Cell walls, Fibres and Pectin

1
Cell walls, Fibres and Pectin
? Gene discovery effort - The
glycosyltransferases of GT family GT77 ?
Applied aspects
Bent Larsen Petersen
2
Primary cell wall structure and components

• Pectic
• Polysaccharides
• HomoGalcturonan (HG)
• RhamnoGacaturonan I II
• (RGI and II)

apoplastic fluid
hemicellulose
structural cell wall glycoproteins, e.g..
extensins
cellulose microfibrils
3
Cell wall biosynthetic machinery
Plasma membrane
Transcript
Cell wall
Apoplast
Trans golgi
ER
Trans golgi network
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Formation of GT family GT77 Background
Hypothesis The fine structure of plant the cell
wall (cw) is unique -gt plant cw GTs outside of
CAZY are to be expected
Strategy 2 and 3D structure information included
in the homology searches applied on the
Arabidopsis proteome
A pool of non-CAZy plant cw GTs identified
(Egelund et al. 2004, Plant Physiol. 136
2609-2620)
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CAZy GT family GT77
A
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Characterization of Arabidopsis Clade A and B
members
- cell wall analysis of knock out mutants and
RNAi- transformants
- expression analysis using promoter-GUS
transformants
- determination of catalytic activity of
heterologously expressed GTs

• baculo virus sf9 cells and / or
• Pichia pastoris
• - and tested in the Free Sugar
• Assay (FSA)

7
Progress on Arabidopsis clade B genes
? Initial biochemical charaterisation of a new
member of the RhamnoGalcaturonanXylosylTransfera
se (RGXT) family
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CAZy GT77 - B-clade activities
RhamnoGalacturonan XylosylTransferase
RGXT1 2
?-(1,3) linkage
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
RG II A-chain
O
O
O
O
O
O
O
O
O
O
O
O
O
? RGII-MeXyl is slightly reduced in rgxt1 and -2
mutants ? RGII of mutant rgxt1 and -2 work as
acceptor in enzyme assays
(Egelund et al. 2006, Plant Cell)
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Characterisation of clade B member At1g56550
(expressed in Pichia pastoris)
Free Sugar Assaydonor UDP-D-Xylose
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Initial characterization of RGXT3 (expressed in
Pichia pastoris)
Me ?-L-Fuc
? RGII of rgxt1 and -2 mutants do NOT work as
acceptor
? Activity is only ?5 of that of RGXT1 -2
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Non-denaturing deglycosylation of
RGXT1-3(expressed in Pichia)
Deglycosylated - - -
MW (D) pred. N-Glc sites RGXT1S
36.5 4 RGXT2S 37.3 5 RGXT3S
40.5 7
RGXT3 will be expressed in baculo virus system
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Clade A RRA1 and -2- Initial characterization
Arabidopsis alpha-1,3-Xylosyltransferase
CAZy GT family GT77
Dictyostelium AX3 alpha-1,3-Galactosyltransferase
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GT77 - A-clade
Chlamydomonas
Cell wall made up of extensin-like glycoproteins
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Ostreococcus
Smallest existing eukaryote One chloroplast One
mitochondrion One Golgi apparatus No real cell
wall - just a glycoconjugate covered membrane
Ostreococcus tauri strain OTH95, photo courtesy
of Hervé Moreau, Laboratoire Arago
57 entries in CAZy-GT-families
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Characterisation of A-clade RRA-1 -2
RRA-1Promotor-GUS analysis
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rra1 and -2 mutants display an reduced residual
arabionose content

• Cell walls isolated from shoot apical meristem

- subjected to stepwise enzymatic removal of
xyloglucan, xylan and pectin and the
remaining pellet analysed
(Egelund et al. 2007, Plant Mol. Biol.)
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Reconciling a reduced-arabinosephenotype of a
family 77 GT knock out

• We can predict what type of arabinans a GT77
  enzyme can possibly produce, and it matches
  extensin of higher plants and their homologes in
  the cell wall of Chlamydomonas

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CAZy GT family GT77- Summary

• ? The A-clade RRA1 and -2 genes
• - may encode arabinosyltransferases involved
  in biosynthesis of extensin
• - will be assayed with suitable donor and
  acceptor substrates
• ? The Clade B RGXT1-3 genes
• - Redundancy seem to be a common feature - and
  it appears that
• there is little freedom to change
  RG-II-A-chain structure without
• compromising plant viability
• ? Evolution
• - The gene family is old and pectic
  polysaccharides like RG-II are very old

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Applied aspects
? Engineering bioethanol plants ? Enzyme
mediated extraction of functional pectins
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Miscanthus late summer these shoots are annual
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Engineering bioethanol plants - Cell wall
digestibility

• In monocots, lignins are cross-linked to
  arabinoxylans (hemicellulose)
• Cross-linking of lignin to arabinoxylan has a
  greater effect on cell wall digestibility than
  lignin concentration

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Engineering bioethanol plants- Conditional
disruption of ArabinoXylan
Working hypothesis Heat-treatment of transgenic
plant material with an thermophilic endo-xylanase
stored in the cell walls of lignified cells will
lead to the disruption of the cross-linking
between lignin and arabinoxylan resulting in
improved cell wall digestibility
Benefits ? Heat pretreatment - No biological
adverse effects - Already and an integral
part the process -gt no extra cost ? Plant
material more susceptible - reduce the cost of
external added wall degrading enzymes
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Engineering bioethanol plantsXylanase-storing
grasses
Pretreatment now gives enzyme
more accessible substrate
Heat-treatment
Cellulose
Lignin
Hemicellulose
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Moving beyond standard applications of pectin
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Enzymatic modification - examples
ara
gal
ara
gal
galA
ara
ara
galA
OMe
OMe
gal
ara
galA
ara
OMe
galA
galA
gal
ara
ara
galA
galA
gal
ara
ara
rha
galA
gal
rha
galA
OAc
rha
ara arabinose gal galactose galA
galacturonic acid OAc acetyl ester OMe methyl
ester rha rhamnose
galA
galA
Homogalacturonan (HG)
Rhamnogalacturonan I (RG I)
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Pectin extraction - The goal identify minimal
enzyme mixes to extract as close to intact pectic
polymers as possible
pulling out a barbed wire of the meshwork
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Drinking yogurt and milkshakes must be smooth,
stable and with a tightly controlled viscosity
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Casein micelles in dairy products - Supra
molecular structures within the nannoscale
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People involved

• Jack Egelund
• Kirsten Faber
• Iben Damager
• Bernhard Borkhardt
• Bodil Jørgensen
• Peter Ulvskov

Henrik Clausen Henrik Vibe Scheller William
Willats
Nicolai Obel Markus Pauly
Anthony Bacic
Marcia J. Kieliszewski