PBIO 691: [Plant Cell Walls]
Lignin
Dening Ye
Oct 29th 2010
What is lignin ?
•
An aromatic heteropolymer
•
Monomers Synthesized from Phenylalanine in the cytoplasm
•
Deposition of lignin occurs both within the secondary cell wall and in the middle
lamella
•
Mechanical support
•
Water impermeable surface
•
Unusually stable
contribute to the long life of tree species
terrestrial carbon cycle
•
Protection from pathogen or fungi degradation
Peter Albersheim, A.D., et al, Plant cell walls. 2011.
Novaes, E., et al., Lignin and biomass: a negative correlation for wood formation and lignin
content in trees. Plant Physiol, 2010. 154(2): p. 555-61.
Bonawitz, N.D. and C. Chapple, The Genetics of Lignin Biosynthesis: Connecting Genotype to Phenotype. Annu Rev Genet, 2010.
Monomers of lignin
( H lignin )
( G lignin )
( S lignin )
Three major monomers
Various composition of the monomer among different species
Syringyl lignin (S lignin) may be superior to guaiacyl lignin( G lignin) in its ability to
strengthen cell walls ?b
Vanholme, R., et al., Lignin biosynthesis and structure. Plant Physiol, 2010. 153(3): p. 895-905
b Li
L, et al. Plant Cell , 2001 13:1567–1586.
Synthesis pathway of lignin monomers
PAL: phenylalanine ammonia lyase
C4H: cinnamate 4-hydroxylase
4CL: 4-coumarate CoA ligase
CHS: chalcone synthase
HCT: hydroxycinnamoyl CoA:shikimate/
quinate hydroxycinnamoyl transferase
C3’H: p-coumarate 3-hydroxylase
CCR: cinnamoyl CoA reductase
CCoAOMT:caffeoyl CoA 3-Omethyltransferase
CAD: cinnamyl alcohol dehydrogenase
F5H: cinnamoyl CoA reductase
Li, X., et al., The growth reduction associated with repressed lignin biosynthesis in Arabidopsis thaliana is independent of flavonoids.
Plant Cell, 2010. 22(5): p. 1620-32.
Vanholme, R., et al., Lignin biosynthesis and structure. Plant Physiol, 2010. 153(3): p. 895-905
The linkages of lignin monomers
peroxidases
laccase
Coniferyl alcohol radicals
The linkages of lignin monomers
Bonawitz, N.D. and C. Chapple, The Genetics of Lignin Biosynthesis: Connecting Genotype to Phenotype. Annu Rev Genet, 2010.
The linkages of lignin monomers
Bonawitz, N.D. and C. Chapple, The Genetics of Lignin Biosynthesis: Connecting Genotype to Phenotype. Annu Rev Genet, 2010.
Example of the polymer structure of Lignin
Characterization
•Difficult to analyze due to heterogeneous linkages
•Highly hydrophobic
•Not exacted from plant tissues by any organic solvent
RNAi-mediated suppression of p-coumaroyl-CoA 3'-hydroxylase
Autofluorescence
light microscopy
UV fluorescence
microscopy
WT
RNAiC3H
Coleman, H.D., et al.,. PNAS, 2008. 105(11): p. 4501-6.
Lignin degradation in wood-feeding insects
Asian longhorned beetle (Anoplophora glabripennis)
Pacific dampwood termite (Zootermopsis angusticollis)
13C-tetramethylammonium
hydroxide (TMAH)
Geib, S.M., et al., Lignin degradation in wood-feeding insects. PNAS, 2008. 105(35): p. 12932-7.
Research trends
• Improvement of lignin degradation by copolymerizing alternative units
that derived from the incomplete monomer biosynthesis pathway
• More fundamental research on the biosynthesis pathway.
• Lignin engineering on end-use applications (pulping, saccharification…)
The Growth Reduction Associated with Repressed Lignin
Biosynthesis in Arabidopsis thaliana Is Independent
of Flavonoids
Xu Li, Nicholas D. Bonawitz, Jing-Ke Weng, and Clint Chapple
The Plant Cell, 2010. 22: 1620–1632
Flavonoid Accumulation in Arabidopsis Repressed in Lignin
synthesis Affects Auxin Transport and Plant Growth
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Silencing of HCT results in a strong reduction of plant growth Yes
•
Several flavonol glycosides and acylated anthocyanin were shown to accumulate in Yes
higher amounts in silenced plants
•
Flavonoid-mediated inhibition of auxin transport is responsible for growth reduction in
Yes
HCT-RNA interference (RNAi) plants.
•
Sinapoylmalate levels were barely affected, suggesting that the synthesis of that
phenylpropanoid compound might be HCT independent
•
Suppression of flavonoid accumulation by chalcone synthase repression in HCT-deficient No
plants restored normal auxin transport and wildtype plant growth
•
Phenotype of HCT-silenced plants is not due to the alteration of lignin synthesis but to
No
flavonoid accumulation.
Besseau, S, et al. The Plant Cell, 2007 , 19: 148–162,
No
The growth phenotype of ref8 is independent of flavonoid accumulation. (Figure 2)
Silencing of HCT in either the wild-type or tt4-2 background results in growth
inhibition. (Figure 3 )
wild-type
tt4-2
Silencing of HCT in either the wild-type or tt4-2 background results in growth
inhibition. (Figure 3 )
Plant height is negatively correlated with HCT silencing in both wild-type
and tt4-2 backgrounds. (Figure 4. )
HCT silencing was strongly correlated with plant growth reduction
in both wild-type and tt4-2 backgrounds
Elimination of flavonoids from HCT-RNAi plants does not rescue their
growth phenotype (Figure 5. )
To eliminate the potential position effects of the T- DNA
insertion, they generate wild type and tt4-2 mutants with
identical HCT-RNAi transgene insertions
Restoration of the ability to synthesize flavonoids does not aggravate the growth
phenotype of the tt4-2 HCT-RNAi plants. (Figure 6. )
Reciprocal test
Flavonoids have no effect on the reducedgrowth phenotype associated with RNAi
silencing of HCT.
Soluble Phenylpropanoids Decrease with Time to a Greater Extent in Wild-Type
Than in HCT-RNAi Plants (Figure 7)
These data are consistent with a role for HCT in sinapoylmalate biosynthesis and suggest that the high concentration of
flavonoids and sinapoylmalate in HCT-RNAi plants may be a result of reduced leaf expansion.
Silencing of HCT in wild-type or tt4-2 background results in similar lignin changes.
(Figure 8)
Expressing Sm-F5H in HCT-RNAi Plants Suppresses Flavonoid Accumulation (Figure 9)
Expression of Selaginella F5H Rescues the Growth Phenotype of HCT-RNAi Plants
Independent of Flavonoid Accumulation. ( Figure 10 )
The reasons for the discrepancy between their results and previous paper
• Previous paper made the HCT and CHS RNAi constructs by the same
promoter 35s
• Slight differences in HCT activity lead to dramatic differences in growth
phenotype. Such small changes cannot be distinguished at the protein
level by the immunoblotting assay
Conclusion
• The phenotype of the ref8 tt4-2 double mutant, which lacks
flavonoids, is indistinguishable from that of ref8
• The growth inhibition in HCT-RNAi plants and the ref8 mutant is
independent of flavonoids
• The fact that partially rerouting lignin biosynthesis via expression
of Selaginella F5H leads to significant alleviation of the growth
defects of ref8 or HCT-RNAi supports the lignin biosynthetic
model