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

Incipient freeze-thaw stress in onion bulb scale tissue is known to cause enhanced efflux of K(+), along with small but significant loss of cellular Ca(2+). During the post-thaw period, irreversibly injured cells undergo a cytological aberration, namely, ;protoplasmic swelling.' This cellular symptom is thought to be caused by replacement of Ca(2+) from membrane by extracellular K(+) and subsequent perturbation of K(+) transport properties of plasma membrane. In the present study, onion (Allium cepa L. cv Sweet Sandwich) bulbs were slowly frozen to either -8.5 degrees C or -11.5 degrees C and thawed over ice. Inner epidermal peels from bulb scales were treated with fluorescein diacetate for assessing viability. In these cells, membrane-associated calcium was determined using chlorotetracycline fluorescence microscopy combined with image analysis. Increased freezing stress and tissue infiltration (visual water-soaking) were paralleled by increased ion leakage. Freezing injury (-11.5 degrees C; irreversible) caused a specific and substantial loss of membrane-associated Ca(2+) compared to control. Loss of membrane-associated Ca(2+) caused by moderate stress (-8.5 degrees C; reversible) was much less relative to -11.5 degrees C treatment. Ion efflux and Ca(2+)-chlorotetracycline fluorescence showed a negative relationship. Extracellular KCl treatment simulated freeze-thaw stress by causing a similar loss of membrane-associated calcium. This loss was dramatically reduced by presence of extracellular CaCl(2). Our results suggest that the loss of membrane-associated Ca(2+), in part, plays a role in initiation and progression of freezing injury.