BioEnergy Research

, Volume 9, Issue 2, pp 691–699 | Cite as

Downregulation of p-Coumaroyl Quinate/Shikimate 3′-Hydroxylase (C3′H) or Cinnamate-4-hydrolylase (C4H) in Eucalyptus urophylla × Eucalyptus grandis Leads to Increased Extractability

  • Angela Ziebell
  • Erica GjersingEmail author
  • Maud Hinchee
  • Rui Katahira
  • Robert W. Sykes
  • David K. Johnson
  • Mark F. Davis


Lignin reduction through breeding and genetic modification has the potential to reduce costs in biomass processing in pulp and paper, forage, and lignocellulosic ethanol industries. Here, we present detailed characterization of the extractability and lignin structure of Eucalyptus urophylla × Eucalyptus grandis RNAi downregulated in p-coumaroyl quinate/shikimate 3′-hydroxylase (C3′H) or cinnamate-4-hydroxylase (C4H). Both the C3′H and C4H downregulated lines were found to have significantly higher extractability when exposed to NaOH base extraction, indicating altered cell wall construction. The molecular weight of isolated lignin was measured and lignin structure was determined by HSQC NMR-based lignin subunit analysis for control and the C3′H and C4H downregulated lines. The slight reductions in average molecular weights of the lignin isolated from the transgenic lines (C3′H = 7000, C4H = 6500, control = 7300) does not appear to explain the difference in extractability. The HSQC NMR-based lignin subunit analysis showed increases in H lignin content for the C3′H but only slight differences in the lignin subunit structure of the C3′H and C4H downregulated lines when compared to the control. The greatest difference between the C3′H and C4H downregulated lines is the total lignin content; therefore, it appears that overall lowered lignin content contributes greatly to reduced recalcitrance and increased extractability of cell wall biopolymers. Further studies will be conducted to determine how the reduction in lignin content creates a less rigid cell wall that is more prone to extraction and sugar release.


Nuclear magnetic resonance (NMR) Plant Cell wall Transgenic Spectroscopy Eucalyptus C3′H C4H Lignin 



The authors would like to thank William Rottmann, ArborGen Inc., for manuscript review and for providing the biomass materials studied in this paper. The BESC is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. This work was supported by the U.S. Department of Energy under Contract No. DE-AC36-08-GO28308 with the National Renewable Energy Laboratory.

Supplementary material

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Copyright information

© The Author(s) 2016

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  • Angela Ziebell
    • 1
  • Erica Gjersing
    • 1
    Email author
  • Maud Hinchee
    • 3
  • Rui Katahira
    • 1
  • Robert W. Sykes
    • 1
  • David K. Johnson
    • 2
  • Mark F. Davis
    • 1
  1. 1.National Bioenergy Center, National Renewable Energy LaboratoryGoldenUSA
  2. 2.BioSciences Center, National Renewable Energy LaboratoryGoldenUSA
  3. 3.ArborGen Inc.RidgevilleUSA

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