Survival Under Prolonged Cold: Duration of Stress, an Overlooked Factor in Freezing Tolerance Tests

Freezing tolerance (FT) is widely assessed in laboratories by measuring electrolyte-leakage (EL) from tissues exposed to sub-zero temperatures. This method employs cooling programs which typically result in extracellular freezing in herbaceous tissues causing cellular desiccation, a major determinant of injury manifested in EL, among other dysfunctions. Conventionally, samples are exposed to given sub-zero temperatures (certain freeze-desiccation) and thawed out after holding them for 0 to ~30 min at the target temperature before measuring EL. The higher the EL, the greater the injury. This protocol mostly ignores the effect of prolonged freezing at a given sub-zero temperature on the severity of injuries as well as the potential for post-thaw recovery. Research from the present author's laboratory and a few others shows that prolonged freezing causes greater injury than relatively shorter freezing times at the same temperature, that is, despite 'fixed' freeze-desiccation, resulting in higher EL and water-soaking, lower quantum efficiency of photosystem II and higher oxidative stress. The readers of this minireview are briefly introduced to the basic understanding of the mechanics of the EL assay, its application to assess reversible versus irreversible injury, the process of freezing (ice-nucleation, cooling rates), equilibrium freezing, and freeze-desiccation, all predominantly in the context of herbaceous and thermally homogenous tissues without barriers to ice progression. Within this framework, a data-driven discussion is shared to highlight the significance of including freezing duration (FD) in FT tests. Ideas presented herein may refine methodologies for evaluating FT that validly predict plants' responses to natural freezes. This may also incentivize the exploration of cellular/molecular mechanisms for differential responses to varying FDs at a fixed temperature.