Vacuolar chain elongation of raffinose oligosaccharides in Ajuga reptans

Galactan : galactan galactosyltransferase (GGT) is the key enzyme responsib le for the accumulation of long-chain raffinose family oligosaccharides (RF Os; alpha-D- gal(n) (1,6) alpha-D-glc (1,2) beta-D-fru) in Ajuga reptans L. leaves during autumn and winter. The exact subcellular location of GGT is not known and its elucidation was the aim of this paper. A method for the i solation of vacuoles from A. reptans mesophyll protoplasts was developed us ing a pH and osmotic shock to rupture the plasma membrane selectively. By c omparing protoplasts with vacuoles, GGT was confirmed to be a vacuolar enzy me. By comparing vacuoles with tonoplast vesicles and cell sap fractions, G GT was further shown to reside in the cell sap and not in the tonoplast. Th ese findings suggest the need for a tonoplast-bound mechanism for the trans port of short-chain RFOs such as stachyose or raffinose into the vacuole fo r subsequent chain elongation.