|Reddy, M.s. srinivasa|
Submitted to: Journal of Biological Chemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/3/2005
Publication Date: 3/31/2006
Citation: Ralph, J., Akiyama, T., Kim, H., Lu, F., Schatz, P.F., Marita, J.M., Ralph, S.A., Reddy, M., Chen, F., Dixon, R.A. 2006. Effects of coumarate 3-hydroxylase downregulation on lignin structure. Journal of Biological Chemistry. 281(13):8843-8853. Interpretive Summary: Lignin is a polymer that is mainly present in plant cell walls, where it provides strength, defense, and allows for water transport through the plant. There is wide interest in understanding the process of lignin biosynthesis and deposition because of its economic relevance. Plant varieties with altered lignin content and composition can have improved performance as fodder crops or in the production of paper and pulp. By studying natural mutants and some genetically engineered transgenic plants, we have come to appreciate that plants can make their crucial lignins with considerable flexibility. The last step to be examined in the plant's pathway to making lignins can be addressed in detail now that collaborators at the Noble Foundation, Ardmore, OK, have generated alfalfa plants downregulated in "C3H". Lignins strikingly rich in normally minor "P"-units are produced; P-levels that are typically 1-3% of the lignin rise to ~65% of the lignin in the most heavily down-regulated line. Analysis of the lignins by nuclear magnetic resonance spectroscopy suggests that the minor monomer (building block), p-coumaryl alcohol, undergoes reactions that are for the most part analogous to those of the normally dominant monomers. The absence of certain key structures and a considerable shift in the proportions of others demonstrate that the lignification profile is, however, appreciably different. Such studies provide the foundations for understanding digestibility limitations for current research aimed at improving the utilization of our crop and forest plant resources.
Technical Abstract: Downregulation of the gene encoding 4-coumarate 3-hydroxylase (C3H) in alfalfa massively but predictably increased the proportion of p-coumaryl (P) units relative to the normally dominant guaiacyl (G) and syringyl (S) units. Levels of up to ~65% P (from wild-type (WT) levels of ~1%) resulting from downregulation of C3H to 5% of WT levels were measured by traditional degradative analyses as well as 2D 13C-1H correlative NMR methods. Such levels put these transgenics well beyond the P:G:S compositional bounds of normal plants; p-coumaryl levels are reported to reach a maximum of 30% in gymnosperm severe compression wood zones but are limited to a few percent in dicots. NMR also revealed striking structural differences in the interunit linkage distribution that characterizes a lignin polymer. Lower levels of key b-aryl ether units were relatively augmented by higher levels of phenylcoumarans and resinols. The C3H-deficient alfalfa lignins were devoid of b-1-coupling products, highlighting the significant differences in the reaction course for p-coumaryl alcohol vs the two normally dominant monolignols, coniferyl and sinapyl alcohols. A larger range of dibenzodioxocin structures was evident, in conjunction with an approximate doubling of their proportion. Unprecedented data regarding the nature of each of the structural units was revealed by long-range 13C-1H correlation experiments. For example, although b-ethers result from coupling of all three monolignols with the growing polymer, phenylcoumarans were formed almost solely from coupling reactions involving p-coumaryl alcohol; they result from both coniferyl and sinapyl alcohol in the WT plants. Such structural differences form a basis for explaining differences in digestibility and pulping performance of C3H-deficient plants.