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Lignans/neolignans are generally synthesized from coniferyl alcohol (CA) in the cinnamate/monolignol pathway by oxidation to generate the corresponding radicals with subsequent stereoselective dimerization aided by dirigent proteins (DIRs). Genes encoding oxidases and DIRs for neolignan biosynthesis have not been identified previously. In Arabidopsis thaliana, the DIR AtDP1/AtDIR12 plays an essential role in the 8-O-4′ coupling in neolignan biosynthesis by unequivocal structural determination of the compound missing in the atdp1 mutant as a sinapoylcholine (SC)-conjugated neolignan, erythro-3-{4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-hydroxymethylethoxy]-3, 5-dimethoxyphenyl}acryloylcholine. Phylogenetic analyses showed that AtDP1/AtDIR12 belongs to the DIR-a subfamily composed of DIRs for 8-8′ coupling of monolignol radicals. AtDP1/AtDIR12 is specifically expressed in outer integument 1 cells in developing seeds. As a putative oxidase for neolignan biosynthesis, we focused on AtLAC5, a laccase gene coexpressed with AtDP1/AtDIR12. In lac5 mutants, the abundance of feruloylcholine (FC)-conjugated neolignans decreased to a level comparable to those in the atdp1 mutant. In addition, SC/FC-conjugated neolignans were missing in the seeds of mutants defective in SCT/SCPL19, an enzyme that synthesizes SC. These results strongly suggest that AtDP1/AtDIR12 and AtLAC5 are involved in neolignan biosynthesis via SC/FC. A tetrazolium penetration assay showed that seed coat permeability increased in atdp1 mutants, suggesting a protective role of neolignans in A. thaliana seeds.

Lignin is a complex phenylpropanoid polymer deposited in plant cell walls. Lignin has long been recognized as an important limiting factor for the polysaccharide-oriented biomass utilizations. To mitigate lignin-associated biomass recalcitrance, numerous mutants and transgenic plants that produce lignocellulose with reduced lignin contents and/or lignins with altered chemical structures have been produced and characterised. However, it is not fully understood how altered lignin chemistry affects the supramolecular structure of lignocellulose, and consequently, its utilization properties. Herein, we conducted comprehensive chemical and supramolecular structural analyses of lignocellulose produced by a rice cad2 mutant deficient in CINNAMYL ALCOHOL DEHYDROGENASE (CAD), which encodes a key enzyme in lignin biosynthesis. By using a solution-state two-dimensional NMR approach and complementary chemical methods, we elucidated the structural details of the altered lignins enriched with unusual hydroxycinnamaldehyde-derived substructures produced by the cad2 mutant. In parallel, polysaccharide assembly and the molecular mobility of lignocellulose were investigated by solid-state 13C MAS NMR, nuclear magnetic relaxation, X-ray diffraction, and Simon’s staining analyses. Possible links between CAD-associated lignin modifications (in terms of total content and chemical structures) and changes to the lignocellulose supramolecular structure are discussed in the context of the improved biomass saccharification efficiency of the cad2 rice mutant.

Lignin is a phenylpropanoid polymer produced in the secondary cell walls of vascular plants. Although most eudicot and gymnosperm species generate lignins solely via polymerization of p-hydroxycinnamyl alcohols (monolignols), grasses additionally use a flavone, tricin, as a natural lignin monomer to generate tricin-incorporated lignin polymers in cell walls. We previously found that disruption of a rice 5-HYDROXYCONIFERALDEHYDE O-METHYLTRANSFERASE (OsCAldOMT1) reduced extractable tricin-type metabolites in rice vegetative tissues. This same enzyme has also been implicated in the biosynthesis of sinapyl alcohol, a monolignol that constitutes syringyl lignin polymer units. Here, we further demonstrate through in-depth cell wall structural analyses that OsCAldOMT1-deficient rice plants produce altered lignins largely depleted in both syringyl and tricin units. We also show that recombinant OsCAldOMT1 displayed comparable substrate specificities towards both 5-hydroxyconiferaldehyde and selgin intermediates in the monolignol and tricin biosynthetic pathways, respectively. These data establish OsCAldOMT1 as a bifunctional O-methyltransferase predominantly involved in the two parallel metabolic pathways both dedicated to the biosynthesis of tricin-lignins in rice cell walls. Given that cell wall digestibility was greatly enhanced in the OsCAldOMT1-deficient rice plants, genetic manipulation of CAldOMTs conserved in grasses may serve as a potent strategy to improve biorefinery applications of grass biomass.

The aromatic composition of lignins is considered an important trait that affects the physico-chemical properties of lignocellulosic biomass. However, our knowledge of the relationship between lignin structure and biomass utilization properties remains limited, especially in monocotyledonous grass species, despite their potential as biomass feedstocks. In this study, we used recently produced rice transgenic lines with distinct lignin monomer compositions, i.e., guaiacyl (G)/syringyl (S)/p-hydroxyphenyl (H) aromatic unit ratios, to study the impact of lignin composition on the chemical reactivity, enzymatic saccharification efficiency and calorific value of rice lignocellulose. The H-lignin-enriched rice transgenic line showed significantly enhanced biomass saccharification efficiency after alkali and acid pretreatments and even without any pretreatment, whereas the S-lignin-enriched rice transgenic line displayed enhanced saccharification efficiency after liquid hot water pretreatment. While we detected no significant differences in biomass heating values between the transgenic rice materials tested, analysis of synthetic lignins comprising only G, S or H units suggested that increased ratios of G or H units could increase the heating value of lignin-based solid biofuels.

Lignin, a ubiquitous phenylpropanoid polymer in vascular plant cell walls, is derived primarily from oxidative couplings of monolignols (p-hydroxycinnamyl alcohols). It was discovered recently that a wide range of grasses, including cereals, utilize a member of the flavonoids, tricin (3′, 5′-dimethoxyflavone), as a natural comonomer with monolignols for cell wall lignification. Previously, we established that cytochrome P450 93G1 is a flavone synthase II (OsFNSII) indispensable for the biosynthesis of soluble tricin-derived metabolites in rice (Oryza sativa). Here, our tricin-deficient fnsII mutant was analyzed further with an emphasis on its cell wall structure and properties. The mutant is similar in growth to wild-type control plants with normal vascular morphology. Chemical and nuclear magnetic resonance structural analyses demonstrated that the mutant lignin is completely devoid of tricin, indicating that FNSII activity is essential for the deposition of tricin-bound lignin in rice cell walls. The mutant also showed substantially reduced lignin content with decreased syringyl/guaiacyl lignin unit composition. Interestingly, the loss of tricin in the mutant lignin appears to be partially compensated by incorporating naringenin, which is a preferred substrate of OsFNSII. The fnsII mutant was further revealed to have enhanced enzymatic saccharification efficiency, suggesting that the cell wall recalcitrance of grass biomass may be reduced through the manipulation of the flavonoid monomer supply for lignification.