p-Coumaroylation of lignin occurs outside of commelinid monocots in the eudicot genus Morus (mulberry)

Abstract

The presence of p-coumarate (pCA) in plant cell walls is generally considered to be a trait present only in commelinid monocots. Here, we show that this long-held overgeneralizing assumption is incorrect and that mulberry trees (Morus) are eudicot plants that have lignins derived in part from monolignol pCA esters. As in commelinid monocots, the lignin-bound pCA acylates the sidechain γ-hydroxyl of both coniferyl and syringyl units. This discovery expands mulberry's potential applications to include being a source of p-coumaric acid, a supplier of nutritious berries, a forage crop, a decorative plant, and the main food source for silkworms.

Figure 1

Phylogenetic tree of the Morus genus and quantification of cell-wall-bound pCA. A, Phylogenetic tree for the genus Morus adapted from Nepal and Ferguson (2012). B, Schematic for the saponification of γ-pCA esters, using the predominant β-aryl ether as an example lignin subunit. C, The amount (mg/100 g) of p-coumaric acid released from cell walls using 2 M NaOH at 90°C for 90 min. Error bars indicate Sem for n = 3 technical replicates.

Figure 2

Aromatic regions of 2D ¹H–¹³C correlation (HSQC) spectra. Spectra were obtained from Morus ELs isolated by cellulase treatment of ball-milled cell walls, solubilized in DMSO-d₆/pyridine-d₅ (4:1): A, M. microphylla; B, M. rubra; C, M. indica; D, M. nigra; and E, M. alba. F, Structures of lignin subunits, phenolate esters, and amino acids that are known to be present in plant cell walls. The substructure units and labels are color-coded to match their assignments in spectra A–E. The relative abundances were calculated on a ½S_2/6 + G₂ = 100% basis using ½S_2/6, G₂, and ½pCA_2/6.

Figure 3

Chemical characterization by pyrolysis–GC–MS and DFRC confirming the presence of pCA. A, GC–MS pyrograms of Morus lignins pyrolyzed at 500°C in the presence of TMAH. Labeled peaks correspond to compounds identified and reported in Table 2. B, GC–MRM–MS chromatograms of the released products diagnostic for lignin-bound pCA (quantified as ML-DHpCA) with MRM transitions: G-DHpCA 370 > 131 m/z and S-DHpCA 400 > 161 m/z, and lignin-bound FA (quantified as DHFA) with MRM-transition G-DHFA 358 > 163 m/z. C, Schematic for the DFRC lignin depolymerization process. D, ML pCA abundance as determined by DFRC (detected as G-DHpCA and S-DHpCA then converted to G-pCA and S-pCA), and the calculated amount of lignin-bound pCA as determined from the DFRC product mixture. Error bars indicate Sem for n = 2 technical replicates.