Research ArticlePLANT SCIENCE

Monolignol ferulate conjugates are naturally incorporated into plant lignins

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Science Advances  14 Oct 2016:
Vol. 2, no. 10, e1600393
DOI: 10.1126/sciadv.1600393
  • 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.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/10/e1600393/DC1

    Supplementary Text

    table S1. Chromatography program and MRM parameters for GC-MS/MS characterization of DFRC product mix.

    table S2. Experimental results from the analysis of extract-free whole-cell-wall and enzyme lignin samples of selected eudicots.

    table S3. Experimental results from the analysis of extract-free whole-cell-wall samples of gymnosperms, magnoliids, and noncommelinid (early) monocots.

    table S4. Experimental results from the analysis of extract-free whole-cell-wall samples of commelinid monocots.

    table S5. Experimental results from the analysis of extract-free whole-cell-wall samples of eudicots.

    table S6. Experimental results from the analysis of extract-free whole-cell-wall samples of plants generated in the enzyme expression study.

    table S7. Primers used in this study.

    fig. S1. The monolignol biosynthetic pathway indicating the formation of ML-FAs.

    fig. S2. The phylogenetic reconstruction of BAHD acyl-CoA ATs is consistent with the convergent evolution of the two feruloyl-CoA monolignol transferases, OsAT5/FMT and AsFMT.

    fig. S3. Genomic position and gene expression data for the AT5-D1 rice activation-tagged line.

    fig. S4. OsAt5 expression data and DFRC-released ML-DHpCA conjugates from OsAT5 rice lines.

    fig. S5. The cell wall compositional differences in OsAT5-D1 straw are predominantly due to the (50 mM TFA, 100°C) insoluble fraction.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Text
    • table S1. Chromatography program and MRM parameters for GC-MS/MS characterization of DFRC product mix.
    • table S2. Experimental results from the analysis of extract-free whole-cell-wall and enzyme lignin samples of selected eudicots.
    • table S3. Experimental results from the analysis of extract-free whole-cell-wall samples of gymnosperms, magnoliids, and noncommelinid (early) monocots.
    • table S4. Experimental results from the analysis of extract-free whole-cell-wall samples of commelinid monocots.
    • table S5. Experimental results from the analysis of extract-free whole-cell-wall samples of eudicots.
    • table S6. Experimental results from the analysis of extract-free whole-cell-wall samples of plants generated in the enzyme expression study.
    • table S7. Primers used in this study.
    • fig. S1. The monolignol biosynthetic pathway indicating the formation of ML-FAs.
    • fig. S2. The phylogenetic reconstruction of BAHD acyl-CoA ATs is consistent with the convergent evolution of the two feruloyl-CoA monolignol transferases, OsAT5/FMT and AsFMT.
    • fig. S3. Genomic position and gene expression data for the AT5-D1 rice activation-tagged line.
    • fig. S4. OsAt5 expression data and DFRC-released ML-DHpCA conjugates from OsAT5 rice lines.
    • fig. S5. The cell wall compositional differences in OsAT5-D1 straw are predominantly due to the (50 mM TFA, 100°C) insoluble fraction.

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