Xylan reducing end sequence-containing oligosaccharides function as priming acceptors for Arabidopsis and <i>Setaria viridis</i> IRX10

Xylan, one of the most abundant hemicelluloses in plant cell walls, consists of β-(1 → 4)-linked xylosyl (Xyl) residues and often contains a conserved Reducing End Sequence (RES) in dicots and gymnosperms, comprising β-d-Xylp-(1 → 3)-α-l-Rhap-(1 → 2)-α-d-GalpA-(1 → 4)-d-Xylp. This tetrasaccharide has been proposed to function as a priming module ('primer hypothesis') or a termination signal ('terminator hypothesis') in xylan biosynthesis, yet its precise biochemical role remains unclear. Here, we examined whether the RES-containing oligosaccharide with one additional Xyl residue at the non-reducing end (X-RES) acts as a priming acceptor for IRREGULAR XYLEM10 (IRX10) proteins from a dicot, Arabidopsis thaliana, and a grass, Setaria viridis. Both recombinant AtIRX10L and SvIRX10 utilized fluorescently labeled X-RES and the canonical primer Xyl5 as acceptor substrates. Time-course analyses revealed that X-RES promoted a more efficient transition of +X1 to +X2 product, i.e. with minimal accumulation of +X1 product and enhanced formation of +X2 and longer products, suggesting that the RES motif facilitates seamless elongation. Consistent with these substrate-dependent differences, docking simulations showed that X-RES and its elongated form (X2-RES) bound more stably to the predicted IRX10 active site than the corresponding linear oligosaccharides Xyl5 and Xyl6. Moreover, the ability of SvIRX10 to recognize X-RES, despite RES motif not yet being detected in grass xylan, suggests that the RES-primed elongation may represent an ancestral substrate recognition in grasses. Our findings identify a structurally unique RES-containing oligosaccharide that functions as a primer in vitro, thereby extending current understanding of acceptor substrate flexibility in xylan biosynthesis. (242 words).