Effect of short-term versus prolonged freezing on freeze-thaw injury and post-thaw recovery in spinach: Importance in laboratory freeze-thaw protocols

- The LT50 in 'Bloomsdale' spinach is −5.5 °C by a conventional artificial freezing test.
- Negative 4 and −4.5 °C became LT50 after 10.5 and 5.5 h of freezing, respectively.
- PSII efficiency decreased with prolonged freezing at a given temperature.
- Oxidative stress increased with prolonged freezing at a given temperature.
- Lesions at −4.5 °C changed from reversible injury to irreversible when freezing duration was increased only by 2.5 h.

'Duration of freezing' at a given treatment temperature can significantly influence the laboratory evaluation of plant freeze-thaw stress tolerance (FTST) by potentially affecting the extent of freeze-thaw injury and, therefore, also the ability of post-thaw recovery. The objective of this study was to compare the extent of freeze-thaw injury, estimated by various physiological indicators [electrolyte leakage, photosystem II efficiency (Fv/Fm), accumulation of reactive oxygen species (O2− and H2O2), and malondialdehyde (MDA) accumulation], in the leaves of spinach (Spinacia oleracea L.) when exposed for various durations (0.5, 3.0, 5.5, and 10.5 h) of freezing. Leaf LT50 was first determined (approximately −5.5 °C) and two sub-lethal temperatures, −4.0 and −4.5 °C, were selected as the freezing stress. The ability of post-thaw recovery was compared in leaves stressed for shorter versus longer durations at −4.5 °C. All measurements indicated that injury progressively increased with longer freezing durations. While no injury was observed (based on the electrolyte leakage) at −4 °C for 0.5 h, tissues were ∼50% injured by the same temperature when frozen for 10.5 h. Moreover, −4.5 °C stress appears as sub-lethal at 3 h of freezing (∼30% injury), but, injury doubled (and exceeded the LT50 threshold) after additional 2.5 h at this temperature. Finally, leaves were identically injured to a recoverable point (∼30% injury) when frozen for 3 h at either −4 °C or −4.5 °C while 5.5 h of freezing at −4.5 °C caused irreversible injury but no change for −4.0 °C. Our results and corresponding discussion highlight the importance of considering 'duration of freezing' as one of the factors for objective interpretation of FTST/LT50 data derived from artificial freeze-thaw tests.