12-02: Elucidation of Alfalfa Lignin Structures on Gene Down-regulation

Independently down-regulation of genes encoding 4-coumarate 3-hydroxylase (C3H) and hydroxycinnamoyl transferase (HCT) has shown to reduce the recalcitrance of alfalfa and thereby improving the yield of simple sugars after pretreatment. One-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) techniques were utilized to identify structural elements of importance to the recalcitrance of genetically engineered alfalfa. After C3H and HCT gene down-regulation, significant structural changes had occurred to the alfalfa lignin.  A significant increase of p-hydroxylphenyl unit content was observed in the transgenic alfalfa lignins as well as a concomitant decrease of up to ~70% of the guaiacyl and syringyl units.  Quantitative ¹³C NMR measurement also showed a significant decrease of carboxylic group, methoxyl group and β-O-4 linkage contents in the alfalfa lignins after genetic engineering. ¹³C-¹H HSQC 2D correlation NMR demonstrated an increase of interunit phenylcoumaran and resinol contents for C3H and HCT transgenic alfalfa. In addition, ³¹P NMR measurement revealed that phenyl hydroxyl group in p-hydroxylphenyl unit was dramatically increased for the transgenic lignins, as well as by over ~50% decrease of guaiacyl hydroxyl group content.  The results of these changes in lignin structure and their relationship to recalcitrance will be examined with a perspective to future improvements in plant cell wall design for enhanced sugar production for biofuels.