Development of Three Copper Metalloenzymes in Clover Leaves

Subterranean clover (Trifolium subterraneum L. cv Seaton Park) was grown in solution cultures containing adequate nitrogen both with and without Cu. After Cu deficiency had developed, Cu(2+) was added to some deficient plants and Cu content, protein content, and activities of three Cu metalloenzymes (diamine oxidase [EC1.4.3.6], ascorbate oxidase [EC1.10.3.3] and o-diphenol oxidase [EC1.10.3.1]) were assayed in young and recently matured leaf blades over 11 days during the development of the next three leaves.Copper deficiency had little effect on protein concentrations, but markedly depressed enzyme activities and Cu concentration in all leaf blades assayed. Within 4 d of adding Cu(2+) to Cu-deficient plants, Cu concentrations of all the leaf blades increased to adequate values. Enzyme activities only increased to control levels in leaves which had not yet emerged at the time that Cu(2+) was added.The results suggest that active holoenzymes of diamine oxidase, ascorbate oxidase, and o-diphenol oxidase can only be synthesized in leaf blades during very early stages of their development.