Protoplasmic Swelling as a Symptom of Freezing Injury in Onion Bulb Cells

Abstract

Freezing injury, in onion bulb tissue, is known to cause enhanced K' efflux accompanied by a small but significant loss of Ca2" following incipient freezing injury and swelling of protoplasm during the postthaw secondary injury. The protoplasmic swelling of the cell is thought to be caused by the passive influx of extracellular K' into the cell followed by water uptake. Using outer epidermal layer of unfrozen onion bulb scales (Allium cepa L. cv Big Red), we were able to stimulate the irreversible freezing injury symptoms, by bathing epidermal cells in 50 millimolar KCI. These symptoms were prevented by adding 20 millimolar CaCI2 to the extracellular KCI solution. Our results provide evidence that loss of cellular Ca2' plays an important role in the initiation and the progression of freezing injury.

In Vivo Perturbation of Membrane-Associated Calcium by Freeze-Thaw Stress in Onion Bulb Cells

“…The uniqueness of calcium as a second messenger in mediating many metabolic responses and in membrane stability in plant cells is well established (6,9,10,23). Furthermore, our earlier studies have suggested a role of calcium in freezing injury (1,2). Therefore, in the present study, we examined the role of calcium rather than other divalent cations.…”

“…Medium-sized onion bulbs were frozen by cooling at the rate of 1.5°C/h, to either -8.5°C ± 0.5°C or -11.5°C ± 0.5°C, and then were thawed slowly over ice (2,15). To prevent supercooling, bulbs were nucleated at -1°C.…”

“…The third scale (counting from outermost) of thawed onion bulbs was visually examined for water soaking and later used for microscopic observations of Ca2+-CTC fluorescence. This was then used to measure ion efflux using the conductivity method (2,16). Two pieces (1 x 1 cm) of the third scale were vacuum infiltrated in 20 ml of distilled-deionized H20 and were shaken for 1 h. Conductivity of effusate was measured using a conductivity meter (YSI model 32), and it was again recorded for the same tissue at room temperature after it was heat-killed.…”

“…From these observations, Palta and Li (19) found to regulate ion transport processes (6,(8)(9)(10). Arora and Palta (2) suggested that removal of Ca2" from the membrane by extracellular K+ results in perturbation of structural integrity of plasma membrane and induces cytological aberration similar to that caused by freeze-thaw stress, namely, protoplasmic swelling. In these studies, it was also demonstrated that elevated concentrations ofCa2e in extracellular solution protected cell membrane against the adverse effect of K+ and prevented the cytological alteration from occurring.…”

“…Due to this leakage, cells after thawing experience a high extracellular K+ concentration. The first microscopic symptom observed in irreversibly injured onion bulb scale cells was protoplasmic swelling (2,19). In parallel with K+ efflux, a small but significant Ca2' efflux was also noted, which increased with progress of injury (16).…”