Figure 5.
PI(4,5)P2 Biosensor Was Depleted from the PM and Partially Colocalized with AtTSPO in Golgi Membranes in the Presence of Full-length AtTSPO but Not AtTSPOΔN
(A) Representative confocal images of Arabidopsis suspension cells stably coexpressing the PI(4,5)P2biosensor (mCherry-tagged double pleckstrin homology domain from phospholipase C; magenta) and full-length AtTSPO or AtTSPOΔN (GFP-tagged; green). Arrowheads indicate mCherry and GFP signals colocalizing at Golgi membranes. * indicates GFP fluorescence in the vacuole. Bars = 10 μm.
(B) Solubilized PM-enriched fractions from cells imaged as in (A) were spotted on a nitrocellulose membrane, and PI(4,5)P2 was detected using monoclonal anti-PI(4,5)P2 antibodies. For signal quantification (ImageJ 1.51 software) we used PM proton ATPase (PMA) as a control.
(C) Ten-day-old Arabidopsis seedlings grown on MS agar plates were incubated in liquid MS medium with or without 50 μM ABA for 24 h, and PLC activity was measured in triplicate using an EnzCheck direct phospholipase C assay kit. Statistical significance was analyzed by one-way ANOVA followed by Tukey's test (***p < 0.001; **p < 0.01; *p < 0.05). Data in (B) and (C) are means ± SD from three technical replicates, and all experiments were performed at least twice.