Functional testing of a PF02458 homologue of putative rice arabinoxylan feruloyl transferase genes in Brachypodium distachyon

Abstract

We show that changing the expression of a putative feruloyl transferase gene belonging to the BAHD acyl-transferase family alters the levels of cell wall esterified ferulates and diferulates in Brachypodium distachyon cell walls. While the potential of grass cell walls for biofuel production has been realized, the technology for lignocellulosic biomass conversion for the production of ethanol is still inefficient because of structural mechanisms that plants have evolved to make the cell wall recalcitrant to enzymatic attack. One of these mechanisms in grasses involves the esterification of arabinoxylans in the cell wall with ferulic acid via an ester linkage to arabinose side chains on xylans. These ferulates undergo oxidative coupling reactions to form ferulate dimers, thus crosslinking polysaccharides. Arabinoxylan feruloylation is an important factor that determines cell wall recalcitrance because it directly cross-links xylans and because ferulates act as nucleating sites for the formation of lignin and for the linkage of lignin to the xylan/cellulose network. Here we report on the effects of changing the expression of Bradi2g43520 (BdAT1), a homologue of the rice feruloyl transferase gene Os01g42880 belonging to the Pfam PF02458 family, in Brachypodium distachyon. Down regulation in several independent RNAi::BdAT1 lines, resulted in up to a 35 % reduction of ferulate levels in both leaves and stems compared to control plants, over 2-3 generations of selfing. In contrast, overexpression of putative BdAT1 resulted in an increase of up to 58 and 47 % of ferulate levels in leaves and stems, respectively, compared to control plants and analyzed over 2-3 generations of selfing. These findings suggest that Bradi2g43520 may be a good candidate for feruloylation of AX in Brachypodium.

Response of cell-wall composition and RNA-seq transcriptome to methyl-jasmonate in Brachypodium distachyon callus

“…GT61 family clades A (yellow) B (green) C (grey) are shown (as defined Anders et al (2012)). Named genes on tree have evidence on their role from [1] Karlen et al (2016) [2] Withers et al (2012) [3] Petrik et al (2014) [4] Sibout et al (2016) [5] Buanafina et al (2016) [6] de Souza et al (2018) [7] Bartley et al (2013) [8] Voiniciuc et al (2015) [9] Anders et al (2012) [10] Chiniquy et al (2012) …”

“…However, there is strong evidence that one of the genes in this clade, OsAT10 , is responsible for acylation of AX with p CA in rice as specific up-regulation of this gene increased Ara f - p CA fivefold (Bartley et al 2013). RNAi suppression of other genes in this clade resulted in decreased cell-wall FA (nearly all of which is likely to be AX-FA) (Piston et al 2010; Buanafina et al 2016) with the strongest effect resulting from suppression of a gene we call SvBAHD01 in Setaria viridis (de Souza et al 2018). Genes within glycosyl transferase family 61 (GT61) are responsible for the addition of three-linked Araf on AX (Anders et al 2012) and a knock-out mutant for a GT61 gene xax1 had severely decreased wall-bound FA and p CA (Chiniquy et al 2012).…”

“…This model is not universally accepted; Chiniquy et al (2012) interpreted their data differently and the existence of the putative UDP-Araf-HCA intermediate has not been reported. Nevertheless, there is extensive circumstantial evidence for the involvement of these BAHD and GT61 genes in the addition of HCA to AX (Mitchell et al 2007; Bartley et al 2013; Buanafina et al 2016; de Souza et al 2018). …”

Proteomics Coupled with Metabolite and Cell Wall Profiling Reveal Metabolic Processes of a Developing Rice Stem Internode

“…Both p CA and FA increased from young to old segments but showed different patterns of change, with p CA amounts continuing to increase through development but FA remaining constant in older segments. When integrated with gene expression or proteomics data of these segments, the difference in p CA and FA accumulation may facilitate identification of the transferases and other proteins that function in their incorporation, a topic of considerable recent interest (Molinari et al, 2013; Buanafina et al, 2016; Chateigner-Boutin et al, 2016; Lin et al, 2016). …”

“…Enzymes in the phenylpropanoid pathway synthesize lignin precursors and hydroxycinnamic acids (HCAs). The latter are incorporated into lignin or polysaccharides by so-called BAHD acyl-CoA acyltransferases (ATs) (Withers et al, 2012; Bartley et al, 2013; Molinari et al, 2013; Petrik et al, 2014; Buanafina et al, 2016; Karlen et al, 2016; Lin et al, 2016; Sibout et al, 2016). To date, all cell wall-precursor modifying ATs belong to a subclade of BAHDs dubbed the “Mitchell clade” (Mitchell et al, 2007; Bartley et al, 2013; Karlen et al, 2016).…”