Lipid anchors are necessary for NLD targeting at membranes. (A–F) Pharmacological approach showing the involvement of S-palmitoylation in NLD addressing to the PM of root cells. Confocal imaging of root cells expressing NLD_full:mCitrine (A and B) or transmembrane GFP:LTI6B control (C and D) after 2-h treatment with 50 µM 2-BP, an inhibitor of palmitoylation (B and D) or mock treatment (EtOH; A and C). (E and F) Quantification of drug treatment effect on NLD subcellular localization using the ratio of the fluorescence intensity at PM compared with the fluorescence intensity in cytosol (PM/cyto fluorescence ratio) in root cells (mean ± SD). (G–L) Targeted mutagenesis of predicted NLD lipid anchor sites C10 and C423. Confocal imaging of root cells and mature pollen from the following lines: NLD_C10S:mCitrine (C10 mutated to S; G and H), NLD_C423S:mCitrine (I and J), and NLD_C10S,423S:mCitrine (K and L). (M and N) Quantification of mutagenesis effect on localization using the ratio of the fluorescence intensity at PM compared with the fluorescence intensity in cytosol in root cells (PM/cyto fluorescence ratio; M) and in pollen grain (endo-PM/cyto fluorescence ratio; mean ± SD; N). ***, significant difference as compared with NLD_full:mCitrine (P < 0.001; Wilcoxon test, n = cell number analyzed). Scale bars = 20 µm.