A, In vitro NANS activity assay analyzed by RP-HPLC of DMB-Neu5Ac in extracts from Arabidopsis seedlings: DMB-Neu5Ac standard (S), DMB-labeled extract from wild-type plants after incubation in the presence (wt + ManNAc-6-P) or absence (wt) of ManNAc-6-P, and 35S:NANS transgenic plants after incubation in the presence (NANS + ManNAc-6-P) or absence (NANS) of ManNAc-6-P. The elution position of DMB-Neu5Ac is indicated. B, The eluate in the region of DMB-Neu5Ac was collected, concentrated, and subjected to RP-LC-ESI-MS/MS to acquire a fragment spectrum of the presumed DMB-Neu5Ac peaks. All traces are shown with the same y axis setting.