Improved analysis of arabinoxylan-bound hydroxycinnamate conjugates in grass cell walls

Abstract

Background Arabinoxylan in grass cell walls is acylated to varying extents by ferulate and p-coumarate at the 5-hydroxy position of arabinosyl residues branching off the xylan backbone. Some of these hydroxycinnamate units may then become involved in cell wall radical coupling reactions, resulting in ether and other linkages amongst themselves or to monolignols or oligolignols, thereby crosslinking arabinoxylan chains with each other and/or with lignin polymers. This crosslinking is assumed to increase the strength of the cell wall, and impedes the utilization of grass biomass in natural and industrial processes. A method for quantifying the degree of acylation in various grass tissues is, therefore, essential. We sought to reduce the incidence of hydroxycinnamate ester hydrolysis in our recently introduced method by utilizing more anhydrous conditions. Results The improved methanolysis method minimizes the undesirable ester-cleavage of arabinose from ferulate and p-coumarate esters, and from diferulate dehydrodimers, and produces more methanolysis vs. hydrolysis of xylan-arabinosides, improving the yields of the desired feruloylated and p-coumaroylated methyl arabinosides and their diferulate analogs. Free ferulate and p-coumarate produced by ester-cleavage were reduced by 78% and 68%, respectively, and 21% and 39% more feruloyl and p-coumaroyl methyl arabinosides were detected in the more anhydrous method. The new protocol resulted in an estimated 56% less combined diferulate isomers in which only one acylated arabinosyl unit remained, and 170% more combined diferulate isomers conjugated to two arabinosyl units. Conclusions Overall, the new protocol for mild acidolysis of grass cell walls is both recovering more ferulate- and p-coumarate-arabinose conjugates from the arabinoxylan and cleaving less of them down to free ferulic acid, p-coumaric acid, and dehydrodiferulates with just one arabinosyl ester. This cleaner method, especially when coupled with the orthogonal method for measuring monolignol hydroxycinnamate conjugates that have been incorporated into lignin, provides an enhanced tool to measure the extent of crosslinking in grass arabinoxylan chains, assisting in identification of useful grasses for biomass applications.

Comparison of Peanut Compounds during Roasting and the Effect of Peanut Shells

“…3-Hydroxycinnamate (m-coumarate) in unshelled peanut seeds decreased after 30 min of roasting, whereas no significant change was observed in the relative content of p-coumarate, which was reported to be linked via an ester bond to arabinoxylans (hemicellulose) in plants. 26 p-Coumarate exhibits antioxidant activity in vitro as a major hydroxycinnamic acid. 27 Choline and Its Derivatives.…”

“…Some organic acids decreased during roasting (Figure B). 3-Hydroxycinnamate ( m -coumarate) in unshelled peanut seeds decreased after 30 min of roasting, whereas no significant change was observed in the relative content of p -coumarate, which was reported to be linked via an ester bond to arabinoxylans (hemicellulose) in plants …”

Overexpression of the rice BAHD acyltransferase AT10 increases xylan-bound p-coumarate and reduces lignin in Sorghum bicolor

“…These results are in contrast with previous studies in which expression of AT10 transferases resulted in up to 60% and 90% reduction of FA-Ara in rice and maize, respectively [ 19 , 22 ], while 21–27% reductions of FA released by dilute alkaline were observed in switchgrass [ 23 ]. Again, these observations could lie in variable AT10 expression levels among engineered crops, but the impact of different native patterns of xylan acylation with p CA and FA between these plant species could be another explanation [ 7 , 8 ]. From previous studies, it remains unclear how modification of p CA transfer activity onto xylan often results in modifications of xylan feruloylation levels, and vice-versa [ 17 – 23 , 26 ].…”

“…1 ). Feruloyl-arabinose (FA-Ara) residues are typically more frequent than coumaroyl-arabinose ( p CA-Ara) residues, and substitution patterns also vary depending on grass species and plant organs [ 7 , 8 ]. These grass-specific FA and p CA esters have been implicated in GAX crosslinks that reinforce cell walls [ 9 ].…”

Transgenic rice plants expressing the α-L-arabinofuranosidase of <i>Coprinopsis cinerea</i> exhibit strong dwarfism and markedly enhanced tillering

“…The arabinoxylan in grass cell walls is acylated by hydroxycinnamates such as ferulate and p-coumarate at the 5′-hydroxy position of arabinosyl residues (Chiniquy et al 2012;Sato-Izawa et al 2020). Some of these hydroxycinnamate units subsequently undergo cell wall radical coupling reactions with other hydroxycinnamate units, monolignols, or oligolignols, thereby generating crosslinks between arabinoxylan chains and/or with lignin polymers (Eugene et al 2020). Consequently, CcAbf62A activity contributes to reducing the number of links between lignin and cell wall polysaccharides, and degrades lignin carbohydrate complex (LCC).…”