Destiny of endo-PM during maize fertilization. (A–D) Tracking of NLD:mCitrine marking the endo-PM during pollen tube growth to the maize ovule. Confocal imaging (Z-projections) after pollination of WT female plants with NLD:mCitrine pollen (A–C) or control cross with WT instead of transgenic pollen (D). Calcofluor-white (CFW), mCitrine, and autofluorescence (AF) channels are used to visualize the cell walls, NLD:mCitrine, and autofluorescence, respectively. (A) NLD:mCitrine location during pollen tube elongation inside of the silk (SI) tissues (pollen tube transmitting tract). Arrows show the NLD:mCitrine signal. Sperm cells (SC) could be identified (white lines). Video 1 shows the Z-stack. (B) NLD:mCitrine location in a pollen tube before pollen tube discharge into a synergid cell. Arrows show the NLD:mCitrine signal. Video 2 shows the Z-stack. (C) NLD:mCitrine location within the embryo sac, after pollen tube discharge into a synergid cell. After pollen tube discharge (monitored thanks to the autofluorescence of the degenerating synergid cell, see D), NLD:mCitrine protein localizes at the apical region of the egg cell apparatus (arrow). The arrowhead represents NLD:mCitrine signal from another arriving pollen tube. Videos 3 and 4 show Z-stack and 3D projection, respectively. (D) Control pollination with WT ovule receiving WT pollen. It allows evaluation of autofluorescence (in both mCitrine and AF channels) of the degenerating synergid (DS) without NLD:mCitrine signal. Video 5 shows Z-stack. (E) Schematic illustration of endo-PM localization within the embryo sac after pollen rupture. AP, antipodal cells; CC, central cell; DS, degenerating synergid; EA, egg apparatus cell (egg cell or intact synergid); ES, embryo sac; I, integument; Nc, nuclei central cell; NU, nucellus; OV, ovary tissue. Scale bars = 50 µm.