Disrupting Flavone Synthase II Alters Lignin and Improves Biomass Digestibility

Abstract

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 (39,59-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.

Downregulation of p‐COUMAROYL ESTER 3‐HYDROXYLASE in rice leads to altered cell wall structures and improves biomass saccharification

“…We also analyzed the distribution of cellulosic and hemicellulosic glycans using a two‐step acid‐catalyzed hydrolysis method (Chen et al ., ; Lam et al ., ). It was confirmed that, as is typical in other grasses, both OsC3 ′ H1 ‐KD and wild‐type cell walls were composed mainly of crystalline cellulose and amorphous arabinoxylans.…”

“…Enhanced saccharification in lignin‐depleted cell walls has been well documented in the literature. With rice, our group has reported reduced biomass recalcitrance for CAD ‐ (Koshiba et al ., ), CAldOMT ‐ (Koshiba et al ., ) and FNSII ‐downregulated (Lam et al ., ) rice plants which all displayed decreased lignin content.…”

“…OsC3′H1‐ KD and OsC3′H1‐ KO rice plants were generated by RNAi (Hattori et al ., ) and CRISPR/Cas9 (Mikami et al ., ) approaches, respectively, and grown in a greenhouse or in a growth chamber, as described previously (Koshiba et al ., ; Lam et al ., ) and as further described in Materials and Methods S1. Oligonucleotides used for vector construction, genotyping, off‐target analysis, and gene expression assay are listed in Table S1.…”

Apigenin produced by maize flavone synthase I and II protects plants against UV‐B‐induced damage

“…Consequently, as we previously proposed for Zm FNSI, it is possible that the regulation of both anthocyanin and flavone biosynthesis by the same regulators might be necessary to achieve an accurate balance of these compounds for pigmentation as well as to stabilize anthocyanins. On the other hand, it was recently demonstrated that tricin, a flavone that functions as a nucleation site for lignification in monocot species is not accumulated in fnsII rice mutants resulting in reduced lignin content (Lam et al, ; Lan et al, ). Thus, the possibility remains that either or both maize FNS enzymes can be involved in lignification, supplying apigenin to be further channeled to tricin biosynthesis.…”

Lignin characterization of rice CONIFERALDEHYDE 5‐HYDROXYLASE loss‐of‐function mutants generated with the CRISPR/Cas9 system

“…(). Regenerated plants and isolated OsCAld5H1 ‐KO mutant lines were grown to maturity in potting soil under growth chamber conditions as described previously (Lam et al ., ). OsCAld5H1 ‐KD and OsCAld5H1 ‐OX plants (T 3 generation) were generated and grown to maturity under greenhouse conditions as described previously (Takeda et al ., ).…”