Changes in the Formononetin Content of Detached Mature Leaves of Trifolium Subterraneum

Abstract

Anthocyanin formation is stimulated in the leaves of some plants when the leaves are detached and floated on water or on sucrose solutions and exposed to light (e.g. Eberhardt 1954;Creasy, Maxie, and Chichester 1965). The synthesis of other flavonoids, e.g. flavans, can be increased under these conditions (Creasy and Swain 1966).We have observed that anthocyanin formation can be greatly stimulated by detaching fully expanded leaves of subterranean clover (T. subterraneum) and holding them for several days in diffuse daylight. We then wondered whether the oestrogenic isoflavones formononetin, genistein, and biochanin A, which occur in subterranean clover (Beck 1964), were also increased. If they were, the point would be of some interest, since with entire plants the net increase in the amounts of these three isoflavones after the leaves reach full expansion is negligible (Rossiter and Beck 1967).Several simple experiments were done with detached leaves, mostly using the Northam A strain of T. subterraneum. Increases in biochanin A were usually small, while for genistein the increases were seldom more than 50% and usually much less than this. Results for these two compounds are not presented here, since treatment differences rarely reached statistical significance. However, for formononetin there were large relative increases, and these are reported below. Materials and MethodsThe clover plants were grown in shallow boxes in an open-sided glasshouse during the winter growing season (average day and night temperatures usually more than 15°e and 10 0 e, respectively). Mature leaves showing no visible senescence were harvested between 1100 and 1500 hr as required, mixed before sorting in a cold room, and weighed. Sufficient leaves were used for each sample to provide a total fresh weight of c. 500 mg. The leaves (laminae only) were floated on 30 ml of solution in covered Petri dishes (9 cm diameter). In all instances the dry matter content of the leaves was within the range 20-25%.Unless specified otherwise, incubations were done in diffuse daylight near a laboratory window where the mean temperature was 21°e, and the range about ±2°e. Light intensity at the leaf surface seldom exceeded 350 f.c., as measured with an EEL light-meter. Some incubations were done in an artificially lit type LB cabinet (Morse and Evans 1962).Formononetin was estimated by the thin-layer chromatographic method of Beck (1964) with modifications based on the microtechnique of Francis and Millington (1965).In the first two experiments the treatments were unreplicated, but three replications were used thereafter. Isoflavone contents are expressed on the basis of original fresh weight, rather than per leaf, since leaf size varied between strains and between experiments.