Fig. 3

Visualization of labeled choline phospholipids in seedlings. Arabidopsis seeds were germinated and seedlings were grown for 7 days on agar media containing a, b 0 µM propargylcholine (negative control) or c–i 250 µM propargylcholine. a–i Light microscopy shows morphology of the tissues analyzed. a′–i′ Fluorescence signals are indicated in green. a Untreated cotyledon and b untreated root exhibit no visible background fluorescence. c–f Fluorescence marks the presence of propargylcholine in c cotyledons, d hypocotyl-root junction, and e root. f–i Propargylcholine labeling is removed by Phospholipase C treatment. Arabidopsis seedlings grown on 250 µM propargylcholine were prepared for click chemistry with fluorescein azide followed by treatment of roots with f EDTA alone, g EDTA with a bacterial phospholipase C (PLC), h CaCl₂ alone, and i CaCl₂ with PLC. The PLC enzyme removes choline head groups from phospholipids and requires calcium for activity. i In the presence of PLC and calcium, the fluorescein azide labeling of propargylcholine is markedly reduced. Scale bars shown in a and f are constant for a–e and f–i, respectively