INCREASE OF FREE CA2-PROTOPLASTS IN RESPONSE TO HEAT-STRESS AS RELATED TO CA2+ HOMEOSTASIS( IN THE CYTOSOL OF PLANT)

We used the fluorescent Ca2+ indicator Indo-1 introduced into protopla sts isolated from pea mesophyll (Pisum sativum L.) and cells of sugar beet suspension culture (Beta vulgaris L.) to trace changes of Ca2+ co ncentration in the cytosol during action of stress temperatures on pro toplasts. Corresponding to three- to fourfold increase of [Ca2+], in 3 min, a rapid increase of the fluorescent signal is recorded with incr ease of temperature, the indicated increase of the signal slowing down significantly during exhaustion of the extra- and intracellular pools of Ca2+-induced by pretreatment with the high-affinity chelator BAPTA -Br2 separately or in combination with the Ca2+-ionophore ionomycin. B locking of Ca2+ transport through verapamil-sensitive Ca2+ channels wa s also manifested in slowing of the rate of [Ca2+]c increase induced b y temperature increase. In order to test the hypothesis as to possible toxic action of the high concentrations of Ca2+ arising as a result o f high-amplitude and probably uncontrolled increase of (Ca2+]c during heat stress, cells of sugar beet suspension culture incubated at physi ological (1.25 mM), high (15 mM), and close to zero (3 mM EGTA on a ba ckground of 1.25 mM CaCl2, pH 7.2) concentrations of Ca2+ in the mediu m were subjected to temperature influence. Determination of cell viabi lity from exclusion of the dye Evans Blue indicated that viability was preserved in 75-80% of the cells 2 h after heat stress if the medium contained 1.25 mM Ca2+ during both incubation and cultivation of the s uspension. Only 60-70% of the cells remained viable in the presence of 15 mM Ca2+, whereas reduction of accessible Ca2+ in the medium achiev ed with the aid of EGTA restored cell thermotolerance to the starting level. The data are discussed within the framework of ideas about the Ca2+-stat system functioning in the plant cell.