“Exclusive” update: p-coumaroylation of lignin not restricted to commelinid monocots

Lignin is a complex aromatic polymer that evolved in early vascular plants around 450 million years ago. The incorporation of lignin in the cell wall was a major step in evolution that contributed to the dominance of the terrestrial ecosystem. Lignification provided early tracheophytes with physical rigidity to stand upright and strengthened their vascular tissues to allow efficient water transport (Renault et al., 2019) (Figure 1A).

Figure 1.

Occurrence of lignin p-coumaroylation in plants. A, Phylogenetic tree depicting bryophytes (non-vascular land plants) and tracheophytes (vascular land plants). Lignin acylation with p-coumarate, long believed to be a feature restricted to commelinid monocots, has been shown to be present in eudicots. Note: length of the nodes in phylogenetic tree are not to scale. B, Structure of monolignol p-coumarate conjugates. C, Simplified phylogenetic tree of the five selected eudicot mulberry tree (Morus) species representative of American and Asian lineages that were studied and confirmed for the presence of p-coumarate (pCA) in the lignin polymer. Using DFRC, lignin from the highlighted species was chemically characterized for abundance of monolignol p-coumarate conjugates (G-pCA and S-pCA) and the total amount of lignin-bound pCA. Error bars show SEM, n = 2 technical replicates. Figure 1C is modified from (Hellinger et al., 2022). Schematic generated using Adobe Illustrator. Legend: H, p-hydroxyphenyl; G, guaiacyl; S, syringyl lignin units; OMe, methoxy group.

Lignin is a product of the phenylpropanoid pathway and is classically described as being formed by the polymerization of three main hydroxycinnamyl alcohols (generally known as “monolignols”): p-coumaryl, coniferyl, and sinapyl alcohol. Their incorporation into the polymer results in the lignin units p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S), respectively (Vanholme et al., 2019). While they form the major components of lignin, this is a very simplistic view of the chemical nature of the polymer. Several other phenolics have been described as naturally occurring lignin monomers, including non-conventional monomers derived from the phenylpropanoid pathway and beyond (del Río et al., 2020). The occurrence of these monomers largely depends on the plant species and tissue type, and their contribution to physicochemical properties of the derived lignin polymers remains largely unknown.

Monolignol p-coumarate (pCA) conjugates (Figure 1B) behave as true lignin monomers. Cytosolic BAHD acyltransferases couple p-coumaroyl-CoA to the γ-hydroxyl of monolignols to produce monolignol-pCA conjugates inside the cell prior to their export to the cell wall for polymerization (Marita et al., 2014; Petrik et al., 2014). These conjugates are incorporated by oxidative coupling of each monomer to the phenolic end of lignin polymers (Ralph, 2010; Ralph et al., 2019; Vanholme et al., 2019; del Río et al., 2020). Once incorporated, the monolignol moiety becomes part of the lignin backbone whereas the acylated pCA appears as a terminal pendant group decorating the lignin backbone.

The general scientific consensus has been that lignin p-coumaroylation is a distinctive characteristic of commelinid monocots, including grasses, sedges, bromeliads, and palms (Karlen et al., 2018). However, the recent finding that the eudicot species kenaf (Hibiscus cannabinus) naturally contains p-coumaroylated lignin (Mottiar et al., 2022) advocates for a broader survey for lignin p-coumaroylation in other eudicot groups.

In this issue of Plant Physiology, Hellinger et al. (2022) expand upon their previous findings (Yamamoto et al., 2020) and demonstrate that the eudicot mulberry (Morus spp) produces p-coumaroylated lignin, confirming that the incorporation of monolignol-pCA esters is not restricted to commelinid monocots. In their earlier study, they observed Nuclear Magnetic Resonance (NMR) signals that could be assigned to pCA acylation of lignin in cell wall preparations of two white mulberry (M. alba) cultivars (Yamamoto et al., 2020). Here, the authors used a combination of chemical assays to elucidate the lignin chemical composition of five mulberry species, three representing Asian mulberry species (M. alba, M. nigra, and M. indica) and two representing American mulberry species (M. rubra and M. microphylla) (Figure 1C).

First, the authors tested whether pCA could be released from cell wall preparations of the different mulberry species. Alkaline treatment of extractive-free wood released similar amounts of pCA from all five species, indicating the presence of cell wall-bound pCA. Next, NMR spectroscopy, used to characterize the structure of lignin, confirmed the presence of pCA in enzymatically isolated lignin of all five mulberry samples. The three Asian mulberry species had 3% pCA (M. rubra, M. indica, and M. microphylla), whereas the American species, M. alba and M. nigra, had only 1% pCA. Higher pCA levels were associated with samples with higher content of G lignin units. These results were further confirmed by pyrolysis–gas chromatography mass spectrometry, which showed higher amounts of the diagnostic decomposition product of pCA in the Asian compared with the American mulberry species.

To unequivocally determine that mulberry plants incorporate monolignol-pCA esters into their lignin, the authors used derivatization followed by reductive cleavage (DFRC). This method cleaves lignin β-ethers but leaves γ-esters intact and, thus, is one of the most diagnostic techniques for identification of lignin-bound pCA (Regner et al., 2018). DFRC has been previously employed to authenticate lignin p-coumaroylation in a wide array of species (Karlen et al., 2018; Mottiar et al., 2022). In both extract-free wood and isolated lignin, the amount of the diagnostic monolignol dihydro-pCA diacetate products released upon DFRC followed the same trend as the results of the other assays for all five mulberry species (Figure 1C). Additionally, pCA acylated both G and S units, but the authors observed higher amounts of S-pCA than G-pCA conjugates in all five mulberry species.

In conclusion, the work of Hellinger et al. (2022) demonstrates our long-held assumption that lignin p-coumaroylation is a trait exclusive to commelinid monocots is incorrect (Figure 1A). An in-depth exploration of plant diversity in terms of lignin chemotypes will certainly expand the list of species that show pCA acylation of lignin outside of the commelinid monocots. With these discoveries, important questions arise: What is the role of pCA in eudicot lignin? What physio-chemical properties do p-coumaroylated monolignols provide to the lignin polymer and how does that contribute to lignin biological functions? Does monolignol p-coumaroylation in eudicots also involve the activity of BAHD acyltransferases to produce monolignol conjugates inside the cell prior to lignification in the wall? Exciting discoveries are on the horizon.