TRIACONTANOL AND L(+) ADENOSINE AFFECT STOMATAL MOVEMENT AND FREE CYTOSOLIC CALCIUM-CONCENTRATION IN ISOLATED GUARD-CELL PROTOPLASTS OF VICIA-FABA

L(+) Adenosine given to floated epidermal peels of Vicia Saba during 2 h produced an enhancement of stomatal aperture over a wide range of concentrations (1 pM-100 mu M), such effect being particularly marked in darkness (maximum response at 10 nM). Lanthanum (1 mM) inhibited L(+) adenosine-induced stomatal opening in darkness. A mixture of 100 mu M verapamil and 50 mu M nifedipine alone had almost no effect on stomatal behavior as compared with the control, but, in the presence of L(+) adenosine, this mixture completely reversed the L(+) adenosine-elicited stomatal opening. In darkness, triacontanol markedly stimulated stomatal opening in a concentration-dependent manner over two orders of magnitude (0.1-10 mu g l(-1)), almost reaching a plateau between 10 and 100 mu g l(-1). On the contrary, similar concentrations of triacontanol slightly inhibited stomatal opening in light, with the lowest effect at the highest concentration. Isolated guard cell protoplasts (GCPs) loaded with the calcium-sensitive fluorescence dye Indol and treated with L(+) adenosine (1 mu M) showed a rapid and low increase (6%) in cytosolic calcium concentration ([Ca+2](c)) over control values (ca. 240 nM). This was followed by a pronounced decrease in [Ca+2](c) at 1, 5 and 10 min with a minimum of ca. 150 nM at 5 min. At the same times a lower concentration of L(+) adenosine (10 nM) had a less pronounced effect, showing a 10% difference with respect to controls. A 30 min preincubation of isolated protoplasts with calcium antagonist La+3 (0.5 mM) showed a tendency to attenuate the abovementioned L(+) adenosine effect on [Ca+2](c). Calcium channel blockers verapamil (100 mu M) plus nifedipine (50 mu M) had no effect on the L(+) adenosine action on [Ca+2](c). Treatment of GCPs with 10 mu g l(-1) triacontanol also provoked a decrease in [Ca+2](c) (approximately 20%); this level did not return to its basal value after 10 min incubation.