Lignin part I:Biosynthesis

Dominique Loqué

Advanced School on Biochemistry of Biofuelssponsored by the American, International and

Brazilian Societies for Biochemistry and Molecular Biology (ASBMB, IUBMB and SBBq)

September2010

Where do we find lignin ?

Plant biomass CelluloseHemicellulose

PectinLignin

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-malateSM

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 mutantPhenolics

Staining withSafranin (red)

CelluloseStaining 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

Transp

orters

?

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

CWG

V

Monolignol export

Kaneda M. et.al. Plant Physiol. 2010:147:1750-1760

[3H]Phe of Pine Tracheids

CH: + Protein synthesis inhibitorPA: + phenylpropanoid metabolism inhibitor

Baseline in yeast for phenolic toxicity

coumarate

mM

ferulate

coniferylalcohol

sinapate

sinapylalcohol

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 12A 1A1 1A2 1A3 1A4 1A5 1A6 1A7 1A8 1A9 1A10 1A11 1A12B 1B1 1B2 1B3 1B4 1B5 1B6 1B7 1B8 1B9 1B10 1B11 1B12C 1C1 1C2 1C3 1C4 1C5 1C6 1C7 1C8 1C9 1C10 1C11 1C12D 1D1 1D2 1D3 1D4 1D5 1D6 1D7 1D8 1D9 1D10 1D11 1D12E 1E1 1E2 1E3 1E4 1E5 1E6 1E7 1E8 1E9 1E10 1E11 1E12F 1F1 1F2 1F3 1F4 1F5 1F6 1F7 1F8 1F9 1F10 1F11 1F12G 1G1 1G2 1G3 1G4 1G5 1G6 1G7 1G8 1G9 1G10 1G11 1G12H 1H1 1H2 1H3 1H4 1H5 1H6 1H7 1H8 1H9 1H10    

Plate2

  1 2 3 4 5 6 7 8 9 10 11 12A 2A1 2A2 2A3 2A4 2A5 2A6 2A7 2A8 2A9 2A10 2A11 2A12B 2B1 2B2 2B3 2B4 2B5 2B6 2B7 2B8 2B9 2B10 2B11 2B12C 2C1 2C2 2C3 2C4 2C5 2C6 2C7 2C8 2C9 2C10 2C11 2C12D 2D1 2D2 2D3 2D4 2D5 2D6 2D7 2D8 2D9 2D10 2D11 2D12E 2E1 2E2 2E3 2E4 2E5 2E6 2E7 2E8 2E9 2E10 2E11 2E12F 2F1 2F2 2F3 2F4 2F5 2F6 2F7 2F8 2F9 2F10 2F11 2F12G 2G1 2G2 2G3 2G4 2G5 2G6 2G7 2G8 2G9 2G10 2G11 2G12H 2H1 2H2 2H3 2H4 2H5 2H6 2H7 2H8 2H9      

Plate4

  1 2 3 4 5 6 7 8 9 10 11 12A 4A1 4A2 4A3 4A4 4A5 4A6 4A7 4A8 4A9      

Goal: 200 transportersABCMDR

MATE…

Plate1

  1 2 3 4 5 6 7 8 9 10 11 12A 1A3 1A4 1A5 1A7 1A8 1A9 1A11 1A12B 1B3 1B4 1B5 1B8 1B11 1B12C 1C1 1C2 1C3 1C4 1C5 1C6 1C7 1C8 1C9 1C10 1C11 1C12D 1D1 1D2 1D3 1D4 1D5 1D6 1D7 1D8 1D9 1D10 1D11 1D12E 1E1 1E2 1E3 1E4 1E5 1E6 1E7 1E9 1E11 1E12F 1F1 1F2 1F4 1F9 1F12G 1G1 1G3 1G4 1G5 1G6 1G7 1G8 1G9H 1H1 1H6 1H7 1H8 1H9 1H10    

Plate2

  1 2 3 4 5 6 7 8 9 10 11 12A 2A1 2A2 2A3 2A4 2A5 2A7B 2B1 2B6 2B7 2B9 2B10 2B11 2B12C 2C1 2C2 2C3 2C4 2C5 2C6 2C11 2C12D 2D3 2D5 2D6 2D9 2D11 2D12E 2E1 2E2 2E3 2E4 2E5 2E6 2E7 2E8 2E9 2E10 2E11 2E12F 2F1 2F3 2F4 2F5 2F6 2F7 2F8 2F9 2F10 2F11 2F12G 2G1 2G2 2G3 2G4 2G5 2G6 2G9 2G10 2G11 2G12H 2H1 2H3 2H5 2H8 2H9      

Plate4

  1 2 3 4 5 6 7 8 9 10 11 12A 4A7 4A8      

LR

pDRf1-GW

Yeast expression vector

mRNAcDNAPCRBP

Approx 140 transporters cloned and sequenced

Generation of ABC/MDR Transporter Library

Library screen

> 120 transporters were tested in AD1-10

Cell dilutions Cell dilutions

Ferulate

2F12

1E4

Control

Control

Ferulate screen

Coniferaldehyde screen

Cell dilutions

1A3

2F9

Coniferaldehyde

11 transporters identified

12 transporters identified

Characterization of Isolated transporters

2F12

Control

1E42B11

4A7

4A8

Ferulate mediaCoumarate media

Cell dilutions Cell dilutions Cell dilutions

Coniferyl alcohol media

Exporter

Monolignols or derivates

Monolignols or derivates

Cinnamic acid

Yeast AD1-10

Production of monolignol in yeast cytosol

Monolignols or

derivates

Bases for Monolignol polymerization: oxidative

coupling

Oxidation by H2O2 + Peroxidase or/and O2 + Laccase

Figures from Onnerud et al., (2002) The plant cell 14: 1953-1962

Resonance forms of radicals

Monolignols

Lignin polymer

H

G

S

Monolignol cross-coupling

van Parijs F. R. et.al.,(2010) Plant Physiol. 153: 1332-1344

Monolignols

Monolignol + Lignin polymer

Monolignol cross-coupling

Adler (1977) Wood Science and Technology, 11: 169-218