NLD promoter activity in the vegetative cell starts at the late bicellular stage, just before formation of the two sperm cells. (A) Calibration of anther size according to pollen development stages in A188 background. 35 anthers were measured for each stage. (B) Tassel branches with male flowers at late bicellular stage (top) when NLD promoter activity is starting, and mature pollen stage (bottom). (C and D) Temporal analysis of NLD promoter activity (pNLD::H2B:mCherry) at different stages of pollen development, between early bicellular and mature pollen stages. NLD promoter activity (red signal) starts at late bicellular stage, probably just before or during the second pollen mitosis. White arrows point at the vegetative nucleus, red arrowheads point at the generative cell nucleus, and white arrowheads point at the sperm cell nuclei. The red asterisk indicates a second pollen mitosis in progress. Determination of pollen stages by epifluorescence imaging of pollen after DAPI staining (D). Scale bars = 500 µm (B); 50 µm (C and D).