Figure 5: NBD-PC and -lysoPC are taken up into A. thaliana roots in an ALA10-dependent manner.

(a) Three independent ala10-knockout lines (ala10-1, ala10-5 and ala10-7) were isolated and tested for homozygosity. Numbers under the DNA fragment denote primers used for homozygosity testing. Black boxes, exons; lines, introns; triangles, T-DNA insertion sites. Size on x axis is given in base pairs (bp). (b) Five-day-old wild-type seedlings were incubated in the presence of NBD-PC or -lysoPC, as indicated. Left, confocal microscopy visualization of NBD fluorescence. Scale bar, 25 μm. Right, after 3 h of NBD-lipid incubation, lipids were extracted from root tissues (Seedlings) and from media (M) and subjected to thin-layer chromatography. Root extract, ground roots incubated with NBD-lipid for 30 min and treated in a similar manner. Asterisks (*), unidentified contaminants already present in the original NBD-lipid stock. Lipid structures are given to the right in each figure (circle, choline; square, phosphate group; star, NBD group). (c) Five-day-old A. thaliana wild-type and mutant seedlings were incubated in the presence of NBD-PC or -lysoPC for 1–3 h before visualization by confocal microscopy and quantification. Data represent means±s.d. (n=5–7 from three biological replicates). (d) Five-day-old seedlings corresponding to the wild-type or ala10-knockout lines were incubated in the presence of the endocytic tracker FM4-64 for 90 min before visualization by confocal microscopy and quantification. FM4-64 fluorescence intensity was measured. Data represent means±s.d. (n=22–25 from three biological replicates). ***P≤0.001, **P≤0.01 and *P≤0.05, significantly different with respect to wild type based on Student’s t-test.