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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
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