The Effects on Lignin Structure of Overexpression of Ferulate 5-Hydroxylase in Hybrid Poplar1

Poplar (Populus tremula × alba) lignins with exceedingly high syringyl monomer levels are produced by overexpression of the ferulate 5-hydroxylase (F5H) gene driven by a cinnamate 4-hydroxylase (C4H) promoter. Compositional data derived from both standard degradative methods and NMR analyses of the entire lignin component (as well as isolated lignin fraction) indicated that the C4H∷F5H transgenic''s lignin was comprised of as much as 97.5% syringyl units (derived from sinapyl alcohol), the remainder being guaiacyl units (derived from coniferyl alcohol); the syringyl level in the wild-type control was 68%. The resultant transgenic lignins are more linear and display a lower degree of polymerization. Although the crucial β-ether content is similar, the distribution of other interunit linkages in the lignin polymer is markedly different, with higher resinol (β-β) and spirodienone (β-1) contents, but with virtually no phenylcoumarans (β-5, which can only be formed from guaiacyl units). p-Hydroxybenzoates, acylating the γ-positions of lignin side chains, were reduced by >50%, suggesting consequent impacts on related pathways. A model depicting the putative structure of the transgenic lignin resulting from the overexpression of F5H is presented. The altered structural features in the transgenic lignin polymer, as revealed here, support the contention that there are significant opportunities to improve biomass utilization by exploiting the malleability of plant lignification processes.In the continuing search for improved biomass utilization in processes including chemical pulping, natural ruminant digestibility, and biomass conversion to ethanol, considerable attention has focused on improving lignocellulosic feedstocks through genetic engineering. Perturbing plant biomass deposition by misregulating key genes/enzymes integral to major cell wall pathways can provide rich insights into cell wall development and architecture and also create significant opportunities for improved lignocellulosic utilization (Baucher et al., 2003; Boerjan et al., 2003; Ralph et al., 2004). Lignins comprise the second most abundant polymer class in the biosphere; however, their combinatorial biosynthesis renders them among the more complex biomacromolecules synthesized by plants. Alterations in plant cell wall chemistry or ultrastructure, including lignin content or structure, can have a profound effect on chemical or enzymatic degradability. For example, in chemical pulping, lignin structure and content have been shown to significantly impact delignification efficiency (both pulp yield and residual lignin RL] content) and pulp bleachability (Chang and Sarkanen, 1973; Huntley et al., 2003; Stewart et al., 2006). Similarly, improvements in fermentable sugar yields have been reported in lignin-engineered Medicago sativa (Chen and Dixon, 2007).In recent years, the genes encoding enzymes specific to the lignin branch of the phenylpropanoid pathway have been cloned and their roles evaluated using a combination of forward and reverse genetics (Franke et al., 2002; Baucher et al., 2003; Boerjan et al., 2003). The down-regulation of genes early in the pathway may limit the overall flux of metabolites to lignin synthesis. In contrast, the genes common to the latter part of the pathway generally affect the distribution of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) lignin units resulting from the primary monomers (the three monolignols p-coumaryl, coniferyl, and sinapyl alcohols). And, in several cases when the biosynthesis of a normal monolignol is severely curtailed, lignins appear to incorporate unique phenolics (e.g. 5-hydroxyconiferyl alcohol, hydroxycinnamaldehydes, and ferulic acid) that have not traditionally been considered lignin monomers (Sederoff et al., 1999; Boerjan et al., 2003; Ralph et al., 2004, 2008a, 2008b).Ferulate 5-hydroxylase (F5H), also referred to coniferaldehyde 5-hydroxylase to reflect one of its preferred substrate (Humphreys et al., 1999; Osakabe et al., 1999), is a key enzyme involved in synthesizing the monolignol sinapyl alcohol and, ultimately, S lignin moieties. F5H therefore affects the partitioning between the two major traditional monolignols, coniferyl and sinapyl alcohols, and is fundamental to the evolutionary differences between gymnosperms (with no S components) and angiosperms (with compositions favoring the S monomers). For example, the fah1 Arabidopsis (Arabidopsis thaliana) mutant, deficient in F5H, has little to no S lignin (Meyer et al., 1998; Marita et al., 1999). Like gymnosperms, it produces G-rich lignins, derived almost exclusively from coniferyl alcohol. In contrast, the overexpression of F5H in the mutant background produces plants displaying substantially higher than normal sinapyl alcohol-derived S units and is consequentially severely depleted in coniferyl alcohol-derived G units. Wet chemical analyses of cell wall lignins estimated S contents of up to about 92% in F5H-up-regulated Arabidopsis (Meyer et al., 1998), up to 84% in tobacco (Nicotiana tabacum; Franke et al. 2000), and as high as 93.5% in hybrid poplar (Populus tremula × Populus alba; Huntley et al., 2003; Li et al., 2003). These engineered cell walls, rich in S units, exceed the highest reported in nature to date (Baucher et al., 1998), with kenaf (Hibiscus cannabinus) bast fiber lignin, at 85% S, being among the highest (Ralph, 1996; Morrison et al., 1999). In poplars, the final methylation step, catalyzed by caffeic acid 3-O-methyl transferase (COMT), appears to adequately accommodate the increased flux from coniferaldehyde to 5-hydroxyconiferaldehyde to produce sinapaldehyde and ultimately sinapyl alcohol (Li et al., 2000, 2003). In Arabidopsis, however, evidence suggests that the COMT is not able to keep pace with the increased 5-hydroxyconiferaldehyde generated by F5H overexpression, since the ensuing lignins comprise a significant component derived from 5-hydroxyconiferyl alcohol (Ralph et al., 2001a). Novel 5-hydroxyguaiacyl benzodioxane structures, which result from incorporation of 5-hydroxyconiferyl alcohol into the lignification scheme, were the same as those noted in COMT-deficient plants (Ralph et al., 2001a, 2001b; Marita et al., 2001, 2003).The availability of woody plant material possessing an extreme S lignin concentration affords unique opportunities to explore the mechanisms and consequences of pushing plants toward compositional limits and has both major fundamental and industrial consequences. Engineering lignin composition to extreme levels provides rare and novel insights into the ramifications on the structure of the lignin component and on other cell wall characteristics. Here, we investigate the nature of the chemical modifications to the lignin polymer in up-regulated cinnamate 4-hydroxylase (C4H)∷F5H poplar and propose a model for how such gene perturbation impacts lignin biosynthesis.