Vacuolar protein sorting is compromised in vps38 mutants. A, Seed protein profile of the indicated mutants and transgenic plants. A protein blot stained with Ponceau S is shown in the upper panel with seed storage protein species indicated by arrowheads (precursors) and vertical bars (mature forms). The two immunoblots below were generated with antibodies against 12S globulin (middle panel) and pro-2S albumin (lower panel). Each lane contains the protein extract of five dry seeds. B, Confocal microscopy images of Citrine-VSR2;1 transgenic plants with indicated genotypes. Root tips of seedlings treated for 1 h with either DMSO or 30 μm Wm were observed. C and D, Graphs illustrate the quantification of citrine-VSR2;1 puncta in wild-type and vps38-1 mutant cells (mean ± se; n > 11 regions of interest). E, Confocal images of wild-type and vps38-1 root cells expressing both citrine-VSR2;1 and mCherry-RHA1. Roots were treated with DMSO or Wm as in (B). F, Quantification of colocalization using images similar to one shown in (E) (mean ± se; n > 23 regions of interest). Columns marked with different letters represent significantly different means according to two-way ANOVA followed by Tukey’s test (C and D) or t test (F) (P < 0.01). Bars = 5 μm. WB, western blot; WT, wild type.