RESUMEN
The Poaceae family of plants provides cereal crops that are critical for human and animal nutrition, and also, they are an important source of biomass. Interacting plant cell wall components give rise to recalcitrance to digestion; thus, understanding the wall molecular architecture is important to improve biomass properties. Xylan is the main hemicellulose in grass cell walls. Recently, we reported structural variation in grass xylans, suggesting functional specialisation and distinct interactions with cellulose and lignin. Here, we investigated the functions of these xylans by perturbing the biosynthesis of specific xylan types. We generated CRISPR/Cas9 knockout mutants in Brachypodium distachyon XAX1 and GUX2 genes involved in xylan substitution. Using carbohydrate gel electrophoresis, we identified biochemical changes in different xylan types. Saccharification, cryo-SEM, subcritical water extraction and ssNMR were used to study wall architecture. BdXAX1A and BdGUX2 enzymes modify different types of grass xylan. Brachypodium mutant walls are likely more porous, suggesting the xylan substitutions directed by both BdXAX1A and GUX2 enzymes influence xylan-xylan and/or xylan-lignin interactions. Since xylan substitutions influence wall architecture and digestibility, our findings open new avenues to improve cereals for food and to use grass biomass for feed and the production of bioenergy and biomaterials.
Asunto(s)
Brachypodium , Xilanos , Animales , Humanos , Xilanos/metabolismo , Lignina/metabolismo , Brachypodium/metabolismo , Pared Celular/metabolismoRESUMEN
Xylan is the most abundant non-cellulosic polysaccharide in grass cell walls, and it has important structural roles. The name glucuronoarabinoxylan (GAX) is used to describe this variable hemicellulose. It has a linear backbone of ß-1,4-xylose (Xyl) residues that may be substituted with α-1,2-linked (4-O-methyl)-glucuronic acid (GlcA), α-1,3-linked arabinofuranose (Araf), and sometimes acetylation at the O-2 and/or O-3 positions. The role of these substitutions remains unclear, although there is increasing evidence that they affect the way xylan interacts with other cell wall components, particularly cellulose and lignin. Here, we used substitution-dependent endo-xylanase enzymes to investigate the variability of xylan substitution in grass culm cell walls. We show that there are at least three different types of xylan: (i) an arabinoxylan with evenly distributed Araf substitutions without GlcA (AXe); (ii) a glucuronoarabinoxylan with clustered GlcA modifications (GAXc); and (iii) a highly substituted glucuronoarabinoxylan (hsGAX). Immunolocalization of AXe and GAXc in Brachypodium distachyon culms revealed that these xylan types are not restricted to a few cell types but are instead widely detected in Brachypodium cell walls. We hypothesize that there are functionally specialized xylan types within the grass cell wall. The even substitutions of AXe may permit folding and binding on the surface of cellulose fibrils, whereas the more complex substitutions of the other xylans may support a role in the matrix and interaction with other cell wall components.
Asunto(s)
Celulosa , Xilanos , Xilanos/metabolismo , Celulosa/metabolismo , Lignina/metabolismo , Ácido Glucurónico/metabolismo , Xilosa/metabolismo , Pared Celular/metabolismoRESUMEN
Cell walls are comprised of networks of entangled polymers that differ considerably between species, tissues and developmental stages. The cell walls of grasses, a family that encompasses major crops, contain specific polysaccharide structures such as xylans substituted with feruloylated arabinose residues. Ferulic acid is involved in the grass cell wall assembly by mediating linkages between xylan chains and between xylans and lignins. Ferulic acid contributes to the physical properties of cell walls, it is a hindrance to cell wall degradability (thus biomass conversion and silage digestibility) and may contribute to pest resistance. Many steps leading to the formation of grass xylans and their cross-linkages remain elusive. One explanation might originate from the fact that many studies were performed on lignified stem tissues. Pathways leading to lignins and feruloylated xylans share several steps, and lignin may impede the release and thus the quantification of ferulic acid. To overcome these difficulties, we used the pericarp of the maize B73 line as a model to study feruloylated xylan synthesis and crosslinking. Using Fourier-transform infra-red spectroscopy and biochemical analyses, we show that this tissue has a low lignin content and is composed of approximately 50% heteroxylans and approximately 5% ferulic acid. Our study shows that, to date, maize pericarp contains the highest level of ferulic acid reported in plant tissue. The detection of feruloylated xylans with a polyclonal antibody shows that the occurrence of these polysaccharides is developmentally regulated in maize grain. We used the genomic tools publicly available for the B73 line to study the expression of genes within families involved or suggested to be involved in the phenylpropanoid pathway, xylan formation, feruloylation and their oxidative crosslinking. Our analysis supports the hypothesis that the feruloylated moiety of xylans originated from feruloylCoA and is transferred by a member of the BAHD acyltransferase family. We propose candidate genes for functional characterization that could subsequently be targeted for grass crop breeding.