pH gradients are not associated with tip growth in pollen tubes of Lilium longiflorum

The cytoplasmic pH of growing pollen tubes of Lilium longiflorum Thunb. was measured using the pH-sensitive fluorescent dye 2',7'-bis-(carboxyethyl)-5(6')-carboxyfluorescein and confocal fluorescence ratio imaging, The average cytoplasmic pH in the clear zone of the pollen tube tip was pH 7.11, and no consistent pH gradients were detected in the clear zone, averaging around -1.00 milli pH unit mu m(-1), or along the first 50 mu m of the tube (3.62 milli pH units mu m(-1)). In addition, no correlation was observed between the absolute tip cytoplasmic pH or the pH gradient and the pollen tube growth rates. Shifts of external pH to more acidic pH values (pH 4.5) caused a relatively small acidification by 0.18 pH units, whereas a more alkaline external pH >7.0 caused a dramatic increase in cytoplasmic pH and growth stopped immediately. Stimulation of the plasma membrane H+-ATPase by fusicoccin, resulted in an increase of tube growth but no change in cytoplasmic pH, On the other hand, vanadate (250-500 mu M), a putative inhibitor of the pump, stopped tube growth and a slight cytoplasmic alkalinisation of 0.1 pH units was observed. Vanadate also arrested fusicoccin-stimulated growth and stimulated an increased alkalinisation of around 0.2 pH units, External application of CaCl2 (10 mM) caused a small acidification of less than 0.1 pH units in the clear zone, whilst LaCl3 (250 mu M) caused slight and rather variable perturbations in cytoplasmic pH of no more than 0.1 pH units, Both treatments stopped growth. It was inferred from these data that tip-acid cytoplasmic pH gradients do not play a central role in the organisation or maintenance of pollen tube tip growth.