Lignin acylation, the decoration of hydroxyls on lignin structural units with acyl groups, is common in many plant species. Monocot lignins are decorated with
p-coumarates by the polymerization of monolignol
p-coumarate conjugates. The acyltransferase involved in the formation of these conjugates has been identified in a number of model monocot species, but the effect of monolignol
p-coumarate conjugates on lignification and plant growth and development has not yet been examined in plants that do not inherently possess
p-coumarates on their lignins. The rice (
Oryza sativa)
p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE gene was introduced into two eudicots, Arabidopsis (
Arabidopsis thaliana) and poplar (
Populus alba ×
grandidentata), and a series of analytical methods was used to show the incorporation of the ensuing monolignol
p-coumarate conjugates into the lignin of these plants. In poplar, specifically, the addition of these conjugates did not occur at the expense of the naturally incorporated monolignol
p-hydroxybenzoates. Plants expressing the
p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE transgene can therefore produce monolignol
p-coumarate conjugates essentially without competing with the formation of other acylated monolignols and without drastically impacting normal monolignol production.Lignification of plant cell walls prototypically involves the polymerization of the monolignols (
MLs),
p-coumaryl alcohol, coniferyl alcohol (
CA), and sinapyl alcohol (
SA), predominantly by stepwise radical coupling of each monomer to the phenolic end of the growing polymer (
Sarkanen and Ludwig, 1971;
Boerjan et al., 2003;
Ralph et al., 2004). The contribution of various
MLs to the lignins depends on plant species, cell type, plant tissue, and tissue age. Although the majority of the lignin polymer is derived from these three
MLs, the lignification process has a high degree of metabolic plasticity (
Boerjan et al., 2003;
Ralph et al., 2004;
Ralph, 2007;
Vanholme et al., 2012). Of particular interest are
ML conjugates in which the ester group can be acetate (
Ac;
Sarkanen et al., 1967;
Ralph, 1996;
Ralph and Lu, 1998;
Del Río et al., 2007;
del Río et al., 2008;
Martínez et al., 2008),
p-hydroxybenzoate (
pBz;
Venverloo, 1971;
Monties and Lapierre, 1981;
Landucci et al., 1992;
Tomimura, 1992a,
1992b;
Hibino et al., 1994;
Sun et al., 1999;
Kuroda et al., 2001;
Lu et al., 2004,
2015;
Morreel et al., 2004;
Rencoret et al., 2013),
p-coumarate (
pCA;
Monties and Lapierre, 1981;
Ralph et al., 1994;
Crestini and Argyropoulos, 1997;
del Río et al., 2008,
2012a,
2012b;
Withers et al., 2012;
Rencoret et al., 2013;
Petrik et al., 2014), or ferulate (
FA;
Grabber et al., 2008;
Ralph, 2010;
Wilkerson et al., 2014). In all cases, the
MLs are acylated before polymerization as proven by the presence in the lignins of unique β-β coupling products that only arise when one or both of the
MLs are acylated, preventing the formation of the typical resinols from internal trapping of the quinone methide intermediates by the γ-OH (
Lu and Ralph, 2002,
2008;
Del Río et al., 2007;
Lu et al., 2015).The BAHD acyltransferase, FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT), was recently identified in
Angelica sinensis and transformed into poplar (
Populus alba ×
grandidentata), which naturally incorporates other acylated
MLs, namely
ML-
pBz conjugates, into its lignin (
Wilkerson et al., 2014). Plants that incorporate
ML-
FAs into their lignins have the potential to be particularly important economically, because their lignin backbones are permeated with readily cleavable ester bonds, facilitating lignin breakdown and removal under alkaline pretreatment conditions. Determining the extent to which
ML-
FAs are incorporated into the lignin polymer is, however, extremely difficult because of the diversity of products generated during the polymerization events, which is described in the supplemental information in
Wilkerson et al., 2014.There is currently only one technique, derivatization followed by reductive cleavage (
DFRC), that can release diagnostic chemical marker compounds from lignins containing
ML-
FAs (
Lu and Ralph, 2014;
Wilkerson et al., 2014). The
DFRC method selectively cleaves β-ethers while leaving ester linkages intact. This technique was recently used to show that
ML-
FA conjugates are fully incorporated into the lignin of the
FMT poplar (
Wilkerson et al., 2014), but the extent of incorporation, the spatial distribution, the exact mechanism of delivery to the developing cell wall, and the efficiency of incorporation remain largely unknown.The biological role of
pCA in lignin has been highly speculative. It is hypothesized that the
pCA moieties may function as a radical sensitizer (
Takahama and Oniki, 1996,
1997;
Takahama et al., 1996;
Ralph et al., 2004;
Hatfield et al., 2008;
Ralph, 2010). Peroxidases and/or laccases readily oxidize
pCA to a radical but are poor oxidizers for
SA. Free radicals of
pCA readily undergo radical transfer to
SA, which in turn, forms a homodimer or couples to the end of a growing polymer chain. Conjugating
pCA to an
ML, like
SA, to form
SA-
pCA, the most prevalent
ML-
pCA conjugate in grasses, creates a compound with a built-in radical sensitizer that can participate in the polymerization event. The prevalence of these conjugates in potential biofuel crops and the impact that these ester-linked conjugates have on the lignin polymer during pretreatment and downstream fermentation processes have driven the search to find the genes and their enzymes responsible for acylating
MLs in monocots (
Withers et al., 2012;
Marita et al., 2014;
Petrik et al., 2014;
Wilkerson et al., 2014).In rice (
Oryza sativa), enzymes have been characterized that function specifically in the addition of
pCA onto hemicelluloses (
Bartley et al., 2013) or lignin (
Withers et al., 2012;
Petrik et al., 2014). The
p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (PMT) was identified as one of many grass-specific BAHD acyltransferases produced by rice and found to coexpress with many
ML biosynthetic enzymes (
Withers et al., 2012). The enzyme preferentially forms a γ-ester through its specificity toward
p-coumaroyl-CoA and an
ML, and has kinetic efficiency with
p-coumaryl alcohol >
SA >
CA. In most grasses, the PMT enzyme predominantly produces
SA-
pCA conjugates that are then incorporated into the lignin polymer (
Petrik et al., 2014).To test the role of PMT during cell wall lignification, genetic manipulation of
PMT genes has been performed in
Brachypodium distachyon and maize (
Zea mays), two model monocots. The suppression and overexpression of a BdPMT revealed the PMT to be involved only in the acylation of
MLs before polymerization and not in the acylation of hemicelluloses (
Petrik et al., 2014). RNA interference-mediated suppression of
BdPMT resulted in decreased incorporation of
ML-
pCA conjugates into the cell wall without adversely affecting growth, height, or digestibility of the mature plants. Even deleterious mutations in the
BdPMT gene, which resulted in a complete absence of
pCA-acylating
B. distachyon lignins, did not affect plant growth or development (
Petrik et al., 2014). The arabinose-bound
FA and
pCA levels remained virtually unchanged in the
PMT-misregulated plants, illustrating the specificity of the PMT enzyme for the
p-coumaroyl-CoA substrate and its
ML acylation. The PMT enzyme identified in maize (
pCAT = ZmPMT) also displayed the highest catalytic efficiency with
p-coumaroyl-CoA and
SA as substrates (
Marita et al., 2014). RNAi-mediated suppression of
ZmPMT also resulted in decreased production of the
ML conjugates. The effect on the lignin polymer when introducing PMT into plants that do not normally express a homologous enzyme is, however, unknown.
pCAs, because they favor radical transfer over radical coupling, are overwhelmingly seen as free-phenolic pendant entities on the lignin polymer (
Ralph et al., 1994;
Ralph, 2010). As a result, the
pCA itself can be completely quantified by simple saponification. The units to which the
pCA is attached are, like their normal
ML-derived counterparts, not fully releasable from lignin as identifiable monomers (during degradative reactions), but the
pCA’s terminal location makes
p-coumaroylated units more readily releasable and detectable than if they participated in lignification (as
FAs do). Examining the effect of PMT and its resulting conjugates on lignification in plants that do not naturally produce such conjugates will contribute to our understanding of the role of PMT in lignification in general.In this study, we aimed to assess the ability of the model eudicot plants Arabidopsis (
Arabidopsis thaliana) and poplar, neither of which naturally produces
ML-
pCA conjugates, to express a
PMT gene and incorporate these novel conjugates into their cell wall lignins. We also investigated the effect that the introduction of
PMT has on the native levels of
ML-
pBz conjugates in poplar lignin. Various analytical techniques were optimized and used to examine the cell walls of the transgenic plants for
pCA conjugates and determine whether they were specifically incorporated into the lignin polymer in the cell wall.
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