Fig. 1

Gross morphological changes in HS-treated plant cells do not match hallmarks of apoptosis. a FM4-64 and SO dyes were used to visualize all cells and cells with permeabilized PM, respectively. BY-2 cells were imaged under control conditions (No HS) or after a 10-min HS at 55 °C and were stained according to protocols i and ii, respectively (see “Sytox Orange and FM4-64 staining”). Scanning was performed within 1 h post-HS. The arrows indicate PM; note the PM (red dotted line) being tightly pressed to the cell wall (white dotted line) under control conditions and detached under stress conditions. b Higher magnification of the areas indicated with arrows in a. c BY-2 cells expressing green fluorescent protein (GFP) fused to nuclear localization signal (NLS) were subjected to the same treatments as in a. Images represent maximum intensity projections of z-stack scans acquired 6 h post-HS. d Quantification of nuclear area in samples shown in c. Data from three independent experiments, with ≥ 142 cells counted per treatment. Student’s t test, *p < 0.005. DIC, differential interference contrast microscopy. n, nucleus. IQR, interquartile range. Scale bars, 20 μm (a, c) or 5 μm (b)