Figure 3.

vps38 affects the intracellular distribution of PI3P but not its total levels. A, Root tip images of wild-type and vps38 expressing the PI3P biosensor, citrine-2xFYVE. Wild-type and vps38 seedlings expressing the sensor were incubated with either 30 μm Wm or DMSO for 1 h before confocal imaging. Arrowheads indicate ring-shaped compartments. Bars = 5 μm. B and C, Graphs illustrate area (B) and density (C) of the citrine-2xFYVE puncta in DMSO- or Wm-treated wild type and vps38-1 (mean ± se; n > 46 regions of interest; *, 0.01 < P < 0.05; **, P < 0.01; two-way ANOVA followed by Tukey’s test). D, Autoradiograph of TLC of ³²P-labeled lipid extracts, which were prepared from three biological replicates, each consisting of three seedlings selected from four seed populations of wild type and two vps38 alleles that were ³²P_i-labeled in vivo. E to G, Levels of PI3P (E), PI4P (F), and PI(4,5)P₂ (G) were quantified by phosphoimaging the phosphoinositide spots in (D) and determining the ratios of PI3P to PI4P by HPLC analysis (see “Materials and Methods”). Columns (mean ± sd) marked with asterisks represent significantly different means (*, 0.01 < P < 0.05; **, P < 0.01; one-way ANOVA followed by Tukey’s test). CL, cardiolipin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; WT, wild type; WT#1 and WT#2, wild-type seed populations.