Extensor protoplasts of thePhaseoluspulvinus: light-induced swelling may require extracellular Ca2+influx, dark-induced shrinking inositol 1,4,5-trisphosphate-induced Ca2+mobilization

Abstract

The photonastic upward movement and scotonastic downward movement of the primary leaf of Phaseolus coccineus L. depends on ion fluxes across the plasma membrane of extensor and flexor cells of the laminar pulvinus. Extensor protoplasts cultured in 0.4 M mannitol, 10 mM KCI, 1 mM CaCl 2 , and 5 mM MES-KOH buffer pH 6 were found to swell upon switching on white light at the end of a 15 h dark period and to shrink upon switching off the light at the end of the following 9 h light period, behaviour consistent with that expected in the cells of intact plants. Light-induced swelling requires Ca 2+ in the surrounding medium. Both the Ca 2+ channel blocker verapamil and La 3+ inhibited this reaction, whereas TMB-8, an inhibitor of intracellular Ca 2+ transport, had no effect. When the Ca 2+ ionophore A 23187, the Ca 2+ channel agonist Bay K-8644, or thapsigargin, an inhibitor of Ca 2+ -ATPases at endo-membranes, was added to the medium, extensor protoplasts swelled in the dark. These results suggest that in extensor protoplasts light opens Ca 2+ channels in the plasma membrane and that the influx of extracellular Ca 2+ results in an increased cytoplasmic Ca 2+ concentration which is sufficient to mimic the light-on signal in activating or deactivating the ion transporters required for swelling. Dark-induced shrinking occurred in Ca 2+ -free medium. It was not inhibited by verapamil, but was by TMB-8. Both neomycin and Li + , substances which are known to inhibit the phosphoinositide pathway of transmembrane signalling, inhibited dark-induced shrinking. Myo-inositol nullified the Li + inhibition of dark-induced shrinking. Neither A 23187 nor Bay K-8644 induced shrinking in the light, but were able to nullify the inhibitory effect of TMB-8 on dark-induced shrinking. These results suggest that, in extensor protoplasts, the shrinking signal 'light off' is transduced through phosphoinositide hydrolysis and Ca 2+ release from internal stores. In addition to the inositol 1,4,5-trisphosphate (IP 3 )-induced increase of the cytoplasmic Ca 2+ concentration, further events depending on the light-off signal appear to be required for shrinking.