Abstract
Independent down-regulation of genes encoding p-coumarate 3-hydroxylase (C3H) and hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) has been previously shown to reduce the recalcitrance of alfalfa and thereby improve the release of fermentable sugars during enzymatic hydrolysis. In this study, ball-milled lignins were isolated from wild-type control, C3H, and HCT gene down-regulated alfalfa plants. One- and two-dimensional nuclear magnetic resonance (NMR) techniques were utilized to determine structural changes in the ball-milled alfalfa lignins resulting from this genetic engineering. After C3H and HCT gene down-regulation, significant structural changes had occurred to the alfalfa ball-milled lignins compared to the wild-type control. A substantial increase in p-hydroxyphenyl units was observed in the transgenic alfalfa ball-milled lignins as well as a concomitant decrease in guaiacyl and syringyl units. Two-dimensional 13C- 1H heteronuclear single quantum coherence correlation NMR, one-dimensional distortionless enhancement by polarization transfer-135, and 13C NMR measurement showed a noteworthy decrease in methoxyl group and β-O-4 linkage contents in these transgenic alfalfa lignins. 13C NMR analysis estimated that C3H gene down-regulation reduced the methoxyl content by ~55-58% in the ball-milled lignin, while HCT down-regulation decreased methoxyl content by ~73%. The gene down-regulated C3H and HCT transgenic alfalfa lignin was largely a p-hydroxyphenyl (H) rich type lignin. Compared to the wild-type plant, the C3H and HCT transgenic lines had an increase in relative abundance of phenylcoumaran and resinol in the ball-milled lignins.
Original language | English |
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Pages (from-to) | 198-208 |
Number of pages | 11 |
Journal | Bioenergy Research |
Volume | 2 |
Issue number | 4 |
DOIs | |
State | Published - Nov 2009 |
Funding
Acknowledgment We thank Drs. Richard A. Dixon and Mark Davis for their suggestion of the manuscript. The BioEnergy Science Center (BESC) is a US Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. The authors would like to gratefully acknowledge the financial support from DOE Office of Biological and Environmental Research through the BioEnergy Science Center (DE-AC05-00OR22725).
Keywords
- Alfalfa
- Gene down-regulation
- Lignin structure
- Methoxyl
- NMR
- p-Hydroxyphenyl
- β-O-4 linkage