Figure 1.
NLD is expressed in the vegetative cell of maize pollen, and protein localizes to the endo-PM of the MGU. (A) Schematic representation of the mature pollen grain organization, focusing on the MGU structure. The two sperm cells (yellow) are surrounded by an endo-PM (dark purple) originating from the PM of the vegetative cell. Light blue indicates the extracellular space (polysaccharide) of the MGU. (B) Characterization of NLD promoter activity (pNLD::H2B:mCherry) in mature pollen. Promoter activity (red signal) is found in the vegetative cell nucleus and not in the sperm cells nuclei. Nuclei are stained with DAPI (blue signal); white arrows indicate the sperm cells nuclei. (C) Subcellular localization of NLD protein (yellow signal) together with NLD promoter activity (red signal) in mature pollen. Confocal imaging (Z-stack projection) of mature pollen expressing pNLD::NLD:mCitrine and pNLD::H2B:mCherry constructs shows that NLD:mCitrine protein localizes at the MGU, whereas the promoter activity is found in the vegetative cell. (D–G) Sperm cells are released (white arrows) from mature pollen of pTUBα::YFP:TUBα (D and F) and pNLD::NLD:mCitrine (E and G) lines after osmotic shock (D and E) and sperm cells isolation using Percoll gradient (F and G). YFP:TUBα fusion protein is localized inside the sperm cells, whereas NLD:mCitrine fusion protein is not detected inside isolated sperm cells (white arrows). Asterisk in E shows an MGU before pollen burst (Z-stack projection). Scale bars in B–G = 20 µm. (H and I) Immunogold labeling of NLD:citrine protein in the MGU. (H) TEM pictures of mature pollen grain of the pNLD::NLD:mCitrine line focusing on the ultrastructure of the two sperm cells (SC1 and SC2). The red and green boxes are close-up views to visualize gold particles (red arrowheads) that recognize anti-citrine antibody and localize to the endo-PM of the MGU, and not the PM of sperm cells (SC PM). Scale bar in H = 0.5 µm. (I) Quantification of gold particles supporting preferential localization of NLD:mCitrine fusion protein at the endo-PM. Data are mean particle repartition (± SD) obtained from six different sperm cells, and quantification is detailed in Table S3. Vegetative cell, particles found within the vegetative cell; endo-PM, particles found in the endo-PM; interspace, particles found within the space between sperm cells PM and endo-PM; sperm cell, particles found in sperm cells (cytoplasm, nucleus, and sperm cells PM); NA, particles that could not be assigned to a particular compartment.

NLD is expressed in the vegetative cell of maize pollen, and protein localizes to the endo-PM of the MGU. (A) Schematic representation of the mature pollen grain organization, focusing on the MGU structure. The two sperm cells (yellow) are surrounded by an endo-PM (dark purple) originating from the PM of the vegetative cell. Light blue indicates the extracellular space (polysaccharide) of the MGU. (B) Characterization of NLD promoter activity (pNLD::H2B:mCherry) in mature pollen. Promoter activity (red signal) is found in the vegetative cell nucleus and not in the sperm cells nuclei. Nuclei are stained with DAPI (blue signal); white arrows indicate the sperm cells nuclei. (C) Subcellular localization of NLD protein (yellow signal) together with NLD promoter activity (red signal) in mature pollen. Confocal imaging (Z-stack projection) of mature pollen expressing pNLD::NLD:mCitrine and pNLD::H2B:mCherry constructs shows that NLD:mCitrine protein localizes at the MGU, whereas the promoter activity is found in the vegetative cell. (D–G) Sperm cells are released (white arrows) from mature pollen of pTUBα::YFP:TUBα (D and F) and pNLD::NLD:mCitrine (E and G) lines after osmotic shock (D and E) and sperm cells isolation using Percoll gradient (F and G). YFP:TUBα fusion protein is localized inside the sperm cells, whereas NLD:mCitrine fusion protein is not detected inside isolated sperm cells (white arrows). Asterisk in E shows an MGU before pollen burst (Z-stack projection). Scale bars in B–G = 20 µm. (H and I) Immunogold labeling of NLD:citrine protein in the MGU. (H) TEM pictures of mature pollen grain of the pNLD::NLD:mCitrine line focusing on the ultrastructure of the two sperm cells (SC1 and SC2). The red and green boxes are close-up views to visualize gold particles (red arrowheads) that recognize anti-citrine antibody and localize to the endo-PM of the MGU, and not the PM of sperm cells (SC PM). Scale bar in H = 0.5 µm. (I) Quantification of gold particles supporting preferential localization of NLD:mCitrine fusion protein at the endo-PM. Data are mean particle repartition (± SD) obtained from six different sperm cells, and quantification is detailed in Table S3. Vegetative cell, particles found within the vegetative cell; endo-PM, particles found in the endo-PM; interspace, particles found within the space between sperm cells PM and endo-PM; sperm cell, particles found in sperm cells (cytoplasm, nucleus, and sperm cells PM); NA, particles that could not be assigned to a particular compartment.

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