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.
Proanthocyanidins (PAs) are plant natural products important for agriculture and human health. They are polymers of flavan-3-ol subunits, commonly (−)-epicatechin and/or (+)-catechin, but the source of the in planta extension unit that comprises the bulk of the polymer remains unclear, as does how PA composition is determined in different plant species. Anthocyanidin reductase (ANR) can generate 2,3-cis-epicatechin as a PA starter unit from cyanidin, which itself arises from 2,3-trans-leucocyanidin, but ANR proteins from different species produce mixtures of flavan-3-ols with different stereochemistries in vitro. Genetic and biochemical analyses here show that ANR has dual activity and is involved not only in the production of (−)-epicatechin starter units but also in the formation of 2,3-cis-leucocyanidin to serve as (−)-epicatechin extension units. Differences in the product specificities of ANRs account for the presence/absence of PA polymerization and the compositions of PAs across plant species.
LACCASE8 from the model system Cleome hassleriana possesses the unusual property of oxidizing caffeyl alcohol but not coniferyl alcohol and plays a critical role in initiating C-lignin polymerization.
The factors controlling lignin composition remain unclear. Catechyl (C)–lignin is a homopolymer of caffeyl alcohol with unique properties as a biomaterial and precursor of industrial chemicals. The lignin synthesized in the seed coat of
Cleome hassleriana
switches from guaiacyl (G)– to C-lignin at around 12 to 14 days after pollination (DAP), associated with a rerouting of the monolignol pathway. Lack of synthesis of caffeyl alcohol limits C-lignin formation before around 12 DAP, but coniferyl alcohol is still synthesized and highly accumulated after 14 DAP. We propose a model in which, during C-lignin biosynthesis, caffeyl alcohol noncompetitively inhibits oxidation of coniferyl alcohol by cell wall laccases, a process that might limit movement of coniferyl alcohol to the apoplast. Developmental changes in both substrate availability and laccase specificity together account for the metabolic fates of G- and C-monolignols in the
Cleome
seed coat.
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