Vacuolar chain elongation of raffinose oligosaccharides in Ajuga reptans

Galactan : galactan galactosyltransferase (GGT) is the key enzyme responsible for the accumulation of long-chain raffinose family oligosaccharides (RFOs; 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 isolation of vacuoles from A. reptans mesophyll protoplasts was developed using a pH and osmotic shock to rupture the plasma membrane selectively. By comparing protoplasts with vacuoles, GGT was confirmed to be a vacuolar enzyme. By comparing vacuoles with tonoplast vesicles and cell sap fractions, GGT was further shown to reside in the cell sap and not in the tonoplast. These findings suggest the need for a tonoplast-bound mechanism for the transport of short-chain RFOs such as stachyose or raffinose into the vacuole for subsequent chain elongation.