CHANGES IN INTRACELLULAR PH DURING ZOOSPOROGENESIS IN PHYTOPHTHORA-CINNAMOMI

Changes in intracellular pH (pH(i)) have been examined during zoosporo genesis in Phytophthora cinnamomi in order to elucidate the molecular mechanisms that bring about initiation of the orderly cleavage of the sporangium. By microinjecting a dextran-conjugated pH-sensitive fluore scent indicator, 2',7'-bis(2-carboxy ethyl)-5 (6)-carboxyfluorescein ( BCECF dextran), and using fluorescence ratio image analysis, the pH(i) of a sporangium was monitored throughout the process of zoosporogenes is. The pH(i) of mature sporangia was found to remain constant at pH 6 .84 +/- 0.05 (mean +/- SE, n = 6) at room temperature (24 degrees C), but increased to 7.04 +/- 0.04 (p < 0.001) during a 20 min cold treatm ent at 19 degrees C. As the process of zoosporogenesis proceeded, the increased level of pH(i) was maintained for 30-40 min after initiation of the cold shock. Zoosporogenesis was blocked when the pH(i) of the sporangium was held constant by microinjecting with 10 mM HEPES buffer (pH 7.0) before cytoplasmic cleavage was induced by a cold shock. In contrast, no inhibitory effect was observed when sporangia were microi njected with HEPES buffer after the cold shock. These results indicate that initiation of cytoplasmic cleavage requires a rise in pH(i).