Proteins constitute to about 10% of the cell wall mass; nevertheless they are essential for maintaining the physical and biological functions in a plant cell. Yet, unidentified functional proteins might still exist in the cell wall. The completion of Arabidopsis genome has allowed the identification of cell wall proteins by using mass spectrometry (MS) techniques. However, it should be noted that several constraints arises during the extraction of cell wall proteins (i) proteins may be embedded in the polysaccharide matrix of cellulose, hemi-cellulose and pectin (ii) some proteins are difficult to solubilise (iii) some proteins undergo post-translational modifications and (iv) lack of surrounding membrane may result in a loss of cell wall proteins. So, specific extraction procedure should be used. Our strategies involved cell wall preparation through mechanical grinding (ball miller, mortar and pestle, sonication) followed by purification with increasing concentration of sucrose and sequential extraction using different concentration of salts. In addition, SDS-PAGE followed by western blotting was done to check the purity of cell wall prepared. Finally, proteins from the cell wall fractions (resultant CW5-pellet and 0.1M CaCl2 extraction) were identified using MS analysis and Arabidopsis thaliana database search. Result: During the cell wall preparation, we observed that mechanical disruption of Arabidopsis cell was the most efficient with Freezer Mill method. In consistent to this, we purified the cell sample homogenized through this method. Upon SDS-PAGE and western blotting using anti-tubulin antibody as the primary antibody, we observed a 55kDa tubulin band only in the first washing point of both basal and induced sample. This implied that the purification strategy that we had adopted was efficient. Furthermore, the resultant CW5 pellet and 0.1M CaCl2 extraction were subjected for proteomic analysis. It revealed that 44.3% of the identified proteins were cell wall proteins in the resultant CW5-pellet (induced) compared to 39.3% in the basal sample. It was also found that some of the cell wall proteins were released during 0.1M CaCl2 extraction. Conclusion: This method of preparing cell wall through mechanical disruption, fractionation through increasing density cushions and extraction of proteins with different concentration of salts provides a good cell wall preparation technique. In fact, the principle of this technique can offer a stage for studying cell wall proteome.
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INTRODUCTION..............................................................2 MATERIALS AND METHODS.....................................................3 PLANT MATERIAL.............................................................3 CELL INDUCTION FOR TE DIFFERENTIATION.........................................4 CELL HOMOGENIZATION.........................................................4 CELL WALL FRACTIONATION.....................................................4 PROTEIN EXTRACTION......................................................... 4 PROTEIN MEASUREMENT BY BRADFORD..............................................5 SDS-PAGE AND WESTERN BLOTTING...............................................5 PROTEIN ANALYSIS BY MASS SPECTROMETRY.........................................5 ANALYSIS OF MS DATA........................................................6 RESULTS...................................................................6 CELL CULTURE AND TES HARVEST................................................6 DIFFERENT METHODS FOR GRINDING...............................................6 WESTERN BLOTTING...........................................................8 ANALYSIS OF SDS-PAGE......................................................9 BIOINFORMATICS ANALYSIS.................................................... 10 DISCUSSION...............................................................13 CONCLUSION...............................................................14 ACKNOWLEDGEMENTS.........................................................15 REFERENCES...............................................................15 APPENDIX.................................................................17 1
MATERIALS AND METHODS.................................................................................................................. 3
PLANT MATERIAL...............................................................................................................................................3CELL INDUCTION FOR TE DIFFERENTIATION.......................................................................................................4CELL HOMOGENIZATION.....................................................................................................................................4CELL WALL FRACTIONATION..............................................................................................................................4PROTEIN EXTRACTION........................................................................................................................................4PROTEIN MEASUREMENT BY BRADFORD............................................................................................................5SDS-PAGE AND WESTERN BLOTTING.................................................................................................................5PROTEIN ANALYSIS BY MASS SPECTROMETRY...................................................................................................5ANALYSIS OF MS DATA.....................................................................................................................................6
CELL CULTURE AND TES HARVEST....................................................................................................................6DIFFERENT METHODS FOR GRINDING..................................................................................................................6WESTERN BLOTTING..........................................................................................................................................8ANALYSIS OF SDS-PAGE..................................................................................................................................9BIOINFORMATICS ANALYSIS.............................................................................................................................10
Analysis of Cell Wall Proteins during Xylem Vessel Secondary Cell Wall Formation in Cell Culture
Gurung Jyoti Mohan, Dwivedi Gaurav Dutta and Linlin Gao
Background: Proteins constitute to about 10% of the cell wall mass; nevertheless they are essential for maintaining the physical and biological functions in a plant cell. Yet, unidentified functional proteins might still exist in the cell wall. The completion of Arabidopsis genome has allowed the identification of cell wall proteins by using mass spectrometry (MS) techniques. However, it should be noted that several constraints arises during the extraction of cell wall proteins (i) proteins may be embedded in the polysaccharide matrix of cellulose, hemi-cellulose and pectin (ii) some proteins are difficult to solubilise (iii) some proteins undergo post-translational modifications and (iv) lack of surrounding membrane may result in a loss of cell wall proteins. So, specific extraction procedure should be used. Our strategies involved cell wall preparation through mechanical grinding (ball miller, mortar and pestle, sonication) followed by purification with increasing concentration of sucrose and sequential extraction using different concentration of salts. In addition, SDS-PAGE followed by western blotting was done to check the purity of cell wall prepared. Finally, proteins from the cell wall fractions (resultant CW5-pellet and 0.1M CaCl 2
extraction) were identified using MS analysis and Arabidopsis thaliana database search. Result: During the cell wall preparation, we observed that mechanical disruption of Arabidopsis cell was the most efficient with Freezer Mill method. In consistent to this, we purified the cell sample homogenized through this method. Upon SDS-PAGE and western blotting using anti-tubulin antibody as the primary antibody, we observed a 55kDa tubulin band only in the first washing point of both basal and induced sample. This implied that the purification strategy that we had adopted was efficient. Furthermore, the resultant CW5 pellet and 0.1M CaCl2 extraction were subjected for proteomic analysis. It revealed that 44.3% of the identified proteins were cell wall proteins in the resultant CW5-pellet (induced) compared to 39.3% in the basal sample. It was also found that some of the cell wall proteins were released during 0.1M CaCl2 extraction. Conclusion: This method of preparing cell wall through mechanical disruption, fractionation through increasing density cushions and extraction of proteins with different concentration of salts provides a good cell wall preparation technique. In fact, the principle of this technique can offer a stage for studying cell wall proteome.
The plant cell wall is a vital component of a plant cell which provides both structural integrity and functional role to a plant. There are two core types of cell walls that are found in plants: the primary cell wall that gets accumulated through cell division and growth, which is capable to elongate; and the
secondary cell wall formed after the elongation, providing mechanical sustenance to the entire plant (Borderies G, et al., 2003). The formation of a dense lignified secondary cell wall only occurs once cells have reached their final shape and size.
Xylem is formed by the combination of tracheary elements (TEs), parenchyma cells, and fibers. TEs are the characteristic cells of the xylem that are categorized by the formation of a secondary cell wall with annular, spiral, reticulate, or pitted wall thickenings. On
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maturity, TEs lose their nuclei and cell contents and leave a hollow tube that is part of a vessel or tracheid (Fukuda H, et al., 1996).The best instances of such cell-wall depositions are the even ring-like wall thickenings that are revealed in the TEs of the xylem, the wood-forming tissue of plants.
Plant cell wall proteins are made up of less than 10% of cell wall dry weight (Zhu S, et al., 2006), but play significant roles in cell wall structure, cell wall metabolism, cell enlargement, signal transduction, responses to abiotic and biotic stresses, and many other physiological events. Based on their interactions with cell wall components, Cell Wall Proteins (CWPs) can be categorized into three categories (Jamet E, et al., 2008). The first group is labile proteins, which have minute or no interaction with cell wall components and thus move freely in the extracellular space. Such proteins can be found in liquid culture media of cell suspensions and seedlings or can be extracted with low ionic strength buffers. The second group of CWPs is the weakly bound proteins that bind the matrix by Vander Waals interactions, hydrogen bonds, hydrophobic or ionic interactions; they can be extracted by salts. The final group is the strongly bound CWPs, and there is no efficient procedure to release these proteins from the extracellular matrix, (E. Jamet, H. Canut, et al., 2006).
Since the actual players of cell wall dynamics are proteins, all CWPs other than structural proteins are of importance. Therefore, to better comprehend the cell wall complexity, the challenge is to go further into the identification of the CWPs and their functional relationships. In this context, the last few years saw the rise in search for cell wall proteins at a given time in specific environmental conditions (Albenne C, et al., 2009).
We used Arabidopsis cell culture system, where cells are growing freely in medium. These cells can be induced to form secondary
cell wall with hormones to make them form TEs (Pesquet E, et al., 2010).
The objective of the present study is to perform fractionation of cell wall from normal cells and cells that has secondary cell wall to identify the different proteins involved in the growing of secondary cell wall and lignification. After the formation of the secondary cell walls, the identification of cell wall proteins and the quality of cell wall fractionation was achieved by using MS/MS.
We performed the cell wall preparation and extraction of the proteins bound to the cell wall. Proteins extracted within the cell wall preparation from the cell wall were identified with MS/MS and the results are compared between the Basal and Induced cell wall preparations and also from different extractions.
As the main component of wood and plant fibers, understanding the cell wall proteins during xylem TE secondary cell wall formation has important biological and economic implications.
Materials and methods
Plant material
Suspension cell cultures of Arabidopsis thaliana were generated by growing the cells at MSAR medium, pH 5.7. Cells were agitated on a shaker at 23℃ at 120 rpm maintained on dark. Cells were sub-cultured by transferring 5ml of one week old culture into 45ml of fresh MSAR medium as a safety backup.
Cell induction for TE differentiation
Cell induction was carried out in a sterile Erlenmeyer flask with one week old cell
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culture. Initially, the cell culture was centrifuged at 200 × g for 2 minutes and a known weight of pelleted cells was diluted with MSAR media to a concentration of 0.031g/ml. Then, cell induction was performed by adding 1µl 6-Benzylaminopurine (BAP)/ml, 0.6µl 1-Napthaleneacetic acid (NAA)/ml and 0.8µl Epibras/ml (Pesquet E, 2010). A basal sample was prepared as reference without any addition of hormones. Finally, samples were placed on a shaker for 7-9 days growth time. The induced sample contain between 15-20% of TEs.
Ultimately, the cell culture was harvested with vacuum filtration (using a 100µm nylon filter) and washed with double distilled water and thereafter froze in liquid nitrogen and stored at -80℃ until used.
Cell homogenization
The cells were homogenized by either of the three methods; grinding, sonication or freezer miller. For grinding, the cell sample was placed in a mortar in liquid nitrogen and crushed with a pestle till it was broken into fine powder. Sonication which is the act of converting an electrical energy into physical vibration to rupture cells was performed by mixing the cells with buffer and agitating it with a sonicator. Sonication was conducted for 2 min, 3 min and 4 min at 10 pulses and 5 rests at amplitude of 70% on ice. Likewise, in case of freezer mill 6850, the cell sample was placed in plastic cylinder with metal cap and was grinded to fine powder using a medium sized metal bar. Moreover, the cells were checked intermittently under the microscope to ensure that they had been crushed sufficiently.
Cell wall fractionation
The powder of cell sample ground for 30 cycles by freezer mill was suspended in 40ml cell wall buffer (150mM NaCl and 10% glycerol in 100mM Acetate buffer, pH 4.6) and
centrifuged at 1 000 × g for 15 minutes with the temperature maintained at 4℃. The supernatant was collected and the resulting pellet was further purified with increasing concentration of sucrose. The pellet was purified by three successive centrifugations (1000 × g, 4℃, 15 minutes) with 0.4M sucrose, 0.6M sucrose and 1M sucrose in acetate buffer. All the supernatant of each time was concentrated by using 50mL centrifugal filter with 4 500 × g, until all supernatant was concentrated and change to cell wall buffer, for further protein analysis. Finally, the pellet was solubilized with 5mM MgCl2 in MES-KOH, pH 5.6 (MESbuffer) and centrifuged twice; the first one at 1 000 × g, 4℃, 3 acc for 15 minutes and the later one at 20 000 × g, 4℃, 3 acc for 10 minutes. Finally, the resulting pellet (CW4) was further grinded in liquid nitrogen and stored at -80℃.
Protein extraction
100mg of sample (CW4) was used for the extraction of protein which was performed using the detergent NP40 and different concentration of CaCl2. Initially, resultant pellet (CW4) was solubilised in 1ml of NP40 solution (0.05% NP40 + 10% DMSO in MESbuffer and centrifuged at 20 000 × g, 4℃ for 10 minutes, followed by 4 successive extraction using different concentration of CaCl2: 0.1M CaCl2, 0.5M CaCl2, 2M CaCl2
and 4M CaCl2 in MESbuffer. Between every step the cell wall pellet was vortexed and centrifuged down at 20 000 × g at 4℃ for 10 minutes. All the supernatants from each extraction were concentrated and desalted by using 0.5ml centrifugal filter collected for protein analysis. Finally, the resultant cell wall pellet (CW5) was washed twice with MESbuffer and stored at -20℃.
Protein measurement by Bradford
The protein content from each supernatant was measured using Bradford method. Firstly,
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standard of different concentration (0.1µg/ml to 0.6µg/ml) were prepared using Bovine Serum Albumin (A3294 by Sigma). Then reaction was carried out in an ELISA plate by mixing 5µl of protein extract or standard with 195µl of Bradford solution at room temperature. Finally, after measuring the absorbance at 595nm, the concentration of the protein in the extract was determined with respect to the curve plotted from the standard.
SDS-page and Western Blotting
After determining the protein concentration in the extract, 40µl of sample mixture was prepared using the protein extract, 5× SDS and water and maintaining the total concentration of protein not to exceed 20µg. It was then heated at 95°C for 5 minutes followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Subsequently, hot Coomassie blue based SDS-PAGE without Western blotting was also performed.
For SDS-PAGE, 15µl of samples were loaded and electrophoresis was run at 75V. After completing the electrophoresis, the gel was loaded on blotting apparatus by stacking the gel between the filter paper, PVDF membrane and filter paper that were equilibrated with 1 × Towbin buffer. Finally, electroblotting was carried out on a semi-dry blot (BioRad) at 0.18A for 30 minutes.
For protein detection, the PVDF membrane was initially agitated in blocking solution (1 × PBST with 5% milk powder) overnight which was followed by treatment with primary Tubulin antibody at 1:8 000 (Abcam) for 3h at room temperature. Following successive washing with blocking solution for three times, the PVDF membrane was finally agitated for 1h with secondary antibody (anti-rabbit IgG-HRP conjugate) at 1:10 000 and detected using ECL detection solution (Amersham, ECL plus Western blotting detection system by GE Healthcare).
The different fractions after cell wall preparation were also isolated using Coomassie stained gel electrophoresis. Accordingly, with the completion of SDS-PAGE, the gel was drained in a solution of 0.02% Coomassie R-350 in 10% acetic acid and heated slightly and left the gels in the coomassie solution for 20min. Finally, after leaving the gels overnight in 10% acetic acid on the shaker, the gel was scanned with an ordinary scanner.
Protein analysis by Mass Spectrometry
The CW5 pellet and 0.1M CaCl2 extraction (supernatant) from basal and induced sample was chosen for MS analysis. To the CW5 pellet, 100µl of denaturating solution was added and the sample was vortexed to homogeneity. 45µl of sample was placed in 1.5ml eppendorf tube; not exceeding the concentration of 1mg/ml. To each tube, 5µl of 1M ammonium carbonate solution (pH11) and 50µl of reduction-alkylation cocktail (97.5% acetonitrile, 2% iodoethanol and 0.5% triethylphosphine) was added and incubated at 37℃ for one hour (Hale J.E, et al., 2004). After the samples were uncapped and evaporated in a speedvac, the digestion was performed in 300µl 20mM ammonium hydrogen carbonate solution containing trypsin with a concentration of 2ng/µl (Trypsin Gold mass spectrometry grade, V5280, Promega Biotech AB) overnight at 37℃. Then the trysinated solution was filtered in 10K centrifuge filter (WVR) and evaporated in speed vac. Finally, samples were dissolved with 10µl of 0.1% formic acid and subjected for MS analysis.
Analysis of MS data
Protein identification was performed using an in-house Mascot server (Version 2.3.01, www. Matrixscience.com) with the following setting: Database: Arath-Tair9; Fixed modification: Ethanolyl (C); Variable modifications:
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methylation (DE), oxidation (M); Peptide mass tolerance: 100ppm; MS/MS fragment mass tolerance: 0.05Da; Missed cleavages: 1; Mass values: monoisotopic; Instrument type: ESI-QUAD-TOF.
Search for protein location was done in the database TAIR (www.arabidopsis.org) and SUBA (www.plantenergy.uwa.edu.au).
Workflow used in this project:
Figure 1: Strategies of cell wall protein extraction and analysis. Prior to protein extraction, the cells of A. thaliana were grinded mechanically. Once extracted, proteins were analyzed by SDS-PAGE, Western blotting and LC-MS/MS.
Results
Cell culture and TEs harvest
Arabidopsis thaliana cells cultured in the dark in MS media. After 7 days, 15-20% of the induced cells were TEs, which then was harvested by vacuum filtration.
Different methods for grinding
It is important to receive good quality of cell homogenization by grinding. Three ways of grinding were compared under the microscope. The effect could be seen in the following figures. Grinding by manpower could finally reach the same effect as other methods, but it was time-consuming and caused sample wasted (see Fig.2F-G and Fig.3F-G). Then sonication was used by different time (3 and 4min), the effect of different time can be seen in Fig.2D-E and Fig.3D-E. With the longer time, the comminution degree became better, but some of the TEs were still not completely destroyed. Freezer mill was the best method among these three, with lowest manual labor and highest sample gain. After 30 cycles grinding, we could received suitable cells comminution Fig.2B-C and Fig.3B-C.
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Suspension cell culture of A. thaliana
Cell induction for TE differentiationBasal cells without any hormone induction
Harvesting of cell culture with vacuum filtration
Cell wall preperation by tissue grinding
Subsequent washes in increasing concentration of sucrose
Protein extraction by different concentration of salts(NP40 + CaCl2)
SDS-PAGE and Western BlottingProtein identification by LC-MS/MS
Figure2: Basal sample with different homogenization methods. (A) basal cells before grinding observed under microscopy; (B) by using freezer mill for 15 cycles; (C) by using freezer mill for 30 cycles; (D) sonication for 3min; (E) sonication for 4min; (F) grinding by manpower for 20min; (G) grinding by manpower for another 20min.
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Figure 3: Induced sample with different homogenization methods. (A) TEs before grinding observed under microscopy; (B) by using freezer mill for 15 cycles, TEs were partly destroyed; (C) by using freezer mill for 30 cycles, almost all the cells became fragments; (D) sonication for 3min;(E) sonication for 4min; (F) grinding by manpower for 20min; (G) grinding by manpower for another 20min.
Western Blotting
Western Blotting was used to confirm the purity of the cell wall preparation. The results from Western Blotting show tubulin at 55kDa only in the sample of the first wash step with 150mM NaCl and 10% glycerol in 100mM Acetate buffer (pH 4.6) from both basal and induced sample(Fig.5 and Fig. 6).
After quantifying the amounts of proteins with Bradford reagent, SDS-PAGE was carried out with protein samples with total concentration of protein not exceeding 10µg. Following SDS-PAGE, western blotting was performed to confirm the purification of cell wall preparation by using anti-tubulin antibody as the primary antibody. The result from western blotting show tubulin at 55kDa only the sample of the first wash with 150mM NaCl and 10% Glycerol in 100mM Acetate buffer,
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pH 4.6, from both basal and induced sample (Fig.5 and Fig.6).
Figure 5: In basal sample. tubulin (55kDa) was found in supernatant of first wash before sucrose fractionation with 150mM NaCl and 10% glycerol in 100mM Acetate Buffer.
Figure 6: In induced sample, tubulin (55kDa) was found in supernatant of the first wash before
sucrose fractionation with 150mM NaCl and 10% glycerol in 100mM Acetate Buffer.
Analysis of SDS-PAGE
Subsequently, after SDS-PAGE, gels were also stained with Coomassie which allows the visualization of isolated proteins in the different samples. From Fig.7, it is evident that CW5-pellet (both basal and induced), 0.4M sucrose wash (basal), 0.6M sucrose wash (induced) and 2M CaCl2 extraction (induced) did not reveal the presence of any band. In fact, the absence of band in these samples could be attributed to two factors; (i) The samples either had negligible amount of proteins that is difficult to be visualized (ii) or all the proteins could have been blotted to the PVDF membrane during western blotting. In contrast to this, first washing and 0.1M CaCl2
extraction in both basal and induced sample showed maximum number of bands indicating that these samples contained more number of proteins compared to other (Fig.8). However, compared to basal sample, 0.4M sucrose wash (induced) showed considerable amount of bands during Coomassie-stained SDS-PAGE. The remaining protein samples exhibited similar patterns of bands.
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Figure 7: SDS-PAGE analysis of protein expression in basal (on the left) and induced (on the right) sample.
Figure 8: Comparing the protein expression between basal and induced sample in first washing and 0.1M CaCl2 extraction.
Bioinformatics analysis
Identification of protein in the samples (CW5-pellet and 0.1M CaCl2 extraction) was performed using LC-MS/MS followed by
database searches through www.arabidopsis.org. However, prior to MS analysis, protein samples were denatured, exposed to reduction-alkylation cocktail and digested with trypsin. During the database search, we mainly focused on the location and function of protein identified through MS with respect to Arabidopsis genome. We identified 79 proteins from CW5-pellet (induced) and 94 proteins from CW5-pellet (basal) out of which 44.3% were CWPs in the induced sample and 39.3% were CWPs in basal sample (Table 1 and 2; Appendix). Notably, both the induced and the basal CW5-pellet also revealed the presence of some proteins contaminants accounting from plasma membrane, nucleus, plastid and vacuole to name a few. Conversely, in case of 0.1M CaCl2 extract, we identified 47.1% of CWPs in basal supernatant compared to 31.1% of CWPs in induced supernatant. This implies that many of the CWPs in the basal sample could have been released during 0.1M CaCl2 extraction. In addition, we also identified the functional characterization of CWPs as listed in the Table 1 and Table 2.
Table 1: List of Arabidopsis thalinana cell wall proteins in CW5Name of protein TAIR Accession Protein acc Functionhomolog of nucleolar protein NOP56 Locus:2205270 AT1G56110* NOP56-like protein
S-Adenosymethionine synthetase 1 Locus:2196160 AT1G02500* methionine adenosyltransferase activityRAS-Related nuclear protein Locus:2147700 AT5G20010* GTP binding, protein binding, GTPase activityHeat shock protein 70-15 Locus:2017859 AT1G79920* ATP bindingHeat shock protein 90.1 Locus:2149569 AT5G52640* ATP binding, unfolded protein bindingLuminal binding protein BIP Locus:2182783 AT5G28540# ATP binding Catalase 3 Locus:2034357 AT1G20620# cobalt ion binding, catalase activityS-adenosylmethionine synthetase Locus:2089070 AT3G17390# methionine adenosyltransferase activityCellulase 3 Locus:2825314 AT1G71380# catalytic activity, hydrolase activity, hydrolyzing O-
glycosyl compoundsSKU5 similar 4 Locus:2120648 AT4G22010# oxidoreductase activity, copper ion bindingCalnexin 1 Locus:2159223 AT5G61790# calcium ion binding, unfolded protein bindingGamma subunit of Mt ATP synthase Locus:2046485 AT2G33040# zinc ion bindingAscorbate peroxidase 1 Locus:2026616 AT1G07890 L-ascorbate peroxidase activityAnnexin 1 Locus:2011344 AT1G35720 ATP binding, calcium ion binding, calcium-dependent
phospholipid binding, copper ion binding, zinc ion binding, peroxidase activity, protein homodimerization activity
MPPBETA Locus:2078623 AT3G02090 zinc ion bindingHeat shock protein 70 Locus:2181833 AT5G02500 ATP bindingVoltage dependent anion channel 3 Locus:2147820 AT5G15090 aerobic respiration, anion transport, defense response
to bacterium, regulation of seed germination, response to bacterium, response to cold
Heat shock protein 70 Locus:2101222 AT3G12580 ATP bindingHeat shock protein 70-2 Locus:2181818 AT5G02490 protein bindingGlycereldehyde-3-phosphate dehydrogenase C2
Locus:2010007 AT1G13440 copper ion binding, zinc ion binding
Heat shock protein 90 Locus:2161815 AT5G56030 ATP binding, protein bindingHeat Shock protein 70 Locus:2074984 AT3G09440 ATP bindingTubulin beta-2 Locus:2172254 AT5G62690 GTPase activity, structural molecule activity, GTP
bindingMitochondrial heat shock protein 70-1 Locus:2121022 AT4G37910 ATP binding, zinc ion bindingTubulin alpha-4 chain Locus:2010677 AT1G04820 structural constituent of cytoskeletonTubulin beta-5 chain Locus:2198661 AT1G20010 structural constituent of cytoskeletonADP/ATP carrier 1 Locus:2077778 AT3G08580 binding, copper ion binding, ATP:ADP antiporter
glycotransferase activityCullin-associated and neddylation dissociated 1
Locus:2065279 AT2G02560 Binding
Cell division cycle 48 Locus:2085064 AT3G09840 identical protein binding, ATPase activityF27F5.8 Locus:2028200 AT1G45000 ATP binding, nucleotide binding, ATPase activity,
hydrolase activity, nucleoside-triphosphatase activityT4I9.19 Locus:2139325 AT4G02930 ATP binding, cobalt ion binding, zinc ion binding,
translation elongation factor activityRIBOSOMAL PROTEIN 5B Locus:2049862 AT2G37270 structural constituent of ribosomeRIBOSOMAL PROTEIN 5A Locus:2081546 AT3G11940 structural constituent of ribosomeCYTOSOLIC NADP+-DEPENDENT ISOCITRATE DEHYDROGENASE
Locus:2009759 AT1G65930 copper ion binding, isocitrate dehydrogenase (NADP+) activity
general regulatory factor 3 Locus:2177386 AT5G38480 ATP binding, protein phosphorylated amino acid binding
F17A17.37 Locus:2077467 AT3G08030 Molecular function unknownACONITASE 3 Locus:2063354 AT2G05710 ATP binding, copper ion bindingheat shock protein 70 Locus:2144801 AT5G09590 ATP bindingHEAT SHOCK PROTEIN 89.1 Locus:2077352 AT3G07770 ATP bindingPHOSPHOGLYCERATE KINASE Locus:2206410 AT1G79550 phosphoglycerate kinase activity40S RIBOSOMAL PROTEIN S18 Locus:2199670 AT1G22780 structural constituent of ribosome, RNA binding,
nucleic acid binding
Protein acc followed by *stands for this protein was found only in induced sample;Protein acc followed by # stands for this protein was found only in basal sample;Others stand for the protein both found in induced and basal sample.
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Table 2: List of Arabidopsis thaliana cell wall proteins in 0.1M CaCl2 extraction
Name of protein TAIR Accession Protein acc FunctionHISTONE DEACETYLASE 2 Locus:2162479 AT5G22650* DNA mediated transformation, negative
regulation of transcription, DNA-dependent, polarity specification of adaxial/abaxial axis.
AIMP ALPHA, IMPORTIN ALPHA, Locus:2083313 AT3G06720 intracellular protein transport, protein import into nucleus
T11A7.10 Locus:2054336 AT2G41800 Molecular function unknown
Protein acc followed by *stands for this protein was found only in induced sample;Protein acc followed by # stands for this protein was found only in basal sample;Others stand for the protein both found in induced and basal sample.
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Discussion
Cell wall proteins which constitute to about 10% of the cell wall mass can be categorized into three main functional groups: structural proteins, defense proteins and cell wall modifying proteins. Moreover, it is believed that unidentified proteins with novel functional classes do still exist in the cell wall (Borderies G, et al., 2005). So, in this study: we intended to extract the cell wall protein from Arabidopsis cell culture as well as to analyze them. Even though it is evident that study of cell wall proteome is complex; (i) polysaccharide linkages of cellulose, hemicelluloses and pectin can retain intracellular proteins and contaminate CWPs (ii) some CWPs are difficult to solubilize, and (iii) some proteins undergoes post-translational modifications, (Borderies G, et al., 2005; Jamet E, et al., 2008), we adopt some specific strategies in this study to investigate the cell wall proteomics of Arabidopsis thaliana.
The principle steps of this Arabidopsis cell wall proteomic study involved induction of TE differentiation, cell wall preparation, protein extraction and finally protein analysis using SDS-PAGE and MS/MS. Several studies have shown that different phytohormones like auxin and cytokinin are known to promote the initiation of TE differentiation. (Fukuda H, et al., 1997; Oda Y, et al., 2005) Consistent with this, BAP, NAA and Epibras were implicated for the induction of TE differentiation which is parallel with the study carried out by Pesquet (Pesquet E, et al., 2010). In addition, similar study was carried out by Oda (Oda Y, et al., 2005) in which they used Brassinosteroid for TE differentiation in AC-GT13 cells of Arabidopsis. Similarly, Falconer (Falconer, et al., 1984) showed that Zinnia mesophyll cells could be induced for TE differentiation by the use of BAP and NAA (Faoconer M.M, et al., 1985; Feiz L, et al., 2006).
Likewise, the composition of washing buffer is critical for the extraction of proteins from the cell wall. The presence of NaCl in washing buffer during the early steps of cell wall preparation promotes the release of weakly-bound proteins interlinked by ionic interaction in the cell wall (Borderies G, et al., 2005; Feiz L, et al., 2006). Moreover, the washing buffer with low ionic intensity and an acidic pH was used for the purification of cell wall. This preserves the interaction between the proteins and polysaccharides and prevents the loss of CWPs. (Jamet E, et al., 2008; Feiz L, et al., 2006). Cell wall preparation also included purification by subsequent centrifugation in solution of increasing density. Since the cell wall polysaccharides are relatively dense in nature, this density gradient centrifugation facilitates in elimination of less-dense cell organelles (Feiz L, et al., 2006). Finally, CaCl2
which is considered as the most efficient salt for the extraction of proteins from higher plants is used to release CWPs from purified cell wall (Borderies G, et al., 2005; Jamet E, et al., 2008) However, it should be noted that CWPs that are tightly bound are still resistant to salt extraction (Jamet E, et al., 2008).
Proteins that were sequentially extracted from Arabidopsis cell wall were subjected for SDS-PAGE and western blotting to further confirm the purity of cell wall prepared. Consistent to this, we used anti-tubulin antibody that detects the presence of tubulin in the protein extract. Our result showed the appearance of a band characteristic to tubulin only in the extract from first washing step of both basal and induced sample. Conversely, other washing step did not reveal any tubulin bands. This implies that the tubulin proteins associated with the Arabidopsis cell wall were eliminated in the early washing step. However, upon MS analysis, tubulin proteins were evident in the resultant CW5 pellet which indicated that some of the proteins were not completely released from the cell wall. Accordingly, it can be inferred that the purification strategies that we adopted was not efficient enough to remove all
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the contaminants. Moreover, it should be noted that several constraints arise during CWP purification and analysis; the difficulty to solubilise many CWPs, the complex polysaccharide linkages by which intracellular proteins remain trapped, and post-translational modification of proteins. Likewise, some of the proteins are embedded strongly and interact differently with other cell wall component making the task more challenging. And when the general strategy of cell wall proteomics is purification of cell wall followed by protein extraction with salt, one of the major disadvantages is the contamination by intracellular proteins sticking non-specifically with the cell wall (Jamet E, et al., 2008). So, improvements can be made in the extraction of non-specifically bound intracellular proteins as well as the proteins that are strongly embedded in the cell wall components. The use of hydrolytic enzyme or chemicals to degrade the cell wall matrix yet maintaining the protein integrity could be of paramount importance in studying the CWPs more conveniently.
MS-based proteomics is indispensible technology to analyze and identify proteins. Generally, prior to peptide sequencing by LC-MS/MS, proteins are digested using proteolytic enzymes (Aebersold R, et al., 2003; Hale J.E, et al., 2004). In this context, digestion was performed using Trypsin. However, it should be considered that efficiency of digestion increases with the disruption of tertiary structure of protein. Studies have demonstrated that sulfhydryl group of cysteine residues can form disulfide bonds and highly stabilize the tertiary structure. So, in advance to digestion by trypsin, reduction and alkylation of cysteine residues were carried out using volatile reagent triethylphosphine and iodoethanol. This assists the blockage of sulfhydryl groups, destabilize the tertiary structure and ultimately lead to enhanced protein digestion (Aebersold R, et al., 2003). To disrupt the tertiary structure of proteins in the CW5 pellet sample we used denaturizing solution containing 6M guanidine
to make the reduction, alkylation and digestion possible more efficient.
From the MS analysis and database search, we identified 44.3% of cell wall proteins in induced CW5-pellet compared to 39.3% of cell wall proteins in basal. Contrastingly, the analysis of cell wall proteins in 0.1M CaCl2
extraction showed that 47.1% of cell wall proteins were present in the basal sample compared to 31.1% in the induced sample. This seems reasonable why the CW5-pellet (basal) had relatively fewer amount of proteins than the CW5-pellet (induced). Tentatively, this implies that majority of the cell wall proteins of basal sample were released during the extraction point; one of the reasons could be that cell wall proteins in basal sample, with no TEs were loosely bound to the cell wall. The other explanation could be that some cell wall proteins get tighter bound to the cell wall during secondary cell wall formation. Yet, we cannot be certain since we had no replicates of the sample and we did not perform MS/MS analysis with other extraction samples. As a result, we are unaware about the proteins that may have been released during the other point of extraction.
Conclusion
We prepared cell wall from Arabidopsis thaliana basal cells as well as cells that had been induced with hormones (NAA, BAP and Epibras) to make them form TEs. The cell wall preparation involved mechanical grinding with cells, density gradient cell-fractionation using different concentration of sucrose and sequential extraction of proteins using NP40 and different concentration of CaCl2. We then performed proteomic analysis of proteins in resultant CW5 pellet and proteins extracted with 0.1M CaCl2 using LC-MS/MS. Protein identification, location and functions were predicted using Arabidopsis database search. We were able to identify 44.3% of cell wall
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proteins in the resultant CW5-pellet (induced) compared to 39.3% of cell wall proteins in the resultant CW5-pellet (basal). Moreover, we observed that some of the cell wall proteins were released from cell wall during 0.1M CaCl2 extraction. Since there were some non-resident proteins in resultant CW5-pellet, we assume that some improvements can be made in the purification of cell wall. For instance, use of hydrolytic enzymes or chemicals with the potential to degrade polysaccharide matrix can possible prevent trapping of non-resident proteins and increase purification of cell wall preparation.
Acknowledgements
We are extremely grateful to Irene Granlund for supervising the project in Applied Functional genomics as well as reading the manuscript; she has given her valuable feedback throughout the project and necessary correction as and when needed. We are also deeply indebted to Edouard Pesquet and Jan Karlsson for their guidance and help during the project. The study was supported by the Umea Plant Science Centre (UPSC), Umea University.
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Appendix
MSAR medium for cell suspension culture:
4.4g MS basal salt plus vitamins (Duchefa M0222.0225)
30g sucrose (3%)
pH 5.7 with 1M KOH (for 1 liter)
Buffers used in cell wall preparation:
Buffer 1;
150mM NaCl
10% Glycerol
100mM Acetate buffer (pH 4.6)
Buffer 2;
5mM MES-KOH (pH 5.6)
5mM MgCl2
Buffer 3;
10% DMSO
5mM MES-KOH (pH 5.6)
5mM MgCl2
Other chemicals in cell wall preparation:
0.4M, 0.6M and 1M sucrose
0.05% NP40
0.1M, 0.5M, 2M and 4M CaCl2
Chemicals and reagents used in protein measurement:
Bovine serum albumin (A3294 by Sigma)
Bradford solution
SDS-PAGE and Western blotting:
Resolving gel (12% gel):
30% acrylamide 29:1 - 4.8ml
1M tris-HCL pH8.8 – 4.5ml
10% SDS – 0.120ml
ddH2O – 2.5ml
10%APS – 0.075ml
16
TEMED – 0.0075ml
Stack gel (6% gel)
30% acrylamide 29:1 – 0.8ml
1M tris-HCL pH8.8 – 0.5ml
10% SDS – 0.05ml
ddH2O – 2.615ml
10%APS – 0.03ml
TEMED – 0.005ml
Buffers used in western blotting:
10 × Towbin buffer
0.13M Tris – 15.7g
10% ethanol – 0.1L
1M glycine – 75g
10 × PBS, pH 7.4 (total amount 1L)
10mM Na2HPO4 – 13.8g
3mMKH2PO4 – 4.08g
140mM NaCl – 81.8g
1 × PBST (Tween 20 )- total amount 1L
10 × PBS – 100ml
0.05% Tween 20 – 0.5ml
10 × electrophoresis buffer, pH 8.3, 1L
Tris – 30.3g
Glycine – 144.1g
1%SDS – 10g
Transfer buffer
10 × electrophoresis buffer – 100ml
Isopropanol (99.8% purity) – 100ml
Coomassie staining
0.02% coomassie R-350 in 10% acetic acid
MS analysis:
Denaturating buffer:
6M guanidin, 0.1M Tris, 5mM EDTA, pH 8
57g Guanidin HCl
614mg Trizma-HCl
750mg Trizma base
58mg EDTA
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Dilute to 100ml
Protein solution (for reduction/alkylation)
97.5% acetonitrile (v/v)
2% iodoethanol (130mM)
0.5% triethylphospine (17mM) final pH 10.
Other chemicals:
2mg/µl trypsin
0.1% formic acid
Table 1: List of all proteins in CW5
Protein acc Protein score Protein cover Localization Function
AT5G13450* 39 5.3 chloroplast, membrane, mitochondrion, plasma membrane cobalt ion binding, zinc ion binding, hydrogen ion transporting ATP synthase activity,
