Elucidating the Enzymatic Framework for the Initiation of Pectic Galactan

Abstract Pectins are a major class of plant cell wall polysaccharide crucial for plant survival and adaptation. Despite their importance, the biosynthesis of pectin remains poorly understood due to its exceptional structural and functional diversity. Functional interrogation of pectin biosynthetic enzymes in planta is often limited by severe developmental or difficult to interpret carbohydrate phenotypes resulting from genetic disruption. Using a direct biochemical approach, we report the identification and functional characterization of a Galactan Initiation (GalTi) enzyme from the aquatic monocot Spirodela polyrhiza, a member of CAZy glycosyltransferase family 47, that initiates galactan side‑chain biosynthesis on rhamnogalacturonan‑I. We further show that orthologs from Arabidopsis thaliana exhibit identical catalytic activity, revealing conservation of this biosynthetic initiation mechanism across monocots and dicots. Comprehensive carbohydrate structural analyses demonstrate that GalTi enzymes specifically catalyze the transfer of single β‑D‑galactopyranosyl residues to the O‑4 position of backbone rhamnose residues, establishing their direct role in galactan side‑chain biosynthesis. Co-synthesis experiments show 3-O-acetylation of backbone rhamnose residues inhibits SpGalTi‑mediated galactosylation, suggesting a potential regulatory mechanism by which plants use to modulate side‑chain patterning in vivo. Together, these findings provide a new framework for RG‑I galactan assembly, advancing our mechanistic understanding of plant cell wall biosynthesis.