using MS, protein spots were excised from 2-DE gels and washed with water twice, and digested as described by
Katayama et al., (2001). Extracted lyophilized tryptic peptides were dissolved with 0.1% TFA in 50% acetonitrile, then 1 μL of the peptide mixture was mixed with an equal volume of saturated CHCA solution. Mass spectrometric analysis was performed with a MALDI-TOF/TOF mass spectrometer 4800 (Applied Biosystems, Foster City, CA, USA). Data were acquired in a positive MS reflector. Mass spectra were obtained from each sample spot by accumulating 900 laser shots in a mass range of 800–3500 Da. The 10 most abundant precursor ions per sample were selected for subsequent fragmentation. Both the MS and MS/MS data were interpreted and processed using Global Proteome Server (GPS) Explorer software (V3.6, AB Sciex) and Mascot2.1 (Matrix Science). NCBInr was selected as the database and taxonomy, respectively. Search parameters used fixed cysteine carbamidomethylation and variable methionine oxidation as modifications, trypsin of the digestion enzyme, one missed cleavage site, fixed modification of cysteine carboamidomethylation and partial modifications of Oxidation (M) and Acetyl (N–term), MS tolerance of 0.15 Da, and MS/MS tolerance of 0.25 Da. The protein score C.I.% had to be over the significance (>95) threshold level (
p<0.05).
qPCR analysis
Total RNA for qPCR analysis was extracted from 15-day-seedlings using the Plant RNA Midi Kit (Omega, USA). cDNA was synthesized from the total RNA (≤5μg) with the Prime Script
TM 1st strand cDNA Synthesis Kit according to the manufacturer’s instructions. The primers were designed using Primer 5.0 software according to the cDNA sequences. Reactions were carried out with the CFX96TM Real-Time System (BIO-RAD, USA) using two-step cycling conditions of 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 60°C for 30 s. The reaction mixture (20 μL) contained 1 μL cDNA solution, 10 μL SYB® Premix Ex Taq (Tli RNaseH Plus) (2 × ), 0.5 μL of each primer and 8 μL water. The reactions were performed in triplicate, and the results were averaged. UBC21 (ubiquitin-conjugating enzyme 21) was used as the reference gene, which is a stably expressed reference gene for vegetative tissues (Xue
Chen et al., 2010). And the primer pairs of UBC21 were: 5′-CCTCTGCAGCCTCCTCAAGT (F), and 5′-CATATCTCCCCTGTCTTGAAATGC (R). The relative gene expression was evaluated using the comparative cycle threshold method (
Livak K. and Schmittgen T. 2001). Statistical analysis was performed using ANOVA and Post Hoc test with a
p value<0.05 being accepted as significant. And the primer sequences used in qPCR were listed in Table 2.
Results
Fatty acid composition in rapeseed of Xiangyou 15 variety and M15
The M15 derived from Xiangyou 15 variety, therefore, there has little genetic background difference between them. The fatty acid composition of rapeseed was determined by Gas chromatography (
Zhang et al., 2009) with methyl heptadecanoate as internal standard compound (Table 3). And there has a obviously diference in oleic acid content.
Change in protein abundance between different samples
Two-dimensional electrophoresis analysis of the total protein in seedlings of M15 and Xiangyou 15 variety was performed at least in triplicate and showed a high level of reproducibility. Representative gels are shown in Fig. 2. Among all the tested samples, more than 9702 protein spots were reproducibly detected with PDQuest 8.0 software on CCB G-250-stained gels. Quantitative analysis revealed that 277 protein spots showed a significant (p<0.05) change in intensity by more than 2-fold from M15 to Xiangyou 15 variety.
Identification of differentially expressed proteins
Fifty differentially expressed protein spots (the ratio of M15 to Xiangyou 15 variety or Xiangyou 15 variety to M15 from 3 to 5) were identified, 48 proteins were successfully identified, and the results were summarized in Table 4. Based on putative physiologic functions, the identified proteins can be classified as the following categories, carbohydrate and energy proteins (36 protein spots), stress and defense proteins (4 protein spots), photosynthesis (3 protein spots), protein metabolism (1 protein spot) and other proteins (4 protein spots). Most of the proteins involved in metabolism of carbohydrate and energy have higher expression in M15, which may reveal M15 has higher metabolism ability. But the corresponding genes has no relationship with FAD2 or FAD3 gene, the reason may be that there has no fatty acid synthesized in seedling period.
Gene expression analysis by qPCR
To investigate the changes in gene expression at the mRNA level, qPCR analysis of identified proteins was performed (Fig. 3). For several differentially expressed protein spots were identified as the same one protein, so there only 15 proteins were investigated. The results showed that, compared with Xiangyou 15 variety the transcripts of M15 of ATP synthesis CF1 alpha subunit (0.7 fold), glutamine synthetase (0.87 fold), Rubisco ssu precursor (0.73 fold), fructose-bisphosphate aldolas (0.71 fold)e, β-glucosidase (0.28 fold), Ribulose bisphosphate carboxylase/oxygenase activase (0.09 fold) and actin (0.01 fold) were downregulated, especially the expression of β-glucosidase (0.28 fold), Ribulose bisphosphate carboxylase/oxygenase activase(0.09 fold) and actin (0.01 fold) were significantly downregulated. While compared with Xiangyou 15 variety the transcripts of chlorophyll a/b binding protein (1.67 fold), Rubisco activase (1.72 fold), chlorophyll a/b binding protein type I (1.45 fold) and ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit (1.78 fold) were significantly upregulated. The expression of ATP synthase subunit beta (0.91 fold), myrosinase (1.02 fold), high chlorophyll fluorescence 136(1.05 fold) and chloroplast translation elongation factor EF-Tu (0.96 fold) almost has no change. The mRNA level does not always correlate with the protein level in this study, the reason may be the post-transcriptional regulation (
Yan et al., 2006).
Discussion
In this study, 15-day-seedlings of Xiangyou 15 variety and M15 were selected for proteomic analysis. These proteins were mainly involved in metabolism of carbohydrate and energy (75%), stress and defense (8.3%), photosynthesis (6.3%), protein metabolism (2.1%) and other proteins (8.3%). Several proteins present in multiple spots in this study, the reason may be potential protein isoforms derived from alternatively spliced mRNAs were dispersed in different spots (
Jiang et al., 2007), or post-translational modifications, such as glycosylation and phosphorylation, which altered the molecular weight and/or charge of proteins.
The differences in physiological and chemical characteristics
The M15 derived from Xiangyou 15 variety has little genetic background difference. And the 15-day-seedling has little difference too. However, the fatty acid content has obviously different, especially the oleic acid content, Xiangyou 15 variety was 64.208%, while the M15 was 80.935%.
Carbohydrate and energy proteins
Metabolisms of carbohydrate and energy are basic but essential activities in plant. Approximately 81.3% identified proteins were involved in the group of carbohydrate and energy metabolism. This suggests that altered carbohydrate and energy proteins play an important role in the growth of high oleic acid rapeseed seedling. Most of the identified proteins were associated with the metabolism of carbohydrate and energy (Table 3).
Rubisco activase is a member of the AAA+ family (
Archie et al., 2003), promote and maintain the catalytic activity of rubisco, which catalyzes the first major step of carbon fixation and regulates the activity of rubisco in response to light-induced changes in the redox potential (
Nancy, 2002). It is a major rate-limiting factor of photosynthesis under present atmosphere (
Portis, Parry, 2007). In rapeseed, seed oil content correlated well with rubisco activity in the silique wall but not in the leaf, a finding consistent with the photosynthesis results (
Hua et al., 2012). And the activities of rubisco and phosphoribulokinase were sufficient to account for significant refixation of CO
2 produced during B. napus oil biosynthesis (
Ruuska et al., 2004). And the qPCR analysis showed that the expression of rubisco activase was significantly higher in M15 seedling than Xiangyou 15 variety seedling.
Ribulose bisphosphate carboxylase, ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit, ribulose 1, 5-bisphospate carboxylase (spot 4313, 6516) and ribulose bisphosphate belongs to the rubisco. And we can see that the expression of different rubisco has the different fold change of high oleic acid rapeseed to Xiangyou 15 variety, the reason may be that different rubisco is incompatible with structural and biochemical information (
Delwiche and Palmer, 1996). Rubisco catalyzes the first step in net photosynthetic CO
2 assimilation and photorespiratory carbon oxidation (
Spreitzer and Salvucci, 2002), and it has the function of activation of ribulose-1, 5-bisphosphate carboxylase/oxygenase involves the ATP-dependent carboxylation of the epsilon-amino group of lysine leading to a carbamate structure. In developing embryos of Brassica napus L., rubisco acts without the Calvin cycle to increase the efficiency of carbon use during the formation of oil. In comparison with glycolysis, this metabolic conversion provided 20% more acetyl-CoA for fatty-acid synthesis and resulted in 40% less loss of carbon as CO
2 (Jörg et al., 2004).
β-glucosidase (EC 3.2.1.21) is a glucosidase enzyme that acts upon β1->4 bonds linking two glucose or glucose-substituted molecules. A β-glucosidase was purified from seeds of Brassica napus L. (oilseed rape). β-glucosidase is encoded by a small gene family in B. napus, the genes are expressed in the seed, with a low degree of expression in other tissues. In situ hybridization also detected β-glucosidase mRNA in shoots, young roots, and the basal part of the hypocotyls (
Falk and Rask, 1995). Spot 8705, 8706 were identified as β-glucosidase, and the qPCR analysis showed that the expression in M15 was significantly lower in Xiangyou 15 variety.
Fructose 1, 6-biphosphate aldolase (d-fructose-1, 6-bisphosphate d-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) is a key enzyme in plants, which is involved not only in glycolysis and gluconeogenesis in the cytoplasm, but also in the Calvin cycle in plastids. Research on Fructose 1, 6-biphosphate aldolase in various organisms has been reported (
Lu et al., 2012). And the spot 3113 was identified as fructose-bisphosphate aldolase in this study, the qPCR analysis showed that the expression of fructose-bisphosphate aldolase in M15 was lower in Xiangyou 15 variety. Eight Fructose 1, 6-biphosphate aldolase family genes (AtFBA1–8) were identified and analyzed in
Arabidopsis thaliana, and most of the AtFBA genes, except for AtFBA3, were significantly responsive to drought stress in roots (
Lu et al., 2012). But there has no relatively study in rapeseed, and its function need to be further investigated.
Stress and defense proteins
Myrosinases (β-thioglucoside glucohydroase, E. C. 3.2.3.1) are thioglucosidases that hydrolyze the natural plant products glucosinolates. It is generally believed to be part of the plant’s defense against insects, and possibly also against pathogens (
Rask et al., 2000). Spot 9701, 5806, 7808 (gi|127733) and Spot 3816 (gi|56130949) were identified as glutamate synthase myrosinase. Oilseed rape contains a unique defense system known as the glucosinolate–myrosinase system. Myrosinase is present in specialist cells known as ‘myrosin cells’ and can also be known as toxic mines. The myrosin cell idioblasts of Brassica napus were genetically reprogrammed to undergo controlled cell death (ablation) during seed development (
Ishita et al., 2011). Two different myrosinase proteins were found in oilseed rape (Brassica napus L. cv. Bienvenu) seedlings, they have similar kinetic characteristics, while the physical is different (
David and John, 1991). The qPCR analysis showed there has little difference between two materials.
Photosynthesis
Chlorophyll a/b complexes associated with PSI and PSII in higher plants (Jansson, 1994), in addition to responsible for absorption and conversion of light energy in oxygen-evolving photosynthetic organisms (
Green and Durnford, 1996), it plays an important role in the adaptation processes under excessive light conditions (
Horton et al., 2005), furthermore, it fulfills multiple functions, such as light harvesting and energy dissipation under different illumination (
Liu et al., 2008). Western blotting showed that light-harvesting chlorophyll a/b binding protein declined considerably during senescence, but on regreening it increased back to the levels seen in green leaves (
Zavaleta et al., 1999). Spot 1205 and 3204 were identified as chlorophyll a/b binding protein. But there has no relatively study on rapeseed. The qPCR analysis showed that the expression of chlorophyll a/b binding protein in M15 seedling has 1.6 fold to Xiangyou 15 variety seedling. It indicates that chlorophyll a/b binding protein may be a promising protein for the further study of M15.
Protein metabolism
In higher plants, the GS/glutamate synthase cycle provides the only efficient pathway for the conversion of inorganic nitrogen to the organic form (
Stewart et al., 1980). In this study, the spot 1511 was identified as glutamate synthase, the qPCR analysis showed that the expression of glutamate synthase was significantly lower in M15 seedling than Xiangyou 15 variety seedling. Glutamine synthetase gene has been cloned from Brassica napus (
Ochs et al., 1993), and its overexpression can alter ammonia assimilation and the response to phosphinothricin (
Downs et al., 1994).
Other proteins
The actin cytoskeleton mediates a variety of essential biologic functions in all eukaryotic cells. In addition to providing a structural framework around which cell shape and polarity are defined, its dynamic properties provide the driving force for cells to move and to divide. And the spot 2605 was identified as actin in this study, the qPCR analysis showed that the expression of actin was significantly lower in M15. This gene plays an important role in fiber elongation but not fiber initiation in cotton (
Li et al., 2005). But there has no relatively literature on the study of actin in rapeseed.
Chloroplast translation elongation factor EF-Tu plays a functional role in heat tolerance by acting as a molecular chaperone, it can reduce thermal aggregation of rubisco activase (
Zoran et al., 2007). This protein promotes the GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein biosynthesis (
Ferro et al., 2003). The spot 8608 was identified in this study. In pea, the expression of this gene stimulated by light and it may have an important role in plant adaptation to environmental stresses (
Singh et al., 2004). The chloroplast translation elongation factor (EF-Tu) was upregulated under drought (
Salekdeha et al., 2002; Kamal et al., 2012). But there has no study of chloroplast EF-Tu in rapeseed.
Conclusions
A comparative proteomic approach based on 2-DE and mass spectrometry was adopted to identify proteins that changed in the 15-day-seedlings of M15 compared with Xiangyou 15 variety. 48 spots which has 3.0-fold from M15 compared with Xiangyou 15 variety were successfully identified by MALDI-TOF/TOF MS. All identified proteins were mainly involved in metabolism of carbohydrate and energy (81.3%), stress and defense (8.3%), and other functions (10.4%). Then qPCR analysis was used to verify the expression levels of differentially expressed proteins, which not well agree with the proteomic results. In this work, the proteomic analysis revealed that some proteins, such as rubisco, chlorophyll a/b and rubisco activase may have important roles in the fatty acid synthesis of M15.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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