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

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.