Intracellular transport and vacuolar accumulation of o-coumaric acid glucoside in Melitolus alba mesophyll cell protoplasts

Abstract

Sweet clover leaves accumulate o-coumaric acid glucoside in the vacuole of the mesophyll cells. Protoplasts isolated from these cells are able to synthesize o-coumaric acid glucoside from exogeneous [ 14C]phenylalanine. Using this simplified system, kinetic analyses have shown that the glucoside is synthesized in the cytoplasm and thereafter transferred into the vacuole. No further degradation of the glucoside occurs in the isolated protoplasts after this transfer. Under our conditions, the vacuolar pH is reduced by light/dark transition and by inhibitors of photosynthesis. These treatments also depressed synthesis and transport of o-coumaric acid glucoside. The data support a relationship between the functioning of photosynthesis and pH cytoplasm/vacuole-dependent transport of o-coumaric acid glucoside into the vacuole. However as o-[ 14 C] coumaric acid glucoside is taken up by isolated vacuoles without any energy requirement, no final conclusions can be drawn as to the coupling of o-coumaric acid glucoside transport with the acidification process. In intact plant or isolated protoplasts o-coumaric acid glucoside is synthesized as the trans isomer but accumulated in the vacuole in the cis form. When loaded with trans o-[ 14 C] coumaric acid glucoside, reconstituted tonoplastic vesicles released this compound during molecular sieving on a Sepharose 4B column. In contrast, the labelled cis form was completely retained inside the vesicular lumen in a parallel experiment. These results suggest a selective permeability of the tonoplast to the two isomers which could be involved, in vivo, in the trapping of the glucoside inside the vacuole and its irreversible accumulation.