Comparison of physiological responses of guard cell protoplasts of Nicotiana glauca isolated from leaves collected before dawn or at midday

We observed that guard cell protoplasts isolated from leaves collected at midday from Nicotiana glauca Graham (tree tobacco) did not give the same physiological responses to light as those isolated from leaves collected in early morning. Based on that observation, we attempted to determine whether there were significant differences between the physiological responses of guard cell protoplasts isolated from leaves collected before dawn (with closed stomata) and those isolated from leaves collected at midday (with open stomata). We isolated guard cell protoplasts from leaves collected before dawn and at midday and compared (1) rates of red and blue light‐induced pH changes in weakly buffered media caused by changes in their metabolism, (2) their rates of oxygen consumption in darkness and oxygen evolution in light and (3) relative rates of decay of variable chlorophyll a fluorescence in their chloroplasts. Studies with the vital stain fluorescein diacetate failed to reveal any significant differences in the viabilities of protoplast preparations from leaves collected before dawn and at midday. Furthermore, protoplasts from leaves collected at these times swelled to similar extents in an osmotic medium containing 10 µM fusicoccin and 5 mM KCI. Nevertheless, rates of light‐induced pH changes, rates of oxygen consumption and evolution and rates of decay of variable chlorophyll a fluorescence were all lower in preparations of guard cell protoplasts from leaves collected at midday than in preparations from leaves collected before dawn. Initial volumes of guard cell protoplasts isolated from leaves collected at midday were 150% of those of guard cell protoplasts isolated from leaves collected before dawn. We suggest that the differences in responses of guard cell protoplasts isolated from leaves collected before dawn and at midday may be caused by (1) nonoptimal isolation conditions for guard cell protoplasts prepared from leaves collected at midday, (2) the lower surface‐to‐volume ratio of guard cell protoplasts isolated from leaves collected at midday or (3) diurnal and/or circadian regulation of guard cell metabolism over the course of a day.