# New Insights in Potato Leaf Freezing by Infrared Thermography
## 4 Discussion
After this second freezing event when leaves had thawed, they were water soaked and limp, which indicated frost damage. Given that during the first freezing event extra-tissue water has frozen, during this second freezing event remaining supercooled water inside the living cells must have frozen. This suggests an intracellular freezing process. Preliminary cryo-microscopic results give evidence that mesophyll cells of potato do not show a freezing cytorhysis that is commonly the result of freeze dehydration but freeze intracellularly during the second freezing event [30]. The time span between the two freezing events in potato leaves was usually 10 min but could occasionally be up to 78 min. This may suggest that the second freezing exotherm associated with intracellular freezing is a stochastic event. This is typical of most freezing processes originating from breakdown of supercooling as for instance seen in xylem parenchyma cells [13] or supercooling buds [16; 17; 18]. _S. tuberosum_ is unable to frost harden and gets frost killed around \(-3\)\({}^{\circ}\)C [25]. None of a number of commonly cultivated cultivars of _S. tuberosum_ examined could survive below \(-2.5\)\({}^{\circ}\)C [31]. Our results show that ice formation (first freezing event) per se is not injurious, but consequent processes, i.e., a second freezing exotherm, is responsible for frost damage. Ice nucleation temperature may be more relevant for frost survival than actual frost severity. Still, under field conditions, in leaf and stem tissue of potato ice nucleates at \(-0.5\) to \(-3.0\)\({}^{\circ}\)C [21; 26] being a stochastic event. Thus, control of ice nucleation as suggested earlier [27] must be considered a key for frost protection of potato. The following are available online at [[http://www.mdpi.com/2076-3417/9/5/819/s1](http://www.mdpi.com/2076-3417/9/5/819/s1)]([http://www.mdpi.com/2076-3417/9/5/819/s1](http://www.mdpi.com/2076-3417/9/5/819/s1)). Video S1: First exothermic event: IDTA sequence obtained on a leaf of _S. tuberosum_ during a controlled freezing treatment at a cooling rate of 3 K\(\cdot\)h\({}^{-1}\). Whitening shows the spreading of ice after ice nucleation in the leaf petiole over the entire leaf blade. This first exotherm lasted approximately 1 min (playback speed 4x), Video S2: Second exothermic event: IDTA sequence obtained on a leaf of _S. tuberosum_ during a controlled freezing treatment at a cooling rate of 3 K\(\cdot\)h\({}^{-1}\). The second freezing event is diffuse without a distinct starting point (playback speed 4x). The video depicts only a part of the second freezing event. [PERSON] and [PERSON] designed the research (conceptualization), [PERSON] and [PERSON] were responsible for the performance of the investigation, acquisition of data and analysis and interpretation of the results. [PERSON] and [PERSON] were responsible for the original draft preparation. G.N. acquired funding. This research was funded by the Austrian Science Fund FWF, grant number P30139-B32. We are grateful to the staff of the Botanical Garden of the University of Innsbruck for the cultivation of potato plants. [PERSON], [PERSON] and [PERSON] from the University of Salzburg are thanked for technical support with the construction of the acryl glass lid for the chest freezer. The authors declare no conflict of interest.