Analysis of Cell Wall Proteins during Xylem Vessel Secondary Cell Wall Formation in Cell Culture

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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 …

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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  • 1. INTRODUCTION .......................................................................................................................................... 2MATERIALS 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 ....................................................................................................................................... 6RESULTS ....................................................................................................................................................... 6 CELL CULTURE AND TES HARVEST...................................................................................................................... 6 DIFFERENT METHODS FOR GRINDING ................................................................................................................... 6 WESTERN BLOTTING ........................................................................................................................................... 8 ANALYSIS OF SDS-PAGE ................................................................................................................................... 9 BIOINFORMATICS ANALYSIS .............................................................................................................................. 10DISCUSSION................................................................................................................................................ 13CONCLUSION ............................................................................................................................................. 14ACKNOWLEDGEMENTS .......................................................................................................................... 15REFERENCES ............................................................................................................................................. 15APPENDIX ................................................................................................................................................... 16 1
  • 2. 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 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 CaCl 2 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 CaCl 2 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.________________________________________________________________ secondary cell wall formed after the elongation, providing mechanical sustenance to the entire plant (Borderies G, et al., 2003).Introduction The formation of a dense lignified secondary cell wall only occurs once cells have reached their final shape and size.The plant cell wall is a vital component of a Xylem is formed by the combination ofplant cell which provides both structural tracheary elements (TEs), parenchyma cells,integrity and functional role to a plant. There and fibers. TEs are the characteristic cells ofare two core types of cell walls that are found the xylem that are categorized by the formationin plants: the primary cell wall that gets of a secondary cell wall with annular, spiral,accumulated through cell division and growth, reticulate, or pitted wall thickenings. Onwhich is capable to elongate; and the 2
  • 3. maturity, TEs lose their nuclei and cell cell wall with hormones to make them formcontents and leave a hollow tube that is part of TEs (Pesquet E, et al., 2010).a vessel or tracheid (Fukuda H, et al.,1996).The best instances of such cell-wall The objective of the present study is to performdepositions are the even ring-like wall fractionation of cell wall from normal cells andthickenings that are revealed in the TEs of the cells that has secondary cell wall to identify thexylem, the wood-forming tissue of plants. different proteins involved in the growing of secondary cell wall and lignification. After thePlant cell wall proteins are made up of less formation of the secondary cell walls, thethan 10% of cell wall dry weight (Zhu S, et al., identification of cell wall proteins and the2006), but play significant roles in cell wall quality of cell wall fractionation was achievedstructure, cell wall metabolism, cell by using MS/MS.enlargement, signal transduction, responses toabiotic and biotic stresses, and many other We performed the cell wall preparation andphysiological events. Based on their extraction of the proteins bound to the cellinteractions with cell wall components, Cell wall. Proteins extracted within the cell wallWall Proteins (CWPs) can be categorized into preparation from the cell wall were identifiedthree categories (Jamet E, et al., 2008). The with MS/MS and the results are comparedfirst group is labile proteins, which have between the Basal and Induced cell wallminute or no interaction with cell wall preparations and also from differentcomponents and thus move freely in the extractions.extracellular space. Such proteins can be found As the main component of wood and plantin liquid culture media of cell suspensions and fibers, understanding the cell wall proteinsseedlings or can be extracted with low ionic during xylem TE secondary cell wall formationstrength buffers. The second group of CWPs is has important biological and economicthe weakly bound proteins that bind the matrix implications.by Vander Waals interactions, hydrogenbonds, hydrophobic or ionic interactions; theycan be extracted by salts. The final group is thestrongly bound CWPs, and there is no efficient Materials and methodsprocedure to release these proteins from theextracellular matrix, (E. Jamet, H. Canut, et al.,2006). Plant materialSince the actual players of cell wall dynamicsare proteins, all CWPs other than structuralproteins are of importance. Therefore, to better Suspension cell cultures of Arabidopsiscomprehend the cell wall complexity, the thaliana were generated by growing the cellschallenge is to go further into the identification at MSAR medium, pH 5.7. Cells were agitatedof the CWPs and their functional relationships. on a shaker at 23℃ at 120 rpm maintained onIn this context, the last few years saw the rise dark. Cells were sub-cultured by transferringin search for cell wall proteins at a given time 5ml of one week old culture into 45ml of freshin specific environmental conditions (Albenne MSAR medium as a safety backup.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 3
  • 4. Cell induction for TE differentiation Cell wall fractionationCell induction was carried out in a sterile The powder of cell sample ground for 30Erlenmeyer flask with one week old cell cycles by freezer mill was suspended in 40mlculture. Initially, the cell culture was cell wall buffer (150mM NaCl and 10%centrifuged at 200 × g for 2 minutes and a glycerol in 100mM Acetate buffer, pH 4.6) andknown weight of pelleted cells was diluted centrifuged at 1 000 × g for 15 minutes withwith MSAR media to a concentration of the temperature maintained at 4℃. The0.031g/ml. Then, cell induction was performed supernatant was collected and the resultingby adding 1µl 6-Benzylaminopurine (BAP)/ml, pellet was further purified with increasing0.6µl 1-Napthaleneacetic acid (NAA)/ml and concentration of sucrose. The pellet was0.8µl Epibras/ml (Pesquet E, 2010). A basal purified by three successive centrifugationssample was prepared as reference without anyaddition of hormones. Finally, samples were (1000 × g, 4℃, 15 minutes) with 0.4Mplaced on a shaker for 7-9 days growth time. sucrose, 0.6M sucrose and 1M sucrose inThe induced sample contain between 15-20% acetate buffer. All the supernatant of each timeof TEs. was concentrated by using 50mL centrifugal filter with 4 500 × g, until all supernatant wasUltimately, the cell culture was harvested with concentrated and change to cell wall buffer, forvacuum filtration (using a 100µm nylon filter) further protein analysis. Finally, the pellet wasand washed with double distilled water and solubilized with 5mM MgCl2 in MES-KOH,thereafter froze in liquid nitrogen and stored at pH 5.6 (MESbuffer) and centrifuged twice; the-80℃ until used. first one at 1 000 × g, 4 ℃ , 3 acc for 15Cell homogenization 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 nitrogenThe cells were homogenized by either of thethree methods; grinding, sonication or freezer and stored at -80℃.miller. For grinding, the cell sample wasplaced in a mortar in liquid nitrogen and Protein extractioncrushed with a pestle till it was broken intofine powder. Sonication which is the act of 100mg of sample (CW4) was used for theconverting an electrical energy into physical extraction of protein which was performedvibration to rupture cells was performed by using the detergent NP40 and differentmixing the cells with buffer and agitating it concentration of CaCl2. Initially, resultantwith a sonicator. Sonication was conducted for pellet (CW4) was solubilised in 1ml of NP402 min, 3 min and 4 min at 10 pulses and 5 rests solution (0.05% NP40 + 10% DMSO inat amplitude of 70% on ice. Likewise, in caseof freezer mill 6850, the cell sample was MESbuffer and centrifuged at 20 000 × g, 4℃placed in plastic cylinder with metal cap and for 10 minutes, followed by 4 successivewas grinded to fine powder using a medium extraction using different concentration ofsized metal bar. Moreover, the cells were CaCl2: 0.1M CaCl2, 0.5M CaCl2, 2M CaCl2checked intermittently under the microscope to and 4M CaCl2 in MESbuffer. Between everyensure that they had been crushed sufficiently. step the cell wall pellet was vortexed and centrifuged down at 20 000 × g at 4℃ for 10 minutes. All the supernatants from each 4
  • 5. extraction were concentrated and desalted by was followed by treatment with primaryusing 0.5ml centrifugal filter collected for Tubulin antibody at 1:8 000 (Abcam) for 3h atprotein analysis. Finally, the resultant cell wall room temperature. Following successivepellet (CW5) was washed twice with washing with blocking solution for three times,MESbuffer and stored at -20℃. the PVDF membrane was finally agitated for 1h with secondary antibody (anti-rabbit IgG- HRP conjugate) at 1:10 000 and detected usingProtein measurement by Bradford ECL detection solution (Amersham, ECL plus Western blotting detection system by GEThe protein content from each supernatant was Healthcare).measured using Bradford method. Firstly,standard of different concentration (0.1µg/ml The different fractions after cell wallto 0.6µg/ml) were prepared using Bovine preparation were also isolated usingSerum Albumin (A3294 by Sigma). Then Coomassie stained gel electrophoresis.reaction was carried out in an ELISA plate by Accordingly, with the completion of SDS-mixing 5µl of protein extract or standard with PAGE, the gel was drained in a solution of195µl of Bradford solution at room 0.02% Coomassie R-350 in 10% acetic acidtemperature. Finally, after measuring the and heated slightly and left the gels in theabsorbance at 595nm, the concentration of the coomassie solution for 20min. Finally, afterprotein in the extract was determined with leaving the gels overnight in 10% acetic acidrespect to the curve plotted from the standard. on the shaker, the gel was scanned with an ordinary scanner.SDS-page and Western Blotting Protein analysis by Mass SpectrometryAfter determining the protein concentration inthe extract, 40µl of sample mixture was The CW5 pellet and 0.1M CaCl2 extractionprepared using the protein extract, 5× SDS and (supernatant) from basal and induced samplewater and maintaining the total concentration was chosen for MS analysis. To the CW5of protein not to exceed 20µg. It was then pellet, 100µl of denaturating solution washeated at 95°C for 5 minutes followed by SDS- added and the sample was vortexed topolyacrylamide gel electrophoresis (SDS- homogeneity. 45µl of sample was placed inPAGE) and Western blotting. Subsequently, 1.5ml eppendorf tube; not exceeding thehot Coomassie blue based SDS-PAGE without concentration of 1mg/ml. To each tube, 5µl ofWestern blotting was also performed. 1M ammonium carbonate solution (pH11) and 50µl of reduction-alkylation cocktail (97.5%For SDS-PAGE, 15µl of samples were loaded acetonitrile, 2% iodoethanol and 0.5%and electrophoresis was run at 75V. After triethylphosphine) was added and incubated atcompleting the electrophoresis, the gel was 37℃ for one hour (Hale J.E, et al., 2004).loaded on blotting apparatus by stacking thegel between the filter paper, PVDF membrane After the samples were uncapped andand filter paper that were equilibrated with 1 × evaporated in a speedvac, the digestion wasTowbin buffer. Finally, electroblotting was performed in 300µl 20mM ammoniumcarried out on a semi-dry blot (BioRad) at hydrogen carbonate solution containing trypsin0.18A for 30 minutes. with a concentration of 2ng/µl (Trypsin Gold mass spectrometry grade, V5280, PromegaFor protein detection, the PVDF membrane Biotech AB) overnight at 37℃. Then thewas initially agitated in blocking solution (1 × trysinated solution was filtered in 10KPBST with 5% milk powder) overnight which 5
  • 6. centrifuge filter (WVR) and evaporated in Figure 1: Strategies of cell wall protein extractionspeed vac. Finally, samples were dissolved and analysis. Prior to protein extraction, the cells ofwith 10µl of 0.1% formic acid and subjected A. thaliana were grinded mechanically. Oncefor MS analysis. extracted, proteins were analyzed by SDS-PAGE, Western blotting and LC-MS/MS.Analysis of MS dataProtein identification was performed using anin-house Mascot server (Version 2.3.01, www. ResultsMatrixscience.com) with the following setting:Database: Arath-Tair9; Fixed modification:Ethanolyl (C); Variable modifications: Cell culture and TEs harvestmethylation (DE), oxidation (M); Peptide masstolerance: 100ppm; MS/MS fragment mass Arabidopsis thaliana cells cultured in the darktolerance: 0.05Da; Missed cleavages: 1; Mass in MS media. After 7 days, 15-20% of thevalues: monoisotopic; Instrument type: ESI- induced cells were TEs, which then wasQUAD-TOF. harvested by vacuum filtration.Search for protein location was done in thedatabase TAIR (www.arabidopsis.org) and Different methods for grindingSUBA (www.plantenergy.uwa.edu.au). It is important to receive good quality of cellWorkflow used in this project: 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. 6
  • 7. Figure2: Basal sample with different homogenization methods. (A) basal cells before grindingobserved under microscopy; (B) by using freezer mill for 15 cycles; (C) by using freezer mill for 30cycles; (D) sonication for 3min; (E) sonication for 4min; (F) grinding by manpower for 20min; (G)grinding by manpower for another 20min. 7
  • 8. Figure 3: Induced sample with different homogenization methods. (A) TEs before grinding observedunder microscopy; (B) by using freezer mill for 15 cycles, TEs were partly destroyed; (C) by usingfreezer 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 another20min. After quantifying the amounts of proteins with Bradford reagent, SDS-PAGE was carried outWestern Blotting with protein samples with total concentration of protein not exceeding 10µg. FollowingWestern Blotting was used to confirm the SDS-PAGE, western blotting was performed topurity of the cell wall preparation. The results confirm the purification of cell wallfrom Western Blotting show tubulin at 55kDa preparation by using anti-tubulin antibody asonly in the sample of the first wash step with the primary antibody. The result from western150mM NaCl and 10% glycerol in 100mM blotting show tubulin at 55kDa only theAcetate buffer (pH 4.6) from both basal and sample of the first wash with 150mM NaClinduced sample(Fig.5 and Fig. 6). and 10% Glycerol in 100mM Acetate buffer, 8
  • 9. pH 4.6, from both basal and induced sample sucrose fractionation with 150mM NaCl and 10%(Fig.5 and Fig.6). 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. InFigure 5: In basal sample. tubulin (55kDa) was fact, the absence of band in these samplesfound in supernatant of first wash before sucrose could be attributed to two factors; (i) Thefractionation with 150mM NaCl and 10% glycerol samples either had negligible amount ofin 100mM Acetate Buffer. 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.Figure 6: In induced sample, tubulin (55kDa) wasfound in supernatant of the first wash before 9
  • 10. Figure 7: SDS-PAGE analysis of protein expression in basal (on the left) and induced (on the right) sample. 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 contaminantsFigure 8: Comparing the protein expression accounting from plasma membrane, nucleus,between basal and induced sample in first washing plastid and vacuole to name a few. Conversely,and 0.1M CaCl2 extraction. 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.Bioinformatics analysis This implies that many of the CWPs in the basal sample could have been released during 0.1M CaCl2 extraction. In addition, we alsoIdentification of protein in the samples (CW5- identified the functional characterization ofpellet and 0.1M CaCl2 extraction) was CWPs as listed in the Table 1 and Table 2.performed using LC-MS/MS followed by 10
  • 11. 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 proteinS-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 bindingCatalase 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 activityMPPBETA 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 coldHeat shock protein 70 Locus:2101222 AT3G12580 ATP bindingHeat shock protein 70-2 Locus:2181818 AT5G02490 protein bindingGlycereldehyde-3-phosphate Locus:2010007 AT1G13440 copper ion binding, zinc ion bindingdehydrogenase C2Heat 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 activityDefective glycolysation Locus:2173659 AT5G66680 dolichyl-diphosphooligosaccharide-protein glycotransferase activityCullin-associated and neddylation Locus:2065279 AT2G02560 Bindingdissociated 1Cell 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 Locus:2009759 AT1G65930 copper ion binding, isocitrate dehydrogenaseISOCITRATE DEHYDROGENASE (NADP+) activitygeneral regulatory factor 3 Locus:2177386 AT5G38480 ATP binding, protein phosphorylated amino acid bindingF17A17.37 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 bindingProtein 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. 11
  • 12. Table 2: List of Arabidopsis thaliana cell wall proteins in 0.1M CaCl2 extractionName 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.F28K19.27 Locus:2029391 AT1G78060* Carbohydrate metabolic process, hydrolase activity.BGLU15, BETA GLUCOSIDASE 15 Locus:2050605 AT2G44450* carbohydrate metabolic processGLP10, GERMIN-LIKE PROTEIN 10 Locus:2079582 AT3G62020* Biological process, manganese ion binding, nutrient reservoir activity.MQJ2.5 Locus:2171228 AT5G58450* Binding.F18G18.200 Locus:2145457 AT5G25460* Response to karrikinEXPA4, ATEXP4, ATEXPA4, F17A14_7, Locus:2043240 AT2G39700* Plant-type cell wall loosening, plant-type cellEXPANSIN A4, ATHEXP ALPHA 1.6 wall modification involved in multidimensional cell growth, syncytium formation, unidimensional cell growth.F11F8.22 Loucus:2074904 AT3G09630* Translation, structural constituent of ribosomeF3L24.33 Locus:2074984 AT3G09440* Protein folding, response to cadmium ion, response to heat, response to karrikin, ATP binding.ATTUDOR1, TUDOR-SN PROTEIN 1 Locus:2183359 AT5G07350* Protein secretion, response to cadmium ion, response to stress, RNA binding, nucleic acid binding, nuclease activity.BIP1, T26D3.10 Locus:2182783 AT5G28540# ATP bindingT19D11.4 Locus:2098308 AT3G28200# peroxidase activityF3L24.33 Locus:2074984 AT3G09440# ATP bindingEXLA3,ATEXLA3, F16L2.170 Locus:2077167 AT3G45960# plant-type cell wall loosening, plant-type cell wall organization,unidimensional cell growthMOJ9.4, ATPGIP2, Locus:2169404 AT5G06870# polygalacturonase inhibitor activityPOLYGALACTURONASE INHIBITINGPROTEIN 2MSJ1.10, EXORDIUM LIKE 2 Locus:2173428 AT5G64260# molecular function unknownMOJ9.20 Locus:2169369 AT5G07030# aspartic-type endopeptidase activityF17A17.37 Locus:2077467 AT3G08030 molecular function unknownF28K19.27 Locus:2029391 AT1G78060 Carbohydrate metabolic process, hydrolase activity.T6P5.12 Locus:2064696 AT2G05920 Negative regulation of catalytic activity, proteolysis, identical protein binding.K19M13.1 Locus:2154463 AT5G23400 Defense response, signal transduction,SKS17, MUD21.18, SKU5 SIMILAR 17 Locus:2174954 AT5G66920 copper ion bindingXTH4, T9F8.4, EXGT-A1, Locus:2065821 AT2G06850 hydrolase activity, acting on glycosylENDOXYLOGLUCAN TRANSFERASE bonds, xyloglucan:xyloglucosyl transferase activityCELLULASE 3, Locus:2825314 AT1G71380 Carbohydrate metabolic process, catalytic activity, hydrolase activity.F21F14.7 Locus:2076745 AT3G61820 aspartic-type endopeptidase activityF28K19.27 Locus:2029391 AT1G78060 hydrolase activity, hydrolyzing O-glycosyl compoundsATCS, CSY4, F4I1.16, CITRATE Locus:2050554 AT2G44350 ATP binding, zinc ion bindingSYNTHASE 4ACO3, T3P4.5, ACONITASE 3 Locus:2063354 AT2G05710 ATP binding, copper ion bindingT3H13.3, EXORDIUM Locus:2138753 AT4G08950 response to brassinosteroid stimulusF21F14.190, GERMIN-LIKE PROTEIN 10 Locus:2079582 AT3G62020 manganese ion bindingF8N16.8, Locus:2053215 AT2G28790 Molecular function unknownEXLA1, ATEXPL1, ATHEXP BETA 2.1, Locus:2077177 AT3G45970 plant-type cell wall loosening, unidimensionalEXPANSIN-LIKE A1 cell growthXTH5,MAC12.33, ENDOXYLOGLUCAN Locus:2159118 AT5G13870 hydrolase activity, acting on glycosylTRANSFERASE A4 bonds, hydrolase activity, hydrolyzing O- glycosyl compounds, xyloglucan:xyloglucosyl transferase activityAIMP ALPHA, IMPORTIN ALPHA, Locus:2083313 AT3G06720 intracellular protein transport, protein import into nucleusT11A7.10 Locus:2054336 AT2G41800 Molecular function unknownProtein 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. 12
  • 13. Likewise, the composition of washing buffer isDiscussion critical for the extraction of proteins from the cell wall. The presence of NaCl in washing buffer during the early steps of cell wallCell wall proteins which constitute to about preparation promotes the release of weakly-10% of the cell wall mass can be categorized bound proteins interlinked by ionic interactioninto three main functional groups: structural in the cell wall (Borderies G, et al., 2005; Feizproteins, defense proteins and cell wall L, et al., 2006). Moreover, the washing buffermodifying proteins. Moreover, it is believed with low ionic intensity and an acidic pH wasthat unidentified proteins with novel functional used for the purification of cell wall. Thisclasses do still exist in the cell wall (Borderies preserves the interaction between the proteinsG, et al., 2005). So, in this study: we intended and polysaccharides and prevents the loss ofto extract the cell wall protein from CWPs. (Jamet E, et al., 2008; Feiz L, et al.,Arabidopsis cell culture as well as to analyze 2006). Cell wall preparation also includedthem. Even though it is evident that study of purification by subsequent centrifugation incell wall proteome is complex; (i) solution of increasing density. Since the cellpolysaccharide linkages of cellulose, wall polysaccharides are relatively dense inhemicelluloses and pectin can retain nature, this density gradient centrifugationintracellular proteins and contaminate CWPs facilitates in elimination of less-dense cell(ii) some CWPs are difficult to solubilize, and organelles (Feiz L, et al., 2006). Finally, CaCl2(iii) some proteins undergoes post-translational which is considered as the most efficient saltmodifications, (Borderies G, et al., 2005; for the extraction of proteins from higherJamet E, et al., 2008), we adopt some specific plants is used to release CWPs from purifiedstrategies in this study to investigate the cell cell wall (Borderies G, et al., 2005; Jamet E, etwall proteomics of Arabidopsis thaliana. al., 2008) However, it should be noted that CWPs that are tightly bound are still resistantThe principle steps of this Arabidopsis cell to salt extraction (Jamet E, et al., 2008).wall proteomic study involved induction of TEdifferentiation, cell wall preparation, protein Proteins that were sequentially extracted fromextraction and finally protein analysis using Arabidopsis cell wall were subjected for SDS-SDS-PAGE and MS/MS. Several studies have PAGE and western blotting to further confirmshown that different phytohormones like auxin the purity of cell wall prepared. Consistent toand cytokinin are known to promote the this, we used anti-tubulin antibody that detectsinitiation of TE differentiation. (Fukuda H, et al., the presence of tubulin in the protein extract.1997; Oda Y, et al., 2005) Consistent with this, Our result showed the appearance of a bandBAP, NAA and Epibras were implicated for characteristic to tubulin only in the extractthe induction of TE differentiation which is from first washing step of both basal andparallel with the study carried out by Pesquet induced sample. Conversely, other washing(Pesquet E, et al., 2010). In addition, similar step did not reveal any tubulin bands. Thisstudy was carried out by Oda (Oda Y, et al., implies that the tubulin proteins associated2005) in which they used Brassinosteroid for with the Arabidopsis cell wall were eliminatedTE differentiation in AC-GT13 cells of in the early washing step. However, upon MSArabidopsis. Similarly, Falconer (Falconer, et analysis, tubulin proteins were evident in theal., 1984) showed that Zinnia mesophyll cells resultant CW5 pellet which indicated that somecould be induced for TE differentiation by the of the proteins were not completely releaseduse of BAP and NAA (Faoconer M.M, et al., from the cell wall. Accordingly, it can be1985; Feiz L, et al., 2006). inferred that the purification strategies that we adopted was not efficient enough to remove all 13
  • 14. the contaminants. Moreover, it should be noted to make the reduction, alkylation and digestionthat several constraints arise during CWP possible more efficient.purification and analysis; the difficulty tosolubilise many CWPs, the complex From the MS analysis and database search, wepolysaccharide linkages by which intracellular identified 44.3% of cell wall proteins inproteins remain trapped, and post-translational induced CW5-pellet compared to 39.3% of cellmodification of proteins. Likewise, some of the wall proteins in basal. Contrastingly, theproteins are embedded strongly and interact analysis of cell wall proteins in 0.1M CaCl2differently with other cell wall component extraction showed that 47.1% of cell wallmaking the task more challenging. And when proteins were present in the basal samplethe general strategy of cell wall proteomics is compared to 31.1% in the induced sample.purification of cell wall followed by protein This seems reasonable why the CW5-pelletextraction with salt, one of the major (basal) had relatively fewer amount of proteinsdisadvantages is the contamination by than the CW5-pellet (induced). Tentatively,intracellular proteins sticking non-specifically this implies that majority of the cell wallwith the cell wall (Jamet E, et al., 2008). So, proteins of basal sample were released duringimprovements can be made in the extraction of the extraction point; one of the reasons couldnon-specifically bound intracellular proteins as be that cell wall proteins in basal sample, withwell as the proteins that are strongly embedded no TEs were loosely bound to the cell wall.in the cell wall components. The use of The other explanation could be that some cellhydrolytic enzyme or chemicals to degrade the wall proteins get tighter bound to the cell wallcell wall matrix yet maintaining the protein during secondary cell wall formation. Yet, weintegrity could be of paramount importance in cannot be certain since we had no replicates ofstudying the CWPs more conveniently. the sample and we did not perform MS/MS analysis with other extraction samples. As aMS-based proteomics is indispensible result, we are unaware about the proteins thattechnology to analyze and identify proteins. may have been released during the other pointGenerally, prior to peptide sequencing by LC- of extraction.MS/MS, proteins are digested using proteolyticenzymes (Aebersold R, et al., 2003; Hale J.E, Conclusionet al., 2004). In this context, digestion wasperformed using Trypsin. However, it shouldbe considered that efficiency of digestionincreases with the disruption of tertiary We prepared cell wall from Arabidopsisstructure of protein. Studies have demonstrated thaliana basal cells as well as cells that hadthat sulfhydryl group of cysteine residues can been induced with hormones (NAA, BAP andform disulfide bonds and highly stabilize the Epibras) to make them form TEs. The cell walltertiary structure. So, in advance to digestion preparation involved mechanical grinding withby trypsin, reduction and alkylation of cysteine cells, density gradient cell-fractionation usingresidues were carried out using volatile reagent different concentration of sucrose andtriethylphosphine and iodoethanol. This assists sequential extraction of proteins using NP40the blockage of sulfhydryl groups, destabilize and different concentration of CaCl2. We thenthe tertiary structure and ultimately lead to performed proteomic analysis of proteins inenhanced protein digestion (Aebersold R, et al., resultant CW5 pellet and proteins extracted2003). To disrupt the tertiary structure of with 0.1M CaCl2 using LC-MS/MS. Proteinproteins in the CW5 pellet sample we used identification, location and functions weredenaturizing solution containing 6M guanidine predicted using Arabidopsis database search. We were able to identify 44.3% of cell wall 14
  • 15. proteins in the resultant CW5-pellet (induced) Acknowledgementscompared to 39.3% of cell wall proteins in theresultant CW5-pellet (basal). Moreover, weobserved that some of the cell wall proteinswere released from cell wall during 0.1M We are extremely grateful to Irene GranlundCaCl2 extraction. Since there were some non- for supervising the project in Appliedresident proteins in resultant CW5-pellet, we Functional genomics as well as reading theassume that some improvements can be made manuscript; she has given her valuablein the purification of cell wall. For instance, feedback throughout the project and necessaryuse of hydrolytic enzymes or chemicals with correction as and when needed. We are alsothe potential to degrade polysaccharide matrix deeply indebted to Edouard Pesquet and Jancan possible prevent trapping of non-resident Karlsson for their guidance and help during theproteins and increase purification of cell wall project. The study was supported by the Umeapreparation. Plant Science Centre (UPSC), Umea University.ReferencesAebersold R and Mann M. March 2003. Nature 422: 198-207.Albenne C, Canut H, Boudart G, Zhang Y, Clemente HS,Pont-Lezica R and Jamet E. Molecular Plant. 2009(2):977–989Borderies G, Jamet E, Lafitte C, Rossigol M, Jauneau A, Fukuda, Plant Physiology and Plant Molecular Biology,Boudart G, Monsarrat B, Esquerré-Tugayé M, Boudet A 1996, Volume 47and Pont-Lezica R. Electrophoresis. 2003(24): 3421-3432. Fukuda H. The Plant Cell. 1997( 9):1147-1 156.Chivasa S, Ndimba BK, Simon WJ, Robertson D, Yu XL, Jamet E, Albenne C, Boudart G, Irshad M, Canut H andKnox JP, Bolwell P and Slabas AR. Pont-Lezica R. Proteomics. 2008 (8): 893–908.Electrophoresis. 2002(11):1754-65. Jamet E, Canut H, Boudart G and Pont-Lezica R. TrendsCravatt BF, Simon GM and Yates III JR. Nature. Plant Sci. 2006(11): 33–39.2007(450):991-1000 Hale JE, Butlera JP, Gelfanovaa V, Youa J andDavid MB, Leo AH. Zeef JE, Royston G and Simon R. kniermana MD. Analytical Biochemistry. 2004(1):174-Turn. The Plant Cell. 2005(17): 2281-2295. 181.Falconer M.M and Seagull R.W, Protoplasma. 1985(125): Oda Y, Mimura T and Hasezawa S. Plant190-198. Physiol. 2005(3): 1027–1036.Feiz L, Irshad M, Pont-Lezica R, Canut H and Jamet E. Zhu S, Chen S, Alvarez VS, Asirvatham DP, SchachtmanPlant Methods. 2006(27): 2-10. Y and Wu RS. Plant Physiol.2006(140): 311–32. 15
  • 16. AppendixMSAR medium for cell suspension culture: 0.05% NP404.4g MS basal salt plus vitamins (Duchefa M0222.0225) 0.1M, 0.5M, 2M and 4M CaCl230g sucrose (3%) Chemicals and reagents used in protein measurement:pH 5.7 with 1M KOH (for 1 liter) Bovine serum albumin (A3294 by Sigma)Buffers used in cell wall preparation: Bradford solutionBuffer 1; SDS-PAGE and Western blotting:150mM NaCl Resolving gel (12% gel):10% Glycerol 30% acrylamide 29:1 - 4.8ml100mM Acetate buffer (pH 4.6) 1M tris-HCL pH8.8 – 4.5mlBuffer 2; 10% SDS – 0.120ml5mM MES-KOH (pH 5.6) ddH2O – 2.5ml5mM MgCl2 10%APS – 0.075mlBuffer 3; TEMED – 0.0075ml10% DMSO Stack gel (6% gel)5mM MES-KOH (pH 5.6) 30% acrylamide 29:1 – 0.8ml5mM MgCl2 1M tris-HCL pH8.8 – 0.5mlOther chemicals in cell wall preparation: 10% SDS – 0.05ml0.4M, 0.6M and 1M sucrose ddH2O – 2.615ml 16
  • 17. 10%APS – 0.03ml 10 × electrophoresis buffer – 100mlTEMED – 0.005ml Isopropanol (99.8% purity) – 100ml Coomassie stainingBuffers used in western blotting: 0.02% coomassie R-350 in 10% acetic acid10 × Towbin buffer0.13M Tris – 15.7g MS analysis:10% ethanol – 0.1L Denaturating buffer:1M glycine – 75g 6M guanidin, 0.1M Tris, 5mM EDTA, pH 810 × PBS, pH 7.4 (total amount 1L) 57g Guanidin HCl10mM Na2HPO4 – 13.8g 614mg Trizma-HCl3mMKH2PO4 – 4.08g 750mg Trizma base140mM NaCl – 81.8g 58mg EDTA1 × PBST (Tween 20 )- total amount 1L Dilute to 100ml10 × PBS – 100ml Protein solution (for reduction/alkylation)0.05% Tween 20 – 0.5ml 97.5% acetonitrile (v/v)10 × electrophoresis buffer, pH 8.3, 1L 2% iodoethanol (130mM)Tris – 30.3g 0.5% triethylphospine (17mM) final pH 10.Glycine – 144.1g Other chemicals:1%SDS – 10g 2mg/µl trypsinTransfer buffer 0.1% formic acid 17
  • 18. Table 1: List of all proteins in CW5Protein acc Protein score Protein cover Localization FunctionAT1G75780* 190 12.5 vacuole GTPase activity, structural molecule activity, GTP bindingAT1G56110* 116 6.3 cell wall, nucleolus NOP56-like proteinAT5G22060* 101 9.8 plasma membrane ATP binding, heat shock protein binding, unfolded protein bindingAT1G02500* 88 3.2 cell wall, membrane, plasma membrane methionine adenosyltransferase activityAT3G23990* 80 5.4 cytosol, cytosolic ribosome, mitochondrial matrix, mitochondrion, ATP binding, copper ion binding vacuolar membraneAT1G14320* 72 6.3 chloroplast, cytosolic large ribosomal subunit, cytosolic ribosome, structural constituent of ribosome large ribosomal subunit, nucleolus, vacuolar membrane, vacuoleAT5G20010* 72 6.3 apoplast, cell wall, plasma membrane GTP binding, protein binding, GTPase activityAT1G79920* 59 6.1 cell wall, plasma membrane ATP bindingAT4G05020* 50 2.9 mitochondrion, extrinsic to mitochondrial inner membrane calcium ion binding, flavin adenine dinucleotide binding, disulfide oxidoreductase activity, oxidoreductase activityAT1G79330* 50 28.4 chloroplast cysteine-type endopeptidase activityAT5G60640* 47 5.6 chloroplast, vacuolar membrane protein disulfide isomerase activityAT1G49240* 47 5.6 chloroplast, plasma membrane, vacuole structural constituent of cytoskeleton, copper ion bindingAT1G163008* 47 4.3 Plastid glyceraldehyde-3-phosphate dehydrogenase (NAD+) (phosphorylating) activity, glyceraldehyde-3-phosphate dehydrogenase activityAT5G52640* 45 3.7 cell wall, cytosol, plasma membrane ATP binding, unfolded protein bindingAT3G02090* 43 4.9 cell wall, chloroplast, membrane, mitochondrial inner membrane, zinc ion binding mitochondrial intermembrane space, mitochondrial matrix, mitochondrial outer membrane, mitochondrial respiratory chain complex III, mitochondrion, nucleolus, vacuolar membraneAT5G07440* 39 6.7 mitochondrion, vacuolar membrane ATP binding, cobalt ion binding, copper ion binding, zinc ion binding , glutamate dehydrogenase [NAD(P)+] activity, glutamate dehydrogenase activity, oxidoreductase activityAT5G13450* 39 5.3 chloroplast, membrane, mitochondrion, plasma membrane cobalt ion binding, zinc ion binding, hydrogen ion transporting ATP synthase activity, rotational mechanismAT4G26970* 37 2.7 chloroplast, cytosol, mitochondrion copper ion binding, aconitate hydratase activityAT4G31700* 32 8.2 chloroplast, cytosolic ribosome, cytosolic small ribosomal subunit, structural constituent of ribosome membrane, nucleolus, plasma membraneAT3G04230* 32 2.2 chloroplast, cytosolic ribosome, cytosolic small ribosomal subunit, structural constituent of ribosome membrane, nucleolusAT1G07890* 30 5.7 cell wall, chloroplast, chloroplast stroma, cytosol, plasma L-ascorbate peroxidase activity membraneAT2G36160* 26 2.3 chloroplast, cytosolic ribosome, cytosolic small ribosomal subunit, structural constituent of ribosome membrane, plasma membrane, vacuolar membraneAT5G11170* 25 0.9 Nucleolus helicase activity, ATP-dependent helicase activity, nucleic acid binding, ATP bindingAT5G28540# 304 12.1 cell wall, chloroplast, endoplasmic reticulum lumen, plasma ATP binding membrane, vacuolar membrane, vacuole 18
  • 19. AT3G12110# 161 13.3 cytoskeleton, mitochondrion, plasma membrane structural constituent of cytoskeletonAT2G07698# 106 6.4 membrane, nucleus, plasma membrane, vacuole hydrogen ion transporting ATP synthase activity, rotational mechanism, poly(U) RNA binding, zinc ion bindingAT2G18960# 91 2.2 membrane, nucleus, plasma membrane, vacuole protein binding , ATPase activity, hydrogen-exporting ATPase activity, phosphorylative mechanismAT1G17880# 88 16.4 Unknown sequence-specific DNA binding transcription factor activityAT1G20620# 78 9.3 apoplast, cell wall, chloroplast, chloroplast envelope, chloroplast cobalt ion binding, catalase activity stroma, cytosolic ribosome, membrane, mitochondrion, peroxisome, plasmaAT2G33210# 76 4.1 chloroplast, mitochondrion, plasma membrane, vacuolar ATP binding, copper ion binding membraneAT2G42910# 76 5.3 cytoplasm, plasma membrane magnesium ion binding, ribose phosphate diphosphokinase activityAT4G24830# 75 6.7 chloroplast, chloroplast stroma ATP binding, argininosuccinate synthase activityAT3G11130# 74 3.6 plasma membrane, vacuolar membrane, vacuole binding, structural molecule activityAT3G17390# 69 6.4 cell wall, membrane, nucleolus, plasma membrane methionine adenosyltransferase activityAT5G10840# 67 3.4 plasma membrane BindingAT3G02090# 64 4 cell wall, chloroplast, membrane, mitochondrial inner membrane, metalloendopeptidase activity, zinc ion binding mitochondrial intermembrane space, mitochondrial matrix, mitochondrial outer membrane, mitochondrial respiratory chain complex III, mitochondrion, nucleolus, vacuolar membraneAT1G07790# 60 10.1 chloroplast DNA bindingAT2G44060# 55 3.4 membrane, plasma membrane Molecular function unknownAT1G71380# 54 6.2 cell wall, plant-type cell wall, plasma membrane catalytic activity, hydrolase activity, hydrolyzing O-glycosyl compoundsAT4G22010# 50 3.9 membrane, plant-type cell wall oxidoreductase activity, copper ion bindingAT1G12840# 48 4.3 chloroplast, plant-type vacuole,plasma membrane,vacuolar proton-transporting ATPase activity, rotational mechanism membrane, vacuoleAT1G70710# 45 4.7 chloroplast cellulase activity, hydrolase activity, hydrolyzing O-glycosyl compoundsAT2G07560# 45 2.2 membrane, plasma membrane protein binding, ATPase activityAT5G61790# 44 8.3 chloroplast, endoplasmic reticulum, membrane, microsome, calcium ion binding, unfolded protein binding mitochondrion, plant-type cell wall, plasma membrane, vacuolar membrane, vacuoleAT1G73230# 43 16.4 No data Molecular function unknownAT2G42210# 40 17 chloroplast, membrane, mitochondrial inner membrane P-P-bond-hydrolysis-driven protein transmembrane transporter activity, protein presequence translocase complex, mitochondrion, plastid outer transporter activity membraneAT1G15690# 39 1.4 chloroplast, chloroplast envelope, endosome membrane, ATPase activity, hydrogen-translocating pyrophosphatase activity membrane, mitochondrion, plant-type vacuole, plant-type vacuole membrane, plasma membrane, vacuolar membrane, vacuoleAT3G57290# 39 2.9 cytoplasm, nucleus, plasma membrane, signalosome protein binding, translation initiation factor activityAT1G09100# 36 3.8 membrane, plasma membrane calmodulin binding, ATPase activityAT1G66110# 36 2.1 No data Molecular function unknownAT2G36580# 36 4.7 plasma membrane magnesium ion binding, potassium ion binding, catalytic activity, pyruvate kinase activityAT1G07890# 36 5.6 cell wall, chloroplast, chloroplast stroma, cytosol, plasma L-ascorbate peroxidase activity 19
  • 20. membraneAT5G07640# 36 2.5 No data zinc ion bindingAT3G13870# 34 2.1 cytoplasm, endoplasmic reticulum, plasma membrane, vacuolar GTP binding membraneAT1G07660# 33 7.8 chloroplast, plasma membrane, vacuolar membrane DNA bindingAT1G17710# 33 6.1 No data phosphatase activityAT1G10630# 31 5.5 membrane, plasma membrane, vacuolar membrane GTP binding, copper ion binding, protein binding, phospholipase activator activityAT5G07340# 25 2.1 chloroplast, endoplasmic reticulum, membrane, vacuolar calcium ion binding, unfolded protein binding membraneAT2G44120# 25 13.6 chloroplast, cytosolic large ribosomal subunit, cytosolic transcription regulator activity, structural constituent of ribosome ribosome, large ribosomal subunit, membrane, nucleolus, vacuoleAT2G33040# 23 6.8 cell wall, chloroplast, cytoplasm, membrane, mitochondrion, zinc ion binding nucleolus, nucleusAT1G20260# 22 4.5 chloroplast, vacuolar membrane, vacuole ATP binding, hydrogen ion transporting ATP synthase activity, rotational mechanism, hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances, proton-transporting ATPase activity, rotational mechanismAT5G02500 581 31.2 apoplast, cell wall, chloroplast, cytosol, cytosolic ribosome, ATP binding membrane, nucleolus, nucleus, plasma membrane, vacuolar membraneAT1G35720 49 3.2 apoplast, cell wall, chloroplast, chloroplast stroma, cytosol, ATP binding, calcium ion binding, calcium-dependent phospholipid binding, copper ion membrane, mitochondrion, plasma membrane, thylakoid, vacuolar binding, zinc ion binding, peroxidase activity, protein homodimerization activity membrane, vacuoleAT5G15090 563 43.8 cell wall, chloroplast, chloroplast envelope, membrane, aerobic respiration, anion transport, defense response to bacterium, regulation of seed mitochondrial outer membrane, mitochondrion, nucleolus, plasma germination, response to bacterium, response to cold membrane, plastid, vacuolarAT3G12580 430 28 cell wall, cytosol, mitochondrion, plasma membrane, vacuolar ATP binding membraneAT5G02490 426 20.7 cell wall, cytosol, nucleus, plasma membrane protein bindingAT1G13440 412 40.5 cell wall, chloroplast, cytosol, membrane, mitochondrion, copper ion binding, zinc ion binding nucleolus, nucleus, plasma membraneAT5G56030 395 18.6 cell wall, cytosol, mitochondrion, nucleus ATP binding, protein bindingAT1G07920 394 36.7 chloroplast, membrane, mitochondrion, nucleolus, plasma calmodulin binding membrane, vacuolar membraneAT3G09440 334 21.9 apoplast, cell wall, chloroplast, cytosol, cytosolic ribosome, plasma ATP binding membrane, vacuolar membrane, vacuoleAT5G62690 332 16.7 cell wall, nucleolus, plasma membrane GTPase activity, structural molecule activity, GTP bindingAT1G56070 318 9.5 chloroplast, cytosol, membrane, nucleolus, plasma membrane, copper ion binding, translation factor activity, nucleic acid binding vacuolar membraneAT4G37910 317 11.9 cell wall, mitochondrial matrix, mitochondrion, vacuolar ATP binding, zinc ion binding membraneAT5G23860 315 16.7 membrane structural constituent of cytoskeleton, protein bindingAT4G20890 306 18 chloroplast, plasma membrane, vacuolar membrane GTP binding, GTPase activity, structural molecule activityAT1G04820 266 15.6 cell wall, chloroplast, cytosol, plasma membrane, tubulin complex, structural constituent of cytoskeleton 20
  • 21. vacuolar membraneAT5G08670 265 19.4 chloroplast, mitochondrial proton-transporting ATP synthase ATP binding, cobalt ion binding, copper ion binding, zinc ion binding, hydrogen ion complex, catalytic core F(1), mitochondrion, plasma membrane, transporting ATP synthase activity, rotational mechanism vacuolarAT1G20010 183 16.3 cell wall, chloroplast, membrane, plasma membrane, vacuolar structural constituent of cytoskeleton membraneAT3G08580 175 7.3 cell wall, chloroplast, chloroplast envelope, membrane, binding, copper ion binding, ATP:ADP antiporter activity mitochondrial envelope, mitochondrial inner membrane, mitochondrion, nucleolus, plasma membrane, vacuolar membrane, vacuoleAT4G13940 173 16.7 membrane, plasma membrane, vacuolar membrane, vacuole adenosylhomocysteinase activity, copper ion bindingAT5G66680 146 8.2 endoplasmic reticulum, endoplasmic reticulum membrane, dolichyl-diphosphooligosaccharide-protein glycotransferase activity membrane, nucleolus, plant-type cell wall, vacuolar membrane, vacuoleAT2G02560 122 3.9 cell wall, plasma membrane BindingAT3G09840 117 6.1 cell wall, cytoplasm, cytosolic ribosome, nuclear envelope, identical protein binding, ATPase activity nucleolus, nucleus, phragmoplast, plasma membrane, spindleAT1G45000 90 7.3 cell wall, membrane, nucleolus, plasma membrane ATP binding, nucleotide binding, ATPase activity, hydrolase activity, nucleoside- triphosphatase activityAT4G02930 90 7.3 mitochondrion, cell wall ATP binding, cobalt ion binding, zinc ion binding, translation elongation factor activityAT3G27280 88 5.2 chloroplast, mitochondrion, plant-type cell wall, plasma Part of protein complexes that are necessary for proficient mitochondrial function or membrane, vacuolar membrane, vacuole biogenesis, thereby supporting cell division and differentiation in apical tissuesAT1G09080 88 5.2 endoplasmic reticulum lumen ATP bindingAT2G37270 80 13.5 cell wall, chloroplast, cytosolic ribosome, cytosolic small structural constituent of ribosome ribosomal subunit, membrane, plasma membrane, ribosome, vacuolarAT3G11940 80 13.5 cell wall, chloroplast, cytosolic ribosome, cytosolic small structural constituent of ribosome ribosomal subunit, plasma membrane, ribosome, vacuoleAT3G01280 78 13.4 chloroplast, chloroplast envelope, mitochondrial outer membrane, voltage-gated anion channel activity mitochondrion, plasma membrane, plastid, vacuolar membrane, vacuoleAT2G18450 77 3.8 mitochondrion succinate dehydrogenase activityAT2G04030 77 3.8 chloroplast, chloroplast envelope, chloroplast stroma, ATP binding mitochondrion, vacuolar membraneAT1G65930 73 5 apoplast, cytosol, plasma membrane copper ion binding, isocitrate dehydrogenase (NADP+) activityAT1G35160 70 11.4 cytoplasm, nuclear envelope, plasma membrane protein phosphorylated amino acid bindingAT5G38480 70 7.5 cell wall, chloroplast, mitochondrion, plasma membrane, vacuole ATP binding, protein phosphorylated amino acid bindingAT1G54270 70 7.5 cytosol, plasma membrane, vacuolar membrane ATP-dependent helicase activity, translation initiation factor activityAT3G08030 68 6 cell wall Molecular function unknownAT2G05710 68 6 cell wall, chloroplast, mitochondrion, vacuolar membrane ATP binding, copper ion bindingAT4G24190 59 6.1 chloroplast, endoplasmic reticulum, membrane, mitochondrion, ATP binding, unfolded protein binding nucleus, plasma membrane, vacuolar membrane, vacuoleAT1G55490 55 4.5 chloroplast, endoplasmic reticulum, membrane, mitochondrion, protein binding 21
  • 22. nucleusAT5G09590 55 4.5 cell wall, chloroplast, mitochondrial matrix, mitochondrion, ATP binding vacuolarAT3G09680 52 2.9 cytosolic ribosome, cytosolic small ribosomal subunit, nucleolus, structural constituent of ribosome ribosomeAT4G20150 50 6.3 mitochondrion, vacuolar membrane Molecular function unknownAT2G47650 48 3.4 Golgi apparatus, membrane, vacuolar membrane, vacuole UDP-glucuronate decarboxylase activity, catalytic activityAT2G20140 48 3.7 plasma membrane ATP binding, nucleotide bindingAT2G41840 46 9.5 cytosolic ribosome, cytosolic small ribosomal subunit, membrane, structural constituent of ribosome nucleolusAT3G07770 45 2.5 cell wall, mitochondrion ATP bindingAT1G79550 44 4 apoplast, cytosol, membrane, nucleus, plasma membrane, vacuolar phosphoglycerate kinase activity membraneAT1G22780 38 7.6 cell wall, cytosolic small ribosomal subunit, plasma membrane, structural constituent of ribosome, RNA binding, nucleic acid binding small ribosomal subunit, vacuolar membraneAT1G04690 37 2.7 membrane, plasma membrane oxidoreductase activity, potassium channel activityAT5G13490 36 7.3 chloroplast, chloroplast envelope, membrane, mitochondrial binding, copper ion binding, protein binding, ATP:ADP antiporter activity envelope, mitochondrial inner membrane, mitochondrion, vacuolar membraneAT2G38940 32 5.6 membrane, nucleus, plasma membrane, vacuole carbohydrate transmembrane transporter activity, inorganic phosphate transmembrane transporter activity, phosphate transmembrane transporter activity,AT1G27400 27 7.3 chloroplast, cytosolic large ribosomal subunit, plasma membrane, structural constituent of ribosome ribosome, vacuolar membrane, vacuoleAT5G53460 25 1.8 chloroplast, chloroplast stroma, plastid glutamate synthase (NADH) activityAT3G22310 21 1.6 plasma membrane DNA binding, RNA bindingAT1G18500 21 2.5 chloroplast 2-isopropylmalate synthase activityAT5G42080 21 2.5 chloroplast thylakoid membrane, microtubule, plasma membrane, GTP binding, clathrin binding, protein binding vacuolar membrane, vacuoleAT3G61760 21 2.5 no data GTPase activity, GTP bindingProtein 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. 22
  • 23. Table 2: List of all proteins in 0.1M CaCl2 extractionProtein acc Protein Score Protein cover Localization FunctionAT5G22650* 410 28.4 cell wall, cytosol, nucleolus, vacuolar membrane DNA mediated transformation, negative regulation of transcription, DNA-dependent, polarity specification of adaxial/abaxial axis.AT1G68560* 347 24 apoplast, cell wall, chloroplast, plant-type cell wall Response to cadmium ion, xylan catabolic process, xyloglucan metabolic processAT5G18170* 338 26.5 mitochondrion Nitrogen compound metabolic process, response to absence of light, response to cadmium ion, response to salt stressAT3G12390* 212 18.7 cytosolic ribosome Response to salt stressAT1G32130* 211 5.6 no data Brassinosteroid mediated signaling pathway, regulation of transcription elongation, DNA-dependent.AT1G78060* 192 4.3 Apoplast, cell wall, chloroplast, plant-type cell wall Carbohydrate metabolic process, hydrolase activity.AT2G43710* 165 16 chloroplast, chloroplast stroma, plastid Defense response, defense response to bacterium, defense response to insect, defense response to virus, fatty acid metabolic process, jasmonic acid biosynthetic process, lipid biosynthetic process, regulation of jasmonic acid mediated signaling pathway,AT2G44450* 164 16.2 cell wall, plant-type cell wall carbohydrate metabolic processAT3G44750* 144 25.7 nucleolus DNA mediated transformation, polarity specification of adaxial/abaxial axis), nucleic acid binding, zinc ion binding, histone deacetylase activity.AT5G16240* 139 15.7 No data Fatty acid biosynthetic process, fatty acid metabolic process, oxidation-reduction process, transition metal ion binding, desaturase activity, oxidoreductase activity.AT2G03870* 124 18.2 nucleus, small nucleolar ribonucleoprotein complex small nuclear ribonucleoprotein, putative / snRNP, putative / Sm protein, putative similar to SNRNP-G (PROBABLE SMALL NUCLEAR RIBONUCLEOPROTEIN G)AT3G62020* 118 13.2 cell wall, plant-type cell wall Biological process, manganese ion binding, nutrient reservoir activity.AT5G08670* 86 2.9 chloroplast, mitochondrial proton-transporting ATP synthase Response to oxidative stress, ATP binding, cobalt ion binding, copper ion binding, zinc complex, catalytic coreF(1), mitochondrion, plasma membrane, ion binding, hydrogen ion transporting ATP synthase activity, rotational mechanism. vacuolar membraneAT4G10480* 83 11.3 No data Nascent polypeptide associated complex alpha chain protein, putative / alpha-NAC, putative Identical to Nascent polypeptide-associated complex subunit alpha-like protein 4 [Arabidopsis Thaliana] (GB:Q9SZY1;GB:Q9ZSA6)AT5G58450* 82 2.7 Cellular component Binding.AT5G25460* 81 11.1 plant-type cell wall Response to karrikinAT2G13540* 73 3.1 nucleus RNA splicing, via endonucleolytic cleavage and ligation, long-day photoperiodism, flowering, organ morphogenesis, primary microRNA processing, response to abscisic acid stimulus, translation, RNA cap binding.AT2G39700* 73 4.7 plant-type cell wall Plant-type cell wall loosening, plant-type cell wall modification involved in multidimensional cell growth, syncytium formation, unidimensional cell growth.AT5G60340* 65 5.6 mitochondrion Metabolic process, ATP binding, oxidoreductase activity.AT5G57120* 62 6.1 nucleolus Function unknownAT1G08970* 62 5.6 cytoplasm, nucleus Regulation of transcription, DNA-dependent, DNA binding, sequence-specific DNA binding transcription factor activity.AT3G04500* 58 7.3 No data RNA binding, nucleic acid binding, nucleotide binding.AT4G16210* 58 8.7 peroxisome Metabolic process, catalytic activity. 23
  • 24. AT4G26630* 54 1.8 No data GTP binding / RNA binding similar to unknown protein [Arabidopsis thaliana]AT3G09630* 51 5.9 Cell wall, chloroplast, cytosolic large ribosomal subunit, cytosolic Translation, structural constituent of ribosome. ribosome, membrane, nucleolus, plasma membrane, ribosome, vacuole membrane, plastid, vacuole.AT1G66070* 51 6.6 membrane Expressed in plant structures during growth stages;AT5G03740* 51 4.1 nucleolus Response to abscisic acid stimulus, response to salt stress, response to water deprivation, nucleic acid binding, zinc ion binding, histone deacetylase activity,AT2G19480* 50 2.9 cytoplasm, nucleus, plasma membrane DNA mediated transformation, DNA repair, nucleosome assembly, response to cadmium ion, DNA binding, binding.AT1G61730* 49 3.7 chloroplast, cytosol, nucleolus Transcription regulator activityAT1G30580* 48 4.3 intracellular Response to cadmium ion, GTP bindingAT4G17260* 47 9.9 plasma membrane Response to abscisic acid stimulus, response to salt stress,binding.AT2G38880* 44 14.9 CCAAT-binding factor complex, nucleus Regulation of transcription, DNA-dependent, response to water deprivation, sequence- specific DNA binding transcription factor activity.AT4G24770* 44 8.5 Chloroplast, chloroplast envelope, chloroplast stroma, chloroplast RNA modification, RNA processing, innate immune response, RNA binding, poly(U) thylakoid membrane, thylakoid. RNA binding.AT3G09440* 43 1.7 Apoplast, cell wall, chloroplast, cytosol, cytosolic ribosome, Protein folding, response to cadmium ion, response to heat, response to karrikin, ATP plasma membrane, vacuolar membrane, vacuole binding.AT3G27400* 43 3.4 endomembrane system Pectate lyase activity.AT1G74050* 42 5.2 cytosolic large ribosomal subunit, intracellular, membrane, plasma Translation, structural constituent of ribosome. membrane, ribosomeAT1G55570* 40 1.6 No data Oxidoreductase activity, copper ion binding.AT3G60130* 39 2.1 No data Carbohydrate metabolic process, cation binding, catalytic activity, hydrolase activity, hydrolyzing O-glycosyl compounds.AT1G29340* 37 1.1 No data Apoptosis, defense response to fungus, incompatible interaction, defense response, incompatible interaction, protein ubiquitination, ubiquitin-protein ligase activity.AT3G27460* 37 3 nucleus Response to salt stress.AT2G38410* 37 2.1 Golgi stack, plasma membrane Intra-Golgi vesicle-mediated transport, intracellular protein transport, protein transporter activity.AT1G56170* 35 6.5 Positive regulation of gene-specific transcription, regulation of transcription, DNA- cytoplasm, nucleus dependent, DNA binding.AT4G38400* 33 7.2 endomembrane system, extracellular region Plant-type cell wall loosening, plant-type cell wall organization, unidimensional cell growth, response to cyclopentenone.AT5G55660* 29 1.8 mitochondrion GTP binding / RNA binding similar to unknown protein [Arabidopsis thaliana] (TAIR: AT4G26630.2); similar to unknown protein [Arabidopsis thaliana] (TAIR:AT4G26630)AT3G46750* 26 1.3 No data Function unknownAT5G61290* 26 1.1 cellular component Oxidation-reduction process, NADP binding, flavin adenine dinucleotide binding, flavin-containing monooxygenase activity, monooxygenase activity.AT5G07350* 24 1.1 Cell wall, chloroplast, cytosol, endoplasmic reticulum, nuclear Protein secretion, response to cadmium ion, response to stress, RNA binding, nucleic envelope, plasma membrane acid binding, nuclease activity.AT2G20450* 23 9 Cytosolic large ribosomal subunit, endoplasmic reticulum, Ribosome biogenesis, translation, structural constituent of ribosome. ribosome, vacuoleAT1G13950* 21 5.1 No data Translational initiation, xylem development, RNA binding, ribosome binding, 24
  • 25. translation elongation factor activity, translation initiation factor activity.AT3G62020* 118 13.2 cell wall, plant-type cell wall manganese ion bindingAT3G18070# 119 5.2 cellular component cation bindingAT4G02290# 116 4.5 endomembrane system catalytic activity, hydrolase activity, hydrolyzing O-glycosyl compoundsAT1G68560# 83 8.5 apoplast, cell wall, chloroplast, plant-type cell wall alpha-N-arabinofuranosidase activity, hydrolase activity, hydrolyzing O-glycosyl compounds, xylan 1,4-beta-xylosidase activityAT5G28540# 70 4.8 cellwall, chloroplast, endoplasmic reticulum lumen, plasma membrane, vacuolar membrane, vacuole ATP bindingAT3G28200# 60 5.4 plant-type cell wall peroxidase activityAT3G09440# 57 1.7 apoplast, cellwall, chloroplast, cytosol, cytosolic ribosome, plasma membrane,vacuolar membrane, vacuole ATP bindingAT3G14600# 55 10.1 cytosolic large ribosomal subunit, cytosolic ribosome, membrane, ribosome structural constituent of ribosome 53 4.7 membrane, plant-type cell wall plant-type cell wall loosening, plant-type cell wall organization,unidimensional cellAT3G45960# growthAT5G06870# 48 4.5 cell wall, plant-type cell wall polygalacturonase inhibitor activityAT5G64260# 45 4.3 cell wall, plant-type cell wall molecular function unknownAT5G07030# 42 3.1 cell wall, plant-type cell wall aspartic-type endopeptidase activityAT5G62340# 31 4.9 endomembrane system enzyme inhibitor activity, pectinesterase inhibitor activityAT2G48160# 26 1 no data no dataAT3G08030 1123 67.4 cell wall molecular function unknownAT5G23400 767 35.1 cell wall Defense response, signal transduction,AT1G71380 589 39.7 cell wall, plant-type cell wall, plasma membrane Carbohydrate metabolic process, catalytic activity, hydrolase activity.AT2G05920 604 19.9 cell wall, plant-type cell wall Negative regulation of catalytic activity, proteolysis, identical protein binding.AT1G17880 155 16.4 no data sequence-specific DNA binding transcription factor activity, response to salt stressAT1G71380 589 39.7 cell wall, plant-type cell wall, plasma membrane Carbohydrate metabolic process, catalytic activity, hydrolase activity.AT5G07440 228 18.5 mitochondrion, vacuolar membrane ATP binding, cobalt ion binding, copper ion binding, zinc ion bindingAT5G66920 234 17.9 cell wall, plant-type cell wall copper ion bindingAT2G06850 763 46.3 cellwall, chloroplast, cytoplasm, extracellular hydrolase activity, acting on glycosyl bonds, xyloglucan:xyloglucosyl transferase region, membrane, plant-type cell wall activityAT2G42910 151 24.3 cytoplasm, plasma membrane magnesium ion bindingAT1G28290 327 19.8 plasma membrane Highly expressed in flowers and vascular tissue and is repressed by jasmonic acid.AT1G73230 51 16.4 no data response to salt stressAT1G56070 63 4.2 chloroplast, cytosol, membrane, nucleolus, plasma copper ion binding, translation factor activity, nucleic acid binding membrane, vacuolar membraneAT3G61820 75 8.1 plant-type cell wall aspartic-type endopeptidase activityAT2G44350 367 26.8 cell wall, chloroplast, mitochondrion ATP binding, zinc ion bindingAT2G05710 66 4 Cellwall , chloroplast, cytosol, mitochondrion, vacuolar membrane ATP binding, copper ion bindingAT3G02630 85 10.1 chloroplast, chloroplast stroma transition metal ion bindingAT3G15010 94 4 nucleolus, nucleus RNA binding, nucleic acid binding, nucleotide bindingAT4G16143 37 2.8 cytosol, nucleolus intracellular protein transport, protein import into nucleusAT4G08950 150 9.6 cell wall, plant-type cell wall response to brassinosteroid stimulus 25
  • 26. AT2G18740 57 37.5 nucleus, small nucleolar ribonucleoprotein complex Molecular function unknownAT2G28790 82 8 plant-type cell wall Molecular function unknownAT3G45970 162 26 cell wall, plant-type cell wall plant-type cell wall loosening, unidimensional cell growthAT3G51880 65 7.3 chromatin structural constituent of chromatinAT1G74310 59 2.3 no data ATP binding, protein bindingAT1G70710 87 4.7 chloroplast cellulase activity, hydrolase activity, hydrolyzing O-glycosyl compoundsAT4G25210 34 5.7 chloroplast, nucleolus transcription regulator activityAT5G13870 101 12.3 apoplast, cell wall, endomembrane system hydrolase activity, acting on glycosyl bonds, hydrolase activity, hydrolyzing O-glycosyl compounds, xyloglucan:xyloglucosyl transferase activityAT5G22010 96 1.3 DNA replication factor C complex, intracellular ATP binding, DNA binding, nucleotide bindingAT3G06720 51 2.8 cell wall, cytosol, nuclear envelope, nucleolus intracellular protein transport, protein import into nucleusAT1G09760 28 6 Cajalbody, chloroplast, cytoplasm, nucleolus, nucleoplasm, nucleu nuclear mRNA splicing, via spliceosome, response to cold sAT1G74210 52 3.8 vacuole glycerophosphodiester phosphodiesterase activity, phosphoric diester hydrolase activityAT4G12420 65 2.7 anchored to membrane, anchored to plasma copper ion binding membrane, apoplast, cell wall, membrane, plant-type cell wall, plasma membrane, vacuolar membraneAT3G11400 45 3.4 plasma membrane RNA bindingAT4G37870 51 6.3 cytosol, membrane, nucleolus, nucleus ATP binding, phosphoenolpyruvate carboxykinase (ATP) activityAT1G74210 52 3.8 vacuole glycerophosphodiester phosphodiesterase activity, phosphoric diester hydrolase activityAT5G62530 34 2.2 chloroplast, mitochondrion cobalt ion binding, zinc ion bindingAT2G41800 247 26.8 cell wall, plant-type cell wall Molecular functionAT1G29965 86 14 cytosolic large ribosomal subunit, ribosome structural constituent of ribosomeAT4G22670 94 3.4 no data binding, response to cadmium ionAT1G20580 38 6.9 nucleolus, nucleus, small nucleolar ribonucleoprotein complex Molecular function unknownProtein 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. 26