Freeze-desiccation due to exosmosis to extracellular ice is considered as the major stress during equilibrium freezing. This causes structural / functional perturbations in the plasma membrane which leads to leakage of cellular contents. To gain further insight into the cellular mechanism of freeze-thaw injury, four cations (K , Ca2 , Mg2 , Fe2 ), known for their critical roles in plant growth and development, were measured in the leachate from injured spinach (Spinacia oleracea L. ‘Reflect’) leaves exposed to four freezing-durations (FDs) (0.5, 3.0, 5.5, 10.5 h) at a fixed temperature. In general, leakage of K , Ca2 , Mg2 increased incrementally at longer FDs and leaves sustained greater water-soaking after prolonged freezing. Data indicated a higher abundance of reactive oxygen species (O2− and H2O2) in leaves with greater injury at longer FDs. PSII efficiency was incrementally compromised at longer FDs as determined by chlorophyll fluorescence (Fv/Fm). Total electrolyte leakage from tissues right-after-thaw versus those allowed to recover for 6-d revealed that injury at 0.5 or 3 h FDs was recoverable, but leaves were irreparably injured at 5.5 or 10.5 h FDs. K was the most abundant cation in leachate. Data suggests that K -leakage can be used as proxy for total electrolyte-leakage in determining LT50 and can serve as an ionic marker to delineate moderate (recoverable) versus severe (non-recoverable) freeze-injury. Ca2 - and K -leakage data, together, are compatible with an earlier conjecture that leaked K ions replace membrane-associated Ca2 during post-thaw. It is proposed that thus structurally weakened plasma membrane, together with inhibited active transport functions of plasma membrane (noted in previous studies) lead to enhanced K -leakage from more severely freeze-injured leaves. Unlike other cations, Fe2 -leakage was indeed lower in the injured (0.5 FD) leaves compared to unfrozen control. Moreover, Fe2 was undetectable in the leachate at longer FDs. It is hypothesized that such lack of Fe2 in the leachate could result from Fenton reaction in injured tissues which converts soluble Fe2 into insoluble Fe3 . Enhanced Mg2 -leakage at greater freeze-injury suggests structural/functional impairment of chlorophyll / chloroplast complex, resulting in reduced quantum yield of PSII.
