Monolignol ferulate conjugates are naturally incorporated into plant lignins

Abstract

Angiosperms represent most of the terrestrial plants and are the primary research focus for the conversion of biomass to liquid fuels and coproducts. Lignin limits our access to fibers and represents a large fraction of the chemical energy stored in plant cell walls. Recently, the incorporation of monolignol ferulates into lignin polymers was accomplished via the engineering of an exotic transferase into commercially relevant poplar. We report that various angiosperm species might have convergently evolved to natively produce lignins that incorporate monolignol ferulate conjugates. We show that this activity may be accomplished by a BAHD feruloyl-coenzyme A monolignol transferase, OsFMT1 (AT5), in rice and its orthologs in other monocots.

Keywords: BAHD transferase; DFRC; GC-MS; Grasses; lignin; monocot; monolignol; phylogenetic tree; rice; transgenic.

Fig. 1. Incorporation of ML-FAs into lignin introduces chemically labile esters into the polymer backbone.

(A) FMT enzyme couples feruloyl-CoA and monolignols together to form ML-FA conjugates. The compounds are then transported to the cell wall and undergo radical coupling–based polymerization to form lignin; all the bonds that can be formed when ML-FAs are incorporated into β-ether structures in zip-lignin are shown with dashed lines. (B) Mild base (for example, 0.05 M NaOH at 30°C) cleaves the ML-FA–derived (green) ester bonds dividing the polymer into ≤(n + 1) fragments, where n is the number of ML-FA units. (C) DFRC breaks down the lignin by cleaving β-aryl ethers but leaving the esters intact. (D) Electron impact MS fragmentation pattern for coniferyl and sinapyl DHFA (G-DHFA and S-DHFA). FW, formula weight; m/z, mass/charge ratio. (E) GC-MRM-MS chromatograms of the DFRC product mix reveal the presence of the diagnostic products for ML-FA incorporation into lignin from a number of WT plants. The symbol ♦ indicates the signals corresponding to S-DHpCA, which shares an MRM transition with G-DHFA.

Fig. 2. Comparison of the DFRC-releasable ML-DHFA conjugates among plant species.

(A) A phylogenetic tree of the spermatophytes (“seed plants”), with the orders and families in which plant species were studied. (B) DFRC-released ML-DHFA conjugates; red bars indicate no evidence of ML-DHFAs. Bars indicate SEM for the summation of detected conjugates on duplicate analyses run on a single sample prepared from each plant species.

Fig. 3. Phylogenic reconstruction of BAHD acyl-CoA ATs is consistent with the convergent evolution of the two feruloyl-CoA monolignol transferases, OsAT5/FMT and AsFMT.

Maximum likelihood phylogeny of AsFMT, OsAT5, and biochemically characterized BAHD proteins (11). Branch values are based on 1000 bootstraps. Protein IDs are National Center for Biotechnology Information GenBank identifiers or genome locus identifiers.

Fig. 4. The amounts of DFRC-releasable ML-DHFA conjugates correlate with the expression of FMT genes but not with the expression of PMT.

(A) No significant change was observed between WT Brachypodium and a Bdpmt mutant with no PMT activity. Introduction of BdPMT into Arabidopsis results in detectable ML-DHpCA but no detectable ML-DHFA. (B) Rice overexpressing OsAT5 (OsFMT1), either via activation-tagging in OsAT5-D1 or via a Ubi promoter, and transgenic AsFMT poplar show an increase (five- to sevenfold) in ML-DHFAs. Bars indicate SEM of three to seven biological replicates that were measured with technical replicates for each. *P < 0.05, **0.001 < P < 0.01, and ***P < 0.001, Student’s t test.