Regulation of a gene encoding coniferaldehyde 5-hydroxylase leads to substantial alterations in lignin structure in rice cell walls, identifying a promising genetic engineering target for improving grass biomass utilization. The aromatic composition of lignin greatly affects utilization characteristics of lignocellulosic biomass and, therefore, has been one of the primary targets of cell wall engineering studies. Limited information is, however, available regarding lignin modifications in monocotyledonous grasses, despite the fact that grass lignocelluloses have a great potential for feedstocks of biofuel production and various biorefinery applications. Here, we report that manipulation of a gene encoding coniferaldehyde 5-hydroxylase (CAld5H, or ferulate 5-hydroxylase, F5H) leads to substantial alterations in syringyl (S)/guaiacyl (G) lignin aromatic composition in rice (Oryza sativa), a major model grass and commercially important crop. Among three CAld5H genes identified in rice, OsCAld5H1 (CYP84A5) appeared to be predominantly expressed in lignin-producing rice vegetative tissues. Down-regulation of OsCAld5H1 produced altered lignins largely enriched in G units, whereas up-regulation of OsCAld5H1 resulted in lignins enriched in S units, as revealed by a series of wet-chemical and NMR structural analyses. Our data collectively demonstrate that OsCAld5H1 expression is a major factor controlling S/G lignin composition in rice cell walls. Given that S/G lignin composition affects various biomass properties, we contemplate that manipulation of CAld5H gene expression represents a promising strategy to upgrade grass biomass for biorefinery applications.

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.

Sorghum (Sorghum bicolor) is cultivated worldwide for food, bioethanol, and fodder production. Although nitrogen fixation in sorghum has been studied since the 1970s, N2-fixing bacteria have not been widely examined in field-grown sorghum plants because the identification of functional diazotrophs depends on the culture method used. The aim of this study was to identify functional N2-fixing bacteria associated with field-grown sorghum by using “omics” approaches. Four lines of sorghum (KM1, KM2, KM4, and KM5) were grown in a field in Fukushima, Japan. The nitrogen-fixing activities of the roots, leaves, and stems were evaluated by acetylene reduction and 15N2-feeding assays. The highest nitrogen-fixing activities were detected in the roots of lines KM1 and KM2 at the late growth stage. Bacterial cells extracted from KM1 and KM2 roots were analyzed by metagenome, proteome, and isolation approaches and their DNA was isolated and sequenced. Nitrogenase structural gene sequences in the metagenome sequences were retrieved using two nitrogenase databases. Most sequences were assigned to nifHDK of Bradyrhizobium species, including non-nodulating Bradyrhizobium sp. S23321 and photosynthetic B. oligotrophicum S58T. Amplicon sequence and metagenome analysis revealed a relatively higher abundance (2.9–3.6%) of Bradyrhizobium in the roots. Proteome analysis indicated that three NifHDK proteins of Bradyrhizobium species were consistently detected across sample replicates. By using oligotrophic media, we purified eight bradyrhizobial isolates. Among them, two bradyrhizobial isolates possessed 16S rRNA and nif genes similar to those in S23321 and S58T which were predicted as functional diazotrophs by omics approaches. Both free-living cells of the isolates expressed N2-fixing activity in a semi-solid medium according to an acetylene reduction assay. These results suggest that major functional N2-fixing bacteria in sorghum roots are unique bradyrhizobia that resemble photosynthetic B. oligotrophicum S58T and non-nodulating Bradyrhizobium sp. S23321. Based on our findings, we discuss the N2-fixing activity level of sorghum plants, phylogenetic and genomic comparison with diazotrophic bacteria in other crops, and Bradyrhizobium diversity in N2 fixation and nodulation.