Abstract
Lignin biosynthesis is evolutionarily conserved among higher plants and features a critical 3-hydroxylation reaction involving phenolic esters. However, increasing evidence questions the involvement of a single pathway to lignin formation in vascular plants. Here we describe an enzyme catalyzing the direct 3-hydroxylation of 4-coumarate to caffeate in lignin biosynthesis as a bifunctional peroxidase that oxidizes both ascorbate and 4-coumarate at comparable rates. A combination of biochemical and genetic evidence in the model plants Brachypodium distachyon and Arabidopsis thaliana supports a role for this coumarate 3-hydroxylase (C3H) in the early steps of lignin biosynthesis. The subsequent efficient O-methylation of caffeate to ferulate in grasses is substantiated by in vivo biochemical assays. Our results identify C3H as the only non-membrane bound hydroxylase in the lignin pathway and revise the currently accepted models of lignin biosynthesis, suggesting new gene targets to improve forage and bioenergy crops.
Highlights
Lignin biosynthesis is evolutionarily conserved among higher plants and features a critical 3hydroxylation reaction involving phenolic esters
A previously described protocol[13] with minor modifications (Supplementary Fig. 1) was used to assay coumarate 3hydroxylase (C3H) activity in crude protein extracts prepared from tissues of several plant species, and the highest activities were found in maize and Brachypodium (Fig. 2a), both of which lack caffeoyl shikimate esterase (CSE) orthologs[7]
C3H is the only reported hydroxylase involved in phenylpropanoid biosynthesis that is not a membrane-bound cytochrome P450
Summary
Lignin biosynthesis is evolutionarily conserved among higher plants and features a critical 3hydroxylation reaction involving phenolic esters. We describe an enzyme catalyzing the direct 3-hydroxylation of 4-coumarate to caffeate in lignin biosynthesis as a bifunctional peroxidase that oxidizes both ascorbate and 4-coumarate at comparable rates. A combination of biochemical and genetic evidence in the model plants Brachypodium distachyon and Arabidopsis thaliana supports a role for this coumarate 3hydroxylase (C3H) in the early steps of lignin biosynthesis. Our results identify C3H as the only non-membrane bound hydroxylase in the lignin pathway and revise the currently accepted models of lignin biosynthesis, suggesting new gene targets to improve forage and bioenergy crops. The facts that some grass species lack orthologs of caffeoyl shikimate esterase (CSE)[6,7] and that reduced expression of some ester pathway enzymes has less than expected to no phenotypic effect in grasses[5,8], along with paradoxical results of metabolomics and labeling studies in Arabidopsis[9], support the existence of an alternative pathway to the currently accepted route involving phenolic esters. We identify and characterize a bifunctional cytosolic ascorbate peroxidase as C3H, the missing link in the conventional phenylpropanoid pathway involving free phenolic acids
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