Light-Induced Trans to Cis Conversion of β-d-Glucosyl o -Hydroxycinnamic Acid in Melilotus alba Leaves

Abstract

Isotope studies indicate that o-coumiaric acid glu-coside (,8-D-glucosyl trans-o-hydroxycinnamic acid) is the immediate precursor of coumarinic acid glu-coside (,8-D-glucosyl cis-o-hydroxycinnamic acid) in sweetclover (5, 7). Structures of these compounds are as follows: Preliminary reports (3, 4) H o-Coumaric acid glucoside H GOOH 0-C6H,105 Coumarinic acid glucoside suggest that this trans to cis conversion is a nonenzy-mic photochemical reaction. However, the published data do not preclude the possibility that the reaction is effected by a light-sensitive isomerase system. The present paper deals with an investigation of the conversion of o-coumaric acid glucoside to coumarinic acid glucoside in intact sweetclover leaflets and in leaflet extracts. Evidence to be presented supports the conclusion that in sweetclover leaves this trans to cis conversion is nonenzymic and is induced by ultraviolet irradiation. Materials and Methods Plant Material. Sweetclover (Melilotus alba Desr.) of the CuCuBB genotype was used in these experiments. The derivation of this genotype has been described elsewhere (2). Hot water extracts of CuCuBB sweetclover leaves are high in content of glucosidically bound o-hydroxycinnamic acid, and homogenates of such leaves contain a highly active ,8-glucosidase. Except where otherwise indicated, plants were grown in plant growth chambers (Instrumentation Specialties Company) 3, in 1-pint plastic-coated milk cartons containing a mixture of soil, sand, and vermic-ulite. Some of the plants were subjected to continuous light and others to a photoperiod of 19 hours. Chambers were equipped with cool white fluorescent lamps (General Electric Power Groove tubes) which provided a light intensity of approximately 1,500 ft-c at the level of the plants. The chambers were maintained at a temperature of 270 and at 50 % relative humidity. Plants from the growth chambers ranged in age from 27 to 44 days when sampled. Only the youngest fully expanded leaf was taken from each plant used. Steam Treatment. The aim of this treatment was the inactivation of any trans-cis isomerase that might be present. Leaflets to be treated were held individually in a stream of steam for 10 to 15 seconds. Tests for ,8-glucosidase in the steamed leaflets indicated that this enzyme was effectively inactivated by the treatment. Light Treatment. Ultraviolet light: A Gates MR4 lamp equipped with the TF8 tube was used as a source of ultraviolet light (peak near 360 mp.). In the experiment employing steamed leaflets, the leaflets were placed on glass microscope slides surrounded by moist filter paper during irradiation. The slides prevented extensive leaching which occurred when steamed leaflets were placed directly on moist filter paper. In all other experiments using detached leaflets , the leaflets were placed on moist filter paper or on ice cubes during irradiation. The distance from the light source to the leaflets was approximately 3.5 cm. Leaflets were turned over every 30 minutes during treatment. Leaf extract and solutions of authentic o-coumaric acid and coumarinic acid gluco-sides (approximately 30-ml volumes) were irradiated in 50-ml beakers placed on magnetic stirrers. The distance from the light source to the surface of the solutions was approximately 3.5 cm. Fluorescent light: Solutions were treated with cool white fluorescent light from General Electric Power Groove lamps in the growth chambers where, as previously indicated, the light intensity was approximately 1,500 ft-c. Solutions were stirred by means of magnetic stirrers during irradiation. Sunlight: Experiments utilizing the sun as a light source were conducted on clear summer days 777