Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/28/2007
Publication Date: 1/15/2008
Citation: Ralph, J., Kim, H., Lu, F., Grabber, J.H., Leple, J., Sierra, J.B., Derikvand, M.M., Jouanin, L., Boerjan, W., Lapierre, C. 2008. Identification of the structure and origin of a thioacidolysis marker compound for ferulic acid incorporation into angiosperm lignins and a pseudo-marker compound for cinnamoyl-CoA reductase. Plant Journal. 53(2):368-379.
Interpretive Summary: There is an enzyme, “CCR” used by all plants in the production of monolignols, the building blocks from which the polymer lignin is produced. It is lignin that holds fibers together in woody and forage plants; lignin is essential for the plant, but is a limit to plant cell wall digestibility by ruminant animals and an impediment in industrial pulping to make paper, or bioconversion to alternative fuels. Consequently, there is a lot of effort directed toward selection and genetic methods for altering lignins. Whereas enzyme levels and gene expression can be measured, the effect on the plant's biochemistry has been difficult to ascertain, except by very detailed and laborious methods. Here we report the discovery of a “molecular marker compound” for CCR, and our work elucidating the structure of the marker. We also deduced that it was derived from diagnostic structures in the lignin arising from incorporation of the ferulic acid monolignol precursors. This finding has two rather profound implications. First, it means that ferulic acid itself is a lignin monomer in these (and other) plants, and can incorporate integrally into the lignin polymer. Second, the way it incorporates provides a new branching mechanism in the polymer -- branchpoints are relatively rare in lignin, but are important to the overall structure and properties of this complex polymer. As it turns out, the branchpoint can be relatively easily cleaved by processing, so ferulic acid incorporation may provide a mechanism for altering lignin in a way that improves biomass processing. The availability of the marker allows researchers to rapidly assess the effect of CCR levels on plant lignin composition, and therefore provides a more streamlined tool for research aimed at improving plant cell wall utilization.
Technical Abstract: A molecular marker compound, derived from lignin by the thioacidolysis degradative method, for structures produced during lignification when ferulic acid is incorporated into lignification in angiosperms (poplar, Arabidopsis, tobacco) has been structurally identified as 1,2,2-trithioethyl ethylguaiacol [2-methoxy-4-(1,2,2-tris-ethylsulfonyl-ethyl)-phenol]. Its truncated sidechain and distinctive oxidation state suggest it derives from ferulic acid that has undergone bis-8-O-4-(cross-)coupling during lignification, as validated by model studies. A diagnostic contour for such structures is found in 2D 13C-1H correlated (HSQC) NMR spectra of lignins isolated from CCR-deficient poplar. As low levels of the marker are also released from normal (i.e. non-transgenic) plants in which ferulic acid may be present during lignification, notably in grasses, we term this only a pseudo-marker for CCR-deficiency in general, but a reliable marker in woody angiosperms such as poplar. Its derivation, along with evidence for 4-O-etherified ferulic acid, strongly implies that ferulic acid incorporates into angiosperm lignins. Its endwise radical coupling reactions suggest that ferulic acid should be considered an authentic lignin precursor. Moreover, ferulic acid incorporation provides a new mechanism for producing branch-points in the polymer. The findings sharply contradict those reported in a recent study on CCR-deficient Arabidopsis.