Phosphoinositides and phosphatidic acid regulate pollen tube growth and reorientation through modulation of [Ca2+]c and membrane secretion

The maintenance of a calcium gradient and vesicle secretion in the apex of pollen tubes is essential for growth. It is shown here that phosphatidylinositol-4,5-bisphosphate (PIP2) and D-myo-inositol-1,4,5-trisphosphate (IP3), together with phosphatidic acid (PA), play a vital role in the regulation of these processes. Changes in the intracellular concentration of both PIP2 and IP3 (induced by photolysis of caged-probes), modified growth and caused reorientation of the growth axis. However, measurements of cytosolic free calcium ([Ca2+](c)) and apical secretion revealed significant differences between the photorelease of PIP2 or IP3. When released in the first 50 mu m of the pollen tube, PIP2 led to transient growth perturbation, [Ca2+](c) increases, and inhibition of apical secretion. By contrast, a concentration of IP3 which caused a [Ca2+](c) transient of similar magnitude, stimulated apical secretion and caused severe growth perturbation. Furthermore, the [Ca2+](c) transient induced by IP3 was spatially different causing a pronounced elevation in the sub-apical region. These observations suggest different targets for the two phosphoinositides. One of the targets is suggested to be PA, a product of PIP2 hydrolysis via phospholipase C (PLC) or phospholipase D (PLD) activity. It was found that antagonists of PA accumulation (e.g. butan-1-ol) and inhibitors of PLC and PLD reversibly halted polarity. Reduction of PA levels caused the dissipation of the [Ca2+](c) gradient and inhibited apical plasma membrane recycling. It was also found to cause abolition of the apical zonation. These data suggest that phosphoinositides and phospholipids regulate tip growth through a multiple pathway system involving regulation of [Ca2+](c) levels, endo/exocytosis, and vesicular trafficking.