Lignin Management: Optimizing Yield and Composition in Lignin-Modified Plants

Clint ChappleDepartment of Biochemistry

Purdue University

Outline

• Lignin pros and cons

• Lignin plasticity versus yield penalty

• Insights into lignin homeostasis

• Identification of an unanticipated mechanism linking lignin metabolism and plant growth

• Identifying new opportunities for lignin engineering

Lignin is critical for plant survival

• structural support• water transport

• large commitment of fixed carbon

• important impact on bioenergy and bioprocessing

• high energy density

OOMe

OMeO OH

O

OH

OMe

O

HO

OMe

OH

OMeO

HO

O

HO

HO

OH

O

OH

OMe

OMe

O

MeO

OH

OMe

HO OH

HO

OH

MeO

Lignin is critical for plant survival

• structural support• water transport

• large commitment of fixed carbon

• important impact on bioenergy and bioprocessing

• high energy density

Lignin is critical for plant survival

• structural support• water transport

• large commitment of fixed carbon

• important impact on bioenergy and bioprocessing

• high energy density

wild type

ref3-1

Lignin modification decreases the need for pretreatment

Chapple et al., Nature Biotechnol., 2007

Lignin modification-induced dwarfing (LMID)

Schilmiller et al., Plant J. 2009

The lignin biosynthetic pathway

ring modification

side

cha

in m

odifi

catio

n

S-LIGNN

PHE

G-LIGNIN

Lignin is a biosynthetically plastic polymer

Bonawitz and Chapple, Ann Rev Genetics, 2010

Lignin is a biosynthetically plastic polymer

OOMe

OMeO OH

O

OH

OMe

O

HO

OMe

OH

OMeO

HO

O

HO

HO

OH

O

OH

OMe

OMe

O

MeO

OH

OMe

HO OH

HO

OH

MeO

• Nucleic acid synthesis• Template-dependent

• Protein synthesis• Template-dependent

• Polysaccharide synthesis• Enzyme specificity-directed

• Lignin synthesis• Random radical coupling dependent on precursor supply

Lignin composition can be modified

F5H

PHE

G-LIGNIN S-LIGNIN

Lignin composition can be modified

G-LIGNIN S-LIGNIN

F5H

PHE

Some changes to lignin composition do not affect growth

Lack of F5H eliminates lignin S subunits

Lack of C3'H reduces lignin content andleads to novel lignins

C3'H

PHE

G-LIGNIN S-LIGNINH-LIGNIN

Franke et al., Plant J, 2002

Lack of C3'H reduces lignin content andleads to novel lignins

Franke et al., Plant J, 2002

ref4 mutants deposit less lignin than wild type

wild type ref4-1 ref4-2 ref4-3

% li

gnin

0

2

4

6

8

10

12

14

16

18

Stout et al., Genetics, 2008

ref4 and rfr1 are dispensable and knockouts do not phenocopy ref4-3

ref4 rfr1 plants accumulate more phenylpropanoids than wild type

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

sina

poyl

mal

ate

conc

entra

tion

(nm

ol/ m

g FW

)

Bonawitz et al., JBC, 2012

ref4 and rfr1 are dispensable and knockouts do not phenocopy ref4-3

REF4 and RFR1 are components of theMediator complex

P Cramer Lab, Gene Center, University of Munich

med5a/bref8-1

med5a/bref8-2

wild type ref8-1med5a/b ref8-2

Disruption of MED5 rescues the dwarf phenotype of ref8

Bonawitz et al., Nature, 2014

Disruption of MED5 restores wild-type levels of lignin

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

thio

glyc

olic

acid

lign

in (A

280

mg

fresh

wei

ght-1

)

Bonawitz et al., Nature, 2014

Disruption of MED5 rescues cell wall structural defects of ref8-1

wild type med5a/b ref8-1 med5a/b ref8-1

1 μm

40 μm

Peter Ciesielski, Bryon Donohoe, NREL

med5a/b ref8-1 mutants synthesize predominantly H lignin

syringyl (S)

guaiacyl (G)

p-hydroxyphenyl (H)

wild type med5a/b med5a/b ref8-1

Yuki Tobimatsu, John Ralph, Wisconsin

ref8-1 mutants show widespread MED5-dependent transcriptional reprogramming

med5a/b89

216med5a/b ref8-1

ref8-14441

95

39

43025

med5a/b19

196med5a/b ref8-1

ref8-13360

54

12

37147

up-regulated down-regulated

(DeSeq, false discovery rate <0.05)

Bonawitz et al., Nature, 2014

Dwarfism of the ref8 mutant requires Med5

What genes underpin LMID?

Li et al., unpublished

What genes underpin LMID?

induce Med5 RNAseq

med5a/bref8-1

proximaltargetgenes

Suppressors of ref4-3 may identify other proteins required for phenylpropanoid regulation

Nonsense mutations in Mediator tail subunits restore metabolism or growth in ref4-3

Dolan et al., unpublished

Summary

• The study of Arabidopsis has revealed a previously unknown regulatory circuit for lignin biosynthesis that involves Mediator

• The dwarfing seen in lignin-deficient plants can be suppressed genetically, indicating that it is an active protein-mediated process

• Plants with high H-lignin are viable and could provide novel inputs for the biorefinery

• Active feedback mechanisms sense changes in phenylpropanoid metabolism and/or cell wall architecture

Acknowledgements

Purdue and C3Bio

Nick BonawitzWhitney Dolan

Jake StoutJo Cusumano

Bryon DonohoePeter Ciesielski

Eduardo XimenesMike Ladisch

Brian DilkesCharles Addo-Quaye

GCEP

Sirius Li

Yuki TobimatsuJohn Ralph

Chris McClellanClaire Halpin

Wout Boerjan