Lignin part I:
Biosynthesis
Dominique Loqué
Advanced School on Biochemistry of Biofuels sponsored by the American, International and
Brazilian Societies for Biochemistry and
Molecular Biology (ASBMB, IUBMB and SBBq) September
2010
Where do we find lignin ?
Plant biomass Cellulose
Hemicellulose
Pectin
Lignin
Why Plant cell walls are excellent reserves of sugar?
Hemicellulose (~20-30%)
C6 and C5 sugar types
Lignin (~15-20%)
phenolics
Cellulose (~40-45%)
C6 sugar type
Cellulose (~40-45%)
Hemicellulose (~20-30%) Lignin (~15-30%)
phenolics
Lignin: A complex phenolic polymer
How much lignin in plants
Modified from Vogel 2008 Current Opinion in Plant Biology 20:301-307
Lignin content in Sugarcane bagasse: 15-25%
When did lignin appear ?
Weng and Chapple 2010 New phytologist 187:273-285
Where is lignin produced ?
Lignin: second most abundant biopolymer
Lignin is mainly found in secondary cell walls
Lignin belongs to phenylpropanoid
family
Monolignol biosynthesis
Bonawitz and Chapple 2010
Annual review of Genetic (in press)
Monolignol biosynthesis stepwise
Bonawitz and Chapple 2010
Annual review of Genetic (in press)
Zabala et al 2006, BMC Plant Biol 6
Biosynthetic pathway elucidation
Screen of mutant affected in the phenylpropanoid pathway
Phenylpropanoid pathway mutant screen
Clint Chapple screen: Sinapoyl-malate screen
WT sin1 mutant
UV@365nm UV@365nm
WT
sin1 mutant
Lignin staining (Maule staining) sinapate sinapoyl-glucose sinapoyl-malate
SM
Chapple et al., 1992 Plant Cell 4:1413-1424
Isolation of genes involved in lignin biosynthesis
Clint Chapple screen: Sinapoyl-malate screen
= Isolation of several key genes in the lignin biosynthesis
Ruegger et al., 2001 Genetics 159:1741-1749
Monolignol biosynthesis
C4H
sinapoyl-malate
C3H
F5H ref1
sng1 brt1
Chapple et al. 1992-now
Irregular xylem screen
WT irx4=ccr1
Jones et al., 2001 Plant Journal 26:205-216
Lignin mutants: screen on digestibility
bm1
(cad mutation)
bm2 (unknown mutation)
Barriere et al., 1994 Agronomie (14):15-25
MaizeGDB: http://www.maizegdb.org/phenotype.php
Lignin mutants: screen on digestibility
bm1 wt
Halpin et al., 1998 Plant Journal 14:545-553
Reverse genetics: 4CL silencing
Silencing of 4CL in poplar
Voelker et al., 2010 Plant Physiology (in press)
control pal mutant
Reverse genetics
Voelker et al., 2010 Plant Physiology (in press)
Silencing of 4CL in poplar
control pal mutant
Phenolics Staining with Safranin (red)
Cellulose
Staining with
Astra-blue (blue)
wt
hct- comt-
Reverse genetics: Gene silencing
Chen and Dixon 2007 Nature Biotechnology 25:759-761
Lignin biosynthesis modification in alfalfa
Lignin deposition increases with stem age
Water, Urea, H202, glycerol
channel ammonium
channel
Passive Diffusion versus Carrier-
Mediated Transport
Monolignol export
?
Cytosol
Cell wall
LIGNIN
Cell
Lignin backbones
Vesicular transport Tr an sp or ter s
?
Monolignol export potentially happens at the Plasma membrane
Kaneda M. et.al. Plant Physiol. 2010:147:1750-1760
[3H]Phe feed of Pine Tracheids
* * *
*
Monolignol export potentially happens
at the Plasma membrane [3H]Phe feed of Pine Tracheids
Control
CW
G
V
Kaneda M. et.al. Plant Physiol. 2010:147:1750-1760
+ phenylpropanoid metabolism inhibitor
+ Protein synthesis inhibitor
CW G
V
G CW
V
Monolignol export
Kaneda M. et.al. Plant Physiol. 2010:147:1750-1760
[3H]Phe of Pine Tracheids
CH: + Protein synthesis inhibitor
PA: + phenylpropanoid metabolism inhibitor
Baseline in yeast for phenolic toxicity
coumarate
mM
ferulate coniferyl
alcohol
sinapate sinapyl alcohol
phenolic exporter Yeast Δabc7
Yeast Complementation: Tool to Screen Monolignol/Phenolic
Exporters
Generation of ABC/MDR Transporter Library
Plate1
1 2 3 4 5 6 7 8 9 10 11 12
A 1A1 1A2 1A3 1A4 1A5 1A6 1A7 1A8 1A9 1A10 1A11 1A12 B 1B1 1B2 1B3 1B4 1B5 1B6 1B7 1B8 1B9 1B10 1B11 1B12 C 1C1 1C2 1C3 1C4 1C5 1C6 1C7 1C8 1C9 1C10 1C11 1C12 D 1D1 1D2 1D3 1D4 1D5 1D6 1D7 1D8 1D9 1D10 1D11 1D12 E 1E1 1E2 1E3 1E4 1E5 1E6 1E7 1E8 1E9 1E10 1E11 1E12 F 1F1 1F2 1F3 1F4 1F5 1F6 1F7 1F8 1F9 1F10 1F11 1F12 G 1G1 1G2 1G3 1G4 1G5 1G6 1G7 1G8 1G9 1G10 1G11 1G12 H 1H1 1H2 1H3 1H4 1H5 1H6 1H7 1H8 1H9 1H10 Plate2
1 2 3 4 5 6 7 8 9 10 11 12
A 2A1 2A2 2A3 2A4 2A5 2A6 2A7 2A8 2A9 2A10 2A11 2A12 B 2B1 2B2 2B3 2B4 2B5 2B6 2B7 2B8 2B9 2B10 2B11 2B12 C 2C1 2C2 2C3 2C4 2C5 2C6 2C7 2C8 2C9 2C10 2C11 2C12 D 2D1 2D2 2D3 2D4 2D5 2D6 2D7 2D8 2D9 2D10 2D11 2D12 E 2E1 2E2 2E3 2E4 2E5 2E6 2E7 2E8 2E9 2E10 2E11 2E12 F 2F1 2F2 2F3 2F4 2F5 2F6 2F7 2F8 2F9 2F10 2F11 2F12 G 2G1 2G2 2G3 2G4 2G5 2G6 2G7 2G8 2G9 2G10 2G11 2G12 H 2H1 2H2 2H3 2H4 2H5 2H6 2H7 2H8 2H9 Plate4
1 2 3 4 5 6 7 8 9 10 11 12
A 4A1 4A2 4A3 4A4 4A5 4A6 4A7 4A8 4A9
Goal: 200 transporters ABC
MDR MATE
…
Plate1
1 2 3 4 5 6 7 8 9 10 11 12
A 1A3 1A4 1A5 1A7 1A8 1A9 1A11 1A12
B 1B3 1B4 1B5 1B8 1B11 1B12
C 1C1 1C2 1C3 1C4 1C5 1C6 1C7 1C8 1C9 1C10 1C11 1C12 D 1D1 1D2 1D3 1D4 1D5 1D6 1D7 1D8 1D9 1D10 1D11 1D12 E 1E1 1E2 1E3 1E4 1E5 1E6 1E7 1E9 1E11 1E12
F 1F1 1F2 1F4 1F9 1F12
G 1G1 1G3 1G4 1G5 1G6 1G7 1G8 1G9
H 1H1 1H6 1H7 1H8 1H9 1H10
Plate2
1 2 3 4 5 6 7 8 9 10 11 12
A 2A1 2A2 2A3 2A4 2A5 2A7
B 2B1 2B6 2B7 2B9 2B10 2B11 2B12
C 2C1 2C2 2C3 2C4 2C5 2C6 2C11 2C12
D 2D3 2D5 2D6 2D9 2D11 2D12
E 2E1 2E2 2E3 2E4 2E5 2E6 2E7 2E8 2E9 2E10 2E11 2E12 F 2F1 2F3 2F4 2F5 2F6 2F7 2F8 2F9 2F10 2F11 2F12 G 2G1 2G2 2G3 2G4 2G5 2G6 2G9 2G10 2G11 2G12
H 2H1 2H3 2H5 2H8 2H9
Plate4
1 2 3 4 5 6 7 8 9 10 11 12
A 4A7 4A8
