NLD protein localization is conserved in Arabidopsis pollen, and the C-terminal part of the NLD protein is not sufficient to ensure subcellular localization. (A) Schematic representation of NLD_full and NLD_trunc proteins. Since the NLD C-terminal region contains both a predicted lipid anchor posttranslational modification site (C423) and a stretch of positively charged amino acids (in red in the sequence), targeting of NLD to membrane could be achieved via lipid anchoring and/or electrostatic interactions between the anionic lipids present at the membrane surface and the cationic amino acid present at the NLD C-terminus. (B–K) Confocal imaging of different forms of NLD protein in Arabidopsis root cells and pollen. Confocal imaging of root tips (B–D, H, and I) and mature pollen grains (E–G, J, and K) from nontransformed Columbia 0 (Col 0; D and G) and lines expressing NLD_full:mCitrine (B and E), NLD_trunc:mCitrine (C and F), the C-terminal part of NLD_full (mCitrine:NLD_full-Cter; H and J) fused to mCitrine, and the C-terminal part of NLD_trunc (mCitrine:NLD_trunc-Cter; I and K) fused to mCitrine. Scale bars = 20 µm. (L) Quantification of mutagenesis effect on 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). ***, significant difference between means as compared with NLD_full:mCitrine (P < 0.001; Wilcoxon test, n = cell number analyzed).