Functional genomics

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Analysis of Cell Wall Proteins during Xylem
Vessel Secondary Cell Wall Formation in Cell
Culture
Students Gurung Jyoti Mohan Dwivedi Gaurav
Dutta Linlin Gao Supervisors Irene
Granlund Edouard Pesquet
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Outline

• INTRODUCTION
• MATERIALS METHODS
• RESULTS DISCUSSION
• ACKNOWLEDGEMENT

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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.

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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.
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Components of secondary cell wall

• The principal components of secondary walls are
• Cellulose
• Hemicellulose
• Lignin.

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Hemicellulose
Is in the form of heteropolymers (matrix
polysaccharides), such as arabinoxylans, present
along with cellulose in almost all plant cell
walls.
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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.

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Lignifications in secondary cell wall
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Lignifications in secondary cell wall
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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.

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Material Methods
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Suspension cell culture of A. thaliana
Cell induction for TE differentiation
Basal cells without any hormone induction
Harvesting of cell culture with vacuum filtration
Cell wall preperation by tissue grinding
Subsequent washes in increasing concentration of
sucrose
Protein extraction by different concentration of
salts (NP40 CaCl2)
SDS-PAGE and Western Blotting
Protein identification by LC-MS/MS
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Tracheary Elements (TEs) Differentiation System
in vitro

Hormones
Normal Cells Basal
Normal Cells Basal
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Tracheary Elements (TEs) Differentiation System
in vitro
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Cells harvest by Vacuum filter
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Freezer mill
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Sonication
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Medium mill
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Cell Wall Preparation
Solubize in 150mM NaCl and 10 glycerol in 100mM
Acetate Buffer pH 4.6
1000 g, 4C, 15 min, 3 acc.
Supernatant
Pellet
Solubilize in 0.4M sucrose in acetate buffer pH4.6
1000 g, 4C, 15 min, 3 acc
Supernatant
Pellet
Solubilize in 0.6M sucrose in acetate buffer pH4.6
1000 g, 4C, 15 min, 3 acc
Supernatant
Pellet
Solubilize in 1M sucrose in acetate buffer pH4.6
1000 g, 4C, 15 min, 3 acc
Supernatant
Pellet
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Solubilise in 5mM MES-KOH pH 5.6 with 5 mM MgCl2
1000 g, 4C, 15 min, 3 acc
Supernatant
Pellet
20 000 g, 4C, 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, 4C, 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
4M CaCl2
2M CaCl2
0.1M CaCl2
0.5M CaCl2
20 000 g, 4C, 10 min
Pellet
0.1 M Extraction
0.5M Extraction
2M Extraction
4M Extraction
CW5
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LC-MS/MS
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Mascot search
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Data analysis
www.arabidopsis.org
www. plantenergy.uma.edu.au
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Results Discussion
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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.

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Cell disruption by different methods
Before grinding
sonication
Sonication
Medium mill
Medium mill
Freezer mill
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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.

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Western blotting using anti-tubulin antibody as
the primary antibody
Induced samples
Basal samples
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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.

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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.

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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.

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Thank you
Tack så mycket
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