Heat stress during the bicellular stage decreases content of centromeric histones in maize. Marker lines expressing centromeric histones (CENH3) specifically in sperm cells (GEX3p:mRuby3-CENH3) were grown in control conditions (25 °C/21 °C light/dark period) until they reached the bicellular stage of pollen development and then submitted to heat stress (35 °C/25 °C light/dark period) for 48 h. A parallel set of marker line plants was maintained under optimal conditions and used as controls. A) Confocal images showing CENH3 in mature pollen grains from NS and (B) HS plants. Insets show enlarged sperm cells containing CENH3 signals. Insets were magnified 3×. C) Intensity quantification of mRuby3-CENH3 in both conditions. Asterisks indicate a significant difference at P < 0.001; 1-tailed t test comparing NS and HS samples. Data are presented as the mean ± Sd. Scale bars = 50 μm.