Fig. 7.

NRAMP1 localizes on the vacuole in the nns1 mutant. (A and B) NRAMP1-GFP and TagRFP-SYP22 were expressed in nramp3-1nramp4-1 (A) and nramp3-1nramp4-1atph1-1 (nns1) (B) mutants. Root epidermal cells in the elongation zone were imaged on a spinning disk confocal microscope for GPF and tagRFP. (Scale bars: 10 µm.) (C) NRAMP1 localization on the vacuole was quantified in single-plane images corresponding to individual cells of nramp3nramp4 (n = 48) and nramp3nramp4atph1 (n = 42) as a normalized fraction of NRAMP1-GFP signal colocalizing with the TagRFP-SYP22 signal. Note that because of the close proximity of cytoplasmic NRAMP1 structures with the vacuole, the value of the vacuolar membrane localization index is never null. In the graph, boxes include the two central quartiles, separated by the median. The whiskers extend to the 5th and 95th percentiles, and outliers are represented by a dot. P < 0.0001; Mann–Whitney test. (D) Root epidermal cells of nramp3nramp4 and nns1 mutants expressing NRAMP1-GFP were imaged across vacuolar planes using spinning disk confocal imaging. GPF fluorescence and transmitted-light (DIC) images are displayed. The asterisks denote the position of vacuoles. Plants were placed in darkness for 14 h (Dark) before imaging to reduce GFP degradation in the vacuoles. The contrast of the images was adjusted for the visualization of GFP in vacuoles. (Scale bar: 10 µm.)