Abstract text: Xylan substitution patterns are important determinants of plant cell wall architecture and mechanical function, yet the structure of xylan in primary cell walls remains poorly understood. Here, we identify a previously uncharacterised β-1,2-xylosyl side chain on glucuronoxylan that defines a conserved structural feature of primary walls in both gymnosperms and angiosperms.

Using enzymatic fingerprinting, NMR spectroscopy and mass spectrometry, we show that this modification occurs in a defined spatial relationship to glucuronic acid substitutions, producing an evenly patterned xylan motif enriched in primary wall-rich tissues such as conifer needles, pro-embryogenic masses and Arabidopsis callus. Functional analyses demonstrate that GT61 glycosyltransferases act as β-1,2-xylosyltransferases responsible for generating this structure with positional specificity. In Arabidopsis, three closely related enzymes act redundantly, and their combined loss abolishes the modification.

Genetic evidence indicates that β-1,2-xylosylation influences developmental processes, including mucilage organisation and leaf senescence, suggesting a role in regulating cell wall remodelling. The defined substitution pattern is compatible with xylan–cellulose interaction geometries, supporting a structural function in matrix assembly and mechanical tuning of primary walls.

These findings reveal a conserved and developmentally regulated feature of plant primary wall xylan, providing new insight into how hemicellulose structure contributes to cell wall function and plant growth.