Immunocytochemical studies on the origin and deposition sites of hydrolyzable tannins

Two specific antibodies, directed toward hydrolyzable tannins, i.e. polygalloylated or oxidized derivatives of 1,2,3,4,6-penta-O-galloylglucose, and toward an acyltransferase from oak leaves that catalyzes the biosynthesis of this principal precursor, were used in immunocytochemical studies to determine the intracellular sites of origin and deposition of these polyphenolic plant constituents. Immunostaining of semi-thin sections from leaves and roots of young pedunculate oak (Quercus robur, syn. Quercus pedunculata) plants with marker enzymes and immunogold labeling of ultra-thin sections revealed immunoreactive sites for both enzyme and hydrolyzable tannins in chloroplasts, cell walls and intercellular spaces. The latter non-cytoplasmic (apoplast) compartments displayed characteristic aggregations of these two epitopes, thus indicating an intimate association of the biocatalyst and its products. Identical distribution patterns for hydrolyzable tannins were observed in leaves of Rhus typhina (sumac) and Tellima grandiflora (fringe cups) which, however, displayed no affinity toward the galloyltransferase antibody that had been raised against enzyme from oak. Controls with spinach leaves, known to be devoid of tannins, were inactive in all cases. The conclusion that, besides chloroplasts, cell walls and intercellular space serve as sites for the biosynthesis and deposition of hydrolyzable tannins was confirmed by analyzing extracts from these non-cytoplasmic compartments.