To examine the effect on Mn treatment on the ACO gene family of white clover [Trifolium repens (L.) cv. Grasslands Challenge], rooted stolon cuttings were maintained in modified Hoaglands medium, at pH 5.4, containing either 5.2 μM Mn or 100 μM Mn over a 9-day time course. A significant uptake of Mn was observed in leaf tissue of plants grown in the 100 μM Mn treatment after 24 h and the content increased in these plants to reach 334 mg/kg DW at the conclusion of the time course. The growth of plants, measured as the petiole extension rate (PER), was significantly less in the 100 μM Mn treatment by day 9, while significantly less accumulation of leaf biomass was observed by day 7. The activity of a cell wall-associated H(2)O(2)-generating NADH peroxidase was shown to be higher in the 100 μM Mn treatment after day 5 of the time course while no significant difference in a H(2)O(2)-consuming guaiacol peroxidase activity was observed between the two Mn treatments. The expression of two leaf-associated ACC oxidase (ACO) genes, TR-ACO2 and TR-ACO3 was examined over the 9-day course but no difference between the two treatments was observed. In contrast, TR-ACO2 enzyme activity was measured and shown to decrease in the 100 μM Mn treatment after day 5 of the time course, with a concomitant decrease in TR-ACO2 accumulation, as determined by western analysis. Using 2DE and western analysis, evidence for post-translational modification of TR-ACO2 was observed.