Changes in cytosolic pH and calcium of guard cells precede stomatal movements

Stomatal opening is induced by indoleacetic acid (IAA), cytokinins, and fusicoccin (FC), whereas stomatal closure is induced by abscisic acid (ABA). To test the effect of these growth regulators on guard cell cytosolic Ca²⁺ ([Ca²⁺]_cyt) and pH (pH_cyt), epidermal strips were taken from the lower side of leaves of the orchid Paphiopedilum tonsum and were loaded with acetomethoxy-esterified forms of the Ca²⁺ indicator fluo-3 or the pH indicator 2′,7′-bis(2-carboxyethyl)-5(6)carboxyfluorescein. Basal [Ca²⁺]_cyt ranged from 0.05 to 0.3 μM and was 0.22 ± 0.015 (n - 21). Increases in both [Ca²⁺]_cyt and pH_cyt were observed in guard cells after application of 10-100 μM ABA to open stomata, and these preceded stomatal closure. The increase in [Ca²⁺]_cyt ranged from 1.5- to 3-fold and was seen in 7 of 10 experiments. Guard cell alkalinization began within 2 min of ABA treatment and continued for the next 8 min. The increase ranged from 0.04 to 0.3 pH unit and was seen in 13 of 14 experiments. Guard cell [Ca²⁺]_cyt increased, whereas pH_cyt decreased after treatment of closed stomata with IAA, kinetin, or FC. In response to 50-100 μM IAA, [Ca²⁺]_cyt increased 1.5- to 2-fold in all cases, and pH_cyt decreased 0.2-0.4 unit within 5 min in 7 experiments. Within 12 min, 10-100 μM kinetin caused [Ca²⁺]_cyt to increase in 28 of 34 experiments (1.3- to 2.5-fold) and pH_cyt fell 0.1-0.4 unit in 15 of 17 treatments. The response to 10-50 μM FC was similar in both time and magnitude. These results show that stomatal opening is accompanied by an increase in [Ca²⁺]_cyt and cytosolic acidification in the guard cells, whereas stomatal closure is preceded by an increase in [Ca²⁺]_cyt and cytosolic alkalinization in the guard cells. The order of these events is still uncertain, hut changes in pH_cyt are correlated with stomatal movement, and these changes may he an important factor in the regulation of guard cell movement.