![Page 1: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/1.jpg)
Lignin Management: Optimizing Yield and Composition in Lignin-Modified Plants
Clint ChappleDepartment of Biochemistry
Purdue University
![Page 2: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/2.jpg)
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
![Page 3: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/3.jpg)
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
![Page 4: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/4.jpg)
Lignin is critical for plant survival
• structural support• water transport
• large commitment of fixed carbon
• important impact on bioenergy and bioprocessing
• high energy density
![Page 5: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/5.jpg)
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
![Page 6: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/6.jpg)
Lignin modification decreases the need for pretreatment
Chapple et al., Nature Biotechnol., 2007
![Page 7: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/7.jpg)
Lignin modification-induced dwarfing (LMID)
Schilmiller et al., Plant J. 2009
![Page 8: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/8.jpg)
The lignin biosynthetic pathway
ring modification
side
cha
in m
odifi
catio
n
S-LIGNN
PHE
G-LIGNIN
![Page 9: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/9.jpg)
Lignin is a biosynthetically plastic polymer
Bonawitz and Chapple, Ann Rev Genetics, 2010
![Page 10: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/10.jpg)
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
![Page 11: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/11.jpg)
Lignin composition can be modified
F5H
PHE
G-LIGNIN S-LIGNIN
![Page 12: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/12.jpg)
Lignin composition can be modified
G-LIGNIN S-LIGNIN
F5H
PHE
![Page 13: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/13.jpg)
Some changes to lignin composition do not affect growth
![Page 14: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/14.jpg)
Lack of F5H eliminates lignin S subunits
![Page 15: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/15.jpg)
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
![Page 16: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/16.jpg)
Lack of C3'H reduces lignin content andleads to novel lignins
Franke et al., Plant J, 2002
![Page 17: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/17.jpg)
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
![Page 18: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/18.jpg)
ref4 and rfr1 are dispensable and knockouts do not phenocopy ref4-3
![Page 19: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/19.jpg)
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
![Page 20: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/20.jpg)
ref4 and rfr1 are dispensable and knockouts do not phenocopy ref4-3
![Page 21: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/21.jpg)
REF4 and RFR1 are components of theMediator complex
P Cramer Lab, Gene Center, University of Munich
![Page 22: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/22.jpg)
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
![Page 23: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/23.jpg)
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
![Page 24: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/24.jpg)
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
![Page 25: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/25.jpg)
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
![Page 26: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/26.jpg)
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
![Page 27: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/27.jpg)
Dwarfism of the ref8 mutant requires Med5
![Page 28: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/28.jpg)
What genes underpin LMID?
Li et al., unpublished
![Page 29: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/29.jpg)
What genes underpin LMID?
induce Med5 RNAseq
med5a/bref8-1
proximaltargetgenes
![Page 30: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/30.jpg)
Suppressors of ref4-3 may identify other proteins required for phenylpropanoid regulation
![Page 31: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/31.jpg)
Nonsense mutations in Mediator tail subunits restore metabolism or growth in ref4-3
Dolan et al., unpublished
![Page 32: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/32.jpg)
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
![Page 33: Lignin Management - Optimizing Yield and Composition in ... · metabolism and plant growth • Identifying new opportunities for lignin engineering. Lignin is critical for plant survival](https://reader034.vdocuments.net/reader034/viewer/2022050504/5f964109dc7dcd1728082124/html5/thumbnails/33.jpg)
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