Short versus prolonged freezing differentially impacts freeze - thaw injury in spinach leaves: mechanistic insights through metabolite profiling

Plant tissues subjected to short or prolonged freezing to a fixed sub-freezing temperature are expected to undergo similar freeze-desiccation but the former causes substantially less injury than the latter. To gain metabolic insight into this differential response, metabolome changes in spinach (Spinacia oleracea L.) leaves were determined following short-term (0.5 and 3.0 h) vs. prolonged freezing (5.5 and 10.5 h) at -4.5 degrees C resulting in reversible or irreversible injury, respectively. LD50, the freezing duration causing 50% injury, was estimated to be similar to 3.1 h and defined as the threshold beyond which tissues were irreversibly injured. From 39 identified metabolites, 19 were selected and clustered into 3 groups: (1) signaling-related (salicylic acid, aliphatic and aromatic amino acids), (2) injury-related (GABA, lactic acid, maltose, fatty acids, policosanols, TCA intermediates) and (3) recovery-related (ascorbic acid, alpha-tocopherol). Initial accumulation of salicylic acid during short-term freezing followed by a decline may be involved in triggering tolerance mechanisms in moderately injured tissues, while its resurgence during prolonged freezing may signal programmed cell death. GABA accumulated with increasing freezing duration, possibly to serve as a 'pH-stat' against cytoplasmic acidification resulting from lactic acid accumulation. Mitochondria seem to be more sensitive to prolonged freezing than chloroplasts since TCA intermediates decreased after LD50 while salicylic acid and maltose, produced in chloroplasts, accumulate even at 10.5-h freezing. Fatty acids and policosanols accumulation with increasing freezing duration indicates greater injury to membrane lipids and epicuticular waxes. Ascorbic acid and alpha-tocopherol accumulated after short-term freezing, supposedly facilitating recovery while their levels decreased in irreversibly injured tissues.