p‐Coumaroyl‐CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon

Abstract

Grass lignins contain substantial amounts of p-coumarate (pCA) that acylate the side-chains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no (<0.5%) residual pCA on lignin, and BdPMT RNAi plants had levels as low as 10% of wild-type, whereas the amounts of pCA acylating arabinosyl units on arabinoxylans in these PMT mutant plants remained unchanged. pCA acylation of lignin from BdPMT-overexpressing plants was found to be more than three-fold higher than that of wild-type, but again the level on arabinosyl units remained unchanged. Taken together, these data are consistent with a defined role for grass PMT genes in encoding BAHD (BEAT, AHCT, HCBT, and DAT) acyltransferases that specifically acylate monolignols with pCA and produce monolignol p-coumarate conjugates that are used for lignification in planta.

Naturally p-Hydroxybenzoylated Lignins in Palms

“…), poplars and aspen (Populus spp. ), and palms (family Arecaceae) that have p-hydroxybenzoylated lignins, and that identification of the genes involved will allow p-hydroxybenzoate levels on lignin to be manipulated as recently demonstrated for p-coumarates [23]. To date, however, the in planta pathway to p-hydroxybenzoate (or its CoA derivative) remains ambiguous.…”

“…4a. This is possible as DFRC advantageously cleaves lignin β-ethers but leaves esters fully intact [62,51,18,23,27]. Unfortunately, the acetylated coniferyl and sinapyl phydroxybenzoate products anticipated from this method do not survive GC analysis conditions, so the products were analyzed by HPLC-ESI-MS, Fig.…”

“…The research presented in this paper seeks to answer several key questions regarding the intricacies of acylation of lignins by p-hydroxybenzoate, specifically: whether p-hydroxybenzoates exist as free-phenolic appendages on lignins, akin to the p-coumarates; whether they, like p-coumarates [51,52,23,49] and acetates [18] in most plants, primarily acylate S units; whether the various lignin substructures carry these units and thus implicate the incorporation of preformed monolignol γ-p-hydroxybenzoate conjugates into the lignification process; and whether diagnostic β-β-coupled units can be found to provide proof that lignification utilizes preformed monolignol p-hydroxybenzoate conjugates, thus, also establishing that monolignol p-hydroxybenzoate conjugates are lignin precursors. Although a survey across all palms is beyond the scope of this work, a sago palm lignin previously examined by analytical pyrolysis [35] and prior studies on coconut (Cocos nucifera) coir fibers [36] are used to suggest a degree of generality in palms.…”

“…Rather than suspecting that the lignin side-chains were acylated after the polymer had already formed, p-hydroxybenzoyl monolignol conjugates were proposed as the precursors to these structures in early research [47]; monolignol acylation was further conjectured [25] and eventually proven by the observation of novel β-β-coupling products in lignins that could only have arisen from pre-acylated monolignols [29,14,48,32]. Recent studies have delivered the transferases p-coumaroyl-CoA:monolignol transferase (PMT) in grasses responsible for lignin p-coumarate esters via p-coumaroylation of monolignols [22,23,49,50] and now an exotic feruloyl-CoA:monolignol transferase (FMT) that feruloylates monolignols prior to lignification [27]; the putative p-hydroxybenzoyl-CoA and acetyl-CoA monolignol transferases have not yet been identified.…”

“…Angiosperm trees have long been thought to have low-level lignin acetylation [15], whereas kenaf bast fiber lignins and lignins in various other plants are extensively acetylated [16][17][18][19][20][21]. Grasses produce the enzyme p-coumaroyl-CoA:monolignol transferase (PMT) [22,23], which produces γ-p-coumaroylated monolignols that are incorporated into the growing lignin polymer, particularly at advanced maturity [24][25][26]. Recently, monolignol ferulate conjugates were introduced into poplar trees through the introduction of a gene expressing feruloylCoA:monolignol transferase (FMT) [27], further demonstrating the plasticity of lignification and the role of monolignol conjugates.…”