Fig. 6

85 °C HS prevents protoplast shrinkage in necrotic cells by fixing cellular content. a Cells stained with FDA were exposed to 55 °C or 85 °C and mounted in the normal growth medium or hypertonic medium supplemented with 500 mM D-mannitol. As expected, hypertonic medium induced plasmolysis in the non-stressed cells (No HS) and had no effect on protoplasts shrunken after 55 °C HS. Importantly, high osmotic pressure failed to induce protoplast shrinkage in cells exposed to 85 °C HS, thus confirming that cells were fixed by the high-temperature treatment. b The extent of protoplast shrinkage upon HS followed by increased osmotic pressure was quantified as the percentage of the cell area (outlined by the white dotted line in a) occupied by the protoplast area (outlined by the red dotted line in a). Student’s t test, n = 3 replicates (each replicate representing ≥ 34 cells), *p < 0.0001, ns, non-significant. IQR, interquartile range. Scale bars, 20 μm