Ascerbate oxidase in cucumber calli and enhancement of enzyme activity by copper sulfate

Ascorbate oxidase [AOD, EC 1.10.3.3] activity was detected in callus induced from the peel tissue of cucumber fruit (Cucumis sativus L. cv. Suyo). The optimum hormone combination for callus growth and AOD activity was 5-10 μM (±)-2-(2-naphthoxy)propionic acid and 1 μM benzylaminopurine. Addition of 3 μM CuSO4 to the medium enhanced the AOD activity without inhibition of callus growth. The higher level of the enzyme activity with 3 μM CuSO4 was maintained even after repeated subcultures. Cu content in the cells was increased in the medium with 3 μM CuSO4. When the callus cultured in the medium with 3 μM CuSO4 was transferred to the medium with 0.1 μM CuSO4, the Cu content decreased with the decrease in the enzyme activity. Imminochemical measurement suggests that enhancement of the AOD activity by CuSO4 may be due to both de novo synthesis of the enzyme protein and assembly of AOD apoprotein with Cu. These findings suggest that the Cu concentration in the cells affects the AOD activity. AOD activity in the callus induced from the hypocotyl of an etiolated seedling was also enhanced by CuSO4, although the level of the AOD activity was lower.