Little is known regarding syringyl lignin biosynthesis in rice (Oryza sativa L. cv. Nipponbare). In the present study, the role of rice caffeic acid O-methyltransferase (OsCOMT1, Q6ZD89), was examined. The recombinant OsCOMT1 catalyzed the 5-Omethylation of 5-hydroxyferulate (5-HFA) and 5-hydroxyconiferaldehyde (5-HCAld). 5-HCAld inhibited 5-HFA methylation by this O-methyltransferase (OMT), while 5-HFA mitigated self-inhibition in 5-HCAld methylation. A rice plant in which OsCOMT1 expression was downregulated exhibited weakened cell wall staining with Wiesner reagent in vascular bundle cells and sclerenchyma tissue, compared with wild-type plants. The lignin content of transgenic rice plants was decreased and the syringyl lignin content reduced largely compared with that of the wild type. Taken together, these data indicated that OsCOMT1 functioned as a 5-HCAld OMT (OsCAldOMT1) in the biosynthetic pathway to syringyl lignin. Recently, because of the increased demand for biobased materials as alternatives to fossil carbon resources, gramineous plants that produce large amounts of inedible lignocellulosic biomass have been drawing attention as potential materials for biofuel and industrial feedstock production (Yamamura et al. 2013). Lignocellulose is composed mainly of lignin and polysaccharides. Lignin is a complex phenylpropanoid polymer, and fills the spaces between cell wall polysaccharides and confers mechanical strength and imperviousness to the cell wall (Boerjan et al. 2003). The inherent robust characteristics of lignin present obstacles to enzymatic hydrolysis of plant cell wall polysaccharides for biorefining, chemical pulping, and forage digestion. On the other hand, lignin is a promising raw material for aromatic feedstock production.Lignins are generally classified into three major groups: guaiacyl (4-hydroxy-3-methoxyphenyl), syringyl (3,5-dimethoxy-4-hydroxyphenyl), and p-hydroxyphenyl lignins. Lignin structures affect their reactivity and thermal properties. For example, plants with high syringyl lignin content are more easily delignified in kraft pulping than those with low syringyl lignin content (Chiang and Funaoka 1990;Lourenço et al. 2012;Shimizu et al. 2012). Condensed lignin structures reduce the thermal mobility of lignin (Kubo et al. 1997), indicating that syringyl lignin is beneficial for use in plastics, compared with guaiacyl lignin, because syringyl lignin lacks the condensed structures. Characterization of lignin and its biosynthetic mechanisms is a basis for the practical use of lignocelluloses.Syringyl lignin had long been proposed to be formed from p-coumaric acid (CouA) via caffeic acid (CA), ferulic acid (FA), 5-hydroxyferulic acid (5-HFA), sinapic acid (SA), sinapoyl CoA (SCoA), sinapaldehyde (SAld), and sinapyl alcohol (SAlc) (Figure 1), based on tracer experiments with isotope-labeled phenylpropanoid monomers and associated enzymatic experiments Original Paper DOI: 10.5511/plantbiotechnology.13.0219a Abbreviations: 4CL, 4-hydroxycinnamate CoA ligase; 5-HCAlc, 5-...

p-Coumaroyl ester 3-hydroxylase (C3'H) is a key enzyme involved in the biosynthesis of lignin, a phenylpropanoid polymer that is the major constituent of secondary cell walls in vascular plants. Although the crucial role of C3'H in lignification and its manipulation to upgrade lignocellulose have been investigated in eudicots, limited information is available in monocotyledonous grass species, despite their potential as biomass feedstocks. Here we address the pronounced impacts of C3'H deficiency on the structure and properties of grass cell walls. C3'H-knockdown lines generated via RNA interference (RNAi)-mediated gene silencing, with about 0.5% of the residual expression levels, reached maturity and set seeds. In contrast, C3'H-knockout rice mutants generated via CRISPR/Cas9-mediated mutagenesis were severely dwarfed and sterile. Cell wall analysis of the mature C3'H-knockdown RNAi lines revealed that their lignins were largely enriched in p-hydroxyphenyl (H) units while being substantially reduced in the normally dominant guaiacyl (G) and syringyl (S) units. Interestingly, however, the enrichment of H units was limited to within the non-acylated lignin units, with grass-specific γ-p-coumaroylated lignin units remaining apparently unchanged. Suppression of C3'H also resulted in relative augmentation in tricin residues in lignin as well as a substantial reduction in wall cross-linking ferulates. Collectively, our data demonstrate that C3'H expression is an important determinant not only of lignin content and composition but also of the degree of cell wall cross-linking. We also demonstrated that C3'H-suppressed rice displays enhanced biomass saccharification.

Several brown midrib (bm) mutants have so far been isolated from the C4 grasses, maize, sorghum and pearl millet, but have not been detected in C3 grasses including rice (Oryza sativa). In the present study we characterized the cad2 (cinnamyl alcohol dehydrogenase 2) null mutant isolated from retrotransposon Tos17 insertion lines of Oryza sativa L. ssp. japonica cv. Nipponbare. This mutant exhibited brown-colored midribs in addition to hulls and internodes, clearly indicating both bm and gold hull and internode (gh) phenotypes. The enzymatic saccharification efficiency in the culm of cad2 null mutant was increased by 16.1% than that of the control plants. The lignin content of the cad2 null mutant was 14.6% lower than that of the control plants. Thioacidolysis of the cad2 null mutant indicated the presence of cinnamaldehyde structures in the lignin. Taken together, our results show that deficiency of OsCAD2 causes the bm phenotype in addition to gh, and that the coloration is probably due to the accumulation of cinnamaldehyde-related structures in the lignin. Additionally, this cad2 null mutant was useful to silage purposes and biofuel production. Lignin is a complex phenylpropanoid polymer, and is biosynthesized via oxidative coupling of phydroxycinnamyl alcohols (monolignols) and related compounds that are formed in the cinnamate/ monolignol pathway (Umezawa 2010). Lignin fills the spaces between cell wall polysaccharides and confers mechanical strength and imperviousness to the cell wall (Boerjan et al. 2003). Therefore, lignin biosynthesis is closely related to the evolution of land plants.Lignin has several properties that present obstacles to chemical pulping, forage digestion, and enzymatic hydrolysis of plant cell wall polysaccharides for biorefining. For these processes, it would be beneficial for plant materials to either have less lignin, or to have lignin that is easier to remove. Mutant plants in which genes encoding lignin biosynthetic enzymes are downregulated are generally expected to have lower lignin content and higher enzymatic saccharification efficiency. For these reasons, lignin biosynthesis is an area of great interest (Chiang 2006;Dixon and Reddy 2003;Vanholme et al. 2008;Weng et al. 2008).Several brown midrib (bm or bmr for sorghum) mutants have been isolated in maize (Zea mays), sorghum (Sorghum bicolor), and pearl millet (Pennisetum glaucum) arising by either spontaneous or chemical mutagenesis (Barrière et al. 2004;Cherney et al. 1991;Sattler et al. 2010). The characteristic reddish-brown to tan colored midribs of the mutant leaf blades contrasts with the pale green midribs of the wild type. In addition, the mutants show similar coloration in stalks and generally have reduced lignin content and higher in vitro digestibility compared with wild-type plants. Hence, the mutants have been receiving a lot of interest in relation not only to silage purposes (Barrière et al. 2004;Cherney et al. 1991;Sattler et al. 2010), but also biofuel production (Sattler et al. 2010).In maize, six...