Feruloylation and structure of arabinoxylan in wheat endosperm cell walls from RNAi lines with suppression of genes responsible for backbone synthesis and decoration

Abstract

SummaryArabinoxylan (AX) is the major component of the cell walls of wheat grain (70% in starchy endosperm), is an important determinant of end‐use qualities affecting food processing, use for animal feed and distilling and is a major source of dietary fibre in the human diet. AX is a heterogeneous polysaccharide composed of fractions which can be sequentially extracted by water (WE‐AX), then xylanase action (XE‐AX) leaving an unextractable (XU‐AX) fraction. We determined arabinosylation and feruloylation of AX in these fractions in both wild‐type wheat and RNAi lines with decreased AX content (TaGT43_2 RNAi, TaGT47_2 RNAi) or decreased arabinose 3‐linked to mono‐substituted xylose (TaXAT1 RNAi). We show that these fractions are characterized by the degree of feruloylation of AX, <5, 5–7 and 13–19 mg bound ferulate (g−1 AX), and their content of diferulates (diFA), <0.3, 1–1.7 and 4–5 mg (g−1 AX), for the WE, XE and XU fractions, respectively, in all RNAi lines and their control lines. The amount of AX and its degree of arabinosylation and feruloylation were less affected by RNAi transgenes in the XE‐AX fraction than in the WE‐AX fraction and largely unaffected in the XU‐AX fraction. As the majority of diFA is associated with the XU‐AX fraction, there was only a small effect (TaGT43_2 RNAi, TaGT47_2 RNAi) or no effect (TaXAT1 RNAi) on total diFA content. Our results are compatible with a model where, to maintain cell wall function, diFA is maintained at stable levels when other AX properties are altered.

Suppression of a single BAHD gene in Setaria viridis causes large, stable decreases in cell wall feruloylation and increases biomass digestibility

“…Cell‐wall‐bound hydroxycinnamate (HCA) content was determined essentially as described (Freeman et al ., ) in labs at Embrapa Agroenergy (Supporting Information Table S1; Fig. S1C) and Rothamsted (Figs , S1D; Table S2) and with some variations in protocol as described in Methods S1.…”

“…To determine total ester‐linked HCA following mild acidolysis (Table S4), aliquots of supernatant and the pellet were saponified, dried under vacuum, resuspended in 250 μl 50% methanol : 0.1% formic acid, and HCA content was quantified as in Freeman et al . () but using the HPLC method above used to separate HCA conjugates.…”

Covalent interactions between lignin and hemicelluloses in plant secondary cell walls

“…Family 61 glycosyl transferase enzymes (GT61) add arabinosyl substitutions to the xylan backbone [34]. RNAi knockdown of the GT61 XAT1 reduced the amount of ferulate in soluble arabinoxylan of wheat endosperm [35]. In rice, a GT61 knockout mutant, xax1, was found to have 60% reduced feruloylation and 50% reduced pcoumaroylation of xylan [36].…”

The contribution of fiber components to water absorption of wheat grown in the UK

“…TOT-AX and β-glucan were also determined as arabinoxylan oligosaccharides (AXOS) and gluco-oligosaccharides (GOS), respectively, by enzyme fingerprinting as described by Freeman et al (2017) with minor modifications. Flours were digested with recombinant endo-1,4-xylanase (PRO-E0062) and lichenase (a glucan-hydrolase) (PRO-E0017) (Prozomix), the oligosaccharides separated by HPAEC, and the peak areas expressed relative to a Melibiose internal standard.…”