3. Aim of the study
Objective of the present study is to accomplish
fractionation of cell wall from normal cells and cells
that has secondary cell wall to identify the different
proteins involved in the growing of secondary cell wall
and lignification. After the formation of the secondary
cell walls, the identification of cell wall proteins and
the quality of cell wall fractionation was achieved by
using MS/MS.
4. Introduction
Secondary cell walls are the
major constituent of
tracheary elements (TEs) and
fibers in wood, which is the
most abundant biomass
produced by plants.
The secondary cell walls
provide strong mechanical
strength to tracheary
elements and fibers, and
ultimately to plant organs.
5. Components of secondary cell wall
The principal components
of secondary walls are:
Cellulose
Hemicellulose
Lignin.
6. Hemicellulose
I s i n t he f or mof het er opol ym s (m r i x pol ysacchar i des), such as
er at
ar abi noxyl ans, pr esent al ong w t h cel l ul ose i n al m al l pl ant cel l w l s.
i ost al
7. Lignin synthesis in tracheary elements (TEs)
Lignin is found in the secondary cell wall of tracheary elements and xylem
fibers.
The tracheary elements are water and mineral conducting structures in wood.
They undergo programmed cell death (PCD) to mature and form a hollow tube
with a lignified secondary cell wall.
The lignified secondary cell wall provides a mechanical
stability, hydrophobicity and pathogenic defense.
10. Limitations arises during the
extraction of cell wall proteins
Proteins may be confined in the polysaccharide matrix of
cellulose, hemi-cellulose and pectin.
Some proteins are difficult to solubilize.
Some proteins undergo post-translational modifications.
Lack of surrounding membrane may result in a loss of
cell wall proteins.
11.
12. Suspension cell culture of A. thaliana
Cell induction for TE differentiation Basal cells without any hormone inductio
Harvesting of cell culture with vacuum filtration
Cell wall preperation by tissue grinding
Subsequent washes in increasing concentration of sucrose
Protein extraction by different concentration of salts
(NP40 + CaCl2)
SDS-PAGE and Western Blotting Protein identification by LC-MS/MS
13. Tracheary Elements (TEs) Differentiation System in vitro
Normal Cells Normal Cells Hormones
Basal Basal
14. Tracheary Elements (TEs) Differentiation System in vitro
Normal Cells Tracheary
Basal elements (TEs)
Induced
19. Cell Wall Preparation
Solubize in 150mM NaCl and 10% glycerol in 100mM Acetate Buffer pH 4.6
1000 × g, 4°C, 15 min, 3 acc.
Supernatant
Pellet
Solubilize in 0.4M sucrose in acetate buffer pH4.6
1000 × g, 4°C, 15 min, 3 acc
Supernatant
Pellet
Solubilize in 0.6M sucrose in acetate buffer pH4.6
1000 × g, 4°C, 15 min, 3 acc
Supernatant
Pellet
Solubilize in 1M sucrose in acetate buffer pH4.6
1000 × g, 4°C, 15 min, 3 acc
Supernatant
Pellet
20. Solubilise in 5mM MES-KOH pH 5.6 with 5 mM MgCl2
1000 × g, 4°C, 15 min, 3 acc
Supernatant Pellet
20 000 × g, 4°C, 10 min
Filtrate and freeze in liquid nitrogen
Pellet
Wash two times with 5 mM MES-KOH pH 5.6 with 5 mM MgCl2 with centrifugation in between
CW4
Freeze in liquid nitrogen and grind.
Solubilise in 0.05% NP40 + 10% DMSO in 5mM MES-KOH pH 5.6 with 5mM MgCl2
20 000 × g, 4°C, 10 min
NP40 extraciton Pellet
Wash with 5 mM MES-KOH pH 5.6 with 5 mM MgCl2
solubilise in 0.1M, 0.5M and 2M and 4MCaCl2 in 5mM MES-KOH pH 5.6 with 5mM MgCl2
0.1M CaCl2 0.5M CaCl2 2M CaCl2 4M CaCl2
20 000 × g, 4°C, 10 min
Pellet
0.1 M Extraction 0.5M Extraction 2M Extraction 4M Extraction CW5
23. Data analysis
www.arabidopsis.org
www. plantenergy.uma.edu.au
24.
25. Cell culture, harvest and
homogenization
After 7 days of cell culture, approx. 15-20% of
induced cells had transformed into Trachery
element (TE) formation.
Cell disruption was carried out by either of the
three methods: medium mill, sonication or freezer
mill.
Freezer mill method was the most efficient among
the three.
26. Cell disruption by different methods
Before sonication
grinding Sonication
Medium mill
Medium mill Freezer mill
27. SDS-PAGE and Western blotting
Proteins were seperated using SDS-PAGE.
Western blotting provided the purity of cell wall
isolated.
It was carried out using anti-tubulin antibody as
the primary antibody.
Tubulin are the proteins that make up
microtubule, a cellular component that lie beneath
the secondary cell walls.
28. Western blotting using anti-tubulin
antibody as the primary antibody
Basal samples Induced samples
29. LC-MS/MS and bioinformatic analysis
We chosed 0.1M CaCl2 extraction (Induced and basal) and
CW5 pellet (Induced and basal).
We identified 79 proteins from CW5-pellet (Induced) and
94 proteins from CW5-pellet (basal).
Out of these, 44.3% were CWPs in induced sample and
39.3% were CWPs in basal sample.
Notably, both the induced and basal CW5-pellet revealed
the presence of some protein contaminants.
Conversely, in case of 0.1M CaCl2 extract, we identified
47.1% of CWPs in basal supernatant compared to 31.1% of
CWPs in induced supernatant.
30. Association between induction hormones and
cell wall proteins
This implies that majority of cell wall proteins of basal
sample were released during the extraction point.
Two possible reasons:
CWPs in basal sample with no TEs were loosely bound
to the cell wall.
Some CWPs are tightly associated with the cell wall
during secondary cell wall formation.
31. Concluding remarks
This method of preparing cell wall through
mechanical disruption, fractionation and extraction of
proteins with different salt concentration provides a
good cell wall preperation technique.
In fact, the principle of this technique can offer a stage
for studying cell wall proteome.
Editor's Notes
Lignin is found in the secondary cell wall of tracheary elements and xylem fibersThe tracheary elements are water and mineral conducting structures in woodThey undergo programmed cell death (PCD) to mature and form a hollow tube with a lignified secondary cell wall.The lignified secondary cell wall provides a mechanical stability, hydrophobicity and pathogenic defense.
Solution also contain protease inhibitors and 0.01mM DTT
Protein samples wtih 10microgm protein conc were loaded on sds-page. Microtubule participate in regulation of secondary wall deposition.
Could there be some association between the induction hormones and tighter association of CWPs to the CW?
Even though there were some non-residients proteins present in the resultant CW5-pellet.
