Evidence for intra- and extra-protoplasmic feruloylation and cross-linking in wheat seedling roots

Abstract

The sub-cellular feruloylation and oxidative coupling sites of cell wall polysaccharides were investigated in planta by monitoring the kinetics of appearance of arabinosyl- and feruloyl-radiolabelled polysaccharides in the protoplasmic compartment and their secretion in the wall either in the presence or absence of brefeldin A (BFA). By using root apical segments excised from wheat seedlings (Triticum durum Desf.), incubated with trans-[U-(14)C]cinnamic acid, we demonstrated that [14C]ferulate, likely [14C]diferulate, as well as trimers and larger products of ferulate are incorporated into the protoplasmic polysaccharides very rapidly within 1-3 min of [14C]cinnamate feeding. This agrees with the assumption that (glucurono)arabinoxylans [(G)AX] feruloylation and oxidative coupling occur intracellularly, likely in the Golgi apparatus. Simultaneously, polymer bound radioactive hydroxycinnamic acids appeared to be incorporated into the cell wall of root apical segments as early as 2 min after trans-[U-(14)C]cinnamic acid feeding. On the contrary, starting from L-[1-(14)C]arabinose as tracer, the secretion of the pentose-containing polymers into the wall was between 5 to 10 min. These results indicated that (G)AX feruloylation and oxidative coupling occur both intra-protoplasmically and in muro. The occurrence of in muro feruloylation and oxidative coupling was confirmed by the use of BFA a well known inhibitor of secretion. The drug caused a strong inhibition of the synthesis and secretion into the wall of the 14C-pentosyl-labelled polymers as well as of 14C-feruloyl-polymers. In spite of this, the total amount of 14C-feruloyl-polymers incorporated into the wall was only slightly affected by BFA. This indicates the existence of a mechanism involved into secretion of the activated hydroxycinnamoyl precursors to the wall, alternative to that involved in polysaccharide secretion.

Cell wall deposition and metabolism in wheat grain

“…The site of xylan feruloylation was investigated in wheat seedlings by incubation of root segments with radiolabelled ferulate. This study provided evidence that feruloylation occurs in the Golgi and in the cell wall (Mastrangelo et al, 2009). However the investigated proteins of the BAHD superfamily were located in the cytosol D'Auria, 2006).…”

Grass Cell Walls: A Story of Cross-Linking

“…Early work suggested that either FA-glucose (Obel et al, 2003) or FA-CoA (Yoshida-Shimokawa et al, 2001) could be the source for FA for FA-GAX synthesis. Using metabolic labeling time-courses, the rapid kinetics of GAX feruloylation suggested FA coupling to GAX occurs intracellularly (Mastrangelo et al, 2009). Current models suggest that select classes of BAHD acyltransferases (D'auria, 2006) are responsible for the FA- and p CA-esterification of GAX.…”

Ferulic acid: a key component in grass lignocellulose recalcitrance to hydrolysis

“…It is well established that oxidative coupling of polysaccharides through ester‐linked feruloyl groups might occur both within the protoplast in the Golgi apparatus (Mastrangelo et al ., ; dos Santos et al ., ; Umezawa, ), and after their secretion into the apoplast. This process is catalysed by multiple cell wall bounds and putative Golgi resident isoperoxidases, which use hydrogen peroxide as substrate in a mechanism similar to that of lignin polymerization (Fry, ; Hatfield et al ., ; Lindsay and Fry, ).…”