OsCAldOMT1 is a bifunctional O-methyltransferase involved in the biosynthesis of tricin-lignins in rice cell walls

Pui Ying Lam,Masaomi Yamamura,Clive Lo,Shiro Suzuki,Toshiaki Umezawa,Naoyuki Matsumoto,Masahiro Sakamoto,John Ralph,Wu Lan,Yuki Tobimatsu,Yuri Takeda

doi:10.1038/s41598-019-47957-0

Abstract

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.

Highlights

Grasses, including cereals classified in the major monocot family Poaceae, show great potential as a source of lignocellulosic biomass, which is primarily composed of secondary cell walls produced in vascular tissues
We first performed a phylogenetic analysis to examine the phylogenetic relationship between OsCAldOMT1 and other CAldOMT proteins implicated in lignification
OsCAldOMT1 is concurrently expressed with monolignol biosynthetic genes, such as p-COUMAROYL ESTER 3-HYDROXYLASE (OsC3′H1)[47], CONIFERALDEHYDE 5-HYDROXYLASE (OsCAld5H1)[48,49], CINNAMYL ALCOHOL DEHYDROGENASE (OsCAD2)[50] and p-COUMAROYL-COA:MONOLIGNOL TRANSFERASE (OsPMT1)[51] (Fig. 1), with high expression levels in tissues in which lignification typically occurs (Supplementary Fig. S2)

Summary

Introduction

Grasses, including cereals classified in the major monocot family Poaceae, show great potential as a source of lignocellulosic biomass, which is primarily composed of secondary cell walls produced in vascular tissues. As a natural lignin monomer generated outside the monolignol biosynthetic pathway (Fig. 1), undergoes dehydrogenative co-polymerization with monolignols exclusively via 4′–O–β-type radical coupling upon cell wall lignification in grasses, as in the way lignification takes place solely with monolignols in typical gymnosperms and eudicots[13]. We www.nature.com/scientificreports recently identified some flavone biosynthetic enzymes responsible for the formation of tricin-lignins in rice cell walls by thorough characterization of rice mutant lines; rice mutants deficient in a rice FLAVONE SYNTHASE II (OsFNSII or CYP93G1)[18] and a bifunctional APIGENIN 3′-HYDROXYLASE/CHRYSOERIOL 5′-HYDROXYLASE (OsA3′H/C5′H or CYP75B4)[19] (Fig. 1) produced altered lignins completely devoid of tricin units and partially incorporating naringenin and apigenin, respectively, as a tricin surrogate, demonstrating predominant roles of these enzyme genes in tricin-lignin formation in rice cell walls Both FNSII and A3′H/C5′H sequences appear to be highly conserved among grasses and likely function in the synthesis of tricin-lignins commonly present in grass cell walls[18,19]

Methods

Results

Conclusion