Protein extraction from rice (Oryza sativa L.) root for two-dimensional electrophresis

Abstract

An efficient protein extraction method for two-dimensional gel electrophoresis (2-DE) from plant samples is usually challenging due to the low protein content and high level of interfering compounds. Proteomic analyses of rice (Oryza sativa L.) roots are limited by the lack of an efficient protein extraction method. To establish an effective protocol of protein extraction suitable for 2-DE analysis in rice roots, we evaluated three protein extraction methods (trichloroacetic acid [TCA]/acetone, Mg/NP-40/TCA, and tris-base/acetone). Our results showed that the Mg/NP-40/TCA extraction method had the highest protein yield and is the best resolution of protein separation among the three methods. The TCA/acetone method exhibited clear €protein€ profiles€ and €detected€ more €protein €spots with the highest intensity in the region of high Mr (above 45 kDa) than the other methods. However, this method was unable to detect proteins with low-Mr (less than 24.0 kDa). The Tris-base/acetone method showed the poorest resolution of protein separation. Our results suggest that the Mg/NP-40/TCA method was the most effective among the three methods and may provide enhanced proteomic information for rice and other crop roots.

Keywords

protein extraction / two-dimensional electrophresis (2-DE) / rice (Oryza sativa L.) roots

Introduction

Rice is not only an important crop worldwide but also a model plant for monocots because of its relatively small genome size. A vast number of rice cultivars as well as wild species of rice are widely grown, and their genetic and molecular makeup is being actively investigated. However, most research focuses on the shoot, whereas the rice root is much less investigated.

Two-dimensional gel electrophoresis (2-DE) is a protein separation technique that combines two different electrophoretic methods, that is, gel isoelectric focusing (IEF) in the first dimension (in which proteins are separated according to pI) and SDS-PAGE in the second dimension (separation according to molecular weight (Mr)). The technique of proteome analysis with two-dimensional gel electrophoresis is a powerful tool, which may be used to monitor global changes that occur in the protein expression of tissues and organisms, especially those that occur under stress. However, the protein extraction is a critical step for two-dimensional electrophoresis (2-DE), which can have significant impact on both quantity and quality of protein detection (Hille et al., 2001; Shaw and Riederer, 2003). Different plant samples require different and adaptive protein extraction protocols. Moreover, the extraction protocol most often needs to be optimized for each particular type of plant tissue. Rice root is notoriously recalcitrant to common protein extraction methods due to low protein content and a high level of interfering compounds (especially the lignins and celluloses).

The objective of this study was to establish an optimal protocol of protein extraction for the 2-DE analysis in rice root. We compared three commonly used protein extraction methods: Mg/NP-40, TCA/acetone, and tris-base/acetone. The efficiency of three extraction methods was evaluated; the performance of the three different methods was also evaluated by measuring the protein yield, counting the number of spots, and determining the best pattern resolution on 2-DE gels.

Materials and methods

Chemicals

Acrylamide and bisacrylamide were purchased from Sigma. Standard molecular weight markers were from Fermantas. Carrier ampholytes were purchased from Bio-Rad. SDS, TEMED, ammonium persulfate, β-mercaptoethanol, and NP-40 came from Amresco. All other chemicals were standard reagent-grade laboratory chemicals and were obtained from Sinopharm Chemical Reagent, Co., Ltd, Shanghai, China. Double distilled water was used for all solutions.

Plant material

Rice cultivar 9311 (Oryza sativa L.) was chosen for this study. The plants were grown from seeds in hydroponic culture in the summer of 2008 at the Fujian Agriculture and Forestry University (Fujian Province, China). The pH of the solution was daily adjusted to 5.5, and the entire nutritive solution was changed weekly. After culturing for 25 days, rice roots were harvested, weighed, and immediately frozen in liquid nitrogen and then stored in a -80°C freezer until protein extraction.

Protein extraction

About 2 g root samples were ground in a prechilled mortar containing 10% w/w polyvinylpolypyrrolidone (PVPP) of sample weight. Finely ground powder was immediately transferred into a 40 mL centrifuge tube and then stored in a -80°C freezer until protein extraction.

Method A

Method A is the Mg/NP-40/TCA extraction method: This method was described by Kim et al. (2001), with some modifications. The powder sample was homogenized in 10 mL of ice-cold Mg/NP-40 extraction buffer containing 0.5 mmol·L^-1 Tris-HCl, pH 8.3, 2% v/v NP-40, 20 mmol·L^-1 MgCl₂, 2% v/v β-mercaptoethanol, 1 mmol·L^-1 phenylmethylsulfonyl fluoride (PMSF), and 1% w/v polyvinylpolypyrrolidone (PVPP). The mixture was sonicated in an ice bath for 10 min and shaken on ice for 30 min. After centrifugation at ×12000 g for 15 min at 4°C, proteins in the supernatant were precipitated by adding four volumes of cold acetone containing 10% TCA and 0.07% β-mercaptoethanol at -20°C for at least 3 h. The precipitated proteins were washed with ice-cold acetone containing 0.07% β-mercaptoethanol. The treatment was repeated until the supernatant was colorless. Pellets were vacuum-dried and stored at -80°C.

Method B

Method B is the TCA/acetone precipitation method: This method was according to Damerval et al. (1986), with some modifications. The powder sample was added to 20-mL precipitation solution (10% TCA and 0.07% β-mercaptoethanol in cold acetone), homogenized, and sonicated in an ice bath for 10 min and then precipitated at -20°C overnight. It was centrifuged at ×12000 g, 4°C for 20 min. The precipitated proteins were washed with ice-cold acetone containing 0.07% β-mercaptoethanol to remove pigments and lipids until the supernatant was colorless. Pellets were vacuum-dried and stored at -80°C.

Method C

Method C is the Tris-base/acetone method: This protocol was performed according to Rabilloud (1998), with modification. The powder sample was homogenized in 10 ml of extraction buffer (40 mmol·L^-1 tris-base, 5 mol·L^-1 urea, 2 mol·L^-1 thiourea, 2% w/v CHAPS, 5% w/v polyvinylpyrollidone, and 2% β-mercaptoethanol). The mixture was sonicated in an ice bath for 10 min and shaken on ice for 30 min. After centrifugation at ×12000 g for 15 min at 4°C, the proteins in the supernatant were precipitated by adding four volumes of ice-cold acetone containing 10% TCA and 0.07% (w/v) β-mercaptoethanol, incubated at -20°C for at least 3 h, and then centrifuged for 15 min at ×12000 g. The pellet was washed with ice-cold acetone containing 0.07% β-mercaptoethanol, incubated at -20°C for 2 h, and centrifuged again at 4°C. The washing was repeated twice. The final pellets were vacuum-dried and stored at -80°C.

Protein quantitation

The protein pellets were weighed and then resuspended in lysis buffer (9 mol·L^-1 urea, 4% w/v CHAPS, 65 mmol·L^-1 dithiothreitol [DTT], and 0.5% v/v carrier ampholytes pH 3-10) and sonicated in an ice bath for 20 min. After centrifugation at ×12000 g for 30 min, the supernatant was collected. Protein concentration was determined by the Bradford method (Bradford, 1976).

Protein separation by SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) was according to the method of Laemmli (1970) in 10.0% w/v polyacrylamide gels using DYY III mini electrophoresis equipment (Beijing 61 Instrument Factory, BJ, CN) at 80 V for about 3.5 h, with 15 µg solubilized protein sample in each lane. After electrophoresis, the gel was transferred into fixing solution (50% v/v methanol, 5% v/v acetic acid) for at least 3 h and then washed with distilled water three times for 5 min each time. Finally, the gel was transferred into a staining solution (0.12% w/v Coomassie brilliant blue G-250, 10% w/v ammonium sulfate, 10% v/v phosphoric acid, and 20% v/v methanol) overnight with gentle shaking. After staining, the gel was decolorized with distilled water.

Two-dimensional gel electrophoresis (2-DE) and image analysis

The 2-DE protocol described by O^’Farrell (1975) was performed with some modifications. IEF gel was carried out in glass tubes, 2.0 mm inner diameter and 17 cm in length. The bottom of tube was sealed with parafilm. The four tubes of IEF gels were made as follows: 1.44 g urea, 0.35 mL of acrylamide solution (28.38% w/v acrylamide and 1.62% w/v bisacrylamide), 0.60 mL 10% v/v NP-40, 0.85 mL ddH₂O, and 0.15 mL ampholine (pH 3.5-10∶pH 5-8=1∶4). To polymerize the gel, 5.60 µL 10%v/v ammonium persulfate and 1.40 µL TEMED were added into the gel mixture. The tube was filled with the gel solution up to 17 cm and overlaid with overlay solution (5 mol·L^-1 urea). After the gel polymerization, this overlay solution was removed, and the sample (200 g protein) was loaded. IEF was performed at 200 V, 300 V, 400 V, 500 V, and 600 V for 0.5 h per step, and 800 V for 16.5 h, and then 1000 V for 4 h. The cathode buffer was 20 mmol·L^-1 NaOH, and the anode buffer was 10 mmol·L^-1 H₃PO₄. Upon completion of IEF, the gel was extruded using a syringe and then rinsed with double distilled water. The focused gel was put into a 10 mL centrifuge tube with 5 mL of an equilibration buffer that contained 10% v/v glycerol, 2.5% w/v SDS, 125 mmol·L^-1 Tris-HCl (pH 6.8), and 5% v/v β-mercaptoethanol. It was then agitated and shaken gently at room temperature for 20 min. The second dimensional electrophoresis (SDS-PAGE) was performed as described by Laemmli (1970). The focused gel was transferred onto 10.0% w/v SDS-PAGE self-cast gels. Electrophoresis was carried out at 20 mA per gel for 30 min and 15 mA per gel until the dye had reached the bottom of the gel. The gels were stained by the method of Candiano et al. (2004). Before dying, gels were fixed with 50% v/v methanol and 5% v/v acetic acid for at least 3 h, and then, the gel was transferred into a staining solution (0.12% w/v Coomassie brilliant blue G-250, 10% w/v ammonium sulfate, 10% w/v phosphoric acid, and 20% v/v methanol) overnight with gentle shaking. After staining, the gel was decolorized with distilled water.

The gels were scanned using ScanMaker 8700 (Microtek), and the images were analyzed using Image Master 2D Platinum Version 5.0 Analysis Software (Amersham Pharmacia).

Results

Comparison of the three methods on the protein yield and total number of spots

The protein yields and the protein spots number of the three protein extraction methods are listed in Table 1. The Mg/NP-40/TCA method showed the highest protein yield (0.60±0.004 mg·g^-1fresh wt) and a high protein spots number (757±3.6 protein spots). The TCA/acetone method gave the most protein spots (784±3.2 protein spots) but had lower protein yield. The Tris-base/acetone method had the lowest protein yield (0.30±0.002 mg·g^-1fresh wt) and the fewest protein spots (708±27.0 protein spots).

Comparison of the three methods according to SDS-PAGE patterns

Rice root proteins extracted by the three extraction methods were analyzed by SDS-PAGE (Fig. 1). The loading quantity used was 15 g solubilized protein sample in each lane. The gel revealed that Method A had the highest resolution separation compared to the other two methods. Method B showed the poorest resolution in region II (about 35-116 kDa), and proteins in the lowest molecular mass (region I, about 14.4-21 kDa) area of the gel were lost.

Comparison of the three methods on the basis of the 2-DE patterns

Protein pattern in 2-DE analysis for rice root samples prepared from three methods are presented in Fig. 2. The results show that Method A detected more root protein spots in the region of low- M_r (less than 24.0 kDa) and high-pI region (about pH 9-10). Method B exhibited clear protein profiles and detected more protein spots with the highest intensity in the region of high M_r (above 45 kDa) than the other methods. However, this method was unable to detect proteins with low-M_r (less than 24.0 kDa). The 2-DE image based on Method C showed the poorest resolution of protein spots in the high Mr and low-pI region.

Scatter plots of 2-DE analysis of the three protein extraction methods are presented in Fig. 3. The results show that the correlation coefficients of Method A and Method B, Method B and Method C, and Method A and Method C were 0.657, 0.716, and 0.426, respectively.

Discussion

Protein extraction from plant samples is challenging due to the low protein content and high level of contaminants. In rice roots, the content of lignin, cellulose, and polysaccharides, all commonly found in roots, seriously interfere with protein extraction and affect protein migration in 2-DE. Three methods of protein extraction examined in this study varied in their efficiency to detect protein quantity and molecular profiles using 2-DE for root tissues of rice.

Comparison of protein spots 3D views in 2-DE analysis for samples prepared from three methods are presented in Fig. 4. The advantages and disadvantages, as well as the suitability of each of the three protein extraction methods for 2-DE analysis, are discussed in the following paragraphs.

The Mg/NP-40 protein extraction method has been reported as a superior protein extraction method in 2-DE analysis of rice leaves (Kim et al., 2001). In this study, we found that TCA/acetone more effectively precipitated proteins and removed pigments compared with acetone precipitation. Therefore, we combined the Mg/NP-40 protein extraction buffer and TCA/acetone to enhance the protein extraction from rice roots. Consequently, the Mg/NP-40/TCA protein extraction method exhibited the highest protein yields and more protein spots among the three methods. Moreover, our results also suggested that grinding the sample in liquid nitrogen, followed by adequate homogenization in Mg/NP-40 buffer and sonication in an ice bath, proved to be a simple and efficient system to extract proteins from rice roots. According to our results, this method seems to be the best approach to perform 2-DE analysis of rice root protein. However, this method had poorer separation and lower protein spot intensities in the region of high M_r (above 45 kDa) than the TCA/acetone method.

Protein extraction with TCA/acetone is one of the most common methods used to extract proteins from plant samples for 2-DE (Görg et al., 2004; Xu et al., 2008; Xie et al., 2009). Our results indicate that the TCA/acetone method resulted in clear protein profiles and detected more protein spots with the highest intensity in the region of high M_r (above 45 kDa) than the other methods. However, this method had lower protein yield and lost low mass proteins (less than 24.0 kDa) in the 2-DE gel, which was consistent with the SDS-PAGE (Fig. 1 and Fig. 2B). The principle function of this method is to precipitate the proteins by TCA/acetone, and it is very useful for protein precipitation. However, it cannot remove lignins and celluloses both of which can significantly impact the proteins dissolved in lysis buffer. That is why the method showed lower protein yield and lost low mass proteins (less than 24.0 kDa).

The Tris-base/acetone method has been reported to be an effective protein extraction method for 2-DE analysis in rice roots (Yang et al., 2007), but our results showed that this method exhibited low 2-DE resolution and poor protein separation, particularly for proteins with high M_r (about 40.0-50.0 kDa) and acidic region (about pH 3).

In conclusion, our results show that for optimal separation of proteins in 2-DE, we have to select a suitable protein extraction method for rice root. Rice roots have a low protein content but are rich in lignin and cellulose, which are natural substances that make protein extraction and separation difficult. We tested three published methods, and by a comprehensive consideration of protein yield, the number of migrating spots, the pattern resolution on 2-DE gels, and the volume of sample loading, we determined that the Mg/NP40/TCA method to be a superior, efficient, and reliable method for 2-DE protein extraction for rice root. We expect that this method can also be applicable to the proteomics study of other plant samples.

References

[1] Bradford M M (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72(1-2): 248-254

[2] Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri G M, Carnemolla B, Zardi L, Righetti P G (2004). Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis, 25(9): 1327-1333

[3] Damerval C, De Vienne D, Zivy M, Thiellement H (1986). Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins. Electrophoresis, 7(1): 52-54

[4] Görg A, Weiss W, Dunn M J (2004). Current two-dimensional electrophoresis technology for proteomics. Proteomics, 4(12): 3665-3685

[5] Hille J M, Freed A L, Wätzig H (2001). Possibilities to improve automation, speed and precision of proteome analysis: a comparison of two-dimensional electrophoresis and alternatives. Electrophoresis, 22(19): 4035-4052

[6] Kim S T, Cho K S, Jang Y S, Kang K Y (2001). Two-dimensional electrophoretic analysis of rice proteins by polyethylene glycol fractionation for protein arrays. Electrophoresis, 22(10): 2103-2109

[7] Laemmli U K (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259): 680-685

[8] O’Farrell P H (1975). High resolution two-dimensional electrophoresis of proteins. J Biol Chem, 250(10): 4007-4021

[9] Rabilloud T (1998). Use of thiourea to increase the solubility of membrane proteins in two-dimensional electrophoresis. Electrophoresis, 19(5): 758-760

[10] Shaw M M, Riederer B M (2003). Sample preparation for two-dimensional gel electrophoresis. Proteomics, 3(8): 1408-1417

[11] Xie C J, Wang D, Yang X Y (2009). Protein Extraction Methods Compatible with Proteomic Analysis for the Cotton Seedling. Crop Science Society of America, 49(2): 395-402

[12] Xu C P, Xu Y, Huang B R (2008). Protein Extraction for Two-Dimensional Gel Electrophoresis of Proteomic Profiling in Turfgrass. Crop Science Society of America, 48(4): 1608-1614

[13] Yang Q S, Wang Y Q, Zhang J J, Shi W P, Qian C M, Peng X X (2007). Identification of aluminum-responsive proteins in rice roots by a proteomic approach: cysteine synthase as a key player in Al response. Proteomics, 7(5): 737-749

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