Cytoplasmic free Ca2+ dynamics in single tomato (Lycopersicon esculentum) protoplasts subjected to chilling temperatures

Protoplasts were isolated from leaves of tomato seedlings ( Lycopersicon esculentum Mill., cv. Marmande) at the 2nd to 4th true leaf stage and were loaded with the calcium binding tetraacetoxymethyl⁺] ester of the fluorescent stilbene chromophore, Fura 2. Although the loading efficiency of the dye in these protoplasts was low, many protoplasts loaded only in the cytosol were always obtained. Changes in the cytosolic calcium concentration (Ca²⁺]_cyt) were determined in single protoplasts in a temperature-controlled perfusion chamber by use of fluorescence photometry microscopy after excitation at 340 and 380 nm. When the protoplasts were subjected to chilling temperatures (10–15°C) by a circulating solution, the Ca²⁺]_cyt increased in 64% of the analysed protoplasts. Depending on the initial resting level of Ca²⁺]_cyt, three main types of kinetics were obtained in these protoplasts: (1) In 21% of the protoplasts, Ca²⁺]_cyt increased to a maximum within 10–20 s from the start of temperature decrease, followed by a fast decrease; (2) in 11% of the protoplasts, the Ca²⁺]_cyt both increased and decreased somewhat slower; and (3) in 32% a constant increase of Ca²⁺]_cyt was obtained 1 min after the start of temperature decrease.