Calcium, Reactive Oxygen Species, and Pollen Tube Growth

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0087517RobinsonThe study of tip growth in general and pollen tube growth in particular has been an area of considerable progress in recent years and more and more sophisticated techniques have been brought to bear on the problem. One important conclusion that has emerged is that the pollen tube tip is a highly dynamic structure. Elongation is not steady, but oscillatory, and a number of important physiological variables are also oscillatory, including cytosolic Ca2+, cytosolic pH, net Ca2+ influx, net H+ influx, and net K+ influx. The phase relationships of these variables with respect to growth oscillations are complex, and it is not at all clear how the oscillations are established and maintained, and what are the feedback mechanisms that connect the variables.It is proposed to directly detect vesicle secretion and determine its timing relative to elongation. Attempts to use electrical methods for measuring secretion have proved fruitless, so optical methods are now employed. By these means, accurate measurements of the timing of vesicle secretion will be made.A substantial fraction of the Ca2+ that is required for the oscillations in cytosolic Ca2+ must come from intracellular stores, and the most likely sources are the secretory vesicles themselves. It is proposed to isolate the vesicles and directly determine their ability to take up and release Ca2+. The physiological controls of these events will also be determined.In order to characterize putative Ca2+ channels in pollen tubes, whole cell clamping of pollen grain protoplasts will be done. The working model posits the existence of turgor pressure oscillations, which appear to underlie the growth oscillations. Ultra-high resolution optical measurement by computer vision will be used to determine if the diameter of pollen tubes oscillates due to turgor oscillations. If so, we will determine the phase of these oscillations with respect to other variables.Finally, the production of ROS at the growing tip will be directly observed, using a luminescent probe, pholasin, which increases its light emission by several orders of magnitude in the presence of ROS.