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Environmental and Stress Physiology

In Vivo Perturbation of Membrane-Associated Calcium by Freeze-Thaw Stress in Onion Bulb Cells ¹

Simulation of This Perturbation in Extracellular KCl and Alleviation by Calcium

Department of Horticulture, University of Wisconsin, Madison, Wisconsin 53706

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 Ca²⁺. 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 Ca²⁺ 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°C or –11.5°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°C; irreversible) caused a specific and substantial loss of membrane-associated Ca²⁺ compared to control. Loss of membrane-associated Ca²⁺ caused by moderate stress (–8.5°C; reversible) was much less relative to –11.5°C treatment. Ion efflux and Ca²⁺-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 CaCl₂. Our results suggest that the loss of membrane-associated Ca²⁺, in part, plays a role in initiation and progression of freezing injury.

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