NMR Characterization of C3H and HCT Down-Regulated Alfalfa Lignin
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NMR Characterization of C3H and HCT Down-Regulated Alfalfa Lignin Yunqiao Pu & Fang Chen & Angela Ziebell & Brian H. Davison & Arthur J. Ragauskas
Published online: 20 October 2009 # Springer Science + Business Media, LLC. 2009
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 Electronic supplementary material The online version of this article (doi:10.1007/s12155-009-9056-8) contains supplementary material, which is available to authorized users. Y. Pu Institute of Paper Science and Technology, Georgia Institute of Technology, Atlanta, GA, USA A. J. Ragauskas (*) School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA e-mail: [email protected] F. Chen Samuel Roberts Noble Foundation, Ardmore, OK, USA A. Ziebell National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO, USA B. H. Davison Oak Ridge National Laboratory, Oak Ridge, TN, USA Y. Pu : F. Chen : A. Ziebell : B. H. Davison : A. J. Ragauskas BioEnergy Science Center, Oak Ridge, TN, USA
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 13 C 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 downregulation 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. Keywords Alfalfa . Gene down-regulation . Lignin structure . NMR . p-Hydroxyphenyl . Methoxyl . β-O-4 linkage Abbreviations C3H p-Coumarate 3-hydroxylase HCT Hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyl transferase NMR Nuclear magnetic resonance HSQC Heteronuclear single quantum coherence DEPT Distortionless enhancement by polarization transfer WT Wild-type /Ar Per aromatic ri
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