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

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 ................................................................................................................................................... 16

1

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 of
plant 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 of
are two core types of cell walls that are found
the xylem that are categorized by the formation
in 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. On
which is capable to elongate; and the
2

maturity, TEs lose their nuclei and cell cell wall with hormones to make them form
contents 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 perform
depositions are the even ring-like wall fractionation of cell wall from normal cells and
thickenings that are revealed in the TEs of the cells that has secondary cell wall to identify the
xylem, the wood-forming tissue of plants. different proteins involved in the growing of
secondary cell wall and lignification. After the
Plant cell wall proteins are made up of less formation of the secondary cell walls, the
than 10% of cell wall dry weight (Zhu S, et al., identification of cell wall proteins and the
2006), but play significant roles in cell wall quality of cell wall fractionation was achieved
structure, cell wall metabolism, cell by using MS/MS.
enlargement, signal transduction, responses to
abiotic and biotic stresses, and many other We performed the cell wall preparation and
physiological events. Based on their extraction of the proteins bound to the cell
interactions with cell wall components, Cell wall. Proteins extracted within the cell wall
Wall Proteins (CWPs) can be categorized into preparation from the cell wall were identified
three categories (Jamet E, et al., 2008). The with MS/MS and the results are compared
first group is labile proteins, which have between the Basal and Induced cell wall
minute or no interaction with cell wall preparations and also from different
components and thus move freely in the extractions.
extracellular space. Such proteins can be found
As the main component of wood and plant
in liquid culture media of cell suspensions and
fibers, understanding the cell wall proteins
seedlings or can be extracted with low ionic during xylem TE secondary cell wall formation
strength buffers. The second group of CWPs is has important biological and economic
the weakly bound proteins that bind the matrix implications.
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
Materials and methods
procedure to release these proteins from the
extracellular matrix, (E. Jamet, H. Canut, et al.,
2006).
Plant material
Since the actual players of cell wall dynamics
are proteins, all CWPs other than structural
proteins are of importance. Therefore, to better Suspension cell cultures of Arabidopsis
comprehend the cell wall complexity, the thaliana were generated by growing the cells
challenge is to go further into the identification at MSAR medium, pH 5.7. Cells were agitated
of the CWPs and their functional relationships. on a shaker at 23℃ at 120 rpm maintained on
In this context, the last few years saw the rise dark. Cells were sub-cultured by transferring
in search for cell wall proteins at a given time 5ml of one week old culture into 45ml of fresh
in 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

Cell induction for TE differentiation Cell wall fractionation

Cell induction was carried out in a sterile The powder of cell sample ground for 30
Erlenmeyer flask with one week old cell cycles by freezer mill was suspended in 40ml
culture. 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) and
known weight of pelleted cells was diluted centrifuged at 1 000 × g for 15 minutes with
with MSAR media to a concentration of the temperature maintained at 4℃. The
0.031g/ml. Then, cell induction was performed
supernatant was collected and the resulting
by adding 1µl 6-Benzylaminopurine (BAP)/ml,
pellet was further purified with increasing
0.6µl 1-Napthaleneacetic acid (NAA)/ml and
concentration of sucrose. The pellet was
0.8µl Epibras/ml (Pesquet E, 2010). A basal
purified by three successive centrifugations
sample was prepared as reference without any
addition of hormones. Finally, samples were (1000 × g, 4℃, 15 minutes) with 0.4M
placed on a shaker for 7-9 days growth time. sucrose, 0.6M sucrose and 1M sucrose in
The induced sample contain between 15-20% acetate buffer. All the supernatant of each time
of TEs. was concentrated by using 50mL centrifugal
filter with 4 500 × g, until all supernatant was
Ultimately, the cell culture was harvested with concentrated and change to cell wall buffer, for
vacuum filtration (using a 100µm nylon filter) further protein analysis. Finally, the pellet was
and 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 15

Cell 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 nitrogen
The cells were homogenized by either of the
three methods; grinding, sonication or freezer and stored at -80℃.
miller. For grinding, the cell sample was
placed in a mortar in liquid nitrogen and Protein extraction
crushed with a pestle till it was broken into
fine powder. Sonication which is the act of 100mg of sample (CW4) was used for the
converting an electrical energy into physical
extraction of protein which was performed
vibration to rupture cells was performed by using the detergent NP40 and different
mixing the cells with buffer and agitating it concentration of CaCl2. Initially, resultant
with a sonicator. Sonication was conducted for pellet (CW4) was solubilised in 1ml of NP40
2 min, 3 min and 4 min at 10 pulses and 5 rests solution (0.05% NP40 + 10% DMSO in
at amplitude of 70% on ice. Likewise, in case
of 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 successive
was grinded to fine powder using a medium extraction using different concentration of
sized metal bar. Moreover, the cells were CaCl2: 0.1M CaCl2, 0.5M CaCl2, 2M CaCl2
checked intermittently under the microscope to and 4M CaCl2 in MESbuffer. Between every
ensure 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

extraction were concentrated and desalted by was followed by treatment with primary
using 0.5ml centrifugal filter collected for Tubulin antibody at 1:8 000 (Abcam) for 3h at
protein analysis. Finally, the resultant cell wall room temperature. Following successive
pellet (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 using
Protein measurement by Bradford
ECL detection solution (Amersham, ECL plus
Western blotting detection system by GE
The protein content from each supernatant was Healthcare).
measured using Bradford method. Firstly,
standard of different concentration (0.1µg/ml The different fractions after cell wall
to 0.6µg/ml) were prepared using Bovine preparation were also isolated using
Serum 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 of
195µl of Bradford solution at room 0.02% Coomassie R-350 in 10% acetic acid
temperature. Finally, after measuring the and heated slightly and left the gels in the
absorbance at 595nm, the concentration of the coomassie solution for 20min. Finally, after
protein in the extract was determined with leaving the gels overnight in 10% acetic acid
respect 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 Spectrometry
After determining the protein concentration in
the extract, 40µl of sample mixture was The CW5 pellet and 0.1M CaCl2 extraction
prepared using the protein extract, 5× SDS and (supernatant) from basal and induced sample
water and maintaining the total concentration was chosen for MS analysis. To the CW5
of protein not to exceed 20µg. It was then pellet, 100µl of denaturating solution was
heated at 95°C for 5 minutes followed by SDS- added and the sample was vortexed to
polyacrylamide gel electrophoresis (SDS- homogeneity. 45µl of sample was placed in
PAGE) and Western blotting. Subsequently, 1.5ml eppendorf tube; not exceeding the
hot Coomassie blue based SDS-PAGE without concentration of 1mg/ml. To each tube, 5µl of
Western 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 at
completing the electrophoresis, the gel was
37℃ for one hour (Hale J.E, et al., 2004).
loaded on blotting apparatus by stacking the
gel between the filter paper, PVDF membrane After the samples were uncapped and
and filter paper that were equilibrated with 1 × evaporated in a speedvac, the digestion was
Towbin buffer. Finally, electroblotting was performed in 300µl 20mM ammonium
carried out on a semi-dry blot (BioRad) at hydrogen carbonate solution containing trypsin
0.18A for 30 minutes. with a concentration of 2ng/µl (Trypsin Gold
mass spectrometry grade, V5280, Promega
For protein detection, the PVDF membrane Biotech AB) overnight at 37℃. Then the
was initially agitated in blocking solution (1 ×
trysinated solution was filtered in 10K
PBST with 5% milk powder) overnight which
5

centrifuge filter (WVR) and evaporated in Figure 1: Strategies of cell wall protein extraction
speed vac. Finally, samples were dissolved and analysis. Prior to protein extraction, the cells of
with 10µl of 0.1% formic acid and subjected A. thaliana were grinded mechanically. Once
for MS analysis. extracted, proteins were analyzed by SDS-PAGE,
Western blotting and LC-MS/MS.
Analysis of MS data

Protein identification was performed using an
in-house Mascot server (Version 2.3.01, www.
Results
Matrixscience.com) with the following setting:
Database: Arath-Tair9; Fixed modification:
Ethanolyl (C); Variable modifications: Cell culture and TEs harvest
methylation (DE), oxidation (M); Peptide mass
tolerance: 100ppm; MS/MS fragment mass
Arabidopsis thaliana cells cultured in the dark
tolerance: 0.05Da; Missed cleavages: 1; Mass
in MS media. After 7 days, 15-20% of the
values: monoisotopic; Instrument type: ESI-
induced cells were TEs, which then was
QUAD-TOF.
harvested by vacuum filtration.
Search for protein location was done in the
database TAIR (www.arabidopsis.org) and Different methods for grinding
SUBA (www.plantenergy.uwa.edu.au).
It is important to receive good quality of cell
Workflow 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

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.

7

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.

After quantifying the amounts of proteins with
Bradford reagent, SDS-PAGE was carried out
Western Blotting with protein samples with total concentration
of protein not exceeding 10µg. Following
Western Blotting was used to confirm the SDS-PAGE, western blotting was performed to
purity of the cell wall preparation. The results confirm the purification of cell wall
from Western Blotting show tubulin at 55kDa preparation by using anti-tubulin antibody as
only in the sample of the first wash step with the primary antibody. The result from western
150mM NaCl and 10% glycerol in 100mM blotting show tubulin at 55kDa only the
Acetate buffer (pH 4.6) from both basal and sample of the first wash with 150mM NaCl
induced sample(Fig.5 and Fig. 6). and 10% Glycerol in 100mM Acetate buffer,

8

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. In
Figure 5: In basal sample. tubulin (55kDa) was fact, the absence of band in these samples
found in supernatant of first wash before sucrose could be attributed to two factors; (i) The
fractionation with 150mM NaCl and 10% glycerol samples either had negligible amount of
in 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) was
found in supernatant of the first wash before

9

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 contaminants
Figure 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 also
Identification of protein in the samples (CW5-
identified the functional characterization of
pellet and 0.1M CaCl2 extraction) was
CWPs as listed in the Table 1 and Table 2.
performed using LC-MS/MS followed by

10

Table 1: List of Arabidopsis thalinana cell wall proteins in CW5

Name of protein TAIR Accession Protein acc Function
homolog of nucleolar protein NOP56 Locus:2205270 AT1G56110* NOP56-like protein
S-Adenosymethionine synthetase 1 Locus:2196160 AT1G02500* methionine adenosyltransferase activity
RAS-Related nuclear protein Locus:2147700 AT5G20010* GTP binding, protein binding, GTPase activity
Heat shock protein 70-15 Locus:2017859 AT1G79920* ATP binding
Heat shock protein 90.1 Locus:2149569 AT5G52640* ATP binding, unfolded protein binding
Luminal binding protein BIP Locus:2182783 AT5G28540# ATP binding
Catalase 3 Locus:2034357 AT1G20620# cobalt ion binding, catalase activity
S-adenosylmethionine synthetase Locus:2089070 AT3G17390# methionine adenosyltransferase activity
Cellulase 3 Locus:2825314 AT1G71380# catalytic activity, hydrolase activity, hydrolyzing O-
glycosyl compounds
SKU5 similar 4 Locus:2120648 AT4G22010# oxidoreductase activity, copper ion binding
Calnexin 1 Locus:2159223 AT5G61790# calcium ion binding, unfolded protein binding
Gamma subunit of Mt ATP synthase Locus:2046485 AT2G33040# zinc ion binding
Ascorbate peroxidase 1 Locus:2026616 AT1G07890 L-ascorbate peroxidase activity
Annexin 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 binding
Heat shock protein 70 Locus:2181833 AT5G02500 ATP binding
Voltage 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 binding
Heat shock protein 70-2 Locus:2181818 AT5G02490 protein binding
Glycereldehyde-3-phosphate Locus:2010007 AT1G13440 copper ion binding, zinc ion binding
dehydrogenase C2
Heat shock protein 90 Locus:2161815 AT5G56030 ATP binding, protein binding
Heat Shock protein 70 Locus:2074984 AT3G09440 ATP binding
Tubulin beta-2 Locus:2172254 AT5G62690 GTPase activity, structural molecule activity, GTP
binding
Mitochondrial heat shock protein 70-1 Locus:2121022 AT4G37910 ATP binding, zinc ion binding
Tubulin alpha-4 chain Locus:2010677 AT1G04820 structural constituent of cytoskeleton
Tubulin beta-5 chain Locus:2198661 AT1G20010 structural constituent of cytoskeleton
ADP/ATP carrier 1 Locus:2077778 AT3G08580 binding, copper ion binding, ATP:ADP antiporter
activity
Defective glycolysation Locus:2173659 AT5G66680 dolichyl-diphosphooligosaccharide-protein
glycotransferase activity
Cullin-associated and neddylation Locus:2065279 AT2G02560 Binding
dissociated 1
Cell division cycle 48 Locus:2085064 AT3G09840 identical protein binding, ATPase activity
F27F5.8 Locus:2028200 AT1G45000 ATP binding, nucleotide binding, ATPase activity,
hydrolase activity, nucleoside-triphosphatase activity
T4I9.19 Locus:2139325 AT4G02930 ATP binding, cobalt ion binding, zinc ion binding,
translation elongation factor activity
RIBOSOMAL PROTEIN 5B Locus:2049862 AT2G37270 structural constituent of ribosome
RIBOSOMAL PROTEIN 5A Locus:2081546 AT3G11940 structural constituent of ribosome
CYTOSOLIC NADP+-DEPENDENT Locus:2009759 AT1G65930 copper ion binding, isocitrate dehydrogenase
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 unknown
ACONITASE 3 Locus:2063354 AT2G05710 ATP binding, copper ion binding
heat shock protein 70 Locus:2144801 AT5G09590 ATP binding
HEAT SHOCK PROTEIN 89.1 Locus:2077352 AT3G07770 ATP binding
PHOSPHOGLYCERATE KINASE Locus:2206410 AT1G79550 phosphoglycerate kinase activity
40S 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.

11

Table 2: List of Arabidopsis thaliana cell wall proteins in 0.1M CaCl2 extraction

Name of protein TAIR Accession Protein acc Function
HISTONE 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 process
GLP10, 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 karrikin
EXPA4, ATEXP4, ATEXPA4, F17A14_7, Locus:2043240 AT2G39700* Plant-type cell wall loosening, plant-type cell
EXPANSIN 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 ribosome
F3L24.33 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 binding
T19D11.4 Locus:2098308 AT3G28200# peroxidase activity
F3L24.33 Locus:2074984 AT3G09440# ATP binding
EXLA3,ATEXLA3, F16L2.170 Locus:2077167 AT3G45960# plant-type cell wall loosening, plant-type cell
wall organization,unidimensional cell growth
MOJ9.4, ATPGIP2, Locus:2169404 AT5G06870# polygalacturonase inhibitor activity
POLYGALACTURONASE INHIBITING
PROTEIN 2
MSJ1.10, EXORDIUM LIKE 2 Locus:2173428 AT5G64260# molecular function unknown
MOJ9.20 Locus:2169369 AT5G07030# aspartic-type endopeptidase activity
F17A17.37 Locus:2077467 AT3G08030 molecular function unknown
F28K19.27 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 binding
XTH4, T9F8.4, EXGT-A1, Locus:2065821 AT2G06850 hydrolase activity, acting on glycosyl
ENDOXYLOGLUCAN TRANSFERASE bonds, xyloglucan:xyloglucosyl transferase
activity
CELLULASE 3, Locus:2825314 AT1G71380 Carbohydrate metabolic process, catalytic
activity, hydrolase activity.
F21F14.7 Locus:2076745 AT3G61820 aspartic-type endopeptidase activity
F28K19.27 Locus:2029391 AT1G78060 hydrolase activity, hydrolyzing O-glycosyl
compounds
ATCS, CSY4, F4I1.16, CITRATE Locus:2050554 AT2G44350 ATP binding, zinc ion binding
SYNTHASE 4
ACO3, T3P4.5, ACONITASE 3 Locus:2063354 AT2G05710 ATP binding, copper ion binding
T3H13.3, EXORDIUM Locus:2138753 AT4G08950 response to brassinosteroid stimulus
F21F14.190, GERMIN-LIKE PROTEIN 10 Locus:2079582 AT3G62020 manganese ion binding
F8N16.8, Locus:2053215 AT2G28790 Molecular function unknown
EXLA1, ATEXPL1, ATHEXP BETA 2.1, Locus:2077177 AT3G45970 plant-type cell wall loosening, unidimensional
EXPANSIN-LIKE A1 cell growth
XTH5,MAC12.33, ENDOXYLOGLUCAN Locus:2159118 AT5G13870 hydrolase activity, acting on glycosyl
TRANSFERASE A4 bonds, hydrolase activity, hydrolyzing O-
glycosyl compounds, xyloglucan:xyloglucosyl
transferase activity
AIMP ALPHA, IMPORTIN ALPHA, Locus:2083313 AT3G06720 intracellular protein transport, protein import
into nucleus
T11A7.10 Locus:2054336 AT2G41800 Molecular function unknown


12

Likewise, the composition of washing buffer is
Discussion critical for the extraction of proteins from the
cell wall. The presence of NaCl in washing
buffer during the early steps of cell wall
Cell 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 interaction
into three main functional groups: structural in the cell wall (Borderies G, et al., 2005; Feiz
proteins, defense proteins and cell wall L, et al., 2006). Moreover, the washing buffer
modifying proteins. Moreover, it is believed with low ionic intensity and an acidic pH was
that unidentified proteins with novel functional used for the purification of cell wall. This
classes do still exist in the cell wall (Borderies preserves the interaction between the proteins
G, et al., 2005). So, in this study: we intended and polysaccharides and prevents the loss of
to 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 included
them. Even though it is evident that study of purification by subsequent centrifugation in
cell wall proteome is complex; (i) solution of increasing density. Since the cell
polysaccharide linkages of cellulose, wall polysaccharides are relatively dense in
hemicelluloses and pectin can retain nature, this density gradient centrifugation
intracellular 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 salt
modifications, (Borderies G, et al., 2005; for the extraction of proteins from higher
Jamet E, et al., 2008), we adopt some specific plants is used to release CWPs from purified
strategies in this study to investigate the cell cell wall (Borderies G, et al., 2005; Jamet E, et
wall proteomics of Arabidopsis thaliana. al., 2008) However, it should be noted that
CWPs that are tightly bound are still resistant
The principle steps of this Arabidopsis cell to salt extraction (Jamet E, et al., 2008).
wall proteomic study involved induction of TE
differentiation, cell wall preparation, protein Proteins that were sequentially extracted from
extraction 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 confirm
shown that different phytohormones like auxin the purity of cell wall prepared. Consistent to
and cytokinin are known to promote the this, we used anti-tubulin antibody that detects
initiation 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 band
BAP, NAA and Epibras were implicated for characteristic to tubulin only in the extract
the induction of TE differentiation which is from first washing step of both basal and
parallel 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. This
study was carried out by Oda (Oda Y, et al., implies that the tubulin proteins associated
2005) in which they used Brassinosteroid for with the Arabidopsis cell wall were eliminated
TE differentiation in AC-GT13 cells of in the early washing step. However, upon MS
Arabidopsis. Similarly, Falconer (Falconer, et analysis, tubulin proteins were evident in the
al., 1984) showed that Zinnia mesophyll cells resultant CW5 pellet which indicated that some
could be induced for TE differentiation by the of the proteins were not completely released
use of BAP and NAA (Faoconer M.M, et al., from the cell wall. Accordingly, it can be
1985; Feiz L, et al., 2006). inferred that the purification strategies that we
adopted was not efficient enough to remove all
13

the contaminants. Moreover, it should be noted to make the reduction, alkylation and digestion
that several constraints arise during CWP possible more efficient.
purification and analysis; the difficulty to
solubilise many CWPs, the complex From the MS analysis and database search, we
polysaccharide linkages by which intracellular identified 44.3% of cell wall proteins in
proteins remain trapped, and post-translational induced CW5-pellet compared to 39.3% of cell
modification of proteins. Likewise, some of the wall proteins in basal. Contrastingly, the
proteins are embedded strongly and interact analysis of cell wall proteins in 0.1M CaCl2
differently with other cell wall component extraction showed that 47.1% of cell wall
making the task more challenging. And when proteins were present in the basal sample
the 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-pellet
extraction with salt, one of the major (basal) had relatively fewer amount of proteins
disadvantages is the contamination by than the CW5-pellet (induced). Tentatively,
intracellular proteins sticking non-specifically this implies that majority of the cell wall
with the cell wall (Jamet E, et al., 2008). So, proteins of basal sample were released during
improvements can be made in the extraction of the extraction point; one of the reasons could
non-specifically bound intracellular proteins as be that cell wall proteins in basal sample, with
well 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 cell
hydrolytic enzyme or chemicals to degrade the wall proteins get tighter bound to the cell wall
cell wall matrix yet maintaining the protein during secondary cell wall formation. Yet, we
integrity could be of paramount importance in cannot be certain since we had no replicates of
studying the CWPs more conveniently. the sample and we did not perform MS/MS
analysis with other extraction samples. As a
MS-based proteomics is indispensible result, we are unaware about the proteins that
technology to analyze and identify proteins. may have been released during the other point
Generally, prior to peptide sequencing by LC- of extraction.
MS/MS, proteins are digested using proteolytic
enzymes (Aebersold R, et al., 2003; Hale J.E, Conclusion
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 We prepared cell wall from Arabidopsis
structure of protein. Studies have demonstrated thaliana basal cells as well as cells that had
that sulfhydryl group of cysteine residues can been induced with hormones (NAA, BAP and
form disulfide bonds and highly stabilize the Epibras) to make them form TEs. The cell wall
tertiary structure. So, in advance to digestion preparation involved mechanical grinding with
by trypsin, reduction and alkylation of cysteine cells, density gradient cell-fractionation using
residues were carried out using volatile reagent different concentration of sucrose and
triethylphosphine and iodoethanol. This assists sequential extraction of proteins using NP40
the blockage of sulfhydryl groups, destabilize and different concentration of CaCl2. We then
the tertiary structure and ultimately lead to performed proteomic analysis of proteins in
enhanced protein digestion (Aebersold R, et al., resultant CW5 pellet and proteins extracted
2003). To disrupt the tertiary structure of with 0.1M CaCl2 using LC-MS/MS. Protein
proteins in the CW5 pellet sample we used identification, location and functions were
denaturizing solution containing 6M guanidine predicted using Arabidopsis database search.
We were able to identify 44.3% of cell wall
14

proteins in the resultant CW5-pellet (induced) Acknowledgements
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 We are extremely grateful to Irene Granlund
CaCl2 extraction. Since there were some non- for supervising the project in Applied
resident proteins in resultant CW5-pellet, we Functional genomics as well as reading the
assume that some improvements can be made manuscript; she has given her valuable
in the purification of cell wall. For instance, feedback throughout the project and necessary
use of hydrolytic enzymes or chemicals with correction as and when needed. We are also
the potential to degrade polysaccharide matrix deeply indebted to Edouard Pesquet and Jan
can possible prevent trapping of non-resident Karlsson for their guidance and help during the
proteins and increase purification of cell wall project. The study was supported by the Umea
preparation. Plant Science Centre (UPSC), Umea
University.

References

Aebersold 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):
977–989
Borderies 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 47
and 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 and
Knox 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. Trends
Cravatt 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 and
David 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. Plant
190-198. 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, Schachtman
Plant Methods. 2006(27): 2-10. Y and Wu RS. Plant Physiol.2006(140): 311–32.

15

Appendix

MSAR medium for cell suspension culture: 0.05% NP40

4.4g MS basal salt plus vitamins (Duchefa M0222.0225) 0.1M, 0.5M, 2M and 4M CaCl2

30g 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 solution

Buffer 1; SDS-PAGE and Western blotting:

150mM NaCl Resolving gel (12% gel):

10% Glycerol 30% acrylamide 29:1 - 4.8ml

100mM Acetate buffer (pH 4.6) 1M tris-HCL pH8.8 – 4.5ml

Buffer 2; 10% SDS – 0.120ml

5mM MES-KOH (pH 5.6) ddH2O – 2.5ml

5mM MgCl2 10%APS – 0.075ml

Buffer 3; TEMED – 0.0075ml

10% DMSO Stack gel (6% gel)

5mM MES-KOH (pH 5.6) 30% acrylamide 29:1 – 0.8ml

5mM MgCl2 1M tris-HCL pH8.8 – 0.5ml

Other chemicals in cell wall preparation: 10% SDS – 0.05ml

0.4M, 0.6M and 1M sucrose ddH2O – 2.615ml
16

10%APS – 0.03ml 10 × electrophoresis buffer – 100ml

TEMED – 0.005ml Isopropanol (99.8% purity) – 100ml

Coomassie staining

Buffers used in western blotting: 0.02% coomassie R-350 in 10% acetic acid

10 × Towbin buffer

0.13M Tris – 15.7g MS analysis:

10% ethanol – 0.1L Denaturating buffer:

1M glycine – 75g 6M guanidin, 0.1M Tris, 5mM EDTA, pH 8

10 × PBS, pH 7.4 (total amount 1L) 57g Guanidin HCl

10mM Na2HPO4 – 13.8g 614mg Trizma-HCl

3mMKH2PO4 – 4.08g 750mg Trizma base

140mM NaCl – 81.8g 58mg EDTA

1 × PBST (Tween 20 )- total amount 1L Dilute to 100ml

10 × 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 trypsin

Transfer buffer 0.1% formic acid

17

Table 1: List of all proteins in CW5

Protein acc Protein score Protein cover Localization Function
AT1G75780* 190 12.5 vacuole GTPase activity, structural molecule activity, GTP binding
AT1G56110* 116 6.3 cell wall, nucleolus NOP56-like protein
AT5G22060* 101 9.8 plasma membrane ATP binding, heat shock protein binding, unfolded protein binding
AT1G02500* 88 3.2 cell wall, membrane, plasma membrane methionine adenosyltransferase activity
AT3G23990* 80 5.4 cytosol, cytosolic ribosome, mitochondrial matrix, mitochondrion, ATP binding, copper ion binding
vacuolar membrane
AT1G14320* 72 6.3 chloroplast, cytosolic large ribosomal subunit, cytosolic ribosome, structural constituent of ribosome
large ribosomal subunit, nucleolus, vacuolar membrane, vacuole
AT5G20010* 72 6.3 apoplast, cell wall, plasma membrane GTP binding, protein binding, GTPase activity
AT1G79920* 59 6.1 cell wall, plasma membrane ATP binding
AT4G05020* 50 2.9 mitochondrion, extrinsic to mitochondrial inner membrane calcium ion binding, flavin adenine dinucleotide binding, disulfide oxidoreductase
activity, oxidoreductase activity
AT1G79330* 50 28.4 chloroplast cysteine-type endopeptidase activity
AT5G60640* 47 5.6 chloroplast, vacuolar membrane protein disulfide isomerase activity
AT1G49240* 47 5.6 chloroplast, plasma membrane, vacuole structural constituent of cytoskeleton, copper ion binding
AT1G163008* 47 4.3 Plastid glyceraldehyde-3-phosphate dehydrogenase (NAD+) (phosphorylating) activity,
glyceraldehyde-3-phosphate dehydrogenase activity
AT5G52640* 45 3.7 cell wall, cytosol, plasma membrane ATP binding, unfolded protein binding
AT3G02090* 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 membrane
AT5G07440* 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
activity
AT5G13450* 39 5.3 chloroplast, membrane, mitochondrion, plasma membrane cobalt ion binding, zinc ion binding, hydrogen ion transporting ATP synthase activity,
rotational mechanism
AT4G26970* 37 2.7 chloroplast, cytosol, mitochondrion copper ion binding, aconitate hydratase activity
AT4G31700* 32 8.2 chloroplast, cytosolic ribosome, cytosolic small ribosomal subunit, structural constituent of ribosome
membrane, nucleolus, plasma membrane
membrane, nucleolus
AT1G07890* 30 5.7 cell wall, chloroplast, chloroplast stroma, cytosol, plasma L-ascorbate peroxidase activity
membrane
membrane, plasma membrane, vacuolar membrane
AT5G11170* 25 0.9 Nucleolus helicase activity, ATP-dependent helicase activity, nucleic acid binding, ATP binding
AT5G28540# 304 12.1 cell wall, chloroplast, endoplasmic reticulum lumen, plasma ATP binding
membrane, vacuolar membrane, vacuole
18

AT3G12110# 161 13.3 cytoskeleton, mitochondrion, plasma membrane structural constituent of cytoskeleton
AT2G07698# 106 6.4 membrane, nucleus, plasma membrane, vacuole hydrogen ion transporting ATP synthase activity, rotational mechanism, poly(U) RNA
binding, zinc ion binding
AT2G18960# 91 2.2 membrane, nucleus, plasma membrane, vacuole protein binding , ATPase activity, hydrogen-exporting ATPase activity, phosphorylative
mechanism
AT1G17880# 88 16.4 Unknown sequence-specific DNA binding transcription factor activity
AT1G20620# 78 9.3 apoplast, cell wall, chloroplast, chloroplast envelope, chloroplast cobalt ion binding, catalase activity
stroma, cytosolic ribosome, membrane, mitochondrion,
peroxisome, plasma
AT2G33210# 76 4.1 chloroplast, mitochondrion, plasma membrane, vacuolar ATP binding, copper ion binding
membrane
AT2G42910# 76 5.3 cytoplasm, plasma membrane magnesium ion binding, ribose phosphate diphosphokinase activity
AT4G24830# 75 6.7 chloroplast, chloroplast stroma ATP binding, argininosuccinate synthase activity
AT3G11130# 74 3.6 plasma membrane, vacuolar membrane, vacuole binding, structural molecule activity
AT3G17390# 69 6.4 cell wall, membrane, nucleolus, plasma membrane methionine adenosyltransferase activity
AT5G10840# 67 3.4 plasma membrane Binding
AT3G02090# 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 membrane
AT1G07790# 60 10.1 chloroplast DNA binding
AT2G44060# 55 3.4 membrane, plasma membrane Molecular function unknown
AT1G71380# 54 6.2 cell wall, plant-type cell wall, plasma membrane catalytic activity, hydrolase activity, hydrolyzing O-glycosyl compounds
AT4G22010# 50 3.9 membrane, plant-type cell wall oxidoreductase activity, copper ion binding
AT1G12840# 48 4.3 chloroplast, plant-type vacuole,plasma membrane,vacuolar proton-transporting ATPase activity, rotational mechanism
membrane, vacuole
AT1G70710# 45 4.7 chloroplast cellulase activity, hydrolase activity, hydrolyzing O-glycosyl compounds
AT2G07560# 45 2.2 membrane, plasma membrane protein binding, ATPase activity
AT5G61790# 44 8.3 chloroplast, endoplasmic reticulum, membrane, microsome, calcium ion binding, unfolded protein binding
mitochondrion, plant-type cell wall, plasma membrane, vacuolar
membrane, vacuole
AT1G73230# 43 16.4 No data Molecular function unknown
AT2G42210# 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
membrane
AT1G15690# 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, vacuole
AT3G57290# 39 2.9 cytoplasm, nucleus, plasma membrane, signalosome protein binding, translation initiation factor activity
AT1G09100# 36 3.8 membrane, plasma membrane calmodulin binding, ATPase activity
AT1G66110# 36 2.1 No data Molecular function unknown
AT2G36580# 36 4.7 plasma membrane magnesium ion binding, potassium ion binding, catalytic activity, pyruvate kinase activity
AT1G07890# 36 5.6 cell wall, chloroplast, chloroplast stroma, cytosol, plasma L-ascorbate peroxidase activity
19

membrane
AT5G07640# 36 2.5 No data zinc ion binding
AT3G13870# 34 2.1 cytoplasm, endoplasmic reticulum, plasma membrane, vacuolar GTP binding
membrane
AT1G07660# 33 7.8 chloroplast, plasma membrane, vacuolar membrane DNA binding
AT1G17710# 33 6.1 No data phosphatase activity
AT1G10630# 31 5.5 membrane, plasma membrane, vacuolar membrane GTP binding, copper ion binding, protein binding, phospholipase activator activity
AT5G07340# 25 2.1 chloroplast, endoplasmic reticulum, membrane, vacuolar calcium ion binding, unfolded protein binding
membrane
AT2G44120# 25 13.6 chloroplast, cytosolic large ribosomal subunit, cytosolic transcription regulator activity, structural constituent of ribosome
ribosome, large ribosomal subunit, membrane, nucleolus, vacuole
AT2G33040# 23 6.8 cell wall, chloroplast, cytoplasm, membrane, mitochondrion, zinc ion binding
nucleolus, nucleus
AT1G20260# 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 mechanism
AT5G02500 581 31.2 apoplast, cell wall, chloroplast, cytosol, cytosolic ribosome, ATP binding
membrane, nucleolus, nucleus, plasma membrane, vacuolar
membrane
AT1G35720 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, vacuole
AT5G15090 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, vacuolar
AT3G12580 430 28 cell wall, cytosol, mitochondrion, plasma membrane, vacuolar ATP binding
membrane
AT5G02490 426 20.7 cell wall, cytosol, nucleus, plasma membrane protein binding
AT1G13440 412 40.5 cell wall, chloroplast, cytosol, membrane, mitochondrion, copper ion binding, zinc ion binding
nucleolus, nucleus, plasma membrane
AT5G56030 395 18.6 cell wall, cytosol, mitochondrion, nucleus ATP binding, protein binding
AT1G07920 394 36.7 chloroplast, membrane, mitochondrion, nucleolus, plasma calmodulin binding
membrane, vacuolar membrane
AT3G09440 334 21.9 apoplast, cell wall, chloroplast, cytosol, cytosolic ribosome, plasma ATP binding
membrane, vacuolar membrane, vacuole
AT5G62690 332 16.7 cell wall, nucleolus, plasma membrane GTPase activity, structural molecule activity, GTP binding
AT1G56070 318 9.5 chloroplast, cytosol, membrane, nucleolus, plasma membrane, copper ion binding, translation factor activity, nucleic acid binding
vacuolar membrane
AT4G37910 317 11.9 cell wall, mitochondrial matrix, mitochondrion, vacuolar ATP binding, zinc ion binding
membrane
AT5G23860 315 16.7 membrane structural constituent of cytoskeleton, protein binding
AT4G20890 306 18 chloroplast, plasma membrane, vacuolar membrane GTP binding, GTPase activity, structural molecule activity
AT1G04820 266 15.6 cell wall, chloroplast, cytosol, plasma membrane, tubulin complex, structural constituent of cytoskeleton

20

vacuolar membrane
AT5G08670 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
vacuolar
AT1G20010 183 16.3 cell wall, chloroplast, membrane, plasma membrane, vacuolar structural constituent of cytoskeleton
membrane
AT3G08580 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,
vacuole
AT4G13940 173 16.7 membrane, plasma membrane, vacuolar membrane, vacuole adenosylhomocysteinase activity, copper ion binding
AT5G66680 146 8.2 endoplasmic reticulum, endoplasmic reticulum membrane, dolichyl-diphosphooligosaccharide-protein glycotransferase activity
membrane, nucleolus, plant-type cell wall, vacuolar membrane,
vacuole
AT2G02560 122 3.9 cell wall, plasma membrane Binding
AT3G09840 117 6.1 cell wall, cytoplasm, cytosolic ribosome, nuclear envelope, identical protein binding, ATPase activity
nucleolus, nucleus, phragmoplast, plasma membrane, spindle
AT1G45000 90 7.3 cell wall, membrane, nucleolus, plasma membrane ATP binding, nucleotide binding, ATPase activity, hydrolase activity, nucleoside-
triphosphatase activity
AT4G02930 90 7.3 mitochondrion, cell wall ATP binding, cobalt ion binding, zinc ion binding, translation elongation factor activity
AT3G27280 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 tissues
AT1G09080 88 5.2 endoplasmic reticulum lumen ATP binding
AT2G37270 80 13.5 cell wall, chloroplast, cytosolic ribosome, cytosolic small structural constituent of ribosome
ribosomal subunit, membrane, plasma membrane, ribosome,
vacuolar
AT3G11940 80 13.5 cell wall, chloroplast, cytosolic ribosome, cytosolic small structural constituent of ribosome
ribosomal subunit, plasma membrane, ribosome, vacuole
AT3G01280 78 13.4 chloroplast, chloroplast envelope, mitochondrial outer membrane, voltage-gated anion channel activity
mitochondrion, plasma membrane, plastid, vacuolar membrane,
vacuole
AT2G18450 77 3.8 mitochondrion succinate dehydrogenase activity
AT2G04030 77 3.8 chloroplast, chloroplast envelope, chloroplast stroma, ATP binding
mitochondrion, vacuolar membrane
AT1G65930 73 5 apoplast, cytosol, plasma membrane copper ion binding, isocitrate dehydrogenase (NADP+) activity
AT1G35160 70 11.4 cytoplasm, nuclear envelope, plasma membrane protein phosphorylated amino acid binding
AT5G38480 70 7.5 cell wall, chloroplast, mitochondrion, plasma membrane, vacuole ATP binding, protein phosphorylated amino acid binding
AT1G54270 70 7.5 cytosol, plasma membrane, vacuolar membrane ATP-dependent helicase activity, translation initiation factor activity
AT3G08030 68 6 cell wall Molecular function unknown
AT2G05710 68 6 cell wall, chloroplast, mitochondrion, vacuolar membrane ATP binding, copper ion binding
AT4G24190 59 6.1 chloroplast, endoplasmic reticulum, membrane, mitochondrion, ATP binding, unfolded protein binding
nucleus, plasma membrane, vacuolar membrane, vacuole
AT1G55490 55 4.5 chloroplast, endoplasmic reticulum, membrane, mitochondrion, protein binding

21

nucleus
AT5G09590 55 4.5 cell wall, chloroplast, mitochondrial matrix, mitochondrion, ATP binding
vacuolar
AT3G09680 52 2.9 cytosolic ribosome, cytosolic small ribosomal subunit, nucleolus, structural constituent of ribosome
ribosome
AT4G20150 50 6.3 mitochondrion, vacuolar membrane Molecular function unknown
AT2G47650 48 3.4 Golgi apparatus, membrane, vacuolar membrane, vacuole UDP-glucuronate decarboxylase activity, catalytic activity
AT2G20140 48 3.7 plasma membrane ATP binding, nucleotide binding
AT2G41840 46 9.5 cytosolic ribosome, cytosolic small ribosomal subunit, membrane, structural constituent of ribosome
nucleolus
AT3G07770 45 2.5 cell wall, mitochondrion ATP binding
AT1G79550 44 4 apoplast, cytosol, membrane, nucleus, plasma membrane, vacuolar phosphoglycerate kinase activity
membrane
AT1G22780 38 7.6 cell wall, cytosolic small ribosomal subunit, plasma membrane, structural constituent of ribosome, RNA binding, nucleic acid binding
small ribosomal subunit, vacuolar membrane
AT1G04690 37 2.7 membrane, plasma membrane oxidoreductase activity, potassium channel activity
AT5G13490 36 7.3 chloroplast, chloroplast envelope, membrane, mitochondrial binding, copper ion binding, protein binding, ATP:ADP antiporter activity
envelope, mitochondrial inner membrane, mitochondrion, vacuolar
membrane
AT2G38940 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, vacuole
AT5G53460 25 1.8 chloroplast, chloroplast stroma, plastid glutamate synthase (NADH) activity
AT3G22310 21 1.6 plasma membrane DNA binding, RNA binding
AT1G18500 21 2.5 chloroplast 2-isopropylmalate synthase activity
AT5G42080 21 2.5 chloroplast thylakoid membrane, microtubule, plasma membrane, GTP binding, clathrin binding, protein binding
vacuolar membrane, vacuole
AT3G61760 21 2.5 no data GTPase activity, GTP binding

22

Table 2: List of all proteins in 0.1M CaCl2 extraction

Protein acc Protein Score Protein cover Localization Function
AT5G22650* 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 process
AT5G18170* 338 26.5 mitochondrion Nitrogen compound metabolic process, response to absence of light, response to
cadmium ion, response to salt stress
AT3G12390* 212 18.7 cytosolic ribosome Response to salt stress
AT1G32130* 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 process
AT3G44750* 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 membrane
AT4G10480* 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 karrikin
AT2G13540* 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 unknown
AT1G08970* 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

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 activity
AT1G30580* 48 4.3 intracellular Response to cadmium ion, GTP binding
AT4G17260* 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, ribosome
AT1G55570* 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 unknown
AT5G61290* 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, vacuole
AT1G13950* 21 5.1 No data Translational initiation, xylem development, RNA binding, ribosome binding,
24

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

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