CONCLUSIONS

In Ajuga reptans, raffinose oligosaccharides accumulated during winter. Stachyose, verbascose, and higher RFO oligomers were exclusively found in the vacuole whereas one-fourth of raffinose was localized in the stroma. The evergreen labiate Ajuga reptans L. can grow at low temperature. The carbohydrate metabolism changes during the cold phase, e.g., raffinose family oligosaccharides (RFOs) accumulate. Additionally, A. reptans translocates RFOs in the phloem. In the present study, subcellular concentrations of metabolites were studied in summer and winter leaves of A. reptans to gain further insight into regulatory instances involved in the cold acclimation process and into the function of RFOs. Subcellular metabolite concentrations were determined by non-aqueous fractionation. Volumes of the subcellular compartments of summer and winter leaves were analyzed by morphometric measurements. The metabolite content varied strongly between summer and winter leaves. Soluble metabolites increased up to tenfold during winter whereas the starch content was decreased. In winter leaves, the subcellular distribution showed a shift of carbohydrates from cytoplasm to vacuole and chloroplast. Despite this, the metabolite concentration was higher in all compartments in winter leaves compared to summer leaves because of the much higher total metabolite content in winter leaves. The different oligosaccharides did show different compartmentations. Stachyose, verbascose, and higher RFO oligomers were almost exclusively found in the vacuole whereas one-fourth of raffinose was localized in the stroma. Apparently, the subcellular distribution of the RFOs differs because they fulfill different functions in plant metabolism during winter. Raffinose might function in protecting chloroplast membranes during freezing, whereas higher RFO oligomers may exert protective effects on vacuolar membranes. In addition, the high content of RFOs in winter leaves may also result from reduced consumption of assimilates.