1. Lignin Dening Ye Oct 29 th 2010 PBIO 691: [Plant Cell Walls]

2. 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 What is lignin ? Peter Albersheim, A.D., et al, Plant cell walls. 2011.

3. 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.

4. Bonawitz, N.D. and C. Chapple, The Genetics of Lignin Biosynthesis: Connecting Genotype to Phenotype. Annu Rev Genet, 2010.

5. Monomers of lignin Three major monomers ( H lignin ) ( G lignin ) ( S lignin )

6. Various composition of the monomer among different species Vanholme, R., et al., Lignin biosynthesis and structure. Plant Physiol, 2010. 153(3): p. 895-905 Syringyl lignin (S lignin) may be superior to guaiacyl lignin( G lignin) in its ability to strengthen cell walls ? b b Li L, et al. Plant Cell, 2001 13:1567–1586.

7. Synthesis pathway of lignin monomers 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. 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-O- methyltransferase CAD: cinnamyl alcohol dehydrogenase F5H: cinnamoyl CoA reductase

8. Vanholme, R., et al., Lignin biosynthesis and structure. Plant Physiol, 2010. 153(3): p. 895-905

9. The linkages of lignin monomers peroxidases laccase Coniferyl alcohol radicals

10. The linkages of lignin monomers Bonawitz, N.D. and C. Chapple, The Genetics of Lignin Biosynthesis: Connecting Genotype to Phenotype. Annu Rev Genet, 2010.

11. The linkages of lignin monomers Bonawitz, N.D. and C. Chapple, The Genetics of Lignin Biosynthesis: Connecting Genotype to Phenotype. Annu Rev Genet, 2010.

12. Example of the polymer structure of Lignin

13. Characterization Difficult to analyze due to heterogeneous linkages Highly hydrophobic Not exacted from plant tissues by any organic solvent

14. RNAi-mediated suppression of p-coumaroyl-CoA 3'-hydroxylase Autofluorescence light microscopy UV fluorescence microscopy RNAi- C3H WT Coleman, H.D., et al.,. PNAS, 2008. 105(11): p. 4501-6.

15. Lignin degradation in wood-feeding insects Geib, S.M., et al., Lignin degradation in wood-feeding insects. PNAS, 2008. 105(35): p. 12932-7. 13 C-tetramethylammonium hydroxide (TMAH) Asian longhorned beetle (Anoplophora glabripennis) Pacific dampwood termite (Zootermopsis angusticollis)

16. 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…)

17. The Growth Reduction Associated with Repressed Lignin Biosynthesis in Arabidopsis thaliana Is Independent of Flavonoids The Plant Cell, 2010. 22: 1620–1632 Xu Li, Nicholas D. Bonawitz, Jing-Ke Weng, and Clint Chapple

19. Flavonoid Accumulation in Arabidopsis Repressed in Lignin synthesis Affects Auxin Transport and Plant Growth Silencing of HCT results in a strong reduction of plant growth Several flavonol glycosides and acylated anthocyanin were shown to accumulate in higher amounts in silenced plants Flavonoid-mediated inhibition of auxin transport is responsible for growth reduction in 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 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 flavonoid accumulation. Besseau, S, et al. The Plant Cell, 2007, 19: 148–162, Yes No Yes

20. The growth phenotype of ref8 is independent of flavonoid accumulation. (Figure 2)

21. Silencing of HCT in either the wild-type or tt4-2 background results in growth inhibition. (Figure 3 ) wild-typett4-2

22. Silencing of HCT in either the wild-type or tt4-2 background results in growth inhibition. (Figure 3 )

23. 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

24. 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

25. 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.

26. 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.

27. Silencing of HCT in wild-type or tt4-2 background results in similar lignin changes. (Figure 8)

28. Expressing Sm-F5H in HCT-RNAi Plants Suppresses Flavonoid Accumulation (Figure 9)

29. Expression of Selaginella F5H Rescues the Growth Phenotype of HCT-RNAi Plants Independent of Flavonoid Accumulation. ( Figure 10 )

30. 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

31. 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