Abstract text: Biosynthesis of cell wall polysaccharides is carried out by membrane-bound glycosyltransferases through sequential transfer of monosaccharides from nucleotide-sugar donors to acceptor molecules. Acceptors can be single monosaccharide residues, carbohydrate sequences, or glycoconjugates that significantly differ from the structural components of the final polysaccharide.

Using the model system of flax phloem fibers, where the stage of tertiary cell wall formation is accompanied by rhamnogalacturonan I (RG-I) biosynthesis, we developed an original toolkit to obtain all the basic components of the "biosynthetic machinery" of this polysaccharide and to reproduce the early stages of its biosynthesis in vitro. Analysis of the new products formed during co-incubation of membrane-bound enzymes, UDP-sugar precursors, and potential acceptors – which we hypothesized based on the redistribution patterns of ¹⁴CO₂ among various RG-I-specific monomers in high- and low-molecular-weight fractions (pulse-chase experiments) – demonstrated that initiation of rhamnogalacturonan I biosynthesis in flax fibers requires an acceptor containing a non-carbohydrate component located not at the reducing end of the acceptor, and also revealed the starting monomers attached to the acceptor sequence.

These breakthrough results expand our understanding of the molecular logic of polysaccharide biosynthesis, opening new avenues for exploring initiation strategies.

The work was partially supported by the RSF #26-16-00294.