ACC oxidase (ACO) genes in Trifolium occidentale (L.) and their relationship to ACO genes in white clover (T. repens L.) and T. pallescens (L.).

The identification and expression of two ACC oxidase (ACO) genes during leaf development in Trifolium occidentale (L.), one of the putative ancestral genomes of the allotetraploid, T. repens (L.; white clover), is described. In common with observations made in T. repens, the ACO genes displayed differential expression, with a TR-ACO2-like gene (designated TO-ACO2) confined to developing and early mature-green leaf tissue while expression of a TR-ACO3-like gene (designated TO-ACO3) is highest in leaves at the onset and during senescence. Biochemical analysis of TO-ACO2 revealed that both accumulation of the protein (determined by western analysis with a TR-ACO2 antibody) and enzyme activity matched the transcriptional activity of TO-ACO2. Western analysis also revealed that the Tr-ACO2 antibody recognised a protein of 37 kDa as a putative TP-ACO2 in T. pallescens. The 3'-UTRs of TO-ACO2 and TO-ACO3 were then compared with the 3'-UTRs of a TR-ACO2-like and TR-ACO3-like gene from T. pallescens, the other proposed ancestral genome (or closely related to the ancestor) of T. repens, with identity values of 87.8% for the ACO2-like genes and 94.8% for the ACO3-like genes. Comparison of the 3'-UTRs of TO-ACO2 with a TO-ACO2-like gene in T. repens (designated TR(O)-ACO2) and TP-ACO2 with a TP-ACO2-like gene in T. repens (designated TR(P)-ACO2) revealed identities of 100% and 96.6%, respectively, lending good support to T. occidentale as one of the ancestral genomes of T. repens. A similar comparison of the 3'-UTRs of TO-ACO3 with a TO-ACO3-like gene in T. repens (designated TR(O)-ACO3) and TP-ACO3 with a TP-ACO3-like gene in T. repens (designated TR(P)-ACO3) revealed identities of 99.5% and 97.9%, respectively, again supporting T. occidentale as one of the ancestral genomes. Further, these data confirm that both TO-ACO-like and TP-ACO-like genes are expressed in the allotetraploid T. repens.

Changes in 1-aminocyclopropane-1-carboxlate (ACC) oxidase expression and enzyme activity in response to excess manganese in white clover (Trifolium repens L.).

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.