EXTENSOR PROTOPLASTS OF THE PHASEOLUS-PULVINUS - LIGHT-INDUCED SWELLING MAY REQUIRE EXTRACELLULAR CA2-INDUCED SHRINKING INOSITOL 1,4,5-TRISPHOSPHATE-INDUCED CA2+ MOBILIZATION( INFLUX, DARK)

The photonastic upward movement and scotonastic downward movement of t he primary leaf of Phaseolus coccineus L. depends on ion fluxes across the plasma membrane of extensor and flexor cells of the laminar pulvi nus. Extensor protoplasts cultured in 0.4 M mannitol, 10 mM KCI, 1 mM CaCl2,, and 5 mM MES-KOH buffer pH 6 were found to swell upon switchin g on white fight at the end of a 15 h dark period and to shrink upon s witching off the light at the end of the following 9 h fight period, b ehaviour consistent with that expected in the cells of intact plants. Light-induced swelling requires Ca2+ in the surrounding medium. Both t he Ca2+ channel blocker verapamil and La3+ inhibited this reaction, wh ereas TMB-8, an inhibitor of intracellular Ca2+ transport, had no effe ct. When the Ca2+ ionophore A 23187, the Ca2+ channel agonist Bay K-86 44, or thapsigargin, an inhibitor of Ca2+-ATPases at endomembranes, wa s added to the medium, extensor protoplasts swelled in the dark. These results suggest that in extensor protoplasts light opens Ca2+ channel s in the plasma membrane and that the influx of extracellular Ca2+ res ults in an increased cytoplasmic Ca2+ concentration which is sufficien t to mimic the light-on signal in activating or deactivating the ion t ransporters required for swelling. Dark-induced shrinking occurred in Ca2+-free medium. It was not inhibited by verapamil, but was by TMB-8. Both neomycin and Li+, substances which are known to inhibit the phos phoinositide pathway of transmembrane signalling, inhibited dark-induc ed shrinking. Myo-inositol nullified the Li+ inhibition of dark-induce d shrinking. Neither A 23187 nor Bay K-8644 induced shrinking in the l ight, 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 phosphoinosit ide hydrolysis and Ca2+ release from internal stores. In addition to t he inositol 1,4,5-trisphosphate (IP3)-induced increase of the cytoplas mic Ca2+ concentration, further events depending on the light-off sign al appear to be required for shrinking.