Using leaf epidermis from Vicia faba, we tested whether auxin-induced stomatal opening was initiated by auxin-induced ethylene synthesis. Epidermis was dark-incubated in buffered KNO3 containing 0.1 mM alpha-napthalene acetic acid or 1 mM indole-3-acetic acid. Maximum net opening was ca. 4 micron after 6 h. Opening was reversed by 20 microM ABA, 0.1 mM CaCl2. 1-Aminocyclopropane carboxylic acid (ACC) synthase catalyzes synthesis of ACC, the immediate precursor to ethylene. Auxin-induced stomatal opening was fully inhibited by 10 microM 1-aminoethoxyvinylglycine (AVG), an ACC synthase inhibitor. In solutions containing AVG, auxin-induced opening was restored in a concentration-dependent manner by exogenous ACC, but not in control solutions lacking an auxin. ACC-mediated reversal of AVG-inhibition of stomatal opening was inhibited by alpha-aminoisobutyric acid (AIB), an inhibitor of ACC oxidase, the last enzyme in the ethylene biosynthetic pathway, by 10 microM silver thiosulfate (STS), an inhibitor of ethylene action, and by 20 microM ABA, 0.1 mM CaCl2. CoCl2, an inhibitor of ethylene synthesis, also inhibited auxin-induced opening. Both STS and CoCl2 inhibited opening induced by light or by fusicoccin, but neither light- nor fusicoccin-induced opening was inhibited by AVG. These results support the hypothesis that auxin-induced stomatal opening is mediated through auxin-induced ethylene production by guard cells.

Cuard cell protoplasts isolated from leaves of Nicofiana g h c a (Craham) were cultured. Conditions were sought that would maximize survival and maintain cells i n their differentiated state. Temperature was an important determinant of survival, growth, and differentiation. As temperatures were increased from 24 to 32'C, survival for 1 week in culture increased from approximately 20% to approximately 80% of cells used to initiate cultures. At all of these temperatures, approximately 90% of surviving cells divided to form callus tissue. "Footprint" areas of cells cultured for 1 week at 32°C increased almost 30-fold. Cells cultured for 1 week at 34 t o 40°C also survived i n high percentages (approximately 80%), but they retained a morphology similar to that of guard cells and they did not divide. Footprint areas of cells cultured for 1 week at 38°C increased 6-fold. Cells cultured at 36 t o 4OoC in media containing 0.1 or 1.0 W M abscisic acid survived in high percentages and did not divide. At 38°C their footprint areas did not increase, but cells so cultured increased in diameter when treated with fusicoccin. Morphologies and electrophoretic profiles of total sodium dodecyl sulfate-extractable proteins suggest that cells cultured at 38°C in media containing abscisic acid remain differentiated. ~-a-(2-Aminoethoxyviny1)-glycine reduced survival t o <1 O/O at 26 or 32°C but had no effect at 38OC. At lower temperatures, cell growth and survival appear to be ethylene dependent.